Critical review of methods for isothermal amplification of nucleic acids for environmental analysis

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.

Download Full-text

Recent advances in the construction of functional nucleic acids with isothermal amplification for heavy metal ions sensor

Microchemical Journal ◽

10.1016/j.microc.2021.107077 ◽

2021 ◽

pp. 107077

Author(s):

Chen Liu ◽

Yujie Li ◽

Jinquan Liu ◽

Lifu Liao ◽

Renlong Zhou ◽

...

Keyword(s):

Heavy Metal ◽

Nucleic Acids ◽

Metal Ions ◽

Heavy Metal Ions ◽

Isothermal Amplification ◽

Recent Advances ◽

Functional Nucleic Acids

Download Full-text

A Sensitive Potentiometric Sensor for Isothermal Amplification-Coupled Detection of Nucleic Acids

Sensors ◽

10.3390/s18072277 ◽

2018 ◽

Vol 18 (7) ◽

pp. 2277

Author(s):

Kang-Ho Lee ◽

Dongkyu Lee ◽

Jongsu Yoon ◽

Ohwon Kwon ◽

Jaejong Lee

Keyword(s):

Nucleic Acids ◽

Real Time ◽

Surface Potential ◽

Pathogenic Bacteria ◽

Field Effect Transistors ◽

Potentiometric Sensor ◽

Isothermal Amplification ◽

Ring Oscillator ◽

Oxide Semiconductor ◽

High Signal

A disposable potentiometric sensor was newly developed for the amplification-coupled detection of nucleic acids. The hydrogen-ion is generally released during isothermal amplification of nucleic acids. The surface potential on the oxide-functionalized electrode of the extended gate was directly measured using full electrical circuits with the commercial metal-oxide semiconductor field-effect transistors (MOSFETs) and ring oscillator components, which resulted in cost-effective, portable and scalable real-time nucleic acid analysis. The current-starved ring oscillator changes surface potential to its frequency depending on the square of the variation in pH with a high signal-to-noise ratio during isothermal amplification. The device achieves a conversion rate of 20.5 kHz/mV and a detection resolution of 200 µV for the surface potential. It is demonstrated that the sensor successfully monitors in real-time isothermal amplification of the extracted nucleic acids from Salmonella pathogenic bacteria. The in situ variations in the frequency of the pH-sensitive sensor were compared with the results of both a conventional optical device and pH-meter during isothermal amplification.

Download Full-text