Multifunctional CMOS Microchip Coatings for Protein and Peptide Arrays

Complementary metal oxide semiconductor (CMOS) microelectronic chips fulfill important functions ... We present a novel surface coating for CMOS micro...
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Multifunctional CMOS Microchip Coatings for Protein and Peptide Arrays Volker Stadler,*,† Mario Beyer,† Kai Ko1 nig,†,‡ Alexander Nesterov,†,‡ Gloria Torralba,‡ Volker Lindenstruth,‡ Michael Hausmann,‡ F. Ralf Bischoff,† and Frank Breitling† German Cancer Research Center, Im Neuenheimer Feld 280, D-69120 Heidelberg, Germany, and Kirchhoff Institute for Physics, University of Heidelberg, Im Neuenheimer Feld 227, D-69120 Heidelberg, Germany Received March 8, 2007

Complementary metal oxide semiconductor (CMOS) microelectronic chips fulfill important functions in the field of biomedical research, ranging from the generation of high complexity DNA and protein arrays to the detection of specific interactions thereupon. Nevertheless, the issue of merging pure CMOS technology with a chemically stable surface modification which further resists interfering nonspecific protein adsorption has not been addressed yet. We present a novel surface coating for CMOS microchips based on poly(ethylene glycol)methacrylate graft polymer films, which in addition provides high loadings of functional groups for the linkage of probe molecules. The coated microchips were compatible with the harshest conditions emerging in microarray generating methods, thoroughly retaining structural integrity and microelectronic functionality. Nonspecific adsorption of proteins on the chip’s surface was completely obviated even with complex serum protein mixtures. We could demonstrate the background-free antibody staining of immobilized probe molecules without using any blocking agents, encouraging further integration of CMOS technology in proteome research. Keywords: ATRP • microchips • array support • immunoassay • surface coating • protein adsorption

Introduction Because of low power requirements and miniaturization, CMOS (complementary metal oxide semiconductor) technology has emerged as a key technology in nowadays electronics, exemplified by microprocessors, storage, and sensor elements. From there, CMOS technology has a considerable impact not only on today’s life, but also on life sciences. To an increasing extent, CMOS microchips participate in the generation and analysis of DNA, protein, and peptide arrays, and hence, considerably contribute to great progress in biomolecular and biomedical research. The most prominent examples are CMOS photosensor arrays for the detection of fluorescence1,2 or chemiluminescence3,4 signals on DNA chips. The detection of DNA probes labeled with gold nanoparticles has also been accomplished by CMOS active pixel image sensors.5 Other CMOS-based analysis technologies employ MOSFET charge sensors,6,7 conduction,8 or impedance sensors,9 as well as electrochemical detection methods.10,11 With respect to microarray generation for high-throughput screenings, the use of passive electrodes only provides low complexities.12,13 In contrast, higher electrode densities and thus enhanced array complexities can be achieved by active CMOS circuitry. This was demonstrated by Caillat et al. using a CMOSbased array of gold-coated electrodes for electroimmobilization * To whom correspondence should be addressed. Tel., +49-6221-424744; fax, +49-6221-421744; e-mail: [email protected]. † German Cancer Research Center. ‡ University of Heidelberg. 10.1021/pr0701310 CCC: $37.00

