Tracking the rapid spread of infectious diseases such as COVID-19 in communities poses a significant global health challenge, mainly because traditional testing methods struggle to detect asymptomatic carriers and effectively manage outbreaks. To address this, we present a scalable, networked molecular testing framework for wastewater-based epidemiology that, for the first time, integrates portable nucleic acid testing (NAT) devices with long-range wireless communication and cloud-based data management. Each battery-powered node can perform Reverse Transcription - Loop-mediated Isothermal Amplification (RT-LAMP), a rapid nucleic acid amplification method, on-site directly on raw wastewater samples for SARS-CoV-2 detection. A node executes up to eight independent tests on a microfluidic cartridge, monitoring fluorescence signals from the RT-LAMP reactions in real time and transmitting the results via long-range radio (LoRa) to a gateway, which uploads structured data to a NoSQL cloud database. An interactive dashboard enables bidirectional communication with nodes and provides spatiotemporal visualization of both live and historical data, including hotspot identification through geotagging and mapping of metrics such as 7-day positivity rates. Rigorous system testing with four nodes under varied transmission scenarios confirmed a packet delivery ratio exceeding 99%, ensuring accurate and reliable reconstruction of time-series RT-LAMP curves. By integrating NAT with IoT communication and cloud analytics, this system supports unattended, long-term wastewater monitoring and can provide public health officials with timely, actionable insights. This framework demonstrates the potential of IoT-enabled molecular testing to advance decentralized surveillance beyond conventional laboratory-based workflows.

Abstract Effective antiretroviral therapy has transformed HIV into a manageable chronic condition, provided viral suppression is maintained through routine viral load (VL) monitoring. Access to frequent VL testing remains limited, particularly outside centralized clinical settings. Decentralized and at-home HIV VL testing requires low-volume systems designed for patient operation. These systems must distinguish true viral suppression from test failure. Many approaches lack internal process verification, which makes negative results ambiguous. Here, we present ViraLite, an ultracompact, battery-powered HIV VL monitoring system that integrates reverse transcription loop-mediated isothermal amplification (RT-LAMP) with an RNase P internal process control, machine learning–assisted fluorescence analysis for one-pot multiplexing, and smartphone-guided operation. We evaluated ViraLite using 45 clinically archived plasma samples and benchmarked it against reverse transcription quantitative polymerase chain reaction (RT-qPCR). The internal process control identified 17 inconclusive tests that would otherwise be misclassified as negative. Among valid tests, ViraLite achieved 93.3% sensitivity and 100% specificity versus RT-qPCR. ViraLite enables decentralized HIV VL testing with low sample volume and interpretable negative results. This capability can expand monitoring beyond traditional clinic workflows and addresses a key barrier to patient-operated VL testing.

Most children with SARS-CoV-2 have mild or asymptomatic symptoms, but some develop severe complications. Early identification of high-risk cases is crucial for timely intervention. Alterations in salivary microRNA (miRNA) levels serve as biomarkers for severity prediction. However, a rapid, non-invasive method is needed to quantify miRNA level changes as an alternative to sequencing. Here, we developed a highly specific and sensitive ligation-recombinase polymerase amplification (RPA) assay for quantifying severe and non-severe miRNAs on a portable platform. The assay begins with a miRNA-templated annealing and ligation-RPA reaction of miR-1273, miR-296, and miR-29. We quantified 100 pM to 1 fM, resolving 1 fM, with 100% specificity. Next, we validated portable extraction against benchtop extraction, achieving R-square > 0.85 and r > 0.92 in clinical samples. Finally, testing 154 clinical samples revealed severe miRNA downregulation compared to non-severe cases. The assay achieved high diagnostic accuracy with an AUC of 0.98. This platform enables informed clinical decisions and optimizes resources, especially in resource-limited settings.

