FlexPCR: A streamlined multiplexed digital mRNA quantification platform with universal primers and limited fluorescence channels.

Accurate quantification of multiple messenger RNA (mRNA) targets is essential for biomedical research and disease diagnosis. Current PCR-based methods for mRNA analysis are limited by the number of fluorescent labels and the complexities associated with multiple target-specific primers, leading to amplification bias and limited multiplexing capability. Here, we introduce Fluorescence-coding extension PCR (FlexPCR), a novel digital PCR-based assay that overcomes these limitations by employing a universal primer and probe strategy in conjugation with oligo extension.

Multiplex digital profiling of DNA methylation heterogeneity for sensitive and cost-effective cancer detection in low-volume liquid biopsies.

Molecular alterations in cancerous tissues exhibit intercellular genetic and epigenetic heterogeneity, complicating the performance of diagnostic assays, particularly for early cancer detection. Conventional liquid biopsy methods have limited sensitivity and/or ability to assess epigenetic heterogeneity of rare epiallelic variants cost-effectively. We report an approach, named REM-DREAMing (Ratiometric-Encoded Multiplex Discrimination of Rare EpiAlleles by Melt), which leverages a digital microfluidic platform that incorporates a ratiometric fluorescence multiplex detection scheme and precise digital high-resolution melt analysis to enable low-cost, parallelized analysis of heterogeneous methylation patterns on a molecule-by-molecule basis for the detection of cancer in liquid biopsies.

Exploiting β-Lactams-Induced Lysis and DNA Fragmentation for Rapid Molecular Antimicrobial Susceptibility Testing of Neisseria Gonorrhoeae via Dual-Digital PCR.

The evolution of antimicrobial resistance (AMR) presents substantial challenges to global medical health systems. Neisseria gonorrhoeae (N. gonorrhoeae), in particular, has developed resistance to all currently available antimicrobials.

Enhanced CRISPR/Cas-Based Immunoassay through Magnetic Proximity Extension and Detection.

Clustered regularly interspaced short palindromic repeats (CRISPR)/Cas-associated systems have recently emerged as a focal point for developing next-generation molecular diagnosis, particularly for nucleic acid detection. However, the detection of proteins is equally critical across diverse applications in biology, medicine, and the food industry, especially for diagnosing and prognosing diseases like cancer, Alzheimer's and cardiovascular conditions. Despite recent efforts to adapt CRISPR/Cas systems for protein detection with immunoassays, these methods typically achieved sensitivity only in the femtomolar to picomolar range, underscoring the need for enhanced detection capabilities.

Highly Sensitive Serum Protein Analysis Using Magnetic Bead-Based Proximity Extension Assay.

Many protein biomarkers are present in biofluids at a very low level but may play critical roles in important biological processes. The fact that these low-abundance proteins remain largely unexplored underscores the importance of developing new tools for highly sensitive protein detection. Although digital enzyme-linked immunosorbent assay (ELISA) has demonstrated ultrahigh sensitivity compared with conventional ELISA, the requirement of specialized instruments limits the accessibility and prevents the widespread implementation.

A vacuum-assisted, highly parallelized microfluidic array for performing multi-step digital assays.

There remains an unmet need for a simple microfluidic platform that can perform multi-step and multi-reagent biochemical assays in parallel for high-throughput detection and analysis of single molecules and single cells. In response, we report herein a PDMS-based vacuum-driven microfluidic array that is capable of multi-step sample loading and digitalization. The array features multi-level bifurcation microchannels connecting to 4096 dead-end microchambers for partitioning liquid reagents/samples.

Magnetofluidic immuno-PCR for point-of-care COVID-19 serological testing.

Serological tests play an important role in the fight against Coronavirus Disease 2019 (COVID-19), including monitoring the dynamic immune response after vaccination, identifying past infection and determining community infection rate. Conventional methods for serological testing, such as enzyme-linked immunosorbent assays and chemiluminescence immunoassays, provide reliable and sensitive antibody detection but require sophisticated laboratory infrastructure and/or lengthy assay time. Conversely, lateral flow immunoassays are suitable for rapid point-of-care tests but have limited sensitivity.

A fast nucleic acid extraction system for point-of-care and integration of digital PCR.

Digital PCR is a powerful amplification method for absolute quantification of nucleic acids. The systems that integrated the nucleic acid extraction and amplification can reduce detection time, improve accuracy, and reduce labor costs. However, current nucleic acid extraction systems cannot be integrated well with integrated fluidic circuit (IFC) dPCR or droplet digital PCR chips perfectly and limit the application of digital PCR.

Proximity ligation assays for precise quantification of femtomolar proteins in single cells using self-priming microfluidic dPCR chip.

The quantification of low concentration proteins can facilitate the discovery of some significant biomarkers, and provide us a more profound understanding of cell heterogeneity when applied to single cell analysis. However, most state-of- art single cell protein detection platforms are bulky, expensive and complicated. Here we report a simple and low cost microfluidic dPCR (digital polymerase chain reaction) chip-based proximity ligation assay (PLA) for the quantification of low concentration proteins.

Power-free polydimethylsiloxane femtoliter-sized arrays for bead-based digital immunoassays.

