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Microfluidic Linear Hydrogel Array for Multiplexed Single Nucleotide Polymorphism (SNP) Detection Yun Kyung Jung, Jungkyu Kim, and Richard A Mathies Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/ac5048696 • Publication Date (Web): 12 Feb 2015 Downloaded from http://pubs.acs.org on February 18, 2015

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Analytical Chemistry

Microfluidic Linear Hydrogel Array for Multiplexed Single Nucleotide Polymorphism (SNP) Detection Yun Kyung Jung,a,b,* Jungkyu Kim,a,c and Richard A. Mathies a,* a

Department of Chemistry, University of California, Berkeley, CA 94720, USA School of Natural Science, Ulsan National Institute of Science and Technology, Ulsan, 689798, Republic of Korea c Department of Mechanical Engineering, Texas Tech University, Lubbock, TX 79409 b

*Corresponding Authors: [email protected]. Phone: (510) 642-4192. Fax: (510) 6423599. [email protected]. Phone: 82-52-217-3015.

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ABSTRACT A PDMS-based microfluidic linear hydrogel array is developed for multiplexed single nucleotide polymorphism (SNP) detection. A sequence of three-dimensional (3D) hydrogel plugs containing the desired DNA probes is prepared by UV polymerization within a PDMS microchannel system. The fluorescently labeled target DNA is then electrophoresed through the sequence of hydrogel plugs for hybridization. Continued electrophoresis provides an electrophoretic wash that removes nonspecific binders. The capture gel array is imaged after washing at various temperatures (temperature gradient electrophoresis) to further distinguish perfect matches from mismatches. The ability of this microdevice to perform multiplex SNP genotyping is demonstrated by analyzing a mixture of model E. coli bacterial targets. This microfluidic hydrogel array is ~1000 times more sensitive than planar microarrays due to the 3D gel capture, the hybridization time is much shorter due to electrophoretic control of the transport properties, and the stringent wash with the temperature gradient electrophoresis enables analysis of single nucleotide mismatches with high specificity.

KEYWORDS: PDMS microfluidics, SNP detection; bacterial diagnostics, clinical diagnostics, point of care detection, microarray


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The detection of DNA variation is of great importance for disease diagnosis, pathogen identification and personalized medicine.1-5 The most conventional platform for highthroughput single-nucleotide polymorphism (SNP) genotyping is the two-dimensional (2D) planar microarray.1-5 This technology allows for the parallel identification of SNPs by hybridization of target DNAs onto glass-tethered oligonucleotide probes on planar solid supports. However, the 2D planar microarray has several drawbacks such as the relatively large sample volume,5 the limited capacity of oligonucleotide probes in a flat surface element,6,7 low sensitivity,8 and slow hybridization of targets to probes.9 By adapting three-dimensional (3D) probe architectures such as polymer-coated surfaces

6

or hydrophilic gel drops,7,10-12 the probe capacity of a DNA microarray can be

increased. For example, Ӧzkumur et al. showed that polymer-coated chips enhance the functional probe density significantly.6 In addition, Rubina et al. made a hydrogel drop microchip by illuminating a gel-forming solution containing 2 mM oligonucleotides with UV light.11 This hydrogel drop formed a 3D gel matrix which had ~2700 times higher binding capacity due to its porous structure compared to a 2D planar surface, and 30-fold better hybridization sensitivity. However, 12 hrs of assay time was still required to deliver target samples to the gel due to their slow diffusion into the gel. Thus, the 2D planar microarrays as well as the existing 3D capture gel structures are limited by the transport of target molecules to the capture probe. Accordingly, it is desirable to develop a method for actively controlling the transport properties. Microfluidic systems have been widely used for many types of DNA analysis because the small channel dimensions and volume enable decreased reagent consumption, faster analysis, parallel operation, and the ability to control fluid and sample transport.13-17 Incorporation of a 3D hydrogel into the micron-sized channels of a microfluidic device would be useful to improve the capacity of the capture element compared to 2D formats while maintaining the activity of the DNA probe and a small footprint. Recently, our group reported 3 ACS Paragon Plus Environment


