Loop-Mediated Isothermal Amplification-Coupled Glass Nanopore Counting Toward Sensitive and Specific Nucleic Acid Testing.

Solid-state nanopores have shown great promise and achieved tremendous success in label-free single-molecule analysis. However, there are three common challenges in solid-state nanopore sensors, including the nanopore size variations from batch to batch that makes the interpretation of the sensing results difficult, the incorporation of sensor specificity, and the impractical analysis time at low analyte concentration due to diffusion-limited mass transport. Here, we demonstrate a novel loop-mediated isothermal amplification (LAMP)-coupled glass nanopore counting strategy that could effectively address these challenges. By using the glass nanopore in the counting mode (versus the sizing mode), the device fabrication challenge is considerably eased since it allows a certain degree of pore size variations and no surface functionalization is needed. The specific molecule replication effectively breaks the diffusion-limited mass transport thanks to the exponential growth of the target molecules. We show the LAMP-coupled glass nanopore counting has the potential to be used in a qualitative test as well as in a quantitative nucleic acid test. This approach lends itself to most amplification strategies as long as the target template is specifically replicated in numbers. The highly sensitive and specific sensing strategy would open a new avenue for solid-state nanopore sensors toward a new form of compact, rapid, low-cost nucleic acid testing at the point of care.

Arbitrarily Accessible 3D Microfluidic Device for Combinatorial High-Throughput Drug Screening.

Microfluidics-based drug-screening systems have enabled efficient and high-throughput drug screening, but their routine uses in ordinary labs are limited due to the complexity involved in device fabrication and system setup. In this work, we report an easy-to-use and low-cost arbitrarily accessible 3D microfluidic device that can be easily adopted by various labs to perform combinatorial assays for high-throughput drug screening. The device is capable of precisely performing automatic and simultaneous reagent loading and aliquoting tasks and performing multistep assays with arbitrary sequences. The device is not intended to compete with other microfluidic technologies regarding ultra-low reaction volume. Instead, its freedom from tubing or pumping systems and easy operation makes it an ideal platform for routine high-throughput drug screening outside traditional microfluidic labs. The functionality and quantitative reliability of the 3D microfluidic device were demonstrated with a histone acetyltransferase-based drug-screening assay using the recombinant Plasmodium falciparum GCN5 enzyme, benchmarked with a traditional microtiter plate-based method. This arbitrarily accessible, multistep capable, low-cost, and easy-to-use device can be widely adopted in various combinatorial assays beyond high-throughput drug screening.

Monitoring electron and proton diffusion flux through three-dimensional, paper-based, variable biofilm and liquid media layers.

The goal of this work is to pursue analytical approaches that elucidate electron and proton diffusion inside the Shewanella oneidensis biofilm and bulk liquid, which will inevitably promote the translation of Microbial Fuel Cell (MFC) technology for renewable, "green energy" solutions that are in demand to sustain the world's ever-increasing energy demands and to mitigate the depletion of current resources. This study provides a novel strategy for monitoring electron/proton fluxes in 3-D multi-laminate structures of paper as a scaffold to support S. oneidensis biofilms and bulk media liquid. Multiple layers of paper containing bacterial cells and/or media are stacked to form a layered 3-D model of the overall biofilm/bulk liquid construct. Mass transport of electrons and protons into this 3-D system can be quantified along with the exploration of microbial energy production. Assembly of a 3D paper stack can be modular and allows us to control the thickness of the overall biofilm/bulk liquid construct with the different diffusion distances of the electrons/protons through the stack. By measuring the current generated from the 3-D stack, the electron and proton diffusivity through biofilms were quantitatively investigated. We found that (i) the diffusion length of the electrons/protons in the S. oneidensis biofilm/bulk liquid is a determinant factor for the MFC performance, (ii) the electron transfer through the endogenous mediators of S. oneidensis can be a more critical factor to limit the current/power generation of the MFCs than the proton transfer in the MFC system and (iii) the thicker biofilm allows higher and longer current generation but requires more time to reach a peak current value and increases the total energy loss of the MFC system.

A paper-based microbial fuel cell array for rapid and high-throughput screening of electricity-producing bacteria.

