Development of aptamer-based lateral flow devices for rapid detection of SARS-CoV-2 S protein and uncertainty assessment.

The outbreak and spread of COVID-19 have highlighted the urgent need for early diagnosis of SARS-CoV-2. Nucleic acid testing as an authoritative tool, is cumbersome, time-consuming, and easy to cross-infect, while the available antibody self-testing kits are deficient in sensitivity and stability. In this study, we developed competitive aptamer-based lateral flow devices (Apt-LFDs) for the quantitative detection of SARS-CoV-2 spike (S) protein. Molecular docking simulation was used to analyze the active binding sites of the aptamer to S protein, guiding complementary DNA (cDNA) design. Then a highly efficient freezing strategy was applied for the conjugation of gold nanoparticles (AuNPs) and DNA probes. Under optimal conditions, the linear range of the constructed Apt-LFDs was 0.1-1 µg/mL, and the limit of detection (LOD) was 51.81 ng/mL. The cross-reactivity test and stability test of the Apt-LFDs showed good specificity and reliability. The Apt-LFDs had recoveries ranging from 89.45 % to 117.12 % in pharyngeal swabs. Notably, the uncertainty of the analytical result was evaluated using a "bottom-up" approach. At a 95 % confidence level, the uncertainty report of (453.37±54.86) ng/mL with k = 2 was yielded. Overall, this study provides an important reference for the convenient and reliable detection of virus proteins based on LFDs.

Highly sensitive detection of carbendazim in agricultural products using colorimetric and photothermal lateral flow immunoassay based on plasmonic gold nanostars.

The wide use and high toxicity of carbendazim (CBD) in agriculture pose unprecedented demands for convenient, sensitive, and cost-effective on-site monitoring. Herein, we propose a novel colorimetric and photothermal dual-mode lateral flow immunoassay (LFIA) based on plasmonic gold nanostars (AuNSs) for CBD detection in agricultural products. The AuNSs were synthesized via a rapid seed-mediated growth method (with growth time of ∼5 s). A stable immunoprobe was formed by adsorbing CBD antibodies onto AuNSs. This immunoprobe exhibited high conversion efficiency and sensitivity in photothermal detection with a low limit of detection (LOD) of 0.28 ng mL-1. The LOD of the colorimetric mode was higher (0.48 ng mL-1). The results of CBD detection in various agricultural products aligned well with ultra-performance liquid chromatography tandem mass spectrometry. Overall, our LFIA shows excellent sensitivity, specificity, reproducibility, and rapidness in CBD detection, and thus is a highly potential on-site platform in resource-limited environments.

Hue-change coupled with CRISPR-Cas12a lateral flow assay for the semiquantitative detection of Salmo salar adulteration.

Salmo salar is one of the most popular salmon species due to its meaty texture and quality protein. Oncorhynchus mykiss, which has a muscle texture similar to that of Salmo salar and is less expensive, is often used as a substitute for Salmo salar. As Salmo salar and Oncorhynchus mykiss belong to the same subfamily of Salmonidae, traditional methods are ineffective in the specific detection of the two. In this study, we combined hue-change with CRISPR/Cas12a lateral flow assay to detect the Salmo salar adulteration. This method detected S. salar genomic DNA at a vLOD of 5 copies, and was able to accurately identify adulterated samples containing 5 % w/w Salmo salar within one hour. In addition, the detection of Salmo salar in processed food products was achieved with the naked-eye at a concentration range of 0 % âˆ¼ 70 % w/w, and the detection accuracy is between 93.3 % âˆ¼ 100 %.

Smartphone-based lateral flow immunoassay for sensitive determination of lactate dehydrogenase at the point of care.

