Bifunctional M13 Phage as Enzyme Container for the Reinforced Colorimetric-Photothermal Dual-Modal Sensing of Ochratoxin A

Abstract

"Point of care" (POC) methods without expensive instruments and special technicians are greatly needed for high-throughput analysis of mycotoxins. In comparison, the most widely used screening method of the conventional enzyme-linked immunosorbent assay (ELISA) confronts low sensitivity and harmful competing antigens. Herein, we develop a plasmonic-photothermal ELISA that allows precise readout by color-temperature dual-modal signals based on enzymatic reaction-induced AuNP aggregation for highly sensitive detection of ochratoxin A (OTA). The bifunctional M13 phage carrying OTA that mimics the mimotope on the end of p3 proteins and abundant biotin molecules on the major p8 proteins is adopted as an eco-friendly competing antigen and enzyme container for amplifying the signal intensity. Under optimal conditions, both colorimetric and photothermal signals enable good dynamic linearity for quantitative OTA detection with the limits of detection at 12.1 and 8.6 pg mL^-1, respectively. Additionally, the proposed ELISA was adapted to visual determination with a cutoff limit of 78 pg mL^-1 according to a vivid color change from deep blue to red. The recoveries of OTA-spiked corn samples indicate the high accuracy and robustness of the proposed method. In conclusion, our proposed strategy provides a promising method for eco-friendly and sensitive POC screening of OTA. Moreover, it can be easily applied to other analytes by changing the involved specific mimotope sequence.

Keywords: AuNP aggregation; bifunctional M13 bacteriophage; enzyme container; ochratoxin A; plasmonic and photothermal ELISA.

Scheme 1

The principle of dc-ppELISA using a bifunctional M13 phage as a competing antigen for colorimetric–photothermal response arising from enzymatic reaction-induced AuNP aggregation.

Figure 1

Characterization of colorimetric–photothermal dual signal response to AuNP aggregation induced by the HRP-H₂O₂-tyramine system. (A) LSPR spectra of different tests (a: H₂O₂ + TYR + AuNPs, b: H₂O₂ + HRP + TYR + AuNPs, c: HRP + TYR + AuNPs, d: HRP + H₂O₂ + AuNPs, e: H₂O₂ + AuNPs, f: TYR + AuNPs, g: HRP + AuNPs); (B) Temperature rise after 5 min excitation with 808 nm laser of test a, b, c, d, e, f, and g, and (C,D) TEM images of AuNP aggregation induced by the HRP-H₂O₂-tyramine system and dispersion of AuNPs in the absence of H₂O₂.

Figure 2

Dual signal response of the substrate solution to the addition of H₂O₂. (A) Calibration curve plotting OD_{630 nm}/OD_{520 nm} against the concentration of H₂O₂; (B) Calibration curve plotting temperature variation against the concentration of H₂O₂.

Figure 3

Optimization of the experimental conditions for the dc-ppELISA. (A) effect of pH values (6.5–8.0); (B) methanol content (v/v, 0–80%); (C) NaCl concentration (0~80 mM) on the performance of the dc-ppELISA. The error bars represent the standard deviation of the three measurements.

Figure 4

Competitive inhibition curves of the proposed dc-ppELISA based on colorimetric signal (A) and photothermal signal (B). The inset photos are the results of color development from detecting samples with different OTA concentrations.