Temperature and Time-Resolved Total Internal Reflectance

Jun 16, 2010 - Bioengineering Department, 306 Stanley Hall, Berkeley, California 94720- ... Bioengineering Department, University of California, Berke...
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Anal. Chem. 2010, 82, 6124–6131

Temperature and Time-Resolved Total Internal Reflectance Fluorescence Analysis of Reusable DNA Hydrogel Chips Thorsten Neumann,*,†,‡,§ Andrew J. Bonham,‡ Gregory Dame,† Bernd Berchtold,† Thomas Brandstetter,† and Ju¨rgen Ru¨he† University of Freiburg – IMTEK, Department of Microsystems Engineering, Laboratory for Chemistry and Physics of Interfaces, Georges-Koehler-Allee 103, D-79110 Freiburg, Germany, University of California Santa Barbara, Department of Chemistry and Biochemistry, Santa Barbara, California 93106, and University of California, Berkeley Bioengineering Department, 306 Stanley Hall, Berkeley, California 94720-1762 Total internal reflection fluorescence (TIRF) coupled with hydrogel-DNA droplet microarrays covalently bound on PMMA substrates presents a reusable, sensitive platform for evaluating DNA hybridization and for rapid biochip development. Hydrogel microarrays, which contain covalently bound DNA probes, are created via a simple printing and photocross-linking process. TIRF measurements of the arrays display robust reusability, show linear sensitivity down to 5 fmol of fluorescently labeled target DNA, and are sensitive to single basepair mismatches. Additionally, the ability to interrogate larger DNA is shown through studies with PCR amplification hybridization. We conclusively demonstrate an efficient, reproducible, low cost platform for DNA hybridization studies that could be used for fast high-throughput diagnostics as well as biochip development. DNA chips are composed of large numbers of different DNA sequences bound into a defined grid (microarray) with redundant replicates across a substrate. These DNA chips have enabled sensitive hybridization studies1 and have shown great potential usage as highly parallel analytical tools in genomic2,3 and proteomic4 research, particularly in gene discovery,5 disease diagnosis,6 drug discovery, and toxicological research.7 Conventional DNA microarrays attach DNA to glass (e.g., via amino- or epoxy* Corresponding author. E-mail: [email protected]. † University of Freiburg. ‡ Department of Chemistry and Biochemistry, University of California, Santa Barbara. § Bioengineering Department, University of California, Berkeley. (1) Palmisano, G. L.; Delfino, L.; Fiore, M.; Longo, A.; Ferrara, G. B. Autoimmun. Rev. 2005, 4, 510. (2) Phimister, B. Nat. Genet. 1999, 21, 1. Collins, F. S. Nat. Genet. 1999, 21, 2. Lander, E. S. Nat. Genet. 1999, 21, 3–4. Southern, E.; Mir, K.; Shchepinov, M. Nat. Genet. 1999, 21, 5–9. Duggan, D. J.; Bittner, M.; Chen, Y.; Meltzer, P.; Trent, J. M. Nat. Genet. 1999, 21, 10–14. Cheung, V. G.; Morley, M.; Aguilar, F.; Massimi, A.; Kucherlapati, R.; Childs, G. Nat. Genet. 1999, 21, 15–19. Lipshutz, R. J.; Fodor, S. P. A.; Gingeras, T. R.; Lockhart, D. J. Nat. Genet. 1999, 21, 20–24. Bowtell, D. D. L. Nat. Genet. 1999, 21, 25–32. Brown, P. O.; Botstein, D. Nat. Genet. 1999, 21, 33–37. Cole, K. A.; Krizman, D. B.; Emmert-Buck, M. R. Nat. Genet. 1999, 21, 38–41. Hacia, J. G. Nat. Genet. 1999, 21, 42–47. Debouck, C.; Goodfellow, P. N. Nat. Genet. 1999, 21, 48–50. Bassett, D. E., Jr.; Eisen, M. B.; Boguski, M. S. Nat. Genet. 1999, 21, 51–55. Chakravarti, A. Nat. Genet. 1999, 21, 56– 60.