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a glass-based two-wafer microfluidic chip containing a linear hydrogel array and demonstrated its capabilities for SNP detection of bacterial pathogens.18 Fabrication of the glass microfluidic chip using traditional methods required complicated fabrication procedures and expensive special equipment.19 To make this technology more facile, accessible and inexpensive, it would be useful to devise a simple polydimethylsolixane (PDMS) version of this device using standard soft lithography.13-17 We present here a PDMS microfluidic chip containing a linear array of DNA-embedded photopolymerized hydrogel plugs for high-throughput SNP genotyping. In this hydrogel array, electrophoretic force18,20-24 is applied to enable rapid hybridization by driving the negatively charged target DNAs through the anchored capture DNA hydrogels. Because the binding selectivity at room temperature is not sufficient to discriminate the single nucleotide variations, temperature gradient electrophoresis is used to gradually increase the stringency of the hybridization. Single base variation of target oligonucleotides is identified correctly by performing melting analysis based on the principle that the melting temperature (Tm) for perfectly matched allelic targets is higher than that for allelic targets with a mismatch.25-29 As a proof-of-concept, the genotypes of three sets of bacterial target DNAs are simultaneously identified within one hour. We demonstrate low attomolar level sensitivity which is at least 1,000 times better than that of a traditional microarray system. These results demonstrate the accuracy, speed, sensitivity and multiplex capability of our platform for diagnostic assays.


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■ EXPERIMENTAL SECTION Design and fabrication of PDMS microfluidic hydrogel array. The gel capture platform shown in Figure 1A was designed to have a horizontal electrophoretic channel intersected by three vertical gel forming channels each with 100 µm width. A microfluidic interface (MFI) element consisting of a narrow channel with a width of 5 µm, a length of 360 µm, and a depth of 25 µm, was placed at the intersections of the electrophoretic channel with the gel-forming channels. The narrow MFI minimizes leakage of the polymer-forming solutions into the electrophoresis channel before polymerization and plays a key role in preparing each hydrogel in the desired position without cross-contamination.18,19 The overall length of the horizontal and vertical channels is 25 mm and 13 mm, respectively. To form this structure, a monolayer PDMS architecture was designed and fabricated with standard soft-lithography (Figure 1B). A SU-8 polymer negative was first fabricated as described in the SI and a PDMS replica of this mold was polymerized. Holes were then punched in the PDMS replica for access to perform the electrophoresis and to add the prepolymer solutions. The surface of the PDMS was treated with oxygen plasma to make it hydrophilic thereby minimizing non-specific adsorption and maximizing the uniformity of electroosmotic flow (EOF).14,20,21,30,31 The plasma-treated PDMS replica was permanently bonded to 1.1 mm thick borofloat glass wafer to form the enclosed channels.32 Further surface modification was performed on the vertical gel channel with a 0.5% (v/v) 3(trimethoxysilyl)propyl methacrylate (TPM, 98 %) solution to enable a covalent link between the polymerized gel and both the PDMS and glass surfaces.20,33 A 1:1 mixture of DEH-100 polymer (The Gel Company, USA) and methanol was then used to coat the horizontal electrophoretic channel in order to further stabilize the EOF and to decreasing non-specific interactions.20,21,23,30,34 Preparation of gels in PDMS microchannels. Hydrogels containing different probe DNAs were polymerized in the PDMS microchannels as illustrated in Figure 1C. The composition 5 ACS Paragon Plus Environment


