Fabrication of surface enhanced Raman Scattering (SERS

Fabrication of surface enhanced Raman Scattering (SERS) substrates made from nanoparticle printing inks for detection of biological molecules

A Thesis

Submitted to the Faculty

of

Drexel University

by

Manuel Alejandro Figueroa

in partial fulfillment of the

requirements for the degree

of

Doctor of Philosophy

August 2012

ii

© Copyright 2012

Manuel Alejandro Figueroa. All Rights Reserved.

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Dedications

To my parents, Olga Beneas and Jesus Maria Figueroa, who have always encouraged me to pursue my educational goals and gave me the tools to succeed. I will do my best to do the same

for my precious daughter Sofia.

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Acknowledgments

The completion of my research was only possible because of the many relationships I have

formed with professors, graduate students, staff members and industry partners. Their

contributions not only helped my research progress but more importantly my personal growth

as a researcher and teacher matured.

I would like to thank my co-advisors, Dr Tyagi and Dr Pourrezaei for their guidance in this work. I

am very appreciative of Dr Tyagi’s unspoken open door policy because it meant I could come up

to see him at any time of day to discuss new results with him. He was very gracious with his time

and would carefully explain things on paper so I could get visuals of the physical concepts. I

know at times I was ill prepared but he would always give me time to gather my thoughts. I also

appreciate the freedom he gave me to explore new things in the lab. Over time I became better

at predicting the outcome before conducting experiments over and over again.

I am very thankful for Dr Pourrezaei introducing me to the field of nanotechnology, because I

hope I can make it a focal point of my career. His continual motivation to seek novel ways to

combine nanoparticles with biology has equipped me with an arsenal of ideas that I intend to

explore further. I also appreciated that I could always talk to him about personal matters

including family, soccer, etc.

Dr Allen has been a very important mentor during all of my years here at Drexel and helped me

stay motivated even during some rough patches. He always knows the right thing to say and

helped me keep things in perspective. I admire the way he teaches his classes and I have tried

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to emulate his style in class. I have also enjoyed many of the insightful conversations we had in

the classes that we taught together.

I would like to thank Greg Jablonski from PChem Associates and Yuri Didenko from UT Dots

whose expertise in nanoparticle colloids was invaluable in making key decisions for experiments

and developing explanations for observed results. I am also extremely grateful for the multiple

silver and gold nanoparticle ink samples they provided and how forthcoming they were when

we suggested slight modifications to their recipes.

Finally, I give thanks to all of the students listed below who participated in my lab. Their curiosity

and ambition to work in the lab gave me the everyday motivation to continue pushing forward.

Edward Keough, Ravi Chokshi, Sanaz Rezaei, Vera Mayo, Burcu Erdogan, Samuel Park, Jennifer

Pillion, William Stephenson, Pedram Niknam, Saran Kokar, Stephen Schraer, Johnny Tang and

Sina Nassiri.

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Table of Contents

LIST OF TABLES ................................................................................................................................ IX

LIST OF FIGURES ................................................................................................................................ X

ABSTRACT ...................................................................................................................................... XIII

CHAPTER 1: INTRODUCTION ..............................................................................................................1 1.1 OVERVIEW OF THESIS RESEARCH .............................................................................................. 2

1.1.1 Specific aims ..................................................................................................................... 3 1.2 THESIS ORGANIZATION ................................................................................................................. 6

CHAPTER 2: BACKGROUND AND LITERATURE REVIEW OF THE SERS EFFECT AND SERS SUBSTRATE FABRICATION ..................................................................................................................9

2.1 SURFACE ENHANCED RAMAN SCATTERING (SERS)............................................................................ 9 2.1.1 Discovery of the SERS effect ........................................................................................................ 10 2.1.2 Mechanisms of the SERS effect ................................................................................................... 11 2.1.3 Nanoparticle junctions as hot sites ............................................................................................. 15 2.1.4 Fractal clusters as hot sites .......................................................................................................... 18 2.1.5 SERS amplification factors ........................................................................................................... 20

2.2 EVOLUTION OF SERS SUBSTRATES ............................................................................................... 22 2.2.1 Randomly ordered (disordered) surfaces: ................................................................................... 23 2.2.2 Ordered periodic nanostructures: ............................................................................................... 27 2.2.3 Scientific standards for SERS substrate platforms ....................................................................... 29

2.3 COLLOIDAL NANOPARTICLE SYNTHESIS .......................................................................................... 30 2.3.1 Optical properties ........................................................................................................................ 31 2.3.2 Stability of nanoparticles ............................................................................................................. 31

2.4 NANOPARTICLE PRINTING INKS .................................................................................................... 32 2.4.1 Use in industry for flexible circuits .............................................................................................. 32 2.4.2 Sintering behavior ....................................................................................................................... 33

CHAPTER 3: REVIEW OF RAMAN SPECTROSCOPY AS AN ANALYTICAL TECHNIQUE ............................. 38 3.1 RAMAN SPECTROSCOPY ............................................................................................................. 38 3.2 RAMAN SPECTROSCOPY EXPERIMENTAL PROTOCOLS: ...................................................................... 43

CHAPTER 4: MICROWAVE ABSORPTION AS A METHOD TO SENSITIVELY MONITOR SINTERING AND ITS USE FOR THE FABRICATION OF SERS SUBSTRATES. ............................................................... 47

4.1 ABSTRACT ............................................................................................................................... 47 4.2 INTRODUCTION ........................................................................................................................ 47 4.3 MATERIALS AND METHODS ......................................................................................................... 49 4.4 RESULTS & DISCUSSION ............................................................................................................. 52

4.4.1 Microwave absorption vs. temperature ...................................................................................... 52 4.4.2 Formulation of microwave absorption model ............................................................................. 59 4.4.3 Microwave absorption and SERS ................................................................................................. 64

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4.5 CONCLUSION ........................................................................................................................... 65

CHAPTER 5: OPTIMIZATION OF SERS SUBSTRATES FABRICATED FROM NANOPARTICLE PRINTING INKS ................................................................................................................................. 67


5.4.1 Nanoparticle arrangement .......................................................................................................... 72 5.4.2 Heating treatment removes stabilizing ligand ............................................................................. 74 5.4.3 Reproducibility of SERS signal ......................................................................................... 81 5.4.4 Stability of SERS substrates ......................................................................................................... 83

5.5 CONCLUSION ........................................................................................................................... 85

CHAPTER 6: FUNCTIONALIZATION OF SILVER NANOPARTICLES FOR IMPROVED SERS DETECTION OF HYALURONIC ACID (HA) ............................................................................................ 87


6.4.1 Conformation of cysteamine ....................................................................................................... 93 6.4.2 HA conjugation ............................................................................................................................ 96 6.4.3 Limit of detection of HA............................................................................................................. 101

6.5 CONCLUSION ......................................................................................................................... 103

CHAPTER 7: SERS FILTER FOR DETECTION OF MOLECULES IN SOLUTIONS AND AIRBORNE PARTICLES ..................................................................................................................................... 104

7.1 ABSTRACT ............................................................................................................................. 104 7.2 INTRODUCTION ...................................................................................................................... 104 7.3 MATERIALS AND METHODS ....................................................................................................... 106 7.4 RESULTS ................................................................................................................................ 108

7.4.1 Surface properties ..................................................................................................................... 108 7.4.2 Rhodamine 6G detection ........................................................................................................... 110 7.4.3 Albuterol sulfate detection ........................................................................................................ 111 7.4.4 Nicotine detection ..................................................................................................................... 112

7.5 CONCLUSION ......................................................................................................................... 114

CHAPTER 8: CONCLUSIONS AND RECOMMENDATIONS ................................................................... 116 8.1 SCIENTIFIC CONTRIBUTIONS ...................................................................................................... 116 8.2 RECOMMENDATIONS ............................................................................................................... 118

LIST OF REFERENCES ....................................................................................................................... 121

APPENDIX A: GLOSSARY OF TERMS................................................................................................. 126

APPENDIX B: COMPARISON OF SUBSTRATE FABRICATION TECHNIQUES .......................................... 127

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VITA .............................................................................................................................................. 130

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List of Tables

Table 1: Frequencies (cm-1) and assignments of Raman bands in the 800 – 1600 cm-1 range [127-129]. ...... 99 Table 2: Comparison of nanoparticle ink deposition techniques .......................................................... 127

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List of Figures Figure 1: Energy level diagram for a molecule adsorbed on a metallic surface. The charge transfer

excitation from the molecule’s highest occupied molecular orbit jumps to the metal and then jumps back to the lowest unoccupied molecular orbit [20]. ......................................................................... 13

Figure 2: Schematic of the SERS electromagnetic effect. .......................................................................... 14 Figure 3: Representation of electromagnetic enhancement, MI, produced around a nanoparticle dimer by

incident polarization at various angles a) 0o, b) 30o, c) 60o and d) 90o [29]. ........................................ 17 Figure 4: Representative SEM micrograph of silver nanoparticle fractal structure. Scale bar represents 100

nm. ................................................................................................................................................ 19 Figure 5: Schematic of silver or gold nanoparticle immobilization to a glass slide through silanization

protocol [52]. ................................................................................................................................... 27 Figure 6: Electrical resistivity of nanoparticle film as a function of temperature and heating time [66]. ...... 35 Figure 7: Coutts’ schematic representation of sintering of nanoparticles functionalized with a stabilizing

ligand before and after heat treatment [69]. ..................................................................................... 36 Figure 8: Schematic showing the three different types of scattered light. Stokes refers to the inelastic

scattering of photons at a lower energy than the incident photons. Rayleigh is the elastic scattering of photons. Anti-Stokes refers to the inelastic scattering of photons at a higher energy. ................. 39

Figure 9: Raman spectrum of human serum protein with band assignments corresponding to specific molecular bonds and amino acids [94]. ............................................................................................. 42

Figure 10: A photo of the X-band Varian E-12 EPR spectrometer (left). A schematic of the modified EPR heating cavity for microwave absorption measurements (right). ..................................................... 50

Figure 11: a) A plot of microwave absorption versus temperature for an Ink 1 substrate; b) SERS spectra of 10 μM R6G; and c) SEM micrographs at three temperatures as indicated. Scale bar depicts 500 nm. ...................................................................................................................................................... 54

Figure 12: a) A plot of microwave absorption versus temperature for the Ink-2 substrate and for the b) gold nanoparticle ink substrate. ...................................................................................................... 56

Figure 13: (a) SEM micrographs of Ink 2 as-deposited substrate, 0 minutes, and annealed at 180oC for 10, 20 and 30 minutes. Scale bar depicts 500 nm. (b) SERS spectrum of 1 μM R6G on an ink 2 substrate annealed at 180oC for 15 minutes. The inset is a plot of the normalized SERS intensity of the 1364 cm-1 line as a function of annealing time at 180oC. .......................................................................... 57

Figure 14: Microwave absorption measurement of nanoparticle film during heating-cooling cycle. ......... 59 Figure 15: A TEM micrograph of the nanoparticle ink as-deposited on glass. The scale bar represents 50

nm. ................................................................................................................................................ 60 Figure 16: a) Schematic model of three dimer stages, b) typical plot of microwave absorption versus

temperature and c) TEM micrographs of nanoparticles representative of the three dimer stages. Scale bars indicate 5 nm. ................................................................................................................ 60

Figure 17: Microwave absorption versus heating time at three temperatures, 160oC, 180oC and 2000C. The solid line indicates the model fit according to equation 13 while the dotted lines indicate the raw data points. .................................................................................................................................... 62

Figure 18: A) A representative SERS spectrum of a 10 μM folic acid sample shows the main peaks at 1176 cm-1, 1497 cm-1 and 1587 cm-1. B) Intensity of the folic acid’s 1587 cm-1 peak after heating at 180oC and 200oC for various time intervals. .............................................................................................. 65

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Figure 19: a) Particle arrangement on thin film before heating treatment. The scale bar represents 50 nm. b) The frequency and size of the voids after heating indicates a large distribution of clusters in the substrate. ....................................................................................................................................... 73

Figure 20: DTA graph shows an exothermic peak in the silver nanoparticle sample at approximately 189oC while the TGA graph shows a mass loss of 20% immediately following the exothermic peak due to the evaporation of the oleylamine ligands. ..................................................................................... 75

Figure 21: SEM micrographs of Ag nanoparticles deposited on glass cover slips after heating for 15 minutes at A) 125oC, B) 175oC and C) 225oC. Magnification 50 kX and a working distance of 5 mm. Scale bar represents 300 nm. .......................................................................................................... 76

Figure 22: Average SERS signal of the 1364 cm-1 R6G peak on dry drop deposited substrates after heating for 15 minutes at the indicated temperatures. ................................................................................ 76

Figure 23: (a) SEM micrographs of Ag nanoparticles as-deposited on substrate, 0 minutes, and annealed at 180oC for 10, 20 and 30 minutes. Scale bar depicts 500 nm. (b) Rhodamine 6G spectrum after various heating times at 180oC. Each spectrum was shifted up for visual clarity. .......................................... 77

Figure 24: A) Schematic representation of heated nanoparticle surface and the direction of the incident laser beam. B) Raman intensity of 1050 cm-1 nitrate ion band decreases as a function of heating time when collected from the exposed side of the substrate. The signal of the same band remains stable when collected from the glass slide. The dotted lines are only an aid for the eyes. .......................... 79

Figure 25: a) Microscope image of one silver nanoparticle ink printed dot on a glass substrate. The diameter of the dot was approximately 1.2 mm. b) The amplitude of the 1364 cm-1 peak at 20 locations across the diameter of the dot. The center portion intensities vary by less than 10% indicating good reproducibility. ...................................................................................................... 82

Figure 26: a) A printed array of silver nanoparticle ink on a microscope glass slide. The red box indicates the 5x5 array of dots that was analyzed. b) The surface plot shows the reproducibility of the 1364 cm-1 peak amplitude from each dot on the array. ............................................................................ 83

Figure 27: a) Stability of the SERS signal intensity from a 1 μM R6G solution on substrates stored in a vacuum chamber for several weeks. b) Stability of the SERS signal intensity from a 10 μM R6G solution on substrates over a period of 3 weeks after storage. ........................................................ 85

Figure 28: The base unit of hyaluronic acid is composed of D-glucuronic acid and N-acetyl-D-glucosamine. ...................................................................................................................................................... 89

Figure 29: Schematic representation of HA attachment to a functionalized silver nanoparticle substrate. 92 Figure 30: Schematic representation of two possible cysteamine conformers on a silver nanoparticle

surface. Adapted from Kudelski et al. [131]. ...................................................................................... 94 Figure 31: The logarithm of the cysteamine trans to gauche ratio at various concentrations shows a mostly

trans conformation over the substrate when the concentration is above 10 mM. The insets show typical SERS spectra in the trans and gauche conformations along with their schematic representations. ............................................................................................................................. 95

Figure 32: Spectra of the thermally treated substrate, 25mM cysteamine on SERS substrate and 1mg/mL HA immobilized on substrate. Spectra were shifted vertically for clarity. ......................................... 98

Figure 33: SERS spectra of 1mg/mL HA after cross linking to cysteamine linker at different concentrations. Spectra were shifted vertically for clarity. ..................................................................................... 100

Figure 34: Schematic of single HA disaccharide immobilized on Ag substrate. The peaks are assigned according to table 1 to give a visual to how they contribute to the Raman spectrum. .................... 101

Figure 35: HA spectra on cysteamine functionalized SERS substrate. ..................................................... 102 Figure 36: Raman spectra of hyaluronic acid at 50 µg/mL on a cysteamine functionalized SERS substrate.

The labeled peaks correspond to previous HA peak assignments. ................................................. 102

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Figure 37: SERS filter (F) is fitted at the mouth of a plastic chamber by a securing cap (SC). Air particles are introduced into the container through a nebulizer at the aerosol inlet and a vacuum pump transports the particles through the SERS filter. ............................................................................ 108

Figure 38: Left SEM micrograph shows thickness of fibers on an uncoated filter to be approximately 12µm. Center micrograph shows the coverage of Ag nanoparticles on a fiber. Right micrograph shows a magnified region of the arrangement of nanoparticles on the fiber surface. ................................. 109

Figure 39: Microwave absorption as a function of heating time at 200oC for the Ag nanoparticle ink deposited on the filter. ................................................................................................................. 109

Figure 40: 100 pM R6G solution detected on SERS filter (left). Linear dependence of R6G intensity per second with respect to concentration based on the 1362 cm-1 R6G band (right). ........................... 111

Figure 41: Albuterol sulfate SERS signal from a single inhaler dose released into the flask (top). Background SERS signal from filter substrate (bottom). ................................................................ 112

Figure 42: The nicotine 1032 cm-1 peak was detected at concentrations as low as 0.5 ng/µL from a solution diluted in 10 mM NaCl on the SERS substrate................................................................................ 113

Figure 43: SERS nicotine signal on Ag coated filter substrate produced from the smoke of a lit cigarette. The main 1031 cm-1 and 1052 cm-1 bands are clearly visible while the other minor peaks come from the background signal from Ag-Cl and the filter’s fibers. ............................................................... 114

Figure 44: SERS intensity of the three major peaks from a 10 μM solution of folic acid on a dry drop and spin coated substrate. .................................................................................................................. 129

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Abstract Fabrication of surface enhanced Raman Scattering (SERS) substrates made from

nanoparticle printing inks for detection of biological molecules Manuel Alejandro Figueroa

Som Tyagi, PhD, and Kambiz Pourrezaei, PhD

Surface enhanced Raman scattering (SERS) has generated great interest as a surface analytical

technique because it can produce amplification factors between 108-1012. Silver and gold are the

most widely used components as their size and structure allows for light to induce conduction

electrons to oscillate locally within the nanoparticle structure. When a molecule lies in the

interparticle space between two nanoparticles, highly detailed vibrational information becomes

detectable. The objective of this study is to reproducibly fabricate such an arrangement in a

nanoparticle substrate while maintaining stability.

In this work, nanoparticle printing inks -- colloidal nanoparticles encapsulated by a stabilizing

ligand -- are used as the main component of SERS substrates. The ligand shell is partially

removed by controlled heating, which reduces spacing between nanoparticles creating a broad

distribution of interparticle distances. Similar to fractal aggregates this arrangement allows

localized plasmons to naturally resonate over a broad range of spectral frequencies.

Microwave absorption is applied as a non-invasive method to sensitively monitor nanoparticle

sintering in order to gauge the substrates’ tuning for large amplification factors. The global

arrangement of nanoparticles has always been difficult to measure during heating through DC

resistivity measurements and surface imaging techniques. Microwave absorption occurs in the

weak resistive links formed between particles during sintering due to the microwave losses in

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loosely coupled particles. By placing the substrate in a microwave cavity, absorption can be

monitored globally during heating. The largest SERS amplification factors occur at a stage

immediately preceding the largest microwave absorption gains. This provides a useful method

for determining a thermal window for heating when optimizing SERS substrates.

Finally, these optimized SERS substrates are used to detect hyaluronic acid. This complex

molecule is a potential biomarker for inflammatory diseases but it has only been detected at a

concentration of 5 mg/mL on commercially available SERS substrates. Here it is shown that

functionalizing the SERS substrate with a self-assembled monolayer lowers the limit of detection

to 50 μg/mL. SERS analysis also provides structural details about the conformation of the

molecule during adsorption.

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Chapter 1: Introduction In the imminent future there will be greater access to an arsenal of ‘smart’ chemical and

biological sensors that will function to detect irregular substances in our homes and

environments. These non-invasive devices will be able to measure dangerous chemicals in the

air or specific biomarkers in our bodies and provide an alert if there is a legitimate concern. In

regards to diagnosing maladies in the body, interest in point of care testing has grown in the

past decade. The commercial goal is to fabricate a rapid test that could reliably detect abnormal

concentrations of molecules in biological fluids. These could be interpreted by a clinician using a

desktop system or by the individual at home. However, there are still many challenges that

remain common to all biological sensors, for example the specificity of molecule capture to the

substrate, the reproducibility of the signal and the reliability of molecule identification.

Surface enhanced Raman scattering (SERS) based substrates are one promising platform to

quickly identify molecules of interest. The SERS phenomenon occurs due to electromagnetic and

chemical effects, which allow molecular detection in the femtomolar range. Its sensitivity

coupled with the specific vibrational information provided by Raman spectroscopy means that

the technique can yield rapid fingerprint-like results. The SERS effect is limited by surface to

analyte distance so the molecule of interest must be in close proximity to the nanoparticles.

The advent of top-down multi-step lithography methods in the area of circuit design affords the

capability of fabricating nanoparticle arrangements with precisely spaced separations. Such

control of nanoparticle position allows matching the laser excitation frequency to the plasmon

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absorbance created in periodic nanostructures to produce the largest signal amplification.

Although there is much merit to this technique for fabricating reproducible structures it is not

the most practical approach. A more widely used approach is silver or gold colloidal aggregates

which allows localized plasmons to naturally resonate at a broad range of spectral frequencies.

These are inexpensive and can be directly added to a biological fluid. However, reproducibility is

difficult to obtain due to Brownian motion and aggregation effects.

Developing label-free substrates is another common goal of fabricating SERS research in order

to provide direct conformational information rather than the indirect detection of a Raman tag.

This has the potential to provide 1000 fold greater enhancement than fluorescence assays and

much more detailed information. Thus far, the success of this strategy has been limited for the

most part to small molecules that are highly Raman active. Large molecules are difficult to

detect because their complex conformational structures can shield Raman active bonds from

the SERS active surface.

1.1 Overview of thesis research

The goal of this work was to fabricate SERS substrates with high amplification factors using silver

nanoparticle printing inks for the detection of biological molecules. These inks, which have

recently been introduced in the electronics industry to produce conductive pathways for flexible

circuits, are colloidal particles encapsulated by a protective surfactant layer. To this end, it was

important to understand the arrangement of nanoparticles to produce a large SERS effect. A

crucial component was the relationship between heating and the removal of the stabilizing layer

which allowed the manipulation of nanoparticle arrangement. The use of microwave absorption

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was introduced as a new technique to sensitively monitor the sintering process globally. Printed

dots, approximately 1 mm in diameter, provide a large enough surface area in which one can

achieve reproducible SERS signals after heat activation, even after months of storage.

Furthermore, the SERS substrate was tested with numerous chemicals and biological molecules

including hyaluronic acid (HA). HA is a complex glycosaminoglycan found in all parts of the body

and it serves as biomarker for inflammation when expressed at elevated concentrations in

biological fluids. To detect such a large molecule, the substrate was functionalized with a self-

assembled monolayer to force the adhesion at a known site. To accomplish these objectives

three specific aims were proposed which are outlined in the next section.

