Muhammad Asad Ullah Khalid

Single‐reaction multiplex nucleic acid testing (NATs) must both co‐amplify multiple targets and distinguish the resulting amplicons within the product mixture. Most conventional assays achieve this distinction using target‐specific reporters or other external encoding elements. Here, we introduce a primer‐programmed multiplexing strategy in which primer design generates target‐associated amplicons with predefined lengths that are read electrically with nanopores. In a proof‐of‐concept implementation, one‐pot recombinase polymerase amplification (RPA) generated four amplicons with predefined product lengths from Monkeypox (Mpox), Cowpox, Vaccinia, and human RNase P. The resulting amplicons were analyzed with glass nanopores, and individual translocation events were classified by a deep neural network (DNN) based on a six‐dimensional feature set derived from nanopore recordings. Event‐level decoding achieved >95% accuracy, >94.7% sensitivity, and >98.3% specificity, and aggregation of classified events yielded sample‐level calls with >93.3% sensitivity and >93.9% specificity in blinded samples, with qualitative calls matching polymerase chain reaction (PCR) results across the 9 PCR‐benchmarked samples. These results demonstrate a probe‐free multiplexing architecture in which target identity is programmed through primer‐defined amplicon design and decoded from the resulting multidimensional nanopore response using a DNN, eliminating the need for target‐specific reporter chemistry and opening new opportunities for all‐electrical multiplex nucleic acid testing based on unmodified amplification products.

DNA sequencing is a powerful tool for diagnosing conditions such as infectious diseases and cancers. Even though current workflows demand rigorous quality control (QC) of DNA samples, this QC is typically limited to lab settings despite recent advances in portable nanopore sequencers. For personalized health care to truly benefit from the portable sequencer, QC must be performed right where the samples are processed. Here, we present a solid-state nanopore device that provides label-free, controlled quantification and qualification of cell-free DNA (cfDNA). We demonstrated the use of a 1 kbp double-stranded DNA internal marker at a known concentration to measure the concentration of a representative cfDNA target in the presence of genomic DNA. We also found that nanopores with diameters ranging from 6 to 19 nm yield consistent measurements with a maximum coefficient of variation (CV) of less than 15%. Moreover, analyzing data from multiple nanopores over longer acquisition times can reduce the uncertainty to below 10% CV. Finally, we applied our nanopore QC assay to a plasma cfDNA sample and compared the results to those from a capillary electrophoresis (CE) assay. Both methods produced highly correlated measurements, demonstrating the potential of our nanopore QC assay for effective cfDNA assessment at the point of need.

Recombinase Polymerase Amplification (RPA) is a rapid, sensitive, and isothermal method for nucleic acid amplification that has gained widespread use in diagnostic applications. While well-suited for single-target assays, extending RPA to multiplex detection remains technically challenging despite the multiplexed RPA being essential for tasks such as differential diagnosis and inclusion of internal controls. Existing multiplex RPA strategies rely on proprietary probes (e.g., Exo, Fpg, Nfo), which require complex design, are susceptible to cross-reactivity, and often depend on sophisticated optical instrumentation, limiting their scalability. To address these limitations, here, we developed a probe-free multiplex RPA assay that distinguishes targets by amplicon length, using solid-state nanopore detection of single molecules and deep neural network (DNN)-based classification. Using the Monkeypox (Mpox) as a model, we designed and validated a multiplex RPA assay targeting both the Mpox gene and the human RNase P gene as an internal control. We systematically evaluated the nanopore size requirement for detecting DNA amplicons ranging from 50 to 500 base pairs and found that ∼7 nm pores provided optimal performance for amplicons between 75 and 500 bp, balancing high event rates without pore clogging. Using single-molecule translocation events as input, we trained and optimized a DNN to classify amplicons by target. The model achieved 94.2% accuracy at the single-molecule event level, which translated to 100% accuracy at the population-level target call. While our current system demonstrates duplex detection, the strategy is inherently scalable to higher multiplex levels. These findings establish a new framework for multiplex RPA that eliminates the need for complex probe design and optical detection, paving the way for robust, scalable, and accessible molecular diagnostics.

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.