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silane chemistry)8,9 silicon, or gold-coated (e.g., via thiol-gold bonding) materials.10-16 The long-term stability of the probe-to(3) Trent, J. M.; Baxevanis, A. D. Nat. Genet. 2002, 32–462. Van Ingen, C. Nat. Genet. 2002, 32, 463. Duyk, G. M. Nat. Genet. 2002, 32, 465–468. Stoeckert, C. J., Jr.; Causton, H. C.; Ball, C. A. Nat. Genet. 2002, 32, 469– 473. Petricoin, E. F., III; Hackett, J. L.; Lesko, L. J.; Puri, R. K.; Gutman, S. I.; Chumakov, K.; Woodcock, J.; Feigal, D. W., Jr.; Zoon, K. C.; Sistare, F. D. Nat. Genet. 2002, 32, 474–479. Holloway, A. J.; Van Laar, R. K.; Tothill, R. W.; Bowtell, D. D. L. Nat. Genet. 2002, 32, 481–489. Churchill, G. A. Nat. Genet. 2002, 32, 490–495. Quackenbush, J. Nat. Genet. 2002, 32, 496–501. Slonim, D. K. Nat. Genet. 2002, 32, 502–508. Chuaqui, R. F.; Bonner, R. F.; Best, C. J. M.; Gillespie, J. W.; Flaig, M. J.; Hewitt, S. M.; Phillips, J. L.; Krizman, D. B.; Tangrea, M. A.; Ahram, M.; Linehan, W. M.; Knezevic, V.; Emmert-Buck, M. R. Nat. Genet. 2002, 32, 509–514. Pollack, J. R.; Iyer, V. R. Nat. Genet. 2002, 32, 515–521. Cheung, V. G.; Spielman, R. S. Nat. Genet. 2002, 32, 522–525. MacBeath, G. Nat. Genet. 2002, 32, 526–532. Chung, C. H.; Bernard, P. S.; Perou, C. M. Nat. Genet. 2002, 32, 533–540. Reinke, V. Nat. Genet. 2002, 32, 541–546. Gerhold, D. L.; Jensen, R. V.; Gullans, S. R. Nat. Genet. 2002, 32, 547–552. (4) Wulfkuhle, J. D.; Liotta, L. A.; Petricoin, E. F. Nat. Rev. Cancer 2003, 3, 267. (5) Shoemaker, D. D.; Schadt, E. E.; Armour, C. D.; He, Y. D.; Garrett-Engele, P.; McDonagh, P. D.; Loerch, P. M.; Leonardson, A.; Lum, P. Y.; Cavet, G.; Wu, L. F.; Altschuler, S. J.; Edwards, S.; King, J.; Tsang, J. S.; Schimmack, G.; Schelter, J. M.; Koch, J.; Ziman, M.; Marton, M. J.; Li, B.; Cundiff, P.; Ward, T.; Castle, J.; Krolewski, M.; Meyer, M. R.; Mao, M.; Burchard, J.; Kidd, M. J.; Dai, H.; Phillips, J. W.; Linsley, P. S.; Stoughton, R.; Scherer, S.; Boguski, M. S. Nature 2001, 409, 922. (6) Ross, D. T.; Scherf, U.; Eisen, M. B.; Perou, C. M.; Rees, C.; Spellman, P.; Iyer, V.; Jeffrey, S. S.; Van de Rijn, M.; Waltham, M.; Pergamenschikov, A.; Lee, J. C. F.; Lashkari, D.; Shalon, D.; Myers, T. G.; Weinstein, J. N.; Botstein, D.; Brown, P. O. Nat. Genet. 2000, 24, 236. (7) Nuwaysir, M. E.; Bittner, M.; Trent, J.; Barrett, J. C.; Afshari, C. A. Mol. Carcinog. 1999, 24, 153. (8) Wu, P.; Hogrebe, P.; Grainger, D. W. Biosens. Bioelectron. 2006, 21, 1252. (9) Oh, S. J.; Hong, B. J.; Choi, K. Y.; Park, J. W. OMICS J. Integr. Biol. 2006, 10, 327. (10) McGovern, M. E.; Thompson, M. Can. J. Chem. 1999, 77, 1678–1689. Chrisey, L. A.; Lee, G. U.; O’Ferrall, C. E. Nucleic Acids Res. 1996, 24, 3031. Huels, C.; Muellner, S.; Meyer, H. E.; Cahill, D. J. Drug Discovery Today 2002, 7, 119. Balladur, V. V.; Theretz, A.; Mandrand, B. J. Colloid Interface Sci. 1997, 194, 408. Kumar, A.; Larsson, O.; Parodi, D.; Liang, Z. Nucleic Acids Res. 2000, 28, e71. Lenigk, R.; Carles, M.; Ip, N. Y.; Sucher, N. J. Langmuir 2001, 17, 2497. (11) Fang, Y.; Hoh, J. H. Nucleic Acids Res. 1998, 26, 588. (12) Chiu, S. K.; Hsu, M.; Ku, W. C.; Tu, C. Y.; Tseng, Y. T.; Lau, W. K.; Yan, R. Y.; Ma, J. T.; Tzeng, C. M. Biochem. J. 2003, 374, 625. (13) Demers, L. M.; Ginger, D. S.; Park, S.-J.; Li, Z.; Chung, S.-W.; Mirkin, C. A. Science 2002, 296, 1837. (14) Herne, T. M.; Tarlov, M. J. J. Am. Chem. Soc. 1997, 119, 8916. (15) Niemeyer, C. M.; Blohm, D. Angew. Chem. 1999, 111, 3039. (16) Kelley, S. O.; Jackson, N. M.; Hill, M. G.; Barton, J. K. Angew. Chem., Int. Ed. 1999, 38, 941. 10.1021/ac1008578  2010 American Chemical Society Published on Web 06/16/2010

