Lateral Flow Assay Research Articles

A ERA/Cas12f1_ge4.1 biosensor for rapid, sensitive, and cost-effective detection of Chlamydia psittaci via fluorescence and lateral flow assays.

Rapid and accurate detection of Chlamydia psittaci, the causative agent of psittacosis, is crucial for both human and animal health but presents significant challenges, particularly in grassroots health institutions. Our previous PDTCTR fluorescence sensing platform, which combined the engineered Cas12f1_ge4.1 system with recombinase polymerase amplification (RPA), significantly enhanced detection efficiency. However, its requirement for specialized equipment, costly RPA reagents, and absence of visual output restricted its practical application in such environments. To address these limitations, we developed the ERA/Cas12f1_ge4.1 system, integrating Cas12f1_ge4.1 with the cost-effective Enzymatic Recombinase Amplification (ERA). This system enables sensitive detection of Chlamydia psittaci double-stranded DNA within 50min through both fluorescence and colloidal gold lateral flow assay strips. The platform achieves detection limits of 10 copies/μL for fluorescence and 100 copies/μL for lateral flow. Clinical validation involving 93 parrot samples demonstrated high performance in both detection modes. Fluorescence detection achieved 95.4% sensitivity, 100% specificity, a 100% positive predictive value (PPV), and a 90.3% negative predictive value (NPV). Meanwhile, the lateral flow assay exhibited 92.3% sensitivity, 100% specificity, 100% PPV, and an 84.8% NPV. The ERA/Cas12f1_ge4.1 system offers a rapid, accurate, cost-effective, and visually interpretable diagnostic tool suitable for both laboratory and community health centers. This advancement holds significant potential for improving psittacosis diagnosis and control, particularly in resource-limited environments.

Rapid and visual detection of hepatitis B virus using the ERA/Cas12f1_ge4.1-based lateral flow assay system.

Hepatitis B virus (HBV) is a major pathogen posing significant challenges to global public health, making early diagnosis critical for preventing severe liver diseases. We previously developed a fluorescent biosensor named PAM-dependent dsDNA Target-activated Cas12f1 Trans Reporter (PDTCTR). However, its reliance on specialized fluorescence equipment and lack of visual readout limited its application in resource-limited settings. To address these limitations, we developed a lateral flow assay (LFA) that integrates enzymatic recombinase amplification (ERA) with the Cas12f1_ge4.1 system. This approach enables the specific amplification of the HBV target gene through ERA and leverages the precise cleavage activity of Cas12f1_ge4.1 for enhanced signal amplification. The entire detection process is completed within 50 minutes, with results readily interpretable through visual inspection. The assay achieves a minimum detection limit of 100 copies per μL and demonstrates high specificity, showing no cross-reactivity with related viruses. In a validation study involving 71 clinical samples, the system achieved a sensitivity of 94.23%, specificity of 100%, and a kappa value of 0.90 compared to quantitative PCR (qPCR), indicating high reliability. This method thus shows promise as an effective tool for early HBV diagnosis, particularly suited for rapid, on-site detection in resource-constrained environments, and holds broad potential for diverse applications.

A Fully Integrated ICP-Enriched and Nanozyme-Catalyzed Lateral Flow Assay for cfDNA Detection in Whole Blood.

Rapid and sensitive detection of Epstein-Barr virus cell-free DNA (EBV cfDNA) is crucial for early diagnosis and monitoring of nasopharyngeal carcinoma (NPC), but accessibility to screening is limited by complicated and costly conventional DNA isolation and purification approaches. Here, a fully integrated ion concentration polarization (ICP)-enriched and nanozyme-catalyzed lateral flow assay (ICP-cLFA) is developed, enabling total analysis of EBV cfDNA in whole blood samples, with DNA isolation, pre-concentration, and amplification performed on a microfluidic chip, consequently providing the signal readout within 75 min. Specifically, ICP preconcentration and amplification steps, together with target recognition catalyzed by a platinum-decorated mesoporous gold nanosphere (MGNS@Pt) nanozyme, result in an ultralow detection limit of 4 aM in standard cfDNA samples and 100 aM in whole blood from NPC-bearing rats. The high sensitivity and specificity of the ICP-cLFA suggest strong potential for cfDNA screening in resource-limited clinical and field applications.

