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Salmonella species are prominent foodborne microbial pathogens transmitted through contaminated food or water and pose a significant threat to human health. Accurate and rapid point-of-care (POC) diagnosis is gaining attention in effectively preventing outbreaks of foodborne disease. However, the presence of dead bacteria can interfere with an accurate diagnosis, necessitating the development of methods for the rapid, simple, and efficient detection of viable bacteria only. Herein, we used an improved propidium monoazide (PMAxx) to develop a nucleic acid lateral flow (NALF) assay based on recombinase polymerase amplification (RPA) to differentiate viable Salmonella Typhimurium. We selected an RPA primer set targeting the invA gene and designed a probe for NALF. RPA-based NALF was optimized for temperature (30–43 °C), time (1–25 min), and endonuclease IV concentration (0.025–0.15 unit/µL). PMAxx successfully eliminated false-positive results from dead S. Typhimurium, enabling the accurate detection of viable S. Typhimurium with a detection limit of 1.11 × 102 CFU/mL in pure culture. The developed method was evaluated with spiked raw chicken breast and milk with analysis completed within 25 min at 39 °C. This study has potential as a tool for the POC diagnostics of viable foodborne pathogens with high specificity, sensitivity, rapidity, and cost-effectiveness. [ABSTRACT FROM AUTHOR]

Abstract Fluorescent‐magneto nanoemitters have gained considerable attention for their applications in mechanical controlling‐assisted optical signaling. However, the incompatibility between magnetic and fluorescent components often leads to functional limitations in traditional magneto@fluorescence nanostructure. Herein, we introduce a new compact‐discrete spatial arrangement on a “fluorescence@magneto” core–shell nanostructure consisting of a close‐packed aggregation‐induced emission luminogen (AIEgen) core and a discrete magnetic shell. This structural design effectively eliminates the optical and magnetic interferences between the dual components by facilitating AIEgens loading in core region and reducing the magnetic feeding amount through effective exposure of the magnetic units. Thereby, the resulting magneto‐AIEgen nanoparticle (MANP) demonstrates “win‐win” performances: (i) high fluorescent intensity contributed by AIEgens stacking‐enhanced photoluminescence and reduced photons loss from the meager magnetic shell; (ii) marked magnetic activity due to magneto extraposition‐minimized magnetic shielding. Accordingly, the dual functions‐retained MANP provides a proof of concept for construction of an immunochromatographic sensing platform, where it enables bright fluorescent labeling after magnetically enriching and separating procalcitonin and lipoarabinomannan in clinical human serum and urine, respectively, for the clinical diagnosis of bacterial infections‐caused inflammation and tuberculosis. This study not only inspires the rational design of magnetic‐fluorescent nanoemitter but also highlights promising potential in magneto‐assisted point‐of‐care test and biomedicine applications.

Fluorescent‐magneto nanoemitters have gained considerable attention for their applications in mechanical controlling‐assisted optical signaling. However, the incompatibility between magnetic and fluorescent components often leads to functional limitations in traditional magneto@fluorescence nanostructure. Herein, we introduce a new compact‐discrete spatial arrangement on a "fluorescence@magneto" core–shell nanostructure consisting of a close‐packed aggregation‐induced emission luminogen (AIEgen) core and a discrete magnetic shell. This structural design effectively eliminates the optical and magnetic interferences between the dual components by facilitating AIEgens loading in core region and reducing the magnetic feeding amount through effective exposure of the magnetic units. Thereby, the resulting magneto‐AIEgen nanoparticle (MANP) demonstrates "win‐win" performances: (i) high fluorescent intensity contributed by AIEgens stacking‐enhanced photoluminescence and reduced photons loss from the meager magnetic shell; (ii) marked magnetic activity due to magneto extraposition‐minimized magnetic shielding. Accordingly, the dual functions‐retained MANP provides a proof of concept for construction of an immunochromatographic sensing platform, where it enables bright fluorescent labeling after magnetically enriching and separating procalcitonin and lipoarabinomannan in clinical human serum and urine, respectively, for the clinical diagnosis of bacterial infections‐caused inflammation and tuberculosis. This study not only inspires the rational design of magnetic‐fluorescent nanoemitter but also highlights promising potential in magneto‐assisted point‐of‐care test and biomedicine applications. [ABSTRACT FROM AUTHOR]

Abstract Background Goose astrovirus (GoAstV) is an important pathogen that causes joint and visceral gout in goslings. It has been circulating in many provinces of China since 2017. Goose astrovirus genotypes 2 (GoAstV-2) is the main epidemic strain, and its high morbidity and mortality have caused huge economic losses to the goose industry. An accurate point-of-care detection for GoAstV-2 is of great significance. In this study, we developed a real-time reverse transcription recombinase polymerase amplification (RT-RPA) method for the on-site detection of GoAstV-2 infection. Results The real-time RT-RPA reaction was carried out at a constant temperature of 39 °C, and the entire detection time from nucleic acid preparation to the end of amplification was only 25 min using the portable device. The results of a specificity analysis showed that no cross-reaction was observed with other related pathogens. The detection limit of the assay was 100 RNA copies/μL. The low coefficient of variation value indicated excellent repeatability. We used 270 clinical samples to evaluate the performance of our established method, the positive concordance rates with RT-qPCR were 99.6%, and the linear regression analysis revealed a strong correlation. Conclusions The established real-time RT-RPA assay showed high rapidity, specificity and sensitivity, which can be widely applied in the laboratory, field and especially in the resource-limited settings for GoAstV-2 point-of-care diagnosis.

Salmonella species are prominent foodborne microbial pathogens transmitted through contaminated food or water and pose a significant threat to human health. Accurate and rapid point-of-care (POC) diagnosis is gaining attention in effectively preventing outbreaks of foodborne disease. However, the presence of dead bacteria can interfere with an accurate diagnosis, necessitating the development of methods for the rapid, simple, and efficient detection of viable bacteria only. Herein, we used an improved propidium monoazide (PMAxx) to develop a nucleic acid lateral flow (NALF) assay based on recombinase polymerase amplification (RPA) to differentiate viable Salmonella Typhimurium. We selected an RPA primer set targeting the invA gene and designed a probe for NALF. RPA-based NALF was optimized for temperature (30–43 °C), time (1–25 min), and endonuclease IV concentration (0.025–0.15 unit/µL). PMAxx successfully eliminated false-positive results from dead S. Typhimurium, enabling the accurate detection of viable S. Typhimurium with a detection limit of 1.11 × 102 CFU/mL in pure culture. The developed method was evaluated with spiked raw chicken breast and milk with analysis completed within 25 min at 39 °C. This study has potential as a tool for the POC diagnostics of viable foodborne pathogens with high specificity, sensitivity, rapidity, and cost-effectiveness.

