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I
Name: Qin Weijie
Degree: Ph.D.
Department: Chemical and Biomolecular Engineering
Thesis title: Fabrication of gold nanoparticle-DNA conjugates
bearing specific number of DNA for quantitative detection and
well-defined nanoassembly
Year of submission: 2007
II
FABRICATION OF GOLD NANOPARTICLE-DNA
CONJUGATES BEARING SPECIFIC NUMBER OF DNA
FOR QUANTITATIVE DETECTION AND WELL-
DEFINED NANOASSEMBLY
QIN WEIJIE
NATIONAL UNIVERSITY OF SINGAPORE
2007
III
Acknowledgements
I would like to sincerely express my greatest gratitude to my supervisor, Dr. Yung Lin Yue Lanry, for his unreserved support and guidance throughout the course of this research project. His continues guidance, constructive criticisms and insightful comments have helped me in getting my thesis in the present form. He has shown enormous patience during the course of my Ph.D. study and constantly gives me encouragements to think positively. More importantly, his rigorous research methodology, objectivity and enthusiasm in scientific discovery will be a model for my life and career. I wish to express my heartfelt thanks to all my friends and colleagues in the research group, Mr. Zhong Shaoping, Miss Zhao Haizheng, Mr. Jia Haidong, Miss Tan Weiling, Mr. Deny Hartono, and Miss Duong Hoang Hanh Phuoc and other staffs of the Department of Chemical and Biomolecular Engineering, especially Miss Li Xiang, Miss Li Fengmei, Mr. Han Guangjun, and Mr. Boey Kok Hong. Without their assistance, this work could not have been completed on time. Special acknowledgements are also given to National University of Singapore for its financial support. I deeply appreciate my girl friend, Miss Liu Ying. Her love and encouragement light up many lonely moments in my life as a graduate student away from home. Last, but not least, I would like to dedicate this thesis to my parents. Without their love, support and understanding, I would not have completed my doctoral study.
IV
Table of contents
Acknowledgements ...................................................................................................... III Table of contents ..........................................................................................................IV Summary..................................................................................................................... VII List of tables .................................................................................................................XI List of figures ............................................................................................................. XII Chapter 1 Introduction....................................................................................................1
1.1 Background...........................................................................................................1 1.2 Aims and scope of this project .............................................................................3
Chapter 2 Literature review............................................................................................6
2.1 Synthesis, stabilization and characterization of metallic nanoparticles ...............6 2.1.1 Reaction mechanism and kinetics .................................................................6 2.1.2 Mechanism of particle formation ..................................................................7 2.1.3 Synthesis of metallic nanoparticles ...............................................................7 2.1.4 Stabilization of nanoparticles ........................................................................8 2.1.5 Characterization of nanoparticles ................................................................12
2.2 Formation of gold nanoparticle-DNA conjugates (nAu-DNA)..........................13 2.2.1 Introduction to synthetic DNA ....................................................................13 2.2.2 Formation of gold nanoparticle-DNA conjugates (nAu-DNA)...................14
2.3 Applications of nAu in nanoassembly and ultrasensitive DNA detection .........15 2.4 Study on the plasmon coupling of metallic nanoparticle dimers .......................25 2.5 Gel electrophoresis study on nanoparticle-DNA conjugates..............................27
2.5.1 Electrophoretic isolation of discrete nAu-DNA conjugates........................27 2.5.2 Conformation study of nanoparticle-bound DNA.......................................29
2.6 Formation of discrete nanoparticle-DNA conjugate groupings .........................31 2.7 Enzyme manipulation of the nanoparticle-bound DNA.....................................32
2.7.1 Introduction to restriction endonuclease .....................................................32 2.7.2 Effect of steric hindrance on DNA hybridization and enzymatic reaction efficiency ..............................................................................................................33 2.7.3 Enzyme manipulation of nanoparticle bound DNA ....................................34
Chapter 3 Synthesis of gold nanoparticles (nAu).........................................................39
3.1 Materials and methods........................................................................................39 3.2 Results and discussion........................................................................................40
3.2.1 Synthesis and characterization of nAu ........................................................40 3.3 Conclusions ........................................................................................................42
Chapter 4 Efficient manipulation of nanoparticle-bound DNA via restriction endonuclease.................................................................................................................46
4.1 Introduction ........................................................................................................46 4.2 Materials and methods........................................................................................48 4.3 Results and discussion........................................................................................53
V
4.3.1 Importance of short ssDNA modification on the surface of nAu for achieving high enzyme digestion efficiency ........................................................53 4.3.2 Effect of ssDNA surface coverage on enzyme digestion nanoparticle-bound DNA .....................................................................................................................55 4.3.3 Enzyme digestion efficiency of nanoparticle-bound DNA .........................57 4.3.4 Effect of ionic strength on dT-ssDNA surface coverage on nAu and enzyme digestion efficiency ..............................................................................................61
4.4 Conclusions ........................................................................................................63 Chapter 5 Fabrication of nanoparticle-DNA conjugates bearing specific number of short DNA strands by enzymatic manipulation of nanoparticle-bound DNA .............65
5.1 Introduction ........................................................................................................65 5.2 Materials and methods........................................................................................67 5.3 Results and discussion........................................................................................71
5.3.1 Enzyme digestion efficiency of nanoparticle-bound dsDNA by polyacrylamide gel electrophoresis ......................................................................71 5.3.2 Agarose gel electrophoresis of the nanoparticle-DNA conjugates..............73 5.3.3 Restriction endonuclease digestion/cleavage of nanoparticle-dsDNA conjugates bearing definite number of DNA strands ...........................................75
5.4 Conclusions ........................................................................................................81 Chapter 6 Nanoparticle based quantitative DNA detection with single nucleotide polymorphism (SNP) discrimination selectivity ..........................................................82
6.1 Introduction ........................................................................................................82 6.2 Materials and methods........................................................................................85 6.3 Results and discussion........................................................................................90
6.3.1 Formation of nAu-DNA conjugate dimers using linker DNA of different lengths...................................................................................................................90 6.3.2 Quantification of target DNA through the formation of nAu-DNA conjugate dimers ...................................................................................................................93 6.3.3 Hybridization efficiency of strand A, strand revA with target DNA without nAu .......................................................................................................................97 6.3.4 Single nucleotide polymorphism (SNP) discrimination using nAu-DNA conjugate groupings............................................................................................100
6.4 Conclusions ......................................................................................................104 Chapter 7 Fabrication of gold nanoparticle based nano-groupings with well-defined structure ......................................................................................................................106
7.1 Introduction ......................................................................................................106 7.2 Materials and methods......................................................................................108 7.3 Results and discussion......................................................................................114
7.3.1 Grouping percentage of nAu-DNA conjugates using linker DNA of different lengths..................................................................................................114 7.3.2 Effect of hybridization conditions on final grouping percentage..............120 7.3.3 Electrophoretic mobility of conjugate groupings linked by various length of linker DNA .........................................................................................................123
VI
7.4 Conclusions ......................................................................................................125 Chapter 8 Conclusions................................................................................................127 Chapter 9 Suggestions for future work.......................................................................130
9.1 Further study on the hybridization of nAu-DNA conjugates ...........................130 9.2 FRET based quantitative DNA detection using nAu and quantum dot as efficient fluorescent acceptor and donor...............................................................................132 9.3 Application of nAu-DNA conjugates in chip-based DNA detection ...............133 9.4 Fabrication of multiple functionalized nAu-DNA conjugates bearing different DNA sequences and its application in SNP discrimination ...................................134
Reference....................................................................................................................136 Appendix I Complete sequences of DNA used in Chapters 6 and 7..........................155 Appendix II List of Publications ................................................................................157
VII
Summary
Self-assembling of gold nanoparticles to form well-defined nano-structures is a field
that has been receiving considerable research interests in recent years. In this field,
DNA is a commonly used linker molecule to direct the assembly of nanoparticles
because of its unique recognition capabilities, mechanical rigidity, enzyme
processibility as well as physicochemical stability and has shown great potential in
fabrication and construction of nanometer-scale assemblies and devices. This Ph.D.
work aims to fabricate gold nanoparticles bearing definite number and length of DNA
strands using gel electrophoresis isolation and restriction endonuclease manipulation
of the nanoparticle-bound DNA. These specially designed nanoparticles are then
applied for quantitative DNA detection and construction of well tailored nano-
groupings.
Topically this thesis is divided into 9 chapters. Chapter 1 is the introduction and
outlines, the specific aims and scope of this thesis. Chapter 2 reviews the current
development in the literature. The main results and findings are discussed through
Chapter 3 to Chapter 7. The conclusions and suggestions for further work are covered
in Chapter 8 and Chapter 9 respectively.
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In Chapter 3, we describe the synthesis of mono-dispersed gold nanoparticles of
various sizes by the reduction of hydrogen tetrachloroaurate (III) tetrahydrate by
trisodium citrate dihydrate and tannic acid. The size and size distribution of the gold
nanoparticles were analyzed by UV-Vis spectroscopy and transmission electron
microscopy (TEM).
In Chapter 4, we demonstrate a strategy for efficient manipulation of gold
nanoparticle-bound DNA using restriction endonuclease. The digestion efficiency of
this restriction enzyme was studied by varying the surface coverage of stabilizer, the
size of nanoparticles as well as the distance between the nanoparticle surface and the
enzyme cutting site of nanoparticle-bound DNA. We found that the surface coverage
of stabilizer is crucial for achieving high digestion efficiency. In addition, the surface
coverage of this stabilizer can be tailored by varying the ion strength of the system.
Based on the results of polyacrylamide gel electrophoresis and fluorescent study, a
high digestion efficiency of 90+% for nanoparticle-bound DNA was achieved for the
first time. This restriction enzyme manipulation can be considered as an additional
level of control on the nanoparticle-bound DNA and is expected to be applied to
manipulate more complicated nanostructures assembled by DNA.
In Chapter 5, we report our novel approach to generate gold nanoparticle-DNA
conjugates bearing specially designed DNA linker molecules that can be used as
nanoprobes for quantitative DNA sequence detection analysis or as building blocks to
IX
construct nano-groupings with precisely controlled structure. In our approach, gold
nanoparticle-DNA conjugates bearing definite number of long dsDNA strands were
prepared by gel electrophoresis. A restriction endonuclease enzyme was then used to
manipulate the length of the nanoparticle-bound DNA. This enzymatic cleavage was
confirmed by gel electrophoresis, and digestion efficiency of 90% or more was
achieved. With this approach, nanoparticle conjugates bearing definite number of
strand of short DNA with less than 20-base can be achieved.
Sequence-specific DNA detection is important in various biomedical applications such
as gene expression profiling, disease diagnosis and treatment, drug discovery and
forensic analysis. In Chapter 6, we develop a gold nanoparticle-based method that
allows DNA detection and quantification and is capable of single nucleotide
polymorphism (SNP) discrimination. The precise quantification of single stranded
DNA is due to the formation of defined nanoparticle-DNA conjugate groupings in the
presence of target/linker DNA. Conjugate groupings were characterized and quantified
by gel electrophoresis. A linear correlation between the amount of target DNA and
conjugate groupings was obtained at lower target DNA concentration and can further
be exploited for target DNA quantification. For SNP detection, single base mismatch
discrimination was achieved for both the end-and center-base mismatch. The method
holds promise for creating a quantitative and highly specific DNA detection method
for biomedical applications.
X
Many interesting properties of nanoparticle-based materials are highly dependent upon
their structural parameters. In Chapter 7, we describe the fabrication of DNA induced
gold nanoparticle nano-groupings with well-defined structures (dimers, trimers and
other higher order multimers) using gold nanoparticles bearing definite number and
length of DNA. These nano-conjugate groupings were analyzed using gel
electrophoresis and discrete gel bands corresponding to groupings with defined
structures were obtained. Various factors that affect the formation of nano-groupings
were explored as well. The results show that direct linkage of two nanoparticle-DNA
conjugates without linker DNA, longer hybridization time and higher ion strength
buffer lead to higher degree of grouping. For nano-grouping formation, a minimum
length of linker DNA of 24-base is needed for our nanoparticle-DNA conjugate
system. Further increase in the linker length results in little improvement in the
grouping percentage. Furthermore, it was found that the number of nanoparticles
involved in the grouping structure is more effective in deciding its electrophoretic
mobility than the length of linker DNA. TEM characterization further demonstrated
that conjugate groupings extracted from each gel band consist of the expected
grouping structure. This confirms that gel electrophoresis is an efficient tool for
isolation of small grouping structures of nanoparticle-DNA conjugates.
XI
List of tables
Table 4.1 Digestion efficiency of free DNA and 10nm nAu-bound dsDNA by endonuclease EcoR V. 59
Table 4.2 dT-ssDNA surface coverage on 7nm nAu under different NaCl concentration and digestion efficiency of particle-bound dsDNA by endonuclease EcoR V. *Determined from fluorescent study using FITC labeled dT-ssDNA and reported in both units – number of strands/nAu and pmol/cm2. 63
Table 5.1 Digestion efficiency of free DNA and nanoparticle-bound DNA by endonuclease EcoR V. 73
Table 6.1 Hybridization efficiency of strand & strand revA with various ratios of target DNA. 98
Table 7.1 Relative mobility of conjugate groupings linked by different length of linker DNA. 124
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List of figures
Figure 2.1 Electrostatic stabilization of nanoparticles 10
Figure 2.2 Steric hindrance stabilization of nanoparticles 11
Figure 2.3 Organization of nAu into spatially defined structure 16
Figure 2.4 Scheme showing the DNA induced nAu assembly process. The scheme is not meant to imply the formation of a crystalline lattice but rather an aggregate structure 18
Figure 2.5 Comparison of UV spectra of nAu functionalized with 5’ thiol 12-base ssDNA before and after treatment with a complementary 24-base ssDNA 18
Figure 2.6 Comparison of the melting transitions for a 30bp dsDNA (squares) and nanoparticles linked with the same 30bp dsDNA (circles). Absorbance changes are measured at 260 nm. The insets are the derivative curves of each set 19
Figure 2.7 Schematic showing of the scanometric DNA array detection with nanoparticle probes 20
Figure 2.8A The structure of nAu-DNA-dye conjugate molecular beacon 22
Figure 2.8B Schematic drawings of the two conformations of the nAu-DNA-dye conjugate molecular beacon. On the left, the hairpin structure brings the dye and the nAu in close proximity (within a few angstroms) and the fluorescent of the dye molecule is quenched. Through sequence-specific hybridization to a target DNA, the hairpin structure changes to a rod-like dsDNA structure (on the right), which separates the dye and the quencher far apart and thus restores the fluorescence 22
Figure 2.9 nAu-based molecular beacon and its operating process. DNA molecules self-assemble into an arch conformation on the nAu (2.5 nm diameter). Single-stranded DNA is represented by a single line and double stranded DNA by a cross-linked double line. In the assembled (closed) state, the dye is quenched by the nAu. Upon target binding, the constrained conformation opens, the dye leaves the surface because of the structural rigidity of dsDNA, and the fluorescence is restored. Au: nAu; F: dye 24
Figure 2.10 Representative scattering spectra of single particles and particle pairs for silver (top) and gold (bottom). Silver particles show a larger spectral shift (102 nm)
XIII
than gold particles (23 nm), stronger light scattering and a smaller plasmon line width. Gold, however, is chemically more stable and is more easily conjugated to biomolecules via –SH, –NH2 or –CN functional groups 26
Figure 2.11 Electrophoretic mobility of 5 nm Au/100b HS-ssDNA conjugates (3% gel). The first lane (left to the right) corresponds to 5 nm particles (single band). When 1 equiv of DNA is added to the nAu (second lane), discrete bands appear (namely 0, 1, 2, 3, ...). When the DNA amount is doubled (third lane), the intensity of the discrete bands change and additional retarded bands appear (4, 5). Because of the discrete character, each band can be directly assigned to a unique number of DNA strands per particle 29
Figure 2.12 Different possible configurations of DNA molecules attached to the surface of nAu 30
Figure 2.13 Patterns of DNA cutting by restriction endonucleases 33
Figure 2.14 Hybridization and extension of nAu-bound DNA. In step 1, the nAu-bound DNA is annealed to the template strand followed by extension in step 2 accomplished by the addition of DNA polymerase 34
Figure 2.15 Enzyme digestion of DNA immobilized on gold surface 35
Figure 2.16 (A) Double stranded nanoparticle-DNA conjugates with the EcoR I recognition site, (B) The conjugates with a 59 ° bending due to the binding of M.EcoRI enzyme, (C) The conjugates after the cutting of the DNA at the R.EcoRI recognition site 36
Figure 2.17 (A) Nanoparticles derivatized with double-stranded DNA are treated with restriction enzyme EcoR I, which cleaves the DNA to yield cohesive ends. The part between the arrowheads represents the recognition site of the enzyme; (B) Two cohesive ends hybridize, which leads to a weak association of particles; (C) The DNA backbones are covalently joined at the hybridized site by DNA ligase to yield a stable 40-base-pair double-stranded link between particles 38
Figure 3.1 UV-visible spectrum of an aqueous solution of 9.7 nm nAu. 41
Figure 3.2a TEM image of nAu with a mean diameter of 9.7nm. 43
Figure 3.2b Size distribution of nAu with 0.001% tannic acid concentration condition. Mean diameter = 9.7nm, standard deviation = 0.64nm. 43
Figure 3.3a TEM image of nAu with a mean diameter of 7.0nm. 44
XIV
Figure 3.3b Size distribution of nAu with 0.01% tannic acid concentration condition. Mean diameter = 7.0nm, standard deviation = 0.56nm. 44
Figure 3.4a TEM image of nAu with a mean diameter of 6.3nm. 45
Figure 3.4b Size distribution of nAu with 0.05% tannic acid concentration condition. Mean diameter = 6.3 nm, standard deviation = 0.67 nm. 45
Figure 4.1 DNA sequences used in this chapter. Underlined sequences are the recognition site of EcoR V and the arrows indicate the enzyme cleavage site. 49
Figure 4.2 Schematic picture of preparation of nanoparticle-DNA conjugates. 51
Figure 4.3 PAGE characterization of enzyme digested 10nm nAu-bound dsDNA without dT-ssDNA saturation. Lanes 1-5 correspond to: (1) 20bp DNA ladder, (2) intact Strand A without enzyme treatment, (3) enzyme treated particle-bound Strand A, (4) intact Strand B without enzyme treatment, (5) enzyme treated particle-bound Strand B. 54
Figure 4.4 dT-ssDNA surface coverage on 10nm nAu as well as corresponding enzyme digestion efficiency of particle-bound Strand A and non-thiol Strand A. 56
Figure 4.5 PAGE characterization of enzyme digested 10nm nAu-bound DNA. Lanes 1-7 correspond to: (1) 10bp DNA ladder, (2) enzyme digested particle-bound Strand A, (3) enzyme digested free Strand A, (4) intact Strand A without digestion, (5) enzyme digested particle-bound Strand B, (6) enzyme digested free Strand B, (7) intact Strand B, (8) particle-bound Strand A, (9) Strand A displaced by DTT in the digestion buffer and (10) 10bp DNA ladder. 58
Figure 4.6 Emission spectrum of FITC Strand A used in enzyme digestion efficiency study. Enzyme digested nAu-bound FITC Strand A (real line), nAu-bound FITC Strand A without enzyme treatment (broken line). 61
Figure 5.1 Schematic picture of preparation of nanoparticle-DNA conjugates. 69
Figure 5.2 DNA sequences used in this chapter. Arrows indicate the enzyme cleavage sites. 70
Figure 5.3 PAGE characterization of enzyme digested particle-bound DNA. Lanes 1-7 correspond to: (1) the 10bp DNA ladder, (2) enzyme digested particle-bound Strand B, (3) enzyme digested free Strand B, (4) intact Strand B without digestion, (5) enzyme digested particle-bound Strand A, (6) enzyme digested free Strand A, and (7) intact Strand A. 72
XV
Figure 5.4 Agarose gel electrophoresis of nanoparticle-dsDNA conjugates bearing Strand A. Lanes A1, B1 and C1 correspond to EcoR V digested conjugates bearing 4, 3 and 2 strands of Strand A respectively; Lanes A2, B2, C2 correspond to the conjugates bearing 4, 3 and 2 strands of Strand A without EcoR V digestion respectively; Lane D corresponds to mixed conjugates bearing different strands of Strand A and Lane E correspond to the conjugate bearing only 5-base ssDNA without Strand A. 74
Figure 5.5 Comparison of the electrophoretic mobility of the enzyme treated and non-enzyme treated 5-base ssDNA modified nanoparticles. Lanes F-H correspond to enzyme treated nanoparticles, non-enzyme treated nanoparticles, and nanoparticle-dsDNA mixture respectively. 76
Figure 5.6 Agarose gel electrophoresis of nanoparticle-dsDNA conjugates bearing Strand B. Lanes I1, J1 and K1 correspond to EcoR V digested conjugates bearing 4, 3 and 2 strands of Strand B respectively; Lanes I2, J2 and K2 correspond to the conjugates bearing 4, 3 and 2 strands of Strand B without EcoR V digestion respectively; Lane L corresponds to mixed conjugates bearing different strands of Strand B and Lane M correspond to the conjugate bearing only 5-base ssDNA without Strand B. 78
Figure 5.7 Comparison of electrophoretic mobility of the digested nanoparticle-dsDNA conjugates bearing Strand A or strand B. Lane P1 and Q1 correspond to the conjugates bearing 5 strands of digested Strand A and Stand B respectively; Lane P2 and Q2 correspond to conjugates bearing 5 strands of intact Strand A and B respectively; Lane N corresponds to mixed conjugates bearing different strands of Strand A and Lane O correspond to the conjugate bearing only 5-base ssDNA without Strand A or Strand B. 79
Figure 6.1 DNA sequences used in this chapter. For strand A' and revA', the underlined sequences are the recognition site of EcoR V and the arrows indicate the enzyme cleavage site. For the mismatched DNA sequences, the underlined base is the mismatch. SM1/SM2/SM3 refer to single-base mismatched DNA, DM refers to double-base mismatched DNA, and NC refers to non-complementary DNA. 87
Figure 6.2 Schematic picture of the formation of nanoparticle-DNA conjugate groupings. 89
Figure 6.3 PAGE characterization of enzyme digested 9.7 nm nAu-bound DNA. Lanes 1-8 correspond to: 10bp DNA ladder, strand A' (free), strand A' (bound), strand A' (no enzyme), strand revA' (free), strand revA' (bound), strand revA' (no enzyme) and 10bp DNA ladder. 91
XVI
Figure 6.4 Formation of nAu-DNA conjugate dimers with various length of DNA linker. Lane A corresponds to nAu without strand A or revA modification (control); Lanes B corresponds to nAu-A + nAu-revA conjugates with no linker; Lanes C, D, E and F correspond to nAu-A + nAu-revA conjugates with 26, 24, 22, and 20 bases of linker DNA respectively. 92
Figure 6.5 Grouping percentage of nAu-DNA conjugates with various ratios of target DNA. Lanes G-O correspond to nAu-A: nAu-revA: target DNA ratio of 1:1:1, 1:1:0.8, 1:1:0.5, control (nAu without strand A/revA conjugation), 1:1:0, 1:1:0.3, 1:1:0.25, 1:1:0.15, and 1:1:0.1 respectively. Gel picture shows the combined results from two experiments. 94
Figure 6.6 Grouping percentage of nAu-DNA conjugates with different ratios of target DNA. 95
Figure 6.7 Grouping percentage of nAu-DNA conjugates with different ratios of target DNA. Lanes P-T correspond to nAu-A & nAu-revA with target DNA at a ratio of 2:1:2 (nAu-A: nAu-2x revA: target DNA), 2:1:5, control (nAu without strand A/revA conjugation), 2:1:20, and 2:1:100, respectively. Gel picture shows the combined results from two experiments. 96
Figure 6.8 Hybridization efficiency of pure DNA (strand A and strand revA with different ratios of target DNA). Lanes 9-13 and 6 correspond to strand A & strand revA with target DNA at a ratio of 1:1:0.2, 1:1:0.4, 1:1:0.6, 1:1:0.8, 1:1:1 and 10bp DNA ladder respectively. 98
Figure 6.9 TEM images of nAu-DNA conjugate dimers. 100
Figure 6.10 SNP discrimination using nAu-DNA conjugates. Lanes U-AA correspond to, nAu-A & nAu-revA plus 24-base perfectly matched DNA (PM), single base matched DNA (SM1/SM2/SM3), double base matched DNA (DM), non-complementary DNA (NC) linker and no target DNA, respectively. 102
Figure 6.11 Melting transition of matched and mismatched DNA samples. Square (brown): PM DNA, Plain curve (blue): SM3 DNA, Triangle (Black): SM2 DNA, Cross (red): SM1 DNA, Diamond (green): DM DNA. 104
Figure 7.1 DNA sequences used in this chapter. For strand A', revA', C', revC1' and revC2', the underlined sequences are the recognition site of EcoR V and the arrows indicate the enzyme cleavage site. 111
Figure 7.2 Schematic picture of the formation of nAu-DNA conjugate groupings using either the pair nAu-A + nAu-revA or nAu-C + nAu-revC1 with linker DNA. 116
XVII
Figure 7.3 Conjugate grouping of nAu-DNA conjugates with various length of DNA linker. Lane A corresponds to conjugate without DNA modification, and lanes B, C, D, and E correspond to conjugates nAu-A + nAu-revA with no linker, with 26-, 24-, and 22-base linker DNA respectively. The stoichiometric ratio of nAu-A, nAu-revA, and linker DNA is 1:1:1. 116
Figure 7.4 Schematic picture of the direct pairing of nAu-DNA conjugate using nAu-C + nAu-revC2 without linker DNA. 118
Figure 7.5 Conjugate grouping of nAu-DNA conjugates with various length of DNA linker. Lanes F, H and J corresponds to conjugate without DNA modification and lanes G, I and K correspond to conjugates nAu-A & nAu-revA with 26-base, 48b+22bp and nAu-C & nAu-revC1 with 60-base linker DNA, respectively. Gel pictures shown are combined results from different sets of experiments. 119
Figure 7.6 Conjugate groupings formation via direct linkage of two nAu-bound DNA. Lanes L, M, N and O correspond to nAu-C, nAu-C + nAu-revC2, nAu-C + nAu-2XrevC2 and nAu-C + nAu-3XrevC2, respectively. 119
Figure 7.7 Study on the grouping percentage of nAu-DNA conjugates under various hybridization time. 121
Figure 7.8 Study on the effect of ion strength of hybridization buffer on the nAu-DNA conjugates grouping percentage. Lane P is corresponding to conjugate without DNA modification and lanes Q-T correspond to hybridization buffer with zero, 2, 10, and 30 mM MgCl2. 122
Figure 7.9 TEM images of nAu-DNA conjugate groupings. 125
Figure 9.1 Fabrication of nAu-DNA conjugates bearing specific number of short DNA with multiple sequences. 135
Figure A1 Complete sequences of Strand A’ and Strand revA’. 155
Figure A2 Complete sequences of Strand C’, Strand revC1’ and Strand revC2’. 156
Chapter 1_____________________________________________________________
1
Chapter 1
Introduction
1.1 Background
The integration of nanotechnology with biology has led to the development of a new
research discipline, nanobiotechnology, where it incorporates the unique optic,
electronic and catalytic features of nanomaterials with the highly specific recognition
and catalytic properties of biomolecules and has led to numerous of fascinating
applications1, 2. As an important characteristic found in many nanobiotechnology
disciplines, the conjugation of nanoparticles (metallic or semiconductive) with
biomolecules for applications in ultra-sensitive bioanalysis and fabrication of
organized nanoassemblies have received particular emphasis in recent years3.
