An ultrasensitive NIR-IIa' fluorescence-based multiplex immunochromatographic strip test platform for antibiotic residues detection in milk samples

Abstract

Introduction: Widely used in livestock breeding, residues of antibiotic drugs in milk have become a threat to food safety and human health. Current rapid detection technologies using colorimetric immunochromatographic strip tests (IST) lack the necessary sensitivity for on-site trace monitoring. Fluorescence-based detection in the near-infrared IIa' (NIR-IIa') region (1000 ∼ 1300 nm) has enormous potential due to greatly minimized auto-fluorescence and light scattering.

Objectives: The aim of this work is to develop an ultrasensitive IST platform using NIR-IIa' fluorescent nanoparticles as labels for multiplex antibiotic residues detection in milk.

Methods: NIR-IIa' fluorescent nanoparticles were assembled by encapsulating synthesized NIR-IIa' fluorophores into carboxyl - modified polystyrene nanoparticles. The NIR-IIa' nanoparticles were subsequently used as labels in an IST platform to detect sulfonamides, quinolones, and lincomycin simultaneously in milk. A portable fluorescent reader was fabricated to provide on-site detection. To further validate the developed IST platform, the detection was compared with LC-MS/MS in 22 real milk samples.

Results: Fluorescent nanoparticles were synthesized with low energy emission (1030 nm) and large Stokes shift (>250 nm) showing a much higher signal-to-noise ratio compared with fluorophores emitting in the NIR-I region. The developed IST platform yielded a highly sensitive, simultaneous quantification of sulfonamides, quinolones, and lincomycin in milk with detection limits of 46.7, 27.6 and 51.4 pg/mL, respectively, achieving a wide detection range (up to 50 ng/mL). The IST platform showed good accuracy, reproducibility, and specificity with the portable fluorescent reader which could rapidly quantify in 10 s. These results were better than reported immunochromatographic assays using fluorescent labels, and remarkably, showed a higher recognition ability than LC-MS/MS for real samples.

Conclusion: The utility of NIR-IIa' fluorescence-based IST platform for the fast, sensitive, and accurate detection of antibiotics in milk was demonstrated, successfully verifying the potential of this platform in detecting trace materials in complex matrices.

Keywords: Drug; Fluorescent nanoparticles; Food safety; Lateral flow test; Second near-infrared region.

Graphical abstract

Scheme 1

Schematic illustration of the multiplex NIR-II-FNPs-based immunochromatographic strip test (IST) platform. (A) Schematic synthesis of the NIR-II-FNPs detection probes. (B) Detection process of the platform. (C) The designed portable reader with internal structure. (D) Quantitative detection principle of the platform.

Fig. 1

Optical characterization of NIR-II-FNPs. (A) TEM image. (B) SEM image. (C) DLS measurement. (D) Optical images of NIR-II-FNPs in water under natural light (left) and NIR excitation (right). (E) Fluorescence excitation spectrum. (F) Fluorescence emission spectrum.

Fig. 2

Representative spectra of fluorescent labels and autofluorescent agents in milk in the UV, visible, and near infrared (NIR) regions. Gold NPs: gold nanoparticles, Cy3: Cy3 fluorescent dye, Eu NPs: lanthanide chelated NPs, VA: vitamin A, OxiP: fluorescent oxidation products, NADH: reduced nicotinamide adenine dinucleotide, NC membrane: nitrocellulose membrane.

Fig. 3

Comparison of NIR-I and NIR-IIa’ labels. (A) Dot-blot images of PBS, NIR-I (785/820 nm) and NIR-IIa’ (785/1030 nm) dyes, pure milk (100 % milk), milk doubly diluted by PBS (50 % milk), and dyes mixed with milk on a nitrocellulose (NC) membrane. (B) The ratio of the fluorescence intensity of NIR-I and NIR-IIa’ dyes in PBS, 50 % milk, and 100 % milk to the corresponding matrix intensity (signal to noise ratio). ** represents significant differences at p < 0.01 (mean ± SD, n = 3).

Fig. 4

Performance evaluation of the multiplex NIR-II-FNPs-based immunochromatographic strip test (IST) platform. (A) Typical optical images obtained using the platform for the detection of sulfonamides (SA) – T₁, quinolones (QN) – T₂, and lincomycin (LIN) – T₃. (B) Fluorescence intensity curves of the strips. (C) Calibration curves of the three antibiotics (mean ± SD, n = 3). For 1–8, the concentrations of the three antibiotics were 0, 0.012, 0.048, 0.195, 0.781, 3.125, 12.5, and 50 ng/mL, respectively.