Background: Goose astrovirus (GoAstV) is an important pathogen that causes joint and visceral gout in goslings. It has been circulating in many provinces of China since 2017. Goose astrovirus genotypes 2 (GoAstV-2) is the main epidemic strain, and its high morbidity and mortality have caused huge economic losses to the goose industry. An accurate point-of-care detection for GoAstV-2 is of great significance. In this study, we developed a real-time reverse transcription recombinase polymerase amplification (RT-RPA) method for the on-site detection of GoAstV-2 infection. Results: The real-time RT-RPA reaction was carried out at a constant temperature of 39 °C, and the entire detection time from nucleic acid preparation to the end of amplification was only 25 min using the portable device. The results of a specificity analysis showed that no cross-reaction was observed with other related pathogens. The detection limit of the assay was 100 RNA copies/μL. The low coefficient of variation value indicated excellent repeatability. We used 270 clinical samples to evaluate the performance of our established method, the positive concordance rates with RT-qPCR were 99.6%, and the linear regression analysis revealed a strong correlation. Conclusions: The established real-time RT-RPA assay showed high rapidity, specificity and sensitivity, which can be widely applied in the laboratory, field and especially in the resource-limited settings for GoAstV-2 point-of-care diagnosis. [ABSTRACT FROM AUTHOR]

Surra is a wasting disease triggered by infection with Trypanosoma evansi, a protozoan blood parasite that causes mortality and morbidity in a broad spectrum of wild and domestic animals and occasionally humans. Trypanosoma evansi has the widest geographical spread among all pathogenic trypanosomes, inflicting significant worldwide economic problems due to its adverse effects on meat and milk production. For diagnosis, most endemic countries continue to rely on traditional parasitological and serological techniques, such as the analysis of blood smears by microscopy and the Card Agglutination Test for T. evansi (CATT/T. evansi). Although these techniques suffer from a limited positive predictive value (PPV), resource constraints in endemic countries often hinder the adoption of more advanced diagnostic tools such as PCR. This paper addresses diverse diagnostic approaches for identifying T. evansi and assesses their viability in field settings. Moreover, it underscores the urgency of transitioning towards molecular diagnostic techniques such as Loop-Mediated Isothermal Amplification (LAMP) and Recombinase Polymerase Amplification (RPA) for dependable high-PPV point-of-care (POC) diagnostics. Finally, this review delves into strategies to enhance and refine next-generation diagnostics for Surra as part of a One Health approach. [ABSTRACT FROM AUTHOR]

Accurate and efficient classification and quantification of CD34+ cells are essential for the diagnosis and monitoring of leukemia. Current methods, such as flow cytometry, are complex, time-consuming, and require specialized expertise and equipment. This study proposes a novel approach for the label-free identification of CD34+ cells using a deep learning model and lens-free shadow imaging technology (LSIT). LSIT is a portable and user-friendly technique that eliminates the need for cell staining, enhances accessibility to nonexperts, and reduces the risk of sample degradation. The study involved three phases: sample preparation, dataset generation, and data analysis. Bone marrow and peripheral blood samples were collected from leukemia patients, and mononuclear cells were isolated using Ficoll density gradient centrifugation. The samples were then injected into a cell chip and analyzed using a proprietary LSIT-based device (Cellytics). A robust dataset was generated, and a custom AlexNet deep learning model was meticulously trained to distinguish CD34+ from non-CD34+ cells using the dataset. The model achieved a high accuracy in identifying CD34+ cells from 1929 bone marrow cell images, with training and validation accuracies of 97.3% and 96.2%, respectively. The customized AlexNet model outperformed the Vgg16 and ResNet50 models. It also demonstrated a strong correlation with the standard fluorescence-activated cell sorting (FACS) technique for quantifying CD34+ cells across 13 patient samples, yielding a coefficient of determination of 0.81. Bland–Altman analysis confirmed the model's reliability, with a mean bias of −2.29 and 95% limits of agreement between 18.49 and −23.07. This deep-learning-powered LSIT offers a groundbreaking approach to detecting CD34+ cells without the need for cell staining, facilitating rapid CD34+ cell classification, even by individuals without prior expertise. [ABSTRACT FROM AUTHOR]

Herpes simplex virus type 1 (HSV-1) Keratitis (HSK) is a highly prevalent eye disease worldwide, characterized by lifelong recurrent episodes and a major risk of leading to blindness. Detecting HSV-1 promptly and accurately can initiate a timely and appropriate therapeutic regimen, minimizing tissue damage and preventing vision impairment. Currently, PCR is the most reliable method for identifying HSV-1, but its utilization for point-of-care (POC) HSV-1 detection is limited due to the need for sophisticated equipment, particularly in areas with limited resources. Here, we propose a new method for on-site HSV detection by using LAMP-Cas12 diagnostic technology and gold nanoparticles. This technique possesses comparable sensitivity to qPCR, and its detection results could be easily read and interpreted without the need for complex equipment. In detecting HSV in clinical tear specimens, this strategy achieved a 93.9 % consistency in positive detection and a 100 % consistency in negative detection compared to qPCR. Our strategy innovates the technique of current HSV-1 detections and is expected to play a crucial role in POC diagnosis of HSK in the future.

Current methods for the diagnosis of urinary tract infections with antimicrobial susceptibility testing take 2-3 days and require a clinical laboratory. The lack of a rapid, point-of-care antibiotic susceptibility test (AST) has contributed to the misuse of antibiotics when treating urinary tract infections (UTIs) and consequently to the rise of multi-drug-resistant organisms. The current clinical approach has led to reduced treatment options and increased costs of diagnosis and therapy. To address this issue, novel diagnostics are needed for the timely determination of antimicrobial susceptibility. We present a rapid, point-of-care, phenotypic AST device that can report the antibiotic susceptibility/resistance of a uropathogen to a panel of antibiotics in as few as 3 h by utilizing fluorescent-labelling chemistry and a highly sensitive particle-counting instrument. We analysed 744 urine samples from the outpatients and inpatients of two Italian hospitals. The 130 UTI-positive patient urine samples we found were measured using a panel of six common UTI antibiotics plus a growth control. By comparing our results to hospital laboratory urine cultures, we obtained an overall sensitivity = 81%, a specificity = 83%, an SPV (sensitivity predicted value) = 95%, and an RPV (resistance predicted value) = 54%. According to our preliminary data, the sensitivity predicted value for a single antibiotic agent was 95%, thus allowing (in the vast majority of cases) an early (within 3 h) recognition of an effective agent for a single patient.