1.1.1 Specific aims

Specific Aim 1: Monitor nanoparticle aggregation during heating treatments and correlate to SERS intensity

- Measurement of optical absorption in colloids is the most-widely used method to characterize the quality of nanoparticle synthesis because optical absorption can be related to nanoparticle size

- Once the colloid is dry deposited, aggregation takes place and the plasmonic resonance will shift.

- Aggregation will change in response to many different variables, for example: time, temperature, pH, etc.

- A non-contact method is needed to monitor the process of sintering globally on planar substrates

- Preliminary experiments show the difficulty of o Resistance measurements (point dependent) o Optical absorption (before and after) o SEM/TEM micrographs (before and after)

Hypothesis: Microwave absorption measurements can be used to sensitively monitor the sintering process and define a thermal treatment for SERS amplification.

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Evidence for specific aim 1: A. Microwave absorption versus increasing temperature on various nanoparticle printing

inks shows increase in absorption is temperature dependent B. SEM micrographs show sintering correlation between various points on the microwave

absorption curve C. Absorption is irreversible just like sintering D. Microwave absorption versus isothermal heating shows sintering behavior E. Descriptive model developed to explain microwave absorption through the change of

interparticle separation of a nanoparticle dimer F. Microwave absorption correlated to SERS intensity of folic acid

Contribution of specific aim 1: Introduced microwave absorption as an alternative to DC resistivity method to sensitively monitor the sintering process. Portions of the work in specific aim 1 were presented at the SPIE Biomedical Optics Photonics West Conference in 2012 [1]. In addition a manuscript was published in the Journal of Raman Spectroscopy in 2012 [2]. Specific Aim 2: Optimize the performance of surface enhanced Raman scattering (SERS) substrates through an annealing treatment.

- Preliminary evidence showed a relationship between heating and SERS intensity - The most widely used SERS substrates are made from Ag or Au colloids. Unfortunately

these are unstable, aggregate and do not firmly adhere to substrates - Metallic nanoparticles in printing inks have a protective layer that prevents aggregation

o Currently studied by electronic industry to make conductive surface paths at low annealing temperatures

o The sintering process modifies and partially removes the protective shell allowing neighboring particles to make contact with each other

Hypothesis: Partial removal of the encapsulant through a short annealing treatment creates an environment of nanoparticles or nanoparticle clusters with nanometer interparticle separations ideal for the SERS effect. Evidence for specific aim 2:

A. Differential thermal analysis shows a prominent peak in the 180-200oC temperature range indicating the oxidative decomposition of encapsulant.

B. Thermogravimetric analysis shows a significant mass loss in the 180oC – 450oC temperature range immediately following the DTA peak. The mass loss is attributed to the evaporation of the encapsulant.

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C. SEM micrographs show nanoparticles sintering through heating treatment D. SERS signal intensity rises after heating but with prolonged heating it eventually decays E. SERS limit of detection for Rhodamine 6G in planar substrate F. Reproducible across printed dot and across arrays of dots G. Seven month signal stability H. Adheres to glass

Contribution of specific aim 2: First time sintering process is used to tune the interparticle separation in the fabrication of highly sensitive SERS substrates using nanoparticle printing inks. Portions of the work in specific aim 2 were presented at the SPIE Biomedical Optics Photonics West Conference in 2008 and 2010 and at the International Workshop on Functional Materials in Berhampur, India in December 2011 [3-5]. In addition, a patent application was filed through Drexel University. Specific Aim 3: Gain an understanding of the adsorption of hyaluronic acid to improve detection using SERS substrate

- Although enzyme-linked immunosorbent assay (ELISA) kits exist for detecting biomarkers for liver disease, a biopsy is still the gold standard in the staging of fibrosis

- If cirrhosis, the last stage of liver disease, is not detected early it can lead to severe liver complications and the need of a liver transplant

- Hyaluronic acid in the serum is elevated during liver disease and the concentration has been shown to be correlated to the stage of fibrosis

- It is considered by many to be the most accurate biomarker for the staging of fibrosis - Previous attempts to detect hyaluronic acid using SERS have only been achieved at high

concentrations most likely due to the repulsion of the molecule due to electrostatic forces.

Hypothesis: By functionalizing the nanoparticles with a short alkanethiol linker molecule, such as cysteamine it is possible to anchor HA to the substrate thus increasing the SERS intensity at low concentrations. Evidence for specific aim 3:

A. Established a protocol for functionalization of nanoparticles with cysteamine. B. Optimized cysteamine coverage on SERS substrate for HA binding C. Change in cysteamine peak ratio correlates to HA binding event D. Raman peaks can be used to determine orientation of HA molecule E. Detected HA to a concentration as low as 50 μg/mL

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Contributions of specific aim 3: Developed proof of ability to observe molecule binding to functionalized nanoparticles and improved SERS detection limit of hyaluronic acid Portions of the work in specific aim 3 were presented at the IEEE Northeast Bioengineering Conference 2011 [6] as well as the Biomedical Engineering Society (BMES) Conference in 2011.

1.2 Thesis organization

This thesis is divided into 8 chapters composed of an introduction, backgrounds, specific aims

and conclusions. The first chapter gives a broad overview of the SERS effect and its potential as

the medium for a biological sensor capable of detecting trace concentrations of various

analytes. The chapter also outlines the specific aims and goes over the content of the thesis.

The second chapter provides a background of various aspects of the research. It begins with the

discovery of the SERS effect and the mechanisms that lead to the large enhancement factors. A

review follows on the most prominent techniques used to fabricate SERS substrates and gives an

overview of the advantages and disadvantages of each approach. It ends with the introduction

of the idea of using the sintering process in nanoparticle printing inks to tune the interparticle

spacing and produce SERS surfaces with high amplification factors. The third chapter is a

technical background of the important analytical techniques performed for this work including

Raman spectroscopy, microwave absorption and thermal analysis.

The fourth chapter introduces the first aim of this project which developed out of a need to non-

invasively characterize the film morphology during the heating. Microwave absorption is

introduced as a novel technique to sensitively monitor the sintering process of a nanoparticle

thin film during heating. The technique is based on the fact that microwave absorption occurs at

nanoparticle junctions where weak resistive links are formed during heating and then destroyed

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upon sintering. A descriptive model was developed to explain the microwave absorption as a

function of heating for the simple case of nanoparticle dimers.

Chapter five describes the second aim of this thesis which involves efforts to fabricate SERS

substrates with high amplification factors using silver nanoparticle printing inks through a

controlled thermal annealing process. The main advantage of printing inks over traditional

metallic colloids is that they adhere to the surface during deposition and the stabilizing layer can

be removed at relatively low heating temperatures. The latter reduces interparticle spacing

thereby increasing SERS amplification factors. This chapter also discusses substrate stability,

reproducibility and nanoparticle adhesion.

Chapter six describes the third aim of this thesis, which involves the SERS detection of

hyaluronic acid (HA) at low concentrations. HA is a glycosaminoglycan found in various places in

the human body. The molecule is overexpressed in the serum in individuals diagnosed with liver

cirrhosis because the endothelial cells cannot properly clear the molecule. It has recently been

proposed as a biomarker to discriminate between the different stages of liver disease. The

current gold standard is a liver biopsy, which can take several days to analyze whereas a SERS

measurement can be made non-invasively and yield results rapidly. To detect the molecule on

the SERS substrate it was necessary to functionalize the surface with a linker molecule that

would directly attach to the carboxyl group of the HA. Raman spectra were collected at

progressively lower concentrations to establish a limit of detection and from its major peaks the

orientation was determined.

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Chapter seven describes a novel three-dimensional SERS substrate made from cellulose fibers

coated with silver nanoparticle printing inks. This work does not form part of the three specific

aims but it lays the foundation for further research. The SERS filter, as it is called, can be used to

detect molecules in solution or airborne particles. The metallic coating on the fibers forms

nanocavities and fractal-like clusters on its surface. This, combined with its three-dimensional

structure provides greater SERS amplification factors than a planar substrate as molecules have

more binding sites and the geometry amplifies the electromagnetic enhancement.

Chapter eight summarizes the entire work and re-emphasizes some of the most salient

conclusions of this thesis. It also places the scientific contributions in a broader context so one

can see the potential applications. Recommendations are also provided for new areas of

research that this work can help explore.

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Chapter 2: Background and literature review of the SERS effect and SERS substrate fabrication

2.1 Surface enhanced Raman scattering (SERS)

Since its discovery in 1974, the SERS effect has captivated the imagination of many scientists

who see its potential as an analytical tool for detecting chemicals at trace concentrations and

collecting detailed vibrational information from single molecules. Moreover, its particular

strength lies in its sensitivity to molecular surface interactions. Akin to IR spectroscopy, the

bands in the spectra correspond to specific molecular bonds that arise from changes in the

polarizability of the molecule rather than dipole changes, making it possible to determine the

precise orientation of a molecule. Average enhancement factors between 106 and 108 are

regularly observed and a few, although difficult to reproduce, have reported factors of 1014 for

single molecule detection [7, 8].

Over the years, SERS research has branched into three main areas: 1) fundamental aspects of

the SERS effect, 2) plasmonics and related spectroscopy techniques, and 3) fabrication and

applications of SERS substrates. The latter topic has been called one of the first true challenges

in the field of nanoscience [9, 10] and is the primary interest of this thesis. It involves the control

of nanoscale metallic materials to produce large electromagnetic fields and the manipulation of

surface characteristics that allow it to intimately interact with specific molecules. The

applications span across multiple areas, from homeland security objectives (detection of

explosives, airborne particles, contamination in water) and pharmaceuticals (detection of

polymorphs, contamination, drug active sites) to biological detection (DNA, bacteria, disease

biomarkers, and illicit drugs) and analytical chemistry (chirality of molecules, trace detection of

molecules). In order to commercialize this technology for its many applications, inexpensive

10

substrates must be fabricated that can be both sensitive and reproducible for detecting trace

concentrations. As this becomes possible, SERS substrates will be able to provide enhancements

1000 times greater than current fluorescent techniques for a single molecule [11].

2.1.1 Discovery of the SERS effect

The discovery of the SERS effect was accomplished by the separate contributions from three

groups headed by Martin Fleischmann, Richard P. Van Duyne and J Alan Creighton. In 1974,

Fleischmann observed a large Raman signal from a monolayer of pyridine molecules adsorbed

on a roughened silver electrode [12]. From the personal published accounts of Fleischmann’s

collaborators, Pat Hendra and James McQuillan, it is clear they were studying the effect of

electrolyte adsorption on electrode behavior and they believed Raman could provide novel

structural information [13, 14]. During their famous experiment we can ascertain that their group

was attempting to increase the Raman signal from electrolytes by using highly roughened

electrodes. However, they did not realize they had observed a new phenomenon but instead

attributed the signal intensity to a superposition effect from the high concentration of

molecules adsorbed to the surface layer of the roughened electrode [10, 15]. In other words the

rough surface created a higher surface area where more molecules could adsorb and thereby

more light could scatter. In a way, their observations were understandable, as Raman scattering

was already known to be a weak process and many groups were already trying to optimize the

inelastic scattering. Nevertheless, the surface area explanation did not fully account for the

magnitude of the observed enhancement.

By the late 1970s, both Creighton and Van Duyne independently repeated Fleishchmann’s

experiment and proposed their own mechanisms for the enhancement. Van Duyne attributed

11

the enhancement to an increase in the electromagnetic field at the roughened surface whereas

Creighton attributed it to the creation of a charge transfer absorption band between the

molecule and the metal [15]. Martin Moskovits also made a large contribution to the

development of the electromagnetic mechanism by postulating the roughened surfaces were

actually sustaining localized plasmons, which were responsible for the large Raman

enhancement [16]. Over the years these two theories have been explored vigorously and it is

generally agreed that the two effects are intimately connected with the electromagnetic effect

providing the largest contribution while the charge transfer effect being strongly dependent on

the adsorption of the molecule to the metal.

2.1.2 Mechanisms of the SERS effect

Before delving into the mechanisms it is important to understand a few concepts involved in

Raman scattering. A more thorough explanation of the Raman effect can be found in Chapter 3.

Classically, the Raman effect is described by the inelastic scattering of photons as they interact

with a molecular system. The energy shifts are collected as wavenumbers (cm-1) and correspond

to vibrational frequencies in the molecule. The combination of the wavenumber bands gives a

fingerprint or signature of the molecule. Unfortunately, Raman scattering is a very weak process

as it only occurs in approximately 1 in 106 photons and it is limited to measuring signals from

molecules that change polarizability during vibrations. Raman selection rules have been

developed to classify the molecular bonds likely to give a Raman signal and density-functional

theory (DFT) calculations are often performed to predict the wavenumbers where bands will

appear. The application of surface enhancement allows the analytical chemist to extract highly

detailed information about potentially any molecular system at trace concentrations as long as

the analyte interacts with the metallic surface. For many years the exact mechanisms of

12

enhancements were debated but most agreed it occurred due to a combination of the chemical

and electromagnetic enhancement.

The chemical enhancement or charge transfer effect stems from the electronic interaction

between the adsorbate and the metallic nanostructure, which yields information about its

orientation and it vibrational properties [17]. In general, there are four types of energy transitions

that can occur in metal-molecular complexes when interrogated with photons: 1) An electron is

excited within the metal, 2) An electron is excited within the molecule, 3) An electron on the

surface of the metal is excited to the adsorbed molecule and 4) an electron in the molecule is

excited to an empty orbital in the surface of the metal above the Fermi level [18]. The charge

transfer process normally ascribed to SERS occurs only for certain adsorbates that exhibit shifts

in molecular energy levels [19]. In this case, the metal acts as an intermediate step between the

highest occupied molecular orbital (HOMO) and the lowest unoccupied molecular orbital

(LUMO) of the adsorbate allowing the electrons to get excited with half the energy usually

required to reach an excited state [20] (Figure 1). It is estimated the charge transfer effect

contributes a factor of 100 overall [20].

13

Figure 1: Energy level diagram for a molecule adsorbed on a metallic surface. The charge transfer

excitation from the molecule’s highest occupied molecular orbit jumps to the metal and then jumps back to the lowest unoccupied molecular orbit [20].

The larger contribution of the SERS effect comes from the electromagnetic (EM) enhancement,

which has been estimated to yield a maximal factor between 1010 to 1012 for a molecule

adsorbed on the nanoparticle surface [11, 21, 22]. In general, light can cause various types of

excitations when interacting with a surface, i.e. scattering and absorption being the most well-

known but also surface plasmon resonance (SPR) [23, 24], and second order nonlinear

hyperscattering [25]. The latter two were developed after the discovery of SERS and are used in

other spectroscopies that are beyond the scope of this work.

In the case of SERS, the electric field component of the incident light interacts with a surface

composed of metallic nanoparticles causing the displacement of free electrons. The

uncompensated charge on the nanoparticle surface leads to a polarization causing more

electrons to oscillate [19]. The electrons oscillate at a frequency known as the localized surface

plasmon resonance (LSPR) [24]. The dipolar plasmon moves back and forth perpendicular to the

14

Figure 2: Schematic of the SERS electromagnetic effect.

direction of the incident beam of light (Figure 2A and Figure 2B). For this to occur, the wavelength

of the incident light has to be much larger than the radius of the nanoparticles. Typically

nanoparticles are chemically synthesized to a size ranging between 10 nm to 100 nm while the

incident wavelengths are in the visible range (400 nm – 750 nm). Since the nanoparticles are

smaller than the wavelength by at least one scale of magnitude then the plasmons remain

localized to the nanoparticle surface. In contrast, if the nanoparticles are larger or on the same

scale as the incident wavelength then the free electrons simply propagate on the conduction

band of the metal surface. The large EM field produced by the plasmon resonance induces a

dipole in the adsorbed analyte thereby amplifying the Raman scattering [15] (Figure 2C). In short,

the EM enhancement comes from the coupling of the electric field of the incident light with the

localized surface plasmon surrounding a metallic nanoparticle.

The EM enhancement has been described mathematically for various arrangements of

nanoparticles including single nanoparticles, two neighboring nanoparticles (dimers) or trimers.

Generally, for a system of nanoparticles the EM enhancement factor, MEM, can be estimated by

multiplying the local electric field enhancement, EL(ωI), by the scattered field enhancement,

EL(ωI-ωs), divided by the corresponding values in the absence of the metal particles, EI(ωI) and

EI(ωI-ωs), respectively (equation 1).

15

𝑀𝐸𝑀 = �𝐸𝐿(𝜔𝐼)𝐸𝐼(𝜔𝐼)

�2

∙ �𝐸𝐿(𝜔𝐼−𝜔𝑠)𝐸𝐼(𝜔𝐼−𝜔𝑠)

�2

, (1)

where ωI and ωs are the frequency of incident light and the frequency of scattered light,

respectively. When ωs is much smaller than ωI then the enhancement factor can be estimated by

a power of 4. This means that the SERS intensity is proportional to the enhancement factor of

the incident local electric field [9].

Furthermore, the EM enhancement is strongly dependent on the relationship between the

distance between the nanoparticle and the molecule of interest, d, and the radius of the

nanoparticle, r. The effect rapidly decays by a factor of [𝑟 (𝑟 + 𝑑)⁄ ]12 [26]. Equation 1 can be

combined with nanoparticle size to the molecule distance relationship to get equation 2.

𝑀𝑒𝑚(𝑣𝑠) = � (𝜀(𝑣𝐿)− 𝜀0)(𝜀(𝑣𝐿)+ 2𝜀0)�

2� (𝜀(𝑣𝑆)− 𝜀0)

(𝜀(𝑣𝑆)+ 2𝜀0)�2� 𝑟𝑟+ 𝑑

�12

, (2)

Equation 2 shows that in theory the greatest enhancement will come when a molecule is

directly adsorbed to a single nanoparticle. However, there are other factors that must be

considered when designing SERS substrates with high amplification factors, for example,

excitation wavelength, type of metal, ligand coating, nanoparticle size, nanoparticle shape, and

interparticle distance.

2.1.3 Nanoparticle junctions as hot sites

The spacing between two nanoparticles in aggregated clusters will have a large influence on the

magnitude of the SERS enhancement [27]. In fact, junctions between neighboring particles that

16

produce large electric fields were termed “hot particles” by Nie and Emory, who estimated an

amplification factor of 1014 at these sites for single molecule detection [1]. Although this estimate

is now considered an over estimation and not even practical [22], the idea of hot active sites still

remains an area of continued research. Applying electromagnetic theory to a nanoparticle dimer

separated by the distance of one hemoglobin molecule, Xu et al showed the largest

electromagnetic enhancement occurred at the midpoint between two particles and only when

the incident polarization was parallel to the spacing [28, 29]. This was four orders of magnitude

larger than an isolated particle and eight orders of magnitude greater than two neighboring

particles with the polarization perpendicular to the dimer axis. Michaels et al went on to confirm

this as they reported minimal enhancement from single particles and high amplification factors

in dimers [27]. Figure 3 shows a computer model of the magnitude of the electrical fields around

a nanoparticle dimer with the strongest fields occurring when the polarization is parallel to the

dimer axis. This was calculated by Xu et al using the generalized Mie theory [29]. The incident

polarization dependence has been confirmed in trapped dimer nanoparticles [30] as well as at the

clefts of bifurcated nanowires [31].

17

Figure 3: Representation of electromagnetic enhancement, MI, produced around a nanoparticle dimer by

incident polarization at various angles a) 0o, b) 30o, c) 60o and d) 90o [29]. Taking the model one step further, as the interparticle distance is decreased for a given size

nanoparticle the magnitude of the electric field increases. Calculations show a four order of

magnitude increase for the electric field produced at the midpoint between two 90 nm particles

when the separation reduced from 5.5 nm (M = 6 x 106) to 1 nm (M = 2.5 x 1010) [28]. In a similar

fashion a five order of magnitude increase was calculated for 60 nm particles with the

separation reduced from 9 nm (M = 1.5 x 104) to 1 nm (M = 5.5 x 109) [11]. On the other hand it

follows that the effect will negate itself when the interparticle distance is increased to a point

where two low magnitude electric fields appear, each corresponding to a single isolated particle

[32].

From these simulations it is clear there is a relationship between the size of the nanoparticle

and the interparticle distance for maximizing the electric field. Garcia-Vidal et al showed the

electromagnetic enhancement factor significantly increased when the center to center distance

between particles was less than three times the radius of the particle [33]. By simple geometry

18

the local field between nanoparticles varies by E=Ei [(D+d)/d] where D is the nanoparticle

diameter and d is the interparticle distance from the respective centers of each particle [34]. This

relationship explains why the EM enhancement becomes so large when the interparticle

distance becomes very small.

While the nanogap structure seems ideal for studying single molecules it remains difficult to

fabricate a large-throughput ordered structure with consistently spaced dimers and

reproducible large amplification factors. Alexander et al used a complicated soft lithography

technique to make an array of nanoholes on a silicon substrate. A stabilized gold colloid was

deposited on the surface and dimers randomly settled into position. To collect a SERS signal the

stabilizer was replaced with a thiophenol molecule which would strongly bind to gold while at

the same time producing a strong SERS signal from the phenol ring [30]. Although this technique

was useful to prove the SERS enhancement exists at the junctions between nanoparticles, it is

not practical for detection of biological molecules as it is limited to chemicals that can replace

the stabilizer while still maintaining interparticle distance.

2.1.4 Fractal clusters as hot sites

If one is not particularly interested in controlling the interparticle distance for single molecule

detection then a much simpler platform is a colloidal suspension deposited on a planar

substrate where the nanoparticles will naturally aggregate into fractal structures. A fractal is a

structure that exhibits self-similarity or scale invariance and cannot be properly characterized by

conventional geometry [35, 36]. In colloidal solutions, fractal clusters are oftentimes found

because the nanoparticle fragments at a smaller scale resemble the structure at a larger scale.

Due to the inherent self-similarity of a nanoparticle substrate the fluctuations in space will be

19

the same over the entire surface [37]. This can be beneficial for finding the average SERS signal

since the surface morphology will resemble itself everywhere. Figure 4 shows an example of a

silver nanoparticle fractal structure made through a roll-to-roll deposition process. In such

fractal clusters it is easy to conceive multiple sites of closely spaced nanoparticle groups. Due to

a lack of translational invariance fractal clusters do not transmit running waves, instead EM

excitations get localized to the clusters. These sites can produce high SERS amplification factors

and are termed ‘hot spots’. Amplification factors as high as 1012 have been reported in such

clusters [19]. It is also true that fractal clusters will have inhomogeneities at all scales. The broad

distribution of local geometries makes it possible to induce surface plasmon resonance with

wavelengths anywhere in the visible to the near IR spectrum [19]. This last point is worthy to note

because in a lot of SERS substrates one will be limited to only one laser wavelength. This is not

the case for fractal clusters where multiple wavelengths can excite plasmons.