 2007 American Chemical Society

of probe molecules.14 With chips of 400 and 1600 electrodes as well as a biotin-mediated immobilization strategy, researchers from Nanogen, Inc. generated DNA and antibody arrays, respectively.15,16 Combinatorial microarray synthesis on CMOS chips was primarily reported by Dill and co-workers employing the CombiMatrix electrode array biochip with 1024 electrodes of 100 µm in diameter.17 Moreover, they demonstrated the utilization of these electrodes for the enzyme-amplified electrochemical detection of DNA-labeled proteins, which have been immobilized by hybridization with complementary strands on the chip.18 Despite elaborated techniques for microarray generation and analysis, little attention has been paid to an appropriate surface modification of CMOS chips. In particular, this might be attributed to the corrosion sensitivity of aluminum in microelectronic devices, prohibiting some of the most common methods for surface processing.19 In most cases, well established silane monolayers were used for functionalizing aluminum6 or SiO2 faces,3,5,8,9 as well as thiolated molecules for noble metals.7,10,11 Higher loadings of probe molecules were obtained by three-dimensional layer geometries, as shown for spincoated agarose films15,16 and modified controlled pore glass (CPG) membranes.17 Since in most array applications proteins are involved, nonspecific adsorption due to hydrophobic interactions20 actually is a crucial point in terms of signal noise and clarity. In a multiplicity of biochemical assays, proteins are used for blocking these unwanted interactions, with the advantageous side effect that probe or target molecules can Journal of Proteome Research 2007, 6, 3197-3202

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research articles be stabilized by the protein solution. However, a substantial background adsorption in immunoassays was reported even in the presence of blocking proteins.21 In applications like the analysis of binding partners by mass spectrometry, or the screening for enzyme substrates, blocking with proteins would, in addition, massively disturb the analysis by superimposing specific signals. Nevertheless, the issue of nonspecific protein adsorption hitherto either was not addressed or just tolerated on CMOS chips. This is exemplified by the agarose coatings, where signalto-noise ratios of only 4.2:1 and below were achieved.16 Therefore, we herewith report the corrosion-free generation of functionalized and protein repelling CMOS chip coatings based on poly(ethylene glycol)methacrylate [PEGMA] graft polymer films via atom transfer radical polymerization [ATRP]. The modified microchips provided a high loading of functional groups and chemical stability with respect to harshest conditions in DNA, protein, or peptide array generation, exemplified by the side chain deprotection of peptides with concentrated trifluoroacetic acid (TFA). Furthermore, no objectionable nonspecific adsorption of proteins was observed when PEGMAcoated CMOS chips were incubated with BSA, lysozyme, fibrinogen, γ-globulin, and human serum, as shown with X-ray photoelectron spectroscopy (XPS) and fluorescence microscopy. As a consequence, the coated microchips could be used without any blocking agent, as illustrated by the backgroundfree antibody staining of spotted peptides. This should allow the unrestricted use of such coated CMOS chips in microarray generation or analysis, making them an ideal substrate for proteome research.

Materials and Methods Surface modification and analysis were performed on polished single-crystal silicon (100) wafers (Silicon Sense, Nashua, NH), silicon nitride wafers (IMS Chips, Stuttgart, Germany), and aluminum plates (Sigma-Aldrich, Taufkirchen, Germany). The CMOS chip was realized by the AMIS C07M I2T100 high-voltage CMOS process.22 X-ray photoelectron spectra and ellipsometric data were recorded as described elsewhere.23 The same applies for the functionalization of PEGMA films, peptide spotting, and FITC-labeling of proteins. A LEO 1530 scanning electron microscope with an e-beam energy of 3 keV was used for chip surface characterization. Fluorescence microscopy was done with a ZEISS Axiovert 35 microscope equipped with a digital CCD camera system (pixelfly 270 XS, PCO AG, Kelheim) and appropriate filter sets for fluorochromes. Initiator Synthesis. A mixture of freshly distilled triethylamine (2.02 g, 20 mmol) and aminopropyltriethoxysilane (4.21 g, 19 mmol) was dissolved in anhydrous methylene chloride (65 mL) under nitrogen atmosphere and cooled to -80 °C. 2-Bromo-2-methyl-N-propyltrimethoxysilyl isobutyramide was then synthesized by adding a solution of bromoacetyl bromide (4.60 g, 20 mmol) in anhydrous methylene chloride (30 mL) dropwise under stirring. When the addition was complete, the mixture was allowed to warm to 20 °C. The solvent was removed under reduced pressure, followed by addition of 50 mL anhydrous n-hexane. After 30 min of stirring under nitrogen atmosphere, the ammonium salts were filtered and washed with anhydrous n-hexane. The filtrate was concentrated to a red oil, which was distilled twice under vacuum to yield 6.19 g (16.7 mmol, 88.0%) of a colorless oil. Bp0.4mbar 127 °C; 1H NMR (300 MHz, CDCl3): δ ) 0.63 (t, 3J ) 7.5 Hz, 2H), 1.19 (m, 9H), 1.65 (m, 2H), 1.91 (s, 6H), 3.23 (m, 2H), 3.75 (m, 6H), 6.85 ppm 3198