A significant bottleneck exists for mass-production of ion-selective electrodes despite recent developments in manufacturing technologies. Here, we present a fully-automated system for large-scale production of ISEs. Three materials, including polyvinyl chloride, polyethylene terephthalate and polyimide, were used as substrates for fabricating ion-selective electrodes (ISEs) using stencil printing, screen-printing and laser engraving, respectively. We compared sensitivities of the ISEs to determine the best material for the fabrication process of the ISEs. The electrode surfaces were modified with various carbon nanomaterials including multi-walled carbon nanotubes, graphene, carbon black, and their mixed suspensions as the intermediate layer to enhance sensitivities of the electrodes. An automated 3D-printed robot was used for the drop-cast procedure during ISE fabrication to eliminate manual steps. The sensor array was optimized, and the detection limits were 10–5 M, 10–5 M and 10–4 M for detection of K+, Na+ and Ca2+ ions, respectively. The sensor array integrated with a portable wireless potentiometer was used to detect K+, Na+ and Ca2+ in real urine and simulated sweat samples and results obtained were in agreement with ICP-OES with good recoveries. The developed sensing platform offers low-cost detection of electrolytes for point-of-care applications. [ABSTRACT FROM AUTHOR]

Detecting and appropriately diagnosing a Mycobacterium tuberculosis infection remains technologically difficult because the pathogen commonly hides in macrophages in a dormant state. Described here is novel near-infrared aggregation-induced-emission luminogen (AIEgen) labeling developed by the current authors' laboratory for point-of-care (POC) diagnosis of an M. tuberculosis infection. The selectivity of AIEgen labeling, the labeling of intracellular M. tuberculosis by AIEgen, and the labeling of M. tuberculosis in sputum samples by AIEgen, along with its accuracy, sensitivity, and specificity, were preliminarily evaluated. Results indicated that this near-infrared AIEgen labeling had satisfactory selectivity and it labeled intracellular M. tuberculosis and M. tuberculosis in sputum samples. It had a satisfactory accuracy (95.7%), sensitivity (95.5%), and specificity (100%) for diagnosis of an M. tuberculosis infection in sputum samples. The current results indicated that near-infrared AIEgen labeling might be a promising novel diagnostic tool for POC diagnosis of M. tuberculosis infection, though further rigorous verification of these findings is required. [ABSTRACT FROM AUTHOR]

Surra is a wasting disease triggered by infection with Trypanosoma evansi, a protozoan blood parasite that causes mortality and morbidity in a broad spectrum of wild and domestic animals and occasionally humans. Trypanosoma evansi has the widest geographical spread among all pathogenic trypanosomes, inflicting significant worldwide economic problems due to its adverse effects on meat and milk production. For diagnosis, most endemic countries continue to rely on traditional parasitological and serological techniques, such as the analysis of blood smears by microscopy and the Card Agglutination Test for T. evansi (CATT/T. evansi). Although these techniques suffer from a limited positive predictive value (PPV), resource constraints in endemic countries often hinder the adoption of more advanced diagnostic tools such as PCR. This paper addresses diverse diagnostic approaches for identifying T. evansi and assesses their viability in field settings. Moreover, it underscores the urgency of transitioning towards molecular diagnostic techniques such as Loop-Mediated Isothermal Amplification (LAMP) and Recombinase Polymerase Amplification (RPA) for dependable high-PPV point-of-care (POC) diagnostics. Finally, this review delves into strategies to enhance and refine next-generation diagnostics for Surra as part of a One Health approach.

Introduction: Community-acquired pneumonia (CAP) is lethal in elderly individuals who are more vulnerable to respiratory failure and require more emergency ventilation support than younger individuals. Interleukin-6 (IL-6) plays a crucial role and has predictive value in CAP; high serum IL-6 concentrations in adults are associated with high respiratory failure and mortality rates. Early detection of IL-6 concentrations can facilitate the timely stratification of patients at risk of acute respiratory failure. However, conventional enzyme-linked immunosorbent assay (ELISA) IL-6 measurement is laborious and time-consuming. Methods: The IL-6 rapid diagnostic system combined with a lateral flow immunoassay-based (LFA-based) IL-6 test strip and a spectrum-based optical reader is a novel tool developed for rapid and sequential bedside measurements of serum IL-6 concentrations. Here, we evaluated the correlation between the IL-6 rapid diagnostic system and the ELISA and the efficacy of the system in stratifying high-risk elderly patients with CAP. Thirty-six elderly patients (median age: 86.5 years; range: 65--97 years) with CAP were enrolled. CAP diagnosis was established based on the Infectious Diseases Society of America (IDSA) criteria. The severity of pneumonia was assessed using the CURB-65 score and Pneumonia Severity Index (PSI). IL-6 concentration was measured twice within 24 h of admission. Results: The primary endpoint variable was respiratory failure requiring invasive mechanical or non-invasive ventilation support after admission. IL-6 rapid diagnostic readouts correlated with ELISA results (p < 0.0001) for 30 samples. Patients were predominantly male and bedridden (69.4%). Ten patients (27.7%) experienced respiratory failure during admission, and five (13.9%) died of pneumonia. Respiratory failure was associated with a higher mortality rate (p = 0.015). Decreased serum IL-6 concentration within 24 h after admission indicated a lower risk of developing respiratory failure in the later admission course (Receiver Operating Characteristic [ROC] curve = 0.696). Conclusion: Sequential IL-6 measurements with the IL-6 rapid diagnostic system might be useful in early clinical risk assessment and severity stratification of elderly patients with pneumonia. This system is a potential point-of-care diagnostic device for sequential serum IL-6 measurements that can be applied in variable healthcare systems. [ABSTRACT FROM AUTHOR]

Diabetes is well established as a widespread, incurable, and fatal disease with glucose monitoring and tight glycaemic control vital for effective illness prevention and management. Hydrogel‐based holographic sensors serve as a low‐cost and label‐free colorimetric sensing platform, directly identifiable by the naked eye and spectroscopy for quantitative monitoring. Herein, a cost‐effective and reusable holographic glucose sensor is developed via single pulse UV‐induced dual‐photopolymerization of boronic acid functionalized hydrogels for point‐of‐care (POC) diagnosis. Computational modeling of holographic sensors response is conducted following Braggs law alongside the study of fabrication parameter optimization and sensor swelling dynamics. Fabrication conditions, responsive and interference hydrogel compositions of holographic sensors are investigated to improve response time, sensitivity in urine (13.03 nm mmol−1 L−1), limit of detection (0.06 mmol L−1), and reusability. Photolithographic patterning of hydrogel‐based holographic sensors permits the inscription of additional information into the sensors for qualitative measurement. Selectivity, reversibility, and continuous monitoring of urine samples are conducted over a physiological glucose concentration range (0.0–9.4 mmol L−1) to demonstrate the viability for diabetic risk identification. The simple incorporation of glucose sensors in a reusable urinary analysis prototype is validated in human urine, showing potential for POC to reduce patient dependency on invasive diabetic monitoring procedures. [ABSTRACT FROM AUTHOR]