There is a large global effort to improve microbial fuel cell (MFC) techniques and advance their translational potential toward practical, real-world applications. Significant boosts in MFC performance can be achieved with the development of new techniques in synthetic biology that can regulate microbial metabolic pathways or control their gene expression. For these new directions, a high-throughput and rapid screening tool for microbial biopower production is needed. In this work, a 48-well, paper-based sensing platform was developed for the high-throughput and rapid characterization of the electricity-producing capability of microbes. 48 spatially distinct wells of a sensor array were prepared by patterning 48 hydrophilic reservoirs on paper with hydrophobic wax boundaries. This paper-based platform exploited the ability of paper to quickly wick fluid and promoted bacterial attachment to the anode pads, resulting in instant current generation upon loading of the bacterial inoculum. We validated the utility of our MFC array by studying how strategic genetic modifications impacted the electrochemical activity of various Pseudomonas aeruginosa mutant strains. Within just 20 minutes, we successfully determined the electricity generation capacity of eight isogenic mutants of P. aeruginosa. These efforts demonstrate that our MFC array displays highly comparable performance characteristics and identifies genes in P. aeruginosa that can trigger a higher power density.

Advances in RT-LAMP for COVID-19 testing and diagnosis.

INTRODUCTION: The SARS-CoV-2 pandemic, and the subsequent limitations on standard diagnostics, has vastly expanded the user base of Reverse Transcription Loop-mediated isothermal Amplification (RT-LAMP) in fundamental research and development. RT-LAMP has also penetrated commercial markets, with emergency use authorizations for clinical diagnosis. AREAS COVERED: This review discusses the role of RT-LAMP within the context of other technologies like RT-qPCR and rapid antigen tests, progress in sample preparation strategies to enable simplified workflow for RT-LAMP directly from clinical specimens, new challenges with primer and assay design for the evolving pandemic, prominent detection modalities including colorimetric and CRISPR-mediated methods, and translational research and commercial development of RT-LAMP for clinical applications. EXPERT OPINION: RT-LAMP occupies a middle ground between RT-qPCR and rapid antigen tests. The simplicity approaches that of rapid antigen tests, making it suitable for point-of-care use, but the sensitivity nears that of RT-qPCR. RT-LAMP still lags RT-qPCR in fundamental understanding of the mechanism, and the interplay between sample preparation and assay performance. Industry is now beginning to address issues around scalability and usability, which could finally enable LAMP and RT-LAMP to find future widespread application as a diagnostic for other conditions, including other pathogens with pandemic potential.

Handheld Purification-Free Nucleic Acid Testing Device for Point-of-Need Detection of Malaria from Whole Blood.

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.

An Ultracompact Real-Time Fluorescence Loop-Mediated Isothermal Amplification (LAMP) Analyzer.

Low-cost access to the highly sensitive and specific detection of the pathogen in the field is a crucial attribute for the next generation point-of-care (POC) platforms. In this work, we developed a real-time fluorescence nucleic acid testing device with automated and scalable sample preparation capability for field malaria diagnosis. The palm-sized battery-powered analyzer equipped with a disposable microfluidic reagent compact disc described in the companion Chap. 16 which facilitates four isothermal nucleic acid tests in parallel from raw blood samples to answer. The platform has a user-friendly interface such as touchscreen LCD and smartphone data connectivity for on-site and remote healthcare delivery, respectively. The chapter mainly focuses on describing integration procedures of the real-time fluorescence LAMP analyzer and the validation of its subsystems. The device cost is significantly reduced compared to the commercial benchtop real-time machine and other existing POC platforms. As a platform technology, self-sustainable, portable, low-cost, and easy-to-use analyzer design should create a new paradigm of molecular diagnosis toward a variety of infectious diseases at the point of need.

Sample-to-Answer Microfluidic Nucleic Acid Testing (NAT) on Lab-on-a-Disc for Malaria Detection at Point of Need.