Metallic and semiconductive nanoparticles are generally defined as isolable particles
between 1 and 50 nm in diameter and are prevented from agglomeration by attaching
stabilizers to the particle surface4. Due to the unique chemical and physical properties
and different methods available for preparing nanoparticles with controlled size and
shape, nanoparticles have been attracting considerable attention especially as building
blocks for the assembly of nanoscale structures and devices5, 6. Numerous potential
applications involving these nanoparticles are quantum computers7 and devices8, 9,
Chapter 1_____________________________________________________________
2
industrial lithography10, nanoelectronic & nanowire11, 12, precursors for new types of
catalysts13-16, and biological related applications17-19.
Biomolecules possess several unique fundamental features that make them very
attractive for the construction of nanoassemblies1, 20. First, biomolecules have highly
specific molecular recognition capabilities, such as the recognition between
complementary DNA, antigen-antibody and ligand-receptor. For nanoparticle
assembly directed via biomolecules, this recognition ability enables the accurate
arrangement of nanoparticles in a parallel process, rather than assembling them in a
sequential way. Second, there are a variety of enzymes available which can be used as
catalytic tools for the manipulation of biomolecules. For example, the hydrolysis of
protein and the endonuclease scission/ligase ligation of DNA molecules provide
efficient tools for controlling the structure of nanoparticle-biomolecule hybrid
materials.
Among the commonly used biomolecules, DNA is one of the most promising one due
to several key advantages21, 22. First, complementary single stranded DNA can bind
with each other based on Watson–Crick base pairing pattern. This lock-and-key
pattern shows very high selectivity and specificity17. Second, DNA of any desired
sequences can be conveniently and reliably synthesized by solid support synthesis and
with various modifications, such as attachment modifications with biotin or thiol and
fluorescent labels. Third, DNA can be manipulated by a variety of enzymes with
atomic level accuracy. Such enzymes include restriction endonucleases, ligases and
Chapter 1_____________________________________________________________
3
polymerase. Fourth, DNA has high physicochemical stability and mechanical rigidity
and can be used as efficient linker or spacer molecules for guiding the nanoparticle
assembly.
Among the nanoparticle-biomolecule based hybrid materials, gold nanoparticle-DNA
conjugates are of particular interest19, 23. Gold nanoparticles can be readily synthesized
from commercially available starting materials, such as hydrogen tetrachloroaurate,
sodium citrate and tannic acid. The sizes of the resulting nanoparticles can be well
controlled by simply adjusting the stoichiometric ratio of the reagents. Gold
nanoparticles have strong UV absorption in the visible range (around 520 nm) which
make them particularly suitable as labeling tags in colorimetric assay. There are two
major applications of gold nanoparticles-DNA conjugates. First, they can be used as
building blocks for preparing nano, meso and macroscopic architectures with well-
defined structures2, 17, 24-29. Second, the DNA functionalized nanoparticles and
sequence-specific hybridization reactions can be used for ultra-sensitive and highly
specific biological & biomedical detections30, 31. Some of these strategies have already
been used in generating novel nanostructure materials32-35, in templating the growth of
nanocircuitry11, 12, and in developing DNA sequence detection36, 37.
1.2 Aims and scope of this project
This Ph.D. work aims to fabricate gold nanoparticles bearing definite number and
length of DNA strands. The scope of this work includes studying the enzyme
manipulation efficiency of gold nanoparticle-bound DNA, preparing well defined
Chapter 1_____________________________________________________________
4
nanoparticle-DNA conjugates. These specially designed nanoparticles are then applied
for quantitative DNA detection and construction of well tailored nano-groupings.
The specific objectives of this thesis include:
1. To investigate the feasibility of enzymatic manipulation of the gold
nanoparticle-bound DNA. A systematic study on various factors that affect the
enzyme manipulation efficiency of nanoparticle-bound DNA is conducted,
including (i) stabilizer surface coverage on nanoparticle, (ii) distance between
nanoparticle surface and enzyme-cutting site of particle-bound DNA, and (iii)
size of nanoparticles.
2. To fabricate gold nanoparticles bearing definite number of DNA strands with
predetermined length. Nanoparticle-DNA conjugates bearing definite number
of long double-stranded DNA strands are prepared by gel electrophoresis. A
restriction endonuclease enzyme is then used to manipulate the length of the
nanoparticle-bound DNA.
3. To conduct quantitative DNA detection and SNP discrimination study using
the well defined gold nanoparticle-DNA conjugates fabricated in the previous
experiments. Establish a reliable correlation between the amount of target
DNA and conjugate groupings formed and study the selectivity of SNP
discrimination using single base mismatched DNA.
Chapter 1_____________________________________________________________
5
4. To construct DNA induced gold nanoparticle nano-groupings with precisely
controlled structures (dimers, trimers and other higher order multimers) using
the well defined nanoparticle-DNA conjugates. Various kinds of factors that
affect the formation of nanoparticle assemblies are explored.
Chapter 2_____________________________________________________________
6
Chapter 2
Literature review
Definitions:
Nanoparticle: “Nanoparticulate metal colloids are generally defined as isolable
particles between 1 and 50 nm in size that are prevented from agglomerating by
protecting shells.”4
2.1 Synthesis, stabilization and characterization of metallic
nanoparticles
2.1.1 Reaction mechanism and kinetics
The formation of neutral metallic atoms, the element of nanoparticles, is the result of
redox reaction in which electrons from a reducing agent are transferred to the oxidized
metallic ions, according to the following chemical reaction equation38:
m Men+ + n Red →m Me0 + n Ox (1)
where Me=Metal; Red=Reducing agent; Ox=Oxidizing agent.
The driving force of this reaction is the difference of redox potential between the two
half-cell reactions, ∆E=EMe-ERed. Reduction is thermodynamically feasible only when
Chapter 2_____________________________________________________________
7
∆E>0. Strongly electropositive metals, e.g., Au, Pt, and Ag (E>0.7V), can readily
react with mild reducing agent38.
2.1.2 Mechanism of particle formation
Metallic atoms generated by the reduction reaction are essentially insoluble in the
solution so they gradually aggregate into clusters, which are named embryos. As the
metal atoms further condense on the embryos, they reach a critical size and separate
from the solution as solid particles, named nuclei. The nuclei will further grow to
larger particles (from sbumicrometers to micrometers) either by continuous atom
addition or by particle aggregation. In order to produce nanosized metal particles, the
particle growth must be stopped in the early stages of particle formation. This
objective can be achieved by using electrostatic or steric stabilization39.
2.1.3 Synthesis of metallic nanoparticles
Metallic nanoparticles can be synthesized from metallic materials, such as gold39-49,
silver50, 51 or platinum52, 53. There are five general methods for transition metal
nanoparticles synthesis: 1. Chemical reduction of transition metal salts, 2. Thermal
decomposition and photochemical methods, 3. Ligand reduction and displacement
from organometallics, 4. Metal vapor synthesis, 5. Electrochemical synthesis54.
Faraday55 first published the transition metal salt reduction method in 1857. This
method involved using chemical reduction of transition metal salts in the presence of
stabilizing reagents to generate zerovalent metal colloids in aqueous or organic
Chapter 2_____________________________________________________________
8
solution. Because of its simplicity, reliability and narrow particle size distribution, this
method has become and still is one of the most common and powerful approaches in
this field54, 56, 57.
The most prominent material for metallic nanoparticles is certainly gold, which can be
synthesized with high quality in organic39, 41, 42 as well as in aqueous solution40, 46-48, 58.
For biological applications, gold nanoparticle (nAu) needs to be soluble in aqueous
environment, which precludes many of the protocols carried out in organic solution.
Handley40 reported a simple synthetic method for size controlled nAu preparation
using hydrogen tetrachloroaurate as the precursor and sodium citrate and tannic acid
as reducing agents. The reaction reagents are simply combined in a reaction flask
under proper temperature, and by adjusting the stoichiometric ratio of the reagents, the
sizes of the resulting nAu could be controlled. Though sodium borohydride is also
commonly used as reducing reagent, the disadvantage is that transition metal borides
are often found alongside the nAu59, 60.
2.1.4 Stabilization of nanoparticles
Metallic nanoparticles are only kinetically stable. They have a tendency towards
aggregation and finally form bulk material, which is thermodynamically more stable54.
Therefore, stabilizers are needed to prevent particle aggregation. There are two general
approaches to accomplish this purpose, electrostatic charge stabilization and steric
hindrance stabilization4.
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9
Electrostatic stabilization is based on the adsorption of ions (e.g. sodium citrate) on the
surface of nanoparticles. As shown in Figure 2.1, the absorbed ions as well as the
counterions form an electrical double layer around the particle surface, which create
coulombic repulsion force between particles4. This kind of stabilizers includes various
ligands (sodium citrate, 4, 4’-(phenylphosphinidene) bis-(benzenesulfonic acid))17, 61-67
as well as thiol derivatives (4-mercaptoberzoic acid, mercaptoethanesulfonate,
mercaptopropionate, mercaptosuccinic acid, 4-hydroxythiophenol and tiopronin). Both
of these stabilizers can ionize in solution49, 68-82. Electrostatic stabilized nanoparticles
can last for months under proper temperature and concentration. However,
electrostatic stabilization cannot generate nanoparticles in dry form, nor can it stabilize
the nanoparticles in high salt/electrolyte concentration. The nanoparticles aggregate
and the color of the solution changes from red to black as soon as a significant amount
of electrolyte is introduced to the system17. The addition of electrolyte to the
electrostatic stabilized nanoparticle solution increases the charge screening effect,
which destroys the electrical double layer around the nanoparticles. The decrease in
interparticle distance (i.e. particle aggregation) leads to a red shift in the particle
plasmon band, which can be detected easily by color change of solution18.
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10
Figure 2.1 Electrostatic stabilization of nanoparticles4
Steric hindrance stabilization can generate more stable metallic nanoparticles. This is
achieved by surrounding the particle with a protective shield made of sterically bulky
materials4. The protective shield forms a steric barrier, which prevents close contact of
the metal particles (Figure 2.2). Materials commonly used as protective shields include
polymers56, 83-88, such as poly (vinylpyrrolidone) (PVP), poly(vinyl alcohol) (PVA),
poly(methylvinylether) and polyethylene glycol (PEG), as well as silanes89-99 and long
chain thiol derivatives100-107.
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11
Figure 2.2 Steric hindrance stabilization of nanoparticles4
For nAu, besides the stabilizers mentioned above, DNA molecules have been used as
another effective stabilizer. Storhoff et al.108 found that nAu modified with DNA
(from 5 to 20 bases) show exceptional stability in electrolytic media. The
nanoparticles are stable in electrolyte solutions with concentration as high as 1M NaCl.
The stability of the nanoparticles is determined by the chain length, composition and
surface coverage of DNA. Higher stability can be reached using DNA with longer
chain length, richer of thymidine (dT) in composition and higher surface coverage.
Since DNA are highly charged and with high molecular weight, the enhanced stability
observed in this case should be attributed to the increase in both surface charge and
steric hindrance.
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12
2.1.5 Characterization of nanoparticles
Common techniques for characterization of nanoparticles include transmission
electron microscopy (TEM), UV–Visible spectroscopy (UV–Vis), nuclear magnetic
resonance spectroscopy (NMR), infrared spectroscopy (IR), energy dispersive
spectroscopy (EDS), scanning tunneling microscopy (STM), and atomic force
microscopy (AFM). TEM and UV–Visible spectroscopy are most often used for
particle size characterization.
Transmission electron microscopy or TEM is the most widely used technique for
nanoparticle characterization. This technique gives direct visual information on the
particle size, shape, dispersity and structure. Though TEM is a useful technique, it has
a few drawbacks109. First of all, no direct information can be gained on how the
nanoparticles exist in solution, since the samples must be dried and vacuumed before
TEM examination. High energy electron beam may induce nanoparticle structural
rearrangement, aggregation, or even decomposition. In addition, only a few particles
can be examined and counted from each TEM micrograph, and thus the size and size
distribution data obtained may not represent the whole sample. Finally, three-
dimensional structural information is difficult to be obtained using two dimensional
micrographs.
UV–Vis spectroscopy is another widely used characterization method for
nanoparticles whose plasmon resonance lies in the visible range. Wilcoxon et al.110, 111
and Chestnoy et al.112 demonstrated a correlation between the size of a semiconductor
Chapter 2_____________________________________________________________
13
nanoparticle and its UV–Vis spectrum. As the particle size decreases, the
characteristic plasmon band (λmax) shifts to shorter wavelength. For nanoparticles with
an absorption band in the visible region, such as nAu, the λmax is dependent not only
on the average size and the shape of the particles, but also how close the particles are
relatively to each other113. Hence, this technique can also be used to determine degree
of particle aggregation. For example, Mirkin et al.114 reported a DNA sequence
detection method based on colorimetric change (from red to blue) induced by the
particle aggregation of DNA-modified nAu solution.
2.2 Formation of gold nanoparticle-DNA conjugates (nAu-
DNA)
2.2.1 Introduction to synthetic DNA
With the progress of whole genome sequencing projects, demand for custom synthetic
DNA has expanded dramatically. Synthetic DNA becomes the fuel that drives the
engine of molecular biology. Today, most molecular biology experiments, including
polymerase chain reaction (PCR), DNA sequencing, site directed mutagenesis, single-
nucleotide polymorphism (SNP) assays and microarray technology115-117, employ
chemically synthesized DNA. In 1996, Alivisatos et al.118 and Mirkin et al.32 first
introduced synthetic DNA to nAu system and expand its application in the area of
biological & biomedical detection as well as nanomaterial assembly.
Chapter 2_____________________________________________________________
14
Methods of synthesizing DNA molecules were first developed more than 30 years ago
and now reach the stage where the DNA of desired sequence can be conveniently
obtained from commercial suppliers. Basically, the strategy of DNA synthesis is the
coupling of a protected nucleotide to the growing end of a DNA chain followed by the
removal of the protecting group. The process is repeated until the desired sequence is
obtained117. The automation of DNA synthesis, the development of versatile
phosphoramidite reagents, and efficient scale-up have expanded the application of
synthetic DNA from fundamental to applied biological research115.
2.2.2 Formation of gold nanoparticle-DNA conjugates (nAu-DNA)
Although DNA can be attached to the surface of nAu by simple adsorption119 or via a
biotin–avidin linkage120, the most commonly used method is through gold-
thiol/disulfide group bonding. Nuzzo et al. first reported the attachment of long chain
ω-substituted dialkyldisulfide molecules on gold substrate121. The strong bonding
between sulfur group and gold surface is in the form of a metal thiolate (~ 44
kcal/mol)21. The introduction of thiolated DNA to the nAu system was first reported
by Alivisatos and Mirkin in 1996 and has been widely adopted18, 63, 64, 114, 118, 122-129.
Since the thiol group has high affinity to gold surfaces, the thiol-modified DNA binds
to the surface of nanoparticles spontaneously. This chemical linkage between DNA
and nAu is much stronger than the nonspecific adsorption63. Since thiol-modified
DNA is already commercially available, one can get any desired sequence easily. The
functionalization of DNA with various chromophores makes the application of nAu-
DNA conjugates more diverse18.
Chapter 2_____________________________________________________________
15
2.3 Applications of nAu in nanoassembly and ultrasensitive
DNA detection
The ability to generate nanoparticle assemblies, in which the relative spatial
arrangement of two or more distinct particles is controlled, would allow for a
systematic investigation of the physical properties of these novel structures18, 130. A
number of methods have been reported for organizing nanoparticles into defined
structures131, 132. Recently, methods using biomolecules to assemble nanoparticles
have appeared in the literature32, 118. Several advantages can be gained from using
biomolecules as linker for construction of nanoassembly: 1) The diversity of
biomolecules allows the selection of linker molecules of predesigned size, shape, and
functionality. 2) The availability of chemical and biological means to modify and
synthesize biomolecules. 3) Enzymes may act as biocatalytic tools for the
manipulation of biomolecules. The hydrolysis of proteins as well as the scission,
ligation or polymerization of DNA can be employed as tools for the assembly of
nanoparticle architectures. Among commonly used biomolecules, DNA is a very
versatile linker and attracts most attention for the controlled assembly of
nanoparticles133 due to its unique molecular recognition property, mechanical rigidity,
and enzyme processibility134.
In 1996, Alivisatos et al.118 and Mirkin et al.32 first reported the DNA induced
sequence specific assembly of nAu into organized structures. Alivisatos demonstrated
the alignment of discrete numbers of nAu into spatially defined structures. In this
work, 1.4 nm nAu functionalized with DNA of defined length and sequence was
Chapter 2_____________________________________________________________
16
aligned on the single stranded DNA (ssDNA) template based on the Watson-Crick
base-pairing between complementary DNA molecules. A “head-to-head” or “head-to-
tail” configuration (Figure 2.3) of this nanoparticle-DNA assembly was confirmed by
transmission electron microscopy (TEM).
In Mirkin’s work, the authors described the sequence-specific assembly of nAu into
macroscopic aggregates using DNA as the linker molecule (Figure 2.4). Due to the
molecular recognition properties and mechanical rigidity of DNA molecules, this
strategy allows precise control over the nanoparticle periodicity, interparticle spacing
and strength of the particle interconnection in the final macroscopic structure.
Figure 2.3 Organization of nAu into spatially defined structure118
Chapter 2_____________________________________________________________
17
The collaborative oscillation of conductive electrons in metal nanoparticles leads to a
surface plasmon resonance/coupling which is very sensitive to the interparticle
distance32, 135. Therefore, the DNA induced nanoparticles aggregation mentioned
above is accompanied by a significant red shift in the UV-Vis spectra, as clearly
shown in Figure 2.5. A distinct red shift from 520 to 600 nm is observed as the
nanoparticles are assembled by the linker DNA into extended structures. This striking
red to blue color change in the solution can be easily identified by naked eyes, and
therefore can be applied as fast and sensitive colorimetric detection of target DNA
molecules. This detection method was further developed to discriminate imperfectly
matched DNA targets36, 37. In addition, it is found that DNA-guided nanoparticle
aggregates exhibits an exceptionally sharp melting transition which is higher than the
Tm of duplex DNA linker alone. This melting transition is much sharper while
nanoparticles are involved compared with pure DNA system (Figure 2.6), and this
leads to a highly selective discrimination of target DNA. Based on the obvious
difference of the melting transition profiles, one can easily distinguish a perfectly
matched target strand from a strand with a single base mismatch, regardless of the
mismatch position on the target DNA. This sharp melting transition mainly originates
from the cooperative dehybridization/melting of multiple duplex DNA linkers between
each pair of nanoparticles.
