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 %.

Carbapenemase-Producing Escherichia coli: Comparison of a Novel Rapid Lateral Flow Assay With the Polymerase Chain Reaction (PCR) and Antimicrobial Resistance Pattern.

Background In critically ill patients, carbapenems are often used as the last line of treatment. Carbapenem-resistant Enterobacterales (CRE) present an extreme challenge to treatment due to their resistance to various antibiotics. Optimal therapy for patients and infection control relies on the early and accurate diagnosis of these infections. The K.N.I.V.O. Detection K-Set is a newly developed immunological rapid test developed to identify the presence of carbapenemase in Gram-negative bacteria resistant to multiple drugs. Objectives This study evaluates a new K.N.I.V.O. Detection K-Set and its application for the rapid detection of isolates of multidrug-resistant Escherichia coli (MDR E. coli) that produce carbapenemase. This test aims to compare the test's performance to the polymerase chain reaction (PCR) method. Methods The study included 150 MDR E. coli isolates that were confirmed to be resistant to at least three groups of antibiotics, including carbapenems. The test followed the manufacturer's instructions using the K.N.I.V.O. Detection K-Set. The outcomes were compared with carbapenemase gene detection (bla-KPC, bla-NDM, bla-OXA-48, bla -VIM, and bla -IMP) using the PCR. The K-Set's sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) were calculated and studied. Results The K.N.I.V.O. Detection K-Set showed highly effective diagnostic performance with a 97.1% sensitivity, 97.5% specificity, 97.1% positive predictive value, and 98.7% negative predictive value. Seventy-eight of the 150 isolates were proven to be producers of carbapenemase, with 68 of those cases having an accurate identification. The remaining isolates were found to be non-producers. Within 15 minutes, the rapid test provided results. Conclusion The K.N.I.V.O. Detection K-Set is an effective and rapid method for identifying carbapenemase producers among MDR E. coli isolates. Its rapid processing time, associated with its high sensitivity and specificity, indicates that it can increase the effectiveness of diagnostic laboratories and better patient care in clinical settings. Implementing such rapid screenings could be vital for controlling the spread of drug-resistant infections and enhancing antimicrobial stewardship. This also ensures that patients receive timely treatment and effective care.

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.

On-site detection of OTA and AFB1 based on branched hybridization chain reaction coupled with lateral flow assay.

Mycotoxins are widely prevalent in various agricultural commodities, whose excessive consumption can pose significant risks to human health. In this study, we developed a facile mycotoxin detection platform based on branched hybridization chain reaction coupled with lateral flow assay. Ochratoxin A/Aflatoxin B1 bind to aptamers triggering the release of initiators, which leads to bHCR amplification and forms three-dimensional dendritic DNA nanostructures. Using the functionalized quantum dots as a fluorescent label, by leveraging smartphones and handheld ultraviolet lamps, the qualitative and quantitative detection of OTA and AFB1 can be achieved with a significantly enhanced sensitivity level, surpassing that of commercial test strips by 2-3 orders of magnitude. The visual detection limits for OTA and AFB1 were 30 pg/mL and 4 pg/mL, respectively. This approach eliminates the necessity for enzyme catalysis or the preparation and purification of antibodies and/or hapten, thereby reducing testing expenses and streamlining operational procedures. Moreover, substituting aptamer and nucleic acid sequences can effectively expand the scope of detection targets. Consequently, the as-proposed strategy exhibits great potential as a versatile technique, suitable for various analytical scenarios due to its sensitivity, accuracy, simplicity, and portability.

Perspectives in Aptasensor-Based Portable Detection for Biotoxins.

