Time-resolved fluorescence microspheres-antibody-penicillin-binding protein assisted construction of immunochromatographic assay for sensitive detection of 22 ß-lactams in milk.

A sensitive immunochromatographic assay (ICA) using time-resolved fluorescence microspheres (TRFMs) coupled with an indirect-labeling mode was developed for simultaneously determining 22 kinds of ß-lactams in milk samples. The TRFMs labeled anti-receptor monoclonal antibodies (mAbs) conjugated to penicillin-binding proteins (PBPs) as ternary TRFMs-mAb-PBPs (TMP) nanoscaffolds provide excellent solubility, brightness, and stability. Thanks to the fact that they not only fully expose the binding sites of PBPs, thereby enhancing the biological affinity of PBPs towards the target, but also generated superb fluorescence signals, the versatile TMP manifested unique possibilities as efficient probes for ICA with remarkable enhancement in sensitivity in ß-lactams screening. The results showed that the standard curves of the 22 varying ß-lactams displayed linearity in their respective concentration ranges (R2 > 0.98), with the cutoff values of 1-100 ng/mL. The constructed TMP-ICA was successfully applied to the analysis of real milk, with consistent results compared with liquid chromatography-tandem mass spectrometry (LC-MS), providing an effective method for sensing ß-lactams in food matrices.

A time-resolved fluorescence microsphere-lateral flow immunochromatographic strip for quantitative detection of Pregnanediol-3-glucuronide in urine samples.

Introduction: Pregnanediol-3-glucuronide (PdG), as the main metabolite of progesterone in urine, plays a significant role in the prediction of ovulation, threatened abortion, and menstrual cycle maintenance. Methods: To achieve a rapid and sensitive assay, we have designed a competitive model-based time-resolved fluorescence microsphere-lateral flow immunochromatography (TRFM-LFIA) strip. Results: The optimized TRFM-LFIA strip exhibited a wonderful response to PdG over the range of 30-2,000 ng/mL, the corresponding limit of detection (LOD) was calculated as low as 8.39 ng/mL. More importantly, the TRFM-LFIA strip was innovatively used for the quantitative detection of PdG in urine sample, and excellent recovery results were also obtained, ranging from 97.39% to 112.64%. Discussion: The TRFMLFIA strip possessed robust sensitivity and selectivity in the determination of PdG, indicating the great potential of being powerful tools in the biomedical and diagnosis region.

Rapid, on-site quantitative determination of higenamine in functional food using a time-resolved fluorescence microsphere test strip.

Higenamine (HIG), a benzyltetrahydroisoquinoline alkaloid found naturally in plants, is classified as an S3 Prohibited Substance in the 2020 World Anti-Doping Agency (WADA) report. To avoid problems such as doping violations in competitive events, it is necessary to develop rapid and sensitive detection methods. In this study, a highly-sensitive anti-HIG monoclonal antibody (mAb) was prepared and a time-resolved fluorescent microsphere immunochromatographic test strip (TRFM-ICTS) was established for the rapid quantitative detection of HIG in functional foods. Under optimized conditions, the TRFM-ICTS was compared with colloidal gold immunochromatographic test strip (CG-ICTS), and the half-maximal inhibitory concentration (IC50) of TRFM-ICTS was 1.37 ng/mL. The spiked recoveries ranged from 86.4% to 105.3%, which was consistent with the results of liquid chromatography-tandem mass spectrometry (LC-MS/MS) in the detection of real functional food. Therefore, TRFM-ICTS can be a candidate method for doping monitoring in functional foods and a powerful tool for HIG quantification.

One-step time-resolved fluorescence microsphere immunochromatographic test strip for quantitative and simultaneous detection of DON and ZEN.

Deoxynivalenol (DON) and zearalenone (ZEN) are mycotoxins that contaminate a wide range of grains and crops. In this study, a one-step time-resolved single-channel immunochromatographic test strip based on europium ion polystyrene fluorescence microspheres was first developed for sensitive and quantitative detection of DON and ZEN. The concentration of the artificial antigen and the mass ratio of the monoclonal antibody to fluorescent microspheres for conjugation were optimized to simplify the sample addition process during immunochromatographic assay and improve the on-site detection efficiency. The limits of detection (LOD) of the single-channel immunochromatographic test strip for DON and ZEN detection were 0.17 and 0.54 µg/L, respectively. Meanwhile, the dual-channel immunochromatographic test strip was designed to simultaneously detect DON and ZEN, with LODs of 0.24 and 0.69 µg/L achieved for DON and ZEN, respectively. The developed test strips also yielded recovery results consistent with that obtained by LC-MS/MS for DON and ZEN detection in real samples of wheat and corn flour, confirming the practicability and reliability of the test strip. The developed immunochromatographic test strips realize quick and sensitive detection of DON and ZEN, exhibiting potential for broad applications in the point-of-care testing platform of multiple mycotoxins in agricultural products. Graphic abstract.

A smartphone-based dual detection mode device integrated with two lateral flow immunoassays for multiplex mycotoxins in cereals.

In this study, a smartphone-based quantitative dual detection mode device, integrated with gold nanoparticles (GNPs) and time-resolved fluorescence microspheres (TRFMs) lateral flow immunoassays (LFIA) for multiplex mycotoxins in cereals were established. The most frequently used visible light and fluorescence detection modes were integrated in one device. A user-friendly application was self-written to rapidly quantify results. GNPs-LFIA and TRFMs-LFIA were used to detect aflatoxin B1 (AFB1), zearalenone (ZEN), deoxynivalenol (DON), T-2 toxin (T-2), and fumonisin B1 (FB1). The visible limits of detection (vLODs) were 10/2.5/1.0/10/0.5, 2.5/0.5/0.5/2.5/0.5 µg/kg for the two methods, respectively. The quantitative limits of detection (qLODs) were 0.59/0.24/0.32/0.9/0.27, 0.42/0.10/0.05/0.75/0.04 µg/kg, respectively. The recoveries of both LFIAs ranged from 84.0%-110.0%. A parallel analysis in 30 naturally contaminated cereal samples was conducted by liquid chromatography-tandem mass spectrometry (LC-MS/MS), the results showed good consistency, indicating the practical reliability of the established methods. The developed two smartphone-based LFIAs provide a promising technique for multiplex, highly sensitive, and on-site detection of mycotoxins.