surface bonds formed strongly varies from method to method, and these bonds are in many cases labile under UV-light and subject to oxidation and hydrolysis.17-19 Additionally, traditional printing strategies necessitate blocking steps to prevent nonspecific binding of analyte molecules to the reactive surface,20 can give a quite strongly varying quantity of bound molecules per spot, and have a poor signal-to-noise ratio. To overcome these shortcomings, chip analysis often requires replicate chips and multiple reactions to generate high quality data. General requirements for high quality analytical procedures are highly precise measurements with limited print runs, quick substrate turnaround, and the ability to rapidly screen a changing and diverse selection of sequences. To address these challenges, which mainly derive from the surface chemistry of tethering biomolecules on self-assembled monolayers, hydrogel microarrays have been generated, which have shown improvements in terms of binding stability and number of bound molecules per surface area while minimizing interactions between probe molecules due to the spacing of probes throughout the volume.21-24 In most cases these hydrogels are based on acrylamide mixed or copolymerized with a cross-linker (e.g., bis-acrylamide) to control cross-link density and swelling of the network, which in turn allows penetration of DNA strands into the gel. Hydrogels based on poly(N-hydroxysuccinimidylmethacrylate)25 or SU826 are also reported as useful for DNA analysis. The DNA molecules are only physically trapped in the polymer network until a binding reaction is performed which can be either chemical (e.g., amine-modified DNA binding to epoxy-modified hydrogels) or photochemical in nature (e.g., with a radical initiating photo-cross-linker).27,28 However, we found several limitations of this system, including uneven spot diameters when different DNA concentrations are used and the nonhomogenous distribution of DNA molecules within a spot area due to rapid evaporation of water.29-31 Instead of using a two step process where the hydrogel is formed and then the DNA is printed on top of it, a single step procedure can be used where the spot consisting of DNA and hydrogel is formed at the same time. For example, a one step (17) Li, Z.; Jin, R.; Mirkin, C. A.; Letsinger, R. L. Nucleic Acids Res. 2002, 30, 1558. (18) Letsinger, R. L.; Elghanian, R.; Viswanadham, G.; Mirkin, C. A. Bioconjugate Chem. 2000, 11, 289. (19) Mirkin, C. A.; Letsinger, R. L.; Mucic, R. C.; Storhoff, J. J. Nature 1996, 382, 607. (20) Blanchard, A. P.; Friend, S. H. Nat. Biotechnol. 1999, 17, 953. (21) Zourob, M.; Gough, J. E.; Ulijn, R. V. Adv. Mater. 2006, 18, 655. (22) Teles, F. R. R.; Fonseca, L. P. Mater. Sci. Eng. 2008, 28, 1530. (23) Vainrub, A.; Pettitt, B. M. Phys. Rev. E 2002, 66, 041905. (24) Vainrub, A.; Pettitt, B. M. J. Am. Chem. Soc. 2003, 125, 7798–7799. (25) Feng, C. L.; Vancso, G. J.; Scho ¨nherr, H. Adv. Funct. Mater. 2006, 16, 1306. (26) Marie, R.; Schmid, S.; Johansson, A.; Ejsing, L.; Nordstroma, M.; Hafliger, D.; Christensen, C. BV; Boisen, A.; Dufva, M. Biosens. Bioelectron. 2006, 21, 1327. (27) Freidank, D. 3D-DNA-Chips: Surface Attached Functional Polymer Networks as Matrix for Nucleic Acid Microarrays. Dissertation, Insitute for Microsystem Technology (IMTEK), University of Freiburg, 2005. (28) Toomey, R.; Freidank, D.; Ru ¨ he, J. Macromolecules 2004, 37, 882–887. (29) Mirzabekov, A. D. Trends Biotechnol. 1994, 12, 27–32. (30) Proudnikov, D.; Timofeev, D.; Mirzabekov, A. D. Anal. Biochem. 1998, 259, 34–41. (31) Barsky, V. E.; Kolchinsky, A. M.; Lysov, Yu. P.; Mirzabekov, A. D. Mol. Biol. 2002, 36, 437.

