A novel microfluidic device for rapid melanoma diagnosis
Final Project Paper Bioengineering 121A Novel Microfluidic Device for Rapid Melanoma DiagnosisDanielle Beeve, Luke Cassereau, Regine Labog, and Tomoya SaitoUniversity of California, Berkeley, Department of Bioengineering Over 3 million Americans a year are diagnosed with skin cancer and 1 in 5 will bediagnosed in their lifetime making skin cancer the most common cancer. While usually benign, asmall percentage of people are diagnosed with a potentially lethal metastatic melanoma.Metastatic melanoma causes over 70% of skin cancer related deaths. The threat from melanomacan be eliminated if diagnosed early enough allowing treatment to start prior to the developmentof aggressive metastatic tumors. We are proposing a microfluidic device that can generate fastbedside quantitative results while also reducing costs and need for invasive biopsies. Successfuldesign and application of this device would not only improve melanoma diagnosis but also serveas a proof of concept for similar devices for other cancer types.Introduction the skin cancer-related deaths, and it requires Skin cancer is the most common type of the most immediate attention. Fortunately,cancer, with 1 in 5 American diagnosed with only 5% of patients have melanoma.skin cancer in their lifetime. There are 3.5 However, due to its rarity and the fact thatmillion cases of skin cancer per year in the few people regularly visit dermatologists,United States alone, and skin-cancer related potential melanomas are often overlooked.deaths are as high as 200,000 per year Current diagnostics are not sufficient, as theyworldwide. are too slow, qualitative, and expensive. It is All cancer is based on aberrant cell usually a 4-step process and there arebehavior leading to uncontrolled growth. associated issues with each step:Treatment effectiveness decreases with tumor 1. Patient visual examination: depends onprogression. Therefore, early diagnosis and ABCDE rule (Fig 2), qualitative, not alltreatment onset are essential for survival and skin areas are easy to see for self-limitations of complications (metastases). examinationSkin cancer is the most common type of 2. Dermatologist visit, visual examinationcancer, with 1 in 5 American diagnosed with redone: Still qualitative, invasive fullskin cancer in their lifetime. There are 3.5 biopsy taken if any doubt.million cases of skin cancer per year in the 3. Pathologist: H&E staining and sectioningUnited States alone, and skin-cancer related followed by image analysis, stilldeaths are as high as 200,000 per year qualitative and subject to error, dependsworldwide. on images used, level of staining is prone Skin cancer types consist of Basal Cell to variability, Expensive to have a fullCarcinoma, Squamous Cell Carcinoma, and time pathologist.Malignant Melanoma. Melanoma is the mostdangerous of all skin cancers, causing 90% of Basal Cell Squamous Cell Malignant Carcinoma Carcinoma Melanoma Figure 1: Skin Cancer Types Figure 2: ABCDE Rule for Visual Melanoma Diagnosis
Final Project Paper Bioengineering 121 4. Full-body imaging: only occurs if isolated 5 key genes, which in a DNA chip pathologist determines growth is format were able to distinguish melanoma potentially a melanoma, used to search for from benign skin growths. other metastases, repeated regularly to A microfluidic device would allow insure no reoccurrence. Also prone to mRNA measurements in a faster format. Only error as it is a qualitative analysis. a small sample size is needed and no At present, more advance medical amplification is required. There is also an facilities have transitioned to additional added benefit of no full biopsy, which is biochemical testing. Nevertheless, the better for patients. There is less risk of mRNA development of new approaches to improve degradation: having an enclosed device means existing cancer diagnostics and therapeutics there is no RNAase exposure besides that on has proven to be insufficient. the outside of the sample. For further details on biochemistry, refer to the supplementary Potential of Biochemistry paper by Luke Cassereau. Promising diagnostic designs have arisen using biochemical analysis. Properly applied Device Design biochemical assays could provide faster Our proposed device involves direct results in a quantitative manner. It would mRNA measurements of five relevant genes allow more accurate diagnosis in less time to melanoma: TRYP1, Melan-A, KIT, which would allow treatment to begin MYO5A, and ENDRB. This method provides immediately thus increasing change of much faster results than traditional methods, survival. Many different targets can be while still being able to accurately predict selected including proteins, DNA methylation melanoma/skin growth severity. For more patterns, and mRNA, but the best choice of information on the five genes, refer to the biomarker is mRNA. mRNA is indicative of supplementary paper by Luke Cassereau. future behavior of potential tumor cells, The overview of our device design can be which is a prognosis/potential risk of seen in Figure 3. The device design is based melanoma or metastases. Previous work has on PDMS-based fluid flow physics. It Figure 3: Device design overview
