fM to aM nucleic acid amplification for molecular diagnostics in a non-stick-coated metal microfluidic bioreactor

Abstract

A sensitive DNA isothermal amplification method for the detection of DNA at fM to aM concentrations for pathogen identification was developed using a non-stick-coated metal microfluidic bioreactor. A portable confocal optical detector was utilized to monitor the DNA amplification in micro- to nanoliter reaction assays in real-time, with fluorescence collection near the optical diffraction limit. The non-stick-coated metal microfluidic bioreactor, with a surface contact angle of 103°, was largely inert to bio-molecules, and DNA amplification could be performed in a minimum reaction volume of 40 nL. The isothermal nucleic acid amplification for Mycoplasma pneumoniae identification in the non-stick-coated microfluidic bioreactor could be performed at a minimum DNA template concentration of 1.3 aM, and a detection limit of three copies of genomic DNA was obtained. This microfluidic bioreactor offers a promising clinically relevant pathogen molecular diagnostic method via the amplification of targets from only a few copies of genomic DNA from a single bacterium.

Isothermal Amplification of Nucleic Acids

“…For instance, the integration of LAMP and electrophoresis separation in microfluidic chips for nucleic acid assays has been reported. − Optical end-point analysis was subsequently employed for simple detection with improved sensitivity. − The introduction of paper into the microfluidic device for LAMP reactions permitted stable test results for longer than 2 months . By integrating miniaturized optical detectors into the chips − or using potable optical readers, , real-time monitoring of LAMP reactions has been demonstrated for the POC detection of pathogens. In particular, the simultaneous analysis of multiple viruses in one system and the integration of a flow-through membrane for nucleic acid preparation (isolation, concentration, and purification) were achieved.…”

“…589−591 The introduction of paper into the microfluidic device for LAMP reactions permitted stable test results for longer than 2 months. 589 By integrating miniaturized optical detectors into the chips 592−595 or using potable optical readers, 596,597 real-time monitoring of LAMP reactions has been demonstrated for the POC detection of pathogens. In particular, the simultaneous analysis of multiple viruses in one system 593 and the integration of a flow-through membrane for nucleic acid preparation (isolation, concentration, and purification) 597 were achieved.…”

A smart device for label-free and real-time detection of gene point mutations based on the high dark phase contrast of vapor condensation

“…The product of RCA is a concatemer containing 10s to 100s of tandem repeats, making them easier to detect. Moreover, RCA process can be carried out easily at room temperature, which makes complex temperature control system unnecessary 31 .…”

“…Moreover, the RCA process can be carried out easily at room temperature, which makes a complex temperature control system unnecessary. 33 A 30 min incubation at 60 °C and another 30 min at 25 °C are needed to bind the reporter DNA and the pre-loop template DNA. Then, 5 μL of 4.5 μM pre-loop template DNA, 8 U μL −1 T4 DNA ligase, and a buffer mixture were added to each microarray.…”

Fast infectious diseases diagnostics based on microfluidic biochip system

“…18 These micro°uidic chips decrease the time necessary for DNA ampli¯cation, consume less sample and reagents, and increase portability, compactness, and the potential for automation of analyses. [19][20][21][22][23] Despite the fact that various LAMP-based micro°uidic chips have been developed for rapid detection and identi¯cation of bacteria and viruses, portable platforms for point-of-care testing are still in the initial stages of development. Indeed, micro°uidic chips for real-time multiple samples and multiple index diagnosis in a single detection run have seldom been reported.…”