Immobilizing enzymes onto electrode arrays by hydrogel photolithography to fabricate multi-analyte electrochemical biosensors

Abstract

This paper describes a biomaterial microfabrication approach for interfacing functional biomolecules (enzymes) with electrode arrays. Poly (ethylene glycol) (PEG) hydrogel photopatterning was employed to integrate gold electrode arrays with the enzymes glucose oxidase (GOX) and lactate oxidase (LOX). In this process, PEG diacrylate (DA)-based prepolymer containing enzyme molecules as well as redox species (vinylferrocene) was spin-coated, registered, and UV cross-linked on top of an array of gold electrodes. As a result, enzyme-carrying circular hydrogel structures (600 microm diameter) were fabricated on top of 300 microm diameter gold electrodes. Importantly, when used with multiple masks, hydrogel photolithography allowed us to immobilize GOX and LOX molecules on adjacent electrodes within the same electrode array. Cyclic voltammetry and amperometry were used to characterize biosensor electrode arrays. The response of the biosensor array was linear for up to 20 mM glucose with sensitivity of 0.9 microA cm(-2) mM(-1) and 10 mM lactate with sensitivity of 1.1 microA cm(-2) mM(-1). Importantly, simultaneous detection of glucose and lactate from the same electrode array was demonstrated. A novel strategy for integrating biological and electrical components of a biosensor described in this paper provides the flexibility to spatially resolve and register different biorecognition elements with individual members of a miniature electrode array. Of particular interest to us are future applications of these miniature electrodes for real-time monitoring of metabolite fluxes in the vicinity of living cells.

Figure 1

(A) Layout of an electrode array consisting of five 300 µm diameter Au electrodes, 15 µm wide leads and 4 mm² contact pads. (B) Micropatterning PEG hydrogel to insulate the leads. This hydrogel layer does not have redox polymer or enzyme molecules. (C) Micropatterning enzyme-carrying hydrogel microstructures in alignment with microfabricated Au electrodes. This process can be repeated with multiple masks and different prepolymer formulations to immobilize distinct enzyme-carrying gel structures on different electrodes of the array.

Figure 2

(A) A portion of five electrode array with 300 µm diameter Au electrodes and 15 µm diameter leads. Contact pads are not shown in this image. (B) Cyclic voltammetry characterization of bare vs. photoresist protected electrodes after silane modification. Ferricyanide was used as diffusible redox species to test the electron transfer. The scan rate was 20 mV/sec Ag/AgCl reference and Pt counter electrodes were used. Electrodes modified with silane or coated with hydrogel were similarly resistive and CV curves for these two conditions overlap. Insulating PEG hydrogel coating did not contain redox polymer. (C) Hydrogel microstructures (600 µm diameter) fabricated on top of the 300 µm Au electrodes. The hydrogel structures were made larger than electrodes to ensure attachment of gel to the glass substrate modified with acrylated silane.

Figure 3

(A) A prepolymer formulations containing either GOX or LOX was spun onto the surface and exposed through a photomask. Subsequently, the second prepolymer formulation was spin-coated on the surface and registered with Au electrodes that remained unmodified. These sequential two mask process sequential two mask process resulted in fabrication of glucose- and lactate-sensitive electrodes in the same array. (B–C) Alignment and deposition of hydrogel microstructures of varying content on microfabricated Au electrodes. Hydrogel type 1 contains peroxidase/Amplex Red and becomes fluorescent when challenged with 5 mM H₂O₂ whereas hydrogel type 2 deposited on an adjacent electrode does not have these reporter molecules and remains insensitive to analyte.

Figure 4

(A) Characterization of redox activity of vinylferrocene-containing hydrogel electrodes using cyclic voltammetry. Scan rate was varied from 10 mV/s to 100 mV/s. Ag/AgCl reference and Pt counter electrodes were used. (B) Linear relationship of anodic peak current vs. scan rate suggests fast and reversible kinetics. (C) Demonstration that adjacent hydrogel electrodes were individually addressable. Comparison of redox activity of single hydrogel/Au electrode vs. two electrodes connected together.

Figure 5

(A) Amperometric response of miniature enzyme electrodes to glucose. Microfabricated GOX-containing hydrogel/Au electrodes were poised at 0.4 mV vs. Ag/AgCl reference electrode and were challenged with glucose in 2 mM increments. A current vs. time response was recorded and averaged for five different devices (n=5). (B) The calibration curve of GOX-based electrodes for 0 to 20 mM glucose range. (C) Amperometric response of LOX-containing hydrogel/Au electrodes to 2 mM aliquots of lactic acid. Conditions for testing the electrodes were identical to those discussed for glucose. (D) The calibration curve of lactate electrode showing response in the 0 to 10 mM range of analyte.

Figure 6

Connecting together two members of an electrode array, one GOX-containing hydrogel/Au electrode another LOX-carrying hydrogel/Au electrode. This electrode pair was challenged with lactic acid followed by glucose. Signals were recorder by amperometry by posing electrodes at 0.4 V vs. Ag/AgCl reference. The response of an electrode pair to each analyte was comparable to that of individual glucose and lactate electrodes. This result shows that glucose and lactate can be detected in the same array of hydrogel/Au electrodes.

Figure 7

(A) Testing stability of glucose electrodes. Electrodes where stored at 4C in PBS and were tested daily by exposure to 6 mM glucose. Response to glucose was recorded as current vs. time plot for five different devices (n=5). (B) Response of GOX-carrying hydrogel/Au electrodes to 2 mM aliquots of glucose dissolved in oxygen-containing (21% oxygen) and oxygen-free PBS. Commercial oxygen electrode was used to verify oxygen tension of electrolyte solution. Electrode was poised at 0.4V vs. Ag/AgCl reference.