Electrochemical Immunosensor Using Electroactive Carbon Nanohorns for Signal Amplification for the Rapid Detection of Carcinoembryonic Antigen

Abstract

In this work, a novel sandwich-type electrochemical immunosensor was developed for the quantitative detection of the carcinoembryonic antigen, an important tumor marker in clinical tests. The capture antibodies were immobilized on the surface of a gold disk electrode, while detection antibodies were attached to redox-tagged single-walled carbon nanohorns/thionine/AuNPs. Both types of antibody immobilization were carried out through Au-S bonds using the novel photochemical immobilization technique that ensures control over the orientation of the antibodies. The electroactive SWCNH/Thi/AuNPs nanocomposite worked as a signal tag to carry out both the detection of carcinoembryonic antigen and the amplification of the detection signal. The current response was monitored by differential pulse voltammetry. A clear dependence of the thionine redox peak was observed as a function of the carcinoembryonic antigen concentration. A linear detection range from 0.001-200 ng/mL and a low detection limit of 0.1385 pg/mL were obtained for this immunoassay. The results showed that carbon nanohorns represent a promising matrix for signal amplification in sandwich-type electrochemical immune assays working as a conductive and binding matrix with easy and versatile modification routes to antibody and redox tag immobilization, which possesses great potential for clinical diagnostics of CEA and other biomarkers.

Keywords: carbon nanohorns; carcinoembryonic antigen; electrochemical immunosensor; redox-tag.

Figure 1

(I–V) Preparation of the nanoprobe consisting of SWCNH/Thi/AuNPs/AntiCEA₂. (VI–X) Immobilization of the AntiCEA₂ on the Au-disk electrode by PIT and assembly of the electrochemical immunosensor. The electrochemical detection is achieved by a dependence on the thionine redox peak as a function of the CEA concentration.

Figure 2

(A,B) HRTEM images of pristine SWCNH, (C,D) HRTEM images of SWCNH/Thi/AuNPs, (E,F) STEM images of SWCNH/Thi/AuNPs.

Figure 3

(A) Energy-dispersive X−ray spectroscopy (EDS) mapping of SWCNH/Thi/AuNPs, including SEM image (B) Carbon (C) element, (C) Oxigen (O) element, (D) Sulfur (S) element, (E) Gold (Au) element and (F) Cyclic voltammetry of glassy carbon electrode (GCE) modified SWCNH/Thi/AuNPs in H₂SO₄ 0.5 M at 50 mVs⁻¹.

Figure 4

(A) CV of Au−disk with (a) 0, (b) 5 µg/mL, (c) 15 µg/mL, (d) 30 µg/mL, (e) 50 µg/mL of AntiCEA₁ in Fe(CN)₆^3-/Fe(CN)₆^4- in PBS 0.01 M at pH 7.4. (B) DPV of Au−disk/AntiCEA₁/CEA/NaPro with (a) 5 µg/mL, (b) 15 µg/mL, (c) 30 µg/mL, (d) 50 µg/mL of AntiCEA₁ in PBS 0.01 M at pH 7.4.

Figure 5

DPV of (a) GCE/SWCNH/Thi/AuNPs/0 µg/mL of AntiCEA₂, (b) GCE/SWCNH/Thi/AuNPs/50 µg/mL of AntiCEA₂, (c) GCE/SWCNH/Thi/AuNPs/100 µg/mL of AntiCEA₂ in PBS 0.01 M at pH 7.4.

Figure 6

(A) CV and (B) EIS of (a) Au−disk, (b) Au−disk/AntiCEA₁, (c) Au−disk/AntiCEA₁/BSA, (d) Au−disk/AntiCEA₁/BSA/CEA, (e) Au−disk/AntiCEA₁/BSA/CEA/NaPro in 10 mM of Fe(CN)₆^3-/Fe(CN)₆^4- in PBS 0.01 M at pH 7.4.

Figure 7

(A) DPV of Au−disk/AntiCEA₁/BSA/CEA/NaPro with different concentrations of CEA in PBS 0.01 M at pH 7.4. (B) The linear relationship between the current peak and the log concentration of CEA.

Figure 8

Current responses of the immunosensor to CEA (50 ng/mL) and interfering substances BSA (50 ng/mL), HSA (50 ng/mL), CA15-3 (50 U/mL) and blank in PBS 0.01 M at pH 7.4 (n = 3).