Colored Cellulose Nanoparticles with High Stability and Easily Modified Surface for Accurate and Sensitive Multiplex Lateral Flow Assay.

Decentralized testing using multiplex lateral flow assays (mLFAs) to simultaneously detect multiple analytes can significantly enhance detection efficiency, reduce cost and time, and improve analytic accuracy. However, the challenges, including the monochromatic color of probe particles, interference between different test lines, and reduced specificity and sensitivity, severely hinder mLFAs from wide use. In this study, we prepared polydopamine (PDA)-coated dyed cellulose nanoparticles (dCNPs@P) with tunable colors as the probe for mLFAs. Cellulose nanoparticles (CNPs) were synthesized with uniform spheric shapes and tunable sizes. Dye molecules were loaded on CNPs via a mature industrial dyeing method. The PDA shell provided a reactive surface for facile receptor conjugation and protected the dye from leaking. dCNPs@P displayed a higher signal intensity than gold nanoparticles. They also had higher stability to tolerate salt and varied pH. The dCNP@P-based mLFAs were successfully applied to detect multiple mycotoxins in cereals and determine the levels of inflammatory biomarkers to differentiate between viral and bacterial infections. The tests represented high specificity and accuracy and were more sensitive than the tests using gold nanoparticles. The quantified detection was accessible by measuring the intensities of the colorimetric or photothermal signals. Overall, this study provides a practical system solution for mLFAs based on colored dCNPs@P.

Innovative Ricin Toxin Detection: Unraveling Apurinic/Apyrimidinic Lyase Activity and Developing Fluorescence Sensors.

Ricin toxin (RT) is a potential bioterrorism agent because of its high potency, extremely small lethal dose, ease of preparation, and notable stability. Therefore, a portable method is urgently required to efficiently detect and determine the presence of toxicity of RT and evaluate its potency for public health monitoring and counter-bioterrorism responses. Currently, enzyme-based assays for detecting RT mainly focus on its N-glycosidase activity. In this study, we demonstrated that RT exhibits apurinic/apyrimidinic (AP) lyase activity using several methods. Characterization of the enzyme reaction and kinetics revealed that AP lyase activity is optimal at 59 °C and pH 4.0. This activity is highly pH-sensitive, remaining active between pH 3.0 and pH 4.6. Furthermore, we developed a portable fluorescence-based lateral flow assay (FLFA) that detects RT much faster than existing assays based on its N-glycosidase activity. Moreover, this assay can efficiently detect RT at nanogram levels from complex matrix samples within 1.5 h while simultaneously determining its biological activity. In conclusion, the discovery of the AP lyase activity of RT and the development of FLFA represent novel approaches for studying the enzymatic profiles of other ribosome-inactivating proteins.

Novel Isothermal Amplification Integrated with CRISPR/Cas13a and Its Applications for Ultrasensitive Detection of SARS-CoV-2.

We herein developed an ultrasensitive and rapid strategy to identify genomic nucleic acids by integrating a clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 13a (Cas13a) into our recently developed isothermal technique, nicking and extension chain reaction system-based amplification (NESBA) reaction. In this technique, named CESBA, the NESBA reaction isothermally produces a large amount of RNA amplicons from the initial target genomic RNA (gRNA). The RNA amplicons bind to the crispr RNA (crRNA) and activate the collateral cleavage activity of Cas13a, which would then cleave the reporter probe nearby, consequently producing the final signals. Based on this design principle, we successfully detected SARS-CoV-2 gRNA as a model target very sensitively down to even a single copy (0.05 copies/μL) in both fluorescence- and lateral flow assay (LFA)-based modes with excellent specificity against other human coronaviruses (H-CoVs). We further validated the clinical applicability of CESBA by testing the 20 clinical samples with 100% clinical sensitivity and specificity. This work represents a potent and innovative strategy for the identification of genomic nucleic acids in molecular diagnostics, delivering exceptional levels of sensitivity.

