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Loop-Mediated Isothermal Amplification (LAMP) is a useful technique for detecting infectious microorganisms in human fluids since it performs similarly to conventional PCR, the results are obtained faster and no thermocyclers or complex devices are required. Since only two isothermal blocks (95 °C to lyse cells and 65 °C for DNA amplification) are needed, LAMP is particularly suited for applications in Low- and Middle-Income Countries (LMICs). To validate such assumption, we first designed and tested Arduino-controlled LAMP thermoblocks to process a considerable number of samples simultaneously with a low-energy consumption to enable routine use under worst-case conditions (no main power source and low ambient temperatures). The thermoblocks were tested when battery-powered at temperature down to 5 °C, showing high stability in well temperatures (<0.8 °C). The charge required for both thermoblocks to simultaneously achieve the target temperatures after switching on and to keep their working temperatures were 4.1 A·h and 2.4 A·h/h, respectively. Second, we implemented a low-cost viewer with LEDs and filters to detect the fluorescent LAMP reaction. All the components required for the instrument are for general purpose and readily available by e-commerce. Thus, the LAMP device allows for considerable autonomy by using a typical car battery in rural and itinerant healthcare or field hospitals in LMICs, even under difficult environmental conditions.

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As a promising liquid biopsy biomarker, exosomes have demonstrated great potential and advantages in the noninvasive tumor diagnosis. However, an accurate and sensitive method for tumors-associated exosomes detection is scarce. Herein, we presented an easy-operation aptasensor which simultaneously detect multiple exosomal proteins by using multicolor fluorescent DNA nanoassemblies (FDNs) and CD63 aptamer-modified magnetic beads (MNPs-AptCD63). In this system, the FDNs were firstly constructed by encapsulating different quantum dots (QDs) into rolling circle amplification (RCA) products that contained different aptamer sequences. Thus, the FDNs could selectively recognize the different exosomal proteins captured by the MNPs-AptCD63, and achieve the multiplex and sensitive detection according to the fluorescence of QDs. Benefiting from the signal amplification capacity and high selectivity of FDNs, this aptasensor not only could detect exosomes as low as 650 particles/µL, but also showed accurate analysis in clinical samples. In addition, we can also achieve point-of-care testing (POCT) due to the simple analysis steps and naked-eye observable fluorescence of QDs under the ultraviolet irradiation. We believe that our aptasensor could provide a promising platform for exosomes-based personalized diagnosis and precise monitoring of human health.

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Pathogens and contaminants in food and the environment present significant challenges to human health, necessitating highly sensitive and specific diagnostic methods. Traditional approaches often struggle to meet these requirements. However, the emergence of the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) system has revolutionized nucleic acid diagnostics. The present review provides a comprehensive overview of the biological sensing technology based on the CRISPR/Cas system and its potential applications in public health-related analysis. Additionally, it explores the enzymatic cleavage capabilities mediated by Cas proteins, highlighting the promising prospects of CRISPR technology in addressing bioanalysis challenges. We discuss commonly used CRISPR-Cas proteins and elaborate on their application in detecting foodborne bacteria, viruses, toxins, other chemical pollution, and drug-resistant bacteria. Furthermore, we highlight the advantages of CRISPR-based sensors in the field of public health-related analysis and propose that integrating CRISPR-Cas biosensing technology with other technologies could facilitate the development of more diverse detection platforms, thereby indicating promising prospects in this field.

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Vascular diseases are a leading cause of death and disability worldwide. Despite having precursor conditions like peripheral arterial disease (PAD), they are often only diagnosed after the onset of stroke or heart attack. Low-cost, portable, noninvasive, point-of-care (POC), label-free assessment of deep vascular function benefits PAD diagnosis, especially in resource poor settings of the world. Doppler ultrasound-based blood flow measurements can diagnose PAD, albeit with limited sensitivity and specificity. To overcome this, here, we propose the first-of-its-kind dual-modality photoacoustic-and-ultrasound (PAUS) imaging system that integrates a multiwavelength pulsed laser diode (PLD) with a compact ultrasound data acquisition unit. The mesoscopic imaging depth of the portable PLD-PAUS system was validated using tissue phantoms, and its multispectral photoacoustic imaging capabilities were validated using an atherosclerosis-mimicking phantom. Furthermore, we demonstrated high-contrast volumetric in vivo photoacoustic imaging of rodent abdominal vasculature and quantified vessel reactivity due to hypercapnia stimulation. The multiparametric functional and molecular imaging capabilities of the PLD-PAUS system holds promise for POC applications.

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Small extracellular vesicle-derived microRNAs (sEV-miRNAs) have emerged as promising noninvasive biomarkers for early cancer diagnosis. Herein, we developed a molecular probe based on three-dimensional (3D) multiarmed DNA tetrahedral jumpers (mDNA-Js)-assisted DNAzyme activated by Na+, combined with a disposable paper-based electrode modified with a Zr-MOF-rGO-Au NP nanocomplex (ZrGA) to fabricate a novel biosensor for sEV-miRNAs Assay. Zr-MOF tightly wrapped by rGO was prepared via a one-step method, and it effectively aids electron transfer and maximizes the effective reaction area. In addition, the mechanically rigid, and nanoscale-addressable mDNA-Js assembled from the bottom up ensure the distance and orientation between fixed biological probes as well as avoid probe entanglement, considerably improving the efficiency of molecular hybridization. The fabricated bioplatform achieved the sensitive detection of sEV-miR-21 with a detection limit of 34.6 aM and a dynamic range from100 aM to 0.2 µM. In clinical blood sample tests, the proposed bioplatform showed results highly consistent with those of qRT-PCRs and the signal increased proportionally with the NSCLC staging. The proposed biosensor with a portable wireless USB-type analyzer is promising for the fast, easy, low-cost, and highly sensitive detection of various nucleic acids and their mutation derivatives, making it ideal for POC biosensing.