Figure 4: Representative SEM micrograph of silver nanoparticle fractal structure. Scale bar represents 100

nm.

20

Besides the aggregates, voids in such fractal clusters can act as resonant nano-cavities where

molecules can get sampled repeatedly and further contribute to SERS amplification. Incident

light will be confined to the cavity and if it has a high Q factor then the incident light will

internally reflect thousands of times thereby sampling the molecule on each pass [38, 39]. In

contrast, a molecule adsorbed to a metallic surface will only get sampled twice.

2.1.5 SERS amplification factors

To characterize the effectiveness of SERS substrates it is customary to report the amplification

factors [24, 40]. This is normally described as the ratio of the molecule’s SERS intensity to its

normal Raman intensity [41]. Different methods for calculating SERS enhancement factors have

been reported in the literature which has made comparison of substrate performance difficult.

For comparison purposes we use the analytical enhancement factor (AEF) described by Le Ru et

al. [40]. It can be typically determined by equation 3,

𝐴𝐸𝐹𝑆𝐸𝑅𝑆 = � 𝐼𝑆𝐸𝑅𝑆 𝑁𝑠𝑢𝑟𝑓 ⁄ �[ 𝐼𝑁𝑅𝑆 𝑁𝑣𝑜𝑙⁄ ]

(3)

where the SERS intensity of a particular band, ISERS, is normalized by the number of molecules

bound to the metallic surface, Nsurf. This value is divided by the normal Raman intensity of the

same band, INRS, normalized by the number of molecules in the excitation volume, Nvol. Of

course, many different types of SERS substrates have been studied so it is important to

categorize amplification factors by the type of substrate used - colloidal, planar, inverted

pyramid, sol-gel, etc. Nevertheless, the difficulty in comparing amplification factors comes from

the variations of calculating Nsurf and Nvol.

21

For the work described here on planar substrates the following method was used. The

amplitude of the 1364 cm-1 Rhodamine 6G (R6G) - - a very good Raman scatterer and well-

studied molecule - - band was used to characterize the amplification factor. The 1364 cm-1 band

is one of the three “cathedral” peaks found in the R6G molecule and corresponds to the

aromatic C-C stretching bond [42]. A standard curve of the absorption of R6G in solution was

determined from dilutions performed down to nanomolar concentrations. For the normal

Raman measurement the number of molecules was calculated from the approximate number of

molecules found in the top layer formed on the surface inside the laser spot size. This was

calculated assuming a homogenous monolayer of Rhodamine molecules.

The number of molecules found on the nanoparticle surface was calculated after incubating the

substrate in a R6G solution of known concentration. After air-drying, the substrate was washed

in water and the runoff liquid was collected in a separate container. This represents the

unbound molecules. The optical absorption from the unbound molecules was collected in the

spectrometer and then matched to the standard curve. This number was subtracted from the

initial concentration to determine what concentration was left attached to the substrate.

Assuming an even distribution of adsorbed analyte the concentration was used to determine

how many molecules remained on a monolayer inside the laser target area.

It is important to reiterate here that due to the vast diversity of planar SERS substrates and the

complexity in calculating the number of molecules adsorbed to the SERS active sites, the

calculated SERS enhancement factor is in reality a very rough estimate of amplification. As

Michael J Natan noted in his concluding remarks at the Royal Society of Chemistry’s first Faraday

22

Discussion Meeting on SERS in 2005, it is more meaningful to report the thresholds of detection

for individual molecules [43].

2.2 Evolution of SERS substrates

The ambitions for fabricating SERS substrates have changed over the years. The early efforts

involved roughening metal surfaces by means of repeated chemical oxidation-reduction cycles.

Here the goal was to have a platform to perform surface chemistry experiments to better

understand the mechanisms of the surface enhancement. Next came the use of colloidal

nanoparticles in solution, which offered a resourceful protocol for any chemistry laboratory to

produce but not reliably reproduce from lab to lab. It quickly became apparent that silver or

gold nanoparticles produced the greatest amplification due to their dielectric properties. These

were widely deposited on planar substrates and produced enhancement factors ranging from

106 – 1012 when compared to normal Raman scattering [7]. Studies were published

demonstrating the detection of just about anything, trace chemicals, biological molecules,

peptides, proteins and airborne particles. Once the potential for commercialization was realized

the goal became to develop fabrication methods for reliable, reproducible and inexpensive SERS

substrate. Overall, it could be said the design of the substrate itself has been the main driver for

the advancements in the understanding of the SERS effect.

As instrumentation improved and nanoscale resolution became possible new fabrication

methods emerged to make highly ordered metallic structures through chemical or electro-

chemical etching, lithography or deposition of metal over inert templates. These methods offer

the advantage of clearly defined features but fabrication tends to be more expensive, requires

multiple preparation steps and the substrates do not yield the highest enhancement factors. To

23

date it has become clear that nanopatterned substrates actually are reproducible for SERS

intensities but at the expense of losing some amplification.

Overall, there is now a diverse arsenal of techniques to produce SERS substrates and they all

have their advantages and disadvantages. So depending on the application, it falls on the user to

define the type of substrate to use. The following section describes some of the most well-

known techniques.

2.2.1 Randomly ordered (disordered) surfaces:

The first SERS substrates were made to duplicate the Raman enhancement observed by

Fleischmann et al on roughened silver electrodes. The substrate was fabricated by successive

electrochemical oxidation and reduction cycles. In short, the first step in the process is to clean

the surface to remove any organics and then immerse it into an electrolyte solution. Then the

oxidation potential is applied to the electrode which produces silver ions on the surface, Ag

Ag+ + e-. This is followed by the reduction potential which reduces the species to form silver

again, Ag+ + e- Ag. This cyclic process forms a disordered surface consisting of rough

protrusions typically in the size range of 25 nm – 500 nm [44, 45] and voids of various sizes. The

broad distribution of features forces the analyst to assess the topography first in order to locate

SERS active areas before sampling. This technique can give large enhancements but the

substrates are not reproducible from batch to batch.

Vacuum deposition is an alternative to the application of electrochemical oxidation-reduction

cycles for making rough metal nanostructures. Here metal atoms are evaporated on a thermally

controlled surface in a vacuum to create thin metal films [44, 46]. By varying the deposition rate

24

and the temperature one can produce surfaces with a range of features, from discontinuous

metal islands with high SERS activity to thick metal films that can be made conductive by further

heating. Recently, this technique has been used in combination with structured substrates.

Pillars, pyramids, fibers and other solid supports are made through various lithography

techniques and then the metal nanoparticles are vapor deposited on the top surface [45].

The most widely used medium for SERS studies is a silver or gold colloid because the chemical

synthesis procedure is fairly straightforward, the reagents are inexpensive and variability in size

and shape can be controlled. They are commonly synthesized through the reduction of metal

ions with sodium borohydride [47], citric acid [48] or other reducing agents [49]. Unless capping

agents are added the colloidal nanoparticles are very unstable as they will naturally aggregate

into large clusters. Therefore, surfactants, such as cetyl trimethylammonium bromide (CTAB)

and polyvinyl pyrrolidone (PVP), are added to the solution in order to control the growth of the

colloid through aggregation [50]. The surfactant prevents further aggregation permitting the

synthesis of nanoparticles of particular sizes. Adjusting the concentrations of the metal salt, the

reducing agents and the surfactants allows further control of the nanoparticle shape. Thus many

journal articles describe their methods for synthesizing nano-spheres, -cubes, -rods, -triangles, -

stars, and -diamonds.

Regardless of the shape there are a few problems associated with colloidal solutions. Even

though high amplification factors have been reported, this has been for high Raman scatterer

molecules such as R6G [42] and pyridine. Generally the SERS signal is unstable primarily due to

the varying interparticle spacing between nanoparticles and the constant Brownian motion of

the molecules in solution. Additionally, the signal is weak due to the surfactant layer, preventing

25

the molecules of interest from being in contact with the nanoparticles. Furthermore addition of

the analyte itself can cause aggregation.

Out of the need to increase the stability of colloidal solutions, particles are commonly deposited

on a flat surface such as a glass slide or a silicon wafer. The particles are allowed to dry and then

the molecule of interest is deposited on top of the nanoparticles. The SERS amplification comes

from the top layer of the nanoparticles which interact with the analyte. One widely observed

problem with this approach is that the dry nanoparticles would lift off when the analyte solution

was added. This causes a rearrangement of nanoparticles leading to problems with

reproducibility of signal intensities.

Silanization is a widely used method to fix nanoparticles and molecules to a glass surface.

Initially the glass slides must be thoroughly cleaned to remove any organics and contaminants

while leaving exposed hydroxyl groups on the surface. Common chemical methods include

incubations in piranha baths, water acid mixtures, alcohol washes and hydroxide baths followed

by rinsing in some solvent and heating [51]. The clean glass is then functionalized with a silane

base linker which physisorbs to the glass surface through the thiol head group. These linkers

also have a functional group on the tail end allowing it to bind to the silver or gold nanoparticles.

For example, 3-aminopropyltriethoxysilane (APTES) or 3-aminopropyltriethoxysilane (APTMS)

form a Si-O-Si bond with the glass surface and the amino group on the other end binds with the

nanoparticles [52-54]. The glass thiol bond is strong enough to leave the nanoparticles fixed on the

glass surface even after repeated washes. This technique provides a fairly uniform single

monolayer of nanoparticles that can yield amplification factors of 108 even though they have

26

been shown to vary from spot to spot by as much as two orders of magnitude [46]. Figure 5

shows a schematic of the immobilization procedure.

Label free SERS detection has worked well for small analytes but for some larger molecules an

indirect detection method is sometimes more appropriate. In this case, the nanoparticles are

labeled with a molecular dye known to produce high Raman scattering, i.e. R6G, crystal violet,

etc. It has been shown that by tagging the nanoparticles with reporter dyes it is possible to

create a detection platform similar to biological assays. Vo-Dinh et al were able to detect a

labeled breast cancer probe (BRCA1) after hybridization with a complementary single stranded

DNA segment. In this work, the probe is tagged with R6G, which is the actual analyte that is

detected. The DNA segment is specific to a sequence on the gene probe so when it is captured

on the substrate the SERS spectrum of R6G will appear. Any labeled probe that is not captured

by the DNA segment is washed off [55]. In this case the nanoparticles are attached to a secondary

antibody which binds directly to a primary antibody. The substrate is washed to remove any

unbound nanoparticles and then interrogated with a Raman spectrometer. If a SERS signal is

detected then it comes from bound particles. This method has allowed the detection of trace

concentration of biological molecules. Although this method certainly has its advantages for

developing highly specific SERS immunoassays but it does not achieve the label free sensor.

27

Figure 5: Schematic of silver or gold nanoparticle immobilization to a glass slide through silanization

protocol [52]. 2.2.2 Ordered periodic nanostructures:

The development of lithography techniques – such as photolithography, electron beam

lithography, focused ion beam milling and nanosphere lithography - have allowed the

fabrication of ordered nanostructures that can produce reasonably reproducible SERS

intensities. However, the equipment for these techniques is expensive and the process requires

multiple manufacturing steps thus lithography techniques are somewhat scarce in conventional

laboratories. Nevertheless, silver or gold nanoparticles have been deposited on arrays of posts,

island pillars, inverted pyramids and cavities [56] formed by photolithography methods.

Photolithography - the most widely used technique – involves the removal of bulk material with

UV light. By using a mask, selected material can be removed to produce the desired features.

However, due to a light beam’s diffraction limit resolution of λ/2 this technique alone cannot

produce the nanoscale gaps necessary for high SERS amplification factors. Oftentimes it is

combined with another technique such as chemical etching to produce smaller sized features.

28

For example, the structure for the Klarite substrate (Renishaw Diagnostics, UK) consists of an

array of square pits made from a silicon dioxide mask with apertures of approximately 1 μm [57].

The surface is etched with KOH to produce inverted pyramids which are then coated with a 300

nm layer of gold. The SERS amplification comes from the progressively smaller spacing between

the gold layers along the vertices of the pyramid. In 2006 the substrate was sold for $100 and

was designed for single use making it an expensive tool for routine analytical studies.

Electron beam lithography (EBL) and focused ion beam (FIB) milling are considered the best for

making reproducible SERS substrates with nanoscale resolution [46, 58]. Unfortunately, the time

required to make such features is not appropriate for mass production. EBL involves the removal

of material from silicon wafers covered with an electron-resist, which can be followed directly

by metal vapor deposition or a further chemical etch to make deeper cavities which are then

covered by metal deposition [59]. FIB milling uses a Gallium ion beam to make nanosized holes

directly on a silver or gold material with a spatial resolution of approximately 10 nm. Both of

these techniques have been mostly used to study the SERS effect and its variations due

nanoparticle geometry and interparticle distance.

Nanosphere lithography is another laboratory technique used to make substrates to study the

SERS effect. It involves the deposition of a layer of closely packed polystyrene beads on the

surface followed by vapor deposition of a thin silver nanoparticle film over the surface. The

interstices between the polystyrene beads serve as a mask and as the beads are chemically

lifted off the surface only the silver particles that fell through the interstices remain on the

surface forming a single-layered periodic array of triangle shaped nanoparticles [60]. Haynes et al

reported SERS amplification factors of 108 using this technique [61].

29

2.2.3 Scientific standards for SERS substrate platforms

It is stressed again that no method is superior to the others in all aspects, so it becomes the

user’s responsibility to have a well-defined problem and then choose the substrate fabrication

technique most appropriate for their application. Unfortunately there is an oversaturation of

fabrication methods in the literature and no guarantee of reproducibility across different

laboratories so it makes it difficult for scientists from a different discipline to follow a technique

and achieve their intended results. Due to misinterpretation of amplification factors, lack of

reproducibility and stability, there exists some confusion in the literature about the

performance of various methods. To remedy some of these issues, Michael Natan’s group

proposed a set of standards for fabricating SERS substrates and reporting the data in the

literature [43]:

1. Less than 20% spot-to-spot variation over 10 mm2.

2. Less than 20% substrate-to-substrate variation over 10 substrates.

3. Measurement on three non-resonant analytes

4. Less than 20% variation in signal measured weekly over one month

5. Bulk enhancement factors measured over regions corresponding to the size of the laser

beam.

These standards present useful criteria for groups working on techniques for fabricating

substrates but it also gives users reasonable expectations for interpreting results during a SERS

experiment.

30

2.3 Colloidal nanoparticle synthesis

As mentioned earlier metal colloids are the most widely used form of SERS substrates due to

their ease in preparation and favorable nano-scale features. Most colloidal particles below 100

nm in diameter are made by a chemical reduction method involving silver nitrate (AgNO3),

sodium borohydride (NaBH4) or sodium citrate and water as the solvent. Sodium citrate and

NaBH4 are reducing agents that isolate the silver atoms by creating a surface charge around its

mass and thus forming silver nanoparticles. These particles alone are stable for approximately

one week in solution. However, if the reducing agent is not carefully measured then the colloid

becomes unstable causing nanoparticles to aggregate after a time as short as 30 minutes [47].

Small clusters naturally form during metal deposition as colloidal particles aggregate into

‘dimers’, ‘trimers’, and eventually larger groups. This leads to a randomly organized

arrangement of occupied areas and voids which are smaller than the visible wavelength of light.

When a laser beam interacts with a nano-cluster the optical excitation, or a plasmon, will be

localized to the particle cluster creating a large local electrical field, hence the term “hot spot”

[19]. In a cluster there will be many ‘hot spots’ making it a suitable structure for large

enhancements. It should be noted that large enhancements can make it difficult to accurately

measure the concentration of an analyte as the intensity corresponds more to the EM effect at a

hot spot rather than the number of molecules attached at the surface. There is a tradeoff

between large enhancements and measurement of analyte concentration so statistical

measures are necessary to correlate signal intensity to analyte concentration.

31

2.3.1 Optical properties

The aggregation of nanoparticles can be observed visually by the color of the solution as it

changes from yellow to orange to violet and to grey [47]. The change in color occurs due to the

changing dipolar plasmon resonance of the colloid. Plasmon resonance is the collective

oscillation of free electrons on the nanoparticle surface at a resonant frequency due to the

incident light. Well-synthesized silver nanoparticles have a plasmon absorption band at

approximately 380 nm and when the particles begin to aggregate the absorption band red shifts

[62]. Aggregation can be accelerated by a variety of methods including heating effects, changes in

pH or addition of electrolytes. Therefore, plasmon absorbance is commonly used as a measure

of particle/cluster size. Tuning the plasmon resonance has become a research area of great

interest as specially designed nanostructures can be made for a variety of applications.

2.3.2 Stability of nanoparticles

Unfortunately colloidal aggregation gives nanoparticles a limited utility. It is not practical to

synthesize nanoparticles that need to be used immediately. To prevent aggregation an organic

stabilizer or capping agent is used during the synthesis process. These include surfactants or

ligands containing functional groups that adsorb easily to the metal such as thiol (-SH), cyano (-

CN), carboxyl (-COOH) and amino (-NH2) [63]. Not only does the stabilizer prolong the shelf life of

the colloid but it will also help control the growth of nanoparticles as well as keep a fairly

consistent interparticle distance [64]. For industry related applications, Natan recommends SERS

substrates produce stable amplification factors for at least 8 months [43]. This implies that a SERS

nanoparticle colloid must have a protective shell that can be removed upon use.

32

2.4 Nanoparticle printing inks

2.4.1 Use in industry for flexible circuits

The miniaturization movement led by the need for faster data processors and driven by the

advances in nanotechnology has made it possible to fabricate circuits on unconventional

surfaces (i.e. clothing fabrics, curved surfaces, paper, polymers, etc). Ink jet printing is an

attractive technology to fabricate electronic devices such as thin film transistors and radio

frequency identification (RFID) tags because it allows the deposition of precise patterns of

conductive materials. Additionally, conductive pathways can be directly printed on the substrate

in one step avoiding the multi-step processes of photolithography and vacuum deposition [65, 66].

Furthermore, the ink can be printed on large-scale organic sheets made out of plastic or paper

significantly reducing manufacturing costs.

Current research in this field has been devoted to making conductive lines in one single step by

developing ink formulations that can sinter at room temperature [67-69]. Nanoparticle printing

inks are defined as colloidal particles below 100 nm in diameter, suspended in a non-viscous

solution enabling deposition on various surfaces. The ink is usually composed of silver or gold

nanoparticles and is rendered conductive through a thermal annealing process. The nanometer

size gives the nanoparticles different characteristics from those of the bulk state allowing them

to sinter at low temperatures. For example silver nanoparticles have been shown to sinter at

temperatures below 150oC whereas bulk silver melting occurs at 960oC [66, 70-72]. Low

temperature sintering allows nanoparticles to be printed on polymers and paper without

damaging or deforming the surface.

33

The particles are encapsulated by a stabilizing ligand that acts as a barrier for oxidation and

agglomeration. The head group of the ligand is usually a thiol, amine or carboxylic acid, which

allows it to directly adsorb to the metal nanoparticle, while the free end is composed of a long

alkyl chain [73]. The alkyl chain can be adapted to modify the surface hydrophobicity as needed.

The particles also have a binder that improves adhesion with the surface.

There are a few types of printing technologies widely used to deposit ink on a surface. Drop-on-

demand systems store the ink in cartridges and a pressure pulse delivers droplets out of the

nozzle. In these devices low viscosity and high surface tension are important parameters that

will directly affect the printed patterns [74, 75]. Cantilever tips are also used to deposit solutions

on a substrate. In the case of tips, there is a hydrophilic reservoir containing the ink and an open

flow channel on the cantilever. As the tip makes contact with the surface there is a transfer of

solution through capillary action thereby depositing the desired amount on to the substrate [76].

Similar to this is dip-pen nanolithography in which an atomic force microscope tip is coated with

a molecular solution and then through capillary transport the tip deposits the molecules on the

surface [77].

2.4.2 Sintering behavior

Once deposited, the solvent naturally evaporates over time and the nanoparticles settle into a

close packed system. The stabilizing ligand layer, which encapsulates the nanoparticles, prevents

electrons from conducting so a relatively high resistivity can be measured on the film with a 4-

point probe ohmmeter. During thermal annealing the resistivity of the film decreases as the

nanoparticles lose the stabilizing ligands and begin to sinter. Figure 6 shows the temporal

relationship between heating temperature and resistivity loss in a DC circuit [66]. In more detail,

34

as the heating temperature increases there comes a point where the disassociation energy is

large enough to cause the stabilizing ligands to debond from the nanoparticle surface [69, 73]. This

is commonly referred to as the curing temperature and it depends on the length of the ligand

layer. Thus, shorter ligand layers will require lower temperatures to induce the sintering process

[78, 79]. Furthermore, if there was no ligand present and no reducing agent keeping charges apart

then sintering of nanoparticles less than 20 nm in diameter would almost occur immediately at

temperature below room temperature [80].

Upon further heating at higher temperatures, the free encapsulant goes through a sublimation

phase [78] allowing the nanoparticles to coalesce. The sintering process begins shortly after the

encapsulant burns off and the nanoparticle surface is exposed [69, 81]. Since nanoparticles have a

large surface area, the surface energy at the interfaces is quite high so some atoms will become

dislodged and interact with the neighboring particle [82]. Nanoparticles begin to interconnect in

the form of necking and upon further heating the surface area decreases leading to dense

connections. In fact the temperature of sintering will depend on particle size; small particles

sinter at lower temperatures.

35

Figure 6: Electrical resistivity of nanoparticle film as a function of temperature and heating time [66].

Figure 7 shows a simple schematic of two neighboring nanoparticles undergoing curing and then

the initial stage of sintering. Although not in the scope of this work, there are 3 stages in the

sintering process. The initial stage is characterized by neck growth between particles loosely in

contact. The intermediate stage, also known as densification, corresponds to onset of grain

growth and the formation of pores between particles [82]. The final stage occurs when grain

growth is impeded and densification increases. Conductivity becomes measurable as the

particles undergo densification through the sintering process and eventually form continuous

percolation networks. In a practical application, to make conductive lines, silver nanoparticles

are typically sintered for approximately 1 hour [74].

36

Figure 7: Coutts’ schematic representation of sintering of nanoparticles functionalized with a stabilizing

ligand before and after heat treatment [69].

It is important to note, the sintering temperature does not refer to a single temperature

because the kinetic process is continual over the entire temperature range of densification [80].

This is often confused in the literature. For comparison purposes the starting temperature

should be used in reference to the initiation of the rapid densification process. The sintering

temperature can be between 200 – 400oC lower for nanoparticles as compared to microspheres.

It is understood that both depositing greater silver nanoparticle concentration to achieve

greater coverage and applying a long annealing treatment will increase film conductivity.