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of the prepolymer solution was 5% (w/v) acrylamide monomer (AAM), 5% (w/v) crosslinker poly(ethyleneglycol) diacrylate (PEG-DA), 0.109% (w/v) photoinitiator 2-hydroxy-2methylpropionophenone (HMPP), and 20 µM of 5’ acrydite probe DNA. Each solution was sparged with nitrogen for 30 min in a 1 mL amber vial prior to injection into the channel. Before polymerization, a glove box was filled with nitrogen. Each channel of the PDMS gel capture device was completely filled with 1×TTE buffer (50 mM Tris base, 50 mM TAPS acid, and 1 mM Na2EDTA, pH 8.4) by capillary action and vacuum. After that, the inlet of the gel channel was emptied and refilled with capture DNA-containing prepolymer solution. Vacuum was applied to the opposite end of the gel channel until the prepolymer solution was evenly distributed. The chip was aligned with the butterfly chrome mask (500 µm long and 100 µm wide) for exposure on an inverted Zeiss Axioskop. A vacuum chuck on the microscope stage held the mask and the wafer in place. After a 2 min equilibrium period, the intersection zone was irradiated through a chrome mask for 6 min (330 – 365 nm wavelength and 13.3 mW/cm2) to form each gel. The formation of successful hydrogels in PDMS microchannels depends on the concentration of reagents, the intensity of UV radiation, deoxygenation conditions, and control of internal flow within the capillaries. The density of the hydrogel is a key factor for obtaining optimal electrophoretic transport: Low-concentration gels can easily collapse or be damaged under high electrophoretic force, whereas high-concentration gels have high resistance and interfere with the migration of target DNAs through the gel matrix. By increasing the concentrations of cross-linker PEG-DA and photoinitiator HMPP 10-fold (5%, w/v) and 15.6-fold (0.109%, w/v) compared to the composition of the gel made in the previous glass-based chip,18 we were able to fabricate the fully swollen gel matrix in a welldefined region of the monolayer PDMS channel (Figure 1C). Additionally, since the PEG-DA portion of hydrogel is covalently linked with the TPM monolayer of the chip surface during the photopolymerization,21,33 the gel is tightly and stably fixed in the channel. After 2 min of 6 ACS Paragon Plus Environment

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equilibration and 6 min of polymerization, consistently sized and rectangular shaped gels were prepared reproducibly in the middle of the channel with a width of 100 µm, a height of 500 µm, and a depth of 25 µm, consisting of only 1.25 nL volume with a high projected probe activity (500 fmol/mm2, 1.1×1010 probes). DNA capture and allele-specific hybridization. To demonstrate genotyping of single-stranded (ss) DNA targets, equimolar samples (30 fmol of each DNA) of FAM-labeled perfectly matched target and Texas Red-labeled single base mismatched target against K12-probe-1, the M13mp18 probe, and K12-probe-2 DNAs (Table S-1) were prepared in 1×TE buffer (10 mM Tris, 0.1 mM EDTA, pH 7.5). The targets, except for the FAM-labeled matched target complementary to K12-probe-2, were loaded simultaneously into the sample inlet of the MFI. A constant voltage of 46 V/cm was applied from the inlet well to the outlet well for 5 min at room temperature to drive the DNAs through the capture gels. The FAM-labeled matched target complementary to K12-probe-2 was then loaded and electrophoresed into the device for another 15 min. Residual DNA within the channel was washed out electrophoretically for 5 min with 1×TTE buffer. For melting analysis, the same field strength was applied for 5 min at 5°C temperature increments after waiting for 2 min (Figure S-1). Epi-fluorescence images of the gels were acquired using a microscope (Nikon Eclipse E800, objective ×20/0.45) with a CCD camera (250 ms exposure for 488 nm excitation, and 90 ms for 595 nm). Melting curve analysis. To generate a melting curve, the fluorescence intensity was quantified by calculating the mean intensity of a defined region at the entrance of the capture gel (50 pixel width ×10 pixel height). After collecting the data from independent triplicate capture experiments, the mean and standard deviation of the fluorescence intensity were determined. The experimental dissociation temperatures (Td) is the temperature at which 50% of the initial signal is washed off. It is analogous to the predicted melting temperature (Tm).25-29 Free energy changes (∆G°) were calculated using the published thermodynamic database for matched and mismatched duplexes which was derived from UV melting experiments.35-37 ∆G° 7 ACS Paragon Plus Environment


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in the hybridization buffer, was calculated using 37°C since the experimental Td was determined near that temperature (Table S-1). Limit of detection. To determine the limit of detection (LOD), 1 µL of various concentrations of FAM-labeled target oligonucleotides were loaded into the sample inlet of a PDMS capture chip containing 20 µM probe oligonucleotide gel that is a perfect match. Samples were electrophoresed into the capture gels for 20 min at a field strength of 46 V/cm at room temperature. Epi-fluorescence images of the trapped FAM-labeled target were obtained using a 500 ms exposure time of the CCD camera and analyzed as described in the melting curve analysis. We characterized the LOD using the 3sb/m criterion,38 where sb is the standard deviation of a negative control (blank) and m is the slope in the linear range. The signal-tonoise (S/N) ratio was calculated using µsig/σbg, where µsig is average signal value and σbg is standard deviation of the background.39,40