process was described in the literature that includes mixing the DNA probe with a polymerizable monomer and printing this solution directly on a chip surface followed by a radical-initiated polymerization reaction.32 The attachment reaction to the substrate and the binding of DNA happen in situ. However, the swelling behavior of these resulting polymer-DNA droplets is difficult to control due to several possible side reactions, and it is unclear how many DNA strands are accessible and unaffected by the radical polymerization process.33 Here, we utilize a process, with a precursor polymer based on dimethylacrylamide (DMAA) that contains photo-cross-linkable units 4-methacryloyloxybenzophenone (MABP), to better control hydrogel formation. In our approach, the polymer precursor is mixed with the probe DNA molecules, and this solution is printed on the chip to generate a microarray. This is followed by a photoinduced cross-linking step that besides gel formation links polymer and DNA molecules to each other and the whole assembly to the chip surface.34 For read-out, optical microarray scanning is widely performed. Fluorescently labeled analyte molecules that bind to the surfacebound probes are measured, which requires careful of washing and drying steps before scanning.35,36 This method generates only one data point; the end point equilibrium which requires long analysis times, especially at low DNA concentrations. To overcome this limitation, a variety of different detection systems have been used; these include surface plasmon resonance (SPR), which enables collection of binding kinetics on a single chip but which cannot follow the full melting process with temperature ramping.37 Other methods which have been used for such analyses are electrochemical detection,38,39 microcantilever or atomic force microscopy(AFM)basedmethods,40,41 capacitancemeasurements,42,43 or even analysis by changes in structure of liquid crystals.44 Use of these techniques on microarrays has been largely confined to proof of concept studies in specialized applications. Here, we present a technique which uses total internal reflectance fluorescence (TIRF)45,46 and allows simultaneous fluorescence readout of all spots in the array at one time, over a range of temperatures, in continuous fashion and without the need for previous sample purification. TIRF instruments are well(32) Rubina, A. Y.; Pankov, S. V.; Dementieva, E. I.; Penkov, 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. (33) Olsen, K. G.; Ross, D. J.; Tarlov, M. J. Anal. Chem. 2002, 74, 1436–41. (34) Bonham, A. J.; Neumann, T.; Tirrell, M.; Reich, N. O. Nucleic Acids Res. 2009, 37, e94. (35) DeRisi, J. L.; Iyer, V. R.; Brown, P. O. Science 1997, 278, 680. (36) Nagl, S.; Schaeferling, M.; Wolfbeis, O. S. Microchim. Acta 2005, 151, 1. (37) Fiche, J. B.; Buhot, A.; Calemczuk, R.; Livache, T. Biophys. J. 2007, 92, 935. (38) Peng, H.; Soeller, Ch.; Vigar, N. A.; Caprio, V.; Travas-Sejdic, J. Biosens. Bioelectron. 2007, 22, 1868. (39) Tokuda, T.; Tanaka, K.; Matsuo, M.; Kagawa, K.; Nunoshita, M.; Ohta, J. Sens. Actuators, A 2007, 135, 315. (40) Biswal, S. L.; Raorane, D.; Chaiken, A.; Majumdar, A. JALA 2006, 11, 222. (41) Koev, S. T.; Powers, M. A.; Yi, H.; Wu, L.-Q.; Bentley, W. E.; Rubloff, G. W.; Payne, G. F.; Ghodssi, R. Lab Chip 2007, 7, 103. (42) Gooding, J. J. Electroanalysis 2002, 14, 1149. (43) Zhang, R.; Xu, X. Appl. Phys. Lett. 2004, 85, 2423. (44) Lai, S. L.; Huang, S.; Bi, X.; Yang, K.-L. Langmuir 2009, 25, 311–316. (45) Lehr, H.-P.; Brandenburg, A.; Sulz, G. Sens. Actuators, B 2003, 92, 303. (46) Bally, M.; Halter, M.; Vo ¨ro ¨s, J.; Grandin, H. M. Surf. Interface Anal. 2006, 38, 1442. (47) Turro, N. J.; Gould, St. I. R.; Liu, J.; Jenks, W. S.; Staab, H.; Alt, R. J. Am. Chem. Soc. 1989, 111, 6378. (48) Turro, N. J.; Aikawa, M.; Gould, I. R. J. Am. Chem. Soc. 1982, 104, 856.

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established in confocal microscopy applications where they are used to detect fluorescence in regions of cells within the field of view.49,50 We have built a TIRF instrument based on the first setup which was described by Lehr et al.,54 which extends this analysis to microarrays in a flow-cell (Figure 1a). The instrument irradiates the fluorescently labeled biomolecules directly from the chip surface by using an evanescent field which detects only fluorescently labeled molecules that are close to the surface (few nanometers in aqueous solution) or which are within a media of higher refractive index (higher than the surrounding media and lower than the chip substrate) that is bound to the surface. In the following, we elucidate how the hydrogel/TIRF biochip platform can be used as a robust method for the analysis of DNA oligomers and PCR products. MATERIALS AND METHODS Substrate. Commercially available PMMA slides (polymethylmethacrylate) with the dimensions of 75 × 25 × 1 mm3 were used. Fluorescence measurements indicated that these PMMA substrates have a lower background than conventional glass substrates (BK 7; Marienfeld, VWR, Schott; excitation 635 nm, emission 670-710 nm) (Supporting Information). Polymer Synthesis. Polymers were synthesized by a statistical radical polymerization of 98 wt % DMAA (Merck; distilled 30 °C at 0.1 mbar), 1 wt % vinyl phosphonic acid (VPA, Fluka), and 1 wt % 4-methacryloyloxybenzophenone (MABP) with 0.1 wt % AIBN (azobisisobutyronitrile, Sigma-Aldrich) in toluene at 60 °C for 16 h. The copolymers were precipitated in diethylether and dried in vacuum before use. MABP was synthesized from 19.82 g (0.1 mol) of 4-hydroxybenzophenone (Fluka), 11.1 g (0.11 mol) of triethylamine (Fluka), and 9.6 mL (0.1 mol) of chloromethacrylic acid (Sigma-Aldrich) in 100 mL of diethylether. After 18 h, the organic phase was washed with water and dried under vacuum. All polymers were characterized by titration with 0.01 M sodium hydroxide solution, elemental analysis, NMR, UV-vis, and FTIR spectroscopy as well as static light scattering (SI). Array Printing. An “Omnigrid 100” (Arrayit SMP 5 pin) was used to print premixed DNA-copolymer solutions on cleaned PMMA substrates at ambient conditions. The printing solutions were prepared with a final concentration of 1 mg/mL PDMAAco-1%MABP-co-1%VPS copolymer, 50 µM of DNA probes, and 100 mM sodium phosphate buffer (NaPi; pH 7). For scanning electron microscopy the hydrogel arrays were allowed to dry and then coated with gold via vapor deposition (Hummer 6.2). Samples were analyzed on a Philips LX-30 SEM. DNA. DNA oligonucleotides were purchased from TIB MOLBIOL and resuspended in water. Detailed DNA sequences are available in the Supporting Information. PCR reactions were performed in an MJ-Research PTC 200 using 50 cycles. A 0.1 µL portion of Taq polymerase (Hotstar, Qiagen) and 2.5 µL of buffer (10×, Qiagen) were added to 1 µL of 10 µM primer and 2 µL template and water to a final volume of 25 µL. The primers were used in a 1:24 ratio for asymmetric PCR to generate a single(49) Turro, N. J.; Gould, St. Ian R.; Liu, J.; Jenks, W. S.; Staab, H.; Alt, R. J. Am. Chem. Soc. 1989, 111, 6378. (50) Turro, N. J. Modern Molecular Photochemistry; University Science Books: Sausalito, CA, 1991 .

