Search Results (159)

Highly infectious and pathogenic viruses seriously threaten global public health, underscoring the need for rapid and accurate diagnostic methods to effectively manage and control outbreaks. In this study, we developed a comprehensive Surface-Enhanced Raman Scattering–Lateral Flow Immunoassay (SERS-LFIA) detection system that integrates SERS scanning imaging with artificial intelligence (AI)-based result discrimination. This system was based on an ultra-sensitive SERS-LFIA strip with SiO₂-Au NSs as the immunoprobe (with a theoretical limit of detection (LOD) of 1.8 pg/mL). On this basis, a negative–positive discrimination method combining SERS scanning imaging with a deep learning model (ResNet-18) was developed to analyze probe distribution patterns near the T line. The proposed machine learning method significantly reduced the interference of abnormal signals and achieved reliable detection at concentrations as low as 2.5 pg/mL, which was close to the theoretical Raman LOD. The accuracy of the proposed ResNet-18 image recognition model was 100% for the training set and 94.52% for the testing set, respectively. In summary, the proposed SERS-LFIA detection system that integrates detection, scanning, imaging, and AI automated result determination can achieve the simplification of detection process, elimination of the need for specialized personnel, reduction in test time, and improvement of diagnostic reliability, which exhibits great clinical potential and offers a robust technical foundation for detecting other highly pathogenic viruses, providing a versatile and highly sensitive detection method adaptable for future pandemic prevention. Full article

Heavy metal ions pose a significant threat to the environment and human health due to their high toxicity and bioaccumulation. Traditional instrumentations, although sensitive, are often complex, costly, and unsuitable for on-site rapid detection of heavy metal ions. Lateral flow assay technology has emerged as a research hotspot due to its rapid, simple, and cost-effective advantages. This review summarizes the applications of lateral flow assay technology based on nucleic acid molecules and antigen–antibody interactions in heavy metal ion detection, focusing on recognition mechanisms such as DNA probes, nucleic acid enzymes, aptamers, and antigen–antibody binding, as well as signal amplification strategies on lateral flow testing strips. By incorporating these advanced technologies, the sensitivity and specificity of lateral flow assays have been significantly improved, enabling highly sensitive detection of various heavy metal ions, including Hg²⁺, Cd²⁺, Pb²⁺, and Cr³⁺. In the future, the development of lateral flow assay technology for detection of heavy metal ions will focus on multiplex detection, optimization of signal amplification strategies, integration with portable devices, and standardization and commercialization. With continuous technological advancements, lateral flow assay technology will play an increasingly important role in environmental monitoring, food safety, and public health. Full article

Arterial hypertension (HTN) is a growing global health concern, with limited tools available for early detection. Previous studies identified the overexpression of the epithelial sodium channel (ENaC) as a potential biomarker for HTN. In this work, we optimized and clinically validated a lateral flow immunoassay (LFIA) using gold nanoparticles (AuNPs) functionalized with anti-ENaC antibodies. The test strips were prepared with 10 µL of each component and performed in a 9-point herringbone format. For validation, a double-blind study was conducted using platelet lysates from 200 individuals, classified based on real-time blood pressure measurements. ENaC expression was assessed via both LFIA and Western blotting, which served as the reference method. Receiver operating characteristic (ROC) analysis yielded an AUC of 0.7314 for LFIA and 0.6491 for the Western blot, with LFIA demonstrating higher sensitivity (76.24%) and comparable specificity (61.54%) compared to the Western blot (68.31% and 60.34%, respectively). These results support LFIA as a practical, rapid, and moderately accurate tool for screening ENaC levels and identifying individuals at risk of hypertension. Full article