Regardless of the process, the SERS enhancement will be attenuated upon increased

conductivity [41, 83, 84] due to the propagation of the localized surface plasmon. On the other

hand, if the deposited surface coverage is below the percolation threshold the SERS

enhancement has been shown to be quite high [83, 85]. In this case, a surface remains with a

random arrangement of nanoparticles with a distribution of large voids as well as a distribution

37

of very small gaps between neighboring nanoparticles. This is also shown during the sintering

process. If the heating is stopped early, at a point slightly before the necking stage, a

morphology will exist where the interparticle spacing is in the single unit nanometer scale. As

explained earlier, this condition of minimal interparticle distance between aggregates of at least

two nanoparticles has been shown to produce the largest SERS amplification factors.

38

Chapter 3: Review of Raman spectroscopy as an analytical technique 3.1 Raman spectroscopy

Raman spectroscopy is an optical technique that provides detailed vibrational information about

the structure and function of a molecule in much the same way as infrared (IR) spectroscopy.

While the IR spectrum comes from the absorption of the incident light, the Raman effect comes

from the inelastic scattering of photons as they interact with a molecular system. The

phenomenon is named after Indian physicist Sir Chandrasekhra Venkata Raman, who in 1928

first published evidence of the effect; the fringes of a benzene molecule were produced by the

scattered light of sunlight passing through a telescope. Sir Raman was able to prove that elastic

(Rayleigh) scattering did not alone account for the scattered light coming from the sample but

rather there was a shift in energy from the incident wavelength, which was directly related to

the sample’s molecular structure.

Classically, when light irradiates a diatomic molecule, the electric field of the laser beam induces

an electric dipole moment in the molecule [86]. The dipole moment, P, is equal to the

polarizability of the molecule, α, and the electric field strength, E, of the incident beam, P = αE.

The incident beam’s frequency is denoted as v0 while the molecule’s vibrational frequency is

written as vm. If inelastically scattered light has a smaller frequency than the incident frequency,

v0 - vm, then it is called Stokes scattering. This occurs when the incident photon interacts with a

molecule in the ground state thereby losing energy as it excites a vibration in the molecule [87].

On the other hand if the scattered light has a larger frequency, v0 + vm, then it is referred to as

anti-Stokes scattering. This occurs when the incident photon interacts with a molecule in an

39

excited vibrational state and it gains energy from the molecular vibrations. Equation 4 can be

used to describe scattering from a molecule in simple terms.

𝑃 = 𝛼𝑜𝐸𝑜 cos 2𝜋𝑣𝑜𝑡 + 12�𝛿𝛼𝛿𝑞�𝑜𝑞𝑜𝐸𝑜[cos{2𝜋(𝑣𝑜 + 𝑣𝑚)𝑡} + cos{2𝜋(𝑣𝑜 − 𝑣𝑚)𝑡}] (4)

where the first term represents an oscillating dipole characteristic of Rayleigh scattering while

the second term corresponds to the anti-Stokes (v0 + vm) and Stokes (v0 - vm) scattering [86]. If

there is no change in polarizability, (δα/δq)o = 0, then there will be no Raman signal. Figure 8

shows an energy diagram of the types of scattering that can occur in a sample. The dominant

scattering process comes from elastic vibrations in the molecule. A cut-off notch filter in the

Raman spectrometer blocks the elastically scattered light. The Anti-Stokes scattering is weak

because the excited electrons start from a higher energy level but still return to the ground

state. Thus, a Raman spectrum only shows the larger Stokes contributions.

Figure 8: Schematic showing the three different types of scattered light. Stokes refers to the inelastic scattering of photons at a lower energy than the incident photons. Rayleigh is the elastic scattering of

photons. Anti-Stokes refers to the inelastic scattering of photons at a higher energy.

40

The narrow bands in a Raman spectrum have a Lorentzian shape and correspond to particular

vibrations in the molecule, which depend on the arrangement of atoms and bond strengths in a

molecule. The bands are referred to by their wavenumber, �̅�, which is defined as the frequency

of the vibration, vvib, divided by the velocity of light, c, �̅� = 𝑣𝑣𝑖𝑏 𝑐 ⁄ and are expressed in cm-1.

The frequency of vibration of a diatomic molecule is given by equation 5.

𝑣𝑣𝑖𝑏 = 12𝜋𝑐 �

𝐾𝜇

(5)

where K is the force constant of a spring representing the strength of a molecular bond between

two atoms and μ is the reduced mass of those atoms. Thus, the wavenumber in the Raman

spectrum will depend on the strength of the bond and the mass of the atoms. The intensity of

the bands will depend on the change in polarizability of the molecule and is given by equation 6.

𝐼𝑅𝑎𝑚𝑎𝑛 = 𝐼𝐿𝑎𝑠𝑒𝑟�̅�𝐿𝑎𝑠𝑒𝑟4 |𝑒0𝛼�𝑒𝑠|2𝑑Ω (6)

where eo and es are unit vectors representing the laser polarization and direction of observation,

respectively, whereas dΩ is the solid angle of light collection [88]. In a molecule the intensity of

the Raman bands will also depend on which mode of vibration contributes the most. The typical

vibrational modes in a bond are symmetric stretching, anti-symmetric stretching and bending

vibrations. The collection of all the wavenumber bands and respective intensities gives the

unique signature of the molecule. Most identifier wavenumbers are contained in a portion of

the spectrum between 700 cm-1 and 1900 cm-1, which is commonly referred to as the fingerprint

region [89]. Minor shifts in wavenumber are indicative of slight changes in the molecule’s

41

orientation, which is the main reason why Raman spectroscopy is a very powerful

characterization technique.

Unfortunately, the Raman signal is very weak because of the extremely small cross sections

(10-30 cm2 per molecule) compared to fluorescence cross sections (10-16 cm2 per molecule) [26, 90].

As Kneipp et al pointed out, with such a low Raman cross section and an excitation source of

100 mW focused to 1 μm2, one Stokes photon would be scattered from a single molecule once

every 16 minutes [90]. In the past, it was common for one to have to wait more than one hour for

one collection so naturally other spectroscopy measurements were preferred. With advances in

the development of versatile instruments, efficient light filters and sensitive charge-coupled

device (CCD) detectors, collection times for high quality spectra have been reduced to a few

minutes. Another common problem is the fluorescent impurities in a sample which can interfere

with Raman bands or completely mask them out [91]. These can now be avoided by using a

wavelength in the near infrared or even in the ultra-violet region.

Applications of Raman Spectroscopy

Although the Raman effect is considered weak it does provide some advantages over other

spectroscopy techniques. For instance, X-Ray diffraction and NMR spectroscopy can only be

used in specific mediums, whereas Raman spectroscopy is applicable to the solid, liquid and

gaseous states [86]. Infrared (IR) spectroscopy is often described as a complementary technique

to Raman scattering as it also gives highly specific information about vibrational frequencies in

molecules. In other words, molecules not Raman active are IR active and vice versa. Some of the

most intense Raman bands can result from totally symmetric vibrations, covalent bonds and

stretching vibrations [86]. For example, C=C, C-C, P=S and S-S are strong Raman scattering bonds.

42

One advantage of Raman scattering over IR spectroscopy is that IR light is strongly absorbed by

water. Such absorption results in a large fluorescence signal that masks important bands in the

fingerprint region of the spectrum. In contrast, water is a weak scatterer making applications in

biology such as cellular composition and classification of bacteria in the near infrared region

common [92]. Thus, Raman scattering is commonly used to study larger molecules such as

proteins, nucleic acids, carbohydrates and lipids. For instance the Raman bands in human serum

protein can be attributed to the molecular vibrations of their amino acids (Figure 9).

Raman spectroscopy is non-invasive so it can be used to analyze substances contained in

transparent packaging. This makes it particularly well suited for the pharmaceutical industry

during the manufacturing process. Some applications include identification of raw materials,

quantitative analysis of active ingredients, identifying polymorphs and quality assurance during

production [93].

Figure 9: Raman spectrum of human serum protein with band assignments corresponding to

specific molecular bonds and amino acids [94].

43

It is also non-destructive when using low powered lasers making it useful for determining

differences in the composition of in-vivo healthy tissue versus diseased tissue without causing

further damage. Differences between cancerous and healthy tissue have been observed in the

Raman bands related to lipids, proteins and nucleic acid [95]. Most of these studies have been

conducted on skin as it is easily accessible. On this front, Lieber et al demonstrated the ability to

discriminate between inflamed scar tissue and nonmelanoma skin cancer [96]. However, due to

the existence of multiple peaks many samples need to be collected and post signal analysis

techniques, such as multivariate statistical analysis and regression analysis must be employed to

discriminate important peaks.

By far its most common use has been in analytical chemistry for the determination of how

molecules interact with each other and how they interact with surfaces. The interaction of

ligands to proteins is widely studied with resonant Raman scattering. Perhaps analytical

chemists are the most enthusiastic about SERS as it could offer a tool to detect a single molecule

as well as its chemical structure.

3.2 Raman spectroscopy experimental protocols: For all experiments described in this thesis two Raman spectrometers were used: 1) the DeltaNu

Advantage NIR system (Intevac, Inc. Santa Clara, CA) with a 785 nm wavelength and 2) the InVia

Raman Microscope (Renishaw Hoffman Estates, IL) with four laser wavelengths 488 nm, 514 nm,

633 nm and 785 nm.

44

Intevac Spectrometer

The Intevac spectrometer is a benchtop system that easily records spectra from substances in a

cuvette. The spectrometer is equipped with a NuScope attachment which allows the collection

of Raman scattering from planar substrates. The focal plane was approximately 0.5 inches so

two cover slips were placed on the X-Y stage and one microscope glass slide on top of the cover

slips so the sample would be on the right focal plane. Focus on the surface was achieved by

interrogating a silica chip and adjusting the coarse focus until the intensity of the 520 cm-1 peak

was at a maximum. Analytical samples were placed on the same plane on top of the microscope

slide. The wavelength of the laser was 785 nm and the power at the focused plane was

measured to be 26 mW at a low power setting. The spot size was approximately 50 μm. The X-Y

stage allowed the horizontal and vertical movement of the sample. Each tick mark on the turn

wheels corresponds to an interval of 100 μm. For best results, spectra were collected in the

dark.

Depending on the analyte, each acquisition was taken at intervals of 3, 5 or 10 seconds under

polarized light with low power. If the CCD sensor was oversaturated then the acquisition time

was reduced. When necessary, an interval of 2 seconds was used between collections to allow

movement to another location. The acquisition software automatically removes background

noise by taking a reference spectra at given intervals. For most experiments, an interval of 5

collections was chosen. Longer intervals would cause spurious peaks or interference peaks from

cosmic rays to appear in the spectra. These can be removed when one is certain they are not

part of the spectrum but it is much easier to avoid them altogether by choosing a small enough

interval. Each acquisition was repeated at least 10 times to remove any spurious peaks and to

attain a good average. In some cases, 30 to 40 spectra were collected over a sample to ensure

45

statistical significance. In addition the software automatically corrects the baseline of the signal

with 162 points. Each individual spectrum along with the average spectrum of a series of

collections were saved as .spc files and .txt files so they could later be analyzed with the

Grams/AI software (ThermoScientific, Philadelphia, PA) and Matlab (MathWorks, Natick, MA),

respectively.

InVia Raman Microscope

The Renishaw spectrometer is a more powerful spectroscopy tool as it has an inverted

microscope that allows one to focus on a particular area and then collect a spectrum from the

same target area. It has 4 excitation sources to choose from and one has more control of the

power, focus, post processing, etc., when compared to the Intevac. However, we did not gain

access to this instrument until November 2011 so it was only used for the experiment dealing

with the reproducibility of the printed dot arrays provided by UT Dots, InC. Since the microscope

objective is inverted the substrate must be turned upside down to interrogate its surface. Focus

on the surface was achieved by interrogating a silica chip facing downward and adjusting the

fine focus until the intensity of the 520 cm-1 peak was at a maximum during a repeated 5 second

acquisition.

For the reproducibility study, an excitation wavelength of 785 nm was used with a microscope

objective of 20x. The laser spot size was approximately 1 μm and the power was set at 0.1% for

Rhodamine samples. In general, high power is avoided for biological samples and chemicals

because it could damage the structure of the molecule. Mapping scans were used to

characterize the surface of printed dots with at least 10 points equally spaced over the diameter

of the dot. A spectrum from each location was collected 6 times for 10 seconds each to reduce

46

noise. The cosmic ray removal setting was used to remove any spurious peak during acquisition.

A Matlab script was used to interpret the data and find the intensity of each peak. Since an

annular area close to the perimeter of the printed dot varied in homegeniety The average signal

was overlayed over an image of each dot to display the low variance over the center of the dot.

47

Chapter 4: Microwave absorption as a method to sensitively monitor sintering and its use for the fabrication of SERS substrates.

4.1 Abstract

One of the most widely used methods for surface enhanced Raman scattering (SERS) employs

silver or gold nanoparticles either in colloidal suspension or in the dry-drop form. In such

substrates the SERS amplification factors (AFs) depend critically on the interparticle distances.

Here we demonstrate that microwave absorption as a function of temperature in dry-drop

substrates can be used as a probe to demarcate temperature regions for thermal annealing to

produce SERS substrates with very high AFs. Under isothermal heating an optimal annealing

time can be determined to improve SERS performance. A descriptive model is developed to

conceptually explain how microwave absorption monitors the transient process of dimers from

a resistive weak-link state to an electrically conductive state. The model and experiments show

that the strongest SERS signal precede a nanoparticle state with large microwave absorption

gains.

Keywords: SERS, thermal annealing, microwave absorption, silver nanoparticle inks

4.2 Introduction

Although a variety of nanolithographic methods have been employed with some success to

fabricate SERS substrates [97, 98], metallic colloids are still widely used due to the ease with which

silver and gold nanoparticles can be produced. However, SERS signals obtained using colloidal

suspensions are not highly reproducible. In colloidal suspensions, most of the SERS fluctuations

are substrate related and are largely due to the Brownian motion of the nanoparticle clusters,

48

the analyte molecules or a combination of the two. Fluctuations can also occur due to the

inherent poly-dispersity of the colloids. Although some of these motional fluctuations may be

expected to be absent in ‘fixed’ or dried-colloid substrates, several other factors can make such

substrates less than stable. For example, changes at the nanometer level in the interparticle

spacing can sensitively influence the coupled-localized surface plasmon (LSP) resonance

condition and hence the SERS amplification. Evidence has been reported for both heating and

thermal annealing under Raman laser irradiation [5, 99, 100]. Such thermal effects can change the

substrate morphology and dislodge the analyte molecules from under the laser beam giving rise

to fluctuations in the SERS signal.

Nanoparticle inks are colloidal suspensions of silver or gold nanoparticles in water or some other

suitable organic solvent. Adhesion promoting chemicals are usually added to such ink

formulations. The nanoparticles in these inks can be sintered at comparatively low temperatures

(70 – 200oC) to fabricate electrically conducting circuit elements on a variety of flexible

substrates [65, 66, 101]. The aim of thermal annealing in such applications is to create electrically

robust contacts between nanoparticles that are initially separated by surfactant polymer shells

in the as-prepared ink. When properly annealed, conductivities approaching bulk silver values

have been reported[101]. However, if the annealing is interrupted before the formation of robust

connections between nanoparticles over large areas, it is possible to obtain a sample that

contains particle clusters of varying sizes and with a distribution of interparticle distances. Such

substrates exhibit relatively high (108 – 109) SERS amplification factors (AFs) [3, 5]. The reason for

the high AFs in nanoparticle clusters is a lack of translational symmetry which does not support

propagating electromagnetic (em) waves. Thus em excitations get localized to very small regions

that can create SERS ‘hot spots’ – regions with very high SERS amplification. Voids in these

49

clusters can also act as resonant nano-cavities and further contribute to SERS amplification. AFs

as high as 1012 have been reported in such clusters [21, 28, 39].

Measurement of the dc-resistivity of thick film samples is usually employed to monitor the

sintering process in nanoparticle inks. In these measurements, the resistance of the film is

interpreted by measuring the current flow and the electric potential drop between two points.

In inhomogeneous films the dc method can be highly unreliable since the measurements are

indicative of some isolated percolation path and not of the whole film. The non-contact

microwave measurements do not rely on any single percolation path and are global in nature

thus they yield a more detailed picture of the sample. Unlike the dc-method the microwave

method is also sensitive to the interparticle separation, which is an important parameter in

determining the SERS amplification factor. In this chapter it is shown that by terminating

sintering in the early stages – as indicated by microwave absorption – the nanoparticle inks can

be used to fabricate SERS substrates with AFs exceeding 108.

4.3 Materials and methods

Substrate Preparation Silver nanoparticle inks were supplied by Pchem Ink (Bensalem, PA – Ink 1) and UT Dots, Inc

(Champaign, IL – Ink 2). Both inks contained 20% Ag by weight. Ink 1 has an average particle size

in the 30-50 nm range and its composition (surfactant and adhesion promoters) is proprietary.

The nanoparticles in Ink 2 are suspended in toluene, have an average particle size in the 10-15

nm range and are encapsulated by an oleylamine surfactant. Samples were fabricated by

depositing 10 μL of nanoparticle ink over square glass cover slips using a micropipette followed

by a two-step spin coating process (1 min at 700 rpm followed by 1 min at 2000 rpm).

50

Microwave Absorption Measurements The microwave absorption measurements were made using a modified X-band Varian E-12

electron paramagnetic resonance (EPR) spectrometer with an associated heater accessory. The

samples (~ 3 mm x 3 mm) were supported at one end of a sapphire rod with a small dab of

Apiezon H grease (SPI Supplies, West Chester, PA). The grease serves as an excellent sticking

agent and it performs well at temperatures between -10oC and 240oC. The sapphire rod was

positioned at the center of a rectangular TE102 cavity resonant at 9.5 GHz. The microwave power

was set between 5-20 mW depending on the surface area of the sample. A smaller sample

would require a higher microwave power to see observable changes in the absorption. Figure 10

shows an image of the modified EPR spectrometer and a schematic drawing of the sample

placement in the microwave cavity. At various points along the microwave absorption curve the

sample was removed from the cavity to characterize its properties after heating conditions.

Figure 10: A photo of the X-band Varian E-12 EPR spectrometer (left). A schematic of the modified EPR

heating cavity for microwave absorption measurements (right).

51

Sample Characterization Each sample was cut in two pieces using a diamond tip pen. One half of the sample was

characterized under the SEM while the other half was used as a SERS substrate. Under SEM,

samples were not sputter coated as the silver itself was conductive after partial removal of the

stabilizer during heating. Samples were placed on circular pegs and carbon tape was used to

secure the sample to the peg. Micrographs were taken with the Zeiss Supra 50VP scanning

electron microscope (SEM) at the Centralized Research Facility (CRF) at Drexel. For SERS

samples, a 5 μL drop of Rhodamine 6G was drop deposited on top of the substrate and allowed

to dry for 10 minutes. The SERS spectra were collected with the Advantage near infrared Raman

spectrometer at a 785 nm excitation wavelength as previously described in Chapter 3.

Sample Heating Dry nitrogen gas (4 psi) passing over a resistively heated filament was used to heat the sample in

the cavity. A voltage regulator attached to the heating element could be adjusted to change the

temperature. Ten to fifteen volts would yield temperatures between 150oC to 200oC. A t-type

thermocouple (TC) was used to monitor the temperature inside the cavity. It was inserted from

the top side of the cavity and rested 5 mm above the sample. The thermocouple was connected

to a cold junction compensator (Omega Engineering, Inc., Stamford, CT) to calibrate the

temperature. An online t-type TC conversion chart was used to monitor the temperature during

experiments and a conversion fomula was used in Excel to convert the voltages to temperature

during analysis.

Data Acquisition Thermocouple and microwave voltages were recorded using two channels on a DI-710 data

logger (DataQ Instruments, Akron, OH) which interfaced with WinDAQ software. Data was

acquired at 20 Hz and the gain for each channel was set to 100. The data was viewed under

52

scroll mode and upon reaching the heating time (under isothermal conditions it was 10 min) or

the maximum temperature (240oC) the acquisition was stopped. Raw data was transferred to

Excel for post analysis and then graphed using a Matlab script.

4.4 Results & Discussion

4.4.1 Microwave absorption vs. temperature

A typical plot of microwave absorption versus temperature for Ink 1 deposited on a glass cover

slip as described earlier is shown in Figure 11a. The samples are heated at a rate of

approximately 6oC/min. The temperature dependence of the microwave absorption can be

described qualitatively as follows. The skin depth δ = [π f μm σ] -1/2, where f is the microwave

frequency, σ = 6 *10 7 mho/m is the conductivity of silver and, since silver is diamagnetic, μm = μ0

= 4 π * 10 -7 T m/A is the permeability of free space. At f = 10 GHz, the skin depth is

approximately 700 nm, which is greater than the average size of the nanoparticles by nearly two

orders of magnitude. This makes the individual nanoparticles separated from each other by

surfactant molecules (oleylamine) nearly transparent to the microwaves in the as-deposited

SERS substrates at room temperature. As the temperature increases, the nanoparticles begin to

get closer together as the surfactant molecules are partially removed by the thermal treatment.

With a further increase in temperature, weak resistive connections are formed between the

metallic nanoparticles. The microwave currents between these loosely coupled particles give

rise to microwave losses resulting in an initial rise in the slope of the microwave absorption. The

microwave absorption continues to increase as more weak links are formed with further

heating. As the sintering process continues and the weak links become more robust with the

resulting decrease in the weak link resistance, the microwave absorption begins to decrease.

When the nanoparticle cluster size exceeds the microwave skin depth, increasingly larger parts

53

of the clusters are shielded from the microwaves. The combined effect of robust interparticle

connections and increasing cluster size on the microwave absorption gives the characteristic

temperature dependence of the microwave absorption at high temperatures.

In three separate runs, samples were removed from the microwave cavity at 100, 110 and

120oC. SERS spectra for aqueous solutions of 10 μM Rhodamine 6G (R6G) are shown in Figure

11b and the scanning electron microscopy (SEM) micrographs are shown in Figure 11c. For the

substrate heated to 100oC the microwave absorption is low, as explained earlier, because of the

fact that the nanoparticles are still isolated from each other by the surfactant molecules.

Additionally, the surfactant prevents the analyte from adsorbing to the Ag nanoparticle surface,

thus resulting in low SERS amplification. As explained in Chapter 2, the magnitude of the

electromagnetic component of the SERS effect is sensitive to the distance between the analyte

and the metal nanoparticle or cluster.

As the temperature increases, there is a further loss of surfactant so the nanoparticles begin to

form small clusters. It is now well known that ‘dimers’ (two nanoparticles separated by a few

nanometers) and small size clusters can give rise to hot spots and very high SERS amplification

[28]. In a cluster, as shown by the SEM micrograph at 110oC, there are numerous sites between

the constituent nanoparticles where plasmonic excitations can be localized. Under this condition

the largest SERS signal magnitude were found.

