Efficient double-quenching of electrochemiluminescence from CdS:Eu QDs by hemin-graphene-Au nanorods ternary composite for ultrasensitive immunoassay

Abstract

A novel ternary composite of hemin-graphene-Au nanorods (H-RGO-Au NRs) with high electrocatalytic activity was synthesized by a simple method. And this ternary composite was firstly used in construction of electrochemiluminescence (ECL) immunosensor due to its double-quenching effect of quantum dots (QDs). Based on the high electrocatalytic activity of ternary complexes for the reduction of H2O2 which acted as the coreactant of QDs-based ECL, as a result, the ECL intensity of QDs decreased. Besides, due to the ECL resonance energy transfer (ECL-RET) strategy between the large amount of Au nanorods (Au NRs) on the ternary composite surface and the CdS:Eu QDs, the ECL intensity of QDs was further quenched. Based on the double-quenching effect, a novel ultrasensitive ECL immunoassay method for detection of carcinoembryonic antigen (CEA) which is used as a model biomarker analyte was proposed. The designed immunoassay method showed a linear range from 0.01 pg mL(-1) to 1.0 ng mL(-1) with a detection limit of 0.01 pg mL(-1). The method showing low detection limit, good stability and acceptable fabrication reproducibility, provided a new approach for ECL immunoassay sensing and significant prospect for practical application.

Figure 1. Schematic illustration of the fabrication process the CEA biosensing platform based on double-quenching of H-RGO-AuNRs.

Process (A,B) respectively represents the preparation of hemin-reduced graphene oxide-Au nanorods (H-RGO-AuNRs) and CEA biosensing platform.

Figure 2

TEM images of CdS:Eu QDs (A), GO (B), H-RGO-Au NRs (C) and the EDX analysis of H-RGO-AuNRs (D). Insert of (C) shows the corresponding partly enlarged view of H-RGO-Au NRs.

Figure 3

(A) ECL spectrum of the CdS:Eu QDs (a),and the UV-vis absorption spectra of GO (b), Hemin (c), H-RGO (d) and H-RGO-AuNRs (e); (B) Cyclic voltammograms of 3 mm H₂O₂ with addition of (b) hemin; (c) GO; (d) H-RGO, and (e) H-RGO-Au NRs with the same concentration in 3 mm H₂O₂ on GCE. Curve a is the CV response of 3 mm H₂O₂ without addition of any material.

Figure 4. ECL emission from CdS:Eu QDs film on GCE in 7.5 mM H₂O₂ + 0.1 M KCl + 0.1 M PBS (pH = 7.4) under continuous cyclic potential scan for 10 cycles.

The PMT voltage: −600 V. Potential: −1.3 V (vs Ag/AgCl). Scan rate, 100 mV/s.

Figure 5

Effects of concentrations on quenching efficiency: optimization of H₂O₂ concentrations (A), CEA incubation temperature on GCE-CdS:Eu QDs/Ab₁ (B), CEA incubation time on GCE-CdS:Eu QDs/Ab₁ (C) and volumes of H-RGO-AuNRs/Ab₂ in the ECL biosensor (D). The ECL peak intensity for analysis was obtained at −1.3 V (vs. Ag/AgCl) in 0.1 M PBS (pH 7.4), CV Scan rate: 0.1 V/s, the PMT voltage: −600 V.

Figure 6

(A) The normalized ECL-potential curves of (a) GCE-CdS:Eu QDs, (b) GCE-CdS:Eu QDs/Ab₁, (c) GCE-CdS:Eu QDs/Ab₁/CEA and (d) GCE-CdS:Eu QDs/Ab₁/CEA/Ab₂/H-RGO-Au. Inset: the normalized ECL-time curves. The ECL peak intensity for analysis was obtained at −1.3 V (vs. Ag/AgCl) in 0.1 M PBS (pH 7.4) containing 7.5 mM H₂O₂. CV Scan rate: 0.1 V/s. The PMT voltage: 600 V. (B) Electrochemical impedance spectra of the proposed biosensing platform: (a) bare GCE; (b) GCE-CdS:Eu QDs; (c) GCE-CdS:Eu QDs/Ab₁; (d) GCE-CdS:Eu QDs/Ab₁/CEA; (e) GCE-CdS:Eu QDs/Ab₁/CEA/H-RGO-Au/Ab₂ in 0.1 M KCl solution containing 5.0 mM K₃[Fe(CN)₆]/K₄[Fe(CN)₆]. The PMT voltage: −600 V. Potential: −1.3 V (vs Ag/AgCl), Scan rate: 100 mV/s.

Figure 7. ECL signals response for detection of different concentrations of CEA.

The concentrations of CEA: (a) 0, (b) 1.0 × 10⁻¹⁴ g/mL, (c) 1.0 × 10⁻¹³ g/mL, (d) 1.0 × 10⁻¹² g/mL, (e) 1.0 × 10⁻¹¹ g/mL, (f) 5.0 × 10⁻¹¹ g/mL, (g) 1.0 × 10⁻¹⁰ g/mL, (h) 5.0 × 10⁻¹⁰ g/mL, (i) 1.0 × 10⁻⁹ g/mL and (j) 2.0 × 10⁻⁹ g/mL, inset: Linear relationship between quenching efficiency and the logarithm of CEA concentration, three measurements for each point. The PMT voltage: −600 V. Potential: −1.3 V (vs Ag/AgCl).