HIV and HCV Co-infection continues to be a significant public health problem globally especially within high-risk groups. Monitoring viral loads with precision helps direct treatment choices and measure treatment success while preventing resistance to drugs. Traditional laboratory-based testing faces limitations due to restricted accessibility and dependence on centralized facilities along with the complex process of quantifying both HIV and HCV viral loads which impedes worldwide control measures for these viruses. We developed a portable self-testing device that measures HIV and HCV viral loads from a 100 μL finger-prick blood sample at the same time. The system combines RNA extraction with rapid multiplex RT-PCR to provide semi-automated testing capabilities and generate results in under 1 h. The system extracts RNA at 80 % efficiency and employs a three-channel optical detection system that detects as low as 5 copies per reaction while delivering high sensitivity and accuracy. Validated studies found a robust connection with Bio-Rad benchtop systems (R2 = 0.97-0.99) which verified that detection sensitivity and accuracy matched standard laboratory testing standards. The testing device enables parallel processing of four patients which results in enhanced efficiency and access to testing services. Individuals affected by HIV/HCV co-infection can use this self-testing solution to track their viral loads on their own to enable prompt treatment changes and lower transmission risks. The technology delivers an effective self-monitoring option for viral load management through its combination of precision, portability and an easy-to-use design which advances HIV and HCV treatment outcomes.

Loop-mediated isothermal amplification (LAMP) is a promising method for point-of-care nucleic acid testing due to its simplicity, rapidity, and high sensitivity. Coupling LAMP with solid-state nanopores enables label-free, single-molecule sensing, enhancing diagnostic accuracy. However, conventional LAMP-coupled nanopore protocols require high-salt buffers (>1 M) to improve signal strength and translocation frequency, complicating workflows and increasing contamination risks. In native LAMP buffers (50 mM KCl), electroosmotic flow (EOF) hinders amplicon transport in sub-10 nm pores, while large amplicons increase the risk of clogging. These challenges limit event rates, data throughput, and device reliability. To address these limitations, we developed a glass nanopore device optimized for direct sensing of amplicons in native buffers, featuring integrated declogging capabilities. Our results revealed that 200 nm pores provided the best balance between minimizing EOF interference and maintaining strong signal strength, achieving the highest event rates. Smaller pores (<100 nm) had low event rates due to EOF effects, while larger pores (>1 μm) showed weakened signal strength. We discovered that clogging in low-salt conditions differs from high-salt environments, with physical vibration effectively resolving clogging in low-salt settings. This led to the integration of an automated vibration motor, extending nanopore lifespan and ensuring continuous data acquisition. Our clog-free, native-buffer sensing platform demonstrated a sensitivity of 0.12 parasite/μL using Plasmodium vivax (P. vivax) as a model organism, exceeding the threshold for detecting asymptomatic infections. These advancements highlight the potential of our nanopore device for rapid, reliable, and user-friendly diagnostics for point-of-care testing.

Respiratory viral infections pose a significant global public health challenge, partly due to the difficulty in rapidly and accurately distinguishing between viruses with similar symptoms at the point of care, hindering timely and appropriate treatment and limiting effective infection control and prevention efforts. Here, we developed a multiplexed, non-invasive saliva-based, reverse transcription loop-mediated isothermal amplification (RT-LAMP) test that enables the simultaneous detection of three of the most common respiratory infections, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), Influenza (Flu), and respiratory syncytial virus (RSV), in a single reaction via specific probes and monitored in real-time by a machine-learning-enabled compact analyzer. Our results demonstrate that the multiplexed assay can effectively detect three target RNAs with high accuracy. Further, testing with spiked saliva samples showed strong agreement with reverse transcription polymerase chain reaction (RT-PCR) assay, with area under the curve (AUC) values of 0.82, 0.93, and 0.96 for RSV, Influenza, and SARS-CoV-2, respectively. By enabling the rapid detection of respiratory infections from easily collected saliva samples at the point of care, the device presented here offers a practical and efficient tool for improving outcomes and helping prevent the spread of contagious diseases.

Solid-state nanopores, known for their label-free detection and operational simplicity, face challenges in accurately sizing the short nucleic acids due to fast translocation and a lack of enzyme-based control mechanisms as compared to their biological counterparts with sizing resolutions still limited to ≥100 bp. Here, we present a facile polyethylene glycol-dimethacrylate (PEG-DMA) hydrogel interfaced glass nanopore (HIGN) system by inserting glass nanopore into the hydrogel to achieve sub-100 base pair (bp) resolution in short DNA sizing analysis. We systematically investigated the effects of hydrogel mesh size, spatial configurations of glass nanopores about the hydrogel, applied bias voltage, and analyte concentration on the transport dynamics of 200 bp double-stranded DNA (dsDNA). A 7.5 w/v% PEG-DMA hydrogel induced ∼11x increase in the mean dwell times compared with bare solution nanopore system. The insertion locations and depths of the glass nanopore into the hydrogel resulted in 7.16% and 5.28% coefficients of variation (CV) for mean normalized event frequencies. This enhancement of dwell times and invariability in translocation characteristics enables precise sizing of dsDNA fragments under 400 bp using HIGN, with an achieved size resolution of 50 bp with observable mean normalized peak amplitude (ΔI/Io) of ∼0.005. Furthermore, we have demonstrated the capability of HIGN to perform multiplex detection of influenza A virus (IAV) and severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) through reverse transcriptase-polymerase chain reaction (RT-PCR). These results demonstrated the potential of HIGN as a versatile tool in nucleic acid analysis and multiplexed label-free molecular diagnostics.