■ RESULTS AND DISCUSSION Figure 1A presents the PDMS hydrogel capture array for multiplexed SNP detection. The platform fabricated on one wafer (Figure 1B) consists of one horizontal electrophoretic channel and three vertical gel-forming channels separated by a narrow MFI unit. Three hydrogels with different probe oligonucleotides are prepared at the intersections between the horizontal and vertical channels by UV irradiation (Figure 1C). Fluorophore-labeled target oligonucleotides are transported to their capture hydrogels by electrophoresis and the stringency is increased step-wise by increasing the temperature and continuing electrophoretic washing. The processes of hybridizing the labeled targets and electrophoretic washing were performed to analyze for perfect matches and mismatches of diagnostic E. coli markers. FAM-labeled matched and Texas-Red labeled mismatched target DNAs were electrophoresed into the concentrated probe DNA-embedded hydrogels at a field strength of 46 V/cm. Figure 8 ACS Paragon Plus Environment

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2A presents the conical-shaped images of both the FAM-labeled matched and the Texas Redlabeled one base mismatched targets captured in each capture gel containing 20 µM acryditemodified oligonucleotides K12-probe-1 (5’-CCAGTAATCATCGTCTGGAT-3’), K12-probe2

(5’-CCAGTGCTTCGCATATTCTG-3’),

and

the

M13mp18

probe

DNAs

(5’-

ACTGGCCGTCGTTTTACTA-3’). The one-base mismatched targets complementary to K12probe-1, K12-probe-2, and M13mp18 contain a T G substitution at position 3, a G T substitution at position 10, and a G T substitution at position 10, respectively (Table S-1). After 2 min equilibration at each elevated temperature (Figure S-1), an electrophoretic wash was performed for 5 min resulting in release of the mismatched oligonucleotides due to the weaker binding of the mismatched base pairs without affecting the perfect duplexes. Significant FAM signals from the matched sequence were maintained at five to ten degrees higher temperatures than for the Texas Red labeled mismatch (Figure 2B). The dynamics of microfluidic electrophoresis enabled much faster hybridization (within 20 min) compared to diffusion mediated binding to probes on a planar glass surface which can require overnight reaction.40 Furthermore, a hydrogel without probe DNA did not capture any fluorophorelabeled DNA, as evidence by a complete lack of fluorescence in either the green or red channels under the same exposure condition. To quantitatively compare the thermal stability of matched targets with that of mismatched targets, melting curve profiles were obtained by measuring relative fluorescence intensities within a defined region in the center of capture area vs. dissociation temperatures (Td).25-29 The data in Figure 2B demonstrates that there is a significant difference between the dissociation rates of the perfectly matched duplex and the mismatched duplex, representing their dissimilar duplex stabilities. The melting curves for the mismatched sequences are always to the left of the perfect match especially when the mismatch is near the middle of the probe sequence (experiments 2 and 3). The experimentally determined ∆Td is compared with the calculated ∆Tm in Table S-1. Both ∆Td and ∆Tm increased when the mismatched position 9 ACS Paragon Plus Environment


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is located near the center of the sequence. The free energy of the mismatched duplex (∆G°MM) for the K12-probe-1 at 37°C was 2.53 kcal/mol above that of perfectly matched hybrid (∆G°PM).35 For both the M13mp18 probe and the K12-probe-2, the mismatched base pairs increase the free energy by 5.04 kcal/mol.35 The free energy ∆G° increase for the latter two probes is twice as much as the K12-probe-1 because that mutation is located in the center.35-37 Our microfluidic 3D hydrogel array demonstrates that the facile but stringent electrophoretic wash process results in readily distinguishable melting temperatures ∆Td for single base mismatches.25-29 To determine the sensitivity of our gel array for oligonucleotide targets, the limit of detection (LOD) was evaluated (Figure 3). Figure 3A shows that the fluorescence signals detected in the entrance of the capture gels increase according to the target DNA concentration as expected. High signal-to-noise (S/N) ratio of over 15 was achieved with a low fluorescent background.39,40 Using the metric described in the methods,38 we estimated a LOD of ~3 amol (1 pM) which gives us at least 1,000 times higher sensitivity than that of a traditional microarray system.2 These results correspond to the capture of approximately 1.8×106 target DNA molecules within a DNA hydrogel containing 1.1×1010 DNA probes. The excellent sensitivity of our hydrogel array is attributable to the high activity of capture probe in the 3D capture hydrogel. A typical projected probe density for a 2D planar microarray array11 is only 0.19 fmol/mm2; however the projected density of our 3D gel using 20 µM of probe DNA is 500 fmol/mm2. The 2500-fold increase in probe surface density enables high capture efficience and hence sensitivity. An unusual case was revealed when studying both matched and mismatched target DNAs complementary to the K12-probe-2. When the target DNAs were loaded simultaneously at room temperature, no DNA was captured by the probe hydrogels. This failure is presumably due to the formation of weak hybrids between the two DNAs mediated 10 ACS Paragon Plus Environment