The development of rapid, sensitive and cost-effective lateral flow assays is crucial for the detection of mycotoxins, ideally at the point-of-care level. This study presents the design and optimization of a competitive lateral flow assay based on gold nanoparticles (AuNPs) for the detection of zearalenone in food samples. Beginning with the synthesis and functionalization of gold nanoparticles, it proceeds to compare the immobilization of antibodies using chemical conjugation and physical adsorption binding strategies, upon optimizing parameters including the pH, antibody concentration and blocking conditions to enhance the stability of the prepared bioconjugates. The bioconjugates are characterized using UV–visible absorption spectroscopy and dynamic light scattering to monitor changes in the spectra and hydrodynamic size of AuNPs upon the addition of antibodies. The assessment of these bioconjugates is based on their ability to bind and manifest a color, developed due to nanoparticle binding with the test zone on the strip with the toxin–protein conjugate. The lateral flow immunochromatographic assay (LFIA) strips are then prepared by dispensing a control line (IgG) and test line (toxin–protein conjugate) on a nitrocellulose membrane using a lateral flow strip dispenser. The sensitivity of the LFIA strips is evaluated after standardizing the conditions by varying the concentration of zearalenone in the spiked samples and optimizing the running buffer solution. The limit of detection and limit of quantification under optimized conditions are determined to be 0.7 ng/mL and 2.37 ng for zearalenone-spiked samples. Furthermore, the mean pixel intensity and RGB values are plotted against the concentration of zearalenone, which can be used in a colorimetric smartphone-based application for the quantification of the amount of mycotoxin in the sample. Full article

Foot and mouth disease virus (FMDV) is a highly pathogenic virus that mainly infects cloven hooved animals, such as pigs. The establishment of a rapid, sensitive and accurate point-of-care detection method is critical for the timely identification and elimination of infected pigs for controlling this disease. In this study, a RT-RAA-CRISPR/Cas13a method was developed for the detection of FMDV serotype O in pigs. Six pairs of RT-RAA primers were designed based on the conserved gene sequence of FMDV serotype O, and the optimal amplification primers and reaction temperatures were screened. The CRISPR-derived RNA (crRNA) was further designed based on the optimal target band sequence and the most efficient crRNA was screened. The results revealed that FMDV-O-F4/R4 was the optimal primer set, and the optimal temperature for the RT-RAA reaction was 37 °C. Moreover, crRNA4 exhibited the strongest detection signal among the six crRNAs. The established RT-RAA-CRISPR/Cas13a method demonstrated high specificity and no cross-reactivity with other common swine pathogens such as Senecavirus A (SVA), porcine reproductive and respiratory virus (PRRSV), porcine epidemic diarrhea virus (PEDV), porcine circovirus type 2 (PCV2), classical swine fever virus (CSFV), and pseudorabies virus (PRV), additionally, it was observed to be highly sensitive, with a detection limit of 19.1 copies/µL. The repeatability of this method was also observed to be good. This method could produce stable fluorescence and exhibited good repeatability when three independent experiments yielded the same results. A validation test using three types of simulated clinical samples (including swab, tissue, and serum samples) revealed a 100% concordance rate. The detection results could be visualized via a fluorescence reader or lateral flow strips (LFSs). Thus, a highly specific and sensitive RT-RAA-CRISPR/Cas13a detection method was developed and is expected to be applied for the rapid detection of FMDV serotype O in situ. Full article