While most children with COVID-19 experience mild symptoms or remain asymptomatic, some may develop severe complications. Early identification of children at risk for severe outcomes is essential to ensuring timely and effective intervention. Recent studies have identified alterations in salivary microRNA (miRNA) expression levels as promising biomarkers for predicting severe complications in children. However, there remains a need for a rapid, noninvasive, and quantitative method to detect miRNA expression level changes, as their upregulation or downregulation serves as a hallmark of various diseases, providing an alternative to sequencing-based methods. Here, we developed a highly specific and sensitive ligation-coupled recombinase polymerase amplification (RPA) assay for quantitatively detecting multiplex severe and nonsevere miRNAs on a portable platform. The assay begins with an miRNA-templated annealing and ligation reaction of miR-1273, miR-296, and miR-29, followed by an RPA reaction. We quantified 100 pM to 1 fM, resolving 1 fM, with 100% specificity. Next, we validated portable extraction against benchtop extraction, achieving R- 2 > 0.85 and r > 0.92 in clinical samples. Finally, testing 154 clinical samples revealed severe miRNA downregulation compared to nonsevere cases. The assay achieved high diagnostic accuracy with an area under the curve (AUC) of 0.98. This platform would empower clinicians to make informed decisions, optimize resource allocation, and improve outcomes, particularly in point-of-care (POC) settings.

The availability of effective antiretroviral therapy has made HIV manageable, provided patients have consistent access to routine viral load (VL) testing. Nonetheless, access to frequent VL testing remains limited. There is a need for accessible, user-friendly testing systems that allow people living with HIV (PLHIV) to monitor their VL more frequently and empower self-management. Here, we developed ViraLite, a sample-to-answer, compact, accessible, and battery-powered system for HIV viral load monitoring. The system is built upon a probe-based RT-LAMP assay that allows for multiplexed detection and quantification. An internal quality control targeting the RNase P was incorporated to enhance the reliability of the results. A software-reconfigurable real-time sensing system empowered by machine learning and a smartphone-guided protocol was developed in tandem to analyze the multiplexed assay. We analyzed 45 clinically archived samples using ViraLite and benchmarked our results against qRT-PCR, which showed 21 positive and 23 negative samples. Using our process control, ViraLite first identified 17 inconclusive samples that would otherwise be classified as negative. Then, ViraLite classified 14 out of 15 HIV-positive samples (93.3%) and 13 out of 13 HIV-negative samples (100%). The incorporation of RNase P as a process control increased the sensitivity of ViraLite from 66.66% to 93.33%, while maintaining a high specificity (100%). To assess the acceptance of ViraLite among PLHIV, we recruited 480 participants from online and three clinical sites to complete a survey. Over 86% of participants indicated ViraLite had benefits in convenience and privacy, on the other hand 61% of participants indicated concerns with test accuracy. The integration of compact hardware, a reliable assay, and smartphone guidance provides an accurate, easy to use system for PLHIV to self-manage their viral load and update their prescriptions frequently.

There is a significant demand for multiplexed fluorescence sensing and detection across a range of applications. Yet, the development of portable and compact multiplexable systems remains a substantial challenge. This difficulty largely stems from the inherent need for spectrum separation, which typically requires sophisticated and expensive optical components. Here, we demonstrate a compact, lens-free, and cost-effective fluorescence sensing setup that incorporates machine learning for scalable multiplexed fluorescence detection. This method utilizes low-cost optical components and a pretrained machine learning (ML) model to enable multiplexed fluorescence sensing without optical adjustments. Its multiplexing capability can be easily scaled up through updates to the machine learning model without altering the hardware. We demonstrate its real-world application in a probe-based multiplexed Loop-Mediated Isothermal Amplification (LAMP) assay designed to simultaneously detect three common respiratory viruses within a single reaction. The effectiveness of this approach highlights the system's potential for point-of-care applications that require cost-effective and scalable solutions. The machine learning-enabled multiplexed fluorescence sensing demonstrated in this work would pave the way for widespread adoption in diverse settings, from clinical laboratories to field diagnostics.