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by the 6 base-pair sequence underlined in Table S-1. To test this hypothesis, we performed electrophoresis of the matched target alone, showing that it occupied the front region of the DNA probe gel; subsequent loading of the mismatched target showed that it hybridized to a more

distal region of the gel (Figure S-2A). In contrast, when the order of DNA

electrophoresis is reversed, both DNAs share the same gel region (Figure S-2B). Thus, both DNAs are capable of binding, supporting the weak hybrid hypothesis. While this suggests that target sequences that display significant homology either internally or to one another should be avoided, loading at a temperature somewhat above room temperature should eliminate these weak hybrid issues. The conversion of this proof of concept study to a practical diagnostic requires analyzing double-stranded products produced by PCR. This conversion can be done in a number of ways. Amplicons can be generated from clinical samples with a biotinylated reverse primer and a fluorophore-labeled forward primer. Biotinylated amplicons were then incubated with streptavidin magnetic beads and fluorophore-labeled ssDNAs were isolated by strand denaturation. To test this idea, FAM- and biotin-labeled 116 bp dsDNA was produced by PCR and then the FAM-labeled ssDNA was isolated using streptavidin magnetic beads and successfully genotyped with our hydrogel array, exhibiting a Kd value of about 47.8°C (Figure S-3). A more detailed manuscript demonstrating multiplex genotyping of CFTR PCR products as well as the detection of other mismatches (e.g. T:G) is in preparation. Another approach for producing ssDNA targets is the performance of asymmetric PCR in a single or nested format. In summary, we have demonstrated a 3D hydrogel DNA array chip and method enabling analysis of single nucleotide variants with high specificity and sensitivity. One advantage of this approach is the use of electrophoresis to control the transport of target to the gel elements thereby decreasing hybridization times and enabling high efficiency and sensitive target capture. In addition, the use of temperature gradient electrophoresis selectively increases the stringency for capture of target DNAs, so that single base mismatches can be accurately 11 ACS Paragon Plus Environment


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discriminated. Our hydrogel array technology based on PDMS microfluidics provides a simple, rapid, accurate, and ultra-sensitive platform for detecting genetic targets and variation.

■ Acknowledgement The authors thank Dr. Dongho Lee of Samsung for facilitating this research and Dr. Erik Jensen for fruitful scientiﬁc discussions. This work was supported by the the Samsung Corporation and by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (NRF-2012R1A1A3015259).

■ Supporting Information Available Table presenting target sequences, ∆Tm, and ∆Td, and figures presenting temperature gradient curves as a function of heating time, the binding pattern according to the loading order of perfectly matched and mismatched target, and genotyping of duplex DNA. This material is available free of charge via the Internet at http://pubs.acs.org.