Chapter 2_____________________________________________________________
18
Figure 2.4 Scheme showing the DNA induced nAu assembly process. The scheme is not meant to imply the formation of a crystalline lattice but rather an aggregate structure19
Figure 2.5 Comparison of UV spectra of nAu functionalized with 5’ thiol 12-base ssDNA before and after treatment with a complementary 24-base ssDNA37.
Chapter 2_____________________________________________________________
19
Figure 2.6 Comparison of the melting transitions for a 30bp dsDNA (squares) and nanoparticles linked with the same 30bp dsDNA (circles). Absorbance changes are measured at 260 nm. The insets are the derivative curves of each set18.
In 2000, this nAu-DNA base detection methodology was combined with chip based
detection platform by Taton et al.136. In this work, nAu labeled DNA and target DNA
were cohybridized to capture DNA modified glass microscope slides and visualized by
a conventional flatbed scanner (Figure 2.7). Labeling DNA targets with nanoparticles
instead of conventional fluorophore probes substantially improves the selectivity of
the assay due to the exceptionally sharp melting transition of nAu-DNA. This permits
the single nucleotide polymorphism (SNP) discrimination of DNA sequences with a
selectivity that is three times higher than that obtained from fluorophore-labeled
targets. In addition, to facilitate the visualization of nanoparticle labels hybridized to
the DNA array surface, a signal amplification step is adopted in which silver ions are
reduced by hydroquinone to silver metal at the nAu surfaces. Coupled with this signal
Chapter 2_____________________________________________________________
20
amplification step, the sensitivity of this scanometric array detection system can be as
low as 50 fM which is 100 times greater than that of the analogous fluorophore system.
Figure 2.7 Schematic showing of the scanometric DNA array detection with nanoparticle probes136.
nAu has been used as an effective substitute for conventional organic quenchers in the
development of new nanoparticle-based biosensors such as molecular beacons that are
commonly used for DNA detection. The molecular beacons contain DNA molecules
that are functionalized with a fluorescent dye and a quencher molecule at specific
positions. Due to the fluorescence resonance energy transfer (FRET) effect,
fluorescent emission of the dye can be quenched by the quencher when they are in
close proximity. The main drawback of conventional molecular beacons is the low
Chapter 2_____________________________________________________________
21
quenching efficiency of organic quencher which impairs the detection sensitivity137 138.
Dubertret et al.139 reported using nAu as a nano-quencher that can quench the
fluorescent emission of a dye molecule effectively (up to 99.97% under favorable
conditions) in a distance dependent manner. A 25-base hairpin ssDNA with a
fluorescent dye at the 3’ end was covalently attached to a 1.4 nm nAu through thiol
linkage at the 5’ end (Figure 2.8A). The hairpin DNA can adopt two conformations: a
hairpin structure with the dye and nAu held in close proximity (close state), and a rod-
like structure with them far apart (open state). The close state is self-assembled by the
intramolecular complementarity of the hairpin DNA within the same DNA molecule.
However, the conformation of nAu-DNA conjugate switches to open state upon the
introduction of a complementary target DNA which hybridizes with the nAu-bound
DNA. The formation of dsDNA opens the hairpin and increases the distance between
the dye molecule and nAu, therefore the fluorescence emission is regained (Figure
2.8B). This system was successfully applied for the detection of single-base mismatch
in DNA sequences with a 100 times enhancement in detection sensitivity and 8 times
improvement in discrimination of single base mismatch compared with conventional
molecular beacons.
Chapter 2_____________________________________________________________
22
Figure 2.8A The structure of nAu-DNA-dye conjugate molecular beacon139.
Figure 2.8B Schematic drawings of the two conformations of the nAu-DNA-dye conjugate molecular beacon. On the left, the hairpin structure brings the dye and the nAu in close proximity (within a few angstroms) and the fluorescent of the dye molecule is quenched. Through sequence-specific hybridization to a target DNA, the hairpin structure changes to a rod-like dsDNA structure (on the right), which separates the dye and the quencher far apart and thus restores the fluorescence139.
Chapter 2_____________________________________________________________
23
In a continuous study, Maxwell and co-workers140 further developed this nAu based
molecular beacon by exploiting the flexibility of DNA chain which does not require
the DNA hairpin structure to achieve conformation change-induced fluorescent
quenching and restoring. The authors showed that nAu and ssDNA with dye
modification at one end and thiol at the other can spontaneously assemble into an
arch-like conformation on the nAu surface where the thiol covalently binds to nAu and
the dye molecule nonspecifically adsorbs on the nAu surface (Figure 2.9). The
fluorescent emission from dye molecule is completely quenched by nAu due to the
close vicinity of the two. Hybridization of the nAu-bound ssDNA with the
complementary target DNA analyte results in a rigidified dsDNA between the dye and
nAu, and thus frees the dye molecule from the nAu surface to restore fluorescence.
The fluorescence signal change induced by this structure change is found to be highly
sensitive and specific to the sequence of target DNA.
Chapter 2_____________________________________________________________
24
Figure 2.9 nAu-based molecular beacon and its operating process. DNA molecules self-assemble into an arch conformation on the nAu (2.5 nm diameter). Single-stranded DNA is represented by a single line and double stranded DNA by a cross-linked double line. In the assembled (closed) state, the dye is quenched by the nAu. Upon target binding, the constrained conformation opens, the dye leaves the surface because of the structural rigidity of dsDNA, and the fluorescence is restored. Au: nAu; F: dye140.
In a later work, Li et al.141 reported another type of nAu based molecular beacon that
relies on the affinity difference between ssDNA and dsDNA to nAu for achieving
fluorescence quenching and restoring. The authors showed that dye labeled ssDNA
tends to adsorb on negatively charged nAu surface and results in efficient quenching.
While the introduction of target DNA analyte and formation of dsDNA leads to the
release of DNA from nAu and regain of fluorescent signal. This assay was developed
to be finished within 10 min and has a sensitivity of less than 0.1 fM of target DNA.
Chapter 2_____________________________________________________________
25
2.4 Study on the plasmon coupling of metallic nanoparticle
dimers
The plasmon resonance/coupling of metallic nanoparticles can be strongly influenced
by the size and shape of the nanoparticles as well as surrounding media, such as the
presence of nearby nanoparticles142. Besides large scale aggregation of nanoparticles,
the coupling of two nanoparticles in close vicinity can also induce shifting of the
plasmon resonance wavelength in a distance-dependent manner143-147. Considerable
effort has been directed toward investigations on the effect of nanoparticle placement
on the resulting plasmon coupling for potential applications such as molecular ruler145,
146.
Sonnichsen et al.145 demonstrated that the distance dependent property of plasmon
coupling can be used to monitor distances between single pair of gold or silver
nanoparticles using particles bearing 33-base ssDNA with biotin to bind with
streptavidin-coated particles immobilized on surface. The biotin-streptavidin linkage
brings two particles in close proximity and results in a significant red-shift of resonant
wavelength peak compared with that of individual nanoparticles (Figure 2.10).
Furthermore, a clear blue-shift was observed upon the addition of complementary
DNA and formation of dsDNA spacer between the two nanoparticles. As dsDNA is
much stiffer than ssDNA, the nanoparticles are pushed apart. This “plasmon ruler”
offers exceptional photostability and brightness compared with conventional
fluorophore and allows continuously monitoring of separations of up to 70 nm for
>3000 s. Therefore, this plasmon ruler has the potential to become an alternative to
Chapter 2_____________________________________________________________
26
Forster Resonance Energy Transfer (FRET) for in vitro single-molecule study,
especially for applications demanding long observation time or large distance.
Figure 2.10 Representative scattering spectra of single particles and particle pairs for silver (top) and gold (bottom). Silver particles show a larger spectral shift (102 nm) than gold particles (23 nm), stronger light scattering and a smaller plasmon line width. Gold, however, is chemically more stable and is more easily conjugated to biomolecules via –SH, –NH2 or –CN functional groups145.
In a later study, Reinhard et al.146 established a correlation of the plasmon resonance
wavelength versus interparticle distance and applied it as “plasmon rulers” to study the
dynamics of DNA hybridization on the single-molecule level. The authors conducted a
Chapter 2_____________________________________________________________
27
detailed experimental and theoretical study on the distance dependence of the plasmon
resonance using DNA linked nAu dimers as the model system. Nanoparticle dimers
with various interparticle spacing were assembled using dsDNA of 10, 20, 40, 67, 110,
and 250 base pairs as the spacer material. It was found that the shifting of plasmon
resonance wavelength is a function of nanoparticle separation and decays
exponentially with increasing interparticle distance. The shift drops to zero when the
spacing reaches about 2.5 times of the diameter of nanoparticle.
2.5 Gel electrophoresis study on nanoparticle-DNA
conjugates
2.5.1 Electrophoretic isolation of discrete nAu-DNA conjugates
Precise control over the number of DNA strands attached on each nanoparticle is
essential for diagnostics whenever there is a need to quantify the degree of
hybridization events17. Unfortunately, the number of DNA strands bound to each
nanoparticle cannot be directly controlled yet. One can only control the average
number of DNA strands per nanoparticle by adjusting the stoichiometric ratio of DNA
to nanoparticle in the incubation solution. However, the stoichiometric mixing always
yields a population of nanoparticles bound with different number of DNA strands63.
Zanchet et al.129, 148 used agarose gel electrophoresis to successfully isolate nAu bound
with different number of DNA strands (Figure 2.11). Agarose gel is basically a porous
polymer matrix, where the average pore size decreases with the increasing of agarose
Chapter 2_____________________________________________________________
28
concentration62. In the case of nAu-DNA conjugates, agarose gel is the most
appropriate medium for separation because of its characteristic pore size (>50 nm) and
chemical compatibility66, 149-153. In electrophoresis, charged particles migrate in the gel
under an electric field and the particle mobility depends on their charge and size.
When nAu are coated and stabilized with negatively charged stabilizers, they can be
highly negatively charged to the extent that subsequent conjugation with single
stranded DNA (ssDNA) yields negligible increase of overall charge. This makes the
size (volume) increase the dominant reason why the conjugation with DNA makes
nAu show a slower electrophoretic mobility. During the electrophoresis of nAu-DNA
conjugates, discrete bands, which correspond to the conjugates bearing different
strands of DNA, appear in the gel. The number of bands appeared in the gel as well as
the relative intensity of the bands can be controlled by changing the overall
stoichiometry of DNA and nAu. Electrophoresis of nanoparticles modified with
ssDNA of different length (from 50 bases to 100 bases) shows that conjugates bearing
shorter DNA strands migrate faster in the gel. This result further demonstrates that the
particle size, instead of particle charge, controls the mobility of the conjugates. The
authors also noted that it is difficult to isolate discrete bands for DNA sizes below 50
bases since the mobility difference is insignificant. Increasing the gel concentration
usually results in better band resolution, but in this case the separation does not
improve significantly due to the spreading of the bands. Furthermore, increasing the
running time dose not yield better separation as well. TEM characterization of the
conjugates from different bands shows insignificant particle grouping, indicating that
Chapter 2_____________________________________________________________
29
the discrete bands do not correspond to the grouping of dimers, trimers or other higher
order multimers.
Figure 2.11 Electrophoretic mobility of 5 nm Au/100b HS-ssDNA conjugates (3% gel). The first lane (left to the right) corresponds to 5 nm particles (single band). When 1 equiv of DNA is added to the nAu (second lane), discrete bands appear (namely 0, 1, 2, 3, ...). When the DNA amount is doubled (third lane), the intensity of the discrete bands change and additional retarded bands appear (4, 5). Because of the discrete character, each band can be directly assigned to a unique number of DNA strands per particle154.
2.5.2 Conformation study of nanoparticle-bound DNA
The conformation and packing of DNA after conjugating with nanoparticles strongly
influence the accessibility of DNA for hybridization and enzyme manipulation. Gel
electrophoresis became an excellent tool for studying the conformation and packing of
Chapter 2_____________________________________________________________
30
nanoparticle bound-DNA in recent years66. Parak et al.66, 108 were the first to propose
three possible conformations for nanoparticle bound-DNA. DNA can be wrapped
around the nanoparticle, in random coiled shape, or in stretched shape pointing
perpendicular to the surface (Figure 2.12). Agarose gel electrophoresis study showed
that the configuration of the nanoparticle-bound DNA is decided by its length and
composition152. For 10 nm diameter nAu saturated with ssDNA, the inner part of the
DNA (approximately 30 bases) is fully stretched, whereas the outer ones adopt a
random coil configuration. The highest binding affinity of single nucleotides to nAu is
cytosine (C), followed by guanine (G), adenine (A), and the least thymine (T). Hence,
DNA with higher GC content has strong tendency to wrap the nanoparticle and makes
the hybridization of DNA difficult.
Figure 2.12 Different possible configurations of DNA molecules attached to the surface of nAu66.
The conformation of nanoparticle bound-DNA was further studied by Sandstrom et
al.155 The authors performed a series of agarose gel electrophoresis experiments of
nAu modified with either thiol-DNA or non-thiol-DNA of different lengths. It was
found that thiol-DNA modified nanoparticles have a thicker DNA layer since the
Chapter 2_____________________________________________________________
31
DNA molecules are only attached to the nanoparticle in one end and can stand up from
the surface more compared with the non-thiol DNA modified ones, where the DNA
molecules tend to lay down on the nanoparticle surface. However, for the thiol-DNA
modified nanoparticles, the thickness of the DNA layer is still smaller than the length
of a corresponding fully stretched DNA, suggesting the nanoparticle bound-DNA still
adopts a random coil shape and is not fully stretched perpendicular to the particle
surface.
2.6 Formation of discrete nanoparticle-DNA conjugate
groupings
In the continued work by Zanchet et al.62, gel electrophoresis isolated nAu-DNA
conjugates were used to form nanoparticle groupings of defined structure, such as
dimers and trimers. TEM characterization of the extracted gel bands shows that the
majority of nanoparticles from each discrete band participate in a single type of
grouping structure (dimer, trimer, tetramer etc.), suggesting that each discrete band in
the gel corresponds to a specific type of structure. The main advantages of this
approach are that the obtained structure is highly predictable and can be reproduced
accurately and the yield of target structure is significantly improved as well. In a later
study, Fu et al.156 described the synthesis of precise groupings of CdSe/ZnS core/shell
semiconductor quantum dots (QDs) with nAu and demonstrated that gel
electrophoresis purification can be applied for the isolation of a variety of nanoparticle
assemblies. The QD-nAu groupings are fabricated by hybridization of complementary
Chapter 2_____________________________________________________________
32
DNA bound on QD and nAu. The nanostructures obtained have one QD in the center
and a discrete number of nAu attached to it. QDs are useful biological labels because
of their broad excitation spectra and narrow and size tunable emission spectra, as well
as their photostability. By putting nAu around QD using DNA as the linker, both the
distance between nAu and QD as well as the number of nAu around the central QD
can be controlled precisely.
2.7 Enzyme manipulation of the nanoparticle-bound DNA
2.7.1 Introduction to restriction endonuclease
Restriction endonucleases are enzymes which recognize short DNA sequences and
cleave the DNA in both strands157. They are very important research tools in
molecular biology, and to date, over 3000 restriction endonucleases have been
discovered and hundreds of them are commercially available116. Different restriction
endonucleases have different cutting patterns. Some cleave both strands immediately
opposite one another, generating fragments of DNA that carry blunt ends, while others
make slightly staggered cleavage, resulting in fragments of DNA that carry single
stranded termini (cohesive ends or sticky ends, Figure 2.13)114, 128, 150, 158-169,
Chapter 2_____________________________________________________________
33
↓ 5’……G A T A T C……3’ EcoR V 5’……G A T A T C……3’ ∣∣∣∣∣∣ ∣∣∣ + ∣∣∣ 3’……C T A T A G……5’ 3’ ……C T A T A G……5’
↑ Blunt ends ↓
5’……G A A T T C……3’ EcoR I 5’…… G A A T T C……3’ ∣∣∣∣∣∣ ∣ + ∣
3’……C T T A A G……5’ 3’ ……C T T A A G……5’ ↑ Cohesive or sticky ends
Figure 2.13 Patterns of DNA cutting by restriction endonucleases156.
2.7.2 Effect of steric hindrance on DNA hybridization and enzymatic
reaction efficiency
The retention of the biological function of DNA after bound with solid structure, such
as nAu or gold film, has been studied by many groups119, 170-173. Studies on DNA
interactions with nAu have demonstrated that the biological function of DNA is only
partially maintained150.
The hybridization and enzymatic reaction of nanoparticle-bound DNA is to some
extend impeded by steric hindrance. Nicewarner et al.150 conducted a thorough
investigation on this topic with 12-nm diameter nAu (Figure 2.14). In this study, DNA
primers were grafted onto nAu via alkanethiol linkers of different length (from
C6H12SH to C12H24SH). The author found that the steric hindrance from the
neighboring nAu-bound DNA and nAu surface play a key role in achieving high
hybridization and enzymatic extension efficiency (polymerase chain reaction using
Chapter 2_____________________________________________________________
34
enzyme DNA polymerase). Lower surface density of nAu-bound DNA and longer
linker lead to less steric hindrance and obviously improved hybridization and
enzymatic extension efficiency. These results are expected to be applicable for most of
the common enzymatic reactions on nanoparticle-bound DNA.
Figure 2.14 Hybridization and extension of nAu-bound DNA. In step 1, the nAu-bound DNA is annealed to the template strand followed by extension in step 2 accomplished by the addition of DNA polymerase150.
2.7.3 Enzyme manipulation of nanoparticle bound DNA
He et al.174 first reported to use double-stranded DNA to connect nAu to a gold film
(Figure 2.15). The nanoparticles can then be freed from the gold film by cleaving the
double stranded DNA (dsDNA) using a restriction endonuclease (Hinf I). In addition,
the authors observed that the cleavage of surface bound DNA is not complete. Using
atomic force microscope (AFM) O’Brien et al.167 detected a change in the surface
topography resulting from the enzymatic cleavage (EcoR V or Hae III) of the dsDNA
Chapter 2_____________________________________________________________
35
attached to a gold film. Yun and coworkers158 demonstrated the absence of
conformational changes of nanoparticle bound DNA by studying the enzyme
manipulation of the nAu-DNA conjugate dimer. DNA methyltransferases (M.EcoRI)
and restriction endonuclease (R.EcoRI) are known to bind and produce specific
conformational changes in DNA. M.EcoRI recognizes the GAATTC sequence and
methylates the second adenine by bending the DNA approximately 55-59° and
flipping the target adenine out of the DNA duplex (Figure 2.16 A, B). The
endonuclease R.EcoRI cleaves the double stranded DNA at the same recognition site
as M.EcoRI (Figure 2.16 C). When this happens, it results in the separation of the
paired nAu which was confirmed by TEM characterization.
Figure 2.15 Enzyme digestion of DNA immobilized on gold surface174.
Chapter 2_____________________________________________________________
36
A
B
C
Figure 2.16 (A) Double stranded nanoparticle-DNA conjugates with the EcoR I recognition site, (B) The conjugates with a 59 ° bending due to the binding of M.EcoRI enzyme, (C) The conjugates after the cutting of the DNA at the R.EcoRI recognition site158.
Chapter 2_____________________________________________________________
37
No detailed experimental methodology was reported in the literature until Kanaras et
al.175 reported an enzymatic manipulation of nanoparticle-DNA conjugates. 150
strands of 45 bp thiol modified dsDNA were loaded onto 15 nm nAu. This high
loading of DNA on nAu (approximately twice the amount of what previously reported
in literature18) was achieved by gradually increasing the ionic strength of the
incubation buffer during the DNA attaching step. This kind of treatment leads to
exceptionally stable nanoparticle-DNA conjugates, and they do not aggregate in buffer
with very high ionic strength that is required by enzyme digestion reaction. Restriction
enzyme EcoRI was used to cleave the dsDNA on the nanoparticles at a specific
recognition site and to leave a cohesive end (Figure 2.17 A), which was subsequently
covalent-joined together by a T4 DNA ligase (Figures 2.17 B and C) and resulted in
particle aggregation. This process can be monitored by TEM or more easily by a
colorimetric method developed by Mirkin and co-workers. Based on the obvious color
change from red to blue upon particle aggregation, this method is extremely sensitive
in distinguishing aggregated nAu from non-aggregated ones. After EcoRI digestion,
the cohesive ends generated by the same restriction enzyme can be associated together
by complementary base pairing leading to loosely aggregated nanoparticles and result
in a blue solution. Such weak connection between two cohesive ends dissociates at
slightly elevated temperature (45˚C), yielding the original red solution. However, the
covalent joining of DNA at the cohesive end by T4 DNA ligase generates a much
stronger linkage that does not dissociate up to 85˚C.
Chapter 2_____________________________________________________________
38
(A)
(B)
(C)
Figure 2.17 (A) Nanoparticles derivatized with double-stranded DNA are treated with restriction enzyme EcoR I, which cleaves the DNA to yield cohesive ends. The part between the arrowheads represents the recognition site of the enzyme; (B) Two cohesive ends hybridize, which leads to a weak association of particles; (C) The DNA backbones are covalently joined at the hybridized site by DNA ligase to yield a stable 40-base-pair double-stranded link between particles175.
In a later study176, further investigation on the enzyme digestion of nAu-bound DNA
with different restriction enzymes (EcoR I, EcoR V, BamH I, Msp I and Apo I) was
conducted. Though all of the enzymes mentioned above can cleave dsDNA attached
on the nAu surface, 100% cleavage was not observed. In order to get a more
quantitative result of the enzymatic digestion efficiency, fluorescence-labeled DNA
was released from the nanoparticle surface after digestion reaction by ligand exchange
with dithiothreitol (DTT). The released DNA was concentrated with polyacrylamide
gel electrophoresis and the fluorescence measurement yielded approximately 65%
digestion of the nAu-bound dsDNA.
Chapter 3_____________________________________________________________
39
Chapter 3
Synthesis of gold nanoparticles (nAu)
3.1 Materials and methods
Materials
Hydrogen tetrachloroaurate (III) trihydrate, trisodium citrate dihydrate, tannic acid,
were purchased from Sigma-Aldrich. All the reagents were used as received. Milli-Q
water with resistance >18MΩ/cm was used throughout the experiments.
General approach for synthesis of nAu
nAu were synthesized by the reduction of hydrogen tetrachloroaurate (III) tetrahydrate
(HAuCl4 • 4H2O) by trisodium citrate dihydrate (C6H5O7Na3 • 2H2O) and tannic acid40.
Briefly, two initial solutions were prepared: (a) 16 ml of 0.01% (w/v) HAuCl4 solution,
and (b) 4ml of the reducing mixture consisting of 0.2% (w/v) trisodium citrate
dihydrate, 0.005-0.25% (w/v) tannic acid, and 0.0125-0.625 mM K2CO3. Both
solutions were heated to 60°C and solution (b) was rapidly added to HAuCl4 solution
with stirring. After the color of the mixed solution turned to red, the solution was
heated to boiling (110°C) for 10 min with stirring for the reaction to complete. Finally,
the solution was cooled with cold water. The nanogold solution was stored at 4°C for
future usages.