Final Project Paper Bioengineering 121 SDS NaC12H25SO4 http://upload.wikimedia.org/wikipedia/commons/thumb/4/4c/ Sodium_dodecyl_sulfate.svg/800px-Sodium_dodecyl_sulfate.svg.png Figure 5: SDS Structure http://www.molecularstation.com/cell/cell-lysis/ chosen to lyse the skin cells for our device. Figure 4: Cell lysis overview SDS is often used in DNA extraction andinvolves two major steps: 1) Cell Lysis and 2) protein unraveling for polyacrylamide gelDetection. Each will be discussed in detail. electrophoresis (SDS-PAGE). SDS acts as a detergent and begins to break apart the cellCell Lysis membrane on contact. While there are many In order to extract mRNA from the skin detergents that can accomplish cell lysis, SDSsample, cell lysis is clearly necessary. There has additional advantages. Not only does itare countless ways to perform cell lysis, require the least amount of time amongwhich include mechanical, electrical, detergents (30 seconds) to complete cell lysis,chemical, and thermal techniques.1 We but as a strong anionic detergent, it also hasdetermined that a chemical technique would the ability to immediately denature enzymesbe the most practical for our application due such as DNAse and RNAse.3 SDS isto its simplicity and relatively low cost. The purposely used in this manner to inhibitchemical technique utilizes a detergent RNAse and prevent mRNA deterioration insolution, which is used to agitate the cell our device. The original device design wasmembrane (Fig 4). The detergent has going to require an RNAse inhibitor solutionhydrophobic long, linear alkyl chains that separately mixed with the skin sampledisorganize and break the membrane’s lipid solution prior to cell lysis, but SDS made thatbilayer. step unnecessary. There is unfortunately no standard The structure of SDS (Fig 5) shows a tailprotocol for selecting a detergent to use for of 12 carbon atoms, attached to a sulfatemembrane lysis. In general, nonionic and group, giving the molecule the amphiphiliczwitterionic detergents are milder and less properties required of a detergent. Thedenaturing than ionic detergents and are used structure also provides it with a binding that isto solubilize membrane proteins where it is cooperative, which means that the binding ofcritical to maintain protein function and/or one molecule of SDS increases the likelihoodretain native protein:protein interactions for that another molecule of SDS will bind to thatenzyme assays or immunoassays. CHAPS, a protein. This alters most proteins into rigidzwitterionic detergent, and the Triton-X series rods whose length is proportional toof nonionic detergents are commonly used for molecular weight.4 The amount of detergentthese purposes. In contrast, ionic detergents needed for optimal protein extraction dependsare strong solubilizing agents and tend to on the critical micelle concentration (CMC),denature proteins, thereby destroying protein aggregation number, temperature and natureactivity and function.2 of the membrane and the detergent.4 CMC for Sodium dodecyl sulfate (SDS, SDS in pure water at 25°C is 0.0082 M,5 andNaC12H25SO4), an anionic surfactant, was
Final Project Paper Bioengineering 121the aggregation number at this concentration method uses a fluorophore and quencher thatis usually considered to be about 62.6 are attached to each end of the stem structure The SDS is diluted to a 0.2% of the molecular beacons. In the absence ofconcentration solution for our application.7 target mRNA, the quencher is located rightThe channel length required to mix SDS and next to the fluorophore and prevents anysample completely is approximately 8 cm, but fluorescent signal. Once hybridization occursour device has a 15.2 cm length to ensure however the fluorophore is released from thelysis.8 SDS and cell sample flow rate is vicinity of the quencher and you are left withroughly ~0.2 µl/min.8 This process may take a strong fluoresecent signal indicating theanywhere from 30-190 seconds.1,3 presence of target mRNA of interest. For our particular device design we have decided toDetection adhere a set of molecular beacons down to the To detect our particular genes of interest bottom of each of the 5 wells that correspondwe have chosen to use currently existing to our 5 different genes of interest by usingMolecular Beacon technology. This technique avidin-biotin surface adhesion to glass. Biotininvolves the use of a DNA (or RNA in our is attached to the quencher side of the stem ofcase) stem-loop structure where the loop each molecular beacon while avidin iscontains a complementary probe sequence for adsorbed onto a glass slide that will be usedone of our 5 individual target genes of as the substrate to bind our PDMS to. Once ainterest. The stem contains complementary solution of these biotin-enhanced molecularbase pairs that keep the structure together in beacons comes into contact with the avidinthe absence of target mRNA, and it is absorbed onto the glass slide the biotin fitsdesigned in such a way that in the presence of into the avidin like a lock-and-key mechanismtarget mRNA exactly complimentary to the and the molecular beacons are anchored intoprobe sequence the stem-loop will place. Each of the 5 wells will have a set ofspontaneously unfold and hybridize to the molecular beacons containing a differenttarget. If there is even one base pair mismatch complementary probe sequence and as thebetween the target mRNA and probe sample solution from the patient’s cells flowssequences this spontaneous hybridization will along the device, target mRNA of interest (ifnot occur, making this and extraordinarily it is present in the sample) will hybridize tospecific genetic detection method. the molecular beacon probes causing The quantitative aspect of this detection fluorescence. This fluorescent signal can then be detected using a plate reader that has been !"#$%&(#)"#$&&(#!"#$%&(#*+,+(#!"#(#,-../,0,1" specialized to fit our device design, and the presence or absence of fluorescent signal for Figure 6: Molecular Beacon Figure 7: Molecular Beacon Binding in device
