Sandwich-Type Electrochemiluminescence Immunosensor Based on CDs@dSiO2 Nanoparticles as Nanoprobe and Co-Reactant

Abstract

In general, co-reactants are essential in highly efficient electrochemiluminescence (ECL) systems. Traditional co-reactants are usually toxic, so it is necessary to develop new environmentally friendly co-reactants. In this work, carbon dots (CDs) were assembled with dendritic silica nanospheres (CDs@dSiO₂ NPs) to form a co-reactant of Ru(bpy)₃²⁺. Subsequently, a sandwich immunosensor for detecting human chorionic gonadotropin (HCG) was constructed based on CDs@dSiO₂ NPs as co-reactants, the nanoprobe loaded with the secondary antibody, and Ru(bpy)₃²⁺ as a luminophore. In addition, compared to directly as a signal probe, the luminophore Ru (bpy)₃²⁺ as a part of the electrolyte solution is simpler in this work. The immunosensor has an extremely low limit of detection of 0.00019 mIU/mL. This work describes the synthesis of low-toxic, efficient, and environmentally friendly CDs, which have become ideal co-reactants of Ru(bpy)₃²⁺, and proposes an ECL immunosensor with excellent stability and selectivity, which has great potential in clinical applications.

Keywords: Ru(bpy)32+; carbon dots; electrochemiluminescence; immunosensor.

Figure 1

Schematic diagram of the preparation process and mechanism of the HCG immunosensor.

Figure 2

TEM images and EDX of (A, C) dSiO₂, (B, D) CDs@dSiO₂; (E) FTIR spectra of dSiO₂ NPs, CDs, CDs@dSiO₂ NPs, respectively, and (F) UV–vis absorption spectrum and fluorescence spectrum of (a, c) CDs, and (b, d) CDs@dSiO₂ NPs.

Figure 3

(A) CV curves and (B) ECL behaviors of Ru(bpy)₃²⁺ (black curve), dSiO₂-Ru(bpy)₃²⁺ (red curve), CDs-Ru(bpy)₃²⁺ (blue curve), and CDs@dSiO₂-Ru(bpy)₃²⁺ (green curve) in a solution of 0.1 M PBS (pH = 7.4) containing 50 μM Ru(bpy)₃²⁺. The voltage of PMT was set at 600 V.

Figure 4

Electrochemical characterization of the preparation process of sandwich immunosensors: (A) CV and (B) EIS of (a) bare GCE, (b) Ab₁/Au/GCE, (c) BSA/Ab₁/Au/GCE, (d) Ag/BSA/Ab₁/Au/GCE, and (e) Ab₂-CDs@dSiO₂ NPs/Ag/BSA/Ab₁/Au/GCE (inset is the circuit model) measured in a solution containing 5 mM [Fe (CN)₆]^3-/^4- and 0.1 M KCl.

Figure 5

(A) ECL response with HCG of different concentrations (a to i: 0.0005 mIU/mL, 0.001 mIU/mL, 0.01 mIU/mL, 0.1 mIU/mL, 1 mIU/mL, 10 mIU/mL, 100 mIU/mL, 200 mIU/mL, 500 mIU/mL). (B) A linear relationship between the ECL intensities and lgC_HCG. (C) Selectivity of the immunosensor with different interferences. (D) Reproducibility tests of the ECL immunosensor (intra-assays and inter-assays). (Error bars: SD, n = 3) in a solution of PBS (0.01 M, pH = 7.4) containing 50 μM Ru(bpy)₃²⁺. The voltage of PMT was set at 800 V.