Biotoxins are pervasive in food and the environment, posing significant risk to human health. The most effective strategy to mitigate the risk arising from biotoxin exposure is through their specific and sensitive detection. Aptasensors have emerged as pivotal tools, leveraging aptamers as biorecognition elements to transduce the specificity of aptamer-target interactions into quantifiable signals for analytical applications, thereby facilitating the meticulous detection of biotoxins. When integrated with readily portable devices such as lateral flow assays (LFAs), personal glucose meters (PGMs), smartphones, and various meters measuring parameters like pH and pressure, aptasensors have significantly advanced the field of biotoxin monitoring. These commercially available devices enable precise, in situ, and real-time analysis, offering great potential for portable biotoxin detection in food and environmental matrices. This review highlights the recent progress in biotoxin monitoring using portable aptasensors, discussing both their potential applications and the challenges encountered. By addressing these impediments, we anticipate that a portable aptasensor-based detection system will open new avenues in biotoxin monitoring in the future.

Lateral flow assay sensitivity and signal enhancement via laser µ-machined constrains in nitrocellulose membrane.

Lateral flow assay (LFA) is a handful diagnostic technology that can identify severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and other common respiratory viruses in one strip, which can be tested at the point-of-care without the need for equipment or skilled personnel outside the laboratory. Although its simplicity and practicality make it an appealing solution, it remains a grand challenge to substantially enhance the colorimetric LFA sensitivity. In this work, we present a straightforward approach to enhance the sensitivity of LFA by imposing the flow constraints in nitrocellulose (NC) membranes via a number of vertical femtosecond laser micromachined microchannels which is important for prolonged specific binding interactions. Porous NC membrane surfaces were structured with different widths and densities µ-channels employing a second harmonic of the Yb:KGW femtosecond laser and sample XYZ translation over a microscope objective-focused laser beam. The influence of the microchannel parameters on the vertical wicking speed was evaluated from the video recordings. The obtained results indicated that µ-channel length, width, and density in NC membranes controllably increased the immunological reaction time between the analyte and the labeled antibody by 950%. Image analysis of the colorimetric indicators confirmed that the flow rate delaying strategy enhanced the signal sensitives by 40% compared with pristine NC LFA.

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.

Surface Functionalization of Gold Nanoparticles Using Alkyne Derivatives: Applications in Chemical Sensing.

Colloidal gold nanoparticles (AuNPs) are important nanomaterials for chemical sensing and therapeutics. For their application, it is vital to develop a reliable and robust surface functionalization method that can be applied to diverse functional molecules and offer better stability under harsh biological conditions. Currently, thiol (SH) is the most commonly used functional group for forming stable covalent bonds with AuNPs. However, thiolated molecules typically require complicated preparation procedures, are susceptible to oxidation, and are not compatible with many electrophiles and reducing groups. In this study, we report that surface functionalization of AuNPs can be achieved using alkyne derivatives, which exhibit several advantages over classical thiolation and peptide-bond methods, including straightforward preparation of alkyne derivatives, rapid and simple conjugation in buffers and complex media, higher conjugation efficiency, long-term stability, and resistance to decomposition under harsh conditions. Several alkynylated biotin and fluorescein derivatives are prepared, and the alkynylated-AuNPs are characterized using a lateral flow assay, gel electrophoresis, and spectroscopy techniques to investigate the conjugation efficiencies, size distributions, protein interaction properties, and binding mode of the Au-alkyne bond. We also demonstrate that alkynylated-AuNPs can be used for the sensitive detection of hydrogen peroxide and streptavidin proteins.

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.

Validation of Recombinase Polymerase Amplification with In-House Lateral Flow Assay for mcr-1 Gene Detection of Colistin Resistant Escherichia coli Isolates.

