VOLTAGE-PROGRAMMABLE BIOFUNCTIONALITY IN MEMS ENVIRONMENTS USING ELECTRODEPOSITION OF A REACTIVE POLYSACCHARIDE Li-Qun Wu(a, b), Hyunmin Yi(a, c), Sheng Li(d, e), David A. Small(a, c), Jung Jin Park(f), Gary W. Rubloff (d, f), Reza Ghodssi(d, e), William E. Bentley(a, c), and Gregory F Payne(a, b) (a)Center for Biosystems Research, UMBI, (b)Chemical and Biochemical Engineering, UMBC, (c)Chemical Engineering, UMCP, (d)Institute for Systems Research, UMCP, (e)Electrical and Computer Engineering, UMCP, (f)Materials and Nuclear Engineering, UMCP, Tel: (301) 405-8389, Fax: (301) 314-9075, Email: [email protected] ABSTRACT The amino-polysaccharide chitosan has distinct properties that make it an attractive interface material for the assembly of biomolecules onto microfabricated surfaces. Chitosan has pH-responsive electrostatic and solubility properties that allow it to be deposited and retained on cathode surfaces. Deposition is shown to be spatially controllable at µm levels by “templating” the chitosan onto micropatterned gold cathodes. Temporal control of deposition can be achieved depending on when the micropatterned electrodes are polarized. Chitosan also has nucleophilic amine groups that can be easily reacted using standard, amine-specific chemistries. Studies show that the sequence of chitosan deposition and chemical modification is repeatable. Finally, standard chemistries can be exploited to couple biomolecules onto chitosan films that have been “templated” onto the micropatterened gold cathodes. Specifically, we used glutaraldehyde activation to assemble the model protein, green fluorescent protein (GFP) onto a chitosan deposit. These studies demonstrate that chitosan has unique properties that allow it to serve as an interface material for the assembly of biomolecules onto microfabricated surfaces. INTRODUCTION The effective integration of biotechnology and microfabrication will provide robust biosensors to better diagnose disease, rapidly detect chemical/biological agents, and efficiently discover drugs. The biological sensing elements (e.g. nucleic acid probes and protein antibodies) confer the sensitivity/selectivity to these sensors while microfabrication permits miniaturization to them. Therefore, small samples can be rapidly analyzed in parallel processing format. The challenge is to effectively couple the labile bio-molecule to the microfabricated surface in a way that retains biological sensing activity. We believe the amino-polysaccharide, chitosan is a unique material that can serve as an effective interface for the assembly of biomolecules onto microfabricated surfaces. EXPERIMENTAL Fabrication of gold patterned surface. The patterned surfaces were fabricated by depositing 150 Å thick chromium and then 2000 Å thick gold films on 4-inch diameter silicon wafers, which had previously been coated with 1-µm thick thermal oxide film. Patterning was achieved using photolithography in which a primer and then photoresist (Microposit Photoresist S1813) were spin-coated onto the gold surface. After soft-baking the coated wafer at 100 °C for 1 minute, a specially-designed mask was placed over the surface and the wafer was exposed to UV light (total dosage ~ 190 mJ/cm2). After 30 seconds of development, the wafer was then hard-baked at 120 °C for 10 minutes. The exposed areas were then etched away by gold and chromium etchants (TFA for gold and TFD for chromium, Transene Co), and the photoresist was removed using acetone. Electrodeposition. The preparation of chitosan (Sigma-Aldrich Chemicals) solutions, NHS-fluorescein (5-(and 6-)-carboxyfluorescein succinimidyl ester, Molecular Probes) solutions, and Fluorescently-labeled chitosan solutions were described elsewhere [1]. For deposition, the patterned wafers were immersed in solutions (pH=5.6, 0.8 w/w % polymer) containing either fluorescently-labeled chitosan or unlabeled chitosan, and the patterned gold surfaces were polarized to serve as negative electrodes. The positive electrode in these experiments was an un-patterned gold-coated silicon wafer. The two electrodes were connected to a DC power supply (Model 6614C, Agilent Technologies) using alligator clips. Deposition was performed for two minutes by applying a voltage to achieve a current density of 1~2 A/m2. After deposition, the wafers were removed from the solutions, rinsed for one minute with deionized water, disconnected from the power supply, and dried at room temperature. After drying, the wafers were immersed in 1M NaOH for 10 minutes to neutralize the chitosan. After neutralization, the wafers were rinsed with distilled water and dried at room temperature overnight.Chemical reaction of chitosan deposit. NHS-fluorescein was reacted with chitosan films that had previously been deposited onto the micropatterned gold surfaces. For this study, chitosan was first deposited as described above and the dried wafer was placed in a 140 mm diameter petri dish with 35 ml PBS buffer (pH=7.4). The reaction was initiated by adding 20 µl of the dimethylformamide/ ethanol (1/4) solution containing NHS-fluorescein (2.5 mg/ml). After allowing the reaction to proceed for five minutes, the wafer was removed from the solution, rinsed with distilled water and dried at room temperature overnight. Production of Green Fluorescent Protein (GFP). GFP was expressed in E.coli BL21 (Invitrogen) using a pTrcHisB (Invitrogen) expression vector. Cells were grown under standard fermentation conditions and the fusion protein was purified using immobilized metal affinity chromatography as described elsewhere [2,3]. Assembly of model protein onto chitosan deposit. Glutaraldehyde was used to anchor the model protein (green fluorescent protein, GFP) onto the deposited chitosan surface. After chitosan was deposited onto the micropatterned gold electrode, the wafer was immersed in glutaraldehyde solution (0.05%) for 30 minutes. After glutaraldehyde activation, the wafer was extensively washed with 0.1 M PBS (Dulbecco’s Phosphate Buffered Saline, Sigma-Aldrich Chemicals) buffer and then immersed in a GFP solution (≈ 0.4 µg/ml) for 30 minutes. Two control experiments were performed at the same time. One control was a wafer with micropatterned gold lines that lacked chitosan. The second control was a wafer in which chitosan was deposited onto micropatterned gold lines but the deposited chitosan film was not activated with glutaraldehyde.