Lateral flow assay as radiological prognosis factor of pulmonary cryptococcosis: a single center retrospective study in China

Lateral flow assay as radiological prognosis factor of pulmonary cryptococcosis: a single center retrospective study in China

Simultaneous or separate detection of heavy metal ions Hg2+ and Ag+ based on lateral flow assays.

Alateral flow assay (LFA) was developed for the simultaneous or separate detection of mercury ion and silver ion based on isothermal nucleic acid amplification. T-Hg2+-T and C-Ag+-C were utilized in the isothermal nucleic acid amplification strategy to form specific complementary base pairs. Under the action of KF polymerase and endonuclease Nt.BbvCl, trace amounts of Hg2+ and Ag+ were converted to Product-Hg2+ and Product-Ag+ as bridges. Biotin-labeled capture strands (Biotin-DNA1, Biotin-DNA1, and Biotin-DNA3) immobilized on the test strips could capture the Au NPs-DNA nanoprobes by hybridization with the generated bridge products for monitoring of two heavy metal ions simultaneously or separately. Theassembly method of DNAs on the nanoprobes was explored, and the DNA sequences on the nanoprobes were designed so that only one kind of DNA strand was used to bind to all three capture DNA strands on the C, T1, and T2 bands. Under optimal detection conditions, the limits of detection for Hg2+ and Ag+ were 2.19 and 5.41pM, respectively, with desired selectivity and reproducibility.

Recent advances in rapid detection of Helicobacter pylori by lateral flow assay.

Infection with H. pylori (Helicobacter pylori) is the most prevalent human infection worldwide and is strongly associated with many gastrointestinal disorders, including gastric cancer. Endoscopy is mainly used to diagnose H. pylori infection in gastric biopsies. However, this approach is invasive, time-consuming and expensive. On the other hand, serology-based methods can be considered as a non-invasive approach to detecting H. pylori infection. The LFA (lateral flow assay) serves as a rapid point-of-care diagnostic tool. This paper-based platform facilitates the detection and quantification of analytes within human fluids such as blood, serum and urine. Due to ease of production, rapid results, and low costs, LFAs have a wide application in clinical laboratories and hospitals. In this comprehensive review, we examined LFA-based approaches for detection of H. pylori infection from human fluids and compare them with other high-sensitivity methods like ELISA (Enzyme-linked immunosorbent assay). Furthermore, we reviewed methods to elevate LFA sensitivity during H. pylori infection including, CRISPR/Cas system and isothermal amplification approaches. The development and optimization of novel labeling agents such as nanozyme to enhance the performance of LFA devices in detecting H. pylori were reviewed. These innovations aim to improve signal amplification and stability, thereby increasing the diagnostic accuracy of LFA devices. A combination of advances in LFA technology and molecular insight could significantly improve diagnostic accuracy, resulting in a significant improvement in clinical and remote diagnostic accuracy.

Ultra-Sensitive Aptamer-Based Diagnostic Systems for Rapid Detection of All SARS-CoV-2 Variants.

The emergence of numerous SARS-CoV-2 variants, characterized by mutations in the viral RNA genome and target proteins, has presented challenges for accurate COVID-19 diagnosis. To address this, we developed universal aptamer probes capable of binding to the spike proteins of SARS-CoV-2 variants, including highly mutated strains like Omicron. These aptamers were identified through protein-based SELEX using spike proteins from three key variants (D614G-substituted Wuhan-Hu-1, Delta, and Omicron) and virus-based SELEX, known as viro-SELEX. Leveraging these universal aptamers, we created a highly sensitive lateral flow assay (LFA) and an ultra-sensitive molecular diagnostic platform that integrates a novel rapid PCR technique, enabling fast and reliable detection across all SARS-CoV-2 variants.

