Abstract
The sensitive quantification of low-abundance nucleic acids holds importance for a range of clinical applications and biological studies. In this study, we describe a facile microfluidic chip for absolute DNA quantifications based on the digital loop-mediated isothermal amplification (digital LAMP) method. This microfluidic chip integrates a cross-flow channel for droplet generation with a micro-cavity for droplet tiling. DNA templates in the LAMP reagent were divided into ~20,000 water-in-oil droplets at the cross-flow channel. The droplets were then tiled in the micro-cavity for isothermal amplification and fluorescent detection. Different from the existing polydimethylsiloxane (PDMS) microfluidic chips, this study incorporates gold nanoparticles (AuNPs) into PDMS substrate through silica coating and dodecanol modification. The digital LAMP chip prepared by AuNPs-PDMS combines the benefits of the microstructure manufacturing performance of PDMS with the light-to-heat conversion advantages of AuNPs. Upon illumination with a near infrared (NIR) LED, the droplets were stably and efficiently heated by the AuNPs in PDMS. We further introduce an integrated device with a NIR heating unit and a fluorescent detection unit. The system could detect HBV (hepatitis B virus)-DNA at a concentration of 1 × 101 to 1 × 104 copies/μL. The LED-driven digital LAMP chip and the integrated device; therefore, demonstrate high accuracy and excellent performance for the absolute quantification of low-abundance nucleic acids, showing the advantages of integration, miniaturization, cost, and power consumption.
Highlights
Owing to its superior performance over real-time nucleic acid amplification technology in terms of accuracy, specificity, and reproducibility, digital nucleic acid amplification technology is widely used in low-abundance nucleic acid quantification for the diagnosis of cancer, viruses, and bacterial infections [1,2]
According to the temperatures required for nucleic acid amplification, digital nucleic acid amplification technology (dNAAT) can be divided into digital polymerase chain reaction and digital isothermal amplification technology [4]
As a branch of digital isothermal amplification technology (dIAT), digital loop-mediated isothermal amplification permits nucleic acid amplification under isothermal conditions, eliminating the need for the complex thermocycling procedures used in digital polymerase chain reaction (dPCR) [5,6]
Summary
Owing to its superior performance over real-time nucleic acid amplification technology (qNAAT) in terms of accuracy, specificity, and reproducibility, digital nucleic acid amplification technology (dNAAT) is widely used in low-abundance nucleic acid quantification for the diagnosis of cancer, viruses, and bacterial infections [1,2]. For dNAAT-based nucleic acid detection, DNA samples are divided into thousands of microdroplets or microchambers, which are subsequently amplified at specific temperatures. In the QuantStudio 3D (Thermo Fisher Scientific) dNAAT system, DNA samples are dispersed into the microchambers of a silicon-based chip using a sample loading device, and incubated in a Peltier heater prior to fluorescence imaging [11]. These systems perform to a high level, but rely on multiple auxiliary devices leading to complex operating procedure and high-power usage. The future of dNAAT development; focuses on portability, integration, and miniaturization [12,13,14]
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