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stranded product. The forward primers used to form the analyte DNA were ordered with a Cy5 fluorescent label. TIRF and Fluorescence Detection. The home-built TIRF system consists of a laser diode (635 nm) focused by a cylindrical lens on the edge of a PMMA slide. The PMMA chip was mounted at the front of a flow cell device which is temperature-controlled by a Peltier element. The fluorescence signal was detected by a CCD camera (Spectrasource) which was equipped with a Cy 5 fluorescence filter (670-710 nm). The software “biodetect” (Genescan, Freiburg i. Br., Germany) controlled the excitation times (between 0.5 and 60 s) and temperature (between 20 and 90 °C).

RESULTS AND DISCUSSION Here, we present an analysis platform with two novel elements: the DNA chip based on a printing and photocross-linking procedure and the TIRF platform which allows continuous measurements. The process of DNA chip preparation is illustrated in Figure 1d. It starts with a cleaned, but otherwise unmodified, PMMA substrate onto which the mixture of DNA and hydrogel precursor is printed. The hydrogel precursor consists of a PDMAA-co-1%MABP-co-1%VPS polymer which is cross-linked after deposition via irradiation with ultraviolet (UV) light. MABP was chosen as a cross-linking agent due to the long lifetime stability of the biradicaloid triplet state that is formed when the benzophenone molecule absorbs UV light (∼250 nm).47 This extended radical lifetime enables a longer diffusion, thus increasing the statistical chance of a successful reaction.48-50 It has been shown that polymers that contain MABP not only cross-link, but also have the ability to form stable covalent bonds between the forming polymer networks and the underlying polymer substrates (e.g., PMMA). The ratio of MABP to other comonomers present in the polymer controls the cross-link density (“mesh size”) and the swelling degree of the hydrogel in the aqueous medium of the hybridization reaction. High MABP content leads to strong crosslinking, low mesh size, and consequently a low degree of swelling, while a too low content can lead to unstable polymer network formation. A minimal concentration of 1% MABP within the polymer was chosen on the basis of previous studies which showed that a lower content prevents formation of a stable hydrogel after the cross-linking reaction.28 The swelling behavior of the formed polymer networks was analyzed by surface plasmon resonance spectroscopy in various concentrations of single and multivalent buffer systems. The results show linearly decreasing swelling rate with increasing buffer concentration (SI). The hydrogel swelling range lies between 300% and 320% for monovalent buffers (sodium chloride buffer) and between 250% and 320% for bivalent buffers (sodium phosphate buffer). Our standard hybridization sodium phosphate buffer used for the experiments has a concentration of 100 mM salt and gives a swelling ratio of 300%, so that, in the swollen gel, roughly 70% of the volume is taken up by water. Combined with the assumption that the hydrogel behaves like an ideal surface attached polymer network (relying on the Flory-Rehner theory27,28), the estimated statistical pore size was 55-65 nm. As this size is comparable to the dimensions of the DNA, a strand of

Figure 1. (A) Principle of the ATR detection system: a laser beam (λ ) 635 nm) is focused at a certain angle above the critical angle on the edge of a PMMA slide which is used as a waveguide. As the light travels through the waveguide, an evanescent field is formed on its surface.55 An array of several different probe DNA-polymer spots are printed within the are of the constant evanescent field. A CCD camera allows the analysis of hybridization in different time ranges along with the use of a temperature-controlled flow cell in real time. (B) Penetration depth and intensity of the evanescent field starting from the PMMA surface through the hydrogel and into the aqueous solution. The hydrogel DNA spots have a significantly higher optical density (nd ) 1.4) compared to that of the surrounding aqueous analyte DNA solution (nd ) 1.33). Thus, the evanescent field intensity levels off slowly with distance within the hydrogel compared to the aqueous solution, since the refractive index of the PMMA slide (nd ) 1.49) is much closer to that of the hydrogel. Since the evanescent field penetrates the hydrogel completely we assume that all fluorescent labels within the hydrogel are irradiated while fluorescent labels in solution were only detected within a few tens of nanometers in solution (roughly less than 1%). (C) Preparation of the DNA-hydrogel-PMMA chip. Aqueous solutions of polymer precursor PDMAA-co1%MABP-co-1%VPS mixed with DNA probes are printed in arrays on PMMA substrates. UV irradiation of the spots cross-links the polymer chains and attaches the DNA and the substrate to the forming hydrogel. The DNA hydrogel arrays are used for hybridization experiments as the spots have a well-controlled morphology. (D) Image of the printing tip and hydrogel array. SEM images (E, F) show that the printed and cross-linked hydrogel spots show consistent size and spacing after printing. The hydrogel porosity (G), with an average pore size of about 80 nm allowing long DNA analyte molecules to penetrate (50 000× magnification).

