Search Results (17)

Aflatoxin B₁ (AFB₁), a highly toxic and carcinogenic mycotoxin, poses significant public health risks due to its widespread contamination of staple food crops such as peanuts and maize. Although conventional lateral flow immunoassays (LFIAs) are widely employed for rapid on-site screening, their limited sensitivity frequently compromises accurate quantification at trace levels. To improve the analytical performance of LFIAs, we developed a novel time-resolved fluorescence-based lateral flow immunoassay (TRFN-LFIA) by integrating reverse artificial antigen labeling with time-resolved fluorescence signal amplification. This method enhances detection sensitivity and enables rapid, ultra-sensitive, visible, and quantitative determination of AFB₁ in peanut and maize samples. Under optimized conditions, the TRFN-LFIA achieved a visible limit of detection (vLOD) of 0.30 ng/mL (2.22 µg/kg), a quantitative limit of detection (qLOD) of 0.04 ng/mL (0.30 μg/kg), and a half-maximal inhibitory concentration (IC₅₀) of 0.09 ng/mL. Recoveries from spiked peanut and maize samples ranged from 81.33% to 117.86%, with coefficients of variation (CVs) below 13.04%. Analysis of 21 real samples (13 maize and 8 peanut samples) yielded results highly consistent with those obtained by liquid chromatography–tandem mass spectrometry (LC-MS/MS). Moreover, the method demonstrates significant advantages in terms of detection speed, cost-effectiveness, and operational convenience. Therefore, the results established the TRFN-LFIA method as a reliable and practical tool for on-site rapid detection of AFB₁ in contaminated food matrices, providing both a rapid and accurate approach for trace-level quantification and a novel strategy for enhancing the sensitivity of lateral flow immunoassays. Full article

Capsaicinoids (CPCs) are regarded as a typical marker of waste oil, which has emerged as a serious food safety issue in developing countries, necessitating the development of rapid, sensitive, and specific detection methods. In this study, a novel hapten was synthesized to generate a high-affinity monoclonal antibody (mAb) targeting CPCs. Subsequently, aggregation-induced emission fluorescent microspheres (AIEFMs), known for their superior fluorescence intensity, were utilized as an enhanced probe to develop a lateral flow immunoassay (LFIA) based on mAb 8B4 for CPC detection. For comparison, a traditional gold nanoparticle (AuNP)-LFIA was also constructed using the corresponding mAb. The AIEFM-LFIA demonstrated a limit of detection (LOD) of 0.33 µg/kg for CPCs in edible oil samples, which is 4.21 times lower than the LOD of 1.39 µg/kg achieved by the AuNP-LFIA. And the assay effectively identified three additional CPCs, with LODs ranging from 0.26 to 0.99 µg/kg, while exhibiting minimal cross-reactivity with CPC analogs, indicating high specificity. The recovery rates of the AIEFM-LFIA in oil samples ranged from 75.0% to 106.0%, with coefficients of variation ≤ 8.3%, exhibiting excellent accuracy and precision. Furthermore, the results of the AIEFM-LFIA demonstrated a strong degree of correlation with liquid chromatography–tandem mass spectrometry, with a correlation coefficient (R²) of 0.978. Consequently, the developed AIEFM-LFIA shows significant promise as a rapid, sensitive, specific, and reliable method for detecting CPCs in oil samples. Full article

Procymidone (PCM) is an effective, low-toxicity fungicide commonly used to control plant diseases in grains, vegetables, and fruits. Its usage has significantly increased in recent years, resulting in higher residues in vegetables. This study developed a sensitive and rapid immunoassay method utilizing a gold- and fluorescence-labeled monoclonal antibody (mAb) for detecting PCM residues in vegetable samples. Under optimal conditions, the fluorescent microsphere-labeled monoclonal antibody immunochromatographic strips achieved a limit of detection (LOD) of 1.67 ng/mL, with a visual LOD of 50 ng/mL. Intra-batch accuracy ranged from 94.98% to 103.82%, with a coefficient of variation (CV) of 1.97% to 8.26%. Inter-batch accuracy ranged from 96.16% to 102.51%, with a CV of 4.62% to 8.91%. The visual detection range of the gold nanoparticle-labeled monoclonal antibody immunochromatographic strips was 50 to 200 ng/g. The method demonstrated excellent performance in actual vegetable samples, confirming its applicability across various matrices. This dual-method approach enables rapid screening of negative samples with gold test strips, followed by accurate quantitative analysis of positive samples using fluorescent test strips, thereby enhancing efficiency and addressing diverse detection needs. Consequently, this method holds significant market potential for practical applications. Full article