Lactate dehydrogenase (LDH), a prevalent enzyme involved in anaerobic glycolysis, is released into body fluids following cell damage and has long been a general marker of tissue injury. However, due to its lack of selectivity and the advent of more accurate biomarkers, the clinical utility of LDH has been largely limited to confirming hemolysis. LDH has been recognized as a valuable prognostic biomarker for various cancers, making its monitoring crucial during cancer management. Traditional LDH methods include spectrophotometric analysis of NADH at 340 nm, native electrophoresis, or enzyme-linked immunosorbent assay. This study presents the first lateral flow immunoassay (LFIA) for the smartphone-based quantification of serum LDH levels at the point of care. Highly-affinity and specific antibodies have been produced, with 5 nM equilibrium dissociation constant and no cross-reactivity with human serum albumin and human immunoglobulin G. Utilizing carbon nanoparticles as signal transducers significantly enhanced the quantification limit 55-fold, compared to the conventional gold nanoparticles-based LFIA, achieving a quantification limit of 1.5 ng mL-1. The developed assay demonstrated a mean recovery rate of 115 ± 21 % when evaluating LDH-spiked serum samples. This method can be an interesting home-testing tool for monitoring cancer progression or therapy effectiveness.

Rapid visual detection of Helicobacter pylori and vacA subtypes by Dual-Target RAA-LFD assay.

BACKGROUND: Helicobacter pylori (H. pylori) infects over 50% of the global population and is a significant risk factor for gastric cancer. The pathogenicity of H. pylori is primarily attributed to virulence factors such as vacA. Timely and accurate identification, along with genotyping of H. pylori virulence genes, are essential for effective clinical management and controlling its prevalence. METHODS: In this study, we developed a dual-target RAA-LFD assay for the rapid, visual detection of H. pylori genes (16s rRNA, ureA, vacA m1/m2), using recombinase aided amplification (RAA) combined with lateral flow dipstick (LFD) methods. Both 16s rRNA and ureA were selected as identification genes to ensure reliable detection accuracy. RESULTS: A RAA-LFD assay was developed to achieve dual-target amplification at a stable 37 °C within 20 min, followed by visualization using the lateral flow dipstick (LFD). The whole process, from amplification to results, took less than 30 min. The 95 % limit of detection (LOD) for 16 s rRNA and ureA, vacA m1, vacA m2 were determined as 3.8 × 10-2 ng/µL, 5.8 × 10-2 ng/µL and 1.4 × 10-2 ng/µL, respectively. No cross-reaction was observed in the detection of common pathogens including Escherichia coli, Klebsiella pneumoniae, Enterococcus faecalis, Staphylococcus aureus, Pseudomonas aeruginosa, and Bacillus subtilis, showing the assay's high specificity. In the evaluation of the clinical performance of the RAA-LFD assay. A total of 44 gastric juice samples were analyzed, immunofluorescence staining (IFS) and quantitative polymerase chain reaction (qPCR) were used as reference methods. The RAA-LFD results for the 16s rRNA and ureA genes showed complete agreement with qPCR findings, accurately identifying H. pylori infection as confirmed by IFS in 10 out of the 44 patients. Furthermore, the assay successfully genotyped vacA m1/m2 among the positive samples, showing complete agreement with qPCR results and achieving a kappa (κ) value of 1.00. CONCLUSION: The dual-target RAA-LFD assay developed in this study provides a rapid and reliable method for detecting and genotyping H. pylori within 30 min, minimizing dependency on sophisticated laboratory equipment and specialized personnel. Clinical validation confirms its efficacy as a promising tool for effectively control of its prevalence and aiding in the precise treatment of H. pylori-associated diseases.

Multiplex reverse transcription recombinase polymerase amplification combined with lateral flow biosensor for simultaneous detection of three viral pathogens in cattle.

Bovine viral diarrhea virus (BVDV), bovine epidemic fever virus (BEFV), and bovine respiratory syncytial virus (BRSV) cause respiratory symptoms in cattle. The absence of rapid, precise, and easily accessible diagnostic methods poses difficulties for herders and veterinary epidemiologists during outbreaks of major infectious animal diseases. Considering the mixed infection of viruses, a multiple-detection method, reverse transcription recombinase polymerase amplification (mRT-RPA) combined with a lateral flow biosensor (LFB), was established to simultaneously detect the three pathogens. This technique is based on the specific binding of three differently labeled RT-RPA products (DNA sequences) to antibodies on the three test lines of the LFB, achieving multiplex detection through the presence or absence of coloration on the LFB test lines. The fluorescence values of the LFB test lines are recorded by a test strip reader. The mRT-RPA-LFB assay completes detection at a constant temperature of 41 °C within 33 min. The limits of detection (LODs) for BVDV, BEFV and BRSV were 2.62 × 101, 2.42 × 101 and 2.56 × 101 copies/µL, respectively. No cross-reactivity was observed with the other six bovine viruses. The developed method showed satisfactory intra- and inter-assay precision, and the average coefficients of variation were ranged from 2.92 % to 3.99 %. The diagnostic sensitivity and specificity were 98.11 % and 100 %, respectively, which were highly consistent with the RT-qPCR assay, and the kappa value was 0.988 (95 % confidence interval, CI). In general, the mRT-RPA-LFB assay has the potential to become a powerful tool for rapid screening of cattle diseases because of its advantages such as fast detection speed, convenient operation, strong specificity, and high sensitivity.