One of the grand challenges for field-deployable NATs is related to the front end of the assays-nucleic acid extraction from raw samples. The ideal nucleic acid sample preparation should be simple, scalable, and easy-to-operate. In this chapter, we present a lab-on-a-disc NAT device for sample-to-answer malaria diagnosis. The parasite DNA sample preparation and subsequent real-time LAMP detection are seamlessly integrated on a disposable single microfluidic compact disc, driven by energy-efficient, non-centrifuge-based magnetic field interactions. Each disc contains four parallel testing units, which could be configured either as four identical tests or as four species-specific tests. When configured as species-specific tests, it could identify two of the most life-threatening malaria species (P. falciparum and P. vivax). The reagent disc with a 4-plex analyzer (discussed in Chapter 1 ) is capable of processing four samples simultaneously with 40 min turnaround time. It achieves a detection limit of ~0.5 parasites/µl for whole blood, sufficient for detecting asymptomatic parasite carriers. The assay is performed with an automated device described in Chapter 14 . The combination of sensitivity, specificity, cost, and scalable sample preparation suggests the real-time fluorescence LAMP device could be particularly useful for malaria screening in field settings.

Fingerpick Blood-Based Nucleic Acid Testing on A USB Interfaced Device towards HIV self-testing.

HIV self-testing is an emerging innovative approach that allows individuals who want to know their HIV status to collect their own specimen, perform a test, and interpret the results privately. Existing HIV self-testing methods rely on rapid diagnostic tests (RDTs) to detect the presence of HIV-1/2 antibodies, which could miss a significant portion of asymptomatic carriers during the window period. In this work, we present a fully integrated nucleic acid testing (NAT) device towards streamlined HIV self-testing using 100 µL finger-prick whole blood. The device consists of a ready-to-use microfluidic reagent cartridge and an ultra-compact NAT-on-USB analyzer. The test requires simple steps from the user to drop the finger-prick blood sample into a collection tube with lysis buffer and load the lysate onto the microfluidic cartridge, and the testing result can be easily read out by a custom-built graphical user interface (GUI). The microfluidic cartridge and the analyzer automatically handle the complexity of sample preparation, purification, and real-time reverse-transcription loop-mediated isothermal amplification (RT-LAMP). With a turnaround time of ∼60 min, we achieved a limit of detection (LoD) of 214 viral RNA copies/mL of whole blood at a 95% confidence level. Due to its ease of use and high sensitivity, we anticipate the HIV NAT-on-USB device would be particularly useful for the high-risk populations seeking private self-testing at the early stages of exposure.

LAMP Diagnostics at the Point-of-Care: Emerging Trends and Perspectives for the Developer Community.

Introduction: Over the past decade, loop-mediated isothermal amplification (LAMP) technology has played an important role in molecular diagnostics. Amongst numerous nucleic acid amplification assays, LAMP stands out in terms of sample-to-answer time, sensitivity, specificity, cost, robustness, and accessibility, making it ideal for field-deployable diagnostics in resource-limited regions.Areas covered: In this review, we outline the front-end LAMP design practices for point-of-care (POC) applications, including sample handling and various signal readout methodologies. Next, we explore existing LAMP technologies that have been validated with clinical samples in the field. We summarize recent work that utilizes reverse transcription (RT) LAMP to rapidly detect SARS-CoV-2 as an alternative to standard PCR protocols. Finally, we describe challenges in translating LAMP from the benchtop to the field and opportunities for future LAMP assay development and performance reporting.Expert opinion: Despite the popularity of LAMP in the academic research community and a recent surge in interest in LAMP due to the COVID-19 pandemic, there are numerous areas for improvement in the fundamental understanding of LAMP, which are needed to elevate the field of LAMP assay development and characterization.

Microfluidic deformability-activated sorting of single particles.