BACKGROUND/OBJECTIVES: The emergence of the mobilized colistin resistance 1 (mcr-1) gene, which causes colistin resistance, is a serious concern in animal husbandry, particularly in pigs. Although antibiotic regulations in many countries have prohibited the use of colistin in livestock, the persistence and dissemination of this plasmid-mediated gene require effective and rapid monitoring. Therefore, a rapid, sensitive, and specific method combining recombinase polymerase amplification (RPA) with an in-house lateral flow assay (LFA) for the mcr-1 gene detection was developed. METHODS: The colistin agar test and broth microdilution were employed to screen 152 E. coli isolates from pig fecal samples of five antibiotic-used farms. The established RPA-in-house LFA was validated with PCR for mcr-1 gene detection. RESULTS: The RPA-in-house LFA was completed within 35 min (20 min of amplification and 5-15 min on LFA detection) at 37 °C. The sensitivity, specificity, and accuracy were entirely 100% in concordance with PCR results. No cross-reactivity was detected with seven common pathogenic bacteria or other mcr gene variants. CONCLUSIONS: Therefore, the in-house RPA-LFA serves as a point-of-care testing tool that is rapid, simple, and portable, facilitating effective surveillance of colistin resistance in both veterinary and clinical settings, thereby enhancing health outcomes.

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.

Wheat Germ Agglutinin-Modified "Three-in-One" Multifunctional Probe Driven Broad-Spectrum and Flexible Immunochromatographic Diagnosis of viruses With High Sensitivity.

The conventional lateral flow assay (LFA) fails to the demands for the accurate screening of viruses as a result of its low sensitivity of colorimetric signal output and poor universality limited by antibody pairs. Here, a magnetically assisted dual-signal output LFA platform is developed for the ultrasensitive, universal, and flexible detection of viruses. A "three-in-one" multifunctional probe (MAuDQD) is prepared using a 180 nm Fe3O4 core to load numerous Au nanoparticles (NPs) and two layers of QDs, which can substantially improve the sensitivity of LFA through coupling with the effects of magnetic enrichment and colorimetric/fluorescent enhancement. Wheat germ agglutinin (WGA)-modified MAuDQD attained the broad-spectrum capture viral membrane proteins and the colorimetric/fluorescent dual-mode detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and monkeypox virus (MPXV) on the LFA strip. In the colorimetric mode, the target viruses detected directly, with the visual sensitivity reaching 0.1-0.5 ng mL-1 and the fluorescent mode supported quantitative analysis of SARS-CoV-2/MPXV with limits of detection decreasing to pg mL-1 level. Practicability of the MAuDQD@WGA-LFA is verified through the detection of 33 real clinical samples, showing the proposed assay has a great potential to become a sensitive, accurate, and universal tool for on-site monitoring of viral infections.

The development of RT-RPA and CRISPR-Cas12a based assay for sensitive detection of Hirame novirhabdovirus.

Hirame novirhabdovirus (HIRRV) is a highly pathogenic fish virus that poses a significant threat to the farming of a variety of economic fish. Due to no commercial vaccines and effective drugs available, sensitive and rapid detection of HIRRV at latent and early stages is important and critical for the control of disease outbreaks. However, most of the current methods for HIRRV detection have a large dependence on instruments and operations. For better detection of HIRRV, we have established a detection technology based on the reverse transcription and recombinase polymerase amplification (RT-RPA) and CRISPR/Cas12a to detect the N gene of HIRRV in two steps. Following the screening of primer pairs, the reaction temperature and time for RPA were optimized to be 40 °C and 32min, respectively, and the CRISPR/Cas12a reaction was performed at 37 °C for 15min. The whole detection procedure including can be accomplished within 1 h, with a detection sensitivity of about 8.7 copies/µl. The detection method exhibited high specificity with no cross-reaction to the other Novirhabdoviruses IHNV and VHSV, allowing naked-eye color-based interpretation of the detection results through lateral flow (LF) strip or fluorescence under violet light. Furthermore, the proliferation dynamic of HIRRV in the spleen of flounder were comparatively detected by LF- and fluorescence-based RPA-CRISPR/Cas12a assay in comparison to qRT-PCR at the early infection stage, and the results showed that the viral positive signal could be firstly detected by the two RPA-CRISPR/Cas12a based methods at 6 hpi, and then by qRT-PCR at 12 hpi. Overall, our results demonstrated that the developed RPA-CRISPR/Cas12a method is a stable, specific, sensitive and more suitable in the field, which has a significant effect on the prevention of HIRRV. RT-RPA-Cas12a-mediated assay is a rapid, specific and sensitive detection method for visual and on-site detection of HIRRV, which shows a great application promise for the prevention of HIRRV infections.