Dinotefuran and clothianidin belong to the third generation of nicotinic insecticides and are widely used in crop pest control. It is necessary to detect their residues in food. The time-resolved fluorescent microspheres lateral flow immunoassay (TRFMs-LFIA) has the advantages of high sensitivity, short duration, and simple operation and is suitable for rapid field testing. In this study, two haptens (FCA-1, FCA-2) were synthesized in three steps and conjugated to the carrier proteins to obtain artificial antigens, which were subsequently used for monoclonal antibody preparation. A TRFMs-LFIA based on monoclonal antibodies was established to detect dinotefuran and clothianidin residues in food. The limit of detection (LOD) for dinotefuran was 0.045 ng/mL, with an IC₅₀ of 0.61 ng/mL and a linear range (IC₂₀~IC₈₀) of 0.12~3.11 ng/mL. The LOD for clothianidin was 0.11 ng/mL, with an IC₅₀ of 0.94 ng/mL and a linear range (IC₂₀~IC₈₀) of 0.24~3.65 ng/mL. Cross-reactivity rates with seven tested structural analogs were less than 1.5%. The pretreatment method was optimized for wheat, cucumber, and cabbage samples, which was time-saving (20 min) and easy to operate. The average recovery rates ranged from 88.0% to 114.8%, with the corresponding coefficients of variation appearing (CV) between 1.9% and 13.5%. The results of actual wheat, cucumber, and cabbage samples detected by the established TRFMs-LFIA were consistent with those of Ultra-Performance Liquid Chromatography coupled with Tandem Mass Spectrometry (UPLC-MS/MS). These results demonstrate that the established TRFMs-LFIA is sensitive, accurate, rapid, and suitable for real sample detection. Full article

The spread of the FluA virus poses significant public health concerns worldwide. Fluorescent lateral flow immunoassay (LFIA) test strips have emerged as vital tools for the early detection and monitoring of influenza infections. However, existing quantitative virus-detection methods, particularly those utilizing smartphone-based sensing platforms, encounter accessibility challenges in resource-limited areas and among the elderly population. Despite their advantages in speed and portability, these platforms often lack user-friendliness for these demographics, impeding their widespread utilization. To address these challenges, this study proposes leveraging the optical pick-up unit (OPU) sourced from commercial optical drives as a readily available fluorescence excitation module for the quantitative detection of antibodies labeled with quantum-dot fluorescent microspheres. Additionally, we utilize miniaturized and high-performance optical components and 3D-printed parts, along with a customized control system, to develop an affordable point-of-care testing (POCT) device. Within the system, a stepping motor scans the test strip from the T-line to the C-line, enabling the calculation of the fluorescence-intensity ratio between the two lines. This simple yet effective design facilitates rapid and straightforward field or at-home testing for FluA. The proposed prototype platform demonstrates promising performance, achieving a limit of detection (LOD) of 2.91 ng/mL, a total detection time of no more than 15 min, and dimensions of 151 mm × 11.2 mm × 10.8 mm³. We believe that the proposed approach holds great potential for improving access to an accurate influenza diagnosis. Full article

Inflammation caused by viral or bacterial infection is a major threat to human health globally. Blood C-reactive protein (CRP) has been proven to be a sensitive indicator for the occurrence and development of inflammation. Furthermore, a tiny change of blood CRP concentration may portend chronic diseases; therefore, high-sensitivity CRP (hs-CRP) detection in a quantitative, rapid, user-friendly, and low-cost manner is highly demanded. In this paper, we developed a europium-chelate polystyrene microsphere (EuPSM)-based lateral flow immunoassay (LFIA) integrating with a benchtop fluorescence analyzer for hs-CRP detection. The optimization of the EuPSM-based LFIA was implemented through adjusting the antibody density on EuPSM from 100% to 60% of the saturated density. Finally, the limit of detection of 0.76 pg/mL and detection range of 0.025–250 ng/mL were obtained. Moreover, the clinical application capability of the proposed platform was validated through detecting CRP in clinical serum samples, showing high consistency with the results obtained from the clinical standard method. Hence, the proposed EuPSM-based LFIA has been verified to be well suitable for hs-CRP detection, while also showing great applicability for sensitively and rapidly detecting other biomarkers. Full article

