High-throughput microfluidic droplets in biomolecular analytical system: A review.

Droplet microfluidic technology has revolutionized biomolecular analytical research, as it has the capability to reserve the genotype-to-phenotype linkage and assist for revealing the heterogeneity. Massive and uniform picolitre droplets feature dividing solution to the level that single cell and single molecule in each droplet can be visualized, barcoded, and analyzed. Then, the droplet assays can unfold intensive genomic data, offer high sensitivity, and screen and sort from a large number of combinations or phenotypes. Based on these unique advantages, this review focuses on up-to-date research concerning diverse screening applications utilizing droplet microfluidic technology. The emerging progress of droplet microfluidic technology is first introduced, including efficient and scaling-up in droplets encapsulation, and prevalent batch operations. Then the new implementations of droplet-based digital detection assays and single-cell muti-omics sequencing are briefly examined, along with related applications such as drug susceptibility testing, multiplexing for cancer subtype identification, interactions of virus-to-host, and multimodal and spatiotemporal analysis. Meanwhile, we specialize in droplet-based large-scale combinational screening regarding desired phenotypes, with an emphasis on sorting for immune cells, antibodies, enzymatic properties, and proteins produced by directed evolution methods. Finally, some challenges, deployment and future perspective of droplet microfluidics technology in practice are also discussed.

Amplification-Free COVID-19 Detection by Digital Droplet REVEALR.

The COVID-19 pandemic, caused by the SARS-CoV-2 virus, exposed a pressing need for new public health tools for pathogen detection, disease diagnosis, and viral genotyping. REVEALR (RNA-encoded viral nucleic acid analyte reporter) is an isothermal DNAzyme-based point-of-care diagnostic that functions with a detection limit of ∼10 copies/µL when coupled with a preamplification step and can be utilized for viral genotyping of SARS-CoV-2 variants of concern through base pair mismatch recognition in a competitive binding format. Here, we describe an advanced REVEALR platform, termed digital droplet REVEALR (ddREVEALR), that can achieve direct viral detection and absolute sample quantitation utilizing a signal amplification strategy that relies on chemical modifications, DNAzyme multiplexing, and volume compression. Using an AI-assisted image-based readout, ddREVEALR was found to achieve 95% positive predictive agreement from a set of 20 nasal pharyngeal swabs collected at UCI Medical Center in Orange, California. We propose that the combination of amplification-free and protein-free analysis makes ddREVEALR a promising application for direct viral RNA detection of clinical samples.

Digital detection and quantification of SARS-CoV-2 in a droplet microfluidic all-fiber device

Silica fibers and capillaries offer opportunities for compact integration of optics with microfluidics while adding advantages such as;flexibility within a high aspect ratio format, uniaxial arrangements, and measurement-at-a-distance. Here, we describe droplet microfluidics-based nucleic acid detection of SARS-CoV-2 in a lab-in-a-fiber platform. The fiber component integrates three modules with key functions: droplet generation, incubation, and fluorescence detection. Within the scope of this work, we developed the component specifically to target the quantification of SARS-CoV-2 viral RNA through reverse-transcription loop-mediated isothermal amplification (RT-LAMP). The all-fiber component could successfully generate uniform droplets and differentiate pre-amplified positive LAMP reaction from negative sample. © 2021 MicroTAS 2021 - 25th International Conference on Miniaturized Systems for Chemistry and Life Sciences. All rights reserved.

A review of thermal impact of surface acoustic waves on microlitre droplets in medical applications

The surface acoustic waves (SAW) propagate inside the microdroplets resulting in kinetic and thermal impacts. The kinetic drives fluid particles inside the droplet while thermal impact increases the liquid’s temperature. This paper provides a comprehensive review of the research investigations related to internal kinetics and heating inside the microdroplet caused by the acoustic waves. The main factors that affect the kinetics and convection heat transfer are the piezoelectric materials, shape of the interdigital transducer (IDT) and mode of acoustic waves. Internal streaming (kinetic) leads to particle mixing, particle manipulation, cell sorting, cell patterning, cell separation, measuring the concentration of immunoglobulin and so forth. The effect of changing the mode of waves and the shape of IDT on the relevant applications are presented. Internal convection heat transfer is important where heating of the liquid is essential for many applications such as monitoring blood coagulation in the human plasma and an acoustic tweezer for particle trapping. Experimental methods developed by researchers to realise uniform temperature with constant heating and cooling cycles are also discussed. Such methods are widely used in the polymerase chain reaction (PCR) to detect COVID-19 infection. The heating of the droplet can be efficiently controlled by changing the input power and by varying the duty factor. © The Author(s) 2022.

Droplet Cas12a Assay Enables DNA Quantification from Unamplified Samples at the Single-Molecule Level.

DNA quantification is important for biomedical research, but the routinely used techniques rely on nucleic acid amplification which have inherent issues like cross-contamination risk and quantification bias. Here, we report a CRISPR-Cas12a-based molecular diagnostic technique for amplification-free and absolute quantification of DNA at the single-molecule level. To achieve this, we first screened out the optimal reaction parameters for high-efficient Cas12a assay, yielding over 50-fold improvement in sensitivity compared with the reported Cas12a assays. We further leveraged the microdroplet-enabled confinement effect to perform an ultralocalized droplet Cas12a assay, obtaining excellent specificity and single-molecule sensitivity. Moreover, we demonstrated its versatility and quantification capability by direct counting of diverse virus's DNAs (African swine fever virus, Epstein-Barr virus, and Hepatitis B virus) from clinical serum samples with a wide range of viral titers. Given the flexible programmability of crRNA, we envision this amplification-free technique as a versatile and quantitative platform for molecular diagnosis.

Droplet microfluidics on analysis of pathogenic microbes for wastewater-based epidemiology.

Infectious diseases caused by pathogenic microbes have posed a major health issue for the public, such as the ongoing COVID-19 global pandemic. In recent years, wastewater-based epidemiology (WBE) is emerging as an effective and unbiased method for monitoring public health. Despite its increasing importance, the advancement of WBE requires more competent and streamlined analytical platforms. Herein we discuss the interactions between WBE and droplet microfluidics, focusing on the analysis of pathogens in droplets, which is hard to be tackled by traditional analytical tools. We highlight research works from three aspects, namely, quantitation of pathogen biomarkers in droplets, single-cell analysis in droplets, and living cell biosensors in droplets, as well as providing future perspectives on the synergy between WBE and droplet microfluidics.

An Ultralocalized Cas13a Assay Enables Universal and Nucleic Acid Amplification-Free Single-Molecule RNA Diagnostics.

Existing methods for RNA diagnostics, such as reverse transcription PCR (RT-PCR), mainly rely on nucleic acid amplification (NAA) and RT processes, which are known to introduce substantial issues, including amplification bias, cross-contamination, and sample loss. To address these problems, we introduce a confinement effect-inspired Cas13a assay for single-molecule RNA diagnostics, eliminating the need for NAA and RT. This assay involves confining the RNA-triggered Cas13a catalysis system in cell-like-sized reactors to enhance local concentrations of target and reporter simultaneously, via droplet microfluidics. It achieves >10 000-fold enhancement in sensitivity when compared to the bulk Cas13a assay and enables absolute digital single-molecule RNA quantitation. We experimentally demonstrate its broad applicability for precisely counting microRNAs, 16S rRNAs, and SARS-CoV-2 RNA from synthetic sequences to clinical samples with excellent accuracy. Notably, this direct RNA diagnostic technology enables detecting a wide range of RNA molecules at the single-molecule level. Moreover, its simplicity, universality, and excellent quantification capability might render it to be a dominant rival to RT-qPCR.