The significance of low titer serum cryptococcal antigen testing from 2017 to 2023 performed in a tertiary care center.

Several false positive low serum cryptococcal antigen (SCrAg) reports by lateral flow assay (LFA) were identified in late 2016 at our tertiary care hospital. After the recall and correction of the problem in the reagent, we studied the significance of SCrAg LFA ≤ 1:10 from January 2017 to October 2023. Of 20 patients with 31 samples of SCrAg LFA ≤ 1:10, 14 patients (70%) were classified as true positives, four (20%) were indeterminate, and only two (10%) patients were false positives. If a new SCrAg LFA ≤ 1:10 is detected, it should be repeated, and additional workup should be pursued.

We studied the significance of low serum cryptococcal antigen (SCrAg) titer lateral flow assay (LFA) ≤ 1:10 from January 2017 to October 2023. Of 20 patients with SCrAg LFA ≤ 1:10, only two patients (10%) were false positives. If a new SCrAg ≤ 1:10 is detected, it should be repeated, and additional workup should be done.

The relationship between lung CT features and serum cryptococcal antigen titers in localized pulmonary cryptococcosis patients.

BACKGROUND: To explore the associations of computed tomography (CT) image features with the serum cryptococcal antigen (CrAg) titers measured by the lateral flow assay (LFA) in localized pulmonary cryptococcosis patients. METHODS: A retrospective analysis of patients with pathologically confirmed pulmonary cryptococcosis admitted to the First Affiliated Hospital of Xiamen University from January 2016 to December 2022 was performed. Clinical data, CT results, serum CrAg-LFA test results, and follow-up data were collected and analyzed. RESULTS: A total of 107 patients with localized pulmonary cryptococcosis were included, of which 31 had a single lesion in chest CT and the other 76 had multiple lesions. The positivity rate was (94.74% vs 64.52%) and titers of serum CrAg-LFA (1.77 ± 0.87 vs 0.91 ± 0.98) in the multiple lesion group were higher than those in the single lesion group, respectively. Multivariate linear regression analysis showed that the serum CrAg titers were positively associated with the number of lesions (ß, 0.08; 95% CI, 0.05 to 0.12) and the lesion size (ß, 0.40; 95% CI, 0.31 to 0.50) after adjusting other covariates. The serum CrAg-LFA titers of 60 pulmonary cryptococcosis patients showed a decreasing trend with the reduction in pulmonary lesion size after effective therapy. CONCLUSION: In pulmonary cryptococcosis patients, the number and size of lung lesions are positively correlated with the titers of the serum CrAg-LFA test. The CrAg-LFA test could be a useful tool for the diagnosis, severity assessment, and therapeutic monitoring of localized pulmonary cryptococcosis patients.

Hantavirus: Preliminary assessment of lateral flow immune assay prototypes to detect IgM and IgG antibodies.

Three lateral flow immunoassay prototypes developed to detect IgM, IgG and IgM/IgG antibodies against Hantavirus were evaluated. A total of 163 samples were tested: 10 from Hantavirus patients, 103 from related diseases, and 50 from healthy controls. The prototypes exhibited 100 % sensitivity, 97.5 % to 99.3 % specificity, indicating promising improved diagnosis.

Development and evaluation of two rapid lateral flow assays for on-site detection of African swine fever virus.