Chapter 3_____________________________________________________________
40
Characterization of nAu
TEM and UV-Vis spectroscopy characterization were conducted for samples
synthesized at different concentration of tannic acid. TEM characterization was
performed on a Philips CM300 FEG system operating at 200 kV. The samples were
prepared by dropping and evaporating the nanoparticle solution onto a carbon coated
copper grid (200-mesh). The characteristic plasmon band (λmax) was measured on a
Shimadzu-1601 UV-Vis spectrophotometer using quartz cuvettes with 10 mm path
length.
3.2 Results and discussion
3.2.1 Synthesis and characterization of nAu
Since electrostatic stabilization of nanoparticles is pH sensitive, the pH of the system
should be maintained at constant during the reaction process. This was achieved by
using K2CO3 to neutralize the acid effect caused by tannic acid. The rate of particle
formation is decided by the concentration of tannic acid (strong reductant) in the
reaction system. With progressively lower tannic acid concentration, the time required
for complete reaction is proportionally increased. In our experiment, a 12 min period
was required with 0.001% tannic acid reaction condition. Figure 3.1 shows the
plasmon absorption spectrum of nAu with 9.7 nm mean diameter dispersed in water.
The characteristic plasmon band (λmax) at 520 nm was observed in all the samples,
confirming the formation of nAu.
Chapter 3_____________________________________________________________
41
Figure 3.1 UV-visible spectrum of an aqueous solution of 9.7 nm nAu.
Nanoparticle samples synthesized by different tannic acid concentration (0.001, 0.01
and 0.05 %) with fixed trisodium citrate concentration (0.04%) were chosen for TEM
characterization (Figures 3.2a, 3.3a and 3.4a). Average particle size was determined
by counting at least 200 particles on the micrographs. The particle size and size
distribution measured from these well-separated particles is shown Figures 3.2b, 3.3b
and 3.4b. The mean particle diameters of the three samples are 9.7 nm, 7.0 nm and 6.3
nm respectively. The particle size distributions of the three samples are all within 15%,
and this is considered monodispersed based on the common practice reported in the
literature.
Chapter 3_____________________________________________________________
42
From the TEM micrographs, the diameter of nAu decreases (from 9.7 nm to 6.3 nm)
with increasing concentration of tannic acid. This can be explained by the fact that the
final diameter of the nAu is determined by the number of core formed (nucleation) in
the beginning of the reaction and subsequent atom condensation on the core (shell
growth). Higher concentration of strong reductant such as tannic acid induces the
formation of larger number of cores, which subsequently consumes more of reaction
reagent in the system, thereby limits the shell growth. Hence, the size of nAu can be
easily controlled by adjusting the stoichiometric ratio of strong reductant (tannic acid)
to weak reductant (sodium citrate).
3.3 Conclusions
In this chapter, we describe the synthesis of nAu of various sizes by reducing
hydrogen tetrachloroaurate (III) tetrahydrate with trisodium citrate dihydrate and
tannic acid. TEM confirms the formation of mono-dispersed nAu and the diameter of
nAu decreases (from 9.7 nm to 6.3 nm) with the increasing concentration of tannic
acid.
Chapter 3_____________________________________________________________
43
Figure 3.2a TEM image of nAu with a mean diameter of 9.7nm.
Figure 3.2b Size distribution of nAu with 0.001% tannic acid concentration condition. Mean diameter = 9.7nm, standard deviation = 0.64nm.
Chapter 3_____________________________________________________________
44
Figure 3.3a TEM image of nAu with a mean diameter of 7.0nm.
Figure 3.3b Size distribution of nAu with 0.01% tannic acid concentration condition. Mean diameter = 7.0nm, standard deviation = 0.56nm.
Chapter 3_____________________________________________________________
45
Figure 3.4a TEM image of nAu with a mean diameter of 6.3nm.
Figure 3.4b Size distribution of nAu with 0.05% tannic acid concentration condition. Mean diameter = 6.3 nm, standard deviation = 0.67 nm.
Chapter 4_____________________________________________________________
46
Chapter 4
Efficient manipulation of nanoparticle-bound DNA
via restriction endonuclease
4.1 Introduction
In recent years, metallic nanoparticles have been attracting considerable attention as
ideal candidates for ultra-sensitive biomolecule detection or nanometer-scale
assembly31, 145, 147, 177-185. Biomolecules, such as DNA, antigen–antibody and enzymes,
are being investigated as linker molecules for guiding the self-assembly of
nanomaterials due to their specific recognition capability and biochemical functions24,
155, 186, 187. The integration of nanoparticles with biomolecules is currently a focused
topic and has led to numerous applications, such as biological and biomedical
detections140, 188-194, biosensors195-197, and nanoparticle-based molecular switches198-200.
As a particularly promising candidate for programmed assembly of nanoparticles,
DNA shows excellent physical and chemical properties and has been used extensively.
Besides their unique sequence-specific binding property and mechanical rigidity,
DNA molecules can be processed by a variety of enzymes, such as endonucleases,
ligases, and polymerase with the atomic level accuracy. This processibility allows a
more efficient and precise manipulation of DNA and therefore opening up the
Chapter 4_____________________________________________________________
47
possibility to produce monodisperse DNA molecules of precisely known size and
chemical composition134.
Among these DNA-processing enzymes, restriction endonucleases which can
recognize short specific DNA sequences and cleave the DNA201 receive considerable
attention over the past few years. He and co-workers174 first reported using restriction
endonuclease Hinf I to cleave double-stranded DNA molecules that connect nAu to a
gold film. Yun and coworkers158 demonstrated the absence of conformational changes
of nanoparticle-bound DNA by successfully performing the enzyme
(methyltransferases and restriction endonuclease) manipulation of the nAu-DNA
conjugate dimers. Kanaras et al.175 as well as Wang et al.176 reported using restriction
endonucleases to cleave DNA molecules heavily loaded on nAu with 65% digestion
efficiency. Despite these recent studies, high digestion efficiency of nanoparticle-
bound DNA by restriction endonuclease has not been demonstrated yet. This high
efficient control is important for application of these bioconjugates since the
nanoparticles with homogeneous length of DNA is crucial in the application of nano-
assemblies with high precision and medical diagnosis with high reproducibility.
In this chapter we reported a systematic study on the restriction endonuclease
manipulation of nAu-bound DNA, which could contribute to a better understanding on
enzymatic manipulation of nanoparticle-biomolecule conjugates. The digestion
reaction was carried out under various conditions, including (i) stabilizer surface
coverage, (ii) distance between nanoparticle surface and enzyme cutting site of
Chapter 4_____________________________________________________________
48
particle-bound DNA, and (iii) size of nanoparticles. Digestion efficiency as high as
92%, which is similar to free DNA digestion in solution, was achieved for the first
time. The application of this enzyme manipulation would substantially enrich the tool-
box for tailoring nanoparticle-bound DNA or more complicated DNA induced nano-
assemblies and lead to the fabrication of more accurate and complex nanostructures.
4.2 Materials and methods
Materials
Hydrogen tetrachloroaurate (III) trihydrate, trisodium citrate dihydrate, tannic acid, 4,
4’-(phenylphosphinidene) bis-(benzenesulfonic acid) (PPBS), dithiothreitol (DTT),
TEMED (N, N, N’, N’- tetramethylethylenediamine), Glycerol, TBE buffer (Tris-
borate EDTA buffer), NaCl, and MgCl2 were purchased from Sigma-Aldrich. The
synthetic DNA was purchased from Integrated DNA Technologies (Figure 4.1).
Restriction endonuclease EcoR V (500000 unit/ml) and bovine serum albumin (BSA)
were obtained from New England Biolabs. 30% acrylamide/Bis solution (29:1)
ammonium persulfate, bromophenol blue and ethidium bromide were obtained from
Bio-Rad Laboratories. Milli-Q water with resistance >18MΩ/cm was used throughout
the experiments.
Synthesis and characterization of nAu
nAu were synthesized by the reduction of hydrogen tetrachloroaurate (III) trihydrate
by trisodium citrate dihydrate and tannic acid40 as described in Chapter 3. In order to
determine the size and size distribution of the resulting nanoparticles, TEM
Chapter 4_____________________________________________________________
49
characterization was performed on a Philips CM300 FEG system operating at 200 kV.
At least 200 particles were sized from TEM micrographs via graphics software
“Image-Pro Express” (Media Cybernetics). nAu with mean diameters of 10 nm and 7
nm are used in this study.
Figure 4.1 DNA sequences used in this chapter. Underlined sequences are the recognition site of EcoR V and the arrows indicate the enzyme cleavage site.
Chapter 4_____________________________________________________________
50
Preparation of nanoparticle-DNA conjugates
The scheme of preparing nanoparticle-DNA conjugates is shown in Figure 4.2. Two
complementary single stranded DNAs (ssDNAs, sequences shown in Figure 4.1), one
modified with a thiol linker, were mixed at equal molar amount in the hybridization
buffer (50 mM Tris pH 8.0 and 50 mM NaCl). The mixture was briefly heated to 95˚C
and then gradually cooled down to room temperature to allow the formation of double
stranded DNA (dsDNA). The nanoparticle-DNA conjugates were prepared as
previously described by Demers and coworkers202 with modifications. Briefly, nAu
were incubated with 60 mM PPBS to gain necessary stability in mild salt
concentration. Extra PPBS was removed by washing and centrifuging the samples
with 50 mM Tris buffer (pH 8.0). Next, a 3X stoichiometric equivalence of 2 µM
dsDNA was mixed with nAu in 50 mM Tris buffer containing 50 mM NaCl. This
stoichiometric mixing of nanoparticles with thiolated DNA results in a population of
nanoparticles bound with different number of DNA strands (1, 2, 3 and so on). The
samples were incubated in room temperature for 2 hours, and 5’ thiolated TTTTT
ssDNA (dT-ssDNA, Figure 4.1) was then added to the system followed by increasing
the NaCl concentration to 100 mM. The final concentrations of nanoparticles and
ssDNA were 90 nM and 45 µM respectively. After 8 hours incubation, the NaCl
concentration was gradually increased to 500 mM by adding 5 M NaCl stock solution
to the mixture in 6 hours. The samples were incubated under this condition for an
additional 24 hours with intermittent vortex mixing. Finally, excess reagents were then
removed by washing and centrifuging the samples with 50 mM Tris buffer for three
times. For conjugates without ssDNA, the dsDNA conjugation was stopped after 2
Chapter 4_____________________________________________________________
51
hours reaction by wash and centrifugation for one time. Without ssDNA saturation,
repeated wash and centrifuge may completely remove PPBS weakly adsorbed on nAu
and cause nAu aggregation, thus the washing step was only performed two times.
Figure 4.2 Schematic picture of preparation of nanoparticle-DNA conjugates.
Enzyme manipulation of nanoparticle-bound DNA and digestion efficiency
The restriction endonuclease digestion of dsDNA was performed by incubating the
conjugates with 100 unit of EcoR V at 37°C in a 200 µl reaction buffer for 20 hours.
After incubation, the enzyme was deactivated by adding 50 mM EDTA. The digested
conjugates were characterized by polyacrylamide gel electrophoresis (PAGE). For
PAGE characterization, the DNA residues were released from the nanoparticle surface
by ligand exchange reaction with excess DTT. After the removal of the nanoparticles
by centrifuging the samples at 13000g for 15 min, the DNA solution was characterized
by 12% PAGE (1X TBE as running buffer). The DNA bands were stained with
ethidium bromide and captured by a UV/White Light gel documentation system
Chapter 4_____________________________________________________________
52
(GeneGenius, Syngene). The enzyme digestion efficiency was calculated by dividing
the amount of digested DNA fragments by the total amount of DNA in the same lane
(digested + undigested). Each efficiency value reported was the average of three
individual tests.
Fluorescence quantification of DNA surface coverage
The fluorescence measurement on dT-ssDNA surface coverage on nanoparticles was
carried out with fluorescein thioisocyanate (FITC) labeled DNA (Figure 4.1). The
fluorescence measurement was performed using a luminescence spectrometer (Perkin
Elmer LS 50B) with a 1-cm cuvette. The samples were excited at 495 nm and the
emission spectra were recorded from 510 to 535 nm. All the fluorescence
measurements were carried out without the presence of the nanoparticles. For the dT-
ssDNA surface coverage study, the FITC dT-ssDNA was released from nanoparticle
by ligand exchange reaction with excess DTT. After overnight incubation with DTT at
room temperature, the solutions containing displaced FITC dT-ssDNA were separated
from the nAu by centrifugation of the particle aggregates. The maximum fluorescent
emission at 520 nm was converted to molar concentrations of the FITC dT-ssDNA by
interpolation from a standard curve using known concentrations of FITC dT-ssDNA
under exactly the same buffer condition and DTT concentration. Finally, the average
surface coverage of FITC dT-ssDNA on nanoparticle was obtained by dividing the
measured amount of dT-ssDNA by the actual amount of nanoparticle in the sample.
Chapter 4_____________________________________________________________
53
4.3 Results and discussion
4.3.1 Importance of short ssDNA modification on the surface of nAu
for achieving high enzyme digestion efficiency
In our experiments, it was found that the saturation of the nanoparticle surface with 5-
base thiol modified ssDNA (dT-ssDNA, Figure 4.1) is a necessary step to prevent non-
specific adsorption of restriction endonuclease on nanoparticles and to achieve high
DNA digestion efficiency. When nAu without ssDNA modification was used, the
enzyme digestion efficiency of the nAu-bound dsDNA was found to be 0%, even
when a large amount of EcoR V was used. Figure 4.3 shows a 12% polyacrylamide
gel image of 10nm nAu bound with only dsDNA but without dT-ssDNA saturation
after EcoR V digestion. As shown in the next section, if enzyme digestion occurs,
DNA bands with lower molecular weight can be found on the gel. In Figure 4.3,
however, no digested DNA band can be seen in lane 3 or lane 5, indicating that no
digestion of particle-bound DNA was achieved with either Strand A or Strand B. This
surprisingly low digestion efficiency is not likely due to the steric hindrance from
nAu, since the enzyme recognition site of Strand B is located in the middle of the
DNA and is far away from nAu (43 bases away from the thiolated 5’ end). We
attribute this low efficiency to two possible reasons: 1. Due to the high affinity of
nucleotides to gold surface108, 203, 204 and low target dsDNA loading on nAu, the target
dsDNA may wrap around nAu66 and impair its accessibility for enzyme binding and
digestion; 2. The non-specific adsorption of enzyme on the nAu surface may lead to a
significant decrease of the free enzyme in the digestion system, and therefore, result in
Chapter 4_____________________________________________________________
54
a severe drop in the digestion efficiency. This non-specific adsorption of enzyme
molecules on nAu has been observed by other groups205, 206, which causes protein
denaturation and loss of biological functions.
1 2 3 4 5
Figure 4.3 PAGE characterization of enzyme digested 10nm nAu-bound dsDNA without dT-ssDNA saturation. Lanes 1-5 correspond to: (1) 20bp DNA ladder, (2) intact Strand A without enzyme treatment, (3) enzyme treated particle-bound Strand A, (4) intact Strand B without enzyme treatment, (5) enzyme treated particle-bound Strand B.
To prevent or minimize the interaction between target dsDNA and nAu as well as
enzyme adsorption, dT-ssDNA was used to saturate the nanoparticles surface prior to
the enzyme treatment. dT was adopted since it has a much lower binding affinity with
nAu surface and can lead to higher surface coverage108. Another advantage gained
from this ssDNA surface saturation is the significant increase in the stability of
nanoparticle. 10nm nAu without this treatment may aggregate irreversibly in the
digestion buffer required by the enzyme within a few hours. However, the dT-ssDNA
modification leads to a stronger steric repulsion among nAu particles, and thus no
particle aggregation was observed even with the use of prolonged digestion time
aiming to improve the digestion efficiency. Without dT-ssDNA saturation, the
80 bp 80 bp60 bp40 bp
Chapter 4_____________________________________________________________
55
digestion of nAu-dsDNA shown above (Figure 4.3) could only be lasted for less than 8
hours depending on the actual buffer condition.
4.3.2 Effect of ssDNA surface coverage on enzyme digestion
nanoparticle-bound DNA
The dT-ssDNA surface coverage on nAu and its effect to enzyme digestion efficiency
were studied using FITC labeled dT-ssDNA (Figure 4.1). In our experiment, the
fluorescent measurement was conducted after nAu removal by centrifugation because
1) the fluorescence signal of nAu-bound FITC dT-ssDNA can be quenched via the
fluorescence resonance energy transfer (FRET) effect by nAu139-141, and 2) nAu has a
maximum optical absorption at 520nm, and their presence leads to a significant
reduction of fluorescent signal by FITC dT-ssDNA whose emission maximum is also
at the 520nm range. Figure 4.4 shows the dT-ssDNA surface coverage on 10nm nAu
with different molar ratio of dT-ssDNA to nAu as well as the corresponding enzyme
digestion efficiency. This ratio refers to the incubating molar ratio used during the
incubation of dT-ssDNA and nAu mixture for surface conjugation. The surface
coverage of dT-ssDNA increases substantially with the increasing molar ratio of dT-
ssDNA and nAu until 400:1 ratio is reached, which results in a surface coverage of
approximately 136 strands/particle (Determined from fluorescent study using FITC
labeled dT-ssDNA). With the molar ratio goes beyond 400:1, only minor increase in
the surface coverage (less than 10%) as the molar ratio increases to 600:1. The
saturation dT-ssDNA surface coverage obtained at 600:1 molar ratio was found to be
approximately 149 strands/particle (corresponds to a surface density of 78.8
Chapter 4_____________________________________________________________
56
pmol/cm2). This surface coverage is about twice the amount that obtained in the work
by Demers and co-workers202. We attribute this to the fact that shorter ssDNA used in
this study has smaller steric hindrance. In addition, the higher ionic strength condition
(500mM NaCl) used in ssDNA binding may screen the electrostatic repulsion among
ssDNA strands more efficiently.
Figure 4.4 dT-ssDNA surface coverage on 10nm nAu as well as corresponding enzyme digestion efficiency of particle-bound Strand A and non-thiol Strand A.
As shown in Figure 4.4, with the increasing surface coverage of dT-ssDNA, an
obvious increase in the digestion efficiency of nAu-bound Strands A from 0% to 92%
was observed. Similar trend was also found for the digestion of the non-thiol Strand A
by simply mixing the non-thiol Strand A and nAu together prior to the addition of
EcoR V. The presence of nAu without dT-ssDNA modification (ratio of dT-ssDNA
Chapter 4_____________________________________________________________
57
and nAu = 0) results in 0% digestion, even though the target Strand A is not bound on
nAu. This further confirms that the low digestion efficiency can only be attributed to
the presence of bare nAu, which leads to enzyme adsorption on the particles and loss
of activity. Again, higher molar ratios of dT-ssDNA to nAu lead to higher surface
coverage and substantially improve the digestion efficiency. For both cases, more than
90% digestion efficiency, which is comparable to the digestion efficiency of pure
DNA in solution (approximately 95%), can be achieved when the molar ratio between
dT-ssDNA and nAu reaches 400:1. At this ratio, the surface of nAu can be considered
as sufficiently covered by ssDNA, and therefore the enzyme adsorption is virtually
eliminated.
4.3.3 Enzyme digestion efficiency of nanoparticle-bound DNA
The result of enzyme digestion efficiency of 10nm nAu-bound DNA mentioned above
was obtained from a 12% polyacrylamide gel electrophoresis (PAGE). Figure 4.5
shows a typical PAGE image of the enzyme digested and non-enzyme treated dsDNA
(a 500:1 molar ratio of dT-ssDNA to nAu was used in this particular test). It is clearly
shown in the figure that both Strands A and B, bound on nAu (Lanes 2 and 5) or
existed as free molecules in the solution (Lanes 3 and 6), show exactly the same
mobility in the gel after enzyme digestion, which is obviously quicker than the
corresponding intact DNA without enzyme treatment (Lanes 4 and 7). This shows that
the enzyme cutting site is not shifted by the presence of a bulky nanoparticle, even if
the cutting site is located only 18 bases away from the surface of the nAu (Strand A).
Matching the results shown in Figure 4.5, the obvious absence of the 80 bases bands in
Chapter 4_____________________________________________________________
58
Lanes 2 and 5 demonstrates the high efficiency of the enzyme digestion. The digestion
efficiency of nAu-bound DNA was found to be above 90% for both Stands A and B,
which is as high as that of the free DNA digestion (without the presence of nAu) at
similar digestion condition (Table 4.1). This digestion efficiency is substantially
higher than the previously reported results by other groups, which is approximately
65%176. This is most likely due to the low target DNA loading (i.e. Strand A or B) on
nAu surface in our system, which leads to much less steric hindrance for the enzyme
to bind, to move and to process the target DNA.
Figure 4.5 PAGE characterization of enzyme digested 10nm nAu-bound DNA. Lanes 1-7 correspond to: (1) 10bp DNA ladder, (2) enzyme digested particle-bound Strand A, (3) enzyme digested free Strand A, (4) intact Strand A without digestion, (5) enzyme digested particle-bound Strand B, (6) enzyme digested free Strand B, (7) intact Strand B, (8) particle-bound Strand A, (9) Strand A displaced by DTT in the digestion buffer and (10) 10bp DNA ladder.
Chapter 4_____________________________________________________________
59
Digestion efficiency of free DNA (%)
Digestion efficiency of 10nm nAu-bound DNA (%)
Strand A 92.5 92.0
Strand B 93.8 92.7
Table 4.1 Digestion efficiency of free DNA and 10nm nAu-bound dsDNA by endonuclease EcoR V.
It is well known that the gold-thiol (Au-S) linkage, although thermodynamically
stable, is kinetically labile and may be displaced by other thiolated compounds. This
phenomenon is commonly referred to as “thiol exchange”. In our enzyme digestion
experiment, the nAu-bound dsDNA may possibly be detached by dithiothreitol (DTT)
since DTT, as a common reducing agent, was put in the digestion buffer to prevent
possible oxidation of the restriction enzyme. To confirm that the particle-bound
dsDNA was not first released by DTT in the digestion buffer before it was digested by
EcoR V, nAu-bound Strand A was incubated at exactly the same digestion condition
but without the restriction enzyme. Subsequently, the nAu-Strand A conjugates were
centrifuged. The supernatant, which contained dsDNA released by DTT, was
separated from the pellet, which contained nAu-bound DNA. Finally, pellet was
mixed with excess DTT to release all bound Strand A for PAGE quantification. As
shown in Figure 4.5, Lanes 8-10 correspond to nAu-bound Strand A, Strand A
displaced by the digestion buffer, and 10bp DNA ladder respectively. By comparing
Lanes 8 and 9, it was found that only trace amount of Strand A was released by the
digestion buffer (approximately 1%) indicating that the DTT displacement of particle-
bound dsDNA was negligible under this digestion condition. This confirms that the
Chapter 4_____________________________________________________________
60
low DTT concentration condition adopted in our experiment (1 µM) was tolerable for
the particle-bound dsDNA and, at the same time, sufficient to preserve enzyme
activity to achieve the high digestion efficiency of the particle-bound dsDNA.
