Disposable Amperometric Label-Free Immunosensor on Chitosan-Graphene-Modified Patterned ITO Electrodes for Prostate Specific Antigen

Abstract

A facile and highly sensitive determination of prostate-specific antigen (PSA) is of great significance for the early diagnosis, monitoring and prognosis of prostate cancer. In this work, a disposable and label-free electrochemical immunosensing platform was demonstrated based on chitosan-graphene-modified indium tin oxide (ITO) electrode, which enables sensitive amperometric determination of PSA. Chitosan (CS) modified reduced graphene oxide (rGO) nanocomposite (CS-rGO) was easily synthesized by the chemical reduction of graphene oxide (GO) using CS as a dispersant and biofunctionalizing agent. When CS-rGO was modified on the patterned ITO, CS offered high biocompatibility and reactive groups for the immobilization of recognition antibodies and rGO acted as a transduction element and enhancer to improve the electronic conductivity and stability of the CS-rGO composite film. The affinity-based biosensing interface was constructed by covalent immobilization of a specific polyclonal anti-PSA antibody (Ab) on the amino-enriched electrode surface via a facile glutaraldehyde (GA) cross-linking method, which was followed by the use of bovine serum albumin to block the non-specific sites. The immunosensor allowed the detection of PSA in a wide range from 1 to 5 ng mL^-1 with a low limit of detection of 0.8 pg mL^-1. This sensor also exhibited high selectivity, reproducibility, and good storage stability. The application of the prepared immunosensor was successfully validated by measuring PSA in spiked human serum samples.

Keywords: amperometric immunosensor; chitosan; disposable electrode; graphene composite; prostate-specific antigen.

Figure 1

Schematic illustration of the facile fabrication of the immunosensor and amperometric determination of PSA.

Figure 2

CV curves obtained on CS/ITO (a) or CS-rGO/ITO (b) electrode for 20 consecutive scans. The electrolyte solution is Fe(CN)₆^3−/4− (2.5 mM) containing 0.1 M KCl.

Figure 3

(a) UV-Vis absorption spectrum of GO and CS-rGO. Insets are photographs of rGO (left) and GO (right) solutions. (b) TEM image of CS-rGO. (c) FT-IR spectra of CS, rGO synthesized in absence of CS and CS-rGO. (d) SEM image of CS-rGO/ITO.

Figure 4

Cyclic voltammetry (a) with EIS curves (b) from different electrodes. The electrolyte solution was Fe(CN)₆^3−/4− (2.5 mM) containing 0.1 M KCl.

Figure 5

DPV peak current for the PSA-bound immunosensor prepared using different concentrations of GA (a) or different reaction times for the immobilization of the antibody (b). The electrolyte is Fe(CN)₆^3−/4− (2.5 mM) containing 0.1 M KCl. The concentration of PSA was 1 ng mL⁻¹. Error bars represent the standard deviation of three measurements.

Figure 6

(a) Differential pulse voltametric curves of the Ab/GA/CS-rGO/ITO electrode towards various concentrations of PSA. (b) The corresponding calibration curves to determine PSA using the Ab/GA/CS-rGO/ITO electrode. Error bars represent the standard deviation of three measurements.

Figure 7

(a) Relative ratio of peak current before (I₀) and after (I) incubation with different proteins or their mixture c. The concentration of PSA and other proteins was 1 and 10 ng mL⁻¹, respectively. Error bars represent the standard deviation of three measurements. (b) Relative ratio of peak currents before (I₀) and after (I) storing the immunosensor at 4 °C for different times. The peak currents were obtained after the immunosensors bound with 1 ng mL⁻¹ of CEA. Error bars represent the standard deviation of three measurements.