Conventional glassy carbon electrodes (GCE) cannot meet the requirements of future electrodes for wider use due to low conductivity, high cost, non-portability, and lack of flexibility. Therefore, cost-effective and wearable electrode enabling rapid and versatile molecule detection is becoming important, especially with the ever-increasing demand for health monitoring and point-of-care diagnosis. Graphene is considered as an ideal electrode due to its excellent physicochemical properties. Here, we prepare graphene film with ultra-high conductivity and customize the 3-electrode system via a facile and highly controllable laser engraving approach. Benefiting from the ultra-high conductivity (5.65 × 105 Sm−1), the 3-electrode system can be used as multifunctional electrode for direct detection of dopamine (DA) and enzyme-based detection of glucose without further metal deposition. The dynamic ranges from 1–200 µM to 0.5–8.0 mM were observed for DA and glucose, respectively, with a limit of detection (LOD) of 0.6 µM and 0.41 mM. Overall, the excellent target detection capability caused by the ultra-high conductivity and ease modification of graphene films, together with their superb mechanical properties and ease of mass-produced, provides clear potential not only for replacing GCE for various electrochemical studies but also for the development of portable and highperformance electrochemical wearable medical devices. [ABSTRACT FROM AUTHOR]

A new mass-sensitive biosensing approach for detecting circulating tumor cells (CTCs) using a quartz crystal resonator (QCR) has been developed. A mathematical model was used to design a ring electrode-based QCR to eliminate the Gaussian spatial distribution of frequency response in the first harmonic mode, a characteristic of QCRs, without compromising the sensitivity of frequency response. An ink-dot method was used to validate the ring electrode fabricated based on our model. Furthermore, the ring electrode QCR was experimentally tested for its ability to capture circulating tumor cells, and the results were compared with a commercially available QCR with a keyhole electrode. An indirect method of surface immobilization technique was employed via modification of the SiO2 surface of the ring electrode using a silane, protein, and anti-EpCAM. The ring electrode successfully demonstrated eliminating the spatial nonuniformity of frequency response for three cancer cell lines, i.e., MCF-7, PANC-1, and PC-3, compared with the keyhole QCR, which showed nonuniform spatial response for the same cancer cell lines. These results are promising for developing QCR-based biosensors for the early detection of cancer cells, with the potential for point-of-care diagnosis for cancer screening. [ABSTRACT FROM AUTHOR]

Herein, a novel microfluidic-biochip enabled with surface enhanced Raman spectroscopy (SERS) as a readout has been demonstrated for uric acid (UA) detection as point-of care (POC) device. Three different biochip designs (D1, D2 and D3) containing pillars in a microchannel with different bending ratios were conceived and optimized for various mixing parameters using a multiphysics simulation tool. The microchannel, integrated with pillars, provide pressure perturbation, sharp corners, and variation in bending ratio improves phase shift and mixing index. Subsequently the microfluidic-biochips were fabricated by a combination of photo-and soft-lithography, and bonding strength between two Polydimethylsiloxane substrates were found stable up to a flow rate of 1.8 ml minâˆ'1. Further to realize SERS activity in the microfluidic-biochip, cubic shape silver nanoparticles (AgNPs), with an average size ∼68 nm, were synthesized using poly-ol method. The SERS activity was optimized by simultaneously flowing AgNPs and crystal violet (CV) dye of 10âˆ'6M, with double inlet in the reservoir and highest sensitivity was achieved in the D3 biochip. Further, D3 biochip was employed for detection of extended concentrations of CV and UA. The enhancement factor limit of detection and relative standard deviation was found to be 2 Ă— 107, 8.9 Ă— 10âˆ'11 and 2.7% respectively for CV and 3.1 Ă— 103, 3.2 Ă— 10âˆ'7 and 2.9% respectively for UA. Interference of UA with lactic acid has been tested and device was able to detect signature peaks of both biomarkers up to 50 Ă— 10âˆ'9 M concentration. Thus, the developed microfluidic-biochip device has potential to be used in a POC setting for onsite detection of biomarkers. [ABSTRACT FROM AUTHOR]

Abstract Nanomaterial-based artificial enzymes (or nanozymes) have attracted great attention in the past few years owing to their capability not only to mimic functionality but also to overcome the inherent drawbacks of the natural enzymes. Numerous advantages of nanozymes such as diverse enzyme-mimicking activities, low cost, high stability, robustness, unique surface chemistry, and ease of surface tunability and biocompatibility have allowed their integration in a wide range of biosensing applications. Several metal, metal oxide, metal–organic framework-based nanozymes have been exploited for the development of biosensing systems, which present the potential for point-of-care analysis. To highlight recent progress in the field, in this review, more than 260 research articles are discussed systematically with suitable recent examples, elucidating the role of nanozymes to reinforce, miniaturize, and improve the performance of point-of-care diagnostics addressing the ASSURED (affordable, sensitive, specific, user-friendly, rapid and robust, equipment-free and deliverable to the end user) criteria formulated by World Health Organization. The review reveals that many biosensing strategies such as electrochemical, colorimetric, fluorescent, and immunological sensors required to achieve the ASSURED standards can be implemented by using enzyme-mimicking activities of nanomaterials as signal producing components. However, basic system functionality is still lacking. Since the enzyme-mimicking properties of the nanomaterials are dictated by their size, shape, composition, surface charge, surface chemistry as well as external parameters such as pH or temperature, these factors play a crucial role in the design and function of nanozyme-based point-of-care diagnostics. Therefore, it requires a deliberate exertion to integrate various parameters for truly ASSURED solutions to be realized. This review also discusses possible limitations and research gaps to provide readers a brief scenario of the emerging role of nanozymes in state-of-the-art POC diagnosis system development for futuristic biosensing applications.