DNA sequencing is a powerful tool for diagnosing conditions like infectious diseases and cancers. Even though current workflows demand rigorous quality control (QC) of DNA samples, this QC is typically limited to lab settings, despite recent advances in portable nanopore sequencers. For personalized healthcare to truly benefit from the portable sequencer, QC must be performed right where the samples are processed. Here, we present a solid-state nanopore device that provides label-free, controlled quantification and qualification of cell-free DNA (cfDNA). We demonstrated the use of a 1 kbp double-stranded DNA internal marker at a known concentration to measure the concentration of a representative cfDNA target in the presence of genomic DNA. We also found that nanopores with diameters ranging from 6 to 19 nm yield consistent measurements, with a maximum coefficient of variation (CV) of less than 15%. Moreover, analyzing data from multiple nanopores over longer acquisition times can reduce the uncertainty to below 10% CV. Finally, we applied our nanopore QC assay to a plasma cfDNA sample and compared the results with those from a capillary electrophoresis (CE) assay. Both methods produced highly correlated measurements, demonstrating the potential of our nanopore QC assay for effective cfDNA assessment at the point of need. Table of Contents Figure:

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 (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.

The global health threat posed by the Monkeypox virus (Mpox) requires swift, simple, and accurate detection methods for effective management, emphasizing the growing necessity for decentralized point-of-care (POC) diagnostic solutions. The clustered regularly interspaced short palindromic repeats (CRISPR), initially known for its effective nucleic acid detection abilities, presents itself as an attractive diagnostic strategy. CRISPR offers exceptional sensitivity, single-base specificity, and programmability. Here, we reviewed the latest developments in CRISPR-based POC devices and testing strategies for Mpox detection. We explored the crucial role of genetic sequencing in designing crRNA for CRISPR reaction and understanding Mpox transmission and mutations. Additionally, we showed the integration of CRISPR-Cas12 strategy with pre-amplification and amplification-free methods. Our study also focused on the significant role of Cas12 proteins and the effectiveness of Cas12 coupled with recombinase polymerase amplification (RPA) for Mpox detection. We envision the future prospects and challenges, positioning CRISPR-Cas12-based POC devices as a frontrunner in the next generation of molecular biosensing technologies.

Soft (software) sensors are widely used in modern industrial manufacturing and processing units due to their advantages over traditional only hardware-based sensing devices. They rely on the process or plant data, which is carefully selected with high quality. This data is processed in computer software algorithms often termed as “soft sensor models.” A few of the traditional data-driven soft sensor models have also been reviewed. While soft sensors are very common in various industrial sectors, their applications in bioprocess monitoring and control still need to be expanded and are rarely discussed. This work highlights some previously reported key soft sensor approaches for various bioprocesses, mainly focusing on their future needs in bioreactors on-chip devices. Then a brief conclusion of the subject in discussion is provided at the end with the authors’ perspective on the deep learning revolution in soft sensors and future smart bioprocess monitoring systems.

Sensing devices have shown tremendous potential for monitoring state-of-the-art organ chip devices. However, challenges like miniaturization while maintaining higher performance, longer operating times for continuous monitoring, and fabrication complexities limit their use. Herein simple, low-cost, and solution-processible inkjet dispenser printing of embedded electrochemical sensors for dissolved oxygen (DO) and reactive oxygen species (ROS) is proposed for monitoring developmental (initially normoxia) and induced hypoxia in a custom-developed gut bilayer microfluidic chip platform for 6 days. The DO sensors showed a high sensitivity of 31.1 nA L mg⁻¹ with a limit of detection (LOD) of 0.67 mg L⁻¹ within the 0–9 mg L⁻¹ range, whereas the ROS sensor had a higher sensitivity of 1.44 nA μm⁻¹ with a limit of detection of 1.7 μm within the 0–300 μm range. The dynamics of the barrier tight junctions are quantified with the help of an in-house developed trans-epithelial–endothelial electrical impedance (TEEI) sensor. Immunofluorescence staining was used to evaluate the expressions of HIF-1α and tight junction protein (TJP) ZO-1. This platform can also be used to enhance bioavailability assays, drug transport studies under an oxygen-controlled environment, and even other barrier organ models, as well as for various applications like toxicity testing, disease modeling and drug screening.