Hand-Powered Microfluidics for Parallel Droplet Digital Loop-Mediated Isothermal Amplification Assays

Abstract

Droplet digital loop-mediated isothermal amplification (ddLAMP) is an important assay for pathogen detection due to its high accuracy, specificity, and ability to quantify nucleic acids. However, performing ddLAMP requires expensive instrumentation and the need for highly trained personnel with expertise in microfluidics. To make ddLAMP more accessible, a ddLAMP assay is developed, featuring significantly decreased operational difficulty and instrumentation requirements. The proposed assay consists of three simplified steps: (1) droplet generation step, in which a LAMP mixture can be emulsified just by manually pulling a syringe connected to a microfluidic device. In this step, for the first time, we verify that highly monodispersed droplets can be generated with unstable flow rates or pressures, allowing untrained personnel to operate the microfluidic device and perform ddLAMP assay; (2) heating step, in which the droplets are isothermally heated in a water bath, which can be found in most laboratories; and (3) result analysis step, in which the ddLAMP result can be determined using only a fluorescence microscopy and an open-source analyzing software. Throughout the process, no droplet microfluidic expertise or equipment is required. More importantly, the proposed system enables multiple samples to be processed simultaneously with a detection limit of 10 copies/μL. The test is simple and intuitive to operate in most laboratories for multi-sample detection, significantly enhancing the accessibility and detection throughput of the ddLAMP technique.

Agarose-Droplet-Based Digital LAMP Assay for Counting Virus DNA in Single-Particle ICP-MS

“…Based on that, a linear range of 25–10,000 copy μL –1 in the LAMP mixture with an RSD of 6.3% ( n = 7, c = 100 copy μL –1 ) was obtained. A comparison of this work with other works of digital assays ,,− or element-labeling ICP-MS and spICP-MS assays ,− for nucleic acids is listed in Table S3. The lower limit of the linear range of this work is higher than most of the works of digital assay due to the relatively low transport efficiency of 2.6% of ICP-MS.…”

“…By counting the partitions with a positive fluorescent signal, the number of targets could be obtained without the need for a calibration curve. A variety of digital isothermal amplification techniques were developed for fast amplifying the target nucleic acids at a simple constant temperature and avoiding the complex process of thermal cycling of dPCR, such as digital loop-mediated isothermal amplification (LAMP) − or recombinase polymerase amplification (RPA). , By virtue of high sensitivity, high accuracy, satisfactory resistance to inhibitors, less amplification bias, and sample contamination, these “digital” techniques have flourished in the fields of nucleic acid detection at extremely low concentrations for analysis of pathogens, ,, cancer biomarkers, , rare mutations, methylation, gene expression, sequencing assistance, , etc. Hitherto, those digital assays are limited to fluorescence detection, mainly including fluorescence microscopy and flow cytometry, and few other techniques have been introduced to digital assay.…”

Simplifying the complex: accessible microfluidic solutions for contemporary processes within in vitro diagnostics

“…Yuan et al recently exploited step-emulsification to develop a hand-powered droplet-based LAMP assay for detecting several strains of pathogenic bacteria. 114 By combining step-emulsification with a hand-powered syringe to drive flow, the group were able to do away with the majority of the equipment typically required for droplet-based assays. Though the detection aspect of the assay still required a relatively complex fluorescence microscope, this work is certainly a step in the right direction.…”

LAMP-Based Point-of-Care Biosensors for Rapid Pathogen Detection

“…Droplet-based digital LAMP requires a highly trained workforce with experience in microfluidics and sophisticated instruments. Therefore, to make ddLAMP more convenient, Yuan et al [ 154 ] designed a “Handifluidics” device integrating the LAMP mixture emulsification, incubation region, and fluorescence detection of the amplified product ( Figure 8 C). A manually operated syringe was used to generate monodispersed droplets in a PDMS chip.…”