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200 basepairs should be able to penetrate through this given mesh size.51 The hydrogel forms a covalent DNA-hydrogel attachment after the UV cross-linking binding reaction. The DNA base thymine generates radicals when irradiated with UV light, similar to the benzophonone molecule. This reaction is widely known for the photoreaction of two thymine molecules located in close proximity, but binding to other nearby molecules has also been observed. Experiments have shown that 10% of thymine molecules are affected when irradiated with light at 250 nm and 1 J of energy.27 It is also known that radicals generated by UV irradiation by benzophenone can be transferred to a thymine molecule. At the same time, the benzophenone decays back to its ground state and is able to absorb light and bind to another molecule.52 In our experiments the DNA probes were modified with an 18 thymine base tail to ensure stable binding to the hydrogel and chip surface. In addition, it is worth noting that the benzophenone radicals are known to attack alkyl groups such as the sugar units of the DNA, but in general do not react with aromatic moieties such as the base parts of the nucleotides, which are the key component for molecular recognition in hybridization. The stability of covalent attachment between DNA, hydrogel, and PMMA substrate is shown in the fluorescence measurements in Figure 2a-c which will be discussed further. The DNA-hydrogel arrays were printed by a conventional pin printer to yield spots with a diameter of 205 µm ± 9% and a height of 100 nm, as measured in the dry state by white light interferometry. On the basis of the swelling studies on large scale planar films, we estimate that the swollen droplets will not be higher than 400 nm. Interestingly, the dried spots also reveal a consistent morphology (Figure 1e-g), with an open mesh structure and average pore size of 80 nm measured by SEM. Although these images were collected in the dry state, they suggest that the interior of the swollen hydrogel will be easily accessible to even large DNA molecules. The accessibility and hybridization reaction of analyte DNA to the probe DNA bound in the chip-attached hydrogel was elucidated by fluorescence analysis. To generate a chip development platform that allows changing temperature and buffer conditions and continuous tracking of the fluorescence signal, we used a home-built TIRF instrument. As mentioned previously, the chip is used as an optical waveguide. Light from a laser or an LED is coupled into the waveguide, and at the surface of the chip an evanescent field is generated. The depth of this field is dependent on the wavelength of the light, the angle of incidence, and the refractive index ratio between the chip and the surrounding media. In our setup, the PMMA (nd ) 1.49) attached swollen hydrogel has a refractive index of nd ) 1.4, which is significantly higher than the surrounding buffer solution (nd ∼1.3) and allows the evanescent field to extend nearly to the full height of the hydrogel droplet with only a loss of 30% in signal intensity. The rate of field intensity loss per unit of distance in solution is about 10 times greater than the loss in the hydrogel (Figure 1b). Consequently, the hydrogel allows the detection of all fluorescently labeled (51) Tegenfeldt, J. O.; Prinz, C.; Cao, H.; Chou, S.; Reisner, W. W.; Riehn, R.; Wang, Y. M.; Cox, E. C.; Sturm, J. C.; Silberzan, P.; Austin, R. H. Proc. Natl. Acad. Sci. U.S.A. 2004, 101, 10979. (52) Dankbar, D. M.; Gauglitz, G. Anal. Bioanal. Chem. 2006, 386, 1967.