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(m, 1H); 13C NMR (75 MHz, CDCl3): δ ) 7.75, 18.28, 22.69, 32.62, 42.59, 58.46, 63.33, 171.86; C13H28O4NSi calcd: C 42.16, H 7.62, N 3.78, Br 21.57; found: C 41.99, H 7.61, N 3.86, Br 21.71. Surface Modification. Before silanization, wafers and microchips were cleaned and preactivated by irradiation with a 150 W mercury vapor lamp (Heraeus Noblelight, Germany, model TQ 150) positioned in a distance of 4 cm for 1 h in air. Afterward, they were immediately incubated in a 10 mM solution of 2-bromo-2-methyl-N-propyltrimethoxysilyl isobutyramide in anhydrous methylene chloride over night. The reaction was stopped by addition of ethanol (p.a.), followed by extensive rinsing with ethanol and distilled water. After that, wafers and microchips were carefully dried in a stream of dried air. To achieve full condensation of silanol groups, silanised wafers and microchips were heated for 1 h at 100 °C in an oven. Storage was done under nitrogen atmosphere at -20 °C. Microchips and wafers were solely handled with PP tweezers to avoid scratching of the surfaces. Graft polymerization was typically carried out with 5 mL of poly(ethylene glycol)methacrylate (average Mn ∼ 360, SigmaAldrich, Taufkirchen, Germany) in 10 mL of a water/methanol (p.a.) mixture (1:1). If monomer concentrations were varied, the total volume was adjusted by reducing or increasing the water/methanol mixture. Then, 64 mg of CuBr (0.45 mmol) and 141 mg of 2,2′-bipyridine (0.90 mmol) were added as ATRP catalyst. The brown reaction mixture was immediately degassed and sonificated under nitrogen atmosphere to dissolve the CuBr, before the silanized wafers or chips were immersed. Polymerization took place under nitrogen atmosphere for at least 20 h, unless otherwise noted. Afterward, the samples were extensively rinsed with distilled water, blown dry in a stream of dried air and stored under nitrogen atmosphere at -20 °C. PEGMA films of about 80 nm and more were indicated by a blue staining of silicon substrates. Incubation with Protein Solutions. Initially, PEGMA-grafted wafers or microchips were hydrated with PBS buffer for 10 min. The buffer was removed, and the samples were immersed into 10 mL of the respective protein solution (PBS buffer, 2 mg/ mL) in a Petri dish. Human serum was diluted 1:240 in PBS buffer to adjust the final protein concentration to approximately 2 mg/mL. With FITC-labeled proteins, samples were incubated in the dark. After 60 min, the protein solutions were diluted continuously with PBS buffer to remove the proteins and to avoid Langmuir-Blodgett like protein transfer onto the surface upon dehumidification of the sample. Finally, the slides were rinsed with Millipore water to remove residual PBS, blown dry with nitrogen, and stored at -20 °C under nitrogen in the dark. Immunoassays. Antibodies (anti-HA and anti-FLAG) were diluted by a factor of 1:1000 in TBS-buffered solution. Coated microchips were preincubated with Tween in TBS (0.1% per volume) at pH 7.4. After 15 min, the buffer solution was removed, and each chip was covered with 1 mL of the antibody solution for 1 h at room temperature. After washing 5 times for 5 min with Tween in TBS, the microchips were covered with 1 mL of fluorescence dye labeled secondary antibodies [Alexa Fluor 647 goat anti-rabbit IgG (H+L) and Alexa Fluor 546 goat anti-mouse IgG (H+L), 1:1000 in TBS/Tween] for 1 h at room temperature in the dark. Again, microchips were washed 5 times for 5 min with Tween in TBS and rinsed with distilled water in lightproof tubes. The samples were blown dry with nitrogen and processed immediately.

