A novel microfluidic device for rapid melanoma diagnosis

Final Project Paper Bioengineering 121

A Novel Microfluidic Device for Rapid Melanoma Diagnosis Danielle Beeve, Luke Cassereau, Regine Labog, and Tomoya Saito University of California, Berkeley, Department of Bioengineering

Over 3 million Americans a year are diagnosed with skin cancer and 1 in 5 will be diagnosed in their lifetime making skin cancer the most common cancer. While usually benign, a small percentage of people are diagnosed with a potentially lethal metastatic melanoma. Metastatic melanoma causes over 70% of skin cancer related deaths. The threat from melanoma can be eliminated if diagnosed early enough allowing treatment to start prior to the development of aggressive metastatic tumors. We are proposing a microfluidic device that can generate fast bedside quantitative results while also reducing costs and need for invasive biopsies. Successful design and application of this device would not only improve melanoma diagnosis but also serve as a proof of concept for similar devices for other cancer types. Introduction

Skin cancer is the most common type of cancer, with 1 in 5 American diagnosed with skin cancer in their lifetime. There are 3.5 million cases of skin cancer per year in the United States alone, and skin-cancer related deaths are as high as 200,000 per year worldwide.

All cancer is based on aberrant cell behavior leading to uncontrolled growth. Treatment effectiveness decreases with tumor progression. Therefore, early diagnosis and treatment onset are essential for survival and limitations of complications (metastases). Skin cancer is the most common type of cancer, with 1 in 5 American diagnosed with skin cancer in their lifetime. There are 3.5 million cases of skin cancer per year in the United States alone, and skin-cancer related deaths are as high as 200,000 per year worldwide.

Skin cancer types consist of Basal Cell Carcinoma, Squamous Cell Carcinoma, and Malignant Melanoma. Melanoma is the most dangerous of all skin cancers, causing 90% of

the skin cancer-related deaths, and it requires the most immediate attention. Fortunately, only 5% of patients have melanoma. However, due to its rarity and the fact that few people regularly visit dermatologists, potential melanomas are often overlooked. Current diagnostics are not sufficient, as they are too slow, qualitative, and expensive. It is usually a 4-step process and there are associated issues with each step: 1. Patient visual examination: depends on

ABCDE rule (Fig 2), qualitative, not all skin areas are easy to see for self-examination

2. Dermatologist visit, visual examination redone: Still qualitative, invasive full biopsy taken if any doubt.

3. Pathologist: H&E staining and sectioning followed by image analysis, still qualitative and subject to error, depends on images used, level of staining is prone to variability, Expensive to have a full time pathologist.

Basal CellCarcinoma

Squamous CellCarcinoma

MalignantMelanoma

Figure 1: Skin Cancer Types

Figure 2: ABCDE Rule for Visual Melanoma Diagnosis


4. Full-body imaging: only occurs if pathologist determines growth is potentially a melanoma, used to search for other metastases, repeated regularly to insure no reoccurrence. Also prone to error as it is a qualitative analysis. At present, more advance medical

facilities have transitioned to additional biochemical testing. Nevertheless, the development of new approaches to improve existing cancer diagnostics and therapeutics has proven to be insufficient. Potential of Biochemistry

Promising diagnostic designs have arisen using biochemical analysis. Properly applied biochemical assays could provide faster results in a quantitative manner. It would allow more accurate diagnosis in less time which would allow treatment to begin immediately thus increasing change of survival. Many different targets can be selected including proteins, DNA methylation patterns, and mRNA, but the best choice of biomarker is mRNA. mRNA is indicative of future behavior of potential tumor cells, which is a prognosis/potential risk of melanoma or metastases. Previous work has

isolated 5 key genes, which in a DNA chip format were able to distinguish melanoma from benign skin growths.

A microfluidic device would allow mRNA measurements in a faster format. Only a small sample size is needed and no amplification is required. There is also an added benefit of no full biopsy, which is better for patients. There is less risk of mRNA degradation: having an enclosed device means there is no RNAase exposure besides that on the outside of the sample. For further details on biochemistry, refer to the supplementary paper by Luke Cassereau. Device Design

Our proposed device involves direct mRNA measurements of five relevant genes to melanoma: TRYP1, Melan-A, KIT, MYO5A, and ENDRB. This method provides much faster results than traditional methods, while still being able to accurately predict melanoma/skin growth severity. For more information on the five genes, refer to the supplementary paper by Luke Cassereau.