Evaluating Methods for Aflatoxin B1 Monitoring in Selected Food Crops Within Decentralized Agricultural Systems.

Aflatoxin B1 (AFB1) contamination of food crops pose severe public health risks, particularly in decentralized agricultural systems common in low-resource settings. Effective monitoring tools are critical for mitigating exposure, but their adoption is limited by barriers such as cost, infrastructure, and technical expertise. The objectives of this study were: (1) to evaluate common AFB1 detection methods, including enzyme-linked immunosorbent assays (ELISA) and lateral-flow assays (LFA), validated via high-performance liquid chromatography (HPLC), focusing on their suitability for possible applications in decentralized, low-resource settings; and (2) to conduct a barriers-to-use assessment for commonly available AFB1 detection methods and their applicability in low-resource settings. Among four ELISA kits, the AgraQuant Aflatoxin B1 2/50 ELISA Kit demonstrated the highest accuracy and precision, reliably quantifying AFB1 in maize and tortillas across 5-150 ppb with minimal cross-reactivity. For LFA, a smartphone-based algorithm achieved a high presence/absence accuracy rate of 84% but struggled with concentration prediction. The barriers-to-use analysis highlighted the practicality of low-cost tools like moisture readers for field screening but underscored their qualitative limitations. Advanced methods like HPLC and LC-MS offer greater precision but remain impractical due to their high costs and infrastructure requirements, suggesting a potential role for adapted ELISA or LFA methods as confirmatory approaches. These findings support the development of multi-tiered frameworks integrating affordable field tools with regional or centralized confirmatory testing. Addressing systemic barriers through capacity building, partnerships, and improved logistics will enhance AFB1 monitoring in decentralized systems, protecting public health in vulnerable communities.

Multiplex lateral flow test sensitivity and specificity in detecting influenza A, B and SARS-CoV-2 in adult patients in a UK emergency department

BackgroundRapid identification of individuals with acute respiratory infections is crucial for preventing nosocomial infections. For rapid diagnosis, especially in EDs, lateral flow devices (LFDs) are a convenient, inexpensive option with...

DNA Tetrahedron Reformative Lateral Flow Assay for Improved Detection Sensitivity and Anti-Interference.

The lateral flow assay is a strip-based analytical method for the portable and convenient detection of analytes of interest. It has the advantages of visual observation, autonomous sample flow, fast coloration time, minimal tedious operation procedures, and reliance on specialized instruments. However, the rough surface of the nitrocellulose membrane renders it difficult for the immobilized nucleic acids to remain in an ordered arrangement, and the immobilized nucleic acids are also liable to be digested in a complex matrix, inducing limited sensitivity and anti-interference. In this work, we demonstrate that the decoration of DNA nanostructures on lateral flow strips can improve assay sensitivity and anti-interference in comparison with commonly studied single-stranded DNA-disposed strips. DNA nanostructures enable probes to be more orderly and arranged on the strip and provide protection. Using adenosine 5'-triphosphate (ATP) as an analyte, a DNA tetrahedron reformative lateral flow strip has increased sensitivity and improved reliability in detection. The DNA nanostructure-decorated lateral flow strip is further successfully applied for ATP detection in real samples, such as bacterium testing and tableware cleanliness checking, by detection of the ATP content therein.

Substructure-Specific Antibodies Against Fentanyl Derivatives.

Structural variants of the synthetic opioid fentanyl are a major threat to public health. Following an investigation showing that many derivatives are poorly detected by commercial lateral flow and related assays, we created hapten conjugate vaccines using an immunogenic virus-like particle carrier and eight synthetic fentanyl derivatives designed to mimic the structural features of several of the more dangerous analogues. Immunization of mice elicited strong antihapten humoral responses, allowing the screening of hundreds of hapten-specific hybridomas for binding strength and specificity. A panel of 13 monoclonal IgG antibodies were selected, each showing a different pattern of recognition of fentanyl structural variations, and all proving to be highly efficient at capturing parent fentanyl compounds in competition ELISA experiments. These results provide antibody reagents for assay development as well as a demonstration of the power of the immune system to create binding agents capable of both broad and specific recognition of small-molecule targets.