CMOS Microchip Coatings for Protein/Peptide Arrays

research articles halide as surface-initiator, this Cu(I)-catalyzed living radical polymerization of the monomer (Figure 2a) obviates such unwanted reactions and further offers very mild reaction conditions.24,25 From there, we synthesized monolayers of 2-bromo-2-methyl-N-propyltrimethoxysilyl isobutyramide (Figure 2b) on silicon, aluminum, and Si3N4 wafers, as well as on custom-made CMOS chips, which we aim to establish for microarray generation and upcoming immunoassays. The CMOS chip disposes of several arrays of 8 × 8 pixel electrodes with an electrode pitch ranging from 30 to 100 µm, which can be addressed individually with voltages from 30 to 100 V. Furthermore, we also integrated photodiodes underneath each single electrode of some arrays. The surface was mainly coated with a Si3N4 insulation, whereas the aluminum electrodes of two distinctive arrays were uncoated.

Figure 1. Degradation of the Si3N4/Al-surface of a passive chip with UV-induced graft polymerization.

Results and Discussion In an earlier report, we investigated the generation and application of a similar protein repelling PEGMA coating on conventional glass slides.23 UV-induced graft polymerization of PEGMA was carried out after ozonization of a 7-octenyltrichlorosilane SAM, followed by extraction of residual homopolymer by stirring in distilled water at 70 °C. In accordance to that, we initially tried to modify a passive chip. This microchip was mainly covered by aluminum and comprised of an array of pixel electrodes coated with a Si3N4 insulation. Unfortunately, we observed complete degradation of the chip’s surface when heated in distilled water to extract residual homopolymer (Figure 1): The Si3N4 insulation partially exfoliated, while the aluminum was corroded. Thus, since it was mandatory to avoid homopolymerisation, we examined the versatile ATRP method as alternative for the generation of PEGMA graft polymer films. Starting from a immobilized alkyl

XPS revealed the formation of the Br-terminated silane monolayer on all surfaces, as clearly indicated by the Br3dSignal (Figure 2c, gray curves). The average stoichiometric ratio C-C:C-O:CdO:N:Br on silicon, Si3N4, and CMOS chips was evaluated to 4 (4):1.9 (2):1.1 (1):1.1 (1): 0.7 (1), which corresponds well with the theoretical values given in parentheses. A slight loss of bromine in the monolayer compared to the elemental analysis of the pure compound might be attributed to an X-ray sensitivity of this head group, but this issue was not addressed further. Generally, the signals obtained from the CMOS chip were in good accordance with XP spectra from Si3N4 wafers. A large N1s peak and the broad Si2p signal of the CMOS chip complied with the Si3N4 surface of the CMOS chip, which, in addition, is usually covered by a thin silicon oxide layer.26 Since the total area of uncoated electrodes was below the XPS detection limit (>3%), aluminum signals were not observed. The monolayer thickness on silicon wafers was averaged to be (20.0 ( 6.7) Å by ellipsometry. Starting from the immobilized initiator (Figure 2B), CuBr/ bipy mediated graft polymerizations of PEGMA films were carried out on all surfaces mentioned above. As shown in Figure 2c (black curves), XP spectra of PEGMA films with a thickness