The overview of our device design can be seen in Figure 3. The device design is based on PDMS-based fluid flow physics. It

Figure 3: Device design overview


involves two major steps: 1) Cell Lysis and 2) Detection. Each will be discussed in detail. Cell Lysis

In order to extract mRNA from the skin sample, cell lysis is clearly necessary. There are countless ways to perform cell lysis, which include mechanical, electrical, chemical, and thermal techniques.1 We determined that a chemical technique would be the most practical for our application due to its simplicity and relatively low cost. The chemical technique utilizes a detergent solution, which is used to agitate the cell membrane (Fig 4). The detergent has hydrophobic long, linear alkyl chains that disorganize and break the membrane’s lipid bilayer.

There is unfortunately no standard protocol for selecting a detergent to use for membrane lysis. In general, nonionic and zwitterionic detergents are milder and less denaturing than ionic detergents and are used to solubilize membrane proteins where it is critical to maintain protein function and/or retain native protein:protein interactions for enzyme assays or immunoassays. CHAPS, a zwitterionic detergent, and the Triton-X series of nonionic detergents are commonly used for these purposes. In contrast, ionic detergents are strong solubilizing agents and tend to denature proteins, thereby destroying protein activity and function.2

Sodium dodecyl sulfate (SDS, NaC12H25SO4), an anionic surfactant, was

chosen to lyse the skin cells for our device. SDS is often used in DNA extraction and protein unraveling for polyacrylamide gel electrophoresis (SDS-PAGE). SDS acts as a detergent and begins to break apart the cell membrane on contact. While there are many detergents that can accomplish cell lysis, SDS has additional advantages. Not only does it require the least amount of time among detergents (30 seconds) to complete cell lysis, but as a strong anionic detergent, it also has the ability to immediately denature enzymes such as DNAse and RNAse.3 SDS is purposely used in this manner to inhibit RNAse and prevent mRNA deterioration in our device. The original device design was going to require an RNAse inhibitor solution separately mixed with the skin sample solution prior to cell lysis, but SDS made that step unnecessary.

The structure of SDS (Fig 5) shows a tail of 12 carbon atoms, attached to a sulfate group, giving the molecule the amphiphilic properties required of a detergent. The structure also provides it with a binding that is cooperative, which means that the binding of one molecule of SDS increases the likelihood that another molecule of SDS will bind to that protein. This alters most proteins into rigid rods whose length is proportional to molecular weight.4 The amount of detergent needed for optimal protein extraction depends on the critical micelle concentration (CMC), aggregation number, temperature and nature of the membrane and the detergent.4 CMC for SDS in pure water at 25°C is 0.0082 M,5 and

http://www.molecularstation.com/cell/cell-lysis/

SDS

NaC12H25SO4

http://upload.wikimedia.org/wikipedia/commons/thumb/4/4c/Sodium_dodecyl_sulfate.svg/800px-Sodium_dodecyl_sulfate.svg.png

Figure 4: Cell lysis overview

Figure 5: SDS Structure


the aggregation number at this concentration is usually considered to be about 62.6

The SDS is diluted to a 0.2% concentration solution for our application.7 The channel length required to mix SDS and sample completely is approximately 8 cm, but our device has a 15.2 cm length to ensure lysis.8 SDS and cell sample flow rate is roughly ~0.2 µl/min.8 This process may take anywhere from 30-190 seconds.1,3

Detection To detect our particular genes of interest

we have chosen to use currently existing Molecular Beacon technology. This technique involves the use of a DNA (or RNA in our case) stem-loop structure where the loop contains a complementary probe sequence for one of our 5 individual target genes of interest. The stem contains complementary base pairs that keep the structure together in the absence of target mRNA, and it is designed in such a way that in the presence of target mRNA exactly complimentary to the probe sequence the stem-loop will spontaneously unfold and hybridize to the target. If there is even one base pair mismatch between the target mRNA and probe sequences this spontaneous hybridization will not occur, making this and extraordinarily specific genetic detection method.

The quantitative aspect of this detection

method uses a fluorophore and quencher that are attached to each end of the stem structure of the molecular beacons. In the absence of target mRNA, the quencher is located right next to the fluorophore and prevents any fluorescent signal. Once hybridization occurs however the fluorophore is released from the vicinity of the quencher and you are left with a strong fluoresecent signal indicating the presence of target mRNA of interest. For our particular device design we have decided to adhere a set of molecular beacons down to the bottom of each of the 5 wells that correspond to our 5 different genes of interest by using avidin-biotin surface adhesion to glass. Biotin is attached to the quencher side of the stem of each molecular beacon while avidin is adsorbed onto a glass slide that will be used as the substrate to bind our PDMS to. Once a solution of these biotin-enhanced molecular beacons comes into contact with the avidin absorbed onto the glass slide the biotin fits into the avidin like a lock-and-key mechanism and the molecular beacons are anchored into place. Each of the 5 wells will have a set of molecular beacons containing a different complementary probe sequence and as the sample solution from the patient’s cells flows along the device, target mRNA of interest (if it is present in the sample) will hybridize to the molecular beacon probes causing fluorescence. This fluorescent signal can then be detected using a plate reader that has been specialized to fit our device design, and the presence or absence of fluorescent signal for