A new mass-sensitive biosensing approach for detecting circulating tumor cells (CTCs) using a quartz crystal resonator (QCR) has been developed. A mathematical model was used to design a ring electrode-based QCR to eliminate the Gaussian spatial distribution of frequency response in the first harmonic mode, a characteristic of QCRs, without compromising the sensitivity of frequency response. An ink-dot method was used to validate the ring electrode fabricated based on our model. Furthermore, the ring electrode QCR was experimentally tested for its ability to capture circulating tumor cells, and the results were compared with a commercially available QCR with a keyhole electrode. An indirect method of surface immobilization technique was employed via modification of the SiO2 surface of the ring electrode using a silane, protein, and anti-EpCAM. The ring electrode successfully demonstrated eliminating the spatial nonuniformity of frequency response for three cancer cell lines, i.e., MCF-7, PANC-1, and PC-3, compared with the keyhole QCR, which showed nonuniform spatial response for the same cancer cell lines. These results are promising for developing QCR-based biosensors for the early detection of cancer cells, with the potential for point-of-care diagnosis for cancer screening.

Inspired by the programmability and modifiability of nucleic acids, point-of-care (POC) diagnostics for nucleic acid target detection is evolving to become more diversified and intelligent. In this study, we introduce a fluorescent and photothermal dual-mode logic biosensing platform that integrates catalytic hairpin assembly (CHA), toehold-mediated stand displacement reaction (SDR) and a DNA walking machine. Dual identification and signal reporting modules are incorporated into DNA circuits, orchestrated by an AND Boolean logic gate operator and magnetic beads (MBs). In the presence of bispecific microRNAs (miRNAs), the AND logic gate activates, driving the DNA walking machine, and facilitating the collection of hairpin DNA stands modified with FAM fluorescent group and CeO 2 @Au nanoparticles. The CeO 2 @Au nanoparticles, served as a nanozyme, can oxidize TMB into oxidation TMB (TMBox), enabling a near-infrared (NIR) laser-driven photothermal effect following the magnetic separation of MBs. This versatile platform was employed to differentiate between plasma samples from breast cancer patients, lung cancer patients, and healthy donors. The thermometer-readout transducers, derived from the CeO 2 @Au@DNA complexes, provided reliable results, further corroborated by fluorescence assays, enhancing the confidence in the diagnostics compared to singular detection method. The dual-mode logic biosensor can be easily customized to various nucleic acid biomarkers and other POC signal readout modalities by adjusting recognition sequences and modification strategies, heralding a promising future in the development of intelligent, flexible diagnostics for POC testing. [ABSTRACT FROM AUTHOR]

Pre-eclampsia is a serious multi-organ complication that severely threatens the safety of pregnant women and infants. To accurate and timely diagnose pre-eclampsia, point-of-care (POC) biosensing of the specific biomarkers is urgently required. However, one of the key biomarkers of pre-eclampsia, placental growth factor (PlGF), has a reduced level of expression in patients, which challenges the quantification capability and Limit-of-detection (LOD) of biosensors. Herein, we reported a microfiber Bragg grating biosensor for the quantification of PlGF in clinical serum samples. The Bragg grating was inscribed in a unilateral tapered fiber to generate the segmented Fabry-Perot spectrum for improving the capability of detection. Furthermore, a temperature-calibrated Bragg grating was added to enable dual parametric detection of PlGF and temperature simultaneously for removing the crosstalk. Finally, the biosensor was envisaged to be perfectly compatible with microfluidic chips, and thus dramatically reducing the sample consumption to as small as 10 μL. The proposed biosensor can respond to PlGF with concentrations ranging from 5 to 120 pg mL−1, attaining a LOD of 5 pg mL−1 of clinical relevance. More importantly, the biosensor achieved micro volume detection of clinical serum samples from patients, and the ROC curve with an AUC of 0.977 confirmed the viability of the device. Our study paves the way to a new idea for cost-effective and high-precision screening of patients with pre-eclampsia, and hence envisages a promising prospect for point-of-care (POC) diagnosis of patients with pre-eclampsia. [ABSTRACT FROM AUTHOR]

Contact lens sensors have been emerging as point-of-care devices in recent healthcare developments for ocular physiological condition monitoring and diagnosis. Fluorescence sensing technologies have been widely applied in contact lens sensors due to their accuracy, high sensitivity, and specificity. As ascorbic acid (AA) level in tears is closely related to ocular inflammation, a fluorescent contact lens sensor incorporating a BSA-Au nanocluster (NC) probe is developed for in situ tear AA detection. The NCs are firstly synthesized to obtain a fluorescent probe, which exhibits high reusability through the quench/recover (KMnO 4 /AA) process. The probe is then encapsulated with 15 wt% of poly(vinyl alcohol) (PVA) and 1.5 wt% of citric acid (CA) film, and implemented on a closed microfluidic contact lens sensing region. The laser-ablated microfluidic channel in contact lens sensors allows for tear fluid to flow through the sensing region, enabling an in-situ detection of AA. Meanwhile, a smartphone application accompanied by a customized 3D printed readout box is developed for image caption and algorism to quantitative analysis of AA levels. The contact lens sensor is tested within the readout box and the emission signal is collected through the smartphone camera at room temperature with an achieved LOD of 0.178 mmol L-1 (0.0–1.2 mmol L-1). The operational and storage lifetime is also evaluated to characterize the sensor properties and resulted in 20 h and 10 days, respectively. The reusable AA contact lens sensor is promising to lead to an alternative accessible diagnostic method for ocular inflammation in point-of-care settings. [ABSTRACT FROM AUTHOR]

Monitoring biomarkers is a great way to assess daily physical condition, and using saliva instead of blood samples is more advantageous as the process is simple and allows individuals to test themselves. In the present study, we analyzed the titers of neutralizing antibodies, IgG and secretory IgA (sIgA), in response to the SARS-CoV-2 vaccine, in saliva. A total of 19 saliva and serum samples were collected over a 10-month period 3 weeks after the first vaccine, 8 months after the second vaccine, and 1 month after the third vaccine. The ranges of antibody concentrations post-vaccination were: serum IgG: 81–15,000 U/mL, salivary IgG: 3.4–330 U/mL, and salivary IgA: 58–870 ng/mL. A sharp increase in salivary IgG levels was observed after the second vaccination. sIgA levels also showed an increasing trend. A correlation with trends in serum IgG levels was observed, indicating the possibility of using saliva to routinely assess vaccine efficacy. The electrochemical immunosensor assay developed in this study based on the gold-linked electrochemical immunoassay, and the antioxidant activity measurement based on luminol electrochemiluminescence (ECL), can be performed using portable devices, which would prove useful for individual-based diagnosis using saliva samples. [ABSTRACT FROM AUTHOR]