Non-Invasive Point-of-Care Detection of Methamphetamine and Cocaine via Aptamer-Based Lateral Flow Test.

Drug abuse is a major public problem in the workplace, traffic, and forensic issues, which requires a standardized test device to monitor on-site drug use. For field testing, the most important requirements are portability, sensitivity, non-invasiveness, and quick results. Motivated by this problem, a point of care (POC) test based on lateral flow assay (LFA) was developed for the detection of cocaine (COC) and methamphetamine (MET) in saliva which has been selected as the matrix for this study due to its rapid and non-invasive collection process. In the design strategy of an LFA test, the use of gold nanoparticles (AuNPs) with strong optical properties has been combined with the advantages of selecting aptamers under in vitro conditions, making it a highly specific and stable recognition probe for the detection of small molecules in saliva. The developed aptamer-based LFA in a competitive format, was able to detect COC and MET in synthetic saliva at concentrations as low as 5.0 ng/mL. After analytical performance studies, the test system also detected COC and MET in real patient samples, which was verified by chromatographic methods.

Nanobody-Based Lateral Flow Immunoassay for Rapid Antigen Detection of SARS-CoV-2 and MERS-CoV Proteins.

The COVID-19 pandemic has highlighted the critical need for pathogen detection methods that offer both low detection limits and rapid results. Despite advancements in simplifying and enhancing nucleic acid amplification techniques, immunochemical methods remain the preferred methods for mass testing. These methods eliminate the need for specialized laboratories and highly skilled personnel, making home testing feasible. Here, we developed nanobody-based lateral flow assays (LFAs) for the rapid detection of SARS-CoV-2 and MERS-CoV in single and dual formats as point-of-care diagnostic tools. The developed LFAs are highly sensitive and successfully detected analytes at clinically relevant diagnostic cutoff values. Additionally, our results confirmed that the LFAs have a long shelf life and can be produced cost-effectively and with ease.

Quantitative determination of leptin hormone using gold nanoparticle-based lateral flow assay

A lateral flow assay (LFA) has been developed that can be used in point-of-care (PoC) use for the sensitive determination of leptin hormone. The limit of detection value was 0.158 ng/mL and the limit of quantification value was 0.479 ng/mL for leptin hormone. The stability studies showed that the assay response was equal to the initial value even after 6 months. LFA accuracy tests performed in commercial artificial serum samples were compared with the enzyme-linked immunosorbent assay (ELISA). The obtained recovery values between 95 and 110% show that the developed LFA can be used for quantitative determination of leptin hormone. As the result of the study, a fast and disposable assay was developed for the determination of leptin hormone, which can be performed with a small amount of sample in less than 15 min without the need for long-term analysis stages, qualified personnel, expensive equipment and devices.Graphical

Sample-to-answer microfluidic device towards the point-of-need detection of Staphylococcus aureus enterotoxin genes in ruminant milk.