The human immunodeficiency virus (HIV) remains a major global health concern for which accurate viral load monitoring is essential for the management of HIV infection. The advent of antiretroviral therapy (ART) has transformed once-fatal HIV disease into a manageable chronic condition that now makes the need for VL testing which aims to satisfy international suppression targets 95-95-95 al l the more essential. Therefore, considering the complexity and diversity of HIV infection, it is essential to develop rapid diagnostic technologies suitable for different clinical situations. Here, we report on a multiplexed PCR device developed for simple and efficient quantification of HIV-1 or HIV-2 viral loads using finger-pricked whole blood from rural decentralized settings. This device is comprised of a previously developed RNA extraction module combined with an optimized real-time PCR amplification system. Together, these combine to simultaneously detect and differentiate HIV-1 & 2; as well are adopting a testing control of RNase P allowing for full diagnostic analysis from one sample. Our device also includes an intuitive user interface and is completely autonomous so it can serve individuals in remote areas who are unfamiliar with the field of medical testing. They get the results in a very short time of around 70 min and hence save on testing times without leaving accuracy behind. The efficiency and effectiveness of the device were validated through the analysis of 30 clinical samples, yielding a sensitivity of 100% for both HIV-1 and HIV-2. The specificity was found to be 100% for HIV-1 and 90.91% for HIV-2, demonstrating high diagnostic accuracy. One of the most attractive things about this device is that it comes in comparison to all other counterparts. Given that you can run the assay for less than $10, it could be an economically viable way to use this as a broadscale test in regions where healthcare budgets don't allow others. Hence it is quite a useful device to aid HIV management in resource-limited settings, where conventional laboratory facilities are out of reach due its simplicity and affordability with rapid output. The point-of-care test is an effective, low-cost, high quality diagnostic tool-promoting rapid testing for HIV-inexpensively overcoming the barriers to efficient control of and care in resource-limited settings.

Loop-mediated isothermal amplification (LAMP) is a rapid, sensitive, and cost-effective method for developing point-of-care nucleic acid testing due to its isothermal nature. Yet, LAMP can suffer from the issue of false positives, which can compromise the specificity of the results. LAMP false positives typically arise due to contamination, nonspecific amplification, and nonspecific signal reporting (intercalating dyes, colorimetric, turbidity, etc.). While dye-labeled primers or probes have been introduced for multiplexed detection and enhanced specificity in LAMP assays, they carry the risk of reaction inhibition. This inhibition can result from the labeled primers with fluorophores or quenchers and probes that do not fully dissociate during reaction. This work demonstrated a nanopore-based system for probe-free LAMP readouts by employing amplicon sizing and counting, analogous to an electronic version of gel electrophoresis. We first developed a model to explore LAMP kinetics and verified distinct patterns between true and false positives via gel electrophoresis. Subsequently, we implemented nanopore sized counting and calibrated the event charge deficit (ECD) values and frequencies to ensure a fair analysis of amplicon profiles. This sized counting method, integrated with machine learning, achieved 91.67% accuracy for false positive discrimination, enhancing LAMP's reliability for nucleic acid detection.

Accurately diagnosing respiratory infections is paramount, particularly during public health crises such as the COVID-19 pandemic, which has stressed the necessity for rapid and reliable point-of-care testing (POCT) for nucleic acid detection. Our study introduces a lens-free optical system integrated with machine learning to streamline multiplexed nucleic acid tests in real-time, overcoming the limitations of spatial parallelization and optical filters found in conventional POCT devices. Through a neural network, our scalable approach efficiently differentiates between fluorescent signals from a mixture of fluorophores, improving detection and quantification capabilities. Moreover, it showcases adaptability in predicting the concentrations of different fluorophores and the concurrent detection of multiple pathogens, such as RSV, Influenza A, and SARS-CoV-2. Notably, our system has been validated with mock saliva samples, affirming its potential for accurate diagnostics in scenarios with limited sample volume and the need for flexible fluorophore deployment, contributing a robust solution for POCT applications.