Figure 2. Graft polymerization on chip surfaces: (a) PEGMA monomer, (b) 2-bromo-2-methyl-N-propyltrimethoxysilyl isobutyramide as immobilized initiator; (c) XP spectra of Si3N4 wafers (solid line) and CMOS chips (dotted line) with silane monolayers (gray) and PEGMA graft polymer films (black). Journal of Proteome Research • Vol. 6, No. 8, 2007 3199

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stoichiometric ratio C-C:C-O:CdO of 3 (3):9.2 (10):1.4 (1). The C:O ratio was 1.9 (2):1, which is in good accordance with PEGMA films obtained from UV-induced polymerization.23 Figure 3 shows the dependency of PEGMA film thicknesses on reaction time and monomer concentration on silicon wafers. With a polymerization time of 30 h, an increasing monomer concentration caused a linear growth in PEGMA film thicknesses. Nevertheless, we adjusted the standard concentration to a third of the total volume, since sufficiently thick films were obtained with a moderate consumption of monomer that way. With respect to incubation time, the curvature corresponded to typical behavior of ATR polymerizations, indicating firstorder kinetics.24,27 With a standardized polymerization time of at least 24 h, we routinely generated PEGMA films of 85-100 nm with a medium standard deviation of only 2 nm all over a 4′′ silicon wafer.

Figure 3. ATR polymerization on silicon wafers, PEGMA film thickness against polymerization time and monomer concentration (the error bars represent standard deviations).

of 50 nm and above resulted in a total attenuation of substrate signals (Si2p, N1s, Br3d). We also observed considerably increasing C1s signals with characteristic peak shapes and a

In consideration of upcoming microarray applications in molecular biology, we investigated the stability and functionality of PEGMA graft polymer films on CMOS microchips under the harshest conditions emerging in the generation of DNA, protein, or peptide arrays. These are represented by treatments with different organic solvents, buffers, and the final TFAmediated side-group deprotection in combinatorial peptide array synthesis.28 The functionality was examined by the SMCCmediated coupling of peptides as probe molecules, which, in addition, were targeted with corresponding monoclonal antibodies. However, we initially determined the loading of PEGMAcoated CMOS chips; therefore, we coupled Fmoc-β-alanine with the terminal OH groups of the polymer films and determined the NH2 loading by measuring the dibenzofulvenepiperidine adduct of piperidine-mediated Fmoc deprotection at 301 nm. In accordance to PEGMA films from UV-induced polymerization, we obtained densities of up to 40 nmol cm-2, which is several magnitudes higher than typical loadings of rigid array supports in the picomolar range.23 In doing so, the

Figure 4. Structural integrity: PEGMA films on CMOS microchips after TFA treatment without (a and b) and with (c and d) prior curing of silane monolayers; (e and f) Reversible wrinkling of PEGMA films on Si3N4 wafers without prior curing. 3200

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Figure 5. Microelectronic functionality: test patterns written in memory cells of individual microelectrodes. Input (left) and corresponded output (right).

structural integrity and microelectronic functionality of the CMOS chips were unaffected. Unfortunately, this was not the case after TFA treatment. Initially, when the silane monolayers were not cured prior to ATRP, we observed wrinkling of the PEGMA polymer accompanied by partial delamination from the chip’s surface (Figure 4a,b). This effect was also noticed on pristine Si3N4 wafers, but not on silicon and aluminum (Figure 4f). It was supposed that the acidic conditions diminished the adhesion of the organic coating by disturbing the interaction of the monolayer’s silanol groups with the surface. On the molecular level, this probably might originate from the protonation of residual amino groups on the silicon nitride surface, exciting repulsive electrostatic interactions. Despite this deformation, XP spectra revealed compact PEGMA films on Si3N4, since substrate signals were still completely attenuated (data not shown). By virtue of the organic character of the wrinkling, we were able to completely remove the coating by irradiation with a 150 W mercury vapor lamp for 2 h in air (Figure 4e), followed again by ATRP and TFA-mediated wrinkling (Figure 4f).