A novel microfluidic chip employing power-free polydimethylsiloxane (PDMS) femtoliter-sized arrays was developed for the detection of low concentrations of protein biomakers by isolating individual paramagnetic beads in single wells. Arrays of femtoliter-sized wells were fabricated with PDMS using well-developed molding techniques. Paramagnetic beads were functionalized with specific antibodies to capture the antigens.

A new method using machine learning for automated image analysis applied to chip-based digital assays.

Chip-based digital assays such as the digital polymerase chain reaction (digital PCR), digital loop-mediated amplification (digital LAMP), digital enzyme-linked immunosorbent assay (digital ELISA) and digital proximity ligation assay (digital PLA) need high-throughput quantification of the captured fluorescence image data. However, traditional methods that are mainly based on image segmentation using either a fixed threshold or an automated hard threshold failed to extract valid signals over a broad range of image characteristics. In this study, we introduce a new method for automated image analysis to extract signals applied to chip-based digital assays.

A highly integrated real-time digital PCR device for accurate DNA quantitative analysis.

Misclassification of positive partitions in microfluidic digital polymerase chain reaction (dPCR) can cause the false positives and false negatives, which significantly alter the resulting estimate of target DNA molecules. To address this issue, establishing real-time fluorescence interrogation of each partition in microfluidic arrays is an effective way in which false positive and false negative partitions can be eliminated. However, currently available devices for real-time fluorescence interrogation are either not competent for microfluidic digital array, or they are bulky, expensive and entail peripheral equipment due to low integration.

High throughput single cell separation and identification using a self-priming isometric and Equant screw valve-based (SIES) microfluidic chip.

The emergence of various single cell separation and identification platforms has greatly promoted the development of single cell research. Among these platforms, microfluidic chip-based strategies occupy a significant position in single cell separation and identification. Here, we proposed a self-priming isometric and Equant screw valve-based microfluidic chip (SIES chip) for high throughput single cell isolation and identification.

Development of a self-priming PDMS/paper hybrid microfluidic chip using mixed-dye-loaded loop-mediated isothermal amplification assay for multiplex foodborne pathogens detection.

Foodborne pathogen is the primary cause of foodborne disease outbreak. Given its great damage, a sensitive, simple and rapid detection method is demanded. Herein, we described a self-priming polydimethylsiloxane (PDMS)/paper hybrid microfluidic chip, termed SPH chip, with mixed-dye-loaded loop-mediated isothermal amplification (LAMP) for multiplex foodborne pathogens detection.

Smartphone-based mobile digital PCR device for DNA quantitative analysis with high accuracy.

Digital polymerase chain reaction (dPCR) circumventing the external calibration and potentially providing absolute quantification of nucleic acids has become an increasingly popular manifestation of PCR in biological researches. However, currently reported or commercial dPCR devices are not suitable for applications in laboratories or zones with limited infrastructures, due to low function integration, cost-inefficiency, or weak mobility. Herein, in order to enable accurate DNA quantitative analysis in such situations, we have developed a smartphone-based mobile dPCR device integrated with thermal cycling control, on-chip dPCR, data acquisition, and result analysis.

Co-detection of ALDH1A1, ABCG2, ALCAM and CD133 in three A549 subpopulations at the single cell level by one-step digital RT-PCR.

Cancer stem-like cells (CSCs) displaying the properties of normal stem cells have become the main culprit associated with cancer transportation and recurrence. As of now, various CSC functions and marker genes have been identified due to the heterogeneity of cancer, such as aldehyde dehydrogenase (ALDH), the second member of the ABC transporter G-subfamily (ABCG2), activated leukocyte cell adhesion molecule (ALCAM) and CD133. To investigate these markers, most conventional approaches are bulk-based strategies, which may veil the disparity of single cells' gene expression.

Rapid and Quantitative Detection of Vibrio parahemolyticus by the Mixed-Dye-Based Loop-Mediated Isothermal Amplification Assay on a Self-Priming Compartmentalization Microfluidic Chip.

Vibrio parahemolyticus (VP) mostly isolated from aquatic products is one of the major causes of bacterial food-poisoning events worldwide, which could be reduced using a promising on-site detection method. Herein, a rapid and quantitative method for VP detection was developed by applying a mixed-dye-loaded loop-mediated isothermal amplification (LAMP) assay on a self-priming compartmentalization (SPC) microfluidic chip, termed on-chip mixed-dye-based LAMP (CMD-LAMP). In comparison to conventional approaches, CMD-LAMP was advantageous on the limit of detection, which reached down to 1 × 10 CFU/mL in food-contaminated samples without the pre-enrichment of bacteria.

Unusual isothermal multimerization and amplification by the strand-displacing DNA polymerases with reverse transcription activities.

Existing isothermal nucleic acid amplification (INAA) relying on the strand displacement activity of DNA polymerase usually requires at least two primers. However, in this paper, we report an unusual isothermal multimerization and amplification (UIMA) which only needs one primer and is efficiently initiated by the strand-displacing DNA polymerases with reverse transcription activities. On electrophoresis, the products of UIMA present a cascade-shape band and they are confirmed to be multimeric DNAs with repeated target sequences.