Neonicotinoids are the most commonly used insecticides due to their effectiveness. However, non-targeted insects, especially bees, are also affected by neonicotinoids. Therefore, neonicotinoid application can contribute to the declining bee populations worldwide. The presented study describes the development of novel competitive, fluorescent microsphere-based suspension immunoassays for neonicotinoid profiling and their application to bees and essential bee-related matrices, using the Multi-Analyte Profiling (xMAP) technology. For the construction of these neonicotinoid microsphere immunoassays (nMIAs), neonicotinoid–ovalbumin conjugates were coupled to unique fluorescent, paramagnetic microspheres, which competed with the free neonicotinoids that were present in test samples for interacting with the corresponding, specific antibodies. In total, five independent nMIA’s were developed for the detection of imidacloprid, acetamiprid, clothianidin, thiacloprid, thiamethoxam, dinotefuran, nitenpyram and imidaclothiz with the limits of detection being for 0.01 ng/mL, 0.01 ng/mL, 0.02 ng/mL, 0.02 ng/mL, 0.003 ng/mL, 2.95 ng/mL, 0.09 ng/mL and 0.04 ng/mL, respectively. The developed nMIAs were applied to fortified matrices including surface water, pollen, honey and honeybees. All of the neonicotinoids, except dinotefuran, could be sensitively detected in all of the tested environmental matrices and bees, with there being sensitivities of 1 ng/mL in water and 10 ng/g in solid materials. These nMIAs provide a rapid profiling method for all of the common neonicotinoids, including those that are banned by the European Union for outdoor use. The developed method can contribute to healthy and sustainable beekeeping, globally, via its application in the apiary environment. Full article

Virulent Newcastle disease virus (NDV) as well as highly pathogenic avian influenza (HPAIV) subtypes H5 and H7 induce contagious and lethal systemic disease in poultry. In contrast, low pathogenic AIV H5 and H7 may circulate clinically unnoticed in poultry but eventually generate HPAIV. Low pathogenic NDV strains are widely used as live-attenuated vaccines against ND. Serological tools are essential to conduct active surveillance for infections with notifiable AIV-H5, -H7 and to control vaccination against NDV and HPAIV in poultry populations. Here, recombinant nucleocapsid proteins (NP) of AIV and NDV, and haemagglutinin protein fragment-1 (HA1) of AIV subtypes H5 and H7 were expressed in E. coli. Purification and refolding were required before coating fluorescent microspheres via streptavidin-biotin linkage. The tetraplexed inhibition fluorescent microsphere immunoassay (iFMIA) was then assembled for analysis on a Luminex^®-like platform (Bioplex^®) using murine monoclonal antibodies specific for each of the four targets. The assay was evaluated by testing galliform poultry sera derived from experimental infections (n = 257) and from farms (n = 250), respectively. The tetraplex iFMIA compared favorably with commercially available ELISAs and the “gold standard” hemagglutination inhibition assay. Tetraplexed iFMIA provided a specific and sensitive tool to detect and discriminate AIV- and NDV-specific antibodies in the sera of galliform poultry. Full article