54

Figure 11: a) A plot of microwave absorption versus temperature for an Ink 1 substrate; b) SERS spectra of

10 μM R6G; and c) SEM micrographs at three temperatures as indicated. Scale bar depicts 500 nm. Further heating destroys the hot spots as the clusters get increasingly larger. At elevated

temperatures, the clusters get multiply connected and voids begin to appear in the structure,

Figure 11c. As the clusters grow larger, the contribution to SERS amplification from plasmonic

excitations in single particles and small clusters would decrease while the contribution from the

nanocavities may become the primary source of SERS amplification [39]. Thus under these

conditions the lowest signal were found.

Since the SERS amplification depends very sensitively on the interparticle separation, the

thermal treatment can provide a direct control of the SERS amplification factor. It would be

tempting to use the microwave absorption versus temperature plots to find the optimal

conditions for fabricating SERS substrates. However, experiments show that when the ink

samples were heated in the microwave cavity and then withdrawn near the peak of the

55

absorption curve, the SERS amplification was not reproducible from sample to sample. This is

not surprising because at elevated temperatures the sintering will become increasingly

dependent on the annealing time. The most promising method to control the morphology of the

substrates is isothermal annealing at temperatures where the microwave absorption first begins

to rise steeply. In this temperature region sintering proceeds slowly, which makes it possible to

control the interparticle distances or the cluster sizes by varying the annealing time. More of this

is discussed later in this chapter; see Figure 17 for additional information.

Ink-2, which begins to sinter in the 180oC - 190oC temperature range, was used to show the

general applicability of the microwave absorption technique to locate an appropriate heating

temperature region for pretreating a SERS substrate. A plot of the microwave absorption versus

temperature for Ink-2 is shown in Figure 12a. As will be shown later in Chapter 5, sintering will

only take place after the partial removal of the stabilizing layer which explains why the

exothermic peak in the differential thermal analysis occurs slightly before the mass loss

observed in the thermogravimetric analysis. It is noteworthy to remind the reader that for Ink-1

(30 – 50 nm in size) the increase in microwave absorption occurred at a much lower

temperature (Figure 11a) than for Ink-2 (10 – 15 nm in size). It is reasonable to infer from these

results that the onset of the rise in microwave absorption is dependent on the partial removal of

the stabilizer and not the nanoparticle size. Furthermore, the microwave absorption was

measured for a gold nanoparticle substrate, which was also encapsulated with an oleylamine

stabilizer and a similar rise in microwave absorption was observed at approximately the 180oC –

190oC range (Figure 12b). Typically, gold sinters at a higher temperature than silver however in

this case the rise in microwave absorption occurred in the same temperature range as silver.

Since microwave absorption occurs at the site of weak links between two particles it implies that

56

(a) (b)

Figure 12: a) A plot of microwave absorption versus temperature for the Ink-2 substrate and for the b) gold nanoparticle ink substrate.

the gold nanoparticles have lost their protective shell, are in intimate contact with each other

but have not necessarily sintered.

Nevertheless, using the temperature where the onset of microwave absorption rise occurred

(~180oC), a second set of substrates were heated at that temperature for intervals of 5 minutes

up to 30 minutes. Figure 13a shows the SEM micrographs of Ag nanoparticles deposited on glass

cover slips after heating for various time intervals at 180oC. The typical sintering process was

observed, as previously described. It is important to note that with no heating the shape of the

nanoparticles cannot be discerned on the micrograph due to the surfactant layer which acts as

an insulator. On the other hand, at 10 minutes of heating the individual particles are visibly

present although they are isolated from each other. A clear distribution of interparticle

distances is observed. On 20 minutes the individual particles cluster together become larger in

size and changing the interparticle distance once again. At the 30 minute heating time point the

clusters have now becomes large masses and the global surface appears to be well connected.

57

Figure 13: (a) SEM micrographs of Ink 2 as-deposited substrate, 0 minutes, and annealed at 180oC for 10,

20 and 30 minutes. Scale bar depicts 500 nm. (b) SERS spectrum of 1 μM R6G on an ink 2 substrate annealed at 180oC for 15 minutes. The inset is a plot of the normalized SERS intensity of the 1364 cm-1 line

as a function of annealing time at 180oC. A typical SERS spectrum for an aqueous 1 μM R6G sample using a substrate annealed at 180oC

for 15 minutes is shown in Figure 13b. The SERS AFs were calculated as described by Le Ru’s

group [40] after experiments explained in chapter 2 of this thesis. The highest AFs exceeding 108

were obtained for samples annealed at 180oC for 15–20 minutes. The inset in Figure 13b shows

the normalized intensity of the 1364 cm-1 R6G band as a function of annealing time at 180oC

from data points collected from three different substrates for each heating time point. The

intensity in the inset was normalized to the SERS intensity of the 1364 cm-1 line of the spectrum

shown in Figure 13b. As can be seen from the plot, the SERS amplification remains nearly

unchanged with annealing time in the 10-15 minute range while decaying at longer heating

times. Such plots can be useful in determining the annealing time for fabricating SERS substrates

with optimum AFs.

58

Microwave absorption is irreversible

It is well known sintering is an irreversible process as the nanoparticles continually lose surface

energy at their interfaces as they build bonds with their neighbors [82]. Thus, it was important to

confirm that the microwave absorption measurement followed the same behavior. This was

accomplished by applying a heating-cooling cycle consisting of three steps: 1) a steady

temperature increase at a rate of 6oC per minute from room temperature to the point of

microwave absorption rise, 2) a stop in heating until the microwave cavity temperature reached

40oC and 3) an increase in heating at the same rate as step 1 to a temperature well above the

rise in microwave absorption. Figure 14 shows the microwave absorption behavior of the

nanoparticle film during the heating-cooling cycle. One drawback of ramping the temperature

and then stopping the heating after the microwave absorption begins to rise is that the heating

and cooling rates are not the same. This means that the sintering effects do not stop

immediately during the cooling step.

During the first heating cycle the microwave absorption responds as expected with a rise in

absorption as the nanoparticles begin to lose their protective shells and approach each other.

When the heating is stopped, the cooling cycle begins but the sintering continues due to the

high energy still present in the system. As the temperature begins to fall, the absorption decays

to a steady state possessing a value higher than the starting point. This can be explained by the

following. Toward the end of the cooling phase, a large majority of nanoparticles have sintered

while a small minority remains with their shells partially intact but very close to other

nanoparticles. As the second heating cycle gets underway there is a slight increase in microwave

absorption at the same temperature as the initial rise in microwave absorption. This slight

increase is indicative of the destruction of the remaining weakly linked particles.

59

Figure 14: Microwave absorption measurement of nanoparticle film during heating-cooling cycle.

To summarize, microwave absorption is not reversible for two reasons: 1) during the cooling

cycle the microwave absorption did not return to its initial value and 2) during the second

heating cycle the microwave absorption increase was a few orders of magnitude lower than

what was observed during the first heating cycle. This is indicative that microwave absorption is

related to sintering; once the nanoparticles are sintered they cannot return to their original

state.

4.4.2 Formulation of microwave absorption model

Figure 15 shows a typical TEM micrograph of the as-deposited substrate. The film surface is

dominated by single nanoparticles separated from each other by the ligands on the nanoparticle

surface. In the descriptive model, the particles are assumed to be in one of three stages, as

shown in Figure 16a. Stage 1 assumes a dimer composed of a particle and its nearest neighbor

separated by the thickness of two ligand layers. The dimer is transparent to microwaves because

it is much smaller than the microwave skin depth of roughly a micron at 10 GHz.

60

Figure 15: A TEM micrograph of the nanoparticle ink as-deposited on glass. The scale bar represents 50

nm.

(a) (b) (c)

Figure 16: a) Schematic model of three dimer stages, b) typical plot of microwave absorption versus temperature and c) TEM micrographs of nanoparticles representative of the three dimer stages. Scale

bars indicate 5 nm. In stage 2 the ligand layer has been partially removed bringing the nanoparticles closer to each

other so as to form weak resistive links between them. In this configuration the atoms on the

surface are very unstable. Thus, the microwave currents through such weak links give rise to the

observed microwave losses. In stage 3, as a result of sintering the dimer constituents have

essentially merged into each other and produce no microwave absorption due to the

61

disappearance of the weak link. Such particles are again transparent to the microwaves. This

irreversible evolution from stage 1 to stage 3 qualitatively describes the microwave absorption

as a function of temperature shown in Figure 16b. Since the losses occur in stage 2, microwave

absorption can be used to monitor the population of particles transferring in and out of this

stage. In essence this is a method to monitor the creation and destruction of hot spots in a

dimer configuration. Figure 16c shows TEM micrographs of representative nanoparticle dimers

from each stage after heating for various times.

Microwave absorption during isothermal heating

Figure 17 shows the time dependence of microwave absorption in the dry-drop ink substrates at

three temperatures as indicated. Qualitatively, the microwave absorption can be explained as

follows. As the ligand is removed upon heating, the nanoparticles move closer to each other and

start forming interparticle weak links. Microwave currents through these weak links, as

described earlier, give rise to the observed microwave losses. When the heating temperature is

below the sintering temperature of the ink, the formation of dimers in stage 2 follows a logistic

growth with no formation of stage 3 dimers. Under these conditions ligand removal is slow with

minimal sintering in the sample. As the isothermal heating temperature is increased to approach

the ink sintering temperature, the microwave absorption rapidly increases up to a maximum

and then decays to a new baseline. In this case the majority of dimers that reach stage 2 will

continue to stage 3.

62

Figure 17: Microwave absorption versus heating time at three temperatures, 160oC, 180oC and 2000C. The

solid line indicates the model fit according to equation 13 while the dotted lines indicate the raw data points.

Microwave absorption model The population of dimers at each stage at time, t, can be described as N1(t), N2(t) and N3(t) with

the total population, N(t), of the sample at any point in time equal to:

𝑁(𝑡) = 𝑁1(𝑡) + 𝑁2(𝑡) + 𝑁3(𝑡). (7)

Since the microwaves can only “see” dimers in stage 2 then the microwave absorption is

proportional to N2(t). Thus, equation 7 can be rearranged as:

𝑁2(𝑡) = 𝑁0 − 𝑁1(𝑡) − 𝑁3(𝑡) . (8)

where N0 is the initial dimer population.

63

Before heating, it is assumed all dimers are in stage 1 so that N1(0) = N0. The population decays

over time due to a negative rate constant, k1, based on the rate of ligand removal and the

temperature. The population of N1 will not decay to zero as there are many more nanoparticles,

which will not reach stage 2. Thus we assume that the population will reach a lower limit, n1.

Equation 9 shows the logistic equation used to model the depletion of N1(t).

𝛿𝑁1𝛿𝑡

= −𝑘1 𝑁1 �1 − 𝑁1𝑛1� . (9)

Solving equation 9, the population of dimers in stage 1 as a function of time can be expressed

as:

𝑁1(𝑡) = 𝑁0 𝑛1 𝑁0 + (𝑛1 − 𝑁0) 𝑒 𝑘1 𝑡 . (10)

Similarly, the population growth of sintered dimers in stage 3 was modeled as a logistic growth

given by equation 11. The rate constant, k3, depends on the sintering temperature of the

nanoparticles and is positive in this case with n3 as the final population of stage 3 dimers.

𝛿𝑁3𝛿𝑡

= 𝑘3 �1 − 𝑁3𝑛3� . (11)

Thus, the population growth of dimers in stage 3 as a function of time becomes:

𝑁3(𝑡) = 𝑛3 �1 − 𝑒−𝑘3𝑛3

𝑡� . (12)

64

Combining equations 8, 10 and 12, the population of dimers in stage 2 can be expressed as:

𝑁2(𝑡) = 𝑁0 − 𝑁0 𝑛1 𝑁0 + (𝑛1 − 𝑁0)𝑒 𝑘1 𝑡

− 𝑛3 �1 − 𝑒−𝑘3𝑛3

𝑡� . (13)

Equation 13 was used to generate the theoretical fits to the microwave absorption versus

heating time experimental data shown in Figure 17.

4.4.3 Microwave absorption and SERS

Microwave absorption at the weak links between the silver nanoparticles and SERS, due to the

enhanced local em field, are two seemingly unrelated phenomena. However, due to a fortunate

coincidence the microwave absorption can be used as an indicator of a morphology that gives

rise to higher SERS intensities. The correlation arises due to the manner in which the

geometrical configuration of nanoparticles influences both the microwave absorption and the

SERS signal. Maximum SERS enhancement is obtained when the interparticle spacing of a dimer

is a small fraction of the diameter of one of the particles [28]. This configuration closely precedes

the point where weak links between nanoparticles begin to form giving rise to microwave

losses. It is this fact that allows one to correlate the microwave absorption to SERS

enhancement.

Figure 18a shows a representative SERS spectrum from a 10 μM folic acid solution deposited on

a thermally treated substrate. The 1587 cm-1 peak, which is assigned to phenyl ring

deformations [102], was used to track the signal intensity in relation to heating time and

65

Figure 18: A) A representative SERS spectrum of a 10 μM folic acid sample shows the main peaks at 1176 cm-1, 1497 cm-1 and 1587 cm-1. B) Intensity of the folic acid’s 1587 cm-1 peak after heating at 180oC and

200oC for various time intervals. temperature. Figure 18b shows the average SERS peak intensity at 180oC and 200oC at the

various times indicated. Each parameter was tested on three different substrates. Just as in the

case of microwave absorption, for isothermal treatment at 200oC, the SERS peak intensity shows

a rapid rise with increasing time and then a rapid decrease. For isothermal heating at 180oC,

both the rates of initial rise and the fall in the SERS intensity are much lower. This mimics the

microwave absorption data at similar heating conditions.

4.5 Conclusion

Measurements of microwave absorption as a function of temperature can be used to sensitively

monitor the transient process from a weakly linked resistive state to an electrically robust

conductive state in metallic nanoparticle thin films. In the case of silver printing inks this

irreversible process correlates to sintering and is dependent on the partial removal of the

stabilizer surrounding the nanoparticles. During heating, the creation of weakly linked particles

naturally occurs immediately after a state where the interparticle distance has been shown by

66

others to be ideal for the SERS effect. Thus, monitoring the change in microwave absorption can

be used to quickly locate an appropriate temperature range for isothermal annealing to

fabricate SERS substrates. The results presented in this chapter show that isothermal annealing

at a temperature near the onset of sintering allows a fine control over the substrate morphology

and the resulting SERS performance.

67

Chapter 5: Optimization of SERS substrates fabricated from nanoparticle printing inks

5.1 Abstract

This section demonstrates a technique for optimizing surface enhanced Raman scattering (SERS)

substrates using nanoparticle printing inks. These inks are colloidal suspensions of silver

nanoparticles coated with an oleylamine ligand. The stabilizing ligand prevents aggregation,

impedes oxidation, maintains interparticle spacing and can be removed at low temperatures.

The application of an annealing treatment is a common preparation step in the printing industry

to remove the ligand and sinter the nanoparticles. SERS is strongly dependent on the

interparticle spacing so by limiting the sintering process to short heating times (10-15 minutes at

180oC) a surface morphology results in which a large majority of the nanoparticles are nearly

touching. Here we present evidence for the decrease in interparticle spacing by partial heat

removal of the ligand and subsequently an increase in the SERS intensity. We also demonstrate

some of the benefits of the ligand, such as adhesion to the surface and long shelf life. The ink

can easily be printed into arrays of dots and yields a SERS signal with a coefficient of variability

less than 10% across the center of the dots and over an array of printed dots.

Keywords: Surface enhanced Raman scattering, SERS substrates, silver nanoparticles, printing inks, sintering and stabilizing ligand 5.2 Introduction

Colloidal nanoparticle solutions are the most widely used medium for SERS substrates due to its

low cost and ease of preparation. Gold and silver colloids – typically synthesized by the sodium

borohydride or citric acid reduction reaction – can produce large enhancements but suffer from

poor reproducibility due to aggregation and Brownian motion effects in solution. Colloids

68

deposited on planar substrates are also prone to aggregation due to changes in pH and lift-off

effects after analyte deposition. To prevent aggregation oftentimes a stabilizing layer, such as

polyvinyl propylene (PVP), or a surfactant (CTAB) are added during synthesis to keep particles

apart.

It is well known the interparticle distance plays a crucial role in the SERS intensity. In fact, the

largest SERS enhancements are observed when a molecule bridges two particles and the dimer

gap is parallel to the incident electromagnetic field [21, 27, 28, 83]. Under this scenario the

interparticle distance is in the 1 – 10 nm range. These hot sites can be found in colloidal

solutions containing aggregated fractal clusters [19]. Often the aggregation process is allowed to

proceed naturally from a stage of many single nanoparticles to a stage of large aggregates

where the SERS amplification is greater [102]. However, the aggregation process is random so

every batch will have its variability. Other means of controlling aggregation involve chemical

means, for example, addition of anions [27], addition of passive electrolytes [103], changing pH or

heating [104]. Nevertheless, all these methods are time dependent processes so stability and

reproducibility remain major challenges.

Heating methods have been shown to reduce the interparticle spacing and increase the

amplification factors of SERS substrates. Lu et al., for example, reported controlling the distance

between nanoparticles by depositing capped colloidal silver particles on a thermally responsive

polymer surface and then heating it. The polymer surface shrinks under heating thereby moving

the adhered nanoparticles closer together until the plasmon resonance matches the laser

excitation wavelength [105]. In the heat-induced method, developed by Ozaki’s group, an analyte

is mixed with a pH controlled colloidal solution and then heated to form a dry film on a

69

substrate. The heating step allows the analyte to take the place of anions in the small gaps

between aggregated nanoparticles [106].

An alternative is nanoparticle printing ink, which is being developed for depositing conductive

paths on flexible materials such as paper and polymers. Nanoparticle suspensions in such

printing inks are colloidal nanoparticles encapsulated by a stabilizing surfactant that helps

maintain the dispersion of particles and binders that allow the particles to adhere to the surface

once deposited. In some cases the encapsulant can act as both a stabilizer and a binder. Such

encapsulants are composed of an alkane chain tail and a thiol, amine or carbonyl head group.

The latter allows the ligand to chemisorb to the nanoparticle surface and thus maintains spacing

between particles when deposited on a surface. Low temperature heating treatments are

commonly employed for sintering the nanoparticles to create multiple percolation paths to

enhance electrical conductivity [69]. Removing the ligand by heating exposes active nanoparticles

and through thermal motion brings them in close proximity to each other. Optimizing the

heating treatment can produce an ideal particle arrangement for achieving high SERS

amplification factors.


Substrate Preparation Silver nanoparticle inks were supplied by UT Dots, Inc (Champaign, IL). The nanoparticles,

suspended in toluene with an average particle size in the 10-15 nm range, were encapsulated

with an oleylamine surfactant. Substrates were prepared on square glass cover slips or

microscope glass slides in a dust free environment by one of three methods: 1) dry drop

deposition, 2) spin coating and 3) ink printed. The dry drop method involved depositing 10 μL of

70

ink (20% by weight) with a micropipette on a microscope slide and then letting it dry for 1 hour

before use. The spin coating method involved depositing 10 μL of nanoparticle ink over square

glass cover slips using a micropipette followed by spin coating with a G3P-8 spin coater

(Specialty Coating Systems, Indianapolis, IN). The spin coating protocol was a two-step process:

1 min at 700 rpm with a ramp rate of 5.0 followed by 1 min at 2000 rpm with a ramp rate of 5.0.

The spin coated sample was allowed to air dry and stored in a desiccator until use. Dot arrays

were printed on microscope glass slides at room temperature using an AD3200 printing

workstation (Biodot Irvine, CA) with 60% by weight silver nanoink toluene solvent (AgTE). The

syringe pump and solenoid valve dispensed approximately 0.1 μL sized drops through a 100 μm

diameter nozzle. The printed 6 x 6 arrays had dots of an approximate size of 1.2 mm with no

visible coffee drip effect. Microscope images of the dots were taken with a BX51 reflection

microscope (Olympus, Center Valley, PA). Micrographs were taken with the Zeiss Supra 50VP

scanning electron microscope (SEM) and the JEOL JEM2100 transmission electron microscope

(TEM) at the Centralized Research Facility (CRF) at Drexel. Comparison of the three different

substrate fabrication techniques can be found in the Appendix section.

Thermal Characterization The thermal properties of the nanoparticle ink were characterized on an Exstar TG/DTA6000

series instrument (RT Instruments, Inc., Woodland, CA). Using the drop deposition method, ink

was allowed to dry on a glass slide at room temperature for 24-48 hours in a desiccator. The

dried ink turned into flakes and then was scraped off and transferred to weighing dish.

Approximately 20 mg of the flakes were collected and placed on the instrument’s sample pan

and then heated at 120oC for 30 minutes to remove any remaining solvent. The temperature

71

was then increased at a rate of 10oC/min to a final temperature of 750oC under nitrogen gas in

order to record its thermal transitions.

SERS experiments on heated substrates The substrates for SERS experiments were prepared by heating the printed nanoparticle dots in

a conventional oven at 180oC. The oven was preheated to said temperature and then the dots

were placed on top of a solid aluminum slab inside the oven to ensure homogeneous heating of

the sample. The heating time interval (10-15 min) was based on the isothermal microwave

absorption experiments described in detail in chapter 4. Rhodamine 6G (R6G) was used as the

test analyte at varying molar concentrations in a 10 mM NaCl solution. The analyte was drop-

deposited on the heat-treated SERS substrate and allowed to dry for at least 10 minutes before

spectrum collection. At least 40 Raman spectra were collected over the surface of the substrate

with the inVia Raman Microscope and the Advantage Near-Infrared Raman Spectrometer at a

785 nm excitation wavelength following the protocol described in Chapter 3. Spectra were

transferred from the Wire software to Grams/AI software for analysis. For each substrate, the

average spectrum was baseline corrected using a user defined multi-point baseline fitting

routine. An offset correction was used in order to zero the baseline. Raman bands in the spectra

were fitted using a Gaussian/Lorentzian function. An in house Matlab script (RhoPeaks.m) was

used to determine the peak heights of the relevant Raman lines using the function file

(PeakFind.m).

Finding the average SERS signal of the substrate can be challenging and many reports address

this differently. Here, it was recognized that the heating treatment produced a random

distribution of interparticle distances so it was assumed that the SERS signal intensity values

72

over the surface would resemble a normal distribution. The mean was calculated after removing

the few points that did not yield a Rhodamine signal. Points lying below and above the second

standard deviation were also removed. From the points remaining a new mean and standard

deviation were calculated and these were used in the graphs.

