An ultrasensitive electrochemiluminescence immunoassay for carbohydrate antigen 19-9 in serum based on antibody labeled Fe3O4 nanoparticles as capture probes and graphene/CdTe quantum dot bionanoconjugates as signal amplifiers

Abstract

The CdTe quantum dots (QDs), graphene nanocomposite (CdTe-G) and dextran-Fe3O4 magnetic nanoparticles have been synthesized for developing an ultrasensitive electrochemiluminescence (ECL) immunoassay for Carcinoembryonic antigen 19-9 (CA 19-9) in serums. Firstly, the capture probes (CA 19-9 Ab1/Fe3O4) for enriching CA 19-9 were synthesized by immobilizing the CA 19-9's first antibody (CA 19-9 Ab1) on magnetic nanoparticles (dextran-Fe3O4). Secondly, the signal probes (CA 19-9 Ab2/CdTe-G), which can emit an ECL signal, were formed by attaching the secondary CA 19-9 antibody (CA 19-9 Ab2) to the surface of the CdTe-G. Thirdly, the above two probes were used for conjugating with a serial of CA 19-9 concentrations. Graphene can immobilize dozens of CdTe QDs on their surface, which can emit stronger ECL intensity than CdTe QDs. Based on the amplified signal, ultrasensitive antigen detection can be realized. Under the optimal conditions, the ECL signal depended linearly on the logarithm of CA 19-9 concentration from 0.005 to 100 pg/mL, and the detection limit was 0.002 pg/mL. Finally, five samples of human serum were tested, and the results were compared with a time-resolved fluorescence assay (TRFA). The novel immunoassay provides a stable, specific and highly sensitive immunoassay protocol for tumor marker detection at very low levels, which can be applied in early diagnosis of tumor.

Figure 1

TEM image of the CdTe QDs (A); CdTe-G nanosheets (B); Fe₃O₄ NPs (C) and dextran-Fe₃O₄ NPs (D); Magnetization hysteresis for (a) Fe₃O₄ and (b) dextran-Fe₃O₄ nanoparticles recorded at room temperature (E); Dextran-Fe₃O₄ NPs in the absence (right) and presence (left) of an external magnetic field (F).

Figure 2

Electrochemiluminescence (ECL)-potential curves of CdTe QDs (a); CdTe-G nanosheets (b) and CA 19-9 Ab2/CdTe-G bioconjuncture (c) modified SPCE electrode. The voltage of the photomultiplier tube was set at 600 V.

Figure 3

ECL-potential curves of the CA 19-9 Ab1/dextran-Fe₃O₄ modified SPCE electrode (a); (a) incubated in 10 pg/mL CA 19-9 + CA 19-9 Ab2/CdTe-G (b); (a) incubated in 10 pg/mL CA 19-9 + CA 19-9 Ab2/CdTe in 0.1 mol/L PBS (pH 7.0) containing 0.1 mol/L KCl and 0.1 mol/L K₂S₂O₈ (c). The voltage of the photomultiplier tube was set at 600 V.

Figure 4

Electrochemical impedance of screen printed carbon electrode (SPCE) (a); CA 19-9 Ab1/dextran-Fe₃O₄ modified SPCE (b); (b) + 10 pg/mL CA 19-9 (c); (c) + CA 19-9 Ab2/CdTe-G (d) in 0.1 mol/L PBS [containing 2 mmol/L Fe(CN)₆^4−/3− and 0.1 mol/L KCl, pH 7.0]. The frequency range is between 0.01 and 100,000 Hz with a signal amplitude of 5 mV.

Figure 5

Effect of pH (a); incubation temperature (b) and incubation time (c) on the quenched ECL intensity of the immunosensor toward 5 pg/mL CA 19-9. The voltage of the photomultiplier tube was set at 600 V.

Figure 6

ECL profiles of the immunosensor in the absence (a) and presence (b→h) of different concentrations of CA 19-9 in 0.1 mol/L PBS (pH 7.0) containing 0.1 mol/L KCl and 0.1 mol/L K₂S₂O₈. CA 19-9 concentration (pg/mL): 0 (a), 0.005 (b), 0.01 (c), 0.02 (d), 0.05 (e), 0.1 (f), 0.2 (g), 0.5 (h), 1 (i), 3 (j), 10 (k), 30 (l), 100 (m), 140 (n), 150 (o), 160 (p), 200 (q). The voltage of the photomultiplier tube was set at 700 V. Scan rate: 100 mV/s. The inset: calibration curve for Ag determination.

Scheme 1

Schematic processes of the immunosensor fabrication process and detection principle. Dropping of Nafion membrane (a); electrochemical deposition of HAuCl₄ (b); and immobilization of CA19-9 Ab₂ (c) and blocked with BSA (d); immunoreaction of CA 19-9 and capture probes (e), resulting in an amplified signal-generating detection.

Scheme 2

The immunoassay procedure [28].