A Comparable icELISA and Lateral Flow Immunoassay for the Sensitive and Rapid Detection of 4,4'-Dinitrocarbanilide in Chicken

Abstract

4,4'-dinitrocarbanilide (DNC) is a key component and marker residue of nicarbazin, which forms residues in edible tissue and then causes nephrotoxicity and hepatotoxicity in humans if used excessively. To simplify sample preparation and monitor the DNC rapidly and accurately, a comparable icELISA and lateral flow immunoassay (LFIA) was developed in this study. Briefly, the reaction parameters were explored for improving the sensitivity of icELISA and LFIA. Under the optimal conditions, methanol was selected as the extracting solvent for DNC in chicken, and 20- and 10-fold dilutions of sample extraction eliminated the matrix effect for icELISA and LFIA, separately. After sample pretreatment, the analysis properties of icELISA and LFIA were compared. The limit of detection of icELISA for DNC was 0.8 μg/kg, and the visual and quantitative limits of detection of LFIA were 8 and 2.5 μg/kg. Compared with icELISA, LFIA showed lower sensitivity but obvious advantages in terms of matrix tolerance and detection time (within 15 min). The sensitivity, specificity, and accuracy of the developed assays satisfied the detection requirement even if using simple sample pretreatment. This comparable icELISA and LFIA provided mutual verifiability methods for the accurate detection of DNC in chicken.

Keywords: 4,4′-dinitrocarbanilide; chicken; icELISA; lateral flow immunoassay; rapid detection.

Scheme 1

The schematic illustration of (a) icELISA and (b) LFIA for the detection of DNC. TMB indicates tetramethylbenzidine. T and C indicate the test line and control line.

Figure 1

Optimization of icELISA. The concentrations of coating antigen were (a) 0.4 µg/mL, (b) 0.2 µg/mL, (c) 0.1 µg/mL, and (d) 0.05 µg/mL with Ab concentrations ranging from 6.25 to 200 ng/mL. The optimization of (e) pH values, (f) ionic strength, (g) methanol concentration, and (h) acetonitrile concentration (n = 3). Ag indicates coating antigen, Ab indicates mAb, and IR is the abbreviation of inhibition ratio. The yellow and green bar chart in (a–d) shows the OD value in the presence of DNC at 0 and 0.5 ng/mL, respectively. The error for all graphs was within the standard deviation.

Figure 2

Standard curves for DNC in (a) optimal buffer solution and (b) matrix solution. (c) The recovery ratio and coefficient of variation for the spiked chicken (n = 3). CVs was the abbreviation for the coefficient of variation. The error for all graphs was within the standard deviation.

Figure 3

The (a) UV−vis spectra of AuNPs; (b) the transmission electron micrograph of AuNPs; (c) the size distribution of AuNPs and AuNPs–mAb; (d) the zeta potential of AuNPs; photographs of test strips optimizing the (e) buffer type of mAb and (g) Ag concentration, peak area, and IR optimizing the (f) buffer type of mAb and (h) Ag concentration. Ag indicates the coating antigen, and IR is the abbreviation for the inhibition ratio. The dashed box on the test strips presents the optimal condition. The bar chart in (f,h) indicates the peak area, and the green and red lines indicate IR in the presence of DNC at 0.3 and 1 ng/mL, respectively. The error for all graphs was within the standard deviation.

Figure 4

(a) Photographs of test strips and (b) standard curve with the increasing DNC concentrations in buffer solution. (c) Photographs of test strips, (d) peak area, and IR when exploring the matrix effect. (e) Photographs of test strips and (f) standard curve with the increasing DNC concentrations in chicken. (g) Photographs of test strips and the (h) recovery test in the spiked chicken. The bar chart in (d) shows the peak area and the green and red lines indicate IR in the presence of DNC at 0.2 and 0.4 ng/mL, respectively. The error for all graphs was within the standard deviation.