Quantum Dot Nanobeads as Multicolor Labels for Simultaneous Multiplex Immunochromatographic Detection of Four Nitrofuran Metabolites in Aquatic Products

Abstract

A multicolor immunochromatographic assay platform based on quantum dot nanobeads (QBs) for the rapid and simultaneous detection of nitrofuran metabolites in different aquatic products is documented. These metabolites include 3-amino-2-oxazolidinone (AOZ), 1-aminohydantoin (AHD), semicarbazide (SEM), and 3-amino-5-morpholino-methyl-1,3-oxazolidinone (AMOZ). QBs with emission colors of red, yellow, green, and orange were employed and functionalized with the corresponding antibodies to each analyte to develop a multicolor channel. The visual detection limits (cutoff values) of our method for AOZ, AHD, SEM, and AMOZ reached up to 50 ng/mL, which were 2, 20, 20, and 20 times lower than those of traditional colloidal gold test strips, respectively. The test strip is capable of detection within 10 min in real samples while still achieving good stability and specificity. These results demonstrate that the developed multicolor immunochromatographic assay platform is a promising technique for multiplex, highly sensitive, and on-site detection of nitrofuran metabolites.

Keywords: fluorescent test strip; multiplex immunochromatographic assay; nitrofuran metabolites; quantum dot nanobeads.

Figure 1

Schematic illustration of the (A) preparation of QB-mAb probes, (B) analysis of QB-based mICA, and (C) interpretation of test results. (“+” and “−” indicate the “positive” and “negative” results.).

Figure 2

(A) Fluorescence emission spectra of QBs and QB-mAb probes. The inset shows the corresponding photograph of QBs and QB-mAb probes solutions under UV light at 365 nm, (B) fluorescent images of QBs and QB-mAb in 0.5% agarose gel after gel electrophoresis, (C) SEM images of QBs at magnifications of (a) 10,000× and (b) 60,000×, and QB-mAb probes at magnifications of (c) 10,000× and (d) 60,000×.

Figure 3

(A) Optimization of the concentrations of competitive antigen on T lines and amounts of QB-mAb probes, (B) optimization of immunoreaction time, and (C) gray values analysis of T and C lines. T1, T2, T3, T4, and C lines were 2-NPAOZ-BSA, 2-NPAHD-BSA, 2-NPSEM-BSA, 2-NPAMOZ-BSA, and rabbit anti-mouse IgG, respectively. The check mark indicated the optimal condition.

Figure 4

(A) Fluorescent image of mICA strip under UV light for detection of AOZ (T1), AHD (T2), SEM (T3), and AMOZ (T4), (B) calibration curves of analytes, and (C) Commercial colloidal gold test strips for analyte detection.

Figure 5

Cross-reaction and specificity analysis of our developed QB-based mICA strip, (A) fluorescent image of mICA strip under UV light, (B) gray values analysis of four T lines. T1, T2, T3, T4, and C lines were 2-NPAOZ-BSA, 2-NPAHD-BSA, 2-NPSEM-BSA, 2-NPAMOZ-BSA, and rabbit anti-mouse IgG, respectively.

Figure 6

(A) The internal structure of smartphone-based immunochromatography reader, and (B) the interfaces of analysis smartphone application.