Lateral flow tests (LFTs) had a pivotal role in combating the spread of the SARS-CoV-2 virus throughout the COVID-19 pandemic thanks to their affordability and ease of use. Most of LFT devices were based on nitrocellulose membrane strips whose industrial upscaling to billions of devices has already been extensively demonstrated. Nevertheless, the assay option in an LFT format is largely restricted to qualitative detection of the target antigens. In this research, we surveyed the potential of UV nanoimprint lithography (UV-NIL) and extrusion coating (EC) for the high-throughput production of disposable capillary-driven, foil-based tests that allow multistep assays to be implemented for quantitative readout to address the inherent lack of on-demand fluid control and sensitivity of paper-based devices. Both manufacturing technologies operate on the principle of imprinting that enables high-volume, continuous structuring of microfluidic patterns in a roll-to-roll (R2R) production scheme. To demonstrate the feasibility of R2R-fabricated foil chips in a point-of-care biosensing application, we adapted a commercial chemiluminescence multiplex test for COVID-19 antibody detection originally developed for a capillary-driven microfluidic chip manufactured with injection molding (IM). In an effort to build a complete ecosystem for the R2R manufacturing of foil chips, we also recruited additional processes to streamline chip production: R2R biofunctionalization and R2R lamination. Compared to conventional fabrication techniques for microfluidic devices, the R2R techniques highlighted in this work offer unparalleled advantages concerning improved scalability, dexterity of seamless handling, and significant cost reduction. Our preliminary evaluation indicated that the foil chips exhibited comparable performance characteristics to the original IM-fabricated devices. This early success in assay translation highlights the promise of implementing biochemical assays on R2R-manufactured foil chips. Most importantly, it underscores the potential utilization of UV-NIL and EC as an alternative to conventional technologies for the future development in vitro diagnostics (IVD) in response to emerging point-of-care testing demands. Full article

Nipah virus (NiV) is a severe zoonotic pathogen that substantially threatens public health. Pigs are the natural hosts of NiV and can potentially transmit this disease to humans. Establishing a rapid, sensitive, and accurate point-of-care detection method is critical in the timely identification of infected pig herds. In this study, we developed an NiV detection method based on reverse transcription–recombinase polymerase amplification (RT-RAA) and the clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 13a (Cas13a) system for the precise detection of NiV. The highly conserved region of the NiV gene was selected as the detection target. We first designed eleven pairs of RT-RAA primers, and the optimal primer combination and reaction temperature were identified on the basis of RT-RAA efficiency. Additionally, the most efficient crRNA sequence was selected on the basis of the fluorescence signal intensity. The results revealed that the optimal reaction temperature for the developed method was 37 °C. The detection limit was as low as 1.565 copies/μL. Specificity testing revealed no cross-reactivity with nucleic acids from six common swine viruses, including Seneca virus A (SVA), foot-and-mouth disease virus (FMDV), classical swine fever virus (CSFV), porcine epidemic diarrhea virus (PEDV), African swine fever virus (ASFV), and pseudorabies virus (PRV). A validation test using simulated clinical samples revealed a 100% concordance rate. The detection results can be visualized via a fluorescence reader or lateral flow strips (LFSs). Compared with conventional detection methods, this RT-RAA-CRISPR/Cas13a-based method is rapid and simple and does not require scientific instruments. Moreover, the reagents can be freeze-dried for storage, eliminating the need for cold-chain transportation. This detection technology provides a convenient and efficient new tool for the point-of-care diagnosis of NiV and for preventing and controlling outbreaks. Full article

(1) Background: There are many cases of human disease caused by the hepatitis A virus contamination of aquatic products, so the development of the rapid detection of hepatitis A virus in aquatic products is crucial. (2) Methods: In this study, we developed three visual loop-mediated isothermal amplification methods for the rapid and intuitive detection of hepatitis A virus in aquatic products. New specific LAMP primers were designed for the HAV-specific VP1 protein shell. (1) HNB dye was added to the LAMP reaction system. After the reaction, the color of the reaction mixture changed from violet to sky blue, showing a positive result. (2) Cresol red dye was added to the LAMP reaction system, and a positive result was indicated by orange, while a negative result was indicated by purple. (3) By labeling FIP with biotin and LF with 6-FAM, the amplified product simultaneously contained biotin and 6-FAM, which bound to the anti-biotin antibody on the gold nanoparticles on the lateral flow dipstick (LFD). Subsequently, biotin was further combined with the anti-fam antibody on the T-line of the test strip to form a positive test result. (3) Results: The three visual LAMP methods were highly specific for HAV. The sensitivity of the visual assay was 2.59 × 10⁰ copies/μL. The positive detection ratio for 155 bivalve shellfish samples was 8.39%, which was the same as that for RT-qPCR. The three visual LAMP methods established in our work have better sensitivity than the international gold standard, and their operation is simple and requires less time. (4) Conclusions: The results can be obtained by eye color comparison and lateral flow dipsticks. Without the use of large-scale instrumentation, the sensitivity is the same as that of RT-qPCR. The test strips are lightweight, small in size, and easy to carry; they are suitable for emergency detection, on-site monitoring, field sampling, or remote farms and other non-laboratory environments for rapid identification. Full article