research articles Nevertheless, we were able to overcome this Si3N4-based problem just by curing the silane films prior to PEGMA graft polymerization to achieve thorough condensation of silanol groups with the hydroxylated surface and, in doing so, to promote the adhesion of organic coatings. TFA treatment of PEGMA films processed that way resulted in homogeneous and flat polymer surfaces on CMOS chips (Figure 4c,d). The microelectronic functionality of the CMOS chips was examined by verifying the functionality of the digital interface and the memory cells contained in each microelectrode. With a lab PC and LabView virtual instrument as control, data patterns were written into the memory cells and read back using the I2C interface on the chip. Chips were tested for functionality prior to and after TFA exposure. Since identical input and output patterns were observed (Figure 5), the devices retained full functionality in spite of the acid treatment, demonstrating the chemical stability of our PEGMA-coated CMOS chips. Here, each matrix (0-9, A-E) corresponded to an array of pixel electrodes, and each field an individual microelectrode (darkgray ) 0, light-gray ) 1). The utilization of this CMOS chip will involve operating photodiodes as in-chip optical sensor elements, as well as individually switched pixel electrodes, by the majority coated with a Si3N4 insulation. Besides spotting on the chip, the electrodes can be employed to generate electric fields in nonconducting ambience, which enable the electrophoretic addressing of charged molecules to generate DNA, peptide, or antibody arrays.15,16,29,30 Thereby, no current flow will take place outside the integrated circuits that could induce any electrochemical reaction. In this regard, we did not observe any influence of electric fields applied to electrodes on the NH2 loading of the PEGMA film, when Fmoc-β-alanine was coupled consecutively under real Merrifield conditions.28 Because of the high transparency of the PEGMA coating in the visible range,23 we further do not expect any significant negative effect on the performance or detection sensitivity of the CMOS photodiodes. To obtain maximized signal-to-noise ratios in future immunoassay applications, we analyzed the interaction of several proteins (fibrinogen, bovine serum albumin, γ-globulin, and lysozyme) and diluted human serum with PEGMA-grafted silicon wafers and CMOS chips. To investigate the extent of nonspecific protein adsorption, a closer look was taken by

Figure 6. Analysis of specific and nonspecific protein interactions: XP spectra in the N1s region of silicon wafers (a) and CMOS microchips (b) after incubation with protein solutions. The strong N1s signals on pristine surfaces (“reference”) indicated the nonspecific adsorption of fibrinogen, whereas PEGMA coatings prevented from nonspecific interactions. (c) Spotted anti-FLAG and anti-HA peptides on a PEGMA-grafted CMOS chip after staining with fluorescently labeled antibodies. Journal of Proteome Research • Vol. 6, No. 8, 2007 3201

research articles supersensitive XP spectroscopy, where the intensity of the N1s signal could be correlated with amide groups of proteins and thus with the amount of protein adsorbed even in the submonolayer range. The strong N1s signals in Figure 6a,b (“reference”) indicated the nonspecific adsorption of FITClabeled fibrinogen from PBS-buffered solution on a pristine silicon wafer and chip surface, respectively. This was in contrast to corresponding PEGMA-coated surfaces, where absolutely no N1s signals were observed. Consequently, we demonstrated the absence of any nonspecific protein adsorption even on the CMOS chips, irrespective of the protein or protein mixtures employed. To investigate the fundamental suitability of PEGMA-coated CMOS chips for microarray applications, we assayed the specific interactions of two monoclonal antibodies with appropriate probe molecules. For this purpose, cysteine-terminated peptides representing the FLAG and HA epitopes were spotted by a spotting robot on PEGMA-grafted and β-AlaSMCC-functionalized CMOS chips, followed by staining with fluorescently labeled antibodies.23 With fluorescence microscopy, specific interactions of anti-HA and anti-FLAG antibodies with the corresponding peptides were displayed by red and green spots on the chip’s surface (Figure 6c). Besides some diffusion of spot solution into the canals between electrodes and electrode arrays, respectively, the background was negligible in spite of renouncing any blocking agent.