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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.

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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.

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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 CeO2@Au nanoparticles. The CeO2@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 CeO2@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.

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BACKGROUND: Distinguishing between allergic and nonallergic forms of Contact Dermatitis (CD) is challenging and requires investigations based on patch-testing. Early detection of allergy biomarkers in active CD lesions could refine and simplify the management of CD patients. OBJECTIVE: To characterize the molecular signatures of active CD lesions. METHODS: We studied the expression of 12 allergy biomarkers by qRT-PCR in active lesions of 38 CD patients. Allergic CD (ACD) was diagnosed based on patch test (PT) results and exposure assessment. Molecular signatures of active lesions, as well as positive PT reactions, were compared with those of reference chemical allergens and irritants. RESULTS: Nineteen of the 38 CD patients reacted positively upon patch-testing and exposure assessment confirmed ACD diagnosis for 17 of them. Gene profiling of active CD lesions revealed 2 distinct molecular patterns: patients harboring signatures similar to reference allergens (n = 23) or irritants (n = 15). Among the 23 patients with an "allergy signature," we found the 17 patients with confirmed ACD, while no culprit allergen was identified for the 6 other patients. Interestingly, the 15 patients without biomarker induction had negative PT, suggesting that they developed nonallergic CD reactions. CONCLUSION: Molecular signatures from active skin lesions may help to stratify CD patients and predict those suffering from ACD.

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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.

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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 (KMnO4/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.

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SETTING: The diagnosis of Buruli ulcer (BU) is frequently made by experienced health workers in rural regions. This leads to long turnaround times to confirm the diagnosis as it requires specialised laboratory infrastructure to perform confirmatory testing. BACKGROUND: Given the lack of success with protein antigens to detect BU in human sera, the aim of this study was to evaluate a range of single synthetic lipid antigens using an enzyme-linked immunosorbent assay (ELISA). The ELISA system used was initially developed to detect TB using single synthetic lipid antigens. METHODS: Thirty polymerase chain reaction (PCR) positive BU samples and 30 PCR-negative healthy contact samples collected from Asante Akim North and Ahafo Ano North Districts, Ghana, that are endemic for BU between 2013 and 2016 were used to evaluate the synthetic lipid antigen ELISA. A Quantikine ELISA was also conducted on a randomly blinded sub-set of 30 samples. RESULTS: The synthetic lipid ELISA evaluated here outperforms all other ELISA tests using protein antigens to detect BU to date and has shown potential as a fast (2 h) test for BU which may be adapted for use at the point of care. A sensitivity of 63% and specificity of 80% was observed for 30 BU-positive and 30 BU-negative samples, with significantly reduced interleukin-8 (IL-8) levels in a subset of patients with BU. CONCLUSION: A single lipid was shown for the first time to have the ability to distinguish between PCR-positive BU and negative sera using ELISA. The low lipid antibody load detected may be a result of immune suppression caused by the presence of mycolactone in patients with BU, given that levels of IL-8 were significantly reduced in patients with BU compared to the control serum samples.

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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.

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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.

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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.

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[This corrects the article DOI: 10.3389/fphar.2023.1166923.].

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The COVID-19 pandemic has taken the world by surprise and people and organisations worldwide worked in some way or the other to combat the spread; isolate from the infected and get back to normal life, as it was before the pandemic hit. In this regard, the diagnosis of COVID-19 was at the centre of control and prevention and have seen a vehement change in every aspect, especially development of point-of-care testing for better and quick diagnosis. Among different types of techniques developed, the most important was the RT-PCR method of detection which detects nucleic acid of the virus in samples. RT-PCR is a laboratory-based method requiring trained professionals and precise steps for accurate testing. With the advent and spread of the pandemic, number of RT-PCR diagnostic centres rose significantly, and the detection process became less cumbersome, easy to use, ability to handle large volume of samples, more accurate, less time-consuming, and cost-effective. Different industries developed RT-PCR kits, reducing the efforts to prepare laboratory samples. Machines were employed for labour-driven tasks in PCR testing. In addition, new age technologies such as artificial intelligence, IoT, digital systems were combined with RT-PCR for accurate and easy testing. In this review, point-of-care RT-PCR methods, when the COVID-19 started, and the methods now, has been compared on the basis of technological advancements.

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Alpha-fetoprotein (AFP) is an important protein biomarker of liver cancer, as its serum levels are highly correlated with the progression of disease. Conventional immunoassays for AFP detection rely on enzyme-linked immunosorbent assay analyses with expensive and bulky equipment. Here, we developed a simple, affordable, and portable CRISPR-powered personal glucose meter biosensing platform for quantitative detection of the AFP biomarker in serum samples. The biosensor takes advantage of the excellent affinity of aptamer to AFP and the collateral cleavage activity of CRISPR-Cas12a, enabling sensitive and specific CRISPR-powered protein biomarker detection. To enable point-of-care testing, we coupled invertase-catalyzed glucose production with the glucose biosensing technology to quantify AFP. Using the developed biosensing platform, we quantitatively detected AFP biomarker in spiked human serum samples with a detection sensitivity of down to 10 ng/mL. Further, we successfully applied the biosensor to detect AFP in clinical serum samples from patients with liver cancer, achieving comparable performance to the conventional assay. Therefore, this novel CRISPR-powered personal glucose meter biosensor provides a simple yet powerful alternative for detecting AFP and potentially other tumor biomarkers at the point of care.

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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.

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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.

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