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Figure 6: Molecular Beacon Figure 7: Molecular Beacon Binding in device


each of the 5 genes will indicate whether melanoma is present in the patient sample. For more specifics (including pictorial diagrams of the molecular mechanisms and corresponding references) on this entire detection process see Danielle Beeve’s paper. Fabrication

The device is fabricated using soft lithography techniques. A silicon wafer is lithographically patterned with a mask design, using SU-8 negative photoresist. The resulting wafer mold is then patterned onto PDMS. 1mm holes punched at the two inlets and the outlet on the PDMS device. The glass slide is coated with molecular beacons in each well with the sequence of DNA that we are looking for. The PDMS is then bonded on a glass slide. For further details on the fabrication method, refer to the supplementary paper by Regine Labog. Overall design

The full movement of liquid through our device can be seen in Figure 8. The SDS inlet is split into two channels that later meet to flank the sample solution. Once combined, they flow together in a mixing channel composed of a series of S-curves. As the lysate flows to the detection line, pressure valves stops the flow for a certain period of time at each well to ensure proper mixing and detection. After the fifth well containing the relevant gene, the flow goes onto a sixth well that contains a control, and then into an outlet. For more information on the overall design,

refer to the supplementary paper by Regine Labog. Expected Impact

We have designed a point-of-care diagnostic that highlight key factors to significantly improve upon current technologies: 1. Reduces cost, wait time, and invasiveness 2. Provides quantitative results and accurate

prognosis 3. Can be used to quickly decide best

approach for each individual patient in one doctor’s visit

With these potential improvements, we hope that this design will be implemented into standard diagnostic methodology in the near future. Future Work

Many improvements can be made to our current proposed device. These include alternative skin sample acquisition methods, alternative cell lysis methods, and alternative genetic detection methods. Alternative detection methods include Quartz Crystal Microbalance (QCM), Surface Acoustic Waves (SAW), and DNA microchip technologies. RNA isolation and addition of PBS buffer to SDS are also considerations in order to possibly improve detection signal. One long-term goal is to apply this device to other cancers and diseases. This will require a clear understanding of which relevant genes for each disease can be used with the molecular beacon technology.

SDS

Cell Sample

Mixing Channel

TRYPI KIT ControlMELAN-A MYO5A ENDRB Outlet

YES YES YES YES YES NO

Figure 8: Device design with red showing fluid flow. Green wells indicate a positive reading and red wells indicate a negative reading.


Discussion Melanoma is a deadly disease and it is

clear that current diagnostics are far from ideal. Biochemistry and microfluidics provide a potential solution to this problem. Possible benefits include reduced cost, shorter wait times, less invasiveness, quantitative results, and higher accuracy. The potential for extension to other cancers and genetic diseases makes this novel diagnostic device a viable choice for future research. References 1. J. Kim, M. Johnson, P. Hill and B. K.

Gale, Microfluidic sample preparation: cell lysis and nucleic acid purification, Integr. Biol., 2009, 1(10), 574–586.

2. "Cell Lysis Solutions." Protein Purification, Modification and Detection: Pierce Protein Research. Thermo Fisher Scientific. Web. 16 Dec. 2010. <http://www.piercenet.com/browse.cfm?fldID=5559C287-5056-8A76-4E25-8975D8025374>.

3. Pang, Z., Al Mahrouki, A., Berezovski, M., Krylov, S. N., Electrophoresis 2006, 27, 1489–1494.

4. "Detergents for Cell Lysis." Protein Purification, Modification and Detection: Pierce Protein Research. Thermo Fisher Scientific. Web. 16 Dec. 2010. <http://www.piercenet.com/browse.cfm?fldID=5558F7E4-5056-8A76-4E55-4F3977738B63>.

5. P. Mukerjee and K. J. Mysels, "Critical Micelle Concentration of Aqueous Surfactant Systems", NSRDS-NBS 36, US. Government Printing Office, Washington,.D.C., 197 1.

6. N.J. Turro. A. Yekta, J. Am. Chem. Soc., 1978, 100, 5951

7. Yu, L., Huang, H., Dong, X., Wu, D., Qin, J., Lin, B., Electrophoresis 2008, 29, 5055–5060.

8. X. Chen, D. Cui, C. Liu and H. Cai, Chin. J. Anal. Chem., 2006, 34,1656–1660.

Note: References from the papers of group members shall be coupled to this list.

Health & Medicine

A novel microfluidic device for rapid melanoma diagnosis