Colorimetric Nucleic Acid Detection on Paper Microchip Using Loop Mediated Isothermal Amplification and Crystal Violet Dye

Abstract

Nucleic acid detection is of paramount importance in monitoring of microbial pathogens in food safety and infectious disease diagnostic applications. To address these challenges, a rapid, cost-effective label-free technique for nucleic acid detection with minimal instrumentations is highly desired. Here, we present paper microchip to detect and quantify nucleic acid using colorimetric sensing modality. The extracted DNA from food samples of meat as well as microbial pathogens was amplified utilizing loop-mediated isothermal amplification (LAMP). LAMP amplicon was then detected and quantified on a paper microchip fabricated in a cellulose paper and a small wax chamber utilizing crystal violet dye. The affinity of crystal violet dye toward dsDNA and positive signal were identified by changing the color from colorless to purple. Using this method, detection of Sus scrofa (porcine) and Bacillus subtilis (bacteria) DNA was possible at concentrations as low as 1 pg/μL (3.43 × 10 copies/μL) and 10 pg/μL (2.2 × 10 copies/μL), respectively. This strategy can be adapted for detection of other DNA samples, with potential for development of a new breed of simple and inexpensive paper microchip at the point-of-need.

Microfluidic devices for detection of RNA viruses

“…They developed an RT‐LAMP paper microchip assay, which could be used as a simple and affordable method for the detection of HIV‐1 41 . Two other studies have shown that microfluidic‐based RT‐LAMP assay can affordably detect the Zika virus and Bacteriophage MS2 virus 71,72 …”

A highly selective electron affinity facilitated H2S sensor: the marriage of tris(keto-hydrazone) and an organic field-effect transistor

“…4,5 Thus, the methodical development of effective sensors to monitor H 2 S gas levels is of paramount importance and can have a significant impact on our daily lives. The standard tool used in workplaces to sense the presence of toxic gases is the detector tube 6,7 due to its calibration-free measurements, low-cost and high efficiency. 6 However, serious drawbacks such as a slow response, temperature sensitivity, low shelf life and low accuracy may lead to false alarms and other detrimental conditions.…”

“…6 However, serious drawbacks such as a slow response, temperature sensitivity, low shelf life and low accuracy may lead to false alarms and other detrimental conditions. 7 Alternatively, the pellistor-type sensor is considered to be a reliable candidate for the detection of combustible gases, 8 where the catalytic activity oxidizes the target gas, resulting in a voltage change. 9 This was followed by using ultrasonic gas detectors that are extensively used as gas-leak detectors where the device operation is based on a frequency-sensing principle.…”

Label-free pathogen detection by a deoxyribozyme cascade with visual signal readout

“…As a result, both ELISA and molecular diagnostic tests are performed only in specialized diagnostic laboratories. Even though a number of nucleic acid-based tests for pathogen detection with a colorimetric output have been reported [7][8][9][10][11][12][13][14][15][16], molecular tests for "laboratory-free" diagnostics of bacterial or viral infections are not currently used in standard practice.…”