Deoxynivalenol (DON), generally the most widespread mycotoxin in wheat, is regulated by the EU regulation in cereals and cereal-derived products. Its presence can be detected by chromatographic or rapid methods; the latter technique is generally used in control analysis, fulfilling the needs of the stakeholders of the wheat grain chain. Lateral flow strips are often used for the rapid detection of different mycotoxins in several agricultural products; regarding DON determination, different lateral flow immunochromatography strips are currently available, also providing quantitative results. The purpose of this work was to evaluate the accuracy of an innovative lateral flow device coupled to a bench top device, following a digital approach. The proposed method was compared to an LC-MS/MS method, analyzing 50 naturally contaminated wheat samples. The results obtained using the two methods were very similar and, applying a paired t-test, the mean difference between measurements resulted not significantly different (α = 0.003). The correlation between the results showed a slope of the line close to 1 (m = 0.9904) and a regression coefficient (r) of 0.9968. Full article

Ciprofloxacin is metabolized from enrofloxacin for use in poultry to manage respiratory and gastrointestinal diseases, raising concerns due to its widespread tissue distribution and prolonged systemic persistence. This lateral flow immunoassay was designed to detect ciprofloxacin using an alternative IgY antibody binded with gold nanoparticles to detect ciprofloxacin residue in raw pork meat samples. The developed strip test achieved adequate sensitivity and specificity under the optimized conditions for pH, which is 7.8, and 20% of MeOH in 0.01 M phosphate buffer containing 1% Tween-20 was used for the buffer composition. An antibody concentration of 1.25 µg/mL was used to bind with gold nanoparticles as a probe for detection. The concentration of the test line (coating antigen) and control line (anti-IgY secondary antibody) was 0.5 mg/mL and 0.2 mg/mL, respectively. The efficiency of the developed strip test showed sensitivity with a 50% inhibitory concentration (IC₅₀) of ciprofloxacin at 7.36 µg/mL, and the limit of detection was 0.2 µg/mL. The proposed strategy exhibited potential for monitoring ciprofloxacin in raw pork samples. Full article

We present a lateral flow aptasensor for the visual detection of alpha-fetoprotein (AFP) in human serum. Leveraging the precise molecular recognition capabilities of aptamers and the distinct optical features of gold nanoparticles, a model system utilizing AFP as the target analyte, along with a pair of aptamer probes, is implemented to establish proof-of-concept on standard lateral flow test strips. It is the first report of an antibody-free lateral flow assay using aptamers as recognition probes for the detection of AFP. The analysis circumvents the numerous incubation and washing steps that are typically involved in most current aptamer-based protein assays. Qualitative analysis involves observing color changes in the test area, while quantitative data are obtained by measuring the optical response in the test zone using a portable strip reader. The biosensor exhibits a linear detection range for AFP concentrations between 10 and 100 ng/mL, with a minimum detection limit of 10 ng/mL. Additionally, it has been successfully applied to detect AFP in human serum samples. The use of aptamer-functionalized gold nanoparticle probes in a lateral flow assay offers great promise for point-of-care applications and fast, on-site detection. Full article

