Sub-nanomolar detection of prostate-specific membrane antigen in synthetic urine by synergistic, dual-ligand phage

Abstract

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.

Figures

Figure 1

Phage-based ELISA comparing ligand binding to monomeric and dimeric forms of PSMA. This ELISA includes PSMA monomer; all other reported experiments with PSMA apply the cancer-relevant PSMA dimer. Stop-4 provides a negative control with helper phage packaging the phagemid DNA. Throughout this report, error bars for ELISA data represent standard error (n = 3). All experimental data points with the exception of the negative controls (n = 1) include such error bars, though often these are quite small.

Figure 2

Schematic diagram of bidentate binding to PSMA by chemically synthesized (KCS-1, green) and genetically encoded (peptide 2, red) ligands to PSMA (PDB: 1Z8L). The former ligand wraps non-covalently onto the negatively charged P8 proteins found on the phage surface due to conjugation with a positively charged K14 peptide (blue). Simultaneous binding by the two ligands provides higher apparent affinity to PSMA. The inset depicts a simplified version of the schematic appearing in the subsequent figures shown here.

Figure 3

Phage-based ELISAs demonstrating the effectiveness of ligand wrapping. (A) Phage with chemically and genetically encoded ligands bind with much higher apparent affinity to the targeted PSMA. (B) The chemically synthesized KCS-2 wrapper converts helper phage, KO7, lacking a genetically encoded PSMA ligand, into a high affinity binding partner to PSMA. The decrease in apparent binding affinity at the highest phage concentration could be attributed to steric effects. (C) ELISA comparing different ligand wrappers. The wrapper combination of KCS-2 & KCS-1 indicates a 1:1 (w/w) ratio of ligand wrappers.

Figure 4

Biosensing with virus-PEDOT films. (A) Cyclic voltammogram for depositing virus-PEDOT films on gold electrodes. (B) SEM image of a PEDOT film. (C) SEM image of a virus-PEDOT film prepared under the same conditions. (D) Relative change in resistance, ΔR/Ro, versus frequency for phage-displayed ligands targeting PSMA. Data collected at 1 kHz (highlighted), were used for the analysis of PSMA binding. (E) Schematic diagram of the biosensing experiment. (F) ΔR/Ro of the film increases with the PSMA concentration. Throughout this report, error bars for the biosensing data represent standard error (n = 5). Data was fit to the indicated lines using the Hill equation, resulting in an R2 value of >0.99.

Figure 5

Detection of PSMA in synthetic urine using virus-PEDOT film biosensors. (A) ΔR/Ro versus frequency for the detection, in synthetic urine, of PSMA. (B) ΔR/Ro versus PSMA concentration. The inset expands the low PSMA concentration region. Data was fit to the indicated lines using the Hill equation, resulting in an R2 value of >0.99.

Scheme 1

The CuI-catalyzed azide-alkyne cycloaddition reaction to provide the secondary recognition ligand, KCS-2.

Scheme 2

Polymerization of EDOT in the presence of (A) LiClO4 or (B) phage-2, followed by wrapping with KCS-1 (green and blue).