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The article discusses phage display mechanisms and phage-based biosensors. Topics include the: advantages of using the bacteriophage called filamentous phage; manipulation of polypeptides on virus particle surfaces; selection of a polypeptide receptor and affinity screening; methods for immobilizing filamentous phage on biosensor surfaces; antibody binding on a covalent virus surface; prostate-specific membrane antigen--a cancer biomarker; biosensor transducer--electrochemical impedance spectroscopy; and binding of target molecules to macroscopic gold electrodes.

The sensitive detection of cancer biomarkers in urine could revolutionize cancer diagnosis and treatment. Such detectors must be inexpensive, easy to interpret, and sensitive. This report describes a bioaffinity matrix of viruses integrated into PEDOT films for electrochemical sensing of prostate-specific membrane antigen (PSMA), a prostate cancer biomarker. High sensitivity to PSMA resulted from synergistic action by two different ligands to PSMA on the same phage particle. One ligand was genetically encoded, and the secondary recognition ligand was chemically synthesized to wrap around the phage. The dual ligands result in a bidentate binder with high-copy, dense ligand display for enhanced PSMA detection through a chelate-based avidity effect. Biosensing with virus–PEDOT films provides a 100 pM limit of detection for PSMA in synthetic urine without requiring enzymatic or other amplification. [ABSTRACT FROM AUTHOR]

Virus-poly(3,4-ethylenedioxythiophene) (virus-PEDOT)biocompositefilms are prepared by electropolymerizing 3,4-ethylenedioxythiophene(EDOT) in aqueous electrolytes containing 12 mM LiClO4andthe bacteriophage M13. The concentration of virus in these solutions,[virus]soln, is varied from 3 to 15 nM. A quartz crystalmicrobalance is used to directly measure the total mass of the biocompositefilm during its electrodeposition. In combination with a measurementof the electrodeposition charge, the mass of the virus incorporatedinto the film is calculated. These data show that the concentrationof the M13 within the electropolymerized film, [virus]film, increases linearly with [virus]soln. The incorporationof virus particles into the PEDOT film from solution is efficient,resulting in a concentration ratio of [virus]film:[virus]soln≈ 450. Virus incorporation into the PEDOT causesroughening of the film topography that is observed using scanningelectron microscopy and atomic force microscopy (AFM). The electricalconductivity of the virus-PEDOT film, measured perpendicular to theplane of the film using conductive tip AFM, decreases linearly withvirus loading, from 270 μS/cm for pure PEDOT films to 50 μS/cmfor films containing 100 μM virus. The presence on the virussurface of displayed affinity peptides did not significantly influencethe efficiency of incorporation into virus-PEDOT biocomposite films. [ABSTRACT FROM AUTHOR]

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 CYL 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 CYL 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. [ABSTRACT FROM AUTHOR]

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 ≈6.5 close-packed monolayers of the virus. The properties of this "covalent virus surface" or CYS 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 CYS 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 HCI rinsing after each exposure. The mass-concentration relationship was linear with a sensitivity of 0.0 18 μg/(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 ≈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. [ABSTRACT FROM AUTHOR]