13 research outputs found
Emerging Multiplex Nucleic Acid Diagnostic Tests for Combating COVID-19
The COVID-19 pandemic caused by SARS-CoV-2 has drawn attention to the need for fast and accurate diagnostic testing. Concerns from emerging SARS-CoV-2 variants and other circulating respiratory viral pathogens further underscore the importance of expanding diagnostic testing to multiplex detection, as single-plex diagnostic testing may fail to detect emerging variants and other viruses, while sequencing can be too slow and too expensive as a diagnostic tool. As a result, there have been significant advances in multiplex nucleic-acid-based virus diagnostic testing, creating a need for a timely review. This review first introduces frequent nucleic acid targets for multiplex virus diagnostic tests, then proceeds to a comprehensive and up-to-date overview of multiplex assays that incorporate various detection reactions and readout modalities. The performances, advantages, and disadvantages of these assays are discussed, followed by highlights of platforms that are amenable for point-of-care use. Finally, this review points out the remaining technical challenges and shares perspectives on future research and development. By examining the state of the art and synthesizing existing development in multiplex nucleic acid diagnostic tests, this review can provide a useful resource for facilitating future research and ultimately combating COVID-19
Enhancing Throughput of Combinatorial Droplet Devices via Droplet Bifurcation, Parallelized Droplet Fusion, and Parallelized Detection
Combinatorial droplet microfluidic devices with programmable microfluidic valves have recently emerged as a viable approach for performing multiplexed experiments in microfluidic droplets. However, the serial operation in these devices restricts their throughput. To address this limitation, we present a parallelized combinatorial droplet device that enhances device throughput via droplet bifurcation, parallelized droplet fusion, and parallelized droplet detection. In this device, sample droplets split evenly at bifurcating Y-junctions before multiple independent reagent droplets are injected directly into the split sample droplets for robust droplet fusion. Finally, the fused sample and reagent droplets can be imaged in parallel via microscopy. The combination of these approaches enabled us to improve the throughput over traditional, serially-operated combinatorial droplet devices by 16-foldâwith ready potential for further enhancement. Given its current performance and prospect for future improvements, we believe the parallelized combinatorial droplet device has the potential to meet the demand as a flexible and cost-effective tool that can perform high throughput screening applications
Emerging Analytical Techniques for Rapid Pathogen Identification and Susceptibility Testing
Multiplex Digital MethylationâSpecific PCR for Noninvasive Screening of Lung Cancer
Abstract There remains tremendous interest in developing liquid biopsy assays for detection of cancerâspecific alterations, such as mutations and DNA methylation, in cellâfree DNA (cfDNA) obtained through noninvasive blood draws. However, liquid biopsy analysis is often challenging due to exceedingly low fractions of circulating tumor DNA (ctDNA), necessitating the use of extended tumor biomarker panels. While multiplexed PCR strategies provide advantages such as higher throughput, their implementation is often hindered by challenges such as primerâdimers and PCR competition. Alternatively, digital PCR (dPCR) approaches generally offer superior performance, but with constrained multiplexing capability. This paper describes development and validation of the first multiplex digital methylationâspecific PCR (mdMSP) platform for simultaneous analysis of four methylation biomarkers for liquidâbiopsyâbased detection of nonâsmall cell lung cancer (NSCLC). mdMSP employs a microfluidic device containing four independent, but identical modules, housing a total of 40â160 nanowells. Analytical validation of the mdMSP platform demonstrates multiplex detection at analytical specificities as low as 0.0005%. The clinical utility of mdMSP is also demonstrated in a cohort of 72 clinical samples of lowâvolume liquid biopsy specimens from patients with computed tomography (CT)âscan indeterminant pulmonary nodules, exhibiting superior clinical performance when compared to traditional MSP assays for noninvasive detection of earlyâstage NSCLC
Facile Coupling of Droplet Magnetofluidic-Enabled Automated Sample Preparation for Digital Nucleic Acid Amplification Testing and Analysis
Simple and Precise Counting of Viable Bacteria by Resazurin-Amplified Picoarray Detection
Simple, fast, and
precise counting of viable bacteria is fundamental
to a variety of microbiological applications such as food quality
monitoring and clinical diagnosis. To this end, agar plating, microscopy,
and emerging microfluidic devices for single bacteria detection have
provided useful means for counting viable bacteria, but they also
have their limitations ranging from complexity, time, and inaccuracy.