This high enzyme digestion efficiency of nAu-bound DNA was further confirmed
using fluorescein-modified dsDNA (FITC Strand A, Figure 4.1). The digestion
efficiency was obtained from dividing the fluorescent emission measurement (520nm)
of the EcoR V treated sample by that of the identical sample without enzyme
treatment. For the sample without enzyme treatment, the nAu-bound FITC Strand A
was released from particle by ligand exchange reaction with DTT. Figure 4.6 shows
the typical fluorescent spectrum of the FITC Strand A with and without enzyme
treatment. Based on the measurement, the digestion efficiency of nAu-bound FITC
Strand A was found to be 91.4%, a result that is consistent with the one obtained from
PAGE characterization shown above. Thus, we have shown here an efficient way of
controlling the length of nAu-bound DNA (in this case, 18bp or 43bp long) via
endonuclease digestion. This enzyme manipulation should find applications in
preparing a master copy of nanoparticle-DNA conjugates bearing DNA strands with
multiple enzyme cutting sites. When it is required, the length of the nanoparticle-
bound DNA can be adjusted using different endonucleases that cleave DNA at
different recognition sites.
Chapter 4_____________________________________________________________
61
Figure 4.6 Emission spectrum of FITC Strand A used in enzyme digestion efficiency study. Enzyme digested nAu-bound FITC Strand A (real line), nAu-bound FITC Strand A without enzyme treatment (broken line).
4.3.4 Effect of ionic strength on dT-ssDNA surface coverage on nAu
and enzyme digestion efficiency
When the same DNA conjugation and enzyme digestion conditions were applied for
smaller nAu of 7nm diameter, it was found that the digestion efficiencies of particle-
bound Strand A and Strand B drop to 74.4% and 78.7% respectively. This can be
attributed to a lower dT-ssDNA surface coverage on smaller nanoparticle, which leads
to a higher possibility that the enzyme adsorbs on the bare gold surface and therefore,
reduce the digestion efficiency. This lower surface coverage of dT-ssDNA on smaller
particle was not unexpected. Due to the anionic nature of the nAu surface and DNA,
the protocol for dT-ssDNA saturation on nAu requires a gradual increase in NaCl
concentration to screen the electrostatic repulsion between the nAu and ssDNA which
Chapter 4_____________________________________________________________
62
was first reported by Mirkin’s group37. 7nm particles have a higher surface charge
density than the 10nm particle, so the 500mM NaCl concentration, which is sufficient
for dT-ssDNA saturation for 10nm nAu, may not be enough to screen the charge
repulsion between 7nm nAu and dT-ssDNA, resulting in a lower dT-ssDNA surface
coverage. Since electrostatic interaction is strongly dependent on the ionic strength of
the medium, under higher ionic strength conditions, the charged dT-ssDNA and nAu
are expected to be better electrostatically shielded, thus allowing higher surface
coverage of dT-ssDNA on nAu. To determine the effect of ionic strength condition on
the enzyme digestion of nAu-bound dsDNA, we studied the surface coverage of FITC
dT-ssDNA on 7nm nAu and enzyme digestion efficiency as a function of NaCl
concentration used in ssDNA conjugation to nAu. The fluorescent study shows that
the dT-ssDNA surface coverage increases with the increasing NaCl concentration
(Table 4.2). It can clearly be seen from the data that higher ionic strength screens the
electrostatic repulsion between nAu and dT-ssDNA more efficiently and results in
more densely packed ssDNA on nAu surface. This leads to the higher digestion
efficiency of particle-bound DNA. Specifically, a 1.5 M NaCl results in 82 dT-
ssDNA/nAu (corresponding to a surface coverage of 88.4 pmol/cm2) on 7nm nAu as
well as 94.0% and 96.3% digestion of particle-bound Stand A and Strand B
respectively. This result is comparable to the one obtained with 10nm nAu. Thus,
besides using expensive dT-ssDNA to increase the surface coverage of ssDNA by
increasing the molar ratio of dT-ssDNA to nAu, one can adopt an alternative strategy
to increase ssDNA coverage using high concentration of NaCl to achieve the same
degree of high enzyme digestion of particle-bound DNA subsequently. Finally, it is
Chapter 4_____________________________________________________________
63
worth to mention that the labeling of FITC at the 3’ end of the dT-ssDNA may pose
some degree of steric hindrance, but after searching through different fluorophores
available for commercial labeling, we found that the molecular sizes of other
fluorophores are no better than FITC and thus FITC labeling was adopted in this
study.
dT-ssDNA surface coverage on nAu* NaCl concentration
(mol/L) number of strands/nAu pmol/cm2
Enzyme digestion efficiency (%)
Strand A 74.4 0.5 62 66.9
Strand B 78.7
Strand A 83.7 1.0 69 74.4
Strand B 84.2
Strand A 94.0 1.5 82 88.4
Strand B 96.3
Table 4.2 dT-ssDNA surface coverage on 7nm nAu under different NaCl concentration and digestion efficiency of particle-bound dsDNA by endonuclease EcoR V. *Determined from fluorescent study using FITC labeled dT-ssDNA and reported in both units – number of strands/nAu and pmol/cm2.
4.4 Conclusions
In this chapter, we demonstrated the feasibility of enzymatic manipulation of nAu-
bound dsDNA by restriction endonuclease. We found that the 5-base ssDNA surface
coverage on nanoparticle is the key factor that affects the digestion efficiency. Enzyme
Chapter 4_____________________________________________________________
64
digestion efficiency can be greatly improved by increasing ssDNA surface coverage
on nanoparticles, a process that can be facilitated using high ionic strength conditions.
Quantitative study shows that digestion efficiency of particle-bound DNA as high as
90+% which is similar to free DNA digestion can be achieved under optimal
conditions. Although restriction endonuclease and nAu are used in this work, the
above results are expected to be applicable to other enzyme reactions (ligation or
polymerization) with different kinds of nanoparticles (metallic, semiconductor or
magnetic).
Chapter 5_____________________________________________________________
65
Chapter 5
Fabrication of nanoparticle-DNA conjugates bearing
specific number of short DNA strands by enzymatic
manipulation of nanoparticle-bound DNA
5.1 Introduction
Recently, there has been considerable research effort aiming at assembling metallic
nanoparticles into two- and three-dimensional functional structures, because of the
collective physical and chemical properties that can be gained from such assemblies34,
200, 207-213. Various kinds of application have been found with such assemblies, such as
biological and biomedical detections31, 32, fabricating nanodevices such as single
electron transistors214-216, nanoparticles-based molecular switch217 and nanocircuitry11,
218. A variety of assembly schemes have been developed based on DNA, proteins,
small organic molecules, and synthetic polymers as particle linkers32, 118, 219-224. DNA
is no doubt one of the most commonly used linker molecules in nanoparticle assembly.
The attractive feature of using DNA as the linker molecules is that, in principle, one
can synthetically program interparticle distances, particle periodicities, and particle
compositions through choosing of DNA lengths and sequences225.
Chapter 5_____________________________________________________________
66
For fabricating nano-assemblies with well defined structure, the ability to deliberately
organize nanoparticles into pre-designed patterns is necessary for achieving the
desired optical155 and electrical properties218. Therefore, precise control over the
number of DNA strands attached on each nanoparticle is essential. Otherwise, the
nanoparticles tend to form an uncontrolled network structure. Unfortunately, the
number of DNA strands bound to each nanoparticle cannot be directly controlled yet.
One can control the average number of DNA strands per nanoparticle by adjusting the
stoichiometric ratio of DNA to nanoparticle in the incubation solution. But this
stoichiometric mixing always yields a population of nanoparticles bound with
different number of DNA strands17. In order to solve this problem, Alivisatos and
coworkers suggested using agarose gel electrophoresis to isolate the nanoparticle-
DNA conjugates from the stoichiometric mixture, based on the electrophoretic
mobility difference between conjugates bearing different number of DNA strands.
However, this method is not applicable to conjugates with DNA size smaller than 50
bases since the mobility difference is insignificant62, 154.
To achieve the full potential of nanoparticle-DNA conjugates in constructing nano-
assemblies, the presence of short DNA strands is necessary since shorter DNA
molecules can discriminate single nucleotide mismatch more effectively134. In this
chapter we described a new strategy of using restriction endonucleases to manipulate
the nanoparticle-bound DNA to gain full control over the number and length of the
bound DNA strands. The product after this enzymatic manipulation can be the well
designed building blocks for nano-assembly. Using such building blocks as the
Chapter 5_____________________________________________________________
67
starting material, the size and structure of the assembly as well as the distance between
the build blocks can possibly be controlled.
Restriction endonucleases are enzymes that recognize short specific DNA sequences
and cleave the DNA201. He et al.174 first reported using restriction endonuclease (Hinf
I) to cleave double-stranded DNA which connected gold nanoparticles to a gold film.
Yun and coworkers158 demonstrated the absence of conformational changes of
nanoparticle-bound DNA by successfully performing the enzyme (methyltransferases
and restriction endonuclease) manipulate of the gold nanoparticle-DNA conjugate
dimer. Kanaras et al.175 as well as Wang and coworkers176 reported using restriction
endonucleases to cleavage double stranded DNA (dsDNA) molecules which were
heavily loaded on gold nanoparticles.
In our protocol, agarose gel electrophoresis was used to isolate nanoparticles bearing
specific number of DNA molecules, then a restriction endonuclease (EcoR V) was
used to cleave the nanoparticle-bound DNA at the pre-designed site. In this way, the
number, length, and sequence of the DNA strands on the resultant conjugates was
precisely controlled.
5.2 Materials and methods
Materials
Hydrogen tetrachloroaurate (III) trihydrate, trisodium citrate dihydrate, tannic acid, 4,
4’-(phenylphosphinidene) bis-(benzenesulfonic acid) (PPBS), dithiothreitol (DTT),
Chapter 5_____________________________________________________________
68
NaCl, MgCl2 and tris borate EDTA (TBE) buffer were purchased from Sigma-Aldrich.
The synthetic DNA (modified with thiol linker at the 5’ end) was purchased from
Integrated DNA Technologies (IDT). Restriction endonuclease EcoR V (500000
unit/ml) and bovine serum albumin (BSA) were obtained from New England Biolabs.
30% acrylamide/Bis solution (29:1) and ethidium bromide were obtained from Bio-
Rad Laboratories. Agarose was purchased from Cambrex. Dialysis tubing (Float-A-
lyzer, MWCO: 3500) was obtained from Spectra/Por. Milli-Q water with resistance
>18MΩ/cm was used throughout the experiments.
Synthesis and characterization of gold nanoparticles (nAu)
nAu were synthesized by the reduction of hydrogen tetrachloroaurate (III) tetrahydrate
by trisodium citrate dihydrate and tannic acid40 as described in Chapter 3 . In order to
determine the size and size distribution of the resulting nanoparticles, TEM
characterization was performed on a Philips CM300 FEG system operating at 200 kV.
At least 200 particles were sized from TEM micrographs via graphics software
“Image-Pro Express” (Media Cybernetics). The mean particle diameter was 9.7 nm
and the size distribution was within 15% of the mean diameter.
Preparation of nanoparticle-DNA conjugates
The scheme of preparing nanoparticle-DNA conjugates is shown in Figure 5.1. Two
complementary single stranded DNAs (ssDNAs), one modified with a thiol linker,
were allowed to hybridize to form a double stranded DNA (dsDNA). The
nanoparticle-DNA conjugates were prepared as previously described by Demers and
Chapter 5_____________________________________________________________
69
coworkers202 with some modifications. Briefly, nAu were incubated with 60 mM
PPBS to gain necessary stability in mild salt concentration. Extra PPBS was removed
by washing and centrifuging the samples with 0.5X TBE buffer (pH 8.3). After that, a
stoichiometric amount (2, 3, etc. equiv. of nanoparticles) of 2µM dsDNA was mixed
with nAu in 0.5X TBE buffer containing 50mM NaCl. Two dsDNA sequences were
used in this chapter and they are shown in Figure 5.2. The samples were incubated in
room temperature for 2 hours, and 5-base 5’ thiolated ssDNA (5’-thiol-TTTTT) was
then added to the system followed by increasing the NaCl concentration to 100mM.
The final concentration of nanoparticle and ssDNA were 90nM and 45µM
respectively. After 10 hours, the NaCl concentration was gradually increased to
500mM by adding 5M NaCl stock solution to the incubation system in 9 hours. The
samples were incubated under this condition for an additional 20 hours with
intermittent vortex mixing. Finally, excess reagents were then removed by repeated
washing and centrifuging the samples with 0.5X TBE.
Figure 5.1 Schematic picture of preparation of nanoparticle-DNA conjugates.
Chapter 5_____________________________________________________________
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Figure 5.2 DNA sequences used in this chapter. Arrows indicate the enzyme cleavage sites.
Agarose gel electrophoresis isolation and extraction of nanoparticle-DNA
conjugates
Agarose gel electrophoresis (2% agarose at 5 V/cm, 0.5X TBE as running buffer) was
carried out for 4 hours to separate the conjugates bearing from one to six dsDNA
molecules. Discrete bands can easily be identified because of the wine-red color of
nAu. Desired bands were extracted from the gel and the conjugates were then
recovered by electrophoretic dialysis116. After recovered from agarose gel, the
conjugates were purified by repeated washing (with 50 mM tris buffer, pH 8.0) and
centrifuging to ensure complete removal of EDTA which may deactivate the
restriction endonuclease.
Chapter 5_____________________________________________________________
71
Enzyme manipulation of nanoparticle-bound DNA and digestion efficiency
The restriction endonuclease digestion of dsDNA was performed by incubating the
conjugates with 100 unit of EcoR V at 37°C in a 200 µl reaction buffer for 20 hours.
After incubation, the enzyme was deactivated by adding 50 mM EDTA. The digested
conjugates were characterized by agarose and polyacrylamide gel electrophoresis. For
polyacrylamide gel electrophoresis characterization, the DNA residues were released
from the nanoparticle surface by ligand exchange reaction with excess DTT. After the
removal of the nanoparticles by centrifuging the samples at 13000g for 15 min, the
DNA solution was characterized by 12% polyacrylamide gel electrophoresis (0.5 X
TBE as running buffer). The DNA bands were stained with ethidium bromide and
visualized by a Syngene GeneGenius UV and White Light Gel Documentation and
Analysis system. The enzyme digestion efficiency was calculated by quantifying
digested DNA fragments using gel analysis software from manufacturer.
5.3 Results and discussion
5.3.1 Enzyme digestion efficiency of nanoparticle-bound dsDNA by
polyacrylamide gel electrophoresis
The enzyme digestion efficiency of 10nm nAu-bound DNA (recovered from agarose
gel electrophoresis isolation) obtained from 12% polyacrylamide gel electrophoresis
(PAGE) is shown in Figure 5.3. As can be found in the figure, both strands A and B,
bound on nAu (Lanes 2 and 5) or existed as free molecules in the solution (Lanes 3
and 6), show exactly the same mobility in the gel after enzyme digestion. They ran
Chapter 5_____________________________________________________________
72
obviously quicker than the corresponding intact DNA without enzyme treatment
(Lanes 4 and 7). This indicates that the enzyme cutting site is not shifted by the
presence of a bulky nAu, even if the cutting site is located only 18-base away from the
surface of the nAu. The absence of the 80-base bands in Lanes 2 and 5 demonstrates
the high digestion efficiency, which was found to be above 90% for both stands A and
B, which is as high as that of the free DNA digestion (without the presence of
nanoparticle) under similar digestion condition, as shown in Table 1. This high
digestion efficiency demonstrates that the enzyme processibility of nAu-bound DNA
is not impaired by the agarose gel electrophoresis isolation and recovery treatment.
Figure 5.3 PAGE characterization of enzyme digested particle-bound DNA. Lanes 1-7 correspond to: (1) the 10bp DNA ladder, (2) enzyme digested particle-bound Strand B, (3) enzyme digested free Strand B, (4) intact Strand B without digestion, (5) enzyme digested particle-bound Strand A, (6) enzyme digested free Strand A, and (7) intact Strand A.
Chapter 5_____________________________________________________________
73
Digestion efficiency of free DNA (%)
Digestion efficiency of particle-bound DNA (%)
Strand A 92.5 91.8
Strand B 93.8 92.7
Table 5.1 Digestion efficiency of free DNA and nanoparticle-bound DNA by endonuclease EcoR V.
5.3.2 Agarose gel electrophoresis of the nanoparticle-DNA conjugates
Agarose gel electrophoresis is a very useful tool commonly used to separate
biomolecules and was first introduced to the nanoparticle-DNA system by Zanchet
and co-workers154. The binding of dsDNA to a nanoparticle decreases its mobility in
the gel, making it possible to discriminate conjugates bearing different number or
length of DNA. To give a direct comparison of the nAu-DNA conjugates before and
after enzyme treatment and obtain a clear view of the digestion efficiency of nAu-
bound DNA, the digested conjugates and untreated conjugates were characterized by
agarose gel electrophoresis. The electrophoresis images of the conjugates mixture with
Strand A and Strand B are shown in Lane D of Figure 5.4 and Lane L of Figure 5.6
respectively. The stoichiometric ratio between the nAu and Strand A/B in the mixture
is 1 to 3. This image of the conjugates mixture in these two lanes are identical to the
ones obtained previously by Zanchet et al154. Each band in Lane D/L corresponds to
the nAu-DNA conjugates bearing different number of dsDNA strands. Using the band
in Lane E, which contains nAu incubating with 5-base ssDNA without Strand A, we
can identify that first band at the bottom of Lane D contains conjugates bearing one
Chapter 5_____________________________________________________________
74
Strand A. Similarly the second, third, and fourth bands contain conjugates bearing two,
three and four Strand A respectively. Similar tactics can be used to identify conjugates
bearing different number of Strand B in Figure 5.6. The band containing conjugates
bearing one Strand A (or Strand B) appears to be vague in this image because of the
high stoichiometric ratio of nanoparticle:dsDNA (1:3) used in preparing the mixture.
The same band appears more prominent when a ratio of 1:1 was used (data not shown).
Figure 5.4 Agarose gel electrophoresis of nanoparticle-dsDNA conjugates bearing Strand A. Lanes A1, B1 and C1 correspond to EcoR V digested conjugates bearing 4, 3 and 2 strands of Strand A respectively; Lanes A2, B2, C2 correspond to the conjugates bearing 4, 3 and 2 strands of Strand A without EcoR V digestion respectively; Lane D corresponds to mixed conjugates bearing different strands of Strand A and Lane E correspond to the conjugate bearing only 5-base ssDNA without Strand A.
Chapter 5_____________________________________________________________
75
5.3.3 Restriction endonuclease digestion/cleavage of nanoparticle-
dsDNA conjugates bearing definite number of DNA strands
As described in the experimental section, the agarose gel purified conjugates bearing
with different numbers of DNA strands (Lane D of Figure 5.4) were extracted and
treated with restriction enzyme EcoR V and characterized by 2% agarose gel. Since
the surface of the nanoparticles is saturated with highly negatively charged ssDNA,
the overall charge increase of the nanoparticle-DNA conjugates by 80bp dsDNA
attachment is negligible. The size of the conjugates, therefore, becomes the
determining factor controlling the electrophoretic mobility of the conjugates. Figure
5.4 shows the conjugates bearing different number of Strand A. Lanes A2, B2, and C2
correspond to the conjugates bearing 4, 3, and 2 strands of Strand A respectively.
These three conjugates were obtained from gel extraction after the agarose gel
electrophoresis of a mixture of conjugates attaching with different number of Strand A,
which is shown in Lane D of the same figure. The horizontal position of the band in
Lanes A2, B2 and C2 tracks closely with the bands in Lane D. This confirms that the
number of dsDNA on each conjugate remains intact after extraction from the agarose
gel. By treating the conjugates in Lanes A2, B2 and C2 with the endonuclease EcoR V,
we obtained the corresponding conjugates shown in Lanes A1, B1 and C1 respectively.
The enzyme treated conjugates show a significantly higher mobility compared with
the corresponding conjugates without enzyme treatment. This is because these
conjugates now bear only short 18bp dsDNA strands and not the full 80bp Strand A
prior to the enzymatic cleavage. In order to exclude the possibility that the mobility
difference between enzyme treated and non-enzyme treated conjugates is due to the
Chapter 5_____________________________________________________________
76
non-specific adsorption of enzyme on the nanoparticle, a control experiment using
nanoparticles modified with only 5-base ssDNA was carried out. The agarose gel
image of the nanoparticles incubated with the enzyme (Lane F) and without the
enzyme (Lane G) is shown in Figure 5.5. No mobility difference was found between
these two samples. This confirms that incubating ssDNA saturated nanoparticles with
restriction enzyme does not alter the electrophoretic mobility of the particles.
Figure 5.5 Comparison of the electrophoretic mobility of the enzyme treated and non-enzyme treated 5-base ssDNA modified nanoparticles. Lanes F-H correspond to enzyme treated nanoparticles, non-enzyme treated nanoparticles, and nanoparticle-dsDNA mixture respectively.
F G H
Enzyme treated
Non-enzyme treated
dsDNA
Chapter 5_____________________________________________________________
77
Figure 5.6 shows the agarose gel electrophoresis results with nAu-DNA conjugates
bearing different numbers of Strand B. In this case, since the enzyme cleaving site is at
the middle of the dsDNA (see Figure 5.2), the digestion resulted in conjugates bearing
longer DNA residues (43bp). The mobility difference between the enzyme treated
samples (Lanes I1, J1, and K1) and non-enzyme treated samples (Lanes I2, J2, and K2)
is obviously smaller when compared with the cases when Strand A was used for
conjugation. This mobility difference is more clearly shown in the Figure 5.7, when
the conjugates bearing five strands of digested Strand A and Strand B were allowed to
run electrophoresis in parallel. It is obvious that the conjugate bearing digested Strand
A (Lane P1) has a higher mobility and runs faster than the one bearing digested Strand
B (Lane Q1). The conjugates bearing undigested Strand A (Lane P2) and Strand B
(Lane Q2) share the same mobility. The undigested conjugates also track closely to the
band of the mixed conjugates bearing five dsDNA (Lane N).
With the use of two molecular biology tools (endonuclease digestion and
electrophoresis), we demonstrated in this study a new strategy to obtain nanoparticle-
DNA conjugates bearing definite number of short DNA strands. This type of
conjugates is important for nano-assembly formation and quantitative diagnostics.
With the precise control of the number and the length of DNA strands, the formation
of defined nanostructures using DNA as linkers can be more robust, and the signal
quantification in diagnostics can also be more accurate. Although enzymatic digestion
of DNA on nanoparticles have been reported by other groups158, 174-176, the high
efficiency cleavage of dsDNA just 18bp away from the surface of the nanoparticle has
Chapter 5_____________________________________________________________
78
not been shown before. By adjusting the position of the cleavage site via
oligonucleotide synthesis, we also demonstrated the feasibility of controlling the
length of dsDNA (in this case, 18bp or 43bp long) via endonuclease digestion. This
can be useful in preparing a master copy of nanoparticle-DNA conjugates bearing
different number of DNA strands with multiple enzymatic digestion sites. When it is
required, the length of the nanoparticle-bound DNA can be adjusted using different
endonucleases that cleave DNA at different recognition sites.
