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.
The mechanical behavior of individual cells plays an important role in regulating various biological activities at the molecular and cellular levels. It can serve as a promising label-free marker of ...cells’ physiological states. In the past two decades, several techniques have been developed for understanding correlations between cellular mechanical changes and human diseases. However, numerous technical challenges remain with regard to realizing high-throughput, robust, and easy-to-perform measurements of single-cell mechanical properties. In this paper, we review the emerging tools for single-cell mechanical characterization that are provided by microfluidic technology. Different techniques are benchmarked by considering their advantages and limitations. Finally, the potential applications of microfluidic techniques based on cellular mechanical properties are discussed.
Abstract
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.
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
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.
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.
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.
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.
•We reported a novel, non-optical technique to analyse the transportation of bacterial cells in paper.•The technique is based on real-time measurement of the microbial electricity production.•This ...work will determine more efficient cell movement within a paper material.•It will also provide a quantitative understanding of the flow of large organisms for use in paper-based diagnostics.
We reported a novel, non-optical technique to analyse and quantify the transportation of bacterial cells in paper-based microfluidics. This approach was based on real-time measurement of the microbial electricity production. Our device was designed to have three hydrophilic regions linked by a channel on a paper layer by patterning hydrophobic barriers in the paper. Each region came into contact with an anodic electrode layer which was stacked with a cathodic compartment through a proton exchange membrane. When bacterial cells were transported to each region by capillary force, the bacteria cells were promoted to adhere onto the anode and the cells completed their respiration by transferring the electrons to the anode. A conductive load connected the anode and cathode to complete the external circuit through which the electrons flowed. The protons generated by the anodic reactions passed through the proton exchange membrane and travelled to the cathode, where they combined with electrons in the reduction process. By measuring the current through the load, bacterial cells’ transportation through the channels in paper was quantitatively investigated according to the pore size of the paper. Further understanding was made by analysing electron microscope examination of the hydrophilic regions. This work will determine more efficient cell movement within a paper material by controlling the paper's microfluidic dimensions and pore size. It will also provide a quantitative understanding of the flow of large organisms for use in paper-based diagnostics.
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.
By measuring the current generated from the 3-D paper stack, the electron and proton diffusivity through biofilms were quantitatively investigated.
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.
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