Due to the price and demand of Ophiocordyceps sinensis having increased dramatically, adulteration with other fungi is a common problem. Thus, a reliable method of authentic O. sinensis identification is essential. In the present work, a rapid DNA extraction and double-tailed recombinase polymerase amplification (RPA) coupled with nucleic acid hybridization lateral flow strip (NAH-LFS) was developed to distinguish authentic O. sinensis ingredients from other fungi substitutes. In the presence of O. sinensis, the RPA amplicons with two ssDNA tails in the opposite ends, which could simultaneously bind with the SH-probes on gold nanoparticles (AuNPs) and capture the probe on the test line, formed visible red bands. RPA combined with NAH-LFS can efficiently detect O. sinensis DNA down to 1.4 ng/μL; meanwhile, the specificity test validated no cross reaction with common adulterants, including Cordyceps gunnii, Cordyceps cicadae, Cordyceps militaris, yungui Cordyceps, and Ophiocordyceps nutans. The whole RPA-NAH-LFS could be completed within 16 min. The RPA-NAH-LFS results in detecting 20 commercial O. sinensis samples are consistent with PCR-AGE and RT-PCR, confirming the feasibility of the RPA-NAH-LFS method. In conclusion, these results are expected to facilitate the application of RPA-NAH-LFS in the authentication detection of O. sinensis materials, providing a convenient and efficient method for O. sinensis quality control. Full article

Sildenafil is used to treat erectile dysfunction and pulmonary arterial hypertension but is often illicitly added to energy drinks and chocolates. This study introduces a lateral flow strip test using aptamers specific to sildenafil for detecting its illegal presence in food. The process involved using graphene oxide SELEX to identify high-affinity aptamers, which were then converted into molecular gate structures on mesoporous silica nanoparticles, creating a unique signaling system. This system was integrated into lateral flow chromatography strips and tested on buffers and chocolate samples containing sildenafil. The method simplifies the lateral flow assay (LFA) for small molecules and provides a tool for signal amplification. The detection limit for these strips was found to be 68.2 nM (31.8 µg/kg) in spiked food samples. Full article

To ensure the safety of foodstuffs, widespread non-laboratory monitoring for pathogenic contaminants is in demand. A suitable technique for this purpose is lateral flow immunoassay (LFIA) which combines simplicity, rapidity, and productivity with specific immune detection. This study considered three developed formats of LFIA for Salmonella Typhimurium, a priority pathogenic contaminant of milk. Common sandwich LFIA with all immunoreagents pre-applied to the test strip (format A) was compared with incubation of the sample and (gold nanoparticle—antibody) conjugate, preceding the lateral flow processes (format B), and sequential passages of the sample and the conjugate along the test strip (format C). Under the chosen conditions, the detection limits and the assay times were 3 × 10⁴, 1 × 10⁵, and 3 × 10⁵ cells/mL, 10, 15, and 20 min for formats A, B, and C, respectively. The selected format A of LFIA was successfully applied to test milk samples. The sample’s dilution to a fat content of 1.0% causes pathogen detection, with 70–110% revealing and 1.5–8.5% accuracy. The obtained results demonstrate that the developed LFIA allows the detection of lower concentrations of Salmonella cells and, in this way, accelerates decision-making in food safety control. Full article

The development of a novel strategy for the measurement of SARS-CoV-2 IgG antibodies is of vital significance for COVID-19 diagnosis and effect of vaccination evaluation. In this investigation, an SiO₂@Au@CDs nanoparticle (NP)-based lateral flow immunoassay (LFIA) strip was fabricated and coupled with a miniaturized fluorimeter. The morphology features and particle sizes of the SiO₂@Au@CDs NPs were characterized carefully, and the results indicated that the materials possess monodisperse, uniform, and spherical structures. Finally, this system was employed for SARS-CoV-2 IgG antibody test. In this work, the strategy for the SARS-CoV-2 IgG antibody test possesses several merits, such as speed (less than 15 min), high sensitivity (1.2 × 10⁻⁷ mg/mL), broad linearity range (7.4 × 10⁻⁷~7.4 × 10⁻⁴ mg/mL), accurate results, high selectivity, good stability, and low cost. Additionally, future trends in LFAs using quantum dot-based diagnostics are envisioned. Full article