Mechanical properties have emerged as a significant label-free marker for characterizing deformable particles such as cells. Here, we demonstrated the first single-particle-resolved, cytometry-like deformability-activated sorting in the continuous flow on a microfluidic chip. Compared with existing deformability-based sorting techniques, the microfluidic device presented in this work measures the deformability and immediately sorts the particles one-by-one in real time. It integrates the transit-time-based deformability measurement and active hydrodynamic sorting onto a single chip. We identified the critical factors that affect the sorting dynamics by modeling and experimental approaches. We found that the device throughput is determined by the summation of the sensing, buffering, and sorting time. A total time of ~100 ms is used for analyzing and sorting a single particle, leading to a throughput of 600 particles/min. We synthesized poly(ethylene glycol) diacrylate (PEGDA) hydrogel beads as the deformability model for device validation and performance evaluation. A deformability-activated sorting purity of 88% and an average efficiency of 73% were achieved. We anticipate that the ability to actively measure and sort individual particles one-by-one in a continuous flow would find applications in cell-mechanotyping studies such as correlational studies of the cell mechanical phenotype and molecular mechanism.

Microfluidic Time-Division Multiplexing Accessing Resistive Pulse Sensor for Particle Analysis.

Due to its simplicity and robustness, pore-based resistive pulse sensors have been widely used to detect, measure, and analyze particles at length scales ranging from nanometers to micrometers. While multiple pore-based resistive pulse sensors are preferred to increase the analysis throughput and to overcome the clogging issues, the scalability is often limited. In response, by combining the time-division multiple access technique in the telecommunication field with the microfluidics, we reported a microfluidic time-division multiplexing accessing (TDMA) single-end resistive pulse sensor, in which particles can be analyzed through a scalable number of microfluidic channels. With an eight-channel microfluidic device and polystyrene particles as proof-of-principle, we successfully demonstrated this multiplexed technology is effective in measuring the particle size and concentration, in analyzing the particle arriving dynamics, and in discriminating mixed populations. Importantly, the availability of multiple sensing pores provides a robust mechanism to overcome the clogging issue, allowing the analysis to continue even when some of the pores are clogged. We anticipate this TDMA approach could find wide applications and facilitate future development of multiplexed resistive pulse sensing from the microscale to nanoscale.

Sample-to-answer palm-sized nucleic acid testing device towards low-cost malaria mass screening.

The effectiveness of malaria screening and treatment highly depends on the low-cost access to the highly sensitive and specific malaria test. We report a real-time fluorescence nucleic acid testing device for malaria field detection with automated and scalable sample preparation capability. The device consists a compact analyzer and a disposable microfluidic reagent compact disc. The parasite DNA sample preparation and subsequent real-time LAMP detection were seamlessly integrated on a single microfluidic compact disc, driven by energy efficient non-centrifuge based magnetic field interactions. Each disc contains four parallel testing units which could be configured either as four identical tests or as four species-specific tests. When configured as species-specific tests, it could identify two of the most life-threatening malaria species (P. falciparum and P. vivax). The NAT device is capable of processing four samples simultaneously within 50 min turnaround time. It achieves a detection limit of ~0.5 parasites/µl for whole blood, sufficient for detecting asymptomatic parasite carriers. The combination of the sensitivity, specificity, cost, and scalable sample preparation suggests the real-time fluorescence LAMP device could be particularly useful for malaria screening in the field settings.

A field-deployable mobile molecular diagnostic system for malaria at the point of need.

In response to the urgent need of a field-deployable and highly sensitive malaria diagnosis, we developed a standalone, "sample-in-answer-out" molecular diagnostic system (AnyMDx) to enable quantitative molecular analysis of blood-borne malaria in low resource areas. The system consists of a durable battery-powered analyzer and a disposable microfluidic compact disc loaded with reagents ready for use. A low power thermal module and a novel fluorescence-sensing module are integrated into the analyzer for real-time monitoring of loop-mediated isothermal nucleic acid amplification (LAMP) of target parasite DNA. With 10 µL of raw blood sample, the AnyMDx system automates the nucleic acid sample preparation and subsequent LAMP and real-time detection. Under laboratory conditions with whole-blood samples spiked with cultured Plasmodium falciparum, we achieved a detection limit of ∼0.6 parasite per µL, much lower than those for the conventional microscopy and rapid diagnostic tests (∼50-100 parasites per µL). The turnaround time from sample to answer is less than 40 minutes. The AnyMDx is user-friendly requiring minimal technological training. The analyzer and the disposable reagent compact discs are cost-effective, making AnyMDx a potential tool for malaria molecular diagnosis under field settings for malaria elimination.