Proceedings - 19th International Conference - IEEE/EMBS Oct. 30 - Nov. 2, 1997 Chicago, IL.

USA

A MICROCHIP ELECTROCHEMICAL IMMUNOSENSOR FABRICATED USING MICROMACHINING TECHNIQUES

Jin-Woo Choi, Ying Ding*, Chong H. A h , H. Brian Halsall* and William R. Heineman* Center for Microelectronic Sensors and MEMS Department of Electrical & Computer Engineering and Computer Science *Department of Chemistry University of Cincinnati, POBox2 10030, Cincinnati, OH 45221 -0030 E-mail: jchoi@ececs.uc.edu (Jin-Woo Choi)
Abstract: Using micromachining techniques, an electrochemical immunosensor has been designed, fabricated and tested on a Pyrex glass wafer. The sensor has an electrochemical cell that is composed of a reference, an auxiliary and two working electrodes on a spin-coated polyimide film, where the reference electrode is silver and the others are platinum. The polyimide film is used for antibody immobilization instead of a polystyrene sheet which is usually used in conventional immunosensors. A reaction chamber is fabricated separately on a silicon wafer using anisotropic etching techniques. The electrochemical cell on a glass wafer and the reaction. chamber on a silicon wafer are then bonded together to construct the immunosensor using polymer bonding techniques. Fabricated immunosensors are tested by the method of cyclic voltammetry (CV) before analyzing the antigen-antibody reaction. Test antibodies are well attached to the polyimide substrate, and the results of the CV test shows that this immunosensor can be successfully applied for electrochemical immunoassay.
Keywords: microchip, electrochemical immunoassay,

The fabricated immunosensor is tested using the method of cyclic voltammetry (CV) [4] before analyzing the immunoassay. Combining the microfabricated immunosensors with a microfluidic system, a fully integrated smart immunosensor can be realized on a chip. SENSOR DESIGN AND FABRICATION In conventional electrochemical immunosensors, there are three electrodes composing an electrochemical cell which usually consists of a reference, auxiliary and working electrode. In this work, a microchip-type immunosensors has been proposed and designed as shown in Figure I . In this device, however, there are two working electrodes for applying to the dual analytes immunoassay [ 5 ] .
Auxiliary electrode (Pt) Wire bonding pads

immunosensor INTRODUCTION Detection of antigens in air or fluid is very important in biological and biomedical analysis to figure out what kind of antigens and how many antigens are there. Early detection of antigens can give an alarm to protect human beings from hazardous agents or help to diagnose diseases more easily. For these reasons, electrochemical immunoassay techniques have been widely studied and developed to detect low concentrations of antigens by means of the antibody-antigen reaction [ 1-31. Electrochemical enzyme immunoassays have superior advantages over low detection limits and exquisite selectivity for different antigens [2]. However, most of them need a hand-assembled immunoassay reaction cuvette and a macro-size electrochemical cell with free standing electrodes. This may cause poor detection reliability and slow response time in a precise detection. To overcome these drawbacks, we have designed and fabricated an electrochemical immunosensor on a chip using microfabrication techniques.
ference electrode (Ag)

Figure 1. Schematic view o f the designed electrochemical immunosensor Polystyrene substrate generally has been used in conventional immunosensors for antibody immobilization but its process on a glass wafer or a silicon wafer is not compatible with microfabrication techniques based on photolithography. Thus, in this work a spin-coated polyimide film on a glass wafer has been introduced for antibody immobilization. The immunosensor is composed of two parts, one is an electrochemical cell on a polyimide film and the other is a reaction chamber made by an anisotropically etched silicon wafer to prevent analytes from spreading out. They are bonded together to construct the

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Proceedings 19th International Conference - IEEE/EMBS Oct. 30 - Nov. 2, 1997 Chicago, IL. USA
immunosensor. Fabrication steps are shown in Figure 2. On a Pyrex glass wafer, polyimide was spin-coated and then cured. Platinum (Pt) was deposited and patterned by the liftoff technique and used as the auxiliary and working electrodes. Deposition and patterning of silver (Ag) was repeated for the reference electrode. An oxidized silicon wafer was patterned and anisotropically etched in potassium hydroxide (KOH) solution to make reaction chambers, and then it was bonded with the sensing cell on the glass wafer using polymer bonding techniques. The opening of the reaction chamber is 5 mm X 5 mm, and the spacing between electrodes is 2.5 mm. The thickness of each electrode is 1 lOOA and the width of both reference and auxiliary electrode is 350 m. Therefore, the exposed area of the auxiliary electrode is about 1.9 mm '. Figures 2 and 3 show the detailed fabrication steps and the fabricated immunosensor, respectively.