Figure 5.6 Agarose gel electrophoresis of nanoparticle-dsDNA conjugates bearing Strand B. Lanes I1, J1 and K1 correspond to EcoR V digested conjugates bearing 4, 3 and 2 strands of Strand B respectively; Lanes I2, J2 and K2 correspond to the conjugates bearing 4, 3 and 2 strands of Strand B without EcoR V digestion respectively; Lane L corresponds to mixed conjugates bearing different strands of Strand B and Lane M correspond to the conjugate bearing only 5-base ssDNA without Strand B.
Chapter 5_____________________________________________________________
79
Figure 5.7 Comparison of electrophoretic mobility of the digested nanoparticle-dsDNA conjugates bearing Strand A or strand B. Lane P1 and Q1 correspond to the conjugates bearing 5 strands of digested Strand A and Stand B respectively; Lane P2 and Q2 correspond to conjugates bearing 5 strands of intact Strand A and B respectively; Lane N corresponds to mixed conjugates bearing different strands of Strand A and Lane O correspond to the conjugate bearing only 5-base ssDNA without Strand A or Strand B.
The three bands in Lanes A1, B1 and C1 of Figure 5.4 show the conjugates with 4, 3,
or 2 strands of short DNA. Since the horizontal positions of these bands are below the
first band in Lane D (contains the conjugate bearing one intact 80bp dsDNA), it
indicates that none of these three conjugates contains the intact 80bp dsDNA. With the
high digestion efficiency shown in Table 5.1, it further shows that the digestion is a
robust process even the DNA is bound to the nanoparticle, and the three bands in
N O P2 P1 Q2 Q1
ssDNA
dsDNA
Chapter 5_____________________________________________________________
80
Lanes A1, B1 and C1 contain conjugates with 18bp DNA. In addition, the mobility of
these conjugates is similar to that of the conjugates only treated with 5-base ssDNA
(Lane E). This shows that the presence of short 18bp DNA(s) bound to nanoparticles
does not essentially slow down the mobility of the resultant conjugates. The small
mobility difference seen here makes the conjugates bearing different number of short
DNA very difficult to be separated. This is the exact reason why the separation of
nanoparticle bearing short DNA (20bp or below) has not been reported in the literature
before. Such nanoparticle-DNA conjugates, however, are more useful than those
bearing longer DNAs (above 50 bases) in DNA sequence detection assays, since
stringency washes are with shorter DNA lengths more efficient at removing
mismatched labels134.
For the endonuclease digestion to be effective, it is important to notice that the
additional bases next to the EcoR V recognition site are required. In the case of Strand
A, it is TCACT (sequence in italic shown in Figure 5.2). Without these extra 5 bases,
the digestion efficiency appeared to drop significantly. These extra bases most likely
allow a better anchorage for the enzyme to search, recognize, and cleave the
designated sequence. Finally, for the nanoparticle-DNA conjugates to be effectively
used for nano-assembly or diagnostics application, it is imperative that short ssDNA,
not dsDNA, should be bound on the nanoparticle surface. The extra 8 bases
(TCACTCTA) remaining on the digested Strand A may be seen as an obstacle. But due
to the extreme short length of these 8 bases, the hybridization temperature (Tm) is
very low (<10°C). Thus it is very likely that this 8-base remnant dissociates from its
Chapter 5_____________________________________________________________
81
complementary strand even at room temperature, and results in an 18-base ssDNA
chemically bonded to the nanoparticle. Further study is required for confirming the
absence of this 8-base DNA. In the case of Strand B, the length of the double stranded
residue remains to be 30bp after digestion. The Tm for this 30bp is approximately
62ºC and is substantially higher than the room temperature or 37ºC condition, at which
all the experiments were conducted. Thus it is reasonable to believe that the
nanoparticle-bound Strand B remains in double stranded form after the digestion step.
5.4 Conclusions
In this chapter, a new methodology that combines agarose gel electrophoresis isolation
and enzyme manipulation of nanoparticle-bound DNA has been described. This
method successfully obtains nanoparticle-DNA conjugates bearing specific number
and length of short DNA strands. We showed that the particle-bound DNA can be
manipulated by restriction endonuclease and a high digestion efficiency of 91.8% can
be achieved. Such well defined conjugates are expected to be useful as build blocks
for controllable nanostructures assembly or as nanoprobes for quantitative DNA
sequence detection analysis.
Chapter 6_____________________________________________________________
82
Chapter 6
Nanoparticle based quantitative DNA detection with
single nucleotide polymorphism (SNP) discrimination
selectivity
6.1 Introduction
There is a major need to develop fast, cheap, and precise detection methodologies that
detect DNA samples at extremely low concentration. This ability is critical to basic
life sciences, medical diagnosis & treatment, pharmaceutical applications,
identification of biological weapons, as well as forensic analysis226-228. To fulfill this
goal, the scientific community is striving to develop new methods and assays that are
highly specific and sensitive. Optical/colormetric32, 118, 145, fluorescent139, 229, and
electrochemical230-232 based methods have been reported for detection of DNA
samples. Among these new methodologies, optical detection methods, which rely on
the hybridization between target DNA and substrate modified with radioactive,
fluorescent, chemiluminescent, or nanoparticle tags, are of particular interest140, 178, 233.
Among these labeling tags, the use of gold nanoparticles (nAu) receives most attention
in recent years, due to their unique chemical and physical properties234-236 that can be
exploited in biological detection assays. nAu have been used in the development of
Chapter 6_____________________________________________________________
83
highly sensitive detection assays37, 136. Although still in its infancy, the application of
surface-functionalized nAu in sequence recognition has shown great promise in
achieving high sensitivity that is difficult to achieve by conventional methods.
Besides sensitivity, quantification and selectivity are the other two important aspects
for the evaluation of DNA biosensor devices. DNA quantification is critical for gene
expression analysis, detection of DNA mutations or genetic defects, early stage
diagnosis of critical illness such as HIV and cancers, and forensic applications237-239.
Furthermore, diagnosis of pathogenic and genetic diseases requires the device to have
high selectivity that can discriminate single nucleotide mismatches37, 226. Single
nucleotide polymorphisms (SNPs) are the most abundant form of genetic variation that
occur once every 100–300 bases and there are greater than 3 million SNPs in the
human genome191. Identify these SNPs and associate individual SNPs with specific
diseases and pharmacological responses are clinically important for medical
diagnostics, disease prevention and prognostics190, 192. These needs have driven intense
efforts toward the development of new methodologies that enable quantitative,
selective and cost-effective detection of SNP in DNA samples134, 136. Currently, real-
time polymerase chain reaction (RT-PCR) is one of the most frequently used methods
for DNA quantification and SNP discrimination in life science and basic clinical
research. However RT-PCR is a time-consuming and labor intense process, and its
selectivity is not always satisfactory even with sophisticated optimizations240, 241. For
commonly used DNA detection systems such as DNA chips, the selectivity and
quantification are dependent on the dissociation properties of target DNA hybridized
Chapter 6_____________________________________________________________
84
with capture strands immobilized on the chip134. To achieve SNP discrimination, a
stringent wash step has to be performed to remove mismatched DNA binding on the
capture strands. However, the difference in binding affinity between a perfectly
matched target DNA and one with a mismatched base is usually too small to achieve
complete discrimination136.
In chapter 5, we have shown that gold nanoparticle-DNA (nAu-DNA) conjugates
bearing specific number of short DNA (< 20 bases) can be prepared by gel
electrophoresis isolation followed by restriction endonuclease manipulation of the
nAu-bound DNA242. In this chapter we described a novel gold nanoparticle (nAu)
based assay methodology that has reliable quantification ability and SNP
discrimination selectivity. In this assay, two sets of specially designed nAu-DNA
conjugates are fabricated via the gel electrophoresis and restriction endonuclease
manipulation methods. These two sets of conjugates with definite number (1, 2, 3…)
of short single stranded DNA (ssDNA) probes are not directly complementary to each
other. After mixing, these conjugates do not recognize and group each other until a
target DNA that is complementary to both sets of conjugates is introduced. Only
conjugate groupings with defined structure (dimmer or trimer) can form due to
definite number of DNA strands on each nAu. The resulting conjugate groupings are
characterized and quantified by agarose gel electrophoresis. The size differentiation
ability of gel electrophoresis allows strict discrimination between different conjugate
structures (monomer, dimer and trimer) and enables precise quantification of target
DNA samples. Furthermore, a strong discrimination between perfectly matched and
Chapter 6_____________________________________________________________
85
single base mismatched DNA can be achieved since only the presence of perfectly
matched target DNA allows the formation of conjugate groupings. This novel assay
methodology is particularly suitable for quantitative DNA analysis and SNP
discrimination, and is expected to find application in the diagnosis of genetic &
infective disease, forensic analysis as well as biodefense applications.
6.2 Materials and methods
Materials
Hydrogen tetrachloroaurate (III) trihydrate, trisodium citrate dihydrate, tannic acid,
dithiothreitol (DTT), NaCl, MgCl2, and tris-borate-EDTA (TBE) buffer were
purchased from Sigma-Aldrich. The synthetic DNA (modified with thiol linker at the
5’ end) was purchased from Integrated DNA Technologies (IDT). Restriction
endonuclease EcoR V (500000 unit/ml) was obtained from New England Biolabs.
30% acrylamide/Bis solution (29:1) and ethidium bromide were obtained from Bio-
Rad Laboratories. Agarose was purchased from Cambrex. Dialysis tubing (Float-A-
lyzer, MWCO: 3500) was obtained from Spectra/Por. Milli-Q water with resistance
>18MΩ/cm was used throughout the experiments.
Synthesis and characterization of gold nanoparticles (nAu)
nAu were synthesized by the reduction of hydrogen tetrachloroaurate (III) tetrahydrate
by trisodium citrate dihydrate and tannic acid36 as described in Chapter 3. In order to
determine the size and size distribution of the resulting nanoparticles, TEM
characterization was performed on a Philips CM300 FEG system operating at 200 kV.
Chapter 6_____________________________________________________________
86
At least 200 particles were sized from TEM micrographs via graphics software
“Image-Pro Express” (Media Cybernetics). The mean particle diameter was 10 nm and
the size distribution was within 15% of the mean diameter.
Preparation of nAu-DNA conjugates
The sequences of DNA used in this chapter are shown in Figure 6.1 (complete
sequences are shown in Figure A1, Appendix I). Two complementary single stranded
DNA (ssDNA), one modified with a thiol linker, were allowed to hybridize to form a
double stranded DNA (dsDNA). Two dsDNA, strand A' with a 5’ thiol group and
strand revA' with a 3’ thiol group, were used to prepare two sets of conjugates (nAu-A'
and nAu-revA') for conjugate grouping studies. The detailed procedure of these
conjugates preparation can be found in Chapter 4. Briefly, 2 µM dsDNA (strand A' or
revA') was mixed with nAu for 2 hours followed by 5-T ssDNA for surface
passivation. Excess reagents were then removed by repeated washing and centrifuging
the samples with 0.5X TBE.
Agarose gel electrophoresis isolation and extraction of nAu-DNA conjugates
Agarose gel electrophoresis (3% agarose at 5 V/cm, 0.5X TBE as running buffer) was
carried out for 4 hours to separate the conjugates bearing from one to three dsDNA
molecules. Discrete bands can easily be identified because of the wine-red color of
nAu. Desired bands were extracted from the gel and the conjugates were then
recovered by electrophoretic dialysis38. After recovered from agarose gel, the
conjugates were purified by repeated washing (with 50 mM tris buffer, pH 8.0) and
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centrifuging to ensure complete removal of EDTA which may deactivate the
restriction endonuclease.
Figure 6.1 DNA sequences used in this chapter. For strand A' and revA', the underlined sequences are the recognition site of EcoR V and the arrows indicate the enzyme cleavage site. For the mismatched DNA sequences, the underlined base is the mismatch. SM1/SM2/SM3 refer to single-base mismatched DNA, DM refers to double-base mismatched DNA, and NC refers to non-complementary DNA.
Enzyme manipulation of nAu-bound DNA and digestion efficiency
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The restriction endonuclease digestion of nAu-bound DNA (strands A' and revA' to
strands A and revA respectively) was performed by incubating the conjugates with
100 unit of EcoR V at 37°C in a 200 µl reaction buffer for 20 hours. After incubation,
the enzyme was deactivated by adding 50 mM EDTA. The digested conjugates were
analyzed by agarose and polyacrylamide gel electrophoresis to confirm the high
digestion efficiency and high yield of nAu conjugated with definite number of DNA
strands. After the removal of the nAu by centrifuging the samples at 13000g for 15
min, the DNA solution was characterized by 12% polyacrylamide gel electrophoresis
(0.5X TBE as running buffer). The DNA bands were stained with ethidium bromide
and visualized by a Syngene GeneGenius UV and White Light Gel Documentation
and Analysis system. The enzyme digestion efficiency was calculated by quantifying
digested DNA fragments using gel analysis software from manufacturer.
Formation and analysis of nAu-DNA conjugate groupings
Two sets of nAu, each carrying a single DNA probe (nAu-A or nAu-revA, 1.5 pmol of
each) were mixed in a buffer containing 50 mM tris pH 8.0, 100mM NaCl and 2mM
MgCl2 at a 1:1 molar ratio. Target DNA molecules (matched and mismatched, Figure
6.1) were then added for hybridization with strand A or rev A on the nAu surface in a
tail-to-tail configuration (Figure 6.2). The ratio between the two sets of nAu-DNA
conjugates and the target DNA (nAu-A: nAu-revA: target) varied from 1:1:0.1 to
1:1:1. The hybridization of target DNA was carried out by first heating the sample to
70˚C for 2 minutes to ensure complete melting of the DNA strands and then the
samples were slowly cooled down to 25˚C at a rate of 0.2˚C / min to form stable
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duplexes. After formation of conjugate groupings, agarose gel electrophoresis (3%
agarose at 5 V/cm, 0.5X TBE as running buffer) was carried out at 4˚C to characterize
and quantify the conjugate groupings. Desired bands of conjugate groupings were
visualized by a Syngene GeneGenius UV and White Light Gel Documentation and
Analysis system or a Bio-Rad GS-800 Calibrated Densitometer. The grouping
percentage of nAu-DNA conjugates was calculated by quantifying the relative optical
intensity of the gel bands using gel analysis software from manufacturer, more
specifically, dividing the amount of conjugate groupings by the total amount of
conjugates in the same lane (unbound conjugates + conjugate groupings). Each value
reported was the average of three individual tests. After that the conjugate groupings
were extracted from the gel and recovered by electrophoretic dialysis38 for TEM
characterization.
Figure 6.2 Schematic picture of the formation of nanoparticle-DNA conjugate groupings.
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6.3 Results and discussion
6.3.1 Formation of nAu-DNA conjugate dimers using linker DNA of
different lengths
As described in previous chapters, agarose gel electrophoresis was first used to isolate
conjugates (nAu-A' and nAu-revA') bearing different number of DNA molecules.
Subsequently the conjugates with specific number of DNA strands were exposed to
endonuclease EcoR V digestion to cleave strands A' and revA' (80 bp) into Strands A
and revA (18 bases). The enzyme digestion efficiency of 10 nm nAu-bound DNA
obtained from 12% PAGE shows more than 90% digestion efficiency for both stands
A' and revA' (Figure 6.3). The use of endonuclease to cleave nanoparticle-bound DNA
has been reported by several groups158, 174-176. including ourselves242, 243. Our high
digestion efficiency here leads to short and homogeneous DNA on nAu which is
critical for the subsequent quantitative DNA analysis.
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Figure 6.3 PAGE characterization of enzyme digested 9.7 nm nAu-bound DNA. Lanes 1-8 correspond to: 10bp DNA ladder, strand A' (free), strand A' (bound), strand A' (no enzyme), strand revA' (free), strand revA' (bound), strand revA' (no enzyme) and 10bp DNA ladder.
Though nanoparticle assembles have been studied extensively, most studies use
nanoparticles with a high loading of DNA and leads to a coordinate effect from
multiple DNA linkages. To avoid this scenario, nAu-A and nAu-revA conjugates with
single DNA strands are used in this study. nAu-DNA conjugate dimer formed with
various DNA linkers (20 bases to 26 bases) was chosen as a model system. A tail to
tail structure was selected to reduce the steric hindrance and allow maximum grouping
efficiency (Figure 6.2). The conjugate dimers formed by nAu-A and nAu-revA
grouping are shown in Figure 6.4. The stoichiometric ratio of nAu-A, nAu-revA, and
linker DNA used in hybridization is 1:1:1. Since Strand A and strand revA are not
complementary to each other, a linker is needed for the formation of conjugate dimers.
Lane A in Figure 6.4 corresponds to nAu modified without strand A or revA
modification (control). Lanes B, C, D, E and F correspond to nAu-A + nAu-revA
conjugates with no linker, with 26, 24, 22, and 20-base linker DNA respectively.
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Using the band in Lane B, which contains only nAu-A and nAu-revA conjugate
monomers, we can identify that the bands on the bottom of Lanes C/D correspond to
the unbound conjugate monomers (share the same mobility with the band in Lane B)
and the bands on the top (with slower mobility) correspond to the conjugate dimers.
The presence of only a single band in Lane B indicates that there is no nonspecific
interaction between the nAu-A and nAu-revA. Therefore, the hybridization of
complementary DNA linker with these two conjugates, not nonspecific interaction,
drives this conjugate dimer formation.
A B C D E F
Figure 6.4 Formation of nAu-DNA conjugate dimers with various length of DNA linker. Lane A corresponds to nAu without strand A or revA modification (control); Lanes B corresponds to nAu-A + nAu-revA conjugates with no linker; Lanes C, D, E and F correspond to nAu-A + nAu-revA conjugates with 26, 24, 22, and 20 bases of linker DNA respectively.
With DNA linkers of different lengths, the dimer grouping percentages of 26 and 24-
base DNA linkers are 55.6% and 55.3% respectively, even 1:1:1 ratio is used to allow
complete grouping formation. Similar result was previously reported by Alivisatos and
coworkers in which complete grouping of nAu-DNA conjugates could not be achieved
even when a linker with complementary sequence of 100-base was used62, 147. In our
experiment, when the 20- and 22-base DNA linkers are used, only a single band
corresponding to conjugate monomers is unexpectedly found in the gel (Lane E and F).
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With half of the DNA linker hybridizes to each conjugate, reducing the linker from 24
to 22 bases cuts only a single base in conjugate hybridization. This single base cut,
however, results in total absence of dimer groupings. The modification of nAu with 5-
T ssDNA as surface passivation in our experiment leads to a highly negatively charged
surface. The strong electrostatic repulsion between both conjugates and perhaps linker
DNA may destabilize the hybridization of short sequences, and thus no dimer is
formed. Furthermore, while running gel electrophoresis, the resistance from the gel
may facilitate the breakage of the DNA linkage, since there are two bulky nAu
conjugates on each side of the dsDNA bridge. DNA linkers with longer
complementary sequence reduce the electrostatic repulsion between conjugates and
may facilitate more stable grouping of dimers. The critical length of DNA linker for
this particular system is found to be 24 bases.
6.3.2 Quantification of target DNA through the formation of nAu-
DNA conjugate dimers
Our nAu-DNA conjugates carry only a single DNA probe and allow quantitative
analysis of target DNA that acts as a linker. This is because each conjugate dimer
formation represents a hybridization event between a single complementary target
DNA and the two nAu-DNA conjugates. By measuring the dimer formation, in
principle, the amount of the target or linker DNA can then be quantified. In our
experiments, equal molar of the nAu-A and nAu-revA conjugates was mixed with
various ratios of 24-base target DNA (nAu-A: nAu-revA: target DNA ranges from
1:1:0.1 to 1:1:1). The target DNA amount was obtained by quantifying the percentage
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of conjugate dimers after agarose gel electrophoresis. In Figure 6.5, Lane J
corresponds to the control where nAu was conjugated without strand A/revA, and
Lanes K is the mixture of nAu-A & nAu-revA without target DNA. Only one band
corresponding to conjugate monomers is found in the gel, indicating no dimer
formation. Lanes G, H, I, L, M, N, O are samples with various ratios of nAu-A &
nAu-revA to target DNA, from 1:1:1 to 1:1:0.1 respectively. Two bands, which
correspond to conjugate monomers and dimmers, are found in the gel. The amount of
the conjugate dimers increases with the increasing molar amount of target DNA. This
trend is more clearly shown in Figure 6.6 after quantifying the dimer band intensity.
When the molar ratio of the target DNA is between zero and 0.5, the dimer grouping
percentage is directly proportional to the target DNA ratio. Such linear relationship
between the amount of target DNA and dimer formation has good potential for the
development of new target quantification assay.
G H I J K L M N O
Figure 6.5 Grouping percentage of nAu-DNA conjugates with various ratios of target DNA. Lanes G-O correspond to nAu-A: nAu-revA: target DNA ratio of 1:1:1, 1:1:0.8, 1:1:0.5, control (nAu without strand A/revA conjugation), 1:1:0, 1:1:0.3, 1:1:0.25, 1:1:0.15, and 1:1:0.1 respectively. Gel picture shows the combined results from two experiments.
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Figure 6.6 Grouping percentage of nAu-DNA conjugates with different ratios of target DNA.
With the molar ratio goes beyond 1:1:0.5, both Figures 6.5 and 6,6 show that only
minor increase in the grouping percentage (less than 20%) is found. For example,
1:1:0.8 and 1:1:1 can only result in 59% and 64% dimer formation respectively.
Similar grouping percentage is also found when conjugates with two strand revA
(nAu-2x revA) were used. The top bands in Lanes P, Q, and S in Figure 6.7
correspond to the conjugate trimers that have the lowest mobility among all the
conjugates. The second bands from the top are conjugate dimers, and bands at the
bottom are conjugate monomers. The maximum grouping percentage (dimers +
trimers) of 66% is achieved when molar ratio of 2:1:2 is used. Excess target DNA,
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however, does not further improve the grouping percentage as the grouping percentage
falls slightly to 60% when the ratio of target DNA increases to 2:1:5. Further drop in
grouping is observed upon the introduction of large excess of target DNA, as the
Lanes S and T (ratio 2:1:20 and 2:1:100) show fade bands of conjugate groupings with
only 51% and 36% grouping percentage respectively. This decrease in grouping
percentage can be attributed to the conjugate grouping inhibition by the excess target
DNA. Since each nAu-DNA conjugate may hybridize with individual target DNA
molecule separately, less conjugates are available for grouping and thus the
dimer/trimer formation is impeded.
P Q R S T
Figure 6.7 Grouping percentage of nAu-DNA conjugates with different ratios of target DNA. Lanes P-T correspond to nAu-A & nAu-revA with target DNA at a ratio of 2:1:2 (nAu-A: nAu-2x revA: target DNA), 2:1:5, control (nAu without strand A/revA conjugation), 2:1:20, and 2:1:100, respectively. Gel picture shows the combined results from two experiments.