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■ REFERENCES (1) Fodor, S. P. A.; Rava, R. P.; Huang, X. C.; Pease, A. C.; Holmes, C. P.; Adams, C. L. Nature 1993, 364, 555-556. (2) Sassolas, A.; Leca-Bouvier, B. D.; Blum, L. J. Chem. Rev. 2008, 108, 109-139. (3) Katsanis, S. H.; Katsanis, N. Nat. Rev. Genet. 2013, 14, 415-426. (4) Stef, M. A.; Palacios, L.; Olano-Martín, E.; Foe-A-Man, C.; Kerkhof, L.; Klaaijsen, L. N.; Molano, A.; Schuurman, E. J.; Tejedor, D.; Defesche, J. C. J. Mol. Diagn. 2013, 15, 362372. (5) Baaj, Y.; Magdelaine, C.; Ubertelli, V.; Valat, C.; Talini, L.; Soussaline, F.; Khomyakova, E.; Funalot, B.; Vallat, J.-M.; Sturtz, F. G. BioTechniques, 2008, 44, 119-126. (6) Ӧzkumur, E.; Ahn, S.; Yalҫin, A.; Lopez, C. A.; Ҫevik, E.; Irani, R. J.; DeLisi, C.; Chiari, M.; Ünlü, M. S. Biosens. Bioelectron. 2010, 25, 1789-1795. (7) Liu, X.; Li, T.; Liu, D.; Wang, Z. Anal. Methods, 2013, 5, 285-290. (8) Epstein, J. R.; Lee, M.; Walt, D. R. Anal. Chem. 2002, 74, 1836-1840. (9) Rennie, C.; Noyes, H. A.; Kemp, S. J.; Hulme, H.; Brass, A.; Hoyle, D. C. BMC Genomics 2008, 9, 317. (10) Rehman, F. N.; Audeh, M.; Abrams, E. S.; Hamismatchedond, P. W.; Kenney, M.; Boles, T. C. Nucleic Acids Res. 1999, 27, 649-655. (11) Rubina, A. Y.; Pan’kov, S. V.; Dementieva, E. I.; Pen’kov, D. N.; Butygin, A. V.; Vasiliskov, V. A.; Chudinov, A. V.; Mikheikin, A. L.; Mikhailovich, V. M.; Mirzabekov, A. D. Anal. Biochem. 2004, 325, 92-106. (12) Xiao, P. F.; Cheng, L.; Wan, Y.; Sun, B. L.; Chen, Z. Z.; Zhang, S. Y.; Zhang, C. Z.; Zhou, G. H.; Lu, Z. H. Electrophoresis 2006, 27, 3904-3915. (13) Bhagat, A. A. S.; Jothimuthu, P.; Papautsky, I. Lab Chip 2007, 7, 1192-1197. (14) Sun, Y.; Kwok, Y. C. Anal. Chim. Acta 2006, 556, 80-96. (15) Situma, C.; Hashimoto, M.; Soper, S. A. Biomol. Eng. 2006, 23, 213-231. 13 ACS Paragon Plus Environment


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(16) Heo, J.; Crooks, R. M. Anal. Chem. 2005, 77, 6843-6851. (17) Lounsbury, J. A.; Karlsson, A.; Miranian, D. C.; Cronk, S. M.; Nelson, D. A.; Li, J.; Haverstick, D. M.; Kinnon, P.; Saul, D.; Landers, J. P. Lab Chip 2013, 13, 1384-1393. (18) Bromberg, A.; Jensen, E. C.; Kim, J.; Jung, Y. K.; Mathies, R. A. Anal. Chem. 2012, 84, 963-970. (19) Emrich, C. A.; Medintz, I. L.; Chu, W. K.; Mathies, R. A. Anal. Chem. 2007, 79, 7360-7366. (20) Hu, S.; Ren, X.; Bachman, M.; Sims, C. E.; Li, G. P.; Allbritton, N. Electrophoresis 2003, 24, 3679-3688. (21) Pallandre, A.; Lambert, B. De; Attia, R.; Jonas, A. M.; Viovy, J.-L. Electrophoresis 2006, 27, 584-610. (22) Edman, C. F.; Raymond, D. E.; Wu, D. J.; Tu, E.; Sosnowski, R. G.; Butler, W. F.; Nerenberg, M.; Heller, M. Nucleic Acids Res. 1997, 25, 4907-4914. (23) Erickson, D.; Liu, X.; Krull, U.; Li, D. Anal. Chem. 2004, 76, 7269-7277. (24) Sosnowski, R. G.; Tu, E.; Butlter, W. F.; O’Connell, J. P.; Heller, M. J. Proc. Natl. Acad. Sci. U.S.A. 1997, 94, 1119-1123. (25) Gresham, D.; Curry, B.; Ward, A.; Gordon, D. B.; Brizuela, L.; Kruglyak, L.; Botstein, D. Proc. Natl. Acad. Sci. U.S.A. 2010, 107, 1482-1487. (26) Ozel, A. B.; Srivannavit, O.; Rouillard, J.-M.; Gulari, E. Biotechnol. Prog. 2012, 28, 556-566. (27) Ohlander, A.; Zilio, C.; Hamismatchederle, T.; Zelenin, S.; Klink, G.; Chiari, M.; Bock, K.; Russom, A. Lab Chip 2013, 13, 2075-2082. (28) Wick, L. M.; Rouillard, J. M.; Whittam, T. S.; Gulari, E.; Tiedje, J. M.; Hashsham, S. A. Nucleic Acids Res. 2006, 34, e26. (29) Crews, N.; Wittwer, C.; Montgomery, J.; Pryor, R.; Gale, B. K. Anal. Chem.2009, 81, 2053-2058. 14 ACS Paragon Plus Environment

