Project Overview
The overall objective of this work is to create optical/electrochemical hybrid sensor arrays. This multidisciplinary project involves electrical engineering, optics, electrochemistry, and analytical chemistry. We performed a multiplexed electrogenerated chemiluminescence (ECL) sandwich immunoassay. This ECL assay used electrodes that consist of a common metal electrode patterned with an array of wells that hold sensing beads. In addition to the electrode used for the ECL assay, we also created microelectrode arrays with individually addressable electrodes. Some of these microelectrode arrays have on-chip electronics that simulate analytical equipment such as a potentiostat. These arrays show promise for ultrasensitive electrochemical analysis.
Individually Addressable Microelectrode Arrays
Microelectrode arrays are widely used in electro-analytical chemistry due to the numerous advantages they have over conventional electrodes.1 (i) Their size allows experiments to be carried out using very small sample volumes. (ii) As the number of microelectrodes increases, the ratio of faradaic current to background current increases. MEAs provide an increased signal to noise ratio that scales with the square root of the number of electrodes. (iii) The magnitude of current produced by MEAs is very small and is less destructive of samples being analyzed, which makes them very useful for in vivo electrochemistry applications. (iv) MEAs exhibit increased rates of mass transport to the surface of the electrode, which facilitates steady-state electrochemistry, and reduces IR drop, enabling analysis in highly resistive solution, such as organic solvents.
We investigated two classes of MEAs for this project.2 The first MEA was an 8 × 8 array of microelectrodes fabricated on silicon using conventional microfabrication techniques with contact pads for external connections to a potentiostat. The second MEA was an individually addressable 32 × 32 microelectrode array system on CMOS with a built-in VLSI potentiostat. The work was carried out through collaboration with the group of Professor Sameer Sonkusale in the Department of Electrical Engineering and Computer Science. The CMOS chips were fabricated using a deep sub-micron technology provided through MOSIS, and the simpler chips were manufactured at the Center for Nanoscale Systems at Harvard University.
CMOS MEAs
The system, shown in Fig. 1, consists of a 32 × 32 MEA, which serves as working electrodes (WE), an integrated CMOS VLSI transimpedance amplifier (TIA), and interface circuitry. The reference and counter electrodes are external to the CMOS chip. The read-out control circuit is used to individually address the 32 × 32 microelectrodes one at a time. The switch array and control circuit have been placed underneath the MEA layer to save space. The role of an external potentiostat is to record the redox current on the working electrode while also controlling the redox potential on the surface of the working electrode. The potentiostat amplifies the redox current and finally, converts it to voltage. In our design, the TIA with external reference electrode acts as the potentiostat.
Fig. 1. (left) Photograph of the fabricated microelectrode array system. The switch arrays are beneath the 1,024 microelectrodes. The die was wire-bonded into an LCC48 package. (right) Block diagram of the CMOS chip (a), and digital control circuit and microelectrode array (b) and built-in potentiostat (c).
Optoelectrochemical Sensing Arrays
We made electrode arrays using photolithography to perform chemical assays using electrogenerated chemiluminescence (Fig. 2 and Fig. 3). This approach built on our previous results making high aspect ratio electrode arrays.3 ECL is a controllable form of chemiluminescence where light emission is initiated by an electron-transfer reaction occurring at an electrode surface. The most common system used for analytical purposes consists of the luminophore label Ru(bpy)32+, or one of its derivatives, with tri-n-propylamine (TPrA) as a co-reactant. The ECL mechanism involving TPrA with dissolved Ru(bpy)32+ or with the ruthenium complex immobilized onto a bead is an active area of investigation. We used a standard sandwich immunoassay scheme to attach ECL-active species to the surface of the microbeads, which could then emit light following an electrochemical reaction. Using the experimental setup shown in Fig. 4, we have been able to collect images showing luminescence from individual beads.
Figure 2. Photolithographic steps involved in electrode fabrication containing an array of wells.
Figure 3. (A) Electrodes made using photolithography. Glass chip containing three electrodes connected to contact pads. Each electrode contains over 10,000 3 mm diameter wells. (B) An electron micrograph of 3 mm sensing beads in an electrode array.
Figure 4. Experimental setup using a chip like the one in Fig. 3on an inverted microscope using an EM-CCD camera as a detector.
We observed ECL emission from individual 3 mm beads with Ru(bpy)3+2 attached as an immunoassay label. The images in Figure 5 show the sandwich immunoassay scheme and the data collected using the scheme. The sandwich assay was completed by exposing anti-IL-8 antibody coated beads to 2 mg/mL of IL-8, followed by exposure to biotinylated anti-IL-8 detection antibody, which was used to bind Ru(bpy)3+2 labeled streptavidin. After completion of the sandwich, an image was captured while running a cyclic voltammogram in the presence of tripropylamine.
Figure 5. A) Schematic for the ECL sandwich immunoassay, where the final step attaches the Ru(bpy)3+2 ECL-active label. B) White light optical micrograph and C) ECL image of 3 mm beads that show the detection of 2 mg/mL IL-8 using the sandwich scheme depicted in (A).
Multiplexed Optoelectrochemical Sensing Arrays
We have also demonstrated the ability to use ECL imaging as an alternative readout mechanism to detect multiple antigens in a microarray format.4 These results represent the first time that individual sensing beads have been imaged by ECL in a multiplexed sandwich immunoassay. This new multiplexed ECL platform could be extended for the simultaneous analysis of dozens of analytes and for DNA assays.
This work was supported by NSF award: CHE-0518293
References
(1) LaFratta, C. N.; Walt, D. R. Chemical Reviews 2008, 108, 614.
(2) Hwang, S.; LaFratta, C. N.; Agarwal, V.; Yu, J.; Walt, D. R.; Sonkusale, S. IEEE Sensors 2009, 9, 609.
(3) Monk, D. J.; Walt, D. R. Journal of the American Chemical Society 2004, 126, 11416.
(4) Deiss, F.; LaFratta, C. N.; Symer, M.; Blicharz, T. M.; Sojic, N.; Walt, D. R. Journal of the American Chemical Society 2009, 131, 6088.
