H-rGO-Pd NPs Nanozyme Enhanced Silver Deposition Strategy for Electrochemical Detection of Glypican-3

Abstract

Glypican-3 (GPC3), as an emerging biomarker, has been shown to be beneficial for the early diagnosis and treatment of hepatocellular carcinoma (HCC). In this study, an ultrasensitive electrochemical biosensor for GPC3 detection has been constructed based on the hemin-reduced graphene oxide-palladium nanoparticles (H-rGO-Pd NPs) nanozyme-enhanced silver deposition signal amplification strategy. When GPC3 specifically interacted with GPC3 antibody (GPC3_Ab) and GPC3 aptamer (GPC3_Apt), an "H-rGO-Pd NPs-GPC3_Apt/GPC3/GPC3_Ab" sandwich complex was formed with peroxidase-like properties which enhanced H₂O₂ to reduce the silver (Ag) ions in solution to metallic Ag, resulting in the deposition of silver nanoparticles (Ag NPs) on the surface of the biosensor. The amount of deposited Ag, which was derived from the amount of GPC3, was quantified by the differential pulse voltammetry (DPV) method. Under ideal circumstances, the response value was linearly correlated with GPC3 concentration at 10.0-100.0 μg/mL with R² of 0.9715. When the GPC3 concentration was in the range from 0.01 to 10.0 μg/mL, the response value was logarithmically linear with the GPC3 concentration with R² of 0.9941. The limit of detection was 3.30 ng/mL at a signal-to-noise ratio of three and the sensitivity was 1.535 μAμM^-1cm^-2. Furthermore, the electrochemical biosensor detected the GPC3 level in actual serum samples with good recoveries (103.78-106.52%) and satisfactory relative standard deviations (RSDs) (1.89-8.81%), which confirmed the applicability of the sensor in practical applications. This study provides a new analytical method for measuring the level of GPC3 in the early diagnosis of HCC.

Keywords: Glypican-3; H-rGO-Pd NPs nanozyme; electrochemical nanobiosensor; hepatocellular carcinoma; peroxidase-like catalytic silver deposition.

Figure 1

(A) Schematic principle of the GPC3 electrochemical nanobiosensor based on H-rGO-Pd NPs nanozyme. (B) DPV curves for the feasibility of the GPC3 electrochemical nanobiosensor with GPC3 or without GPC3.

Figure 2

(A) UV-vis spectrum of H-rGO-Pd NPs. (B) FT-IR characterization of H-rGO-Pd NPs. (C) Zeta potential analysis diagram of H-rGO-Pd NPs. (D) SEM image of H-rGO-Pd NPs. (E) EDS image of H-rGO-Pd NPs. (F) The peroxidase-like activities of H-rGO-Pd NPs.

Figure 3

(A) CV characterization of Au NPs@rGO/SPE in 5.0 mM [Fe(CN)₆]^3−/4− and 0.1 M KCl solution at 0.01 V/s, 0.025 V/s, 0.05V/s, 0.075 V/s, 0.1 V/s, 0.5 V/s, 0.75 V/s, 1 V/s, 1.25 V/s, 1.5 V/s, and 2 V/s. (B) Raman spectra of electrode for different processing steps. (C) CV characterization. (D) EIS characterization of the electrochemical nanosensor preparation process in 5.0 mM [Fe(CN)₆]^3−/4− and 0.1 M KCl solution at the frequency range from 0.1 Hz to 10 kHz. (a, SPE; b, Au NPs@rGO/SPE; c, GPC3_Ab/Au NPs@rGO/SPE; d, GPC3/GPC3_Ab/Au NPs@rGO/SPE; e, H-rGO-Pd NPs-GPC3_Apt/GPC3/GPC3_Ab/AuNPs@rGO/SPE; f, AgNPs/H-rGO-PdNPs-GPC3_Apt/GPC3/GPC3_Ab/Au NPs@rGO/SPE).

Figure 4

SEM images of the (A) bare SPE, (B) Au NPs@rGO/SPE, (C) GPC3_Ab/Au NPs@rGO/SPE, (D) GPC3/GPC3_Ab/Au NPs@rGO/SPE, (E) H-rGO-Pd NPs-GPC3_Apt/GPC3/GPC3_Ab/Au NPs@rGO/SPE, and (F) Ag NPs/H-rGO-Pd NPs-GPC3_Apt/GPC3/GPC3_Ab/Au NPs@rGO/SPE.

Figure 5

(A) DPV curves of the electrochemical nanobiosensor with different GPC3 concentrations from 0.01 to 100.0 μg/mL in the potential range of −0.2 to 0.4 V at a scan rate of 100 mV s⁻¹. (B) Calibration plot of the GPC3 electrochemical nanobiosensor in two different concentration ranges of 10.0–100.0 μg/mL and 0.01–10.0 μg/mL. (C) Histogram for the specificity investigation of the proposed GPC3 electrochemical nanobiosensor (the concentration of GPC3 was 1.0 μg/mL and the concentration of each interfering agent was 10.0 μg/mL). (D) Histogram for the stability analysis of the proposed GPC3 electrochemical nanobiosensor (the concentration of GPC3 was 1.0 μg/mL). All the above-mentioned values are presented as the median from the analysis of three independent experiments and the error bars indicate the relative standard deviation.