Reproducibility and stability on SERS substrates For the reproducibility study on the dot surfaces the 1364 cm-1 peak from R6G was used to

compare the signal intensity from location to location. Substrates were heated first using the

protocol mentioned above. After cooling for 10 minutes at room temperature the analyte was

added. To ensure an even deposition over the 6x6 array the entire substrate was dipped into a 2

mL R6G bath and left to incubate overnight. The next day the substrate was allowed to dry for at

least 10 minutes before spectrum collection. Spectra were then collected from a mapped area

over the diameter of the dot with the inVia Raman Microscope at a 785 nm excitation

wavelength. Twenty spectra were collected for each dot on spots separated at least 100 μm

from each other. Each collection consisted of 6 acquisitions for 10 seconds each with a 1%

power. The coefficient of variation was used to report the variation from spot-to-spot and

between substrate-to-substrate.


5.4.1 Nanoparticle arrangement

The intensity of the SERS signal produced from printed substrates is highly dependent on the

particle arrangement after an applied heat treatment. Upon ink deposition, the nanoparticles

are isolated from each other in relation to the thickness of the stabilizer. In other words, the

spacing between nanoparticles is random. Figure 19a shows a TEM image of the arrangement of

73

nanoparticles on the surface before heating. After heating the surface, nanoparticles approach

each other forming groups of dimers, trimers and large clusters. Conversely, large voids are also

formed between the clusters and small voids are formed between the nanoparticle components

of the clusters. The sizes of these voids were measured using image analysis techniques with

Image J software. Figure 19b shows the void size versus area after heating for SEM micrographs

at optimal SERS conditions. The distribution of voids follows a scale invariant behavior that is

commonly observed in fractal structures. Such particle arrangement has been shown to produce

large SERS intensities [37, 39]. One of the most important attributes of the random distribution of

these structures is the broad plasmon resonance which allows the use of multiple excitation

wavelengths to produce SERS amplification as opposed to the wavelength matching that is

required for lithography fabricated SERS substrates. Furthermore, the interparticle distances

found within clusters resemble the dimers shown by Xu et al to yield large electromagnetic

enhancements [21, 28, 29]. By optimizing the concentration of these sites within a fractal-like

nanostructure it is possible to achieve very large SERS amplification factors.

(a) (b)

Figure 19: a) Particle arrangement on thin film before heating treatment. The scale bar represents 50 nm. b) The frequency and size of the voids after heating indicates a large distribution of clusters in the

substrate.

100 101 102 103 104 105100

101

102

103

104

Area / nm2

Perim

eter

/ nm

74

5.4.2 Heating treatment removes stabilizing ligand

Silver nanoparticle sintering occurs at temperatures far below the melting point of bulk metal

due to the increased presence of interfacial defects, which collectively have a high surface

energy [70, 107]. Before sintering can occur the stabilizing ligand must debond from the

nanoparticle so the surface area can be exposed [69]. Depending on the length and affinity of the

ligand the removal temperature will vary. For example, if the ligand has groups with strong

affinity to silver or gold such as thiols, amines or carboxyls then the ligand will require more

energy to be removed [73]. To better understand the thermal effects on the nanoparticle ink

several substrates were studied through thermogravimetric analysis (TGA), differential thermal

analysis (DTA), Raman spectroscopy, and SEM.

Thermal analysis revealed the transitions of the stabilized silver nanoparticles during heating.

The DTA data shows a strong exothermic peak at a temperature of 189oC, which is attributed to

a partial oxidative decomposition of the ligand (Figure 20). This peak is immediately followed by

a mass loss of approximately 20% between 190oC and 450oC as shown in the TGA. The mass loss

is typically attributed to the evaporation of the ligand [69, 73]. A similar behavior of an exothermic

peak followed by a rapid weight loss was recently shown in the thermal analysis of an

interconnect paste composed of silver nanoparticles capped with a Myristyl alcohol [108]. It

follows that the sintering process begins immediately after the partial removal of the ligands, as

the particles are able to interact with their neighbors. Between 300oC and 500oC there are

exothermic features that are attributed to the reduction of free surface energy due to the

decrease in surface area during sintering. Kim et al supports this view as the exothermic peaks

during sintering are attributed to the diffusion of interfacial atoms in the necking protrusions

between nanoparticles [109].

75

Figure 20: DTA graph shows an exothermic peak in the silver nanoparticle sample at approximately 189oC

while the TGA graph shows a mass loss of 20% immediately following the exothermic peak due to the evaporation of the oleylamine ligands.

Characterization of the film surface was performed by taking SEM micrographs after 15 minutes

of heating in a conventional oven (Figure 21). The changes in particle arrangement after heating

confirmed that sintering was occurring in the given temperature range found by the DT analysis.

With no heating the nanoparticles are not individually discernible with the SEM because the

protective ligand acts as an insulator. At 125oC, the ligand stabilizer is still bound to the

nanoparticle surface because the temperature is below the removal temperature so the

nanoparticle features are still not clearly distinguishable. However, at 175oC necking behavior is

observed because the ligand layer partially decomposed allowing some nanoparticles to move

into intimate contact with each other. After heating at 225oC, a large population of

nanoparticles sintered forming large clusters and multiple percolation paths.

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Figure 21: SEM micrographs of Ag nanoparticles deposited on glass cover slips after heating for 15

minutes at A) 125oC, B) 175oC and C) 225oC. Magnification 50 kX and a working distance of 5 mm. Scale bar represents 300 nm.

The average SERS signal intensity of R6G was measured after heating dry drop deposited

substrates in the oven for 15 minutes at temperatures between 150oC and 225oC as shown in

Figure 22. The signal intensity was compared to substrates not heated (room temperature)

which shows that some heating was necessary to produce the large SERS enhancements. The

signal intensity compared well to the SEM micrographs as the largest signals occurred in the

175oC to 200oC temperature range just before the nanoparticles began to form large clusters.

Figure 22: Average SERS signal of the 1364 cm-1 R6G peak on dry drop deposited substrates after heating

for 15 minutes at the indicated temperatures.

77

The fact that the SERS intensity increases indicated that the ligand layer is partially removed

allowing more R6G molecules to make intimate contact with the nanoparticle surface. At 225oC

the signal decreased as the nanoparticles began to anneal and the small interparticle distances

disappeared or became too large.

From the microwave absorption data and the thermal analysis a temperature of 180oC was

chosen to study the nanoparticle behavior and SERS intensity under isothermal heating

conditions. Figure 23a shows that adjusting the heating time proved to provide a fine control of

the sintering process. As mentioned previously, with no heating the nanoparticles are not

discernible with the SEM because the protective layer covers all of the nanoparticles. As the

nanoparticles are heated the polymer layer is partially removed and nanoparticles of an average

size of 15 nm become visible. Further heating progressively eliminates ligand molecules allowing

more particles to agglomerate into larger clusters until eventually forming conductive

percolative paths [110].

(a) (b)

Figure 23: (a) SEM micrographs of Ag nanoparticles as-deposited on substrate, 0 minutes, and annealed at 180oC for 10, 20 and 30 minutes. Scale bar depicts 500 nm. (b) Rhodamine 6G spectrum after various

heating times at 180oC. Each spectrum was shifted up for visual clarity.

78

The relationship between the SERS intensity of R6G and the heating time is shown in Figure 23b.

Here multiple substrates were heated at 5 minute intervals at a temperature in the range of the

ligand decomposition -- for oleylamine this is approximately 180oC - 200oC. A 5 μL drop of a 1

μM R6G solution was drop deposited on the substrates and then allowed to dry for 10 minutes.

Raman spectra were collected over the entire surface area and then the average was reported.

On the as-deposited substrate there is no R6G signal presumably because there is too much

polymer, which prevents the analyte from being in close proximity to the nanoparticles. As the

substrate is heated the magnitude of the signal intensity begins to rise. At the 15 minute

interval, the SERS signal reached its maximum magnitude and thereafter, the signal magnitude

decayed. It is reasonable to believe that at this heating temperature there is a thermal window

between 10 and 20 minutes where the spacing between particles is optimal for SERS

amplification.

The removal of the ligand can also be monitored indirectly from the Raman signal of a clean

nanoparticle ink surface. The Raman spectra were recorded from the side of the surface

exposed to the air and from the side making contact with the glass during heating treatments at

180oC in increments of 5 minutes. Figure 24a shows a schematic of the experiment after

prolonged heating in which most of the polymer surrounding the nanoparticles on the top

surface has been removed due to the heating. Figure 24b shows the Raman intensity of the

1050 cm-1 band from both sides of the substrate after heating treatments. The 1050 cm-1 band,

assigned to a vibrational symmetric stretch mode of nitrate ions [111], was detectable from both

sides of the substrate. As the substrate was heated, the band intensity from the exposed side

decayed while the intensity from the bottom side remained constant. After 5 minutes of

heating, the 1050 cm-1 Raman band from the glass side of the substrate was greater in intensity

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(a) (b)

Figure 24: A) Schematic representation of heated nanoparticle surface and the direction of the incident laser beam. B) Raman intensity of 1050 cm-1 nitrate ion band decreases as a function of heating time

when collected from the exposed side of the substrate. The signal of the same band remains stable when collected from the glass slide. The dotted lines are only an aid for the eyes.

when compared to the peak from the exposed side most likely due to a greater concentration of

nitrate ions beneath the nanoparticles. The Raman intensity from the glass side remained

relatively stable throughout the duration of the heating suggesting the ligand remained adhered

to the nanoparticles and to the glass.

The relationship between the nitrate ion Raman peak intensity and the ligand disassociation

from the nanoparticle surface can be explained by chemical means. During nanoparticle

synthesis, silver nitrate is reduced with sodium borohydride. Oleylamine is added to the solution

as a capping agent to restrict silver ion nucleation. Most nitrate ions are removed through a final

washing step but a small fraction remains in the solution. On nanoparticle deposition, Raman

analysis confirms nitrate ions remain on the surface. Heating on the surface has a larger effect

on the superficial surface because it is exposed to the heated space whereas on the glass

surface there is more oleylamine, which acts as an insulator. Therefore, oleylamine ligands

disassociate from the surface allowing nitrate ions to react with the exposed silver nanoparticles

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thus decreasing the intensity of the 1050 cm-1 band. On the glass side the heating effects are not

as pronounced so the nitrate ion peak remains relatively stable. This observation along with the

SEM micrographs, the heat dependence of the R6G SERS signal and the DTA exothermic peak

suggests the ligand debonds from the nanoparticles and partially evaporates leaving them

unprotected on the exposed side. This explains the large SERS signals observed after an

optimized heating treatment.

It was also observed the nanoparticles form a strong adhesion to the glass surface. This is an

important advantage of nanoparticle printing inks compared to colloidal nanoparticles for their

when a solution is deposited the nanoparticles lift off. Due to Brownian motion, colloidal

nanoparticles and analytes constantly move in the sample. Partly due to this constant

movement, SERS band intensities have been shown to fluctuate by factors as large as 20%

during long integration times [27]. On the other hand, our experiments show nanoparticles in

printing inks deposited on glass cover slips free from organics remained firmly adhered to the

surface after heating treatments even after repeated washes in distilled water and ethanol

solutions. Huang et al also observed a strong adhesion of ligand encapsulated printed gold

nanoparticles to polyester at elevated temperatures and suggested the adhesion came from an

interfacial layer formed between the metal nanoparticles and the substrate [78]. It has also been

reported that the polymer between nanoparticles improves cohesion in gold films, even

resisting a Scotch tape peel test [110]. Thus it is reasonable to conclude the ligand on the glass

side of the nanoparticles acts as a binder to the glass surface which allows the particles to

remain fixed to the surface during SERS experiments.

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5.4.3 Reproducibility of SERS signal

Fabricating SERS substrates that yield reproducible spectra over a 3 mm x 3 mm area is a

noteworthy challenge often reported in the literature [43, 44]. The advantage of lithography

techniques is the fabrication of well-tailored nanostructures with translational symmetry.

However, in a SERS analytical experiment such detail is not necessary as the laser spot size,

typically 1 μm – 50 μm, is three orders of magnitude larger than the nanoscale features. An

alternative is printing arrays of nanoparticle dots that can be activated through heat as

described in this report. Although this approach yields a surface morphology of randomly

arranged nanoparticles, over the scale of the laser spot size there will be a large population of

active dimers to produce SERS signals. As shown in Chapter 4, the microwave absorption

measurement provides a fine control to optimize the population of dimers. A printed

homogenous layer is preferred which is controlled by the viscosity, nozzle size, solvent,

humidity, and nanoparticle size. Furthermore, since the laser spot size is so small a 9 mm2

nanoparticle active area is still a relatively large area.

In this report, each printed dot measured approximately 1.2 mm in diameter (or ~ 1.1 mm2 in

area) and the surface was homogenous under a 20x objective, which minimized laser defocusing

during scans (Figure 25a). The SERS spectrum from a 1 μM R6G solution was collected from 20

different locations across the diameter of the spot with a 10 μm laser spot size. Figure 25b

shows the amplitude of the 1364 cm-1 line plotted for every spot collected on a single dot. As

expected the signal at the edges is lower than through the center region of the printed dot

because the edges have a higher concentration of nanoparticles causing a varied scattering

profile. In fact, we have observed the signal at the edges differ by at least one order of

magnitude. By removing the points from the edges we find that the signal reproducibility is

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(a) (b)

Figure 25: a) Microscope image of one silver nanoparticle ink printed dot on a glass substrate. The diameter of the dot was approximately 1.2 mm. b) The amplitude of the 1364 cm-1 peak at 20 locations

across the diameter of the dot. The center portion intensities vary by less than 10% indicating good reproducibility.

within 10% of the mean amplitude. This is appropriate for an application setting, as the

analytical chemist would target a center region of the spot and then take multiple collections

over the entire surface. The low variability is also under the 10% limit that has been cited as a

technology standard specification for a high quality SERS substrate [43].

The measurement of the coefficient of variability across an array of dots was used to compare to

the substrate-to-substrate reproducibility technical standard of less than 20% [43]. The entire

substrate was immersed overnight in a 1 μM R6G solution to ensure that each dot would be

covered by the same concentration of analyte. The Raman spectra were collected over 25 dots

and the average of the amplitude of the 1364 cm-1 peak was measured for each dot. In fact the

variability was consistent with the reproducibility of one dot. The coefficient of variation for the

red highlighted area in Figure 26a was 7.8% indicating the formidable reproducibility of the

printed arrays. Figure 26b shows a color surface plot of the 1364 cm-1 peak amplitudes from

each dot to demonstrate the average intensity was the same over the entire substrate. The

figure is shown at an angle to make the low variability apparent.

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(a) (b)

Figure 26: a) A printed array of silver nanoparticle ink on a microscope glass slide. The red box indicates the 5x5 array of dots that was analyzed. b) The surface plot shows the reproducibility of the 1364 cm-1

peak amplitude from each dot on the array. 5.4.4 Stability of SERS substrates

One of the main drawbacks of colloidal nanoparticles is the formation of aggregates as the pH of

the solution changes over short time periods, which directly impacts the consistency of the

Raman intensity. These changes can cause the signal intensity to decrease by several orders of

magnitude. As Natan described it, in an application setting a “SERS substrate that must be used

immediately after fabrication is of limited utility” [43]. As mentioned previously, nanoparticles

suspended in printing inks have a thermally responsive coating that serves as a protective shell

against aggregation and oxidation. This stabilizing ligand can help nanoparticles retain their

properties and therefore increase their shelf life. To study their longevity, printed substrates

were stored in a glass vacuum desiccator for various time periods (0, 2, 4, 8 weeks). At each time

point three substrates were removed from the desiccator and heated for 15 minutes at 180oC to

remove the stabilizer and to optimize the interparticle spacing. The time and temperature were

determined after DSC thermal analysis and microwave absorption measurements. After heating,

a 1 μM R6G solution was drop deposited on the nanoparticle surface and after 10 minutes of

drying the Raman spectrum was collected. Figure 27a shows the amplitude of the 1364 cm-1 R6G

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Raman band plotted over time. The substrates of this batch remained active for at least eight

weeks. In another batch the printed substrate was stored for 7 months in a vacuum chamber

and upon removal heated to remove the stabilizing polymer. After 7 months the substrate still

performed as well as when the silver nanoparticles were first deposited. This was very close to

the 8 month goal that Natan outlined in his work [43]. The importance here is that printed

substrates can be stored for a long period of time and then rendered active by the appropriate

heat treatment right before use.

In previous studies, stability of a substrate has been tested by measuring the signal intensity

daily and observing the loss over time. Ozaki’s group demonstrated a 33% signal loss over 6 days

on a substrate made by depositing a Ag colloid over an aluminum plate [106]. The signal loss

decreased to 20% after storing the substrate in the refrigerator after each day. In our

experiment a 10 μM R6G solution was drop deposited on the nanoparticle surface and then the

Raman signal was monitored daily. It should be noted the substrate was stored in a desiccator

after each day and the sample area was large enough to interrogate unique areas during each

daily scan. The latter prevented scanning over laser heat affected areas such as locally heated

adsorbed molecules or locally annealed nanoclusters as previously reported [99, 112]. The intensity

of the 1364 cm-1 peak was found to remain relatively stable for at least 10 days before

deteriorating (Figure 27b). In comparison to the Ozaki group the signal intensity decreased by

33% after 13 days. This seems to indicate that the active dimers maintain their small

interparticle distances over time. Also, the deterioration in signal intensity is attributed to the

photobleaching of Rhodamine over time and not the oxidation process as was reported by Zhou

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(a) (b)

Figure 27: a) Stability of the SERS signal intensity from a 1 μM R6G solution on substrates stored in a vacuum chamber for several weeks. b) Stability of the SERS signal intensity from a 10 μM R6G solution on

substrates over a period of 3 weeks after storage. et al [106]. Oxidation of silver nanoparticles is slow and can usually be removed by a cleaning

protocol. With proper storage the substrates should have even longer stability after addition of

the analyte.

5.5 Conclusion

Nanoparticle printing inks stabilizing with an oleylamine layer and deposited on a planar glass

substrate help avoid some of the common problems associated with chemically reduced

colloidal nanoparticles normally used for SERS substrates. The stabilizing layer protects the

nanoparticles from aggregation and oxidation but can be partially removed through a relatively

short heating pretreatment right before use. During heating, a decrease in interparticle distance

naturally occurs in silver nanoparticle films creating idea conditions for the SERS effects. The

appropriate heating temperature was chosen after monitoring the thermal profile of the sample

and the observed partial decomposition of the stabilizing layer. The SERS signal intensity

increased and decreased in relation to the heating times as particles were brought closer

together until sintering. Therefore, interrupting the sintering process shortly after ligand

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removal presents a reasonable method for optimizing SERS amplification factors on substrates

composed of capped silver nanoparticles. In addition, some of the oleylamine remains on the

surface after heating, which acts like a binder preventing nanoparticles from lifting off from the

surface when a solution is added.

The use of printing inks can also help meet the technical standards necessary for fabricating

reproducible SERS substrates. The oleylamine stabilizer helped preserve the SERS performance

of the nanoparticles for up to seven months when stored in a desiccator and then heated before

use. Also, the spot to spot variability was less than 10% along the center region of a printed dot

allowing a large surface area where reproducible signals can be collected. Finally, the substrate

to substrate reproducibility was shown to be less than 10% over multiple dots in the printed

array making them reliable substrates for SERS studies.

Acknowledgments

We greatly acknowledge Yuri Didenko of UT Dots, Inc for the many insightful discussions about

nanoparticle deposition. Also we are grateful for the assistance Kevin Freedman provided in

collecting the TEM micrographs at the Drexel Centralized Research Facility. This work was

supported in part by the Nano-Technology Initiative (NIT) of Southeastern Pennsylvania.

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Chapter 6: Functionalization of silver nanoparticles for improved SERS detection of hyaluronic acid (HA)

6.1 Abstract

Hyaluronic acid (HA) is a promising biomarker for the early detection of liver disease and the

accurate staging of cirrhosis. Elevated levels in the serum can indicate late stage cirrhosis which

usually results in the need for a liver transplant. Currently the gold standard for diagnosing

cirrhosis is a liver biopsy which is a painful and inconvenient procedure. Conventional biological

assays require multiple preparation steps and take a few days to yield results. An alternative is

to use a direct non-invasive technique such as surface enhanced Raman scattering (SERS), which

can give specific vibrational information about the biomarker of interest. In the past, HA was

detected on SERS substrates at concentrations in the low mg/mL range, limiting its use for

clinical diagnosis. In this study, SERS substrates were prepared from a silver nanoparticle

printable ink, which was thermally treated after deposition on glass slides. Functionalization of

the SERS substrates with a cysteamine ligand in the trans conformation allowed immobilization

of HA. HA anchoring through a covalent bond was observed through a Raman shift as the trans

conformer changed to a gauche conformer. By optimizing the concentration of the trans

conformer on the substrate, HA was detected to a concentration as low as 50 μg/mL. Although

this concentration is high for clinical early detection of hyaluronic acid in cirrhotic patients,

which is typically in the ng/mL range, this methods offers a three order of magnitude

improvement over previous SERS studies on HA.

Keywords: SERS substrates, functionalization, hyaluronic acid, cysteamine, cirrhosis, liver disease, and silver nanoparticles

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6.2 Introduction

Cirrhosis is the final phase of liver disease and is characterized by the accumulation of hepatic

fibrosis. It commonly occurs in heavy alcohol drinkers or people infected with the Hepatitic B or

C virus. The damage is irreversible leading to serious complications if not detected in its early

stages. A liver transplant is the usual outcome. Thus, staging the severity of hepatic fibrosis

becomes very important to enable doctors to recommend treatment options. The standard

procedure for diagnosis is a biopsy, which is not favorable for patients because of the associated

pain, possible complications, sampling error and false positives [113]. Furthermore, laparoscopic

and transcutaneous biopsy studies have shown disagreement in the stage of fibrosis as high as

37% [114] and cirrhosis is underestimated in 15% to 30% of cases [115].

Biomarkers in the serum have been identified previously and studied as possible alternatives to

biopsy when diagnosing liver disease. Among the direct markers, those produced in the

extracellular matrix (ECM) usually can be traced in the serum. These include amino-terminal

procollagen type III peptide (PIIIP), collagen type IV, collagen type VI, laminin, fibronectin, YKL-

40, TIMP-1, TIMP-2, MMP-2 and MMP-9 [114, 116]. Hyaluronic acid (HA) has been advocated as the

most promising biomarker in the serum to predict liver disease progression in several studies:

chronic hepatitis C [117, 118], cirrhosis [119-122] and chronic alcoholic liver disease [113, 116].

