A microfluidic chip capable of generating and trapping emulsion droplets for digital loop-mediated isothermal amplification analysis

Abstract

Loop-mediated isothermal amplification (LAMP) is a nucleic acid amplification technique that rapidly amplifies specific DNA molecules at high yield. In this study, a microfluidic droplet array chip was designed to execute the digital LAMP process. The novel device was capable of 1) creating emulsion droplets, 2) sorting them into a 30 × 8 droplet array, and 3) executing LAMP across the 240 trapped and separated droplets (with a volume of 0.22 nL) after only 40 min of reaction at 56 °C. Nucleic acids were accurately quantified across a dynamic range of 50 to 2.5 × 10 DNA copies per μL, and the limit of detection was a single DNA molecule. This is the first time that an arrayed emulsion droplet microfluidic device has been used for digital LAMP analysis. When compared to microwell digital nucleic acid amplification assays, this droplet array-based digital LAMP assay eliminates the constraint on the size of the digitized target, which was determined by the dimension of the microwells for its counterparts. Moreover, the capacity for hydrodynamic droplet trapping allows the chip to operate in a one-droplet-to-one-trap manner. This microfluidic chip may therefore become a promising device for digital LAMP-based diagnostics in the near future.

Droplet and Microchamber‐Based Digital Loop‐Mediated Isothermal Amplification (dLAMP)

“…Recently, droplet microfluidics is further enhanced and functionalized, extending its applications to biochemical analyses, including digital nucleic acid amplifications such as dLAMP . Using droplet microfluidics, the well‐mixed nucleic acid samples and LAMP reagents are emulsified by oils with stabilizing surfactants . Then, the resultant monodisperse droplets from the droplet microfluidics are heated to perform LAMP reactions.…”

“…c) The trapping device for confining different droplet in different holes to prevent droplet coalescence. Reproduced with permission . Copyright 2018, Royal Society of Chemistry.…”

“…In many studies using droplet microfluidic LAMP, off‐chip heating in a PCR tube or a 96 well‐plate is adopted to bypass the problem of droplet evaporation . Droplet heating in PDMS microfluidic chips is more challenging since the liquid droplets are more likely to evaporate inside porous PDMS microchannel during heating . The evaporation of the LAMP emulsions will lead to the loss of the droplets, the increase of the LAMP mixture concentration inside the droplets or even the coalescence of the droplets, which will cause inaccurate results of nucleic acid quantification and inconsistent efficiency of LAMP reactions.…”

“…Therefore, efforts have been made to prevent the evaporation of LAMP emulsions. For instance, a 5% mineral oil‐enriched PDMS layer and a glass coverslip have been introduced to cover the PDMS layer containing microchannels to avoid evaporation of the LAMP emulsions, as shown in Figure b1 . A PDMS layer with microchannels has been sandwiched by two pieces of cover glasses, as shown in Figure b2 .…”

“…A PDMS layer with microchannels has been sandwiched by two pieces of cover glasses, as shown in Figure b2 . The accurate results of nucleic acid quantification from these dLAMP techniques indicate the ability of these strategies to prevent the evaporation issue …”