Conclusions With a diversity of experiments, we demonstrated the generation and application of multifunctional PEGMA coatings on custom-made CMOS chips designed for upcoming immunoassays. Details on surface-initiated graft polymerization as well as on atomic surface composition were investigated, and the chemical stability of the entire system was carefully tested: Wrinkling of PEGMA films due to incubation with TFA could be avoided by an adequate pretreatment of the pristine CMOS chips, whereas the microelectronic functionality was revised by the read out of each individual memory cell. The utility of PEGMA-coated CMOS chips for microarray applications was proven by staining two spotted peptides as probes with specific antibodies as targets. Although usually applied blocking procedures with agents like BSA or milk powder were eliminated, solely specific red and green fluorescence spots were detected without any background signal. This was further emphasized by XP spectra of the nitrogen region after incubation with several protein solutions. Even with complex protein mixtures, we were able to obviate nonspecific interactions within the detection limit of supersensitive XPS. To summarize finally, we introduced a novel combination of sophisticated microchips for complex microarray generation and analysis with an elaborated and versatile surface chemistry for maximized signal-to-noise ratios in immunoassays, which should forward the utilization of CMOS technology in proteome research. Abbreviations: CMOS, complementary metal oxide semiconductor; MOSFET, metal oxide semiconductor field effect transistor; PEGMA, poly(ethylene glycol)methacrylate; ATRP, atom transfer radical polymerization; TFA, trifluoroacetic acid; XPS, X-ray photoelectron spectroscopy; FITC, fluorescein isothiocyanate; SAM, self-assembled monolayer; SMCC, N-succinimidyl 4-(maleimidomethyl)cyclohexanecarboxylate; I2C, interintegrated circuit; PBS, phosphate-buffered saline; BSA, bovine serum albumin; TBS, tris-buffered saline. 3202