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6.3.3 Hybridization efficiency of strand A, strand revA with target
DNA without nAu
Though the target DNA sequences are designed with minimized self-hybridization, to
preclude the possibility that the incomplete conjugate grouping is resulted from the
self hybridization of the target DNA, a control experiment involving only the target
DNA, strand A and strand revA was carried out. In this experiment, the three types of
DNA were annealed using the same protocol as the grouping of nAu-DNA conjugates.
After annealing, polyacrylamide gel electrophoresis (PAGE) was used to visualize and
quantify the amount of the formed DNA duplex. Figure 6.8 is a typical PAGE image
of 12% gel. The two bands appeared in Lanes 9-13 correspond to the annealed dsDNA
(top band) and single stranded strand A & strand revA (bottom). The bottom band
becomes weaker and weaker as the molar ratio of target DNA is raised from 0.2 to 1,
indicating that the strand A & strand revA is annealed and consumed by the increased
target DNA. In Lane 13 where 1:1:1 ratio was used, the bottom band can barely be
seen, confirming complete hybridization. Therefore, the amount of dsDNA formed in
lane 13 is assigned as 100% annealing. Table 6.1 shows quantification of the
hybridization efficiencies and increasing ratio of target DNA results in enhanced
dsDNA formation. The amount of formed dsDNA tracks closely with the amount of
target DNA introduced to the system through the entire test, even at high target DNA
ratio (1:1:1). This further confirms that the incomplete grouping of nAu conjugates
can only be attributed to the presence of highly charged nAu conjugates.
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9 10 11 12 13 14
Figure 6.8 Hybridization efficiency of pure DNA (strand A and strand revA with different ratios of target DNA). Lanes 9-13 and 6 correspond to strand A & strand revA with target DNA at a ratio of 1:1:0.2, 1:1:0.4, 1:1:0.6, 1:1:0.8, 1:1:1 and 10bp DNA ladder respectively.
Molar ratio between strand A, strand revA and target DNA 1:1:1 1:1:0.8 1:1:0.6 1:1:0.4 1:1:0.2
Percentage of dsDNA formed (obtained by dividing the dsDNA amount in each
lane by the amount in Lane 13) 100 79 62 40 18
Table 6.1 Hybridization efficiency of strand & strand revA with various ratios of target DNA.
For the grouping of nAu-DNA conjugates, the highest percentage is found to be
approximately 60-65%. This incomplete grouping can be attributed to the low number
of strand A/revA bound on nAu (1 or 2 strands per nAu) which may result in a much
lower collision rate between the conjugates and target DNA. Thus extended incubation
may be necessary to reach higher degree of hybridization. Furthermore, the relatively
heavy nAu conjugates compared with the target DNA may further reduce the collision
rate and contribute to the observed low hybridization percentage. Hybridization data in
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Figure 6.8 and Table 6.1 shows that without nAu, hybridization between strands A,
revA and target DNA is efficient and over 90%. This further suggests the possibility of
nAu interference in the hybridization process. Further study is needed to obtain clearer
understanding of DNA hybridization on nanoparticles.
To give a direct connection between conjugates with different electrophoretic mobility
and their actual structure, dimer groupings were recovered from the gel and visualized
by TEM. Figure 6.9 shows the structure of the conjugate groupings from the dimer
band extracted from Lane P. The large majority of the conjugates are participated in
the same dimer grouping structure, indicating that each discrete gel band does contain
a single type of groupings and not a mixture of several conjugate structures. A small
number of monomers and high-order groupings were observed in the TEM images,
and this may be due to the interruption by the extraction of conjugate samples from gel
as well as TEM preparation.
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Figure 6.9 TEM images of nAu-DNA conjugate dimers.
6.3.4 Single nucleotide polymorphism (SNP) discrimination using
nAu-DNA conjugate groupings
Conventional techniques for the detection of SNP using mass spectrometry or gel
electrophoresis to discriminate DNA fragments192, 244 are time consuming and
relatively costly. Chip based detection methods using fluorescent dyes or nanoparticles
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as labels have become popular in recent years136, 191. These newly emerging methods
are based on the melting temperature difference between perfectly matched and
mismatched DNA duplex, and involve a stringent wash process with precise
temperature control as well as skillful personnel and long analysis time. Furthermore,
it may be difficult to discriminate DNA targets that exhibit insignificant melting
temperature difference. Compared with the existing techniques mentioned above, our
newly proposed method offers the advantage of straightforward single-base mismatch
discrimination without the need of stringent wash. As shown in Figure 6.10, Lanes U-
AA correspond to nAu-A & nAu-revA plus 24-base perfectly matched DNA (PM),
single base matched DNA (SM1/SM2/SM3), double base matched DNA (DM), non-
complementary DNA (NC) and no target DNA respectively. There is an obvious
difference in Lane U where the PM case shows a top dimer band and a bottom
monomer band. For the DM and NC cases (Lanes Y and Z), only a single band can be
found in the gel, indicating no dimer structure is formed. For SM1, 2, and 3 cases
(Lanes V, W and X), only a single band corresponding to conjugate monomers is
found, and no false positive signal is observed. The monomeric structure in these three
lanes can be further confirmed by the one without target DNA (Lane AA), which
shows exactly the same electrophoretic mobility. Even with end-mismatched SNP
(Lanes X), which introduces least interruption to the duplex DNA structure and is
often difficult to discriminate, this method shows very good discrimination and no
sign of conjugate dimers is observed in Lanes X. The key strategy to achieve such
high selectivity in SNP discrimination is to use unfavorable conditions for
hybridization, so that only target DNA with a perfect match has a chance to hybridize
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with nAu-bound DNA. This unfavorable condition is mainly resulted from the highly
negatively charged nAu surfaces (nAu-A) which repel the target DNA from
hybridizing with the other conjugate (nAu-revA). As the result, only perfectly matched
duplex is energetically stable to form dimer grouping. Furthermore, with gel
electrophoresis, the resistance from the gel during running may assist the breakage of
conjugate groupings linked by DNA with mismatched base/bases. So far, only
synthetic DNA is used in our model system, but we believe that our results can be
extended to real samples such as blood after appropriate extraction and purification of
the genetic material.
U V W X Y Z AA
Figure 6.10 SNP discrimination using nAu-DNA conjugates. Lanes U-AA correspond to, nAu-A & nAu-revA plus 24-base perfectly matched DNA (PM), single base matched DNA (SM1/SM2/SM3), double base matched DNA (DM), non-complementary DNA (NC) linker and no target DNA, respectively.
As a currently widely used method in SNP discrimination, RT-PCR relies on the
difference in melting temperature (Tm) between perfectly matched DNA and single
base mismatched ones for discrimination. To give a direct comparison between our
nanoparticle based method and RT-PCR on SNP discrimination, a control experiment
using RT-PCR was conducted. In this experiment, Strand A, revA (both without nAu)
and matched & mismatched target DNA (PM, SM1/SM2/SM3 and DM DNA) same as
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those used in the nAu based assay were used. The discrimination was carried by
comparing the difference in the melting transition between matched and mismatched
samples. Complete matched samples results in more homogeneous disassociation of
the DNA duplex and therefore leads to a sharper melting transition than the
mismatched ones. Figure 6.11 is the RT-PCR melting transition curve of matched and
mismatched DNA samples. The PM DNA shows an obviously higher peak value and
sharper melting transition than DM DNA, where no peak can be found due to the
absence of DNA duplex. For the single base mismatched DNA, the center mismatched
SM1 and SM2 show wider melting transition and lower peak value which are different
from the PM DNA. However, the end mismatched SM3 shows almost the same
melting transition and is hardly distinguishable compared with PM DNA. This is not
unexpected, since end mismatching introduces least disturbance to the DNA duplex,
the difference in melting behavior between PM and SM3 is too small to support an
accurate discrimination. In our nAu based assay, the introduction of highly charged
and stericly bulky nAu to the DNA duplex significantly reduces its stability, therefore
even single base mismatched at the end position can lead to sufficient disturbance and
results in dissociation of the DNA duplex (Figure 6.10, lane X). This further confirms
the critical role of nAu in achieving high selectivity in SNP discrimination.
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Figure 6.11 Melting transition of matched and mismatched DNA samples. Square (brown): PM DNA, Plain curve (blue): SM3 DNA, Triangle (Black): SM2 DNA, Cross (red): SM1 DNA, Diamond (green): DM DNA.
6.4 Conclusions
In this chapter we have demonstrated a novel nAu-based quantitative DNA assay
method with SNP discrimination sensitivity and can be an ideal detection platform for
bioanalytical systems. This method combines gel electrophoresis isolation and
restriction endonuclease manipulation to produce precisely controlled nAu-DNA
conjugates which allows quantitative analysis of DNA molecules based on the
formation of conjugate groupings by target DNA linkage. A linear correlation between
the amount of target DNA and conjugate groupings was obtained at lower target DNA
concentration and can further be exploited for target quantification. For SNP study,
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single base mismatch discrimination is achieved for both the end and center
mismatched cases. The method is particularly suitable for creating a quantitative and
selective DNA detection for biomedical applications.
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Chapter 7
Fabrication of gold nanoparticle based nano-
groupings with well-defined structure
7.1 Introduction
Driven by the need from nanotechnology, materials science, biological and biomedical
applications, various approaches for the fabrication of defined nanostructures have
been developed in recent years130. Among them, the bottom up approach in which
nanometer-scaled building blocks are assembled into larger entities attract most
attention18. Promising applications of this approach include the programmable
organization of metal and semiconductor nanoparticles133, 220, 245, 246, numerous
bioanalytical techniques140, 178, 190, 247, and nanomechanical devices200, 248. Within these
areas, great efforts have been directed to self-assembly of hybrid materials from
inorganic nanoparticles and biomolecules, such as DNA, RNA and proteins131, 132.
Metallic, semiconductor and magnetic nanoparticles are commonly used components
in the bottom up self-assembly. Due to their small size, nanoparticles show unique
chemical and physical properties and have great potentials in the development of
chemical sensors114, biomolecular electronics11, 218 and biological applications17, 18.
However, the realization of above applications requires deliberate and accurate
arrangement of nanoparticles into pre-designed structures. DNA molecules, which
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possess unique molecular recognition capability, mechanical rigidity as well as high-
precision enzyme processibility, are particular attractive for applications demanding
accurate arrangement and high degree of structural control134, 149, 249. Furthermore,
DNA molecules can be synthesized readily in a predesigned length and sequence with
various functional groups for conjugation or fluorescent detection. Thus, DNA is a
highly promising molecule for directing programmable self-assembly, and has been
extensively used to fabricate nanostructured scaffolds as well as position proteins,
metal or semiconductor nanoparticles, and other molecular devices155, 250, 251. The
combination of DNA molecules with inorganic nanoparticles has led to the
development of novel hybrid materials that incorporate the sequence specific
recognition capability of DNA molecules with the size-dependent optical, electrical,
magnetic and catalytic properties of nanoparticles.
The collective properties of nanoparticles are mainly dependent on their hierarchical
arrangements and interparticle distance. For example, noble metallic nanoparticles
have plasmon resonances in the visible range and do not blink or bleach. The
wavelength of this plasmon resonance shifts upon varying the distance between
nanoparticles. Applying the shifting of plasmon resonance as nano-scale distance
monitoring tool requires precise control over the distance between nanoparticles to
allow the propagation of light along a defined path efficiently145, 146. However, there
have been only a limited number of studies focused on fabricating nanoparticle-DNA
assemblies with very well-defined structure such as dimer, trimer and other higher
order multimers62, 147.
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In previous chapters, we have shown that gold nanoparticle-DNA (nAu-DNA)
conjugates bearing specific number of short DNA can be prepared by gel
electrophoresis isolation followed by restriction endonuclease manipulation of the
nAu-bound DNA242. In this chapter, we further demonstrate how these conjugates can
be used to construct well-defined nano-groupings. The effects of interparticle spacing
as well as nAu-linker DNA interactions on the final conjugate grouping were studied.
In our approach, two sets of specially designed nAu-DNA conjugates are first
modified with definite number (1, 2, 3…) of short single stranded DNA (ssDNA) that
are not directly complementary to each other. These conjugates do not recognize each
other until a complementary linker DNA, which hybridizes to the DNA on both sets of
conjugates, is introduced. Only conjugate groupings with defined structure (dimer or
trimer) can form due to the definite number of DNA on each nAu. The resulting
conjugate groupings are analyzed and quantified by agarose gel electrophoresis. The
size differentiation ability of gel electrophoresis enables strict discrimination between
different nanoparticle grouping structures (monomer, dimer and trimer) and allows
separation of them.
7.2 Materials and methods
Materials
Hydrogen tetrachloroaurate (III) trihydrate, trisodium citrate dihydrate, tannic acid, 4,
4’-(phenylphosphinidene) bis-(benzenesulfonic acid) (PPBS), dithiothreitol (DTT),
NaCl, MgCl2, and tris-borate-EDTA (TBE) buffer were purchased from Sigma-
Aldrich. The synthetic DNA (modified with thiol linker at the 5’ end) was purchased
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from Integrated DNA Technologies (IDT). Restriction endonuclease EcoR V (500000
unit/ml) was obtained from New England Biolabs. 30% acrylamide/Bis solution (29:1)
and ethidium bromide were obtained from Bio-Rad Laboratories. Agarose was
purchased from Cambrex. Dialysis tubing (Float-A-lyzer, MWCO: 3500) was
obtained from Spectra/Por. Milli-Q water with resistance >18MΩ/cm was used
throughout the experiments.
Synthesis and characterization of gold nanoparticles (nAu)
Gold nanoparticles were synthesized by the reduction of hydrogen tetrachloroaurate
(III) tetrahydrate by trisodium citrate dihydrate and tannic acid40 as described in
chapter 3. In order to determine the size and size distribution of the resulting
nanoparticles, TEM characterization was performed on a Philips CM300 FEG system
operating at 200 kV. At least 200 particles were sized from TEM micrographs via
graphics software “Image-Pro Express” (Media Cybernetics). The mean particle
diameter was 10 nm and the size distribution was within 15% of the mean diameter.
Preparation of nAu-DNA conjugates
The DNA sequences used in this chapter are shown in Figure 7.1 (complete sequences
are shown in Figure A1 and A2, Appendix I). Two complementary single-stranded
DNA (ssDNA), one modified with a thiol linker, were allowed to hybridize to form a
double-stranded DNA (dsDNA). Strand A', strand C' and strand revC2' were modified
with a 5' thiol linker, whereas strand revA' and strand revC1' were with a 3' thiol
linker. These dsDNA were used to prepare different sets of conjugates (nAu-A' and
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nAu-revA', nAu-C' and nAu-revC1', and nAu-C' and nAu-revC2') for conjugate
grouping studies. The detailed procedure of conjugate preparation can be found in our
previous work242. Briefly, 2 µM dsDNA was mixed with nAu for 2 hours followed by
5-T ssDNA for surface passivation. Excess reagents were then removed by repeated
washing and centrifuging the conjugates with 0.5X TBE.
Agarose gel electrophoresis isolation and extraction of nAu-DNA conjugates
Agarose gel electrophoresis (3% agarose at 5 V/cm, 0.5X TBE as running buffer) was
carried out for 4 hours to separate the conjugates bearing from one to three dsDNA
molecules. Discrete bands can easily be identified because of the wine-red color of
nAu. Desired bands were extracted from the gel and the conjugates were then
recovered by electrophoretic dialysis38. After recovered from agarose gel, the
conjugates were purified by repeated washing (with 50 mM tris buffer, pH 8.0) and
centrifuging to ensure complete removal of EDTA which may deactivate the
restriction endonuclease.
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Figure 7.1 DNA sequences used in this chapter. For strand A', revA', C', revC1' and revC2', the underlined sequences are the recognition site of EcoR V and the arrows indicate the enzyme cleavage site.
Chapter 7_____________________________________________________________
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Enzyme manipulation of nAu-bound DNA and digestion efficiency
The restriction endonuclease digestion of dsDNA (strand A' to strand A, strand revA'
to strand revA, etc.) was performed by incubating the conjugates with 100 unit of
EcoR V at 37°C in a 200 µl reaction buffer for 20 hours. After incubation, the enzyme
was deactivated by adding 50 mM EDTA. The digested conjugates (nAu-A, nAu-revA,
etc.) were analyzed by agarose and polyacrylamide gel electrophoresis to confirm the
digestion efficiency and yield of nAu conjugates with definite number of DNA strands.
For polyacrylamide gel electrophoresis (PAGE), the digested samples were first
centrifuged (13000g for 15 min) to separate digested DNA from nAu, the digested
DNA was loaded into a 12% gel for electrophoresis (0.5X TBE as running buffer).
The DNA bands were stained with ethidium bromide and visualized by a Syngene
GeneGenius UV and White Light Gel Documentation and Analysis system. The
enzyme digestion efficiency was calculated by quantifying digested DNA fragments
using gel analysis software from manufacturer.
Formation and analysis of nAu-DNA conjugate groupings
A paired set of nAu-DNA conjugates (nAu-A + nAu-revA, nAu-C + nAu-revC1, etc.),
each carrying a single DNA strand (1.5 pmol of each), were mixed in a buffer
containing 50mM tris pH 8.0, 100mM NaCl and 2mM MgCl2 at 1:1 molar ratio.
Different linker DNA molecules (Figure 7.1) were then added to the mixture to allow
the hybridization of linker DNA with the complementary strands attached to the nAu
surface in a tail-to-tail configuration (Figure 7.2). The hybridization of
complementary DNA to link nAu conjugates was carried out by first heating the
Chapter 7_____________________________________________________________
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sample to 70˚C for 2 min to ensure complete melting of the DNA strands followed by
slow cooling down to 25˚C at a rate of 0.2˚C / min. After formation of conjugate
groupings, agarose gel electrophoresis (3% agarose at 5 V/cm, 0.5X TBE as running
buffer) was carried out at 4˚C to characterize and quantify the conjugate groupings.
Desired bands of conjugate groupings were visualized by a Syngene GeneGenius UV
and White Light Gel Documentation and Analysis system or a Bio-Rad GS-800
Calibrated Densitometer. The grouping percentage of nAu-DNA conjugates was
calculated by quantifying the relative optical intensity of the gel bands using gel
analysis software from manufacturer, more specifically, dividing the amount of
conjugate groupings by the total amount of conjugates in the same lane (unbound
conjugates + conjugate groupings). Each value reported was the average of three
individual tests. For the study on relative mobility of conjugate groupings in 3%
agarose gel, the elapsed time and the corresponding position of the conjugate
groupings were monitored and recorded during electrophoresis. The positions of the
gel bands from seven different electrophoretic durations were then measured. The
conjugate groupings were next extracted from the gel and recovered by electrophoretic
dialysis116 for TEM characterization.
Chapter 7_____________________________________________________________
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7.3 Results and discussion
7.3.1 Grouping percentage of nAu-DNA conjugates using linker DNA
of different lengths
As reported earlier by Alivisatos and coworkers154, agarose gel electrophoresis can
only be used to isolate nAu-DNA conjugates bearing definite numbers of long DNA
molecules. For DNA of less than 50 bases, electrophoresis fails since the
electrophoretic mobility difference between conjugates bearing different number of
DNA strands is insignificant. To obtain nAu-DNA conjugates bearing definite
numbers of short DNA, we have used an approach that combines both agarose gel
electrophoresis and endonuclease digestion242. First, agarose gel electrophoresis is
used to isolate nAu-DNA conjugates bearing different number of long DNA strands.
Subsequently the conjugates with definite number of DNA strands are treated with
EcoR V digestion to cleave DNA into shorter strands. In this way, the number, length,
and sequence of the DNA strands on the resultant conjugates is precisely controlled.
Restriction endonucleases are enzymes that recognize short specific DNA sequences
and cleave the DNA201. Their use on nAu-bound DNA has been reported by several
groups158, 174-176 including ourselves242. The enzyme digestion efficiency of 10 nm
nAu-bound DNA obtained from PAGE (data not shown) shows that above 90%
digestion can be achieved for all the five dsDNA (strands A', revA', C', revC1', and
revC2'). The high digestion efficiency leads to short and homogeneous DNA on nAu
that is critical for the subsequent conjugate assembly formation.
Chapter 7_____________________________________________________________
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When nAu-A and nAu-revA conjugates were mixed with linker DNA, a tail-to-tail
dimer configuration (Figure 7.2) was formed to reduce the steric hindrance and allow
maximum grouping percentage. Since both nAu-A and nAu-revA were bound with
only a single DNA strand, other higher order structures (trimers, tetramers, etc.) were
not obtained. The identification of these conjugate groupings is shown in Figure 7.3.
The stoichiometric ratio of nAu-A, nAu-revA, and linker DNA used for assembling is
1:1:1. Since stand A and strand revA are not complementary to each other, a linker is
needed for the formation of conjugate groupings. Lane A corresponds to the
conjugates without strand A or revA modification (control). Lanes B, C, D, and E
correspond to conjugates nAu-A & nAu-revA with no linker, 26-, 24-, and 22-base
linker DNA respectively. The band in lane B shows slightly slower mobility than the
one in lane A, indicating that the 18-base strand A or revA is not released by the
enzyme manipulation and subsequent purification treatment. Using the band in lane B,
which contains only nAu-DNA conjugate monomers, we can identify that the bands
on the bottom of lanes C/D correspond to the unbound conjugate monomers (share
exactly the same mobility with the band in lane B) and the bands on the top (with
slower mobility) correspond to the conjugate dimers. The dimers with two nAu
particles exhibit slower mobility due to the overall larger size that slows their
movement through the gel. The presence of a single band in lane B indicates that there
is no nonspecific interaction between the two sets of conjugates. Therefore, the
bonding of the complementary DNA linker with nAu-A and nAu-revA, not
nonspecific interactions, drives this dimer formation.
Chapter 7_____________________________________________________________
116
Figure 7.2 Schematic picture of the formation of nAu-DNA conjugate groupings using either the pair nAu-A + nAu-revA or nAu-C + nAu-revC1 with linker DNA.
A B C D E
Figure 7.3 Conjugate grouping of nAu-DNA conjugates with various length of DNA linker. Lane A corresponds to conjugate without DNA modification, and lanes B, C, D, and E correspond to conjugates nAu-A + nAu-revA with no linker, with 26-, 24-, and 22-base linker DNA respectively. The stoichiometric ratio of nAu-A, nAu-revA, and linker DNA is 1:1:1.
Inherently, complementary DNA hybridization is highly efficient, however for
dimerization of nAu conjugates via linker DNA, a mixture of monomers and dimers is
found in the gel. As seen in lanes C and D in Figure 7.3, with linker DNA of different
lengths, the grouping percentages of 26- and 24-base linkers are only 55.6% and
55.3% respectively, even the nAu-A:nAu-revA:linker ratio is 1:1:1 to allow complete
dimer formation. Similar results was previously reported by Alivisatos and coworkers
in which complete grouping of nAu-DNA conjugates could not be achieved when a
linker with complementary sequence of 100-base was used62, 147.