Introduction: African swine fever (ASF) is a lethal and highly contagious transboundary animal disease with the potential for rapid international spread. In the absence of a widely available and definitively proven vaccine, rapid and early detection is critical for ASF control. The quick and user-friendly lateral flow assay (LFA) can easily be performed by following simple instructions and is ideal for on-site use. This study describes the development and validation of two LFAs for the rapid detection of ASF virus (ASFV) in pig serum. Methods: The highly immunogenic antigens (p30 and p72) of ASFV Georgia 2007/1 (genotype II) were expressed in plants (Nicotiana benthamiana) and were used to immunize BALB/c mice to generate specific monoclonal antibodies (mAbs) against the p30 and p72 proteins. mAbs with the strongest binding ability to each protein were used to develop p30_LFA and p72_LFA for detecting the respective ASFV antigens. The assays were first evaluated using a spike-in test by adding the purified p30 or p72 protein to a serum sample from a healthy donor pig. Further validation of the tests was carried out using serum samples derived from experimentally infected domestic pigs, field domestic pigs, and feral pigs, and the results were compared with those of ASFV real-time PCR. Results: p30_LFA and p72_LFA showed no cross-reaction with common swine viruses and delivered visual results in 15 min. When testing with serially diluted proteins in swine serum samples, analytical sensitivity reached 10 ng/test for p30_LFA and 20 ng/test for p72_LFA. Using real-time PCR as a reference, both assays demonstrated high sensitivity (84.21% for p30_LFA and 100% for p72_LFA) with experimentally ASFV-infected pig sera. Specificity was 100% for both LFAs using a panel of PBS-inoculated domestic pig sera. Excellent specificity was also shown for field domestic pig sera (100% for p30_LFA and 93% for p72_LFA) and feral pig sera (100% for both LFAs). Conclusion: The results obtained in this study suggest that p30_LFA and p72_LFA hold promise as rapid, sensitive, user-friendly, and field-deployable tools for ASF control, particularly in settings with limited laboratory resources.

Enhancing Colorimetric Detection of Nucleic Acids on Nitrocellulose Membranes: Cutting-Edge Applications in Diagnostics and Forensics.

This study re-introduces a protein-free rapid test method for nucleic acids on paper based lateral flow assays utilizing special multichannel nitrocellulose membranes and DNA-Gold conjugates, achieving significantly enhanced sensitivity, easier protocols, reduced time of detection, reduced costs of production and advanced multiplexing possibilities. A protein-free nucleic acid-based lateral flow assay (NALFA) with a limit of detection of 1 pmol of DNA is shown for the first time. The total production duration of such an assay was successfully reduced from the currently known several days to just a few hours. The simplification and acceleration of the protocol make the method more accessible and practical for various applications. The developed method supports multiplexing, enabling the simultaneous detection of up to six DNA targets. This multiplexing capability is a significant improvement over traditional line tests and offers more comprehensive diagnostic potential in a single assay. The approach significantly reduces the run time compared to traditional line tests, which enhances the efficiency of diagnostic procedures. The protein-free aspect of this assay minimizes the prevalent complications of cross-reactivity in immunoassays especially in cases of multiplexing. It is also demonstrated that the NALFA developed in this study is amplification-free and hence does not rely on specialized technicians, nor does it involve labour-intensive steps like DNA extraction and PCR processes. Overall, this study presents a robust, efficient, and highly sensitive platform for DNA or RNA detection, addressing several limitations of current methods documented in the literature. The advancements in sensitivity, cost reduction, production time, and multiplexing capabilities mark a substantial improvement, holding great potential for various applications in diagnostics, forensics, and molecular biology.

Innovations in one-step point-of-care testing within microfluidics and lateral flow assays for shaping the future of healthcare.

Point-of-care testing (POCT) technology, using lateral flow assays and microfluidic systems, facilitates cost-effective diagnosis, timely treatment, ongoing monitoring, and prevention of life-threatening outcomes. Aside from significant advancements demonstrated in academic research, implementation in real-world applications remains frustratingly limited. The divergence between academic developments and practical utility is often due to factors such as operational complexity, low sensitivity and the need for trained personnel. Taking this into consideration, our objective is to present a critical and objective overview of the latest advancements in fully integrated one-step POCT assays for home-testing which would be commercially viable. In particular, aspects of signal amplification, assay design modification, and sample preparation are critically evaluated and their features and medical applications along with future perspective and challenges with respect to minimal user intervention are summarized. Associated with and very important for the one-step POCT realization are also readout devices and fabrication processes. Critical analysis of available and useful technologies are presented in the SI section.