Milk is commonly screened both for indicators of animal disease and health, but also for foodborne hazards. Included in these analyses is the detection of Staphylococcus aureus, that can produce an enterotoxin, causing staphylococcal food poisoning (SFP), which often leads to sudden onset of significant gastrointestinal symptoms in humans. Epidemiological data on SFP are limited, particularly in low- and middle-income countries. Many conventional assays for the detection of staphylococcal enterotoxins rely on the detection of the genes coding for them, either directly in food samples or after bacterial culture. Currently, many of the nucleic acid-based methods used require specific expertise and equipment, whilst bacterial culture takes 24-48 hours; both are contributory factors that limit efforts either during food safety emergencies or routine screening. Here we present the development of a "sample-to-answer" isothermal nucleic acid loop-mediated amplification (LAMP) assay in a microfluidic device for the detection of Staphylococcus aureus enterotoxin genes in ruminant milk. A multiplex LAMP assay targeting two of the most prevalent S. aureus enterotoxin-encoding genes (A and B) was integrated into a microfluidic device combining simple 1 : 10 dilution for sample preparation and a lateral flow assay for easy readout. We achieved a limit of detection of 104 colony forming units per ml in spiked cow and goat milk samples, an order of magnitude more sensitive than the European recommendation for the maximum allowable presence of coagulase-positive staphylococci in raw milk. The assay showed no cross-reactivity in detecting other tested non-enterotoxigenic S. aureus strains or associated foodborne pathogens. The test integrated the simplicity of use of microfluidic devices with the sensitivity, specificity and rapidity of a nucleic acid-based assay, and a simple lateral flow readout to provide an appropriate device to ensure the safety of milk for human consumption. To illustrate its potential for point-of-need practical applications, the test was performed in agricultural settings in rural Turkey in a limited feasibility exercise.

Progression of the faecal microbiome in preweaning dairy calves that develop cryptosporidiosis.

Cryptosporidiosis is a diarrheal disease that commonly affects calves under 6 weeks old. The causative agent, Cryptosporidium parvum, has been associated with the abundance of specific taxa in the faecal microbiome during active infection. However, the long-term impact of these microbiome shifts, and potential effects on calf growth and health have not yet been explored in depth. Three hundred and forty-six (346) calves from three dairy farms had one faecal swab collected during the first week of life (W1). Thereafter, sampled calves were monitored for diarrhoeal disease and those that suffered a diarrhoea event were tested for C. parvum by lateral flow testing (LFT). Calves that experienced diarrhoea and tested positive for C. parvum by LFT were assigned to the Cryptosporidium-positive (Cp+) group (n = 32). Matched healthy (H) controls with no history of diarrhoea were selected from the remaining cohort (n = 33). The selected subset of calves (n = 65) was observed until weaning, collecting a faecal swab, at approximately Week 5 (W5) and Week 10 (W10) after birth, resulting in a total of 191 samples (W1; n = 65, W5; n = 64, W10; n = 62). 16S rRNA gene amplicon sequencing was performed on all extracted samples. Analysis of the longitudinal microbiome showed significant changes in the microbial diversity and composition across all three time-points. Whilst Firmicutes were elevated in the Cp+ group at W5 compared to the H group, no other significant differences were detected between H and Cp+ groups. Whilst the core microbiota showed some taxa were exclusive to each group, the role of these taxa in health and disease has yet to be determined. Antibiotics were also found to have an impact on the relative abundance of some taxa. Though healthy calves received a significantly higher body condition score than Cp+ calves at W5, the difference did not reach significance at W10, suggesting that Cp+ calves may catch up to their healthy counterparts once the infection has resolved. The findings of this study illustrated the changes in the microbial diversity and composition during the preweaning period in dairy calves. The results also indicated that the faecal microbiome is not predictive of cryptosporidiosis and implied that cryptosporidiosis doesn't cause long-term gut dysbiosis. This study furthered our understanding of the parasite-microbiome relationship and its impact on the bovine host.

Machine Learning-Based Quantification of Lateral Flow Assay Using Smartphone-Captured Images.

Lateral flow assay has been extensively used for at-home testing and point-of-care diagnostics in rural areas. Despite its advantages as convenient and low-cost testing, it suffers from poor quantification capacity where only yes/no or positive/negative diagnostics are achieved. In this study, machine learning and deep learning models were developed to quantify the analyte load from smartphone-captured images of the lateral flow assay test. The comparative analysis identified that random forest and convolutional neural network (CNN) models performed well in classifying the lateral flow assay results compared to other well-established machine learning models. When trained on small-size images, random forest models excelled CNN models in image classification. Contrarily, CNN models outperformed random forest models in classifying noisy images.