Final Project Paper Bioengineering 121 Mixing Channel Cell Sample YES YES YES YES YES NO TRYPI MELAN-A KIT MYO5A ENDRB Control Outlet SDSFigure 8: Device design with red showing fluid flow. Green wells indicate a positive reading and red wells indicate anegative reading. each of the 5 genes will indicate whether refer to the supplementary paper by Reginemelanoma is present in the patient sample. Labog.For more specifics (including pictorialdiagrams of the molecular mechanisms and Expected Impactcorresponding references) on this entire We have designed a point-of-caredetection process see Danielle Beeve’s paper. diagnostic that highlight key factors to significantly improve upon currentFabrication technologies: The device is fabricated using soft 1. Reduces cost, wait time, and invasivenesslithography techniques. A silicon wafer is 2. Provides quantitative results and accuratelithographically patterned with a mask design, prognosisusing SU-8 negative photoresist. The 3. Can be used to quickly decide bestresulting wafer mold is then patterned onto approach for each individual patient inPDMS. 1mm holes punched at the two inlets one doctor’s visitand the outlet on the PDMS device. The glass With these potential improvements, we hopeslide is coated with molecular beacons in each that this design will be implemented intowell with the sequence of DNA that we are standard diagnostic methodology in the nearlooking for. The PDMS is then bonded on a future.glass slide. For further details on thefabrication method, refer to the Future Worksupplementary paper by Regine Labog. Many improvements can be made to our current proposed device. These includeOverall design alternative skin sample acquisition methods, The full movement of liquid through our alternative cell lysis methods, and alternativedevice can be seen in Figure 8. The SDS inlet genetic detection methods. Alternativeis split into two channels that later meet to detection methods include Quartz Crystalflank the sample solution. Once combined, Microbalance (QCM), Surface Acousticthey flow together in a mixing channel Waves (SAW), and DNA microchipcomposed of a series of S-curves. As the technologies. RNA isolation and addition oflysate flows to the detection line, pressure PBS buffer to SDS are also considerations invalves stops the flow for a certain period of order to possibly improve detection signal.time at each well to ensure proper mixing and One long-term goal is to apply this device todetection. After the fifth well containing the other cancers and diseases. This will require arelevant gene, the flow goes onto a sixth well clear understanding of which relevant genesthat contains a control, and then into an outlet. for each disease can be used with theFor more information on the overall design, molecular beacon technology.
Final Project Paper Bioengineering 121Discussion 4. "Detergents for Cell Lysis." Protein Melanoma is a deadly disease and it is Purification, Modification andclear that current diagnostics are far from Detection: Pierce Protein Research.ideal. Biochemistry and microfluidics provide Thermo Fisher Scientific. Web. 16 Dec.a potential solution to this problem. Possible 2010.benefits include reduced cost, shorter wait <http://www.piercenet.com/browse.cfm?times, less invasiveness, quantitative results, fldID=5558F7E4-5056-8A76-4E55-and higher accuracy. The potential for 4F3977738B63>.extension to other cancers and genetic 5. P. Mukerjee and K. J. Mysels, "Criticaldiseases makes this novel diagnostic device a Micelle Concentration of Aqueousviable choice for future research. Surfactant Systems", NSRDS-NBS 36, US. Government Printing Office,References Washington,.D.C., 197 1.1. J. Kim, M. Johnson, P. Hill and B. K. 6. N.J. Turro. A. Yekta, J. Am. Chem. Soc., Gale, Microfluidic sample preparation: 1978, 100, 5951 cell lysis and nucleic acid purification, 7. Yu, L., Huang, H., Dong, X., Wu, D., Integr. Biol., 2009, 1(10), 574–586. Qin, J., Lin, B., Electrophoresis 2008, 29,2. "Cell Lysis Solutions." Protein 5055–5060. Purification, Modification and 8. X. Chen, D. Cui, C. Liu and H. Cai, Chin. Detection: Pierce Protein Research. J. Anal. Chem., 2006, 34,1656–1660. Thermo Fisher Scientific. Web. 16 Dec. 2010. Note: References from the papers of group <http://www.piercenet.com/browse.cfm? members shall be coupled to this list. fldID=5559C287-5056-8A76-4E25- 8975D8025374>.3. Pang, Z., Al Mahrouki, A., Berezovski, M., Krylov, S. N., Electrophoresis 2006, 27, 1489–1494.