Hyaluronan, as it is referred to inside the extracellular matrix (ECM), is a high molecular weight

glycosaminoglycan composed of a repeating disaccharide composed of a D-glucuronic acid and a

N-acetyl-D-glucosamine. Figure 28 shows the chemical structure of the base disaccharide unit in

HA. Besides its main structure, it has three important active sites that allow it to interact with

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Figure 28: The base unit of hyaluronic acid is composed of D-glucuronic acid and N-acetyl-D-glucosamine. surrounding molecules: a carboxyl, an acetamido and a hydroxyl group. These sites can be

modified for functionalization, scaffolding or other biomaterials [123].

The biopolymer is found in all soft connective tissues in the body but notably high

concentrations are found in the skin, umbilical cord, synovial fluid and vitreous humor [Leach

2008]. Its exact biological function is complex and its conformation is not well understood but it

is generally agreed that it provides structure to the ECM as it binds with cells and other

components. Its polyanionic nature makes it highly hygroscopic giving it an important role in

regulating tissue hydration and maintaining osmotic balance [124]. Its high viscosity gives it

stiffness and restricts the movement of water in tissue. In its hydrated state, it acts as a barrier

preventing certain pathogens and plasma proteins from passing through [Leach 2008] [125].

During the inflammatory response to tissue damage, HA has a very important role as it is

expressed in the serum at elevated levels [124, 125]. Its linear repeating structure forms a strong

backbone supporting granulation tissue formation and remodeling. Its interaction with two

hyaladherins (CD44 and RHAMM) triggers cell adhesion and migration at the injured site [Leach

2008]. It also scavenges for free radicals to create an antioxidant effect [Leach 2008]. Thus, it

90

has also been implicated in the healing of acute and chronic dermal wounds [125]. Its prominent

role during the inflammation process makes it a likely candidate as a biomarker for many

pathological disorders, including liver disease.

The concentration of HA in the serum of healthy individuals is very low but increases to levels in

the microgram per liter range in patients with liver cirrhosis [116, 117]. During liver disease hepatic

stellate cells undergo a switch causing them to deposit excess ECM components along the portal

triads of the liver, which leads to fibrosis [115]. This can disrupt the architecture of the liver

eventually causing it to fail. Normally, sinusoidal endothelial cells remove excess ECM

components through endocytosis but during liver disease the ECM turnover is impaired leading

to an increase of HA in the circulation [126].

Enzyme-linked immunosorbent assay (ELISA) kits are available from biomedical companies, such

as Kabi Pharmacia Diagnostics and Chugai Diagnostic Science Company, to measure HA in the

serum. These utilize a specific antibody to immobilize HA followed by a second antibody and a

fluorescent marker. Although these detection schemes are the standard in many biological

laboratories they do require multiple preparation steps and take at least 72 hours to yield

results. In addition, the tests are measurements of the capture of a fluorescent label which has

been shown to sometimes give false positives due to antibody cross reactivity. Furthermore, no

current diagnostic test has been shown to be more accurate in staging than a biopsy [115].

A possible alternative is to use a direct non-invasive optical technique such as SERS, which can

give specific vibrational information about the bonds in a molecule. The Raman peaks of HA

have been identified previously [127-129] and SERS has been used to identify its peaks [130],

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however due to the large molecular weight of HA and its polyanionic nature it does not readily

adsorb to the nanoparticle surface. Its full spectrum was only shown at concentration in 4 to 5

mg/mL range after long collection times.

In this report, SERS substrates made from silver nanoparticle printing inks are functionalized

with a cysteamine monolayer in order to immobilize the negatively charged HA. By anchoring it

to the substrate the molecule is in a favorable orientation relative to the nanoparticle surface

allowing its excitation by the SERS effect. By varying the cysteamine concentration various HA

bands can be identified which can be useful for understand HA’s conformation and detecting it

at low concentrations.


SERS substrates were prepared on glass cover slips using printable silver nanoparticle ink (UT

Dots Inc., Champaign, IL). In brief, cover slips were first cleaned in piranha solution and then

silanized with 1% aminopropyltriethoxysilane (APTES) [52]. A 10 μL drop of the ink solution was

spin coated on the cover slips and then heated at 180oC for 15 minutes to remove any remaining

solvent and to partially remove the stabilizer. The heating step produces a distribution of

interparticle distances and cluster sizes; giving the surface a favorable environment for the SERS

effect (see Chapter 5). A self-assembled monolayer (SAM) was formed on the surface by

immersing the substrate in a cysteamine solution over night at room temperature and then

rinsing with ethanol to remove free cysteamine. Cysteamine adsorption and its orientation were

confirmed by examining the log ratio of the 725 cm-1 to 640 cm-1 SERS band intensities [131] from

multiples spots on the substrate. At this point, it should be noted that the wavenumber of a

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particular mode will have a small tolerance due to slight variations in the orientation of the

silver to thiol bond [132].

HA carboxylic groups were attached covalently to the cysteamine amino group through a

carbodiimide reaction [133, 134]. Briefly, HA was dissolved in a 10 mM hydroxyethylpiperazine

ethane sulfonic acid (HEPES) buffer to make a 1 mg/mL stock solution at a pH of 7.0.

Dimethylaminopropyl carbodiimide and N-hydroxysuccinimide (EDC/NHS in a 4:1 ratio) were

mixed in the activated HA solution for 15 minutes to form an amide linkage. The functionalized

SERS substrate was then immersed in the solution for 16 hours and then washed with ethanol to

ensure only covalently bonded HA remained on the surface. Figure 29 shows the attachment

scheme of HA to the cysteamine linker molecule functionalized to the nanoparticle surface.

Quick SERS spectra were collected with the Advantage Near-Infrared Raman Spectrometer

(Intevac-DeltaNu, Laramie, WY) at a 785 nm excitation wavelength. The spectra were acquired

Figure 29: Schematic representation of HA attachment to a functionalized silver nanoparticle substrate.

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with a 10 second acquisition time, 20 mW laser power and 50 µm spot size. For characterization

of the surface coverage the inVia Raman Microscope (Renishaw Inc, Hoffman Estates, IL) was

employed at 514 nm and 785 nm excitation wavelengths. The spot size was approximately 1 μm

and the acquisition time was 10 seconds on each spot.

Relevant bands in the Raman spectra were analyzed with Grams/AI software (ThermoScientific,

Philadelphia, PA). For each substrate, the average spectrum was baseline corrected using a user

defined multi-point baseline fitting routine. An offset correction was used in order to zero the

baseline. Raman bands in the spectra were fitted using a Gaussian/Lorentzian function. An in

house Matlab script (CysPeaks.m) was used to determine the peak heights of the relevant

Raman lines using the function file (PeakFind.m).


6.4.1 Conformation of cysteamine

Cysteamine was specifically chosen because it has a short two-carbon backbone structure. As

mentioned earlier the SERS effect is distance dependent so the linker molecule had to be short

enough to bring HA in close proximity to the nanoparticles. Cysteamine was also chosen because

it has a free amino functional group which can be cross-linked with various molecules. In

addition, it is widely known the sulfur group of alkanethiols chemically adsorbs to silver or gold

nanoparticles forming a stable metal-sulfur bond [132]. The linkage can form different

conformations based on the solution pH, the concentration of the ligand and the electrolyte

content [131, 135, 136]. Raman spectroscopy is particularly well suited to measure the Ag-S

conformations of cysteamine over the entire surface [131]. The trans conformation is indicated by

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the 725 cm-1 Raman band, which appears when the carbon backbone sits upright and the amino

group is exposed as shown in Figure 30.

The gauche conformation is identified by the 640 cm-1 Raman band, which arises due to the

amino group competing with the sulfur group for available positions on the silver surface. This

leaves a bend in the carbon backbone and the amino group in a position adsorbed to the surface

or very close to it. On a self-assembled monolayer both Raman peaks will be present because

the laser spot size (~ 50 µm) samples a large enough area to include many cysteamine

molecules.

A predominantly cysteamine trans conformer covered surface was desired in order to maximize

bonding of HA. By monitoring the SERS intensity ratio of the 725 cm-1 band to the 640 cm-1 band

from various spots on the substrate the distribution of conformer coverage was determined.

This was repeated at various concentrations of cysteamine (0.5 mM, 1 mM, 10 mM, 25 mM, 50

mM and 100 mM) to identify the optimal conditions to immobilize HA (n = 5). Figure 31 shows

the logarithm of the peak ratios at the aforementioned concentrations and the typical Raman

Figure 30: Schematic representation of two possible cysteamine conformers on a silver nanoparticle

surface. Adapted from Kudelski et al. [131].

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spectra for each conformer. The dashed line at zero indicates the point at which the

concentration of trans conformers is equal to the number of gauche conformers based on the

intensity of the Raman peaks. Based on the experiments, the log ratios indicate a concentration

dependence with the lower concentrations showing a gauche conformation (0.5 mM and 1 mM)

while the higher concentrations showing a trans conformation (10 mM, 25 mM, 50 mM and 100

mM). Thus, it was reasonable to expect the highest HA band intensities at cysteamine

concentrations above 10 mM.

The log ratio of cysteamine peaks demonstrates there is a relationship between the orientation

of cysteamine on the surface and the concentration of cysteamine deposited. From a spatial

point of view there is a limit of space on the substrate as the concentration of the monolayer

increases. At 50 mM and 100 mM, saturation was observed due to the lower peak ratio, as

compared to the 25 mM, most likely due to an overcrowding effect that produced multiple

Figure 31: The logarithm of the cysteamine trans to gauche ratio at various concentrations shows a mostly

trans conformation over the substrate when the concentration is above 10 mM. The insets show typical SERS spectra in the trans and gauche conformations along with their schematic representations.

96

layers of cysteamine. This creates a superficial layer that is not adsorbed to the silver

nanoparticles so it shields the light scattering that occurs at the surface and thus lowers the 725

cm-1 peak contribution. At the lower end of the cysteamine concentrations, 0.5 mM and 1 mM,

there is more open space available over the spot size so the amine group on cysteamine is free

to compete with the thiol group for a position on the silver surface resulting in a gauche

conformation as evident from the significantly larger 640 cm-1 Raman peak. The largest positive

difference between the 725 cm-1 band and the 640 cm-1 band was found at a cysteamine

concentration of 25 mM. At this concentration, the free space within the spot size is limited so

the cysteamine molecule prefers to stand straight up indicating a trans conformation. Thus, a

concentration of 25 mM was used to maximize HA attachment.

6.4.2 HA conjugation

The SERS spectrum was used to monitor the changes of the cysteamine signal as HA was added

to the surface. Figure 32 shows three Raman spectra in the 200 cm-1 - 1200 cm-1 region: 1) a

typical SERS spectrum of the thermally treated substrate, 2) a functionalized substrate made

with 25 mM cysteamine and 3) a cysteamine functionalized substrate with 1 mg/mL of HA

adsorbed on the surface. The focus is on this region of the Raman spectrum because this is

where the vibrational modes of cysteamine’s thiol bond appear and there are very few HA peaks

in this region. The spectra were placed on the same intensity scale and were shifted vertically

for clarity. As shown, the thermally treated nanoparticle substrate displays no prominent peaks

before addition of cysteamine so there were no interfering peaks from the substrate itself.

As mentioned earlier, at 25 mM there is a surface coverage of mostly trans conformers

evidenced from the larger 721 cm-1 band intensity in relation to the 630 cm-1 band intensity.

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After immobilization of HA on the substrate, there was a reversal in the thiol bond ratio (725 cm-

1 to 640 cm-1) from trans to gauche conformation stemming from a change in the bend in

cysteamine’s structure. This can be explained by the carbodiimide reaction, which creates a

covalent bond between the amine group on cysteamine and the carboxyl group on HA. The thiol

bond on the cysteamine molecule in a trans conformation will vibrate at a lower frequency due

to the increased weight at the linkage, which cause the wavenumber to shift to the left. The

physical change to the cysteamine backbone results in a more parallel orientation which can be

detected by the increased intensity at the lower wavenumber (~ 630 cm-1). Since the amine

group on cysteamine is now closer to the Ag surface, it can also loosely interact with Ag. This

results in a dramatic increase in the 225 cm-1 peak intensity after HA immobilization. Usually the

Ag-N vibration occurs at 240 cm-1 [136] but in this case the HA is attached to the nitrogen bond so

the peak will shift to left forming a loose bond with Ag.

In addition to the gauche and trans bands, there are a few other bands that appear in the

spectra of the cysteamine functionalized surface in the 940 cm-1 to 1030 cm-1 range. These are

attributed to skeletal vibrations in the ligand (C-C and C-N stretching vibrations) [131, 136, 137] .

Upon the cross-linkage of HA, the bands around 940 cm-1 and 1016 cm-1 remain present because

they are part of the cysteamine ligand structure [131]. The increased intensity of the 1016 cm-1

peak gives evidence that the skeletal carbon linkage of cysteamine is bent parallel to the surface

thereby bringing parts of HA close to the surface. It is not entirely clear why the 970 cm-1 band

disappeared but it does mean a change has occurred in the polarizability of the molecule.

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Figure 32: Spectra of the thermally treated substrate, 25mM cysteamine on SERS substrate and 1mg/mL

HA immobilized on substrate. Spectra were shifted vertically for clarity. Nevertheless, it seems there is ample evidence to conclude that HA is captured through the

cysteamine ligand. The majority of the ligands in the trans conformation have transformed to a

gauche conformation leaving parts of HA in close proximity to the nanoparticle surface so a

Raman signal should appear. To study this we examined Raman bands that appear in the 800

cm-1 to 1700 cm-1 region of the spectrum. Table 1 shows the Raman peaks of sodium

hyaluronate that apply to the spectrum and their assignments gathered from various sources

[127-129]. In addition it also shows the prominent peaks of cysteamine and HA’s disaccharides,

glucuronic acid and acetyl glucosamine.

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Table 1: Frequencies (cm-1) and assignments of Raman bands in the 800 – 1600 cm-1 range [127-129]. Cysteamine Glucuronic

acid Acetyl

glucosamine Sodium

hyaluronate Assignment

864 865 899 β-linkages 940 941 936 949 C-C stretch, skeletal

C-O-C 964 960

1015 1010 C-C-N stretch ~1030 1025 1059 1055 ~1050 C-O, C-C, C-OH ~1087 ~1096 C-OH ~1120 1128 ~1125 C-C stretch, C-OH and

CH deformations ~1155 1156 ~1153 C-O, C-C, O2 bridge 1205 1206 1210 CH2 twist 1232 1273 1266 1268 ~1327 ~1331 CH bend, Amide III ~1383 ~1375 CH3, ionized carboxyl ~1412 COO- sym, CH bend,

ionized carboxyl 1457 1462 ~1460 CH2 bend ~1556 1565 Amide II 1631 1620 Amide I

Figure 33 shows the HA SERS spectrum after cross-linking with cysteamine at various

concentrations in the 800 - 1600 cm-1 range. In the given region of the spectrum, no discernable

Raman peaks were identified from the control substrate, which consisted of HA without

cysteamine. This indicates that the molecule is not interacting with the silver substrate possibly

due to its size or its polyanionic structure. This was also observed in preliminary experiments

and it is the reason that a linker molecule was chosen to immobilize HA on the surface.

As the cysteamine concentration increased, the relevant bands in the HA spectrum became

apparent with the largest intensity at 25 mM. This agrees with the initial idea that the largest

concentration of trans conformers on the surface would lead to the largest HA peaks (Figure 31).

The detected HA bands include 820 cm-1, 910 cm-1, 944 cm-1, 955 cm-1 (shoulder peak), 1018 cm-

1, 1065 cm-1, 1216 cm-1, 1263 cm-1, 1300 cm-1, 1378 cm-1, 1419 cm-1 and 1454 cm-1.

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Figure 33: SERS spectra of 1mg/mL HA after cross linking to cysteamine linker at different concentrations.

Spectra were shifted vertically for clarity. As mentioned earlier the bands close to 940 cm-1 and 1015 cm-1 are from a skeletal vibration

attributed to the cysteamine ligand. The 1419 cm-1 band is attributed to the COO- symmetric

stretch in HA which is the attachment point where the amine group of cysteamine covalently

bonds to the carboxyl group of HA. Therefore, this peak was chosen as one of the signature

peaks when determining HA attachment. At 50 mM the intensity of the Raman peaks decreases

possibly due to a saturation effect. At this concentration there could be multiple layers of

cysteamine which cover the nanoparticles making the SERS effect weaker.

A possible conformation of a single HA molecule immobilized on the Ag surface through the

cysteamine ligand is shown in Figure 34. The bonds are labeled according to their wavenumber

to give an idea on how they may contribute to the Raman spectrum. As the ligand transforms

into a gauche conformation there will be certain vibrations that are likely become enhanced

more than others. These include 1375 cm-1, ~ 1460 cm-1 and 1620 cm-1, which correspond to the

dangling methyl group, the CH2 bend and the C=O, respectively. However, there are many

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Figure 34: Schematic of single HA disaccharide immobilized on Ag substrate. The peaks are assigned

according to table 1 to give a visual to how they contribute to the Raman spectrum. cysteamine ligands on the surface so the intensity of the peaks will vary from spot to spot. It

would be interesting to minimize the number of ligands to see if the intensity of the mentioned

peaks increases significantly after binding.

6.4.3 Limit of detection of HA

Using a cysteamine concentration of 25 mM, substrates were prepared to find the lowest

concentration of HA detectable on the surface. The substrates were interrogated over 40

different locations and the top ten spectra relative to the 1419 cm-1 band were averaged.

Measuring the intensities of the 1419 cm-1 and ~1610 cm-1 bands, which correspond to the

linkage between cysteamine and HA and the C=O bond, respectively, the lowest concentration

detectable was 50 μg/mL. Figure 35 shows the SERS spectra of HA on the substrate as the

concentration of HA was decreased. Figure 36 shows the Raman spectrum produced from a 50

µg/mL solution of HA immobilized on the functionalized SERS substrate. The peaks highlighted

agree with the literature and their assignments are shown in Table 1. It seems possible that

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these bands could be detected at lower concentrations but this has not been done at this time.

It should be noted that these signals were acquired at 10 seconds, which is approximately 20

times faster than the previous SERS study on HA [130].

Figure 35: HA spectra on cysteamine functionalized SERS substrate.

Figure 36: Raman spectra of hyaluronic acid at 50 µg/mL on a cysteamine functionalized SERS substrate.

The labeled peaks correspond to previous HA peak assignments.

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6.5 Conclusion

Although SERS has been shown to give immense amplification factors for small analytes,

complex molecules can have polyanionic structures that limit their close interaction with a silver

nanoparticle surface. By functionalizing a SERS substrate with a short ligand it was

demonstrated that a large molecule, such as hyaluronic acid, could be isolated in an orientation

to produce SERS bands. Cysteamine coverage was optimized to create a surface of trans

conformers in order to increase the available binding sites of the carboxyl group found in HA.

This scheme immobilized HA in an orientation that allows its molecular structure to be identified

through the SERS effect. HA adhesion to the ligand was indirectly observed by monitoring the

conformational change of cysteamine from trans to gauche through the measurement of the log

ratio between the 725 cm-1 and 640 cm-1 bands.

The intensity of the HA bands were also measured directly and they increased in relation to the

cysteamine trans coverage. Using a cysteamine concentration of 25 mM a carbodiimide reaction

was used to anchor HA to the surface. HA was detected to a concentration as low as 50 µg/mL,

making it a worthwhile technique for diagnosing the presence or absence of cirrhosis. Further

studies on the interaction of HA with SERS substrates could improve its detection to the ideal

goal of staging liver disease. Additional studies could also use Raman spectroscopy to help

understand the conformation of HA under various physiological conditions.

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Chapter 7: SERS filter for detection of molecules in solutions and airborne particles

7.1 Abstract

In order to analyze a sample using SERS, the analyte has to be brought in intimate contact with

the substrate. This can be problematic when sampling large volumes where the molecules of

interest are found in very low concentrations. For example, a biotoxic aerosol in a large room or

a bio-hazardous substance mixed in a large volume of water is difficult to collect for SERS

interrogation. In principle it is possible to filter out the molecules of interest and then deposit

them on the SERS substrate for analysis. In practice this is very cumbersome and therefore is

rarely used. Here we discuss flexible and porous SERS substrates that have been fabricated by

depositing silver nanoparticle inks on cellulose spun fibers followed by thermal annealing at

about 200oC for 10 to 15 minutes. Microwave absorption at approximately 10 GHz is measured

as a means of monitoring the sintering process in the polymer-nanoparticle matrix and to

optimize the SERS effect. By varying the annealing time, different nanoparticle cluster sizes and

nanocavities are formed. These nanostructures along with the innate three dimensional

structure of the filter contribute to ideal conditions for large SERS amplification, even greater

than on planar substrates. Sampling of nicotine and albuterol sulfate in large air samples using

the SERS filter is discussed.

7.2 Introduction

There is a growing interest in the ability to detect trace amounts of toxins in real time in large

volumes of air, water or biological fluids as it pertains to improving security in public spaces,

monitoring contamination in food sources, and responding to chemical or industrial accidents.

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Unfortunately, it is difficult to detect toxins in small concentrations without performing

laboratory analysis, which normally takes a few days. SERS substrates have the potential of

quickly detecting trace concentrations of chemicals with fingerprint like precision. SERS has

been shown to enhance a Raman signal by an amplification factor of 108 so it would be plausible

to use it to detect even the smallest amount of toxins in the air.

It can be a challenge to sample large volumes using SERS analysis since the analyte has to be

brought in intimate contact with the substrate over an active area that is usually about a cm2.

This becomes a difficult problem when, for example, the molecules of interest are in very low

concentrations and are dispersed in large spaces, for example, a chemical agent released in a

large room or in a large volume of water. In principle it is possible to filter out the molecules of

interest and then transfer them to a SERS substrate for further analyses but this can be very

cumbersome especially for dilute specimens.

Filter paper is already used in many analytical techniques as a filtration step or as a collection

medium. From a commercial point of view it is an inexpensive material, simple to use, and easy

to manufacture. Filter paper is made from a random distribution of cellulose or glass fibers

creating a substrate of varying porosity. In general there are two approaches used for coating

filter papers with silver nanoparticles for making SERS substrates. These include, synthesizing

colloidal particles directly on the filter paper [138], and coating filters with colloidal particles [139-

141]. For direct SERS detection of aerosols on filter papers, Ayora et al first retained the analyte

aerosol on the filter paper and then added silver colloid [142]. For the most part all of these

studies have suffered from problems with nanoparticle adsorption, deterioration of the signal

and lack of reproducibility.