Serotonin (5-hydroxytryptamine (5-HT)) is one of the important neurotransmitters which is released from the endocrine system. An abnormal level of this biomarker leads to several neurological diseases. The accurate assessment of serotonin is the utmost option to start treatment in the early stages of the disease. The current work is focused on the development of a disposable, screen-printed electrochemical sensor for the depression biomarker, serotonin in the physiological pH medium (pH 7.4) with the aid of a hexagonal, Ni(OH)2-nanoplate (NH-HNP)-embedded chitosan (Chit) and modified, screen-printed carbon electrode (SPCE). Initially, hexagonal nanoplates of Ni(OH)2 were synthesized by an eco-friendly and simple hydrothermal method. The prepared materials were well characterized by advanced analytical techniques to examine the physicochemical properties of the synthesized Ni(OH)2 hexagonal nanoplates. From the cyclic voltametric (CV) analysis, it was found that the oxidative current response of 5-HT at a NH-HNP-modified SPCE has about fivefold higher current values than over bare SPCE. The scan rate studies of NH-HNP-Chit/SPCE electrodes revealed that the oxidation mechanism of 5-HT is controlled by the diffusion behavior of the analyte. Differential pulse voltammetric tests of the NH-HNP-Chit/SPCE electrode exhibited a linear response in the dynamic concentration range of 0.1 to 30 µM, with a detection limit of about 60 nM. The sensor response is very reproducible from electrode to electrode, and the deactivation or surface-fouling of the sensor was not observed within the several experimental measurements. The sensor exhibited excellent storage stability over a period of twenty days. Finally, the fabricated, disposable SPCE sensor has shown respectable activity for the detection of depression biomarker 5-HT from synthetic urine and saliva samples. [ABSTRACT FROM AUTHOR]

Mycoplasma hominis, which is difficult to culture and identify by ordinary methods, is one of the smallest pathogens in the human genitourinary tract causing urogenital infections. A CRISPR-Cas12a-based detection system might provide a novel application for M. hominis nucleic acid detection in molecular diagnostics. A plasmid containing the glyceraldehyde-3-phosphate dehydrogenase gene of M. hominis (ATCC_27545) as the positive control was constructed by homologous recombination. The active Cas12a protein was purified by affinity chromatography. The primers for recombinase polymerase amplification (RPA), the CRISPR RNA (crRNA), and the ratio of Cas12a to crRNA were further optimized. Finally, the sensitivity, specificity, and clinical effectiveness of the Cas12a detection system were confirmed. We successfully constructed and optimized a novel nucleic acid detection system for M. hominis based on RPA-CRISPR-Cas12a, and the whole process takes only 1 h. The limit of detection for the gap gene of M. hominis was 3 copies/μl and no cross-reactivity with other urogenital pathogens appeared. In the evaluation of 111 clinical samples, the sensitivity and specificity were both 1.000 and the area under the curve of the receiver operating characteristic was 1.000 (p < 0.001), indicating that the RPA-Cas12a-fluorescent assay was fully comparable to the traditional culture method. Finally, the RPA-Cas12a detection system can also be combined with lateral flow strips (LFS) to achieve visual detection. We successfully developed a low-cost and rapid detection method of M. hominis based on RPA-Cas12a technology. This method realized by fluorescence value readout and visual detection by LFS could be applied in population screening and resource-limited conditions.

Monitoring biomarkers is a great way to assess daily physical condition, and using saliva instead of blood samples is more advantageous as the process is simple and allows individuals to test themselves. In the present study, we analyzed the titers of neutralizing antibodies, IgG and secretory IgA (sIgA), in response to the SARS-CoV-2 vaccine, in saliva. A total of 19 saliva and serum samples were collected over a 10-month period 3 weeks after the first vaccine, 8 months after the second vaccine, and 1 month after the third vaccine. The ranges of antibody concentrations post-vaccination were: serum IgG: 81–15,000 U/mL, salivary IgG: 3.4–330 U/mL, and salivary IgA: 58–870 ng/mL. A sharp increase in salivary IgG levels was observed after the second vaccination. sIgA levels also showed an increasing trend. A correlation with trends in serum IgG levels was observed, indicating the possibility of using saliva to routinely assess vaccine efficacy. The electrochemical immunosensor assay developed in this study based on the gold-linked electrochemical immunoassay, and the antioxidant activity measurement based on luminol electrochemiluminescence (ECL), can be performed using portable devices, which would prove useful for individual-based diagnosis using saliva samples.

Serotonin (5-hydroxytryptamine (5-HT)) is one of the important neurotransmitters which is released from the endocrine system. An abnormal level of this biomarker leads to several neurological diseases. The accurate assessment of serotonin is the utmost option to start treatment in the early stages of the disease. The current work is focused on the development of a disposable, screen-printed electrochemical sensor for the depression biomarker, serotonin in the physiological pH medium (pH 7.4) with the aid of a hexagonal, Ni(OH)2-nanoplate (NH-HNP)-embedded chitosan (Chit) and modified, screen-printed carbon electrode (SPCE). Initially, hexagonal nanoplates of Ni(OH)2 were synthesized by an eco-friendly and simple hydrothermal method. The prepared materials were well characterized by advanced analytical techniques to examine the physicochemical properties of the synthesized Ni(OH)2 hexagonal nanoplates. From the cyclic voltametric (CV) analysis, it was found that the oxidative current response of 5-HT at a NH-HNP-modified SPCE has about fivefold higher current values than over bare SPCE. The scan rate studies of NH-HNP-Chit/SPCE electrodes revealed that the oxidation mechanism of 5-HT is controlled by the diffusion behavior of the analyte. Differential pulse voltammetric tests of the NH-HNP-Chit/SPCE electrode exhibited a linear response in the dynamic concentration range of 0.1 to 30 µM, with a detection limit of about 60 nM. The sensor response is very reproducible from electrode to electrode, and the deactivation or surface-fouling of the sensor was not observed within the several experimental measurements. The sensor exhibited excellent storage stability over a period of twenty days. Finally, the fabricated, disposable SPCE sensor has shown respectable activity for the detection of depression biomarker 5-HT from synthetic urine and saliva samples.

A rapid point-of-care (PoC) test as an adjunct to blood culture to aid the diagnosis of sepsis patients with bloodstream infections may decrease the use of broad-spectrum antibiotics and reduce rates of inappropriate antimicrobial therapy (IAAT) use. Through a decision tree model, taking the perspective of the Irish healthcare provider, the cost-effectiveness of such an intervention was evaluated against the current standard of care (SoC) in hospitalized adults in Ireland. The base-case scenario showed that using a rapid PoC test was cost-saving and life-saving, with €8188 saved per death averted. The results were sensitive to the length of hospital stay for patients with true-positive and true-negative results and to the length of stay (LOS) in the intensive care unit. The threshold analysis showed that even at lower sensitivities, the rapid PoC test could be cost-effective due to the substantial impact of starting earlier targeted and appropriate treatment in patients with bloodstream infection (BSI) and sepsis.

