A digital microfluidic system for loop-mediated isothermal amplification and sequence specific pathogen detection

Abstract

A digital microfluidic (DMF) system has been developed for loop-mediated isothermal amplification (LAMP)-based pathogen nucleic acid detection using specific low melting temperature (Tm) Molecular Beacon DNA probes. A positive-temperature-coefficient heater with a temperature sensor for real-time thermal regulation was integrated into the control unit, which generated actuation signals for droplet manipulation. To enhance the specificity of the LAMP reaction, low-Tm Molecular Beacon probes were designed within the single-stranded loop structures on the LAMP reaction products. In the experiments, only 1 μL of LAMP reaction samples containing purified Trypanosoma brucei DNA were required, which represented over a 10x reduction of reagent consumption when comparing with the conventional off-chip LAMP. On-chip LAMP for unknown sample detection could be accomplished in 40 min with a detection limit of 10 copies/reaction. Also, we accomplished an on-chip melting curve analysis of the Molecular Beacon probe from 30 to 75 °C within 5 min, which was 3x faster than using a commercial qPCR machine. Discrimination of non-specific amplification and lower risk of aerosol contamination for on-chip LAMP also highlight the potential utilization of this system in clinical applications. The entire platform is open for further integration with sample preparation and fluorescence detection towards a total-micro-analysis system.

Real-time kinetics and high-resolution melt curves in single-molecule digital LAMP to differentiate and study specific and non-specific amplification

“…Although these methods improve the assay, they do not capture non-specific reactions and thus cannot give insights into the origin of non-specific amplification or the conditions that led to non-specific amplicons. Moreover, probes and beacons do not eliminate non-specific amplification; non-specific amplification still competes for reagents and can limit the extent of the signal generated by specific amplification events (20). Hence, it is highly desirable to distinguish specific from non-specific amplification.…”

Loop-mediated isothermal amplification (LAMP) – review and classification of methods for sequence-specific detection

“…Molecular beacons were used for the detection of a part of the 1031-bp ompW gene from Vibrio cholera 88 and also, on a digital microuidic system, for the detection of the human African trypanosomiasis pathogen Trypanosoma brucei. 89 The hairpins are unstable under LAMP conditions, resulting in high signal-to-noise ratios. An approach to improving the thermal stability of molecular beacons is to use locked nucleic acids to decrease the background uorescence signal.…”

“…), facilitating on-site sample processing. 82,89,158,170 Compared to methods based on uorescence, electrochemical readout seems more amenable to miniaturization due to the independence of optical path lengths, but suffers from the additional costs of disposables with integrated electrodes.…”

A digital microfluidic system with 3D microstructures for single-cell culture

“…Arrays of on-chip electrodes (1 mm × 1 mm) were designed with AutoCAD and patterned on a glass substrate (31.5 mm × 31.5 mm) as the bottom plate. A layer of 10 μm SU-8 photoresist was first coated on the bottom plate as the dielectric layer, followed by a second patterned layer (60 μm thickness SU-8) as fences to prevent droplets from drifting 37,38 and a third patterned layer (10 μm thickness SU-8) as a microstructure array to perform single-cell cultures. During the fabrication, a mask aligner (ABM, California, USA) was used for precise patterning of the dielectric layer, fences and microstructure array on the patterned chromium electrodes.…”