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molecules confined in the hydrogel while excluding similarly labeled molecules in solution from significant excitation. As the hydrogel is negatively charged, it is likely that nonspecifically adsorbing, non-complementary DNA molecules are expelled from the polymer, so that only complementary DNA strands remain in the hydrogel to be analyzed by fluorescence analysis, in agreement with the excellent signal-to-noise ratios observed. To demonstrate the robustness of the new polymer hydrogel DNA chips in combination with the TIRF device, several DNA hybridization reactions were performed where the temperature was repeatedly cycled from 25 °C up to 85 °C at a rate of 5 °C per minute inducing several annealing and melting steps. At low temperatures, complementary DNA analyte strands in solution bind to probes on the chip and give high fluorescence signal. As the temperature increases, hydrogen bonds dissociate (“melting of the DNA”) which leads to fewer bound analyte molecules and a weaker signal observed on the chip. At 75 °C and above, all analyte molecules are removed and returned to solution, giving the minimum fluorescence signal, essentially returning the chip back to the virgin state. When the temperature is decreased, DNA hybridization occurs again. This process of annealing and melting was repeated 50 times over a time span of 1000 min (Figure 2a-c, all 50 cycles shown in Supporting Information). In order to minimize signal loss from photobleaching of the fluorescent dye, the analyte solution was exchanged every 100 min. This entailed removing the analyte solution from the chamber, followed by a heated wash cycle (90 °C) to ensure all bound analyte molecules were melted and removed from the probe molecules on the chip, followed by drying the chamber under nitrogen and then refilling it with new analyte solution. Interestingly, even with frequent exchange of analyte solutions the maximum signal of the hybridization reaction at low temperatures remained nearly constant. The observed slightly lower hybridization efficiency after each initial round of addition of new analyte solution is probably due to the denaturation of preformed secondary structures of the analyte. This interpretation is in agreement with the slightly lower melting temperature observed after each new analyte addition. The results of the repetition experiments demonstrate that the fluorescence signals remain constant over time even during many temperature cycles, that the amount of accessible DNA probe molecules bound in the hydrogel remains constant, and that the hydrogel, DNA, and the covalent bonding between both are stable enough to survive even prolonged heating without degradation. Also, heating up the chip and melting off the DNA analytes from the probes on the surface followed by washing the DNA analyte off the hybridization chamber at high temperature offers an easy way to rescue the chip for further studies, as the hybridization efficiency is retained. Reuse of the chips has important benefits for technology development, as it allows for inexpensive chip fabrication and extensive studies with the use of only a small number of chips, thus decreasing development cost significantly. High sensitivity is another major goal in microarray based analysis processes. In our study the chip sensitivity was measured with a 21-mer DNA probe and different analyte concentrations, from 0.1 nM to 100 nM labeled antisense DNA. The lowest amount of analyte molecules detected within a minute of irradiation was 4 fmol (Figure 2b). The greater the analyte concentration, the more molecules can bind to the probe molecules on the chip and

Figure 2. Stability and sensitivity of the TIRF-DNA chip platform: (A, C) Stability of the DNA hydrogel chip within the hybridization regime. Several hybridization-melting cycles have been performed by cycling the temperature between 25 and 80 °C with 5 °C steps. The measured fluorescent signal increases at low temperatures and decreases with increasing temperatures as labeled analyte DNA molecules melt or reanneal with probe DNA molecules on the chip. Even after several cycles a constant maximum signal was obtained, indicating that the DNA-hydrogel chip is stable for many hybridization reactions even at high temperatures. (B) Sensitivity of the DNA chips performed with 21-mer DNA probe and complementary analyte molecules. Several concentrations of analyte between 0.1 and 100 nM at a given volume of 20 µL were analyzed. The irradiation time-normalized signals of the CCD camera give a linear dependence and allow a detection limit of 4 fmol of the analyte molecules. (D) Melting curves of a 33 base-long probe DNA (standard mixture of 100 mM Napi, 1 mg/mL PDMAAco-1% MABP-co-1% VPS; 50 µM DNA) with 21-mer Cy5 labeled analytes. The wild type probes have a higher melting temperature than the mutant type, caused by the single nucleotide polymorphism (SNP). This mismatch leads to less hydrogen bonding between the analyte and probe DNA and therefore weaker binding between both strands. The difference between both curves gives with its maximum the optimum differential temperature between wild type and mutant. One chip was used to analyze the melting temperature of wild type strands (E) and mutant type strands (F) at a wide range of hybridization buffer concentration (between 50 and 900 mM Napi) which was found to be dependent on the probe length.

the lower the measurement time needed to obtain a processable signal. All fluorescence signals were normalized to an irradiation time of 1 s which was possible due to the linearity of the signal of the CCD camera (data not shown). In our microarray experiments we observed a linear dependence between the amount of analyte

and the signal intensity from 4 fmol to 4 pmol, spanning nearly 3 orders of magnitude. We further applied our system to the detection of single nucleotide polymorphisms (SNP), which consist of a single base change in the DNA leading to a one basepair mismatch in the Analytical Chemistry, Vol. 82, No. 14, July 15, 2010

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Figure 3. (A) TIRF analysis of DNA sample. Firstly DNA strands were extracted and purified from blood samples. These DNA strands were amplified by polymerase chain reactions (PCR) with fluorescently (Cy5) labeled primers for the H63D gene. The obtained PCR product was mixed with hybridization buffer and then injected into the flow cell of the TIRF device without any further purification. The resulting false color fluorescent images of a DNA chip containing wild type and mutant probes incubated in the flow cell with three different blood samples are shown in (B-D). DNA amplification products of a blood sample shown in (B) predominantly bind to mutant probes on the chip indicating a mutant sample or gene mutation in the patient’s DNA (homozygote mutant type). In sample (C) both mutant and wild type probes display fluorescent signals indicating that the blood sample contains both genetically mutated and unmutated material (heterozygote). This means that both genes in the blood cells vary in their genetic information. In (D) only wild type probes bind to labeled analyte molecules which indicate that only unmutated genetic material is present in the blood samples.

