Highly sensitive immunosensing platform for one-step detection of genetically modified crops

Abstract

The wide cultivation of genetically modified (GM) insect-resistant crops has raised concerns on the risks to the eco-environment resulting from a release of Cry proteins. Therefore, it is vital to develop a method for the quantification of GM crops. Herein, A highly sensitive immunosensing platform has been developed for both colorimetric and chemiluminescent (CL) detection of Cry 1Ab using dual-functionalized gold nanoparticles (AuNPs) as signal amplification nanoprobes for the first time. In this work, anti-Cry 1Ab monoclonal antibody and horseradish peroxidase (HRP) are simultaneously functionalized on the surface of AuNPs with an exceptionally simple synthesis method. Combined with immunomagnetic separation, this immunosensing platform based on colorimetric method could detect Cry 1Ab in one step in a linear range from 1.0 to 40 ng mL^-1 within 1.5 h, with a limit of detection of 0.50 ng mL^-1. The sensitivity of fabricated nanoprobes was 15.3 times higher than that using commercial HRP-conjugated antibody. Meanwhile, the fabricated nanoprobes coupled with CL detection was successfully applied for Cry 1Ab detection with a minimum detection concentration of 0.050 ng mL^-1 within a linear range of 0.10-20 ng mL^-1. The proposed approach was validated with genuine GM crops, and the results showed a good correlation coefficient of 0.9906 compared to those of a commercial ELISA kit. Compared with ELISA, the developed immunosensing platform significantly simplified the assay procedure and shortened the analytical time, thus providing a new platform for the detection of genetically modified crops with high sensitivity, rapidity and simplicity.

Figure 1

(A) Particle size distributions of AuNPs. (B) UV-vis absorption spectra of bare AuNPs and AuNP nanoprobes. (C) Absorbance response of H₂O, AuNPs-dissolved buffer, bare AuNPs and AuNP nanoprobes after reaction with TMB solution and H₂SO₄ stopping solution

Figure 2

Schematic illustration of a magnetic bead-based immunosensing platform for the one-step detection of Cry 1Ab using dual-functionalized AuNPs.

Figure 3

Signal-to-blank ratio (A) at the indicated addition volume of immunomagnetic and (B) in the presence of AuNP nanoprobes at the indicated dilution folds. Signal refers to the absorbance of 10 ng mL⁻¹ Cry 1Ab and blank refers to the absorbance of PB buffer containing 0.10% BSA.

Figure 4

(A) Specificity of developed immunosensing platform. (B) Dose-response curves for Cry 1Ab assay based on colorimetric detection using (a) AuNP nanoprobes and (b) HRP-labeled antibody. Error bars are standard deviations of five repeated measurements.

Figure 5

(A) CL responses of the developed immunosensing platform for analyzing Cry 1Ab at concentrations of 0, 0.10, 1.0, 5.0, 10 and 20 ng mL⁻¹ from bottom to top. (B) Calibration curve. Error bars are standard deviations of five repeated measurements