Ionic polymers as double-capture agents in an aptamer lateral flow assay strip for point-of-care detection of ethyl carbamate using peroxidase-like activity of bimetallic NiCo2O4 nanoparticles.

A novel aptamer LFA (Apt-LFA) strip is first constructed for ethyl carbamate (EC) detection, in which cationic polyethyleneimine (PEI) and anionic polyacrylamide (APAM) are used as double-capture agents. The black NiCo2O4 nanoparticles (NCO NPs) encapsulated by EC1-34 aptamer are employed as recognition probes. The added EC will bind to EC1-34 aptamer on the probes, so that the test-line (T-line) immobilized with APAM can electrostatically capture the exposed NCO NPs. The control-line (C-line) sprayed with PEI can capture the excessive black recognition probes. The peroxidase-like activity of bimetallic NCO NPs is employed as signal amplification to improve the sensitivity of Apt-LFA strip. The naked eye discernible concentration of EC is 5 µg L-1 and the detection limit (LOD) is as low as 1.36 µg L-1. The Apt-LFA strip has the advantages of strong stability, simplicity and low cost, which provide a new method for EC detection and offer a new way for designing LFA.

Dual-Fluorescent Quantum Dot Nanobead-Based Lateral Flow Immunoassay for Simultaneous Detection of C-Reactive Protein and Procalcitonin.

Simultaneous detection of C-reactive protein (CRP) and procalcitonin (PCT) at the point of care is crucial for the management of infections in patients with inflammation and in critical care settings. The challenge of detecting high concentrations of CRP alongside low concentrations of PCT in plasma from inflammatory patients has limited the clinical application of multiplexed immunoassays. Herein, we developed a lateral flow immunoassay (LFIA) that employs quantum dot nanobeads (QDNBs) of varying sizes and colors to enable the simultaneous quantification of PCT and CRP in human plasma. To extend the dynamic range of CRP detection, we combined QDNBs with smaller particle sizes with the CRP detection antibodies, thereby increasing the assay's dynamic range and reducing the hook effect. At the same time, the stronger fluorescence emitted by these larger QDNBs, in conjugation with the PCT detection antibodies, allows for the detection of PCT at the nanogram level, meeting the demand for high sensitivity. The results show that this method can detect CRP concentrations from 0.1 to 3 mg/L and PCT with a detection limit of 0.09 ng/mL, which is on par with clinically used methods. By employing this dual-color and dual-size QDNB labeling strategy, we successfully achieved simultaneous detection of CRP with a broad dynamic range and PCT with high sensitivity in a one-step point-of-care rapid test.

Home-Made Lateral Flow Test Strip Versus POC-CCA Assay for Detection of Active Schistosomiasis in Egypt.