Dysregulation of nitric oxide (NO) and it's two relatively stable metabolites, nitrite, and nitrate, in SARS-CoV-2, are reported in infected populations, especially for nitrates levels > 68.4 μmol/L. In this paper, we measure the abnormal presence of nitrite in the saliva by developing a cheap μPAD for colorimetric detection through the modified Griess reaction. This includes a diazotization reaction between nitrite and Griess reagent, including Sulfanilamide and N-Naphthyl-ethylenediamine in an acidic medium, causing a pink Azo compound. The modifications are suggested by a numerical method model that couples the mass flux with the porosity medium equations (convection, diffusion and, dispersion) that improves the mixing process. The mixing index was quantified from the concentration deviation method via simulation of a homogeneous two-phase flow in a porous environment. Five μPAD designs were fabricated to verify the simulation results of mixing enhancement on the Griess reactants in saliva samples. The investigated geometries include straight, helical, zig–zag, square wave, and inclined jagged shapes fabricated by direct laser writing, suitable for low cost, mass fabrication. Inclined jagged micromixer exhibited the best performance with up to 40% improvement compared with the simple straight geometry. Deliberate geometrical modifications, exemplified here in a jagged micromixer on paper, cut the limit of detection (LOD) by at least half without impacting the linear detection range. [ABSTRACT FROM AUTHOR]

Abstract Background Improved point-of-care diagnostic tests for tuberculosis (TB) in severe immune suppressed people living with HIV (PLWH) are needed to decrease morbidity and mortality outcomes. The aim of the study is to evaluate the performance of the lipoarabinomannan antigen test (LAM-test) with and without α-mannosidase pre-treated urine in a cohort of PLWH in primary care clinics in Guatemala. We further determined TB incidence, and mortality rates and its risk factors in PLWH with TB symptoms. Methods Prospective longitudinal study of PLWH with TB symptoms. Urine samples were collected at 2 HIV sites to test the sensitivity of the LAM-test in urine with and without α-mannosidase pre-treatment. A composite reference standard of either a positive Mycobacterium tuberculosis complex culture and/or GeneXpert® MTB/RIF (Xpert, Cepheid, Sunnyvale, CA, USA) results was used in the LAM-test diagnostic accuracy studies. Cox proportional hazards regression was used to study mortality predictors. Results The overall sensitivity of the LAM-test was of 56.1% with 95% CI of (43.3–68.3). There were no differences in the LAM-test sensitivity neither by hospital nor by CD4 T cell values. LAM-test sensitivity in PLWH with

Alkaline phosphatase (ALP) is one of the most versatile biomarkers for early detection of several diseases, such as oral carcinomas and periodontitis; therefore, great efforts have been dedicated for developing an ALP biosensor. Multicolor detection of ALP in saliva is ideal for a point-of-care diagnosis; however, this approach is very challenging since spectral responses over wavelengths of several tens of nanometers have thus far remained difficult to achieve. In this work, a colorimetric biosensor for ALP assay has been developed based on ALP affinity to dephosphorylate glucose phosphate into glucose, which has the affinity to deposit Ag nanoshells onto Au nanobipyramids with a multicolor response. This approach provides a blue shift of localized surface plasmon resonance (LSPR) as large as 190 nm corresponding to distinctive color changes, from yellowish brown to red based on the thickness of the formed Ag shell around the Au nanobipyramids. The change in the LSPR has been conducted for highly sensitive quantitative bioassay of ALP with a detectable multicolor change with linear dynamic range of 0.1–20 U/L and low limit of detection (LOD) of 0.085 U/L. Furthermore, the developed multicolor ALP biosensor exhibits high selectivity with high recovery of 98.6% demonstrating its reliability and suitability for a point-of-care diagnosis. [ABSTRACT FROM AUTHOR]

Highlights: Enzyme-mimicking activities of different nanomaterials (nanozymes) and the recent progress in the construction of nanozyme-based biosensors with various examples are discussed in this review. Physicochemical properties of nanomaterials (size, composition, pH, temperature, surface chemistry) play crucial role in the nanozyme activities. The emerging nanozyme-based biosensors promise great potential for point-of-care diagnostic applications following the ASSURED criteria defined by WHO.Nanomaterial-based artificial enzymes (or nanozymes) have attracted great attention in the past few years owing to their capability not only to mimic functionality but also to overcome the inherent drawbacks of the natural enzymes. Numerous advantages of nanozymes such as diverse enzyme-mimicking activities, low cost, high stability, robustness, unique surface chemistry, and ease of surface tunability and biocompatibility have allowed their integration in a wide range of biosensing applications. Several metal, metal oxide, metal–organic framework-based nanozymes have been exploited for the development of biosensing systems, which present the potential for point-of-care analysis. To highlight recent progress in the field, in this review, more than 260 research articles are discussed systematically with suitable recent examples, elucidating the role of nanozymes to reinforce, miniaturize, and improve the performance of point-of-care diagnostics addressing the ASSURED (affordable, sensitive, specific, user-friendly, rapid and robust, equipment-free and deliverable to the end user) criteria formulated by World Health Organization. The review reveals that many biosensing strategies such as electrochemical, colorimetric, fluorescent, and immunological sensors required to achieve the ASSURED standards can be implemented by using enzyme-mimicking activities of nanomaterials as signal producing components. However, basic system functionality is still lacking. Since the enzyme-mimicking properties of the nanomaterials are dictated by their size, shape, composition, surface charge, surface chemistry as well as external parameters such as pH or temperature, these factors play a crucial role in the design and function of nanozyme-based point-of-care diagnostics. Therefore, it requires a deliberate exertion to integrate various parameters for truly ASSURED solutions to be realized. This review also discusses possible limitations and research gaps to provide readers a brief scenario of the emerging role of nanozymes in state-of-the-art POC diagnosis system development for futuristic biosensing applications. [ABSTRACT FROM AUTHOR]