Monkeypox virus (MPXV) poses a global health emergency, necessitating rapid, simple, and accurate detection to manage its spread effectively. The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) technique has emerged as a promising next-generation molecular diagnostic approach. Here, we developed a highly sensitive and specific CRISPR-Cas12a assisted nanopore (SCAN) with isothermal recombinase polymerase amplification (RPA) for MPXV detection. The RPA-SCAN method offers a sensitivity unachievable with unamplified SCAN while also addressing the obstacles of PCR-SCAN for point-of-care applications. We demonstrated that size-counting of single molecules enables analysis of reaction-time dependent distribution of the cleaved reporter. Our MPXV-specific RPA assay achieved a limit of detection (LoD) of 19 copies in a 50 μL reaction system. By integrating 2 μL of RPA amplifications into a 20 μL CRISPR reaction, we attained an overall LoD of 16 copies/μL (26.56 aM) of MPXV at a 95% confidence level using the SCAN sensor. We also verified the specificity of RPA-SCAN in distinguishing MPXV from cowpox virus with 100% accuracy. These findings suggest that the isothermal RPA-SCAN device is well-suited for highly sensitive and specific Monkeypox detection. Given its electronic nature and miniaturization potential, the RPA-SCAN system paves the way for diagnosing a wide array of other infectious pathogens at the point of care.

To enable faster, easier extraction of viral RNA outside of traditional laboratories, we use a custom, automated, and portable centrifuge for processing Qiagen QIAamp RNA extractions. Viral RNA is eluted into approx. 60 µl of nuclease free water that can then be analyzed by downstream testing (PCR, gel electro, Qubit). Our results showed very strong agreement with benchtop extractions and probit analysis demonstrated a limiting extraction concentration of 0.75 cp/µl at 95% confidence.

Nucleic acid testing (NAT) has revolutionized diagnostics by providing precise, rapid, and scalable detection methods for diverse biological samples. These recent advancements satisfy the increasing demand for on-site diagnostics, yet sample preparation remains a significant bottleneck for achieving highly sensitive diagnostic assays. There is an unmet need for compatible, efficient, and lab-free sample preparation for point-of-care NAT. To address this, we developed a portable, lab-free, and battery-powered device for extracting nucleic acids. We explored using low centrifugal forces with existing commercial chemistry, demonstrating excellent performance. We designed and tested a battery-powered device to enable lab-free extractions, and verified reagents stored out to 6 months, suggesting exceptional deployment capabilities. We evaluated our device, comparing our results against those from a benchtop centrifuge across three types of samples: HIV RNA in buffer, HIV RNA in plasma, and SARS-CoV-2 RNA in saliva. The portable device demonstrated excellent agreement with the benchtop centrifuge, indicating high reliability. By providing an effective on-site sample preparation solution, the widespread adoption of low centrifugal extractions will improve the sensitivity and reliability of NAT and will positively impact other point-of-care technologies such as next generation sequencing (NGS), biomarker detection, and environmental monitoring.

HIV is a major health issue in LMICs, necessitating precise viral load (VL) monitoring for effective management. Antiretroviral therapy (ART) has made HIV manageable, underscoring the need for effective VL testing to achieve the 95-95-95 targets. The diversity of HIV subtypes in LMICs calls for innovative diagnostic solutions. Here, we demonstrate a portable multiplex RT-PCR device designed for fast and cost-effective testing of HIV-1 and HIV-2 using finger-prick blood in LMICs. It features on-site RNA extraction and real-time multiplex RT-PCR targeting HIV-1, HIV-2, and an internal process control of RNase P. The cost per test is approximately $10, based on laboratory-scale material costs. The device offers a sample-to-answer turnaround time of less than 70 minutes. Our internally controlled device exhibited 100% sensitivity and specificity against HIV-1, and 100% sensitivity and 90.91 % specificity against HIV-2.

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World Health Organization's aim to eliminate malaria from developing/resource-limited economies requires easy access to low-cost, highly sensitive, and specific screening. We present a handheld nucleic acid testing device with on-chip automated sample preparation to detect malaria (Plasmodium falciparum) infection from a whole blood sample as a feasibility study. We used a simple two-reagent-based purification-free protocol to prepare the whole blood sample on a piezo pump pressure-driven microfluidic cartridge. The cartridge includes a unique mixing chamber for sample preparation and metering structures to dispense a predetermined volume of the sample lysate mixture into four chambers containing a reaction mix. The parasite genomic DNA concentration can be estimated by monitoring the fluorescence generated from the loop-mediated isothermal amplification reaction in real time. We achieved a sensitivity of ∼0.42 parasite/μL of whole blood, sufficient for detecting asymptomatic malaria parasite carriers.