BACKGROUND: For years, the Kato-Katz (KK) technique has been considered the gold standard for diagnosing schistosomiasis. The aim of this study was to compare the effectiveness of our previously developed gold nanoparticle-based lateral flow test strip (AuNPs-LFTS) for diagnosing active Schistosoma mansoni with that of the commercially available point-of-care Circulating Cathodic Antigen detection (POC-CCA) kit. METHODS: In this study, we collected sixty positive and twenty negative urine samples from patients in endemic hot spots in the Nile Delta, as well as from patients visiting the internal medicine clinic at Theodor Bilharz Research Institute (TBRI). We produced monoclonal antibodies (MAbs) against S. mansoni soluble egg antigen (SEA) from cloned hybridoma cells (4D/1D). These MAbs were conjugated with gold and mesoporous silica nanoparticles, and used to develop the LFTS. RESULTS: The LFTS demonstrated a limit of detection (LoD) of 3 ng/ml. The sensitivity and specificity of the developed LFTS were found to be 96.7% and 95%, respectively, compared to 85% and 90% for the POC-CCA detection kit. The cases were divided into groups based on egg count in the stool, categorized as light, moderate, and heavy infections. The sensitivity of the LFTS in the group with light infection was higher than that of the POC-CCA. When using the KK technique (eggs per gram of stool sample [EPG]) as the reference test, the kappa value for the nano-based strips was 0.902, compared to 0.672 for the CCA strips, indicating an almost perfect agreement between KK and our developed LFTS. CONCLUSION: These results confirm the reliability and effectiveness of the LFTS compared to commercially available kits for rapid, sensitive, and early diagnosis of schistosomiasis. However, it is recommended to conduct further assessments of the developed strip on a larger scale with a broader range of cases before considering its introduction to local or international markets.

Fluorescent lateral flow assay strip for Mycobacterium tuberculosis and Mycobacterium smegmatis based on mycobacteriophage tail protein and aptamer.

Timely and facile monitoring of Mycobacterium tuberculosis (M. tuberculosis) plays an important role for preventing and controlling tuberculosis infection. Mycobacterium smegmatis (M. smegmatis) has long been employed as a safe surrogate for the investigation of M. tuberculosis. In this work, an aqueous soluble tail protein derived from our previously isolated mycobacteriophage was prepared with a recombinant expression technique and noted as GP89, which shows noticeable binding capacity to Mycobacterium genus. GP89 was sprayed as a capture agent onto a nitrocellulose membrane for forming the test line of a lateral flow assay (LFA) strip. Moreover, an aptamer binding M. tuberculosis and M. smegmatis was labeled with fluorescent microspheres to act as the signal tracer of the LFA method. With the GP89 based LFA, M. tuberculosis and M. smegmatis can be detected with the aid of a handheld UV flashlight or a portable fluorescent strip reader within 10 min. The concentration range for quantitating M. tuberculosis and M. smegmatis are both 1.0 × 102 CFU mL-1 - 1.0 × 106 CFU mL-1, and the detection limits for the two mycobacteria are 2.0 and 24 CFU mL-1 (S/N = 3), respectively. The test strip was applied to detect M. tuberculosis and M. smegmatis in different samples such as physiological salt solution, urine, and saliva. This study offers a promising screening tool for diagnosing M. tuberculosis infection in resource-limited institutes.

Novel lateral flow assay to detect H2O2 by utilizing self-biotinylation of G-quadruplex.

We herein describe a novel lateral flow assay (LFA) to detect H2O2 by utilizing self-biotinylation of G-quadruplex (G4). In this strategy, the G4 strand promotes the self-biotinylation of G4 itself in the presence of H2O2, which is then allowed to bind to the FAM-labeled complementary detector probe. The resulting biotin-labeled G4/FAM-detector probe complex is captured on the test line, producing a red-colored band during lateral flow readout. Based on this unique approach, we achieved the naked-eye detection of target H2O2 at concentrations as low as 1 µM, with reliable quantification down to 0.388 µM. This method also demonstrated exceptional specificity in distinguishing H2O2 from other non-target molecules. We further verified its versatile applicability by reliably identifying another biomolecule, choline, by coupling with choline oxidase, which generates H2O2 during oxidation. This novel LFA strategy holds great promise as a powerful point-of-care testing (POCT) platform for detecting a large spectrum of target biomolecules by employing their corresponding oxidases.

Detection of Avian Leukosis Virus Subgroup J (ALV-J) Using RAA and CRISPR-Cas13a Combined with Fluorescence and Lateral Flow Assay.