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In our approach, cellulose filter paper is coated with Ag nanoparticle printing ink and then the

substrate is heated to render a SERS active surface. Previously, we have shown that an

appropriate heating treatment partially removes polymer around the particles, which activates

the SERS substrates [5]. In addition, nanoparticle printing inks are preferred over silver colloids

because the thin stabilizers prevent aggregation and have sticking agents that allow it to adsorb

to clean surfaces. The flexibility of the SERS filter allows it to be placed in different

configurations for the capture of airborne particles as well as molecules in solution. In this

report, the nanoparticle arrangement and the geometry of the SERS filter are discussed as the

main sources of high amplifications factors. In addition, we demonstrate the detection of

albuterol sulfate and nicotine on the SERS filter.


Rhodamine 6G (R6G), nicotine solution and sodium chloride were obtained from Fisher

Scientific. Stock solutions were prepared in 10 mM of NaCl. Chloride ions have been shown to

activate silver nanoparticles and therefore increase Raman enhancement [27, 42]. Ten milliliters of

nicotine solution is equivalent to the extract content of one cigarette. Albuterol sulfate was

collected from a prescription metered-dose inhaler.

Ag nanoparticle ink, 40% by weight (UT Dots, Champaign, IL), was spin coated on glass cover

slips and then heated to remove the surfactant layer. Annealing parameters were determined

by measuring the microwave absorption during heating as previously described [2]. After cooling,

the analyte was drop deposited on the surface and allowed to adsorb for at least 10 minutes

before Raman analysis.

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Cellulose fiber fabric samples were coated with Ag nanoparticle ink and then heated at 180oC for

15 minutes to partially remove the surfactant layer. The filter pore size ranged between 0.7 µm

to 3 µm. The substrates were fitted on a 2 cm wide opening on one side of a 750 mL plastic

chamber and an open end cap was screwed in to secure the substrate. A hole was drilled on the

other side of the chamber to act as an inlet for air particles. Albuterol was introduced through

the inlet by a nebulizer and nicotine particulate by smoke extract from a burning cigarette. The

hose leading to a vacuum pump was placed in contact with the underside of the SERS filter (F) at

the securing cap (SC) aperture as depicted in Figure 37.

SERS spectra were collected with the Advantage Near-Infrared Raman Spectrometer (Intevac-

Delta Nu, Laramie, WY) with a 785 nm excitation wavelength. Microwave absorption

measurements were made using a modified X-band Varian E-12 EPR spectrometer with an

associated heater accessory. The samples are supported at one end of a sapphire rod and

positioned at the center of a rectangular TE102 cavity resonant at 9.5 GHz. Dry nitrogen gas

passing over a resistively heated filament was used to heat the sample. SEM micrographs were

collected using a Supra 40 VP electron microscope (Zeiss, Germany).

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Figure 37: SERS filter (F) is fitted at the mouth of a plastic chamber by a securing cap (SC). Air particles are introduced into the container through a nebulizer at the aerosol inlet and a vacuum pump transports the

particles through the SERS filter. 7.4 Results

7.4.1 Surface properties

The surface of the cellulose filter paper is composed of a network of micrometer fibers that

cross link each other creating a porous mesh. When coated with silver nanoparticles (15 to 20

nm in diameter) the fibers and areas between the fibers can become active areas for SERS

enhancement. Whereas silver colloids often settle into fractal structures before aggregating, the

nanoparticle ink forms at least two geometries ideal for Raman enhancement: nanocavities

(regions without nanoparticles) and tightly packed clusters as shown in Figure 38. Both the

electromagnetic field confinement in the nanocavities and the plasmonic excitation in the

nanoparticle clusters will contribute to the local field enhancement [143]. A molecule residing in

the cavity will get sampled by the light multiple times in contrast to only twice on a reflective

planar surface [38]. The enhancement in the cluster geometry can be improved by applying a heat

treatment to partially remove the shell leading to a reduction in interparticle spacing. It is well

known that as the distance between metal spheres is decreased the plasmonic excitations

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Figure 38: Left SEM micrograph shows thickness of fibers on an uncoated filter to be approximately 12µm.

Center micrograph shows the coverage of Ag nanoparticles on a fiber. Right micrograph shows a magnified region of the arrangement of nanoparticles on the fiber surface.

dramatically increase [28, 144]. In addition to these two cases, the three dimensional structure of

the filter increases the adsorption sites available to the molecule as well as the scattering due to

nanoparticle coated fibers on different planes.

The annealing treatment was optimized by measuring the microwave absorption behavior of the

coated SERS filter as a function of heating time. Figure 39 shows a representative sample and in

this case the microwave absorption peaked after 20 seconds of heating at 200oC. Chapter 4

Figure 39: Microwave absorption as a function of heating time at 200oC for the Ag nanoparticle ink

deposited on the filter.

110

shows more detail on how microwave absorption can be used to monitor nanoparticle sintering

and how it correlates to the SERS intensity. As discussed earlier, heating the substrate at a lower

temperature can slow down the removal of the polymer and therefore create a longer thermal

window for heating.

7.4.2 Rhodamine 6G detection

To determine the feasibility of the SERS filter for detecting analytes in solution, R6G was drop

deposited on the filter and detected at low concentrations. Due to its high cross sectional area it

is commonly used as a probe molecule [42] and it is easily recognized by its three cathedral peaks

1309 cm-1, 1362 cm-1 and 1512 cm-1. In Figure 40a, the SERS signal is shown at a concentration of

100 pM (approximately 2 molecules of R6G per trillion molecules of water). The reported typical

non-resonant SERS limit of detection is usually in the parts per billion range [140]. It was necessary

to collect several spectra over the filter because of its inhomogeneity. The distribution of the

1362 cm-1 line intensities was plotted and the top tier signals were used to find the average

intensity at each concentration. The 1362 cm-1 band intensity for R6G showed a good linear fit in

the range between 100 nM to 100 µM (Figure 40b).

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Figure 40: 100 pM R6G solution detected on SERS filter (left). Linear dependence of R6G intensity per

second with respect to concentration based on the 1362 cm-1 R6G band (right). 7.4.3 Albuterol sulfate detection

The SERS filter was also used to detect a common aerosol. Albuterol sulfate, also known as

salbutamol hemisulphate, is a beta-2 adrenergic bronchodilator found in asthma metered dose

inhalers. Each dose delivers 90 micrograms of albuterol sulfate. One actuation of the medicine

was introduced into the chamber followed by pumping out the air-albuterol mixture through

the SERS filter. The actual amount of albuterol trapped by the filter will depend on the porosity

of the filter and the flow rate of the air-albuterol mixture through the filter. The filter was

removed to collect the SERS signal and to detect the albuterol vibrational bands. The main band

between 970 cm-1 and 990 cm-1 corresponds to the C-OH group [145, 146] and was used for

identification purposes. The other peaks agree well with a recent vibrational spectroscopic study

of albuterol [146]. Three other peaks, 856.6 cm-1, 1132 cm-1 and 1604 cm-1, are attributed to a

polymer found on the filter paper as shown in Figure 41.

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Figure 41: Albuterol sulfate SERS signal from a single inhaler dose released into the flask (top).

Background SERS signal from filter substrate (bottom). 7.4.4 Nicotine detection

The Raman shifts of nicotine are well known [147, 148]. The main band is found at 1030 cm-1

corresponding to the pyridine ring breathing mode and is attributed to the electron pair of

nitrogen interacting with a metal [149]. Spin coated SERS substrates were prepared to detect

nicotine solution at low concentrations. On average nicotine content is estimated to be 10

mg/cigarette therefore our stock solution contained 1 mg/mL of nicotine. This was diluted in a

10 mM NaCl solution to obtain a range between 0.1 ng/µL to 50 ng/µL. Within this range a linear

dependence was obtained between the concentration of nicotine and the intensity per second

as shown in Figure 42. The nicotine 1032 cm-1 band was detected at concentrations as low as 0.5

ng/µL (or 50 ppb).

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Figure 42: The nicotine 1032 cm-1 peak was detected at concentrations as low as 0.5 ng/µL from a solution

diluted in 10 mM NaCl on the SERS substrate. Once nicotine solution was detected on the spin coated heated substrate the SERS filter was

used to detect nicotine from the smoke of a cigarette. A wisp of smoke was allowed to enter

the chamber and was transported through the filter. The filter was then removed and 10 µL of

NaCl was immediately added. The SERS 1032 cm-1 and the 1051 cm-1 nicotine peaks were clearly

detected as shown in Figure 43. From the standard curve, the detection limit of our substrates

was estimated to be 1-2 molecules of nicotine per billion molecules of water. The remaining

peaks come from the filter’s fiber while the 238 cm-1 peak is attributed to the Ag-Cl bond.

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Figure 43: SERS nicotine signal on Ag coated filter substrate produced from the smoke of a lit cigarette. The main 1031 cm-1 and 1052 cm-1 bands are clearly visible while the other minor peaks come from the

background signal from Ag-Cl and the filter’s fibers. 7.5 Conclusion

SERS substrates fabricated by depositing silver colloidal particles over cellulose filters can be

used to sample large volumes that may contain trace concentrations of molecules of interest.

Using the SERS filter two things are achieved: [1] the interparticle distances and the cluster sizes

can be finely adjusted by the thermal treatment and [2] unlike SERS substrates on planar

surfaces, the substrates made using glass-fiber filters are three-dimensional. The dimensionality

provides multiple anchoring sites for a single analyte molecule and is the likely reason for the

very high SERS amplification factors.

A SERS filter attached to the opening of a vacuum chamber was used to detect airborne particle

concentrations of nicotine as well as albuterol sulfate. Nicotine from cigarette smoke was

detected at a concentration of 2 ppb while the Raman peaks of albuterol sulfate were

detectable from one aspiration of a metered inhaler. Deposited samples of R6G on the filter also

115

showed a greater enhancement than on a planar substrate. R6G was detectable at a

concentration as low as 100 pM with no post signal processing. This is one thousand times more

dilute than our best detection efforts on a plan substrate. Although these experiments were

done in isolated volumes of known concentrations, it is quite conceivable that one could coat

the SERS filter with a ligand to target specific molecules from a mixture as it passes through the

porous membrane.

There is no doubt that SERS filters could find many applications especially in public spaces. They

could be incorporated into existing air circulation systems to sample air for possible

contamination. The filters could be removed at regular intervals for later examination or they

could be monitored by a battery operated Raman spectrometer module and the signals

transmitted to a central location. Furthermore, the air-sampling technique could easily be

adapted for water-sampling to test for chemicals, pesticides or prescription drug residues in

waste water treatment plants.

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Chapter 8: Conclusions and recommendations 8.1 Scientific contributions

The research niche of SERS substrate design has been saturated for many years because of the

number of papers published yearly on the use of colloidal particles and the various multi-step

lithography methods. It becomes difficult to compare among studies. To frame the discussion of

my substrate design, I followed the standards proposed by Natan et al [43] and I believe the

substrate described in this work meets those standards.

My approach to substrate design is unique because it takes a common step in circuit fabrication

-- sintering -- and it stops the heating short to create a concentration of nearly touching

nanoparticles that produce large electromagnetic fields in the interparticle spacing. In the past,

annealing was used but it would take the substrate to a point of complete conductivity so a low

SERS signal was collected [41]. In this thesis I show evidence for an increase of the SERS signal in a

substrate composed of nanoparticle printing inks after short heating treatments. The ligand

layer surrounding the nanoparticle prevents them from aggregating prematurely so the heating

treatment is applied to partially remove the ligand layer, allowing the particles to approach each

other. The evidence for ligand removal comes from the thermal analysis study which shows an

oxidative decomposition of the ligand followed by a mass loss stemming from the removal of

the ligand. Furthermore, SEM analysis shows a decrease in interparticle spacing followed by the

formation of large cluster aggregates which altogether correlate with the rise and fall of the

SERS signal throughout the heating process.

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Microwave absorption was introduced as a novel technique to sensitively monitor the sintering

process in a substrate on a global scale. Although microwaves are completely unrelated to the

SERS effect, microwave losses occur at the interfaces between two particles, where they are

almost in intimate contact, due to resistive weak links at the sites. It just so happens that at a

point during the sintering process the interparticle spacing in a dimer is slightly larger than the

condition for microwave absorption so the site produces the large em field normally observed in

the SERS effect. Thus, by monitoring the sintering process, we showed the largest SERS

magnitude occurred at a point preceding the largest microwave absorption. In fact the

microwave absorption measurements were used in all of our SERS experiment to determine an

optimal thermal window where the highest SERS amplification factors could be observed.

Typically for oleylamine capped silver nanoparticles the rise in microwave absorption occurred

in the 180oC to 200oC temperature range for various heating times. This agrees well with the

thermal analysis discussed in Chapter 5, which shows a prominent exothermic peak at

approximately 189oC, which is assigned to the partial decomposition of the ligand layer.

The combined results from Chapters 4 and 5 give us some insight into the mechanism during the

beginning stages of sintering. As the oleylamine ligand decomposes, its residues remain on the

substrate causing a displacement between two neighboring nanoparticles. However, when there

is an increase in spacing at one site there is a decrease in spacing at another site. The

rearrangement of nanoparticle spacing is immediately captured with the microwave absorption

measurements, which according to my descriptive model, monitors the transient process of

dimers moving from a weak link resistive state to an electrically conductive state.

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To show my substrate’s capability at detecting analytes various simple chemicals were detected

on its surface, including R6G, folic acid, pyridine and nicotine. However, for biological

applications it was important to show that more complex molecules could also be detected. In

this case I chose to study hyaluronic acid, which is a glycosaminoglycan with various roles in the

human body including support, response to inflammation, and homeostasis in the extracellular

matrix. It is advocated as a biomarker for the accurate staging of fibrosis in the liver, which

eventually leads to cirrhosis and consequently to the need for a liver transplant. Today only liver

biopsies serve as the gold standard for staging liver disease progression.

Previous attempts at detecting HA through the SERS effect on a Klarite substrate were

disappointing most likely due to the fact that the molecule was not interacting with the gold

surface. Here I chose to functionalize my nanoparticle surface with a two carbon self-assembled

monolayer that could anchor HA to the surface. The ligand layer was optimized to capture the

most HA molecules and numerous vibrational bands became detectable. Determining HA

capture through SERS was an easy task as the change in band intensities gave evidence that the

ligand was changing from a trans conformer to a gauche conformer. On top of that the HA

Raman bands became apparent after the incubation. With this technique I was able to detect

HA to a concentration as low as 50 μg/mL which is a 3000 fold improvement from previous

attempts taking into account concentration used and collection time.

8.2 Recommendations

The work in this thesis shows enough evidence to suggest that microwave absorption

measurements can be used to sensitively monitor the sintering process. This is a non-

contact method that could be used to determine the time it takes for nanoparticle-

119

based circuits to sinter. The current method of choice is DC resistivity which can be

inaccurate at low conductivities and because it makes contact with the surface it can

cause damage. The application of microwave absorption for this application should be

further explored.

As is shown in this work, measuring microwave absorption provides a method to find

the optimal heating conditions of a SERS substrate composed of nanoparticle ink to

produce large amplification factors. What is not so clear is if microwave absorption can

measure the creation and destruction of SERS hot spots. It would be interesting to see if

a model could be developed to explain the transition between a SERS hot dimer and a

high microwave absorption dimer.

In terms of HA detection or for that matter any other complex molecule, it will be very

important to show that the molecule can be detected in a mixture that resembles the

serum. Using cysteamine as a ligand means that any molecule with a carboxyl group

could attach to the substrate. Using a more specific ligand such as an antibody could

guarantee specific binding although to the size of the antibody the HA Raman bands

might not be as apparent if detectable at all. However, a change in antibody

confirmation might be evidence enough to detect antigen binding events. Protein-ligand

interactions on silver SERS substrates has been studied by Drachev et al with some

limited success [150, 151].

120

Finally, the work I began on SERS filters should be continued. The platform has the

potential for producing higher amplification factors than planar substrates due to the

presence of nanocavities and nanoclusters on the fibers’ surfaces. In addition, the three

dimensional structure provides multiple anchoring sites where a molecules vibrational

bands could be detected. By functionalizing the silver nanoparticles on the fibers it

seems plausible to be able to capture analytes of interest as they are filtered through.

121

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Appendix A: Glossary of Terms Annealing – A process of heating a material followed by a slow cool to reduce internal stresses

Sintering – A heating process used to bond adjacent metallic surfaces made from fine powders or nanoparticles. This process strengthens the mass and produces densification and recrystallization

Surface plasmon resonance (SPR) – coherent oscillation of the surface conduction electrons excited by electromagnetic radiation.

Self-similarity/Scale invariance – also called invariance under internal similarity or sometimes dilation invariance. A feature of an object that appears similar to itself at different scales.

Fractal dimension (D) – is a measure of complexity in an object when the unit of measurement changes. An example is the measure of volume which is measured with the unit of length to the third power. Here the exponent is the fractal dimension. In a fractal the dimension will vary as a non-integer between 1 and 2 for an object on a two-dimensional surface and between 2 and 3 for a three-dimensional object.

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Appendix B: Comparison of substrate fabrication techniques

In this work, three main methods were used to deposit silver nanoparticle inks over a

glass surface for the purpose of fabricating SERS substrates: 1) dry drop deposition, 2) spin

coating and 3) ink printed. A comparison of all three methods was done qualitatively based on

the following characteristics: spot diameter, reproducibility of making a spot, uniformity of the

distribution of particles, SERS signal variability and the maximum achievable SERS amplification

factors. The results of the analysis are given in Table 2.

The dry drop method involved depositing 10 μL of ink (20% by weight) with a micropipette on a

microscope slide and then letting it dry for 1 hour before use. This created a coffee drop effect

(~ 5-10 mm in diameter) where the nanoparticles settled on the surface according to a

distribution gradient from high to low from the periphery of the dried spot to the center of the

dried spot. The variability over the substrate varied over the diameter of the spot. It was

common to see coefficients of variability above 50% for this method. However, there were

Table 2: Comparison of nanoparticle ink deposition techniques

128

certain areas on the spot that would produce very large amplification factors due to an ideal

arrangement of nanoparticles. However, this was impossible to reproduce from substrate to

substrate.

The spin coating method involved depositing 10 μL of nanoparticle ink over square glass cover

slips using a micropipette followed by spin coating with a G3P-8 spin coater (Specialty Coating

Systems, Indianapolis, IN). The spin coating protocol was a two-step process: 1 min at 700 rpm

with a ramp rate of 5.0 followed by 1 min at 2000 rpm with a ramp rate of 5.0. The spin coated

sample was allowed to air dry and stored in a desiccator until use. This method had the

potential to produce spot sizes of approximately 5-10 mm in diameter. Of course the size could

be controlled by depositing more or less ink on the cover slip before spinning. The coverage of

nanoparticles was very uniform and the variability of the SERS signal intensity was much lower

compared to the dry drop method. In fact the variability was compared by measuring the

intensity of the three major SERS peaks from a 10 μM solution of folic acid, 1173 cm-1, 1492 cm-1

and 1587 cm-1, on both deposition methods. From Figure 44 it is quite clear that even though

there was a larger average signal on the dry drop deposited substrates, the signal variability was

so large that it was not worth using for reproducibility experiments. On the other hand, for spin

coated substrates the variability was very low but the average intensity dropped significantly. At

this point it was more important to have less variability so the spin coated method was

preferred.

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Figure 44: SERS intensity of the three major peaks from a 10 μM solution of folic acid on a dry drop and

spin coated substrate.

The printed dot arrays were made by UT Dots, Inc and they were shipped to us as needed. By

varying the solvent and nozzle size reproducible SERS dots with high amplification factors were

fabricated. They were printed on microscope glass slides at room temperature using an AD3200

printing workstation (Biodot Irvine, CA) with 60% by weight silver nanoink toluene solvent

(AgTE). The syringe pump and solenoid valve dispensed approximately 0.1 μL sized drops

through a 100 μm diameter nozzle. The printed 6 x 6 arrays had dots of an approximate size of

1.2 mm with no visible coffee drip effect. In other words, they were very uniform and the SERS

signal variability was very low.

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Vita

MANUEL ALEJANDRO FIGUEROA EDUCATION: Drexel University, School of Biomedical Engineering Philadelphia, PA PhD in Biomedical Engineering Sept 2012 Tulane University, Tulane College of Engineering New Orleans, LA Bachelor of Science in Biomedical Engineering May 2004 GRADUATE FELLOWSHIPS AWARDED: Freshman Design Teaching Fellowship Sept 2010 -- present GK-12 Fellowship July 2008 – Aug 2010 Bridge to the Doctorate Fellowship Sept 2006 – Aug 2008 RESEARCH EXPERIENCE: Magnetic Materials Laboratory Philadelphia, PA Drexel University Research Assistant Jan 2007 – Present HEDO, Air Force Research Laboratory (AFRL) San Antonio, TX Associate Research Biomedical Engineer Aug 2004 – Sept 2006 Laboratory for Health, Human Performance and Rehabilitation New Orleans, LA Tulane University Research Assistant Nov 2002 – July 2004 Bascom Palmer Eye Institute, Ophthalmic Biophysics Center Miami, FL University of Miami Research Assistant Summers 2001, 2002 TEACHING EXPERIENCE: Drexel courses taught: Drexe-Haddonfield Summer Pre-Engineering Academy ENGR101, 102 and 103 – Introductory course to engineering ENGR180 – Mathematics for Engineers BMES 303 – Biomedical Electronics BMES 326 – Principles of Biomedical Engineering SELECTED PUBLICATIONS: M Figueroa, K Pourrezaei and S Tyagi. “Microwave monitoring of silver nanoparticle sintering for surface-enhanced Raman scattering substrates.” Journal of Raman Spectroscopy. vol 43 (4) p 588-591, 2012. M Figueroa, K Pourrezaei and S Tyagi. “Fabrication of flexible and porous surface enhanced Raman scattering (SERS) substrates using nanoparticle inks.” AIP Conference Proceedings, vol 1461 p 47-53, 2012. M Figueroa, S Schraer, K Pourrezaei and S Tyagi. “Surface-enhanced Raman scattering and microwave absorption in silver nanoparticle inks.” Plasmonics in Biology and Medicine IX, SPIE Proceedings vol 8234, 2012. B Pelleg, M Figueroa, M VanKouwenberg, A Fontecchio and E Fromm. “Implementing nanotechnology education in the high school classroom.” Proceedings of the 41st ASEE/IEEE Frontiers in Education Conference, Rapid City, SD. October 2011. M Figueroa, K Pourrezaei and S Tyagi, “Detection of hyaluronic acid on a functionalized surface enhanced Raman scattering substrate.” Proceedings of the 37th IEEE Northeast Bioengineering Conference, Troy, NY. April 2011. J Mitchell-Blackwood, M Figueroa, C Kokar, A Fontecchio and E Fromm. "Tracking middle school perception of engineering during an inquiry-based engineering science and design curriculum," Proceedings of the ASEE Annual Conference & Exposition, Louisville, KY. June 2010.

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Documents

Fabrication of surface enhanced Raman Scattering (SERS