We present herein our new method RAPiD (<u>R</u>esazurin-<u>A</u>mplified <u>Pi</u>coarray <u>D</u>etection) for addressing this important problem. In
RAPiD, we employ vacuum-assisted sample loading and oil-driven sample
digitization to stochastically confine single bacteria in Picoarray,
a microfluidic device with picoliter-sized isolation chambers (picochambers),
in <30 s with only a few minutes of hands-on time. We add AlamarBlue,
a resazurin-based fluorescent dye for bacterial growth, in our assay
to accelerate the detection of âmicrocoloniesâ proliferated
from single bacteria within picochambers. Detecting fluorescence in
picochambers as an amplified surrogate for bacterial cells allows
us to count hundreds of microcolonies with a single image taken via
wide-field fluorescence microscopy. We have also expanded our method
to practically test multiple titrations from a single bacterial sample
in parallel. Using this expanded âmulti-RAPiDâ strategy,
we can quantify viable cells in <i>E. coli</i> and <i>S. aureus</i> samples with precision in âŒ3 h, illustrating
RAPiD as a promising new method for counting viable bacteria for microbiological
applications
Quantification of Transcription Factor Binding in Cell Extracts Using an Electrochemical, Structure-Switching Biosensor
Transcription factor expression levels, which sensitively
reflect
cellular development and disease state, are typically monitored via
cumbersome, reagent-intensive assays that require relatively large
quantities of cells. Here, we demonstrate a simple, quantitative approach
to their detection based on a simple, electrochemical sensing platform.
This sensor sensitively and quantitatively detects its target transcription
factor in complex media (e.g., 250 ÎŒg/mL crude nuclear extracts)
in a convenient, low-reagent process requiring only 10 ÎŒL of
sample. Our approach thus appears a promising means of monitoring
transcription factor levels
Rapid Minimum Inhibitory Concentration (MIC) Analysis Using Lyophilized Reagent Beads in a Novel Multiphase, Single-Vessel Assay
Antimicrobial resistance (AMR) is a global threat fueled by incorrect (and overuse) of antibiotic drugs, giving rise to the evolution of multi- and extreme drug-resistant bacterial strains. The longer time to antibiotic administration (TTA) associated with the gold standard bacterial culture method has been responsible for the empirical usage of antibiotics and is a key factor in the rise of AMR. While polymerase chain reaction (PCR) and other nucleic acid amplification methods are rapidly replacing traditional culture methods, their scope has been restricted mainly to detect genotypic determinants of resistance and provide little to no information on phenotypic susceptibility to antibiotics. The work presented here aims to provide phenotypic antimicrobial susceptibility testing (AST) information by pairing short growth periods (~3â4 h) with downstream PCR assays to ultimately predict minimum inhibitory concentration (MIC) values of antibiotic treatment. To further simplify the dual workflows of the AST and PCR assays, these reactions are carried out in a single-vessel format (PCR tube) using novel lyophilized reagent beads (LRBs), which store dried PCR reagents along with primers and enzymes, and antibiotic drugs separately. The two reactions are separated in space and time using a melting paraffin wax seal, thus eliminating the need to transfer reagents across different consumables and minimizing user interactions. Finally, these two-step single-vessel reactions are multiplexed by using a microfluidic manifold that allows simultaneous testing of an unknown bacterial sample against different antibiotics at varying concentrations. The LRBs used in the microfluidic system showed no interference with the bacterial growth and PCR assays and provided an innovative platform for rapid point-of-care diagnostics (POC-Dx)