Avian Leukosis Virus (ALV) is a retrovirus that induces immunosuppression and tumor formation in poultry, posing a significant threat to the poultry industry. Currently, there are no effective vaccines or treatments for ALV. Therefore, the early diagnosis of infected flocks and farm sanitation are crucial for controlling outbreaks of this disease. To address the limitations of traditional diagnostic methods, which require sophisticated equipment and skilled personnel, a dual-tube detection method for ALV-J based on reverse transcription isothermal amplification (RAA) and the CRISPR-Cas13a system has been developed. This method offers the advantages of high sensitivity, specificity, and rapidity; it is capable of detecting virus concentrations as low as 5.4 × 100 copies/µL without cross-reactivity with other avian viruses, with a total testing time not exceeding 85 min. The system was applied to 429 clinical samples, resulting in a positivity rate of 15.2% for CRISPR-Cas13a, which was higher than the 14.7% detected by PCR and 14.2% by ELISA, indicating superior detection capability and consistency. Furthermore, the dual-tube RAA-CRISPR detection system provides visually interpretable results, making it suitable for on-site diagnosis in remote farms lacking laboratory facilities. In conclusion, the proposed ALV-J detection method, characterized by its high sensitivity, specificity, and convenience, is expected to be a vital technology for purification efforts against ALV-J.

An RPA-Based CRISPR/Cas12a Assay in Combination with a Lateral Flow Assay for the Rapid Detection of Shigella flexneri in Food Samples.

Among the pathogens that cause infectious diarrhea in China, Shigella is the most prominent. Shigellosis affects both adults and children, particularly those in developing nations, with nearly 190 million annual cases and a third resulting in fatalities. The recently emerged CRISPR/Cas system has also been increasingly applied for the detection of different biological targets. The lateral flow assay (LFA) has the advantages of short detection time, simple operation, high sensitivity, and low cost, and it provides an ideal platform for on-site detection. In this study, a recombinase polymerase amplification-CRISPR/Cas12a-LFA test for Shigella flexneri was constructed. The established method had good specificity and sensitivity, and the qualitative accuracy of 32 tested strains reached 100%. The detection limit of genomic DNA reached 8.3 copies/µL. With the advantages of high accuracy and portability, this diagnostic apparatus represents a novel method of identification and detection of Shigella flexneri, particularly in settings that lack complex laboratory infrastructure.

Human Papillomaviruses 16 and 18 E6 Oncoprotein Detection Test in Primary Oropharyngeal Carcinomas and Metastatic Lymph Nodes: A Cross-Sectional Study.

PURPOSE: Accuracy in the diagnosis of HPV-associated oropharyngeal carcinoma (OPSCC) of a rapid, low-cost lateral flow immunochromatographic assay for detecting E6 oncoprotein of HPV-16 and HPV-18 was previously evaluated in a small pilot study. This cross-sectional study aimed to assess on a large case series the sensitivity and specificity of E6 oncoprotein as a diagnostic marker for HPV-associated carcinogenesis in OPSCC. METHODS: 137 consecutive patients with histologically confirmed OPSCC were enrolled in two hospitals in Northeast Italy. HPV status was determined by PCR for HPV DNA and p16INK4a immunohistochemistry on primary tumor biopsies. An OPSCC was defined as HPV-associated when double positive for high-risk HPV-DNA and p16INK4a overexpression in primary lesion. Cytological samples from primary tumors and metastatic lymph nodes were obtained and tested for HPV16/18 E6 oncoproteins using the lateral flow immunochromatographic assay, which requires between 90 and 120 min to provide a result. Diagnostic performances were calculated as percentage with confidence intervals (CI). RESULTS: Of the 137 OPSCC cases, 68 (49.6%) were HPV-associated, testing positive for both high-risk HPV-DNA and p16INK4a, with HPV16 predominating (82.4%). An average waiting time of 22 days was observed to obtain the results of p16INK4a and HPV-DNA after primary lesions biopsy. In patients with HPV16/18-associated OPSCC, the HPV16/18 E6 oncoprotein was detected in 59 out of 60 cytological samples from the primary lesion (sensitivity: 98.3%; 95% CI: 91.1-100%) and in 45 out of 51 cytological samples from lymph node metastases (sensitivity: 88.2%; 95% CI: 76.1-95.6%). The E6 oncoprotein assay showed a specificity of 100% in both primary tumors and lymph node metastases. CONCLUSION: The low-cost lateral flow immunochromatographic assay for detecting HPV16/18 E6 oncoproteins confirmed high accuracy for identifying HPV-associated OPSCC, particularly in primary tumors, suggesting its potential as a valuable diagnostic tool in clinical practice. Its rapid diagnostic capability could significantly accelerate the process of treatment decision-making, enhancing the timely management of patients.

