Virus Electrodes for Universal Biodetection

Principle Scientists: Li-Mei Yang and Dr. Phil Tam

Li-Mei C. Yang, Phillip Y. Tam, Benjamin J. Murray, Theresa M. McIntire, Gregory A. Weiss* and Reginald M. Penner*, Virus Electrodes for Universal Biodetection, Analytical Chemistry 78 (2006) 326. NOTE: This work is a collaboration with the research group of Prof. Greg Weiss at UCI

Phage-displayed peptide libraries having 1010 unique members offer the promise of universal biorecognition but this amazing technology has found only limited application in biosensors. In prior work, detecting molecular recognition between phage and target until now has focused on a "sandwich assay" scheme involving the detection of phage binding to immobilized target using quartz crystal microbalance, microelectrode arrays, nanowire field effect transistors, a bead-based electrochemical immunoassay, electric DNA chips, infection of different bacteria, or fluoroimmunoassays. In this work, we took a different approach involving the covalent attachment of the engineered virus directly to a gold electrode surface.

Figure 1. The virus electrode is a 3 mm diameter gold disk electrode onto which engineered M13 phage particles are covalently attached according to the steps 1-3 shown in (e). The state of this electrode was determined by measuring its resistive impedance, ZRe, at high frequency (2 - 500 kHz) versus a large area platinum electrode immersed in the same salt-based buffer solution. Prior to the attachment of phage particles (a), ZRe of this system, at all frequencies, was relatively low. (b) A dense virus layer was then covalently bonded to the gold surface according to the steps labeled 1-3 shown in (e). This produces a dense phage layer that completely electrically insulates the gold surface from contact with the buffer solution. (c) Exposure of this virus electrode to a "negative" antibody (n-Ab, blue) causes no change to either the imaginary component of impedance, ZIm or to ZRe. (d) Exposure to a "positive" antibody (p-Ab, red) that is selectively recognized and bound by the phage causes a significant increase in the high frequency ZRe, but little change in ZIm (at any frequency). Separately, QCM measurements are measure the uptake of the two antibodies by the virus electrode. (e) Schematic view of the step involved in the preparation of a virus electrode investigated here. Step 1. An electrochemically activated gold electrode was exposed to thioctyl NHS ester to form a thiol-Au bonded SAM, Step 2. M13 phage was covalently tethered to the self-assembled monolayer, through formation of amide bonds between free amines on the phage and the activated carboxylate, Step 3. Gaps in the monolayer and unreacted NHS esters were capped with bovine serum albumin (BSA), Step 4. The virus electrode is ready to be used for the analyses.

Figure 1 shows the basic concept, and also how the covalent attachment of virus to a gold electrode surfaces was accomplished in this study. Once these viruses are attached to the gold, do they continue to recognize and bind p-Ab and PSMA? The answer is yes, as demonstrated by the QCM data shown below.

Figure 2.(a) Flow cell-based QCM measurements of mass versus time during the exposure of a virus electrode to n-Ab and p-Ab demonstrating selective binding of p-Ab and the absence of nonselective binding to n-Ab. Shown are the response of a virus electrode prepared according to the steps shown in Fig. 1e (top trace), and a second electrode on which no virus was attached, corresponding to omission of step 2 in Fig 1e. (b) Photomicrograph of a gold electrodes patterned on silicon after the formation of a covalent virus surface. (c) Fluorescence micrograph of similarly patterned gold electrodes exposed to thioctyl NHS ester, followed by consecutive incubation in buffer, BSA, and then fluorescein-labelled p-Ab. In this experiment, which omits the M13 phage, no binding to the electrode is observed. (d) Fluorescence micrograph after the attachment of M13 phage to the thioctyl SAM and binding of fluorescein-labeled p-Ab.

There is too much to know about the electrochemical detection to describe it all here, but the bottom line is the following: We find that the highest sensitivity for the detection of either p-Ab or PSMA binding is obtained by measuring the electrical impednace of the gold electrode in the real channel at high frequencies above 2 kHz. This is surprising because the vast majority of experiments up until now have probes the capacitive impedance at very low frequencies. It is absolutely clear in our experiments that this mode of detection is inferior, but our observations may be specific to our surface chemistry

Figure 3.(A,B) Signal-to-noise (S/N) ratio, defined as Z/Z, plotted as a function of frequency. S/N above 10 is not observed at any frequency for ZIm (C) but relatively high S/N is observed for both PSMA and p-Ab over a broad range of frequencies for ZRe from 2 kHz to 500 kHz. Error bars in figures depict the standard deviation. (C) Measurements of ZRe at four frequencies, as indicated, after exposures to 20 ng ml-1 PSMA and after rinsing with aqueous 100 mM glycine, pH=2.0. The resistance of the rinsed state matched that seen for the freshly prepared virus electrode. Clean modulation between a high resistance "bound state" and a low resistance "rinsed state" was seen only at 10 kHz and 100 kHz. (D) Calibration curve for PSMA detection by virus electrodes prepared as described previously. Measurement of ZRe provides a linear response to variable concentrations of PSMA. Each data point represents the average value obtained from measurements with four identically prepared virus electrodes.


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Copyright 2006 R.M. Penner