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(30) Tan, S. H.; Nguyen, N.-T.; Chua, Y. C.; Kang, T. G. Biomicrofluidics 2010, 4, 032204. (31) Wheeler, A. R.; Trapp, G.; Trapp, O.; Zare, R. N. Electrophoresis 2004, 25, 11201124. (32) Kim, J.; Jensen, E. C.; Megens, M.; Boser, B.; Mathies, R. A. Lab Chip 2011, 11, 3106-3112. (33) Liu, Z.-B.; Zhang, Y.; Yu, J.-J.; Mak, A. F.-T.; Li, Y.; Yang, M. Sens. Actuators, B 2010, 143, 776-783. (34) Chen, Y.; Zhang, L.; Chen, G. Electrophoresis 2008, 29, 1801-1814. (35) SantaLucia, J.; Hicks, Jr., D. Annu. Rev. Biophys. Biomol. Struct. 2004, 33, 415-440. (36) Tulpan, D.; Andronescu, M.; Leger, S. BMC Bioinformatics 2010, 11, 105. (37) Breslauer, K. J.; Frank, R.; Blöcker, H.; Marky, L. A. Proc. Natl. Acad. Sci. U.S.A. 1986, 83, 3746-3750. (38) Wang, Y.; Bao, L.; Liu Z.; Pang, D.-W. Anal. Chem. 2011, 83, 8130-8137. (39) Zhang, J.-H.; Chung, T. D. Y.; Oldenburg, K. R. J. Biomol. Screen. 1999, 4, 6773. (40) Lenigk, R.; Liu, R. H.; Athavale, M.; Chen, Z.; Ganser, D.; Yang, J.; Rauch, C.; Liu, Y.; Chan, B.; Yu, H.; Ray, M.; Marrero, R.; Grodzinski, P. Anal. Biochem. 2002, 311, 40-49.



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Figure 1. (A) Schematic illustration of PDMS-based microfluidic hydrogel array for genotyping consisting of one sample input channel and three capture channels, (B) replica image showing the fabricated PDMS microchannel intersection, and (C) optical image of three-dimensional capture hydrogel (100 µm width × 500 µm height × 25 µm depth) formed in the microchannel intersection zone by UV irradiation.


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Figure 2. Fluorescence images (A) and dissociation curves (B) of FAM-labeled matched and Texas Red-labeled one base mismatched targets captured in each capture gel containing 20 µM K12-probe-1 (1), the M13mp18 probe (2), and K12-probe-2 DNAs (3) at the indicated temperature.

The experimentally determined dissociation temperature (Td) for perfectly

matched and mismatched target oligonucleotides were Td (B-1, Match) = 42.3 ± 0.3°C, Td (B1, Mismatch) = 39.9 ± 0.2°C, Td (B-2, Match) = 42.2 ± 0.3°C, Td (B-1, Mismatch) = 34.1 ± 0.8°C, Td (B-3, Match) = 43.1 ± 0.2°C, Td (B-3, Mismatch) = 38.5 ± 0.5°C.



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Figure 3. Detection limit of the PDMS-based hydrogel array. (A) Epifluorescence images of capture gels according to the concentration of FAM-labeled target oligonucleotides loaded in the inlet well. (B) A plot showing a relationship between the target concentration and fluorescence intensity on a logarithmic scale.


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For TOC only

A linear hydrogel microarray in polydimethylsiloxane (PDMS) microfluidic channels has been developed for multiplexed identification of single-base-pair genetic mutation. With the gel plug microarray, three different targets are typed with 3 amol sensitivity in about an hour.


(SNP) Detection - American Chemical Society

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