Gold/DNA-Cu2+ Complex Nanozyme-Based Aptamer Lateral Flow Assay for Highly Sensitive Detection of Kanamycin.

Aptamer-based lateral flow analysis (Apt-LFAs) has promising applications in many fields. Nanozymes have demonstrated high potential in improving the performance of Apt-LFAs and have been increasingly utilized in recent studies. In this study, we developed a nanozyme-based Apt-LFA for the rapid and sensitive detection of kanamycin by using a novel dual-functionalized AuNPs@polyA-DNA/GpG-Cu2+ nanozyme as a nanoprobe. In the nanoprobe design, the polyA-cDNA strand can discriminate a kanamycin aptamer from the kanamycin/aptamer complex, and the GpG-Cu2+ complex can amplify the detection signal by catalyzing the chromogenic reaction. The nanozyme Apt-LFA can quantify kanamycin in the range of 1-250 ng/mL with an LOD of 0.08 ng/mL, which demonstrated a 4-fold sensitivity improvement and had a wider linear range than the conventional AuNP-based LFA. The Apt-LFA was successfully applied to the detection of kanamycin in honey with good recoveries. Our dual-functionalized AuNP nanoprobe is easily prepared and can be highly compatible with the conventional AuNP-DNA-based LFA platform; thus, it can be extended to the application of Apt-LFAs for other small molecules.

Transitions in Immunoassay Leading to Next-Generation Lateral Flow Assays and Future Prospects.

In the field of clinical testing, the traditional focus has been on the development of large-scale analysis equipment designed to process high volumes of samples with fully automatic and high-sensitivity measurements. However, there has been a growing demand in recent years for the development of analytical reagents tailored to point-of-care testing (POCT), which does not necessitate a specific location or specialized operator. This trend is epitomized using the lateral flow assay (LFA), which became a cornerstone during the 2019 pandemic due to its simplicity, speed of delivering results-within about 10 min from minimal sample concentrations-and user-friendly design. LFAs, with their paper-based construction, combine cost-effectiveness with ease of disposal, addressing both budgetary and environmental concerns comprehensively. Despite their compact size, LFAs encapsulate a wealth of technological ingenuity, embodying years of research and development. Current research is dedicated to further evolving LFA technology, paving the way for the next generation of diagnostic devices. These advancements aim to redefine accessibility, empower individuals, and enhance responsiveness to public health challenges. The future of LFAs, now unfolding, promises even greater integration into routine health management and emergency responses, underscoring their critical role in the evolution of decentralized and patient-centric healthcare solutions. In this review, the historical development of LFA and several of the latest LFA technologies using catalytic amplification, surface-enhanced Raman scattering, heat detection, electron chemical detections, magnetoresistance, and detection of reflected electrons detection are introduced to inspire readers for future research and development.

Use of a Novel Whole Blood Separation and Transport Device for Targeted and Untargeted Proteomics.

BACKGROUND: There is significant interest in developing alternatives to traditional blood transportation and separation methods, which often require centrifugation and cold storage to preserve specimen integrity. Here we provide new performance findings that characterize a novel device that separates whole blood via lateral flow then dries the isolated components for room temperature storage and transport. METHODS: Untargeted proteomics was performed on non-small cell lung cancer (NSCLC) and normal healthy plasma applied to the device or prepared neat. RESULTS: Significantly, proteomic profiles from the storage device were more reproducible than from neat plasma. Proteins depleted or absent in the device preparation were shown to be absorbed onto the device membrane through largely hydrophilic interactions. Use of the device did not impact proteins relevant to an NSCLC clinical immune classifier. The device was also evaluated for use in targeted proteomics experiments using multiple-reaction monitoring (MRM) mass spectrometry. Intra-specimen detection intensity for protein targets between neat and device preparations showed a strong correlation, and device variation was comparable to the neat after normalization. Inter-specimen measurements between the device and neat preparations were also highly concordant. CONCLUSIONS: These studies demonstrate that the lateral flow device is a viable blood separation and transportation tool for untargeted and targeted proteomics applications.

