Phosphate-triggered ratiometric fluoroimmunoassay based on nanobody-alkaline phosphatase fusion for sensitive detection of 1-naphthol for the exposure assessment of pesticide carbaryl

Abstract

The excessive use of carbaryl has resulted in the risk of its exposure. In this study, we isolated six nanobodies (Nbs) from a camelid phage display library against the biomarker of carbaryl, 1-naphthol (1-NAP). Owing to its characteristics of easy genetic modifications, we produced a nanobody-alkaline phosphatase (Nb-CC4-ALP) fusion protein with good stability. A dual-emission system based ratiometric fluoroimmunoassay (RFIA) for quick and highly sensitive determination of 1-NAP was developed. Silicon nanoparticles (SiNPs) was used as an internal reference and for aggregation-induced emission enhancement (AIEE) of gold nanoclusters (AuNCs), while AuNCs could be quenched by MnO₂ via oxidation. In the presence of ALP, ascorbic acid phosphate (AAP) can be transformed into ascorbic acid (AA), the later can etch MnO₂ to recover the fluorescence of the AuNCs. Based on optimal conditions, the proposed assay showed 220-fold sensitivity improvement in comparison with conventional monoclonal antibody-based ELISA. The recovery test of urine samples and the validation by standard HPLC-FLD demonstrated the proposed assay was an ideal tool for screening 1-NAP and provided technical support for the monitoring of carbaryl exposure.

Keywords: 1-Naphthol; Carbaryl; Nanobody; Pesticide exposure; Ratiometric fluoroimmunoassay.

Fig. 1

(A) The titer of camelid antiserum, hapten 1-BSA (1 μg/mL) was employed for coating antigen; (B) Inhibition of free 1-NAP (1 μg/mL) to camelid antiserum; (C) Subtype separation of camelid antibodies, M: marker, lane 1: IgG₁, lane 2: IgG₂ lane 3: IgG₃; (D) Inhibition of camelid IgG₁, IgG₂, IgG₃.

Fig. 2

Schematic diagram of RFIA. Ascorbic acid phosphate (AAP); Ascorbic acid (AA); Alkaline phosphatase (ALP); 1-naphthol (1-NAP)

Fig. 3

The (A)TEM image, (B) emission spectrum, (C) XPS spectrum, and (D) FTIR spectrum of AuNCs. The blue circles indicate the FT-IR spectrum peak of S-H bond. The (E)TEM image, (F) emission spectrum, and (G) FTIR spectrum of SiNPs. (H) The zeta potential of AuNCs, SiNPs, and SiNP-AuNC.

Fig. 4

(A₁) Ex optimizing of SiNP-AuNC; (A₂) The emission spectrum of AuNCs, SiNPs, and SiNP-AuNC; (B₁) TEM image of SiNP-AuNC; The (B₂) HAADF image and (B₃ and B₄) energy dispersive spectroscopy map of SiNP-AuNC.

Fig. 5

(A) The TEM image and absorbance of MnO₂ nanosheet; (B) The spectrum of SiNP-AuNC in the absence or presence of MnO₂ nanosheet; (D) TEM image of SAM mixture; (D) High resolution of Au 4f_7/2 and Au 4f_5/2.

Fig. 6

(A) Optimizing of ratios of SiNPs and AuNCs; (B) Optimizing of various concentrations of AAP with the 1 mU of ALP; (C) The signal inhibition with MnO₂, I0 and I was the value of I₆₁₀/I₄₇₀ without and with MnO₂, respectively; Optimizing of (D) pH and (E) reaction time for ALP. The red circles indicate the optimized parameters that was chose for the development of RFIA; (F) Response of ALP by using optimized SAM system.

Fig. 7

(A) The titer of pNPP-based ELISA and RFIA; (B) The calibration curve of TMB-based ELISA, pNPP-based ELISA and RFIA, with the IC₅₀ value of 8.3 ng/mL, 7.6 ng/mL, and 1.8 ng/mL, respectively; and with the LOD of 1.4 ng/mL, 0.1 ng/mL, and 0.01 ng/mL, respectively; (C) Response of analogues for RFIA; (D) Correlation between HPLC-FLD and RFIA.