Synthesis of a virus electrode for measurement of prostate specific membrane antigen.

Though relatively unexploited in biosensor applications, phage display technology can provide versatile recognition scaffolds for detection of cancer markers and other analytes. This chapter details protocols for covalent attachment of viruses directly to electrodes for reagent-free detection of analytes in real-time. Customization of binding specificity leverages selections with large phage display libraries prior to covalent attachment of the selected virus to the electrode. The methods described here utilize electrochemical impedance spectroscopy (EIS) to detect molecular recognition between M13 phage bound to a Au electrode and the following analytes: prostate specific membrane antigen (PSMA), positive and negative control antibodies (p-Ab and n-Ab, respectively). Because of a thick layer built on the Au electrode, the real impedance (Zre) increases reliably with S/N ratios upon noncovalent binding to PSMA (approximately 14) and p-Ab (approximately 20).

Direct electrical transduction of antibody binding to a covalent virus layer using electrochemical impedance.

Electrochemical impedance spectroscopy is used to detect the binding of a 148.2 kDa antibody to a "covalent virus layer" (CVL) immobilized on a gold electrode. The CVL consisted of M13 phage particles covalently anchored to a 3 mm diameter gold disk electrode. The ability of the CVL to distinguish this antibody ("p-Ab") from a second, nonbinding antibody ("n-Ab") was evaluated as a function of the frequency and phase of the measured current relative to the applied voltage. The binding of p-Ab to the CVL was correlated with a change in the resistance, reducing it at low frequency (1-40 Hz) while increasing it at high frequency (2-140 kHz). The capacitance of the CVL was virtually uncorrelated with p-Ab binding. At both low and high frequency, the electrode resistance was linearly dependent on the p-Ab concentration from 20 to 266 nM but noise compromised the reproducibility of the p-Ab measurement at frequencies below 40 Hz. A "signal-to-noise" ratio for antibody detection was computed based upon the ratio between the measured resistance change upon p-Ab binding and the standard deviation of this change obtained from multiple measurements. In spite of the fact that the impedance change upon p-Ab binding in the low frequency domain was more than 100 times larger than that measured at high frequency, the S/N ratio at high frequency was higher and virtually independent of frequency from 4 to 140 kHz. Attempts to release p-Ab from the CVL using 0.05 M HCl, as previously described for mass-based detection, caused a loss of sensitivity that may be associated with a transition of these phage particles within the CVL from a linear to a coiled conformation at low pH.

Covalent virus layer for mass-based biosensing.

M13 virus particles were covalently attached to a planar gold-coated quartz crystal microbalance (QCM) through reaction with a self-assembled monolayer of N-hydroxysuccinimide thioctic ester, followed by incorporation of the blocking agent bovine serum albumin. This immobilization chemistry produced a phage multilayer having a coverage equivalent to approximately 6.5 close-packed monolayers of the virus. The properties of this "covalent virus surface" or CVS for the mass-based detection of a 148.2 kDa antibody were then evaluated in a phosphate buffer using a flow injection analysis system. The mass of the CVS increased with exposure to an antibody (p-Ab) known to bind the phage particles with high affinity. Bound p-Ab was removed by washing with 0.5 M HCl thereby regenerating the sensor surface. A calibration plot for p-Ab binding was constructed by repetitively exposing the surface to p-Ab at concentrations between 6.6 and 200 nM and HCl rinsing after each exposure. The mass-concentration relationship was linear with a sensitivity of 0.018 microg/(cm2 nM) and a limit of detection of 7 nM or 1.3 pmol. The CVS could be saturated with high doses of p-Ab enabling the determination that an average of approximately 140 binding sites are available per M13 phage particle. Exposure of the CVS to a second, nonbinding antibody (n-Ab) did not cause a measurable mass change. These results demonstrate that the covalent virus layer is a rugged, selective, and sensitive means for carrying out mass-based biodetection.

Virus electrodes for universal biodetection.

A dense virus layer, readily tailored for recognition of essentially any biomarker, was covalently attached to a gold electrode surface through a self-assembled monolayer. The resistance of this "virus electrode", Z(Re), measured in the frequency range from 2 to 500 kHz in a salt-based pH 7.2 buffer, increased when the phage particles selectively bound either an antibody or prostate-specific membrane antigen (PSMA), a biomarker for prostate cancer. In contrast to prior results, we show the capacitive impedence of the virus electrode, Z(Im), is both a noisier and a less sensitive indicator of this binding compared to Z(Re). The specificity of antibody and PSMA binding, and the absence of nonspecific binding to the virus electrode, was confirmed using quartz crystal microbalance gravimetry.