CRISPR/Cas and Argonaute-powered lateral flow assay for pathogens detection.

Pathogens contamination is a pressing global public issue that has garnered significant attention worldwide, especially in light of recent outbreaks of foodborne illnesses. Programmable nucleases like CRISPR/Cas and Argonaute hold promise as tools for nucleic acid testing owning to programmability and the precise target sequence specificity, which has been utilized for the development pathogens detection. At present, fluorescence, as the main signal output method, provides a simple response mode for sensing analysis. However, the dependence of fluorescence output on large instruments and correct analysis of output data limited its use in remote areas. Lateral flow strips (LFS), emerging as a novel flexible substrate, offer a plethora of advantages, encompassing easy-to-use, rapidity, visualization, low-cost, portability, etc. The integration of CRISPR/Cas and Argonaute with LFS, lateral flow assay (LFA), rendered a new and on-site mode for pathogens detection. In the review, we introduced two programmable nucleases CRISPR/Cas and Argonaute, followed by the structure, principle and advantages of LFA. Then diversified engineering detection pattens for viruses, bacteria, parasites, and fungi based on CRISPR/Cas and Argonaute were introduced and summarized. Finally, the challenge and perspectives involved in on-site diagnostic assays were discussed.

Clinical performance of a new lateral flow immunoassay for xylazine detection.

OBJECTIVES: Xylazine is a potent sedative used in veterinary medicine. Recently, recreational drugs such as fentanyl have been found to contain xylazine, increasing the risk of respiratory depression and death. Despite a similar presentation to opioid overdose, patients who ingest xylazine do not respond to treatment with Narcan. Therefore, rapid detection of xylazine could improve patient management and prevent adverse outcomes. METHODS: We evaluated the XYL500 one-step xylazine drug of abuse test for its ability to detect xylazine in 152 urine samples from patients on chronic opioid therapy for pain management or in treatment for substance use disorder. Results were compared to LC-MS/MS as the reference method. Precision, cross-reactivity, interference and stability studies were performed. RESULTS: Pooled patient samples were consistently negative or positive when tested five times on the same day and over three days of testing. The diagnostic sensitivity, specificity and accuracy of the XYL500 assay were 74, 98, and 82 % respectively, as compared with LC-MS/MS. XYL500 detected 77 of the 104 LC-MS/MS positive samples identified in our initial evaluation, including some that contained low levels of xylazine (n=8), <10 ng/mL. Minimal cross-reactivity with other opioid analgesics and commonly encountered drugs was seen with only one false positive result. Interferences by common urine contaminants were negligible. Specimens were stable up to 160 days refrigerated and up to 80 days at room temperature. CONCLUSIONS: XYL500 allows for rapid detection of xylazine, illustrating its utility in monitoring patients who ingested recreational drugs containing the additive, xylazine, and its potential to improve patient management.

Integrating lateral flow device with controllable gold in situ growth for sensitive detection of staphylococcal enterotoxin A in milk.

Gold nanoparticle-based lateral flow immunoassays (AuNP-LFIA) are widely used for pathogen monitoring to prevent foodborne illness outbreaks. However, conventional AuNP-LFIA exhibits poor sensitivity and limited quantitative capacity due to the low colorimetric signal intensity of AuNPs. Herein, we introduced a low-background gold in situ growth (GISG) strategy by lowering the pH of the growth solution to weaken the reducibility of hydroxylamine, thereby enhancing the sensitivity of AuNP-LFIA. Additionally, we developed a universal and manufacturable lateral flow device to streamline the GISG process. We applied this device to detect staphylococcal enterotoxin A (SEA), an exotoxin produced by Staphylococcus aureus. Under optimal conditions, the proposed device demonstrated superior practicality and excellent sensitivity for SEA detection, achieving a detection limit of 0.061 ng/mL with the total detection time of 37 min, showing 311 times more sensitive than the unamplified AuNP-LFIA. Furthermore, SEA detection in milk samples showed a strong correlation (R2 = 0.8845) with results obtained from a conventional ELISA kit. Therefore, this promising LFIA device offers a novel strategy with high sensitivity and practicality for in-field detection of Staphylococcus aureus and can be easily adapted for screening other foodborne pathogens.