EXPERIMENTAL RESULTS AND DISCUSSION The polyimide spin-coated on a glass wafer was tested first for antibody immobilization. 0.3 pgld Rat anti-mouse immunoglobulin G (IgG) alkaline phosphatase, that is enzyme-labeled antibody, was pipetted onto the polyimide film and incubated 12 hours. After incubation, that solution was removed and the polyimide surface was rinsed with acetate buffer (pH 5.5). 4mM p-Nitrophenyl phosphate (PNPP) was then pipetted onto the polyimide surface. In less than 20 minutes, the PNPP solution turned from colorless transparent solution into a yellow-colored solution, indicating that the enzyme-labeled antibody had been coated onto the polyimide surface. This result can provide a promising feasibility of a microchip immunosensor using the polyimide substrate. Cyclic voltammetry was used to evaluate the functionality of this microfabricated immunosensor as an electrochemical cell. p-Aminophenol (PAP) was chosen to be the analyte in the CV test because PAP is the final product in our electrochemical immunoassay that needs to be detected by the cell. PAP can be electrochemically oxidized and its oxidized species, p-quinoneimine, can be electrochemically reduced easily. PAP and p-quinoneimine form a very good redox couple. This electrochemical oxidation mechanism of PAP is shown in Figure 4. Before performing CV experiments, the reference electrode was coated by silver chloride (AgCI) through electrolysis in a 0.1M HCI solution to generate a Ag/AgCI reference electrode that is needed for an electrochemical cell. After coating, PAP solution (in Tris buffer, pH 9.0, saturated with NaN03) was pipetted onto the cell. CV experiments were run on a BAS- 100 workstation (Bioanalytical Systems Inc., West Lafayette, IN). Figure 5 shows cyclic voltammogram of PAP solution in the fabricated sensor.

-PI coating on glass wafer

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Silicon oxidation

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Patterning for Pt electrodes Patterning 8 oxide etching '
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Pt deposition and lift-off

Silicon anisotropic etching

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Ag deposition and lift-off

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Figure 2. Summarized fabrication steps

H2N-L?oH PAP
2l-f + 2e-+ H

0
p-quinoneimine

Figure 4. The oxidation mechanism of p-Aminophenol(PAP) As shown in Figure 5, the microfabricated immunosensor worked well as an electrochemical cell which provides a

Figure 3. Microphotograph of the fabricated immunosensor

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Proceedings - 1 t International Conference - IEEE/EMBS Oct. 30 - Nov. 2,1997 Chicago, IL. USA 9h
promising clue to apply this microfabricated immunosensor to the electrochemical immunoassay. Jour. of Chemical Edu., vo1.60, 1983, pp.702-706. [5] Y. Ding, H. B. Halsall and W. R. Heineman, Dual Analyte Electrochemical Immunoassay, Pittcon97, March 16-2I , Atlanta, Georgia, 1997.

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Potential vs. AgIAgCI [my

Figure 5 . Cyclic voltammogram of PAP in the fabricated electrochemical cell

CONCLUSION

A microchip electrochemical immunosensor has been designed, fabricated and tested in this work. The polyimide film was used as a substrate for antibody immobilization. Test antibodies were successfully attached to the polyimide substrate. Cyclic voltammetry was used to test the fabricated sensor as an electrochemical cell and the experimental results show a promising feasibility to apply the fabricated device to electrochemical immunoassays. This immunosensor can be flexibly combined with microfluidic systems on a chip. A fully integrated smart immunosensor can be envisioned for early detection and diagnostic systems against hazardous biomedical agents.
ACKNOWLEDGMENT The authors would like to thank Craig L. King and Peter M. Bertin in University of Cincinnati for their helps on this work. The authors also would like to gratefully acknowledge DuPont for their donations of polyimide. REFERENCES E. P. Diamandis and T. K. Christopoulos, Eds., Immunoassay, Academic Press, 1996. W. R. Heineman, H. B. Halsall, K. R. Wehmeyer, M. J. Doyle and D. S. Wright, Immunoassay with Electrochemical Detection, Methods of Biochemical Anal., vo1.32, pp.345393. D. S. Hage, Immunoassays, Analytical Chemistry, vo1.65, N0.12, 1993, pp.420R-424R. P. T. Kissinger and W. R. Heineman, Cyclic Voltammetry,

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