The expansion of large-scale aquaculture has exacerbated the challenge of aquatic diseases, resulting in substantial economic losses annually. Currently, traditional laboratory-based diagnostic methods are time-consuming and costly, hindering on-site testing for individual farmers. We address this issue by developing a state-of-the-art handheld isothermal nucleic acid amplification device (WeD-1) capable of fluorescence tracking of reactions and integrating it with an enhanced one-pot Prokaryotic Argonaute based nucleic acid detection method, enabling duplex visual detection of aquatic pathogens. WeD-1 is portable, reusable, user-friendly, and cost-effective, offering real-time smartphone interaction and enabling real-time fluorescence observation during the reaction. The enhanced one-pot Loop-Mediated Isothermal Amplification (LAMP)-PfAgo method, incorporating paraffin-encapsulated lyophilized PfAgo protein, achieves precise target-specific cleavage, significantly enhancing multiplex nucleic acid detection. This innovation streamlines on-site testing, negating the need for specialized laboratory conditions while ensuring an aerosol-free system. With newly developed and highly sensitive LAMP primer sets, our compact WeD-1/LAMP-PfAgo nucleic acid rapid testing system exhibits remarkable sensitivity, readily detecting aquatic pathogens with naked eyes from rapidly prepared fish and shrimp samples within 40 min, even when the Ct values are as high as 34. • On-Site Duplex Pathogen Visualization: Handheld isothermal fluorescence device (WeD-1) for efficient and duplex visual detection of aquatic pathogens in fish and shrimp samples. • One-pot Aerosol-Free Detection with Paraffin Encapsulation: Controlled release of PfAgo protein via paraffin encapsulation ensures simplicity and safety, while preventing aerosol contamination. • Enhanced Pathogen Detection: LAMP-PfAgo detects pathogens with Ct values up to 34 in 40 min, no nucleic acid purification required. • Smartphone-Controlled Real-Time Monitoring: WeD-1 allows users to program heating profiles via smartphones and monitor temperatures in real-time, even at 95 °C. [ABSTRACT FROM AUTHOR]

The Zika virus (ZIKV) is a great concern for global health due to its high transmission, including disseminating through blood, saliva, urine, semen and vertical transmission. In some cases, ZIKV has been associated with microcephaly, neurological disorders, and Guillain–Barré syndrome. There is no vaccine, and controlling the disease is a challenge, especially with the co-circulation of the Dengue virus, which causes a severe cross-reaction due to the similarity between the two arboviruses. Considering that electrochemical immunosensors are well-established, sensitive, and practical tools for diagnosis, in this study we developed a sensor platform with intrinsic redox activity that facilitates measurement readouts. Prussian blue (PB) has a great ability to form electrocatalytic surfaces, dispensing redox probe solutions in voltammetric measurements. Herein, PB was incorporated into a chitosan–carbon nanotube hybrid, forming a nanocomposite that was drop-casted on a screen-printed electrode (SPE). The immunosensor detected the envelope protein of ZIKV in a linear range of 0.25 to 1.75 µg/mL (n = 8, p < 0.01), with a 0.20 µg/mL limit of detection. The developed immunosensor represents a new method for electrochemical measurements without additional redox probe solutions, and it is feasible for application in point-of-care diagnosis.

The Zika virus (ZIKV) is a great concern for global health due to its high transmission, including disseminating through blood, saliva, urine, semen and vertical transmission. In some cases, ZIKV has been associated with microcephaly, neurological disorders, and Guillain–Barré syndrome. There is no vaccine, and controlling the disease is a challenge, especially with the co-circulation of the Dengue virus, which causes a severe cross-reaction due to the similarity between the two arboviruses. Considering that electrochemical immunosensors are well-established, sensitive, and practical tools for diagnosis, in this study we developed a sensor platform with intrinsic redox activity that facilitates measurement readouts. Prussian blue (PB) has a great ability to form electrocatalytic surfaces, dispensing redox probe solutions in voltammetric measurements. Herein, PB was incorporated into a chitosan–carbon nanotube hybrid, forming a nanocomposite that was drop-casted on a screen-printed electrode (SPE). The immunosensor detected the envelope protein of ZIKV in a linear range of 0.25 to 1.75 µg/mL (n = 8, p < 0.01), with a 0.20 µg/mL limit of detection. The developed immunosensor represents a new method for electrochemical measurements without additional redox probe solutions, and it is feasible for application in point-of-care diagnosis. [ABSTRACT FROM AUTHOR]

Abstract Background Glaesserella parasuis (G. parasuis) is an influential pathogen of the pig, which induces high morbidity and mortality in naive pig populations in the pig industry. Accurate and rapid detection of the agent is important for disease control. In this study, a simple recombinase polymerase amplification (RPA) with a Lateral flow (LF) strip (RPA-LF-GPS) was developed to detect G. parasuis. Results The RPA-LF-GPS can specifically detect G. parasuis a limit of 100 CFU from other common related pathogens causing arthritis in the pig. The RPA-LF-GPS assay can use boiled synovial fluid samples as a template with the same sensitivity as other DNA extraction methods. In the detection of clinic positive synovial fluid sample, RPA-LF-GPS is equally sensitive (98.1%) compared with that of PCR (90.4%) (P > 0.05). The whole procedure of the RPA-LF-GPS assay could be finished in 1 hour without professional equipment. Conclusions RPA-LF-GPS assay is a rapid and simple method for point-of-care diagnostic testing for G. parasuis infection.

Background & objectives: Due to the absence of specific drugs or vaccines for Ebola virus disease, rapid, sensitive and reliable diagnostic methods are required to control the transmission chain of the disease and for better patient management. Isothermal amplification of nucleic acids has emerged as a promising alternative in which rapid and efficient amplification is achieved at a constant temperature without the thermal cycling required in PCR. Methods: A one-step single-tube accelerated quantitative reverse trascription loop-mediated isothermal amplification (RT-LAMP) assay was developed by targeting the NP gene of 2014 Zaire Ebola virus (ZEBOV). The RT-LAMP assay was found to be specific for ZEBOV, without having any cross-reactivity with related haemorrhagic fever viral agents. Results: The comparative evaluation of Ebola virus NP gene-specific RT-LAMP assay with reverse transcription (RT) - PCR and TaqMan real-time RT-PCR demonstrated that RT-LAMP was 10-1000 folds more sensitive than TaqMan real-time RT-PCR and conventional RT-PCR, respectively, with a detection limit of 1 copy number. In the absence of real-world clinical samples, the feasibility of Ebola virus RT-LAMP assay for clinical diagnosis was evaluated with different body fluids including serum, urine, saliva, semen and stool samples from healthy human volunteers spiked with gamma-irradiated ZEBOV 2014 obtained from Robert Koch Institute, Berlin, Germany, through the European Network for Diagnostics of Imported Viral Diseases. The Ebola virus RT-LAMP assay could correctly be picked up the spiked samples up to 1 copy of viral RNA without having any matrix interference. The monitoring of gene amplification can also be visualized with the naked eye by using SYBR Green I fluorescent dye. Interpretation & conclusions: Thus, due to easy operation without a requirement of sophisticated equipment and skilled personnel, the RT-LAMP assay reported here is a valuable tool as a point-of-care diagnosis for the rapid and real-time detection of Ebola virus in resource-limited healthcare settings of developing countries. [ABSTRACT FROM AUTHOR]