An LED-Driven AuNPs-PDMS Microfluidic Chip and Integrated Device for the Detection of Digital Loop-Mediated Isothermal DNA Amplification

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

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

Lab on a Chip ◽

10.1039/c7lc01004d ◽

2018 ◽

Vol 18 (2) ◽

pp. 296-303 ◽

Cited By ~ 19

Author(s):

Yu-Dong Ma ◽

Kang Luo ◽

Wen-Hsin Chang ◽

Gwo-Bin Lee

Keyword(s):

Nucleic Acids ◽

Microfluidic Chip ◽

Limit Of Detection ◽

Isothermal Amplification ◽

Loop Mediated Isothermal Amplification ◽

Novel Device ◽

Dna Molecule ◽

Emulsion Droplets ◽

Droplet Array ◽

Single Dna Molecule

A microfluidic droplet array chip was designed to execute the digital LAMP. This novel device was capable of 1) creating emulsion droplets, 2) sorting them into a trapping array, and 3) executing LAMP for only 40 min. Nucleic acids could be accurately quantified and the limit of detection was only single DNA molecule.

Download Full-text

EFFECTIVENESS OF THE LOOP-MEDIATED ISOTHERMAL AMPLIFICATION WITH FLUORESCENT DETECTION IN THE DIAGNOSIS OF PARVOVIRUS ENTERITIS IN CARNIVORES

Journal of microbiology epidemiology immunobiology ◽

10.36233/0372-9311-2019-1-90-95 ◽

2019 ◽

Vol 1 (1) ◽

pp. 90-95 ◽

Cited By ~ 1

Author(s):

O. A. Petrusha ◽

T. L. Chernichenko ◽

I. A. Kofiadi ◽

V. V. Zverev ◽

E. B. Faizuloev

Keyword(s):

Real Time ◽

Point Of Care ◽

Reference Method ◽

Diagnostic Value ◽

Isothermal Amplification ◽

Fluorescent Detection ◽

Lamp Method ◽

Analytical Sensitivity ◽

Loop Mediated Isothermal Amplification ◽

Syto 9

The aim of the study was to evaluate the diagnostic value of the method of loop-mediated isothermal amplification of DNA with real-time fluorescent detection (real-time LAMP, or RT-LAMP) on the model of carnivore parvoviruses. Materials and methods. Samples of feces, blood and swabs from the rectum of different species of predatory animals with parvovirus enteritis (n = 39) and healthy animals (n = 31), as well as laboratory strains of mink enteritis virus, were analyzed by RT-LAMP using SYTO-9 and SYTO-82 dyes. Real-time PCR was used as a reference method. Results. In our study, the LAMP method with real-time fluorescence detection (RT-LAMP) in the carnivore parvovirus enteritis model provides high analytical sensitivity (1.5×103 copies of DNA/ml), diagnostic sensitivity and specificity (up to 100% under optimal conditions). Comparison of the two intercalating dyes showed that the SYTO-82 dye provides a higher signal-to-background ratio (22.6 ± 2.1) than the SYTO-9 dye (6.3 ± 1.5) (p <0.0000001 ). At the same time, SYTO-9 dye at the sensitivity limit (10 copies of DNA) provides an increase in fluorescence in the reaction mixture 13 minutes earlier than for SYTO-82 (23 and 36 minutes, respectively). Conclusion. RT-LAMP is a promising method for rapid and highly sensitive «point-of-care» diagnosis of infectious diseases, as well as in conditions of livestock farms or in field conditions.

Download Full-text

Rapid diagnostics of genital herpes by loop-mediated isothermal amplification method with fluorescent detection

Journal of microbiology epidemiology immunobiology ◽

10.36233/0372-9311-2019-6-40-46 ◽

2019 ◽

pp. 40-46

Author(s):

D. I. Smirnova ◽

O. A. Petrusha ◽

A. V. Gracheva ◽

E. A. Volynskaya ◽

V. V. Zverev ◽

...

Keyword(s):

Real Time ◽

Sensitivity And Specificity ◽

High Efficiency ◽

High Sensitivity ◽

Isothermal Amplification ◽

Fluorescent Detection ◽

Lamp Method ◽

Analytical Sensitivity ◽

Loop Mediated Isothermal Amplification

Introduction. Due to the high clinical significance of herpesvirus diseases, the searching of fast and effective methods for their diagnosis remains relevant.The aim of the study was to evaluate the diagnostic efficiency of the loop-mediated isothermal amplification of DNA with real-time fluorescent detection (RT-LAMP) with SYTO-82 dye on a model of herpes simplex virus (HSV) infection.Materials and methods. A total of 44 urogenital swabs containing type 1 and type 2 HSV DNA and 43 swabs without HSV DNA, including 33 samples containing the DNA of cytomegalovirus, Epstein-Barr virus and herpesvirus type 6, were studied. For RT-LAMP, Bst 2.0 WarmStart DNA polymerase, SYTO-82 dye, LAMP primers were used.Results. The high efficiency of HSV DNA detection in the RT-LAMP reaction with SYTO-82 dye was shown. RT-LAMP in optimal conditions allowed to reduce reaction time for 2-3 times compared to real-time PCR (to 35 minutes). Analytical sensitivity of HSV type 1 and 2 detection in RT-LAMP was 103 copies of DNA/ml. The diagnostic sensitivity and specificity of the RT-LAMP diagnosis of HSV infection were 96% and 100%, respectively.Discussion. RT-LAMP method has a high sensitivity and specificity comparable to RTPCR, while the risk of false positive results obtaining is minimal.Conclusion. Thus, the reaction of RT-LAMP with SYTO-82 dye allows quickly, with high sensitivity and specificity to detect HSV DNA in clinical material and can be considered as a promising point-of-care testing method.

Download Full-text

Transcription of 4′-thioDNA templates to natural RNA in vitro and in mammalian cells

Chemical Communications ◽

10.1039/c4cc08862j ◽

2015 ◽

Vol 51 (37) ◽

pp. 7887-7890 ◽

Cited By ~ 17

Author(s):

Hideto Maruyama ◽

Kazuhiro Furukawa ◽

Hiroyuki Kamiya ◽

Noriaki Minakawa ◽

Akira Matsuda

Keyword(s):

Nucleic Acids ◽

Mammalian Cells ◽

Genetic Material ◽

Rna Polymerases ◽

Chemically Modified ◽

Biological Studies ◽

Modified Nucleic Acids

Synthetic chemically modified nucleic acids, which are compatible with DNA/RNA polymerases, have great potential as a genetic material for synthetic biological studies.

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