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Acknowledgment. We thank Alexander Ku¨ller and Reiner Dahint for providing surface analytical methods, and Ulrich Trunk for his work in the ASIC laboratory. This work was supported by the German Federal Ministry of Education and Research (Grant Nos. 03N8710 and 0313375A), the MaxBuchner-Forschungsstiftung at the DECHEMA, and the Fonds der Chemischen Industrie. References (1) Ardeshirpour, Y.; Deen, M. J.; Shirani, S. Can. J. Electr. Comput. Eng. 2004, 29, 231-235. (2) Maruyama, Y.; Sawada, K.; Takao, H.; Ishida, M. IEEE Trans. Electron. Devices 2006, 53, 553-558. (3) Mallard, F.; Marchand, G.; Ginot, F.; Campagnolo, R. Biosens. Bioelectron. 2005, 20, 1813-1820. (4) Eltoukhy, H.; Salama, K.; El Gamal, A. IEEE J. Solid State Circ. 2006, 41, 651-662. (5) Wang, Y. J.; Xu, C.; Li, J.; Hsing, I. M.; Chan, M. S. Solid State Electron. 2005, 49, 1933-1936. (6) Barbaro, M.; Bonfiglio, A.; Raffo, L. IEEE Trans. Electron. Devices 2006, 53 158-166. (7) Kim, D. S.; Jeong, Y. T.; Park, H. J.; Shin, J. K.; Choi, P.; Lee, J. H.; Lim, G. Biosens. Bioelectron. 2004, 20, 69-74. (8) Li, J.; Xue, M.; Lu, Z. H.; Zhang, Z. K.; Feng, C.; Chan, M. IEEE Trans. Electron. Devices 2003, 50, 2165-2170. (9) Cheng, Y. T.; Pun, C. C.; Tsai, C. Y.; Chen, P. H. Sens. Actuators, B 2005, 109, 249-255. (10) Nebling, E.; Grunwald, T.; Albers, J.; Scha¨fer, P.; Hintsche, R. Anal. Chem. 2004, 76, 689-696. (11) Schienle, M.; Paulus, C.; Frey, A.; Hofmann, F.; Holzapfl, B.; Schindler-Bauer, P.; Thewes, R. IEEE J. Solid State Circ. 2004, 39, 2438-2445. (12) Egeland, R. D.; Marken, F.; Southern E. M. Anal. Chem. 2002, 74, 1590-1596. (13) Heller, M. J.; Tu, E. Methods for electronic synthesis of polymers, U.S. Patent 5,929,208, 1999. (14) Caillat, P.; David, D.; Belleville, M.; Clerc, F.; Massit, C.; RevolCavalier, F.; Peltie, P.; Livache, T.; Bidan, G.; Roget, A.; Crapez, E. Sens. Actuators, B 1999, 61, 154-162. (15) Swanson, P.; Gelbart, R.; Atlas, E.; Yang, L.; Grogan, T.; Butler, W. F.; Ackley, D. E.; Sheldon, E. Sens. Actuators, B 2000, 64, 2230. (16) Yang, J. M.; Bell, J.; Huang, Y.; Tirado, M.; Thomas, D.; Forster, A. H.; Haigis, R. W.; Swanson, P. D.; Wallace, R. B.; Martinsons, B.; Krihak, M. Biosens. Bioelectron. 2002, 17, 605-618. (17) Oleinikov, A. V.; Gray, M. D.; Zhao, J.; Montgomery, D. D.; Ghindilis, A. L.; Dill, K. J. Proteome Res. 2003, 2, 313-319. (18) Dill, K.; Montgomery, D. D.; Ghindilis, A. L.; Schwarzkopf, K. R.; Ragsdale, S. R.; Oleinikov, A. V. Biosens. Bioelectron. 2004, 20, 736-742. (19) Scandurra, A.; Curro`, G.; Frisina, F.; Pignataro, S. J. Electrochem. Soc. 2001, 148, B289-B292. (20) Jeon, S. I.; Lee, J. H.; Andrade, J. D.; De Gennes, P. G. J. Colloid Interface Sci. 1991, 142, 149-158. (21) Renken, J.; Dahint, R.; Grunze, M.; Josse, F. Anal. Chem. 1996, 68, 176-182. (22) Layout Rules CMOS I2T100-Intelligent Interface Technology, DS13350, Revision 8, AMI Semiconductor Belgium BVBA, Oudenaarde, Belgium, 2002. (23) Beyer, M.; Felgenhauer, T.; Bischoff, F. R.; Breitling, F.; Stadler, V. Biomaterials 2006, 27, 3505-3514. (24) Feng, W.; Chen, R.; Brash, J. L.; Zhu, S. Macromol. Rapid Commun. 2005, 26, 1383-1388. (25) Xu, D.; Yu, W. H.; Kang, E. T.; Neoh, K. G. J. Colloid Interface Sci. 2004, 279, 78-87. (26) Tsukruk, V. V.; Bliznyuk V. N. Langmuir 1998, 14, 446-455. (27) Wang, X. S.; Armes, S. P. Macromolecules 2000, 33, 6640-6647. (28) Chan, W. C.; White, P. D. FMOC Solid Phase Peptide Synthesiss A Practical Approach; Oxford University Press: Oxford, 2000. (29) Edman, C. F.; Raymond, D. E.; Wu, D. J.; Tu, E.; Sosnowski, R. G.; Butler, W. F.; Nerenberg, M.; Heller, M. J. Nucleic Acids Res. 1997, 25, 4907-4914. (30) Sosnowski, R. G.; Tu, E.; Butler, W. F.; O’Connell, J. P.; Heller, M. J. Proc. Natl. Acad. Sci. U.S.A. 1997, 94, 1119-1123.

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