Virus-polymer hybrid nanowires tailored to detect prostate-specific membrane antigen

Abstract

We demonstrate the de novo fabrication of a biosensor, based upon virus-containing poly(3,4-ethylene-dioxythiophene) (PEDOT) nanowires, that detects prostate-specific membrane antigen (PSMA). This development process occurs in three phases: (1) isolation of a M13 virus with a displayed polypeptide receptor, from a library of ≈10(11) phage-displayed peptides, which binds PSMA with high affinity and selectivity, (2) microfabrication of PEDOT nanowires that entrain these virus particles using the lithographically patterned nanowire electrodeposition (LPNE) method, and (3) electrical detection of the PSMA in high ionic strength (150 mM salt) media, including synthetic urine, using an array of virus-PEDOT nanowires with the electrical resistance of these nanowires for transduction. The electrical resistance of an array of these nanowires increases linearly with the PSMA concentration from 20 to 120 nM in high ionic strength phosphate-buffered fluoride (PBF) buffer, yielding a limit of detection (LOD) for PSMA of 56 nM.

Figures

Figure 1

Three phases of development of a virus-PEDOT nanowire biosensor for prostate specific membrane antigen (PSMA).

Figure 2

Synthesis of virus-PEDOT nanowires: a) Process flow for the preparation, using the LPEN process, of virus-PEDOT composite nanowires for the detection of PSMA. b) Cyclic voltammograms for the electrodeposition of virus-PEDOT nanowires within the LPNE microfabricated template. Two primer scans were first carried out in aqueous 2.5 mM EDOT, 12.5 mM LiClO4 (black). Then five additional scans in 2.5 mM EDOT, 10 nM PSMA-binding viruses, and 12.5 mM LiClO4 were used to build up a virus-PEDOT composite nanowire 200–300 nm in total width. c, d) Optical micrographs of the resulting nanowire array on glass after the application of silver paste electrical contacts, e, f) Scanning electron micrographs of single nanowires of pure PEDOT (e), and the virus-PEDOT composite (f). The net-like structures observed in (f) are aggregates of filamentous M13 virus particles.

Figure 3

Affinity maturation of phage-displayed, PSMA-binding ligands. (a) Phage-based ELISA with the first generation PSMA-1 and PSMA-2 ligands. Throughout this report, error bars in ELISAs indicate standard error (n=3). (b) The homolog-shotgun scanning library design used the PSMA-2 sequence as a template with the programmed mutations shown in red. The selectants incorporated the mutations highlighted in red. (c) Phage-based ELISA with the affinity-matured, PSMA-binding selectants. (d) Phage-based ELISA comparing PSMA-1,-2, and -3.

Figure 4

Real-time biosensing with PSMA-binding PEDOT nanowires. (a) A real-time trace of biosensing data, with the indicated injections of negative antibody (n-Ab), PSMA, or washes with PBF buffer (black arrows). (b) A compilation of all real-time biosensing data, depicted as a calibration curve with error bars indicating standard deviation (n=3). The change in resistance upon injection is plotted versus analyte concentration. (c) A plot demonstrating the change in resistance upon injection of analytes in a solution of synthetic urine (errors bars indicate standard deviation with n=3).

Scheme 1

(a) Electrochemical oxidation of EDOT to form PEDOT in perchlorate-containing electrolyte. (b) Electrochemical oxidation of EDOT in the presence of negatively-charged M13 virus particles to form PEDOT. The yellow “pacman” symbolizes a displayed polypeptide epitope optimized to bind PSMA.