Pipette-Free and Fully Integrated Paper Device Employing DNA Extraction, Isothermal Amplification, and Carmoisine-Based Colorimetric Detection for Determining Infectious Pathogens

Abstract

A pipette-free and fully integrated device that can be used to accurately recognize the presence of infectious pathogens is an important and useful tool in point-of-care testing, particularly when aiming to decrease the unpredictable threats posed by disease outbreak. In this study, a paper device is developed to integrate the three main processes required for detecting infectious pathogens, including DNA extraction, loop-mediated isothermal amplification (LAMP), and detection. All key reagents, including sodium dodecyl sulfate (SDS), NaOH, LAMP reagents, and carmoisine, are placed on the paper device. The paper device is operated simply via sliding and folding without using any bulky equipment, and the results can be directly observed by the naked eye. The optimized concentrations of sodium dodecyl sulfate (SDS), sodium hydroxide (NaOH), and carmoisine were found to be 0.1%, 0.1 M, and 0.5 mg/mL, respectively. The paper device was used to detect Enterococcus faecium at concentrations as low as 10² CFU/mL within 60 min. Also, E. faecium spiked in milk was successfully detected using the paper device, demonstrating the feasible application in real sample analysis.

Keywords: Enterococcus faecium; Pipette-free and fully integrated paper device; carmoisine-based colorimetric detection; point-of-care testing.

Figure 1

Overview of the paper device fabrication. (a) Pad 1 contains four outer pillars and four inner pillars that carry Whatman paper discs and reagent-containing Whatman paper discs for colorimetric detection, respectively. (b) Pad 2 is fabricated with the ability to be folded into a hollow box. (c) Pad 3 is made from a twelve-hole PDMS-coated felt attached on a Whatman paper of the same size with no holes. (d) Illustration of the paper device.

Figure 2

Operation of the paper device for DNA extraction, LAMP reaction, and colorimetric detection.

Figure 3

Optimization of NaOH (0.1 M, 0.5 M, and 1 M) and SDS (0.1%, 1%, and 10%) concentrations for DNA extraction. M represents the 100 bp DNA ladder. (+) represents samples containing extracted DNA using a commercial kit. (−) represents samples not containing bacteria.

Figure 4

Optimization of carmoisine-based detection. (a) Principle of carmoisine-based detection. (b) Effect of time on the colorimetric detection occurring in liquid form. (c) Ratios (−)/(+) represent the ratios of the mean gray values of the negative and positive samples from 0 min to 20 min that were measured in liquid form. (d) Effect of time on color change on paper over time. (e) Ratios (−)/(+) represent the ratios of the mean gray values of the negative and positive samples that were measured on paper discs.

Figure 5

Compatibility of carmoisine-based colorimetric detection in LAMP reaction. (a) Optimization of carmoisine concentration. (−) samples not containing DNA. (+) samples containing DNA. (b) The ratios of mean gray values of negative and positive samples measured on paper discs. (c) Electrophoresis results showing the compatibility of carmoisine in the LAMP reaction.

Figure 6

Results showing (a) selectivity test and (b) electrophoresis. (c) The results of the sensitivity test performed using a paper device. (d) A graph showing the statistical analyses of the sample containing 10¹–10³ CFU/mL E. faecium. (e) Electrophoresis results displaying the sensitivity performance. (f) Detection of E. faecium in milk using the paper device. (g) A graph showing the statistical analyses of E. faecium detected in the milk sample using the paper device. (h) Electrophoresis results obtained when a spiked milk sample was used.