Carbohydrate antigen 199 (CA199) is a serum biomarker which has certain value and significance in the diagnosis, prognosis, treatment, and postoperative monitoring of cancer. In this study, a lateral flow immunoassay based on europium (III) polystyrene time-resolved fluorescence microspheres (TRFM-based LFIA), integrated with a portable fluorescence reader, has been successfully establish for rapid and quantitative analysis of CA199 in human serum. Briefly, time-resolved fluorescence microspheres (TRFMs) were conjugated with antibody I (Ab1) against CA199 as detection probes, and antibody II (Ab2) was coated as capture element, and a “TRFMs-Ab1-CA199-Ab2” sandwich format would form when CA199 was detected by the TRFM-based LFIA. Under the optimal parameters, the detection limit of the TRFM-based LFIA for visible quantitation with the help of an ultraviolet light was 4.125 U/mL, which was four times lower than that of LFIA based on gold nanoparticles. Additionally, the fluorescence ratio is well linearly correlated with the CA199 concentration (0.00–66.0 U/mL) and logarithmic concentration (66.0–264.0 U/mL) for quantitative detection. Serum samples from 10 healthy people and 10 liver cancer patients were tested to confirm the performances of the point-of-care application of the TRFM-based LFIA, 20.0 U/mL of CA199 in human serum was defined as the threshold for distinguishing healthy people from liver cancer patients with an accuracy of about 60%. The establishment of TRFM-based LFIA will provide a sensitive, convenient, and efficient technical support for rapid screening of CA199 in cancer diagnosis and prognosis. Full article

The pandemic of new coronary pneumonia caused by the COVID-19 virus continues to ravage the world. Large-scale population testing is the key to controlling infection and related mortality worldwide. Lateral flow immunochromatographic assay (LFIA) is fast, inexpensive, simple to operate, and easy to carry, very suitable for detection sites. This study developed a COVID-19 N protein detect strip based on p-toluenesulfonyl modified rare earth fluorescent microspheres. The p-toluenesulfonyl-activated nanomaterials provide reactive sulfonyl esters to covalently attach antibodies or other ligands containing primary amino or sulfhydryl groups to the nanomaterial surface. Antibodies are immobilized on these nanomaterials through the Fc region, which ensures optimal orientation of the antibody, thereby increasing the capture rate of the target analyte. The use of buffers with high ionic strength can promote hydrophobic binding; in addition, higher pH could promote the reactivity of the tosyl group. The detection limit of the prepared COVID-19 N protein strips can reach 0.01 ng/mL, so it has great application potential in large-scale population screening. Full article

A novel rare earth upconversion nanomaterial with a three-layer sandwich core–shell structure was synthesized by an improved thermal decomposition method, and the morphology, fluorescence intensity and diffraction peak position of the new material were characterized by TEM (transmission electron microscope), XRD (Powder X-ray diffraction)and fluorescence spectrophotometer. The inert core/active shell/inert shell design improved the upconversion luminous efficiency of the new material several times. FTIR (Fourier transform infrared spectroscopy)characterization showed that the surface of activated upconversion nanoparticles was modified with silicon shell and amino group. Combined with the characteristics that aminoated polystyrene magnetic microspheres could be separated by the magnetic field, an upconversion magnetic separation immunoassay method for the detection of pyrethroid pesticide residues was established. The capture probe competed with the pyrethroid standard, combined the signal probe, and measured the fluorescence signal value formed by the capture probe signal probe complex at 542 nm under 980 nm excitation light. The LOD (limit of detection)of fenpropathrin was 0.01 μg/L, cypermethrin was 0.015 μg/L, and fenvalerate was 0.011 μg/L. Through the actual sample detection of apple, cabbage and other samples, the recovery rate of pyrethroids was between 83.4~97.8%. The comparison with the HPLC (high performance liquid chromatography)detection results showed that the established method had good accuracy, and could realize the quantitative analysis of pyrethroids in food. Full article

Suspension microsphere immunoassays are rapidly gaining attention in multiplex bioassays. Accurate detection of multiple analytes from a single measurement is critical in modern bioanalysis, which always requires complex encoding systems. In this study, a novel bioassay with Raman-coded antibody supports (polymer microbeads with different Raman signatures) and surface-enhanced Raman scattering (SERS)-coded nanotags (organic thiols on a gold nanoparticle surface with different SERS signatures) was developed as a model fluorescent, label-free, bead-based multiplex immunoassay system. The developed homogeneous immunoassays included two surface-functionalized monodisperse Raman-coded microbeads of polystyrene and poly(4-tert-butylstyrene) as the immune solid supports, and two epitope modified nanotags (self-assembled 4-mercaptobenzoic acid or 3-mercaptopropionic acid on gold nanoparticles) as the SERS-coded reporters. Such multiplex Raman/SERS-based microsphere immunoassays could selectively identify specific paratope–epitope interactions from one mixture sample solution under a single laser illumination, and thus hold great promise in future suspension multiplex analysis for diverse biomedical applications. Full article