Chapter 7_____________________________________________________________
117
There is a number of factors which may affect the grouping percentage of nAu
conjugates, such as length of the complementary part of nAu-bound DNA to linker
DNA, the length of linker DNA (distance between nAu conjugates in the resultant
groupings) as well as the ways of formation of DNA linkage (either through linker
DNA or via direct hybridization of two nAu-bound DNA as shown in Figure 7.4).
Determining which is the dominant factor controlling the nanoparticle grouping is
crucial for achieving precisely organized nanoassemblies with high yield. The
assembly of nAu conjugates using various kinds of linker DNA is shown in Figure 7.5.
Lanes F, H and J correspond to nAu without DNA conjugation (control) and lanes G
and I correspond to conjugates nAu-A & nAu-revA with 26-base and 48b+22bp
linkers respectively. Lane K is nAu-C & nAu-revC1 with 60-base linker. Note that the
48+22bp linker is a double stranded DNA with “sticky” ends that are complementary
to the nAu-bound strands A and revA. The use of 48b+22bp dsDNA and 60-base
ssDNA linkers (lanes I and K) yield similar gel band distribution and grouping
percentage compared with conjugates linked by 26-base ssDNA (lane G). The top
bands in the gel are the conjugate dimers and the maximum grouping percentages of
these three linkers are approximately 55%. Increasing interparticle distance should
reduce the charge repulsion between nAu conjugates and lead to more stable grouping
structure as well as higher grouping percentage. However, no such trend is found in
Figure 7.5 where longer DNA linker was used. Interestingly, an improved grouping
percentage is obtained when direct hybridization between nAu-bound DNA is adopted
without the linker DNA. In lanes M, N and O of Figure 7.6, conjugate grouping
structures (dimers, trimers and tetramers) appear to be the primary product and the
Chapter 7_____________________________________________________________
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monomer bands are extremely faint, implying the higher consumption of the
monomers. The grouping percentages are found to be 70-80% for lanes M, N and O,
which are obviously higher than those obtained in the three-strand hybridization
system shown in Figure 7.5. We attribute this increased grouping percentage to two
possible reasons: 1) Compared with free DNA in solution, the translational mobility of
nAu-bound DNA is relatively low. This may lead to a low collision rate between
complementary DNA. In addition, the low number of DNA bound on nAu (1-3 DNA
per nAu) relative to more than 100 DNA strands per nAu reported previously202 may
further reduce the collision probability. Direct linkage between two nAu-bound DNA
may be more favorable since it may require shorter collision time compared with
grouping via linker DNA where the linkage of two nAu-bound DNA to the same
complementary linker DNA is required. 2) Due to the surface modification of nAu
with highly charged 5-T ssDNA, the strong electrostatic repulsion on the surface of
nAu may force the nAu-bound DNA to adopt a stretching conformation than free
linker DNA and facilitate the hybridization of two nAu-bound DNA.
Figure 7.4 Schematic picture of the direct pairing of nAu-DNA conjugate using nAu-C + nAu-revC2 without linker DNA.
Chapter 7_____________________________________________________________
119
F G H I J K
Figure 7.5 Conjugate grouping of nAu-DNA conjugates with various length of DNA linker. Lanes F, H and J corresponds to conjugate without DNA modification and lanes G, I and K correspond to conjugates nAu-A & nAu-revA with 26-base, 48b+22bp and nAu-C & nAu-revC1 with 60-base linker DNA, respectively. Gel pictures shown are combined results from different sets of experiments.
L M N O
Figure 7.6 Conjugate groupings formation via direct linkage of two nAu-bound DNA. Lanes L, M, N and O correspond to nAu-C, nAu-C + nAu-revC2, nAu-C + nAu-2XrevC2 and nAu-C + nAu-3XrevC2, respectively.
Chapter 7_____________________________________________________________
120
7.3.2 Effect of hybridization conditions on final grouping percentage
A further study on the grouping percentages under different post-annealing
hybridization period at 25°C was carried out using nAu-C, nAu-revC1 and 60-base
linker DNA (ratio of 1: 1: 1) and the results are shown in Figure 7.7. It can be found
that the grouping percentage is proportional to the hybridization time. With increasing
hybridization time, the grouping percentage increases and the maximum grouping of
approximately 60% is obtained after 24 hours. This percentage almost doubles
compared with the case without post-annealing hybridization (hybridization time of
zero). However prolonged hybridization does not further improve the grouping
percentage much. For example, less than 4% increase is observed as the hybridization
time extends from 12 to 24 hours. This increase in grouping percentage further
indicates the low collision rate as argument described in the previous section. Longer
hybridization time increases the collision rate and therefore facilitates conjugate
groupings formation.
Chapter 7_____________________________________________________________
121
Figure 7.7 Study on the grouping percentage of nAu-DNA conjugates under various hybridization time.
Ion strength in the hybridization buffer is another important factor that affects the nAu
conjugate grouping percentage. Buffer with high ion strength is commonly used to
screen the electrostatic repulsion between nAu conjugates and facilitate the assembly
process. To determine the effect of ion strength on the grouping of DNA-linked nAu
conjugates, we investigated the grouping percentage as a function of MgCl2
concentration in hybridization buffer. In this test, nAu-2A, nAu-2XrevA (two revA
strands per nAu) and 26-base linker DNA (nAu-A: nAu-2XrevA: linker DNA= 2:1:2)
and 50 mM NaCl was used in all samples. As shown in Figure 7.8, when 2 mM or
more MgCl2 is used, the nAu conjugates assemble into dimer and trimer structures and
at least 50% grouping percentage can be achieved (lanes R-T). However, no grouping
structure is formed when the salt concentration drops to 50mM NaCl and no MgCl2.
This obvious difference in grouping percentage is evidently shown with the clear
absence of gel band corresponding to conjugate groupings in lane Q. In contrast,
Chapter 7_____________________________________________________________
122
complementary DNA of the same sequences (in the absence of nAu) hybridizes into
dsDNA well in 50mM NaCl or less. Such a significant difference in grouping
percentage was not unexpected. As shown above, the 5-T ssDNA modification leads
to a highly negatively charged surface and stronger electrostatic repulsion among nAu
when they come close to each other during DNA hybridization. Bivalent ions, such as
Mg2+ can efficiently screen this electrostatic repulsion and allow more hybridization
events to take place, leading to an improved grouping percentage. Our results clearly
show that electrostatic interactions between nAu-nAu conjugates and nAu-linker DNA
play a critical role in the conjugates assembly process and these interactions can be
easily tailored by adjusting the ion strength in the hybridization buffer which can
enable reversible control on the grouping and ungrouping of nAu-DNA conjugates. 2
mM Mg2+ is sufficient for efficiently screen the electrostatic repulsion and less than
10% further improvement in grouping percentage is obtained when the Mg2+
concentration increases from 2 mM to 30 mM.
P Q R S T
Figure 7.8 Study on the effect of ion strength of hybridization buffer on the nAu-DNA conjugates grouping percentage. Lane P is corresponding to conjugate without DNA modification and lanes Q-T correspond to hybridization buffer with zero, 2, 10, and 30 mM MgCl2.
Chapter 7_____________________________________________________________
123
7.3.3 Electrophoretic mobility of conjugate groupings linked by
various length of linker DNA
The electrophoretic mobilities of conjugate dimers and trimers linked by different
length of linkers were compared using the relative mobility of gel bands containing
different conjugate groupings. The relative mobility is determined as the ratio of
migration distance of conjugate groupings relative to that of the nAu without DNA
modification. The relative mobility of the conjugate groupings is found to be a
function of the length of linker DNA and the number of nAu involved in the grouping.
As shown in Table 7.1, the difference in the relative mobility between 26-base and 60-
base linker DNA linked grouping dimers/trimers is only 0.1, even there is a 34 bases
difference in the linker length (approximately 11 nm based on Watson-Crick pairing).
The addition of an extra 10 nm nAu and formation of higher order of structure is more
effective in reducing the mobility than using a longer DNA linker. This can be seen
from the approximately 0.2 relative mobility difference between conjugate dimers and
trimers among all linker cases. It is worth to point out that conjugate groupings via
direct linkage of nAu-bound DNA (nAu-C and nAu-revC2) have an actual spacing of
50b between two nAu; however, they exhibit slower mobility (Table 7.1) compared
with conjugate groupings linked by 48+22bp dsDNA (actual spacing between two
nAu is 58b). These phenomena can be seen for both conjugate dimer or trimer cases.
As reported by previous studies, the mobility of conjugate groupings in agarose gel is
decided by their size62, 154. This abnormal mobility behavior can be attributed to the
formation of more rigid DNA duplex by direct linkage compared with the use of linker
DNA. The presence of linker DNA results in additional hinges in the middle of the
Chapter 7_____________________________________________________________
124
DNA duplex and may make the resultant conjugate grouping more flexible in going in
between pores of the gel during electrophoresis.
DNA liner 26-base ssDNA
48+22bp dsDNA
Direct linkage without linker DNA
60-base ssDNA
Relative mobility of conjugate dimer 0.66 0.60 0.57 0.54
Relative mobility of conjugate trimer 0.45 0.41 0.39 0.35
Table 7.1 Relative mobility of conjugate groupings linked by different length of linker DNA.
To give a direct correlation between conjugates with different electrophoretic mobility
and their actual structure, conjugate groupings were recovered from the gel and
visualized by TEM. Figure 7.9 shows the structure of conjugate dimer, trimer and
tetramer extracted from corresponding gel bands. The large majority of the conjugates
are participated in the same grouping structure, indicating that each discrete gel band
does contain a single type of groupings and not a mixture of several conjugate
structures. A small number of monomers and high-order groupings observed in the
TEM images may be due to the interruption by the extraction of conjugate samples
from gel as well as TEM preparation processes.
Chapter 7_____________________________________________________________
125
Dimer Trimer Tetramer
Figure 7.9 TEM images of nAu-DNA conjugate groupings.
7.4 Conclusions
In this chapter, nAu conjugates bearing a definite number of short DNA were used to
construct nanoassemblies with well-defined structure. Various factors that affect the
grouping percentages of nAu-DNA conjugates were investigated. The conjugate
groupings were analyzed by gel electrophoresis and discrete gel bands corresponding
to groupings with defined structures were observed. The results showed that direct
linkage of two nAu-bound DNA, longer hybridization time and higher ion strength
buffer lead to higher grouping percentage of nAu-DNA conjugates. Furthermore, it
was found that the number of nAu involved in the grouping structure is more effective
in deciding its electrophoretic mobility than the length of linker DNA. TEM
characterization further demonstrated that conjugate groupings extracted from each gel
Chapter 7_____________________________________________________________
126
band consist of the expected grouping structure. This confirms that gel electrophoresis
is an efficient tool for isolation of small grouping structures of nAu-DNA conjugates.
The results obtained in this study provide additional characteristics of the
hybridization pattern of nAu-bound DNA and can be a critical step towards achieving
more defined and complex assembly of nanoparticles. Finely tailored nanoparticle
assemblies can find application in a wide range of area, such as fabrication of
nanomaterials, nanoelectronics, and nanomechanical systems.
Chapter 8_____________________________________________________________
127
Chapter 8
Conclusions
In this Ph.D. work, the feasibility of combining gel electrophoresis isolation and
restriction enzymatic manipulation of nAu-bound DNA for fabricating nAu bearing
definite number and length of DNA strands has been demonstrated. By exploiting
these specially designed nAu-DNA conjugates, a quantitative DNA detection method
with SNP discrimination ability has been developed and well organized nano-
groupings have been constructed as well. The properties of these nano-grouping are
exploded and some rather intriguing results were obtained. The major findings of this
thesis study include the following:
1. The feasibility of enzymatic manipulation of nAu-bound DNA by restriction
endonuclease is demonstrated. We found that the 5-base short ssDNA surface
passivation on nAu is the key factor that increases the digestion efficiency since
the ssDNA can effectively prevent the non-specific adsorption of restriction
enzyme as well as the DNA on nAu. The ssDNA surface passivation on nAu was
also found to be facilitated in high ionic strength conditions. Quantitative results
from PAGE and fluorescent study show that the digestion efficiency of nAu-bound
Chapter 8_____________________________________________________________
128
DNA as high as 90+% which is similar to free DNA digestion can be achieved
under optimal conditions.
2. A new methodology that combines agarose gel electrophoresis isolation and
enzyme manipulation of nAu-bound DNA has been developed. We showed that
agarose gel electrophoresis is capable of isolate nAu-DNA conjugates bearing
specific number of 80-base DNA and results in no impair on the enzyme
processibility of nAu-bound DNA. For each of nAu-DNA conjugates recovered
from agarose gel, a high digestion efficiency of 91.8% can be achieved by
restriction endonuclease manipulation on nAu-bound DNA. In this way, both the
number and length of nAu-bound DNA can be precisely controlled.
3. We have demonstrated a novel nAu-based quantitative DNA assay method with
SNP discrimination sensitivity and can be an ideal detection platform for
bioanalytical systems. This method exploits the well defined nAu-DNA conjugates
fabricated by gel electrophoresis isolation and restriction endonuclease
manipulation and allows quantitative analysis of DNA molecules based on the
formation of conjugate groupings by target DNA linkage. A linear correlation
between the amount of target DNA and conjugate groupings was obtained at lower
target DNA concentration and can further be exploited for target DNA
quantification. For SNP study, single base mismatch discrimination is achieved for
both the end and center mismatched cases.
Chapter 8_____________________________________________________________
129
4. Nano-groupings with well-defined structures (dimers, trimers and other higher
order multimers) were successfully constructed using nAu conjugates bearing a
definite number and length of DNA. These nano-conjugate groupings were
analyzed using gel electrophoresis and discrete gel bands corresponding to
groupings with defined structures were obtained. The results showed that direct
linkage of two nAu-DNA conjugates without linker DNA, longer hybridization
time and higher ion strength buffer lead to higher degree of grouping. For nano-
grouping formation, a minimum length of linker DNA of 24-base is needed for our
nAu-DNA conjugate system. Further increase in the linker length cannot improve
the grouping percentage furthermore. Furthermore, it was found that the number of
nAu involved in the grouping structure is more effective in deciding its
electrophoretic mobility than the length of linker DNA. TEM characterization
further demonstrated that conjugate groupings extracted from each gel band
consist of the expected grouping structure. This confirms that gel electrophoresis is
an efficient tool for isolation of small grouping structures of nAu-DNA conjugates.
The results obtained in this study provide additional characteristics of the
hybridization pattern of nAu-bound DNA and can be a critical step towards achieving
more defined and complex assembly of nanoparticles. Such finely tailored
nanoparticle assemblies can find application in a wide range of area, such as
fabrication of nanomaterials, nanoelectronics, and nanomechanical systems.
Chapter 9_____________________________________________________________
130
Chapter 9
Suggestions for future work
9.1 Further study on the hybridization of nAu-DNA
conjugates
Hybridization of DNA molecules has tremendous impacts on fundamental biology &
nanoscience research as well as biomedical & clinical applications, and it has been
extensively studied in the solution phase and at a solid/liquid interface252, 253. The
hybridization between nAu-bound DNA and formation of nAu aggregates have been
extensively investigated by Mirkin and co-workers254. In their systems, multiple DNA
duplex linkages are formed between nAu conjugates to induce a coordinate effect in
the DNA hybridization and melting process. In contrast, few investigations have been
focused on the hybridization between two individual nAu-bound DNA and formation
of defined grouping structure (dimers trimers and other multimers). Due to the
presence of a bulky and highly negatively charged nAu, the hybridization and melting
of nAu-bound DNA may differ from the bulk aqueous environment in many ways. A
thorough investigation on the kinetics and thermodynamics of nAu-bound DNA
hybridization in this nAu grouping system will lead to a better understanding on the
hybridization mechanism and optimization of nAu based DNA biosensors.
Chapter 9_____________________________________________________________
131
We have shown in Chapters 6 and 7 that there is a significant difference in the
grouping percentage between 22 and 24-base DNA linker but no obvious difference
for 24 and 60-base linker DNA. Furthermore, the grouping of nAu-DNA conjugates
via linker DNA is substantially lower than that achieved in solution phase. Since the
presence of nAu may significantly destabilize the nAu-bound DNA duplex and affect
the hybridization thermodynamic equilibrium between bound and free DNA,
excessively long incubation time may be needed to reach equilibrium. Due to the low
DNA loading on nAu and high spectral absorption of nAu within the UV-vis range,
the hybridization/melting process cannot be studied by monitoring the changes of UV
absorption of DNA at 260nm. Therefore, we propose using fluorescence resonance
energy transfer (FRET) to monitor the grouping and disassociation process of nAu-
DNA conjugate groupings and study the kinetics and thermodynamic of hybridization
between two individual nAu-bound DNA. FRET is extremely sensitive to small
(nanometer scale) separation distance changes between the donor and acceptor which
makes it ideal for real-time monitoring of conformation changes of biomolecules, such
as DNA. FRET assay is particularly suitable for our nAu-DNA conjugate system,
since nAu has been demonstrated to be an efficient fluorescent quencher139. The
hybridization and melting of two nAu-bound DNA leads to distance variation between
the dye molecule and nAu and therefore results in detectable fluorescent signal
changes. Since there are a definite number of hybridization/melting events in the
system, highly predictable and reproducible results can be expected.
Chapter 9_____________________________________________________________
132
Besides experiment work, a modulation study on the hybridization/melting kinetics
and thermodynamics will give a clearer understanding of this particular system for
further development and optimization as a DNA detection assay.
9.2 FRET based quantitative DNA detection using nAu and
quantum dot as efficient fluorescent acceptor and donor
We have shown in Chapter 7, nAu-DNA conjugate groupings with defined structure
(dimers, trimers…) can be fabricated. For applications in biomolecule detection, a
more useful grouping structure is the one composed of nAu and quantum dots (QDs).
In recent years, QD have been widely studied due to their unique optical properties
and great potential to overcome the limitations of the conventional fluorophore. QD
has high quantum yields, exceptional photochemical stability, broad excitation, and
size-tunable emission spectra with narrow bandwidth255. Besides its high quenching
efficiency, nAu has wide absorbance spectra, which makes it possible for quenching
of multiple QD with different emission spectra. The combination of nAu with QD as
efficient fluorescent acceptor and donor in the FRET based DNA detection system can
largely improve the detection sensitivity and screen throughput. Our gel
electrophoresis isolation and enzymatic manipulation method can be applied to
produce nAu-QD groupings with defined structure and interparticle distance. Such
grouping strategy has the potential to make the FRET assay more accurate and
quantitative results can be obtained based on the quenching efficiencies. Due to the
Chapter 9_____________________________________________________________
133
strong discrimination ability of nAu conjugates as shown in chapter 6, a high SNP
selectivity can be expected as well.
9.3 Application of nAu-DNA conjugates in chip-based DNA
detection
The chip-based, high throughput DNA screening technique which relies on fluorescent
dye labeling for target identification is currently widely used in medical,
pharmaceutical, and forensic applications256, 257. Due to several intrinsic disadvantages
of fluorescent dye molecules, nAu has been exploited as an alternative color label in
the chip based DNA sequence analysis. Currently, nAu bearing multiple DNA has
been adopted, but it may lead to the formation of indefinite number of DNA duplex
linkage between the nAu and chip surface, making the target DNA quantification
difficult136, 232. The application of our defined nAu-DNA conjugates results in a
precise number of DNA duplex between the nAu and chip which is directly
proportional to the amount of nAu immobilized on the chip surface. Therefore a more
accurate quantification of target DNA can be expected. Furthermore, due to the strong
discrimination ability of this particular kind of nAu-DNA conjugates as shown in
Chapter 6, the stringent wash, which is necessary in most chip based assays for
achieving high selectivity, can be eliminated to simplify the overall procedure.
Chapter 9_____________________________________________________________
134
9.4 Fabrication of multiple functionalized nAu-DNA
conjugates bearing different DNA sequences and its
application in SNP discrimination
The simultaneous detection of multiple DNA targets is an important aspect for the
development of novel DNA biosensor. Currently, in most of the studies on nAu based
DNA biosensor, nAu functionalized with only one DNA sequence is used. This
singularity in the recognition capability of nAu-DNA conjugates limits their
application in the paralleled multiple targets detection. nAu bearing a definite number
of multiple DNA sequences is desired for quantitative analysis of DNA samples with
improved processing ability and throughput. Such multi-functionalized nAu-DNA
conjugates can be fabricated using agarose gel electrophoresis isolation followed by
restriction enzyme manipulation as shown in Figure 9.1. First, DNA of difference
sequences is attached to nAu (these DNA molecules need to be of different lengths to
endow mobility difference to conjugates bearing different sequences of DNA in the
gel for isolation purpose). Then, restriction enzyme is used to cut the nAu-bound DNA
into desired lengths. In this way the number, sequence (recognition functionality), and
length of DNA on each nAu can be precisely controlled. These multiple functionalized
nAu-DNA conjugates can be used in DNA SNP detection in real samples such as
blood after appropriate extraction and purification of the genetic material.
Chapter 9_____________________________________________________________
135
Figure 9.1 Fabrication of nAu-DNA conjugates bearing specific number of short DNA with multiple sequences.
Reference_____________________________________________________________
136
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Appendix I____________________________________________________________
155
Appendix I
Complete sequences of DNA used in Chapters 6 and 7
Figure A1 Complete sequences of Strand A’ and Strand revA’.
Appendix I____________________________________________________________
156
Figure A2 Complete sequences of Strand C’, Strand revC1’ and Strand revC2’.
Appendix II____________________________________________________________
157
Appendix II
List of Publications
Journal publication:
(1) Qin, W. J.; Yung, L. Y. L. Fabrication of gold nanoparticle based nano-groupings with well-defined structure. Manuscript in preparation.
(2) Qin, W. J.; Yung, L. Y. L. Nanoparticle based quantitative DNA detection
with single nucleotide polymorphism (SNP) discrimination selectivity. Manuscript in preparation.
(3) Tan, W. L.; Qin W. J.; Yung, L. Y. L. Difference in "Base Pair to Termini"
affects the enzymatic digestion of nanoparticle-bonded DNA. Biomacromolecules 2007, 8, 750-752.
(4) Qin, W. J.; Yung, L. Y. L. Efficient manipulation of nanoparticle-bound DNA
via restriction endonuclease. Biomacromolecules 2006, 7, 3047-3051.
(5) Qin, W. J.; Yung, L. Y. L. Nanoparticle-DNA conjugates bearing a specific number of short DNA strands by enzymatic manipulation of nanoparticle bound DNA. Langmuir 2005, 21, 11330-11334.
Conference publication:
(1) Qin, W. J.; Yung, L. Y. L. AIChE Annual Meeting, San Francisco, CA, USA, November 2006.
(2) Yung, L. Y. L.; Qin, W. J. Abstracts of Papers of The American Chemical
Society 2006, 231, Atlanta, GA, USA, March 2006.
(3) Qin, W. J.; Yung, L. Y. L. Abstracts of Papers of The American Chemical Society, 230, Washington, DC, USA, August 2005.
(4) Qin, W. J.; Yung, L. Y. L. International Conference on Materials for
Advanced Technologies (ICMAT 2005) Suntec City, Singapore, July, 2005.