DNA double strand (mutant type) compared to a fully complementary double strand (wild type). We focused on the hemochromatosis gene (HFE), as it is well characterized to have SNPs in the 63 and 282 alleles.53-55 A basepair mismatch causes a melting point decrease in DNA annealing, and as the TIRF device can sensitively measure the melting point of analyte DNA to probes of known sequence on the chip, we should be able to differentiate between mutant and wild type analytes. To do this, we generated an array consisting of increasing probe length between 13- and 31-mers of wild type and mutant type sequences and challenged it by adding synthetic Cy5-labeled wild type analyte molecules (21-mer) in solution. After 10 min of incubation at 20 °C, the temperature was ramped up to 85 °C (1 °C/20 s). The kinetics of the melting process for the mutant type and wild type probes are shown in Figure 2d. The data show that the melting point of the wild type (61 °C) is significantly higher than the mutant type probe (52 °C) when wild type analyte is used. Furthermore, the kinetics of the melting process between both probes allows us to determine (53) Feder, J. N.; Gnirke, A.; Thomas, W.; Tsuchihashi, Z.; Ruddy, D. A.; Basava, A. Nat. Genet. 1996, 13, 399. (54) Jazwinska, E. C.; Cullen, L. M.; Busfield, F.; Pyper, W. R.; Webb, S. I.; Powell, L. W.; Morris, C. P.; Walsh, T. P. Nat. Genet. 1996, 14, 249. (55) Carella, M.; D’Ambrosio, L.; Totaro, A.; Grifa, A.; Valentino, M. A.; Piperno, A.; Franco, B.; Gasparini, P.; Camaschella, C. Am. J. Hum. Genet. 1997, 60, 828.

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the optimized temperature where the difference between the fluorescence signal of mutant and wild type DNA is maximized (57 °C). Additionally, we were able to determine the optimal hybridization/melting conditions dependent on probe length and buffer conditions. This was done for the wild and mutant type probes shown separately in Figure 2e,f. We observe that, in general, the melting temperature increases with buffer concentration, with the largest change seen between 50 and 100 mM buffer and with the higher buffer concentrations giving smaller changes in signal. The melting temperature also increased with the probe length, up to the length of the full analyte, 21 bases. Again this demonstrates that the ability to use one chip to optimize the reaction conditions allows easy, fast, and inexpensive chip development and the whole parameter space can be analyzed within a few hours using just one chip. Our platform can also be used to analyze longer (up to 200 bases long) PCR products. This is especially important as the binding of longer DNA pairs is more challenging than simple oligo-oligo hybridization. DNA was purified from blood samples and amplified by PCR with fluorescently labeled primers. The primers were chosen to amplify the HFE 63 allele. After PCR amplification, buffer was added and the solution was injected into the TIRF flow cell across an array with probes for both the point mutation at the 63 allele and wild type sequence. The 63 gene is

present as one copy per chromosome such that three states are possible: mutant type (homozygote), wild type (homozygote), or both wild and mutant type (heterozygote). In Figure 3, fluorescence images of three analyte solutions that were measured at 49 °C are shown: (A) Fluorescence signals only at the mutant type probes while the wild types probes show no signal, indicating that the probe is homozygote mutant type. (B) Both wild and mutant type probes show a signal indicating a heterozygote sample. (C) Only the wild type probes on the chip give a signal, representing a wild type homozygote sample (melting kinetics available in Supporting Information). The complete set of all measurements starting with the PCR products to the final result was performed in less than 1 h, showing the strength of the system for practical on-site screening applications. CONCLUSION In this paper, we present a novel DNA-hydrogel chip platform for the analysis of biological reactions on microarrays with excellent time efficiency, stability, and reproducibility. Our platform is based on the use of a polymer substrate (PMMA) and the printing of mixtures of DNA together with a water-swellable, slightly charged polymer precursor that is able to form a hydrogel upon short UV irradiation. The irradiation process induces, at the same time, covalent attachment of nucleic acids to the hydrogel and the formation of covalent bonds between hydrogel and the chip surface. It is particularly attractive in that surface attachment occurs without the need of any prior surface modification reaction or substrate treatment. We have demonstrated that the use of hydrogel chips combined with TIRF readout gives a system that is highly sensitive, allows on-chip reactions with real time readout, and can be used for the quick and convenient analysis of SNPs via analysis of PCR products. Using

the chip itself as a waveguide and taking advantage of evanescent field illumination results in a higher signal-to-noise ratio as the background is significantly attenuated. In addition, we have shown that the chip can be reused for many cycles without loss of probe molecules or sensitivity, which enables the use of this device for chip development purposes, such as screening buffer conditions or probe length. Furthermore, it permits vastly improved quality control as, after completion of the analysis process, the analyte DNA can be melted off, washed away and replaced by DNA from either positive/negative controls or calibration solutions without removing the chip from the device. Further studies, where we use the platform described here for the analysis of patient samples in various microdiagnostic applications or in food quality control will be reported in upcoming communications. ACKNOWLEDGMENT We would like to thank Dr. Holger Klapproth, Dr. Oswald Prucker, Prof. Matthew Tirrell, and Prof. Norbert Reich for discussions and Dr. Silke Lassmann (Pathological Institute, University of Freiburg) for the provided DNA extracted from the blood samples and for discussions. We would like to thank Surekha Gajria for help in manuscript preparation and Andreas Evers for his comments. SUPPORTING INFORMATION AVAILABLE Additional protocols, controls, and experimental details. This material is available free of charge via the Internet at http:// pubs.acs.org. Received for review April 1, 2010. Accepted May 29, 2010. AC1008578

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