Aflatoxin B1 (AFB1) is a toxic compound naturally produced by the genera Aspergillus. Distillers’ grains can be used as animal feed since they have high content of crude protein and other nutrients. However, they are easily contaminated by mycotoxins, and currently there are no rapid detection methods for AFB1 in distillers’ grains. In this study, a lateral flow immunoassay (LFIA) based on red fluorescent microsphere (FM), is developed for quantitative detection of AFB1 in distillers’ grains. The whole test can be completed within 15 min, with the cut-off value being 25.0 μg/kg, and the quantitative limit of detection (qLOD) being 3.4 μg/kg. This method represents satisfactory recoveries of 95.2–113.0%, and the coefficients of variation (CVs) are less than 7.0%. Furthermore, this technique is successfully used to analyze AFB1 in real samples, and the results indicates good consistency with that of high-performance liquid chromatography (HPLC). The correlation coefficient is found to be greater than 0.99. The proposed test strip facilitates on-site, cost-effective, and sensitive monitoring of AFB1 in distillers’ grains. Full article

Bluetongue virus (BTV) causes internationally reportable hemorrhagic disease in cattle, sheep, and white-tailed deer. The closely related, and often co-circulating, epizootic hemorrhagic disease virus causes a clinically similar devastating disease in white-tailed deer, with increasing levels of disease in cattle in the past 10 years. Transmitted by Culicoides biting midges, together, they constitute constant disease threats to the livelihood of livestock owners. In cattle, serious economic impacts result from decreased animal production, but most significantly from trade regulations. For effective disease surveillance and accurate trade regulation implementation, rapid, sensitive assays that can detect exposure of cattle to BTV and/or EHDV are needed. We describe the development and validation of a duplex fluorescent microsphere immunoassay (FMIA) to simultaneously detect and differentiate antibodies to BTV and EHDV in a single bovine serum sample. Performance of the duplex FMIA for detection and differentiation of BTV and EHDV serogroup antibodies was comparable, with higher sensitivity than commercially available single-plex competitive enzyme-linked immunosorbent assays (cELISA) for detection of each virus antibody separately. The FMIA adds to the currently available diagnostic tools for hemorrhagic orbiviral diseases in cattle as a sensitive, specific assay, with the benefits of serogroup differentiation in a single serum sample, and multiplexing flexibility in a high-throughput platform. Full article

Immunoassay has the advantages of high sensitivity, high specificity, and simple operation, and has been widely used in the detection of mycotoxins. For several years, time-resolved fluorescence immunochromatography (TRFIA) paper-based sensors have attracted much attention as a simple and low-cost field detection technology. However, a traditional TRFIA paper-based sensor is based on antibody labeling, which cannot easily meet the current detection requirements. A second antibody labeling method was used to amplify the fluorescence signal and improve the detection sensitivity. Polystyrene fluorescent microspheres were combined with sheep anti-mouse IgG to prepare fluorescent probes (Eu-IgGs). After the probe fully reacted with the antibody (Eu-IgGs-Abs) in the sample cell, it was deployed on the paper-based sensor using chromatography. Eu-IgGs-Abs that were not bound to the target were captured on the T-line, while those that were bound were captured on the C-line. The paper-based sensor reflected the corresponding fluorescence intensity change. Because a single molecule of the deoxynivalenol antibody could bind to multiple Eu-IgGs, this method could amplify the fluorescence signal intensity on the unit antibody and improve the detection sensitivity. The working standard curve of the sensor was established under the optimum working conditions. It showed the lower limit of detection and higher recovery rate when it was applied to actual samples and compared with other methods. This sensor has the advantages of high sensitivity, good accuracy, and good specificity, saving the amount of antibody consumed and being suitable for rapid field detection of deoxynivalenol. Full article