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Colorimetric nucleic acid detection on paper microchip using loop mediated isothermal amplification and crystal violet dye Sharmili Roy, Noor Faizah Mohd-Naim, Mohammadali Safavieh, and Minhaz uddin Ahmed ACS Sens., Just Accepted Manuscript • DOI: 10.1021/acssensors.7b00671 • Publication Date (Web): 01 Nov 2017 Downloaded from http://pubs.acs.org on November 2, 2017
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Colorimetric nucleic acid detection on paper microchip using loop mediated isothermal amplification and crystal violet dye Sharmili Roy†, Noor Faizah Mohd-Naim §, Mohammadali Safavieh*∥ ∥‡ and Minhaz Uddin Ahmed*† †Biosensors and Biotechnology Laboratory, Chemical Science Programme, Faculty of Science, Universiti Brunei Darussalam. Jalan Tungku Link, Gadong, BE 1410 Brunei Darussalam § PAPRSB Institute of Health Science, Universiti Brunei Darussalam. Jalan Tungku Link, Gadong, BE 1410, Brunei Darussalam. ∥Division of Engineering in Medicine, Brigham and Women’s Hospital-Harvard Medical School, 75 Francis Street, Boston, Massachusetts 02115, United States KEYWORDS Molecular diagnostic, DNA detection, LAMP, Isothermal amplification, Colorimetric sensing, Paper microchip, Point of Care Diagnostic
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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 utilising colour change in presence of nucleic acid. The extracted DNA from food samples of meat as well as microbial pathogens was amplified utilising 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 utilising crystal violet dye. The affinity of crystal violet dye towards dsDNA and positive signal were identified by changing the colour 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
-1
copies/µl)
and 10 pg/µl (2.2 × 103 copies/µl), respectively. This strategy can be adapted for detection of other DNA samples, with potential for development of new breed of simple and inexpensive paper microchip at the point-of-need.
Food safety and security are one of the top health concern globally with a profound impact on quality of human life1. Unsafe foods containing various microbial pathogens cause more than 200 diseases ranging from diarrhea to cancer2. Food adulteration, misbranding and food contamination are one of the main problems that plagued the food industry in recent years. Current high-profile cases include contamination of milk-based products with melamine to artificially increase protein values of products in China, the adulteration of beef with undeclared horse meat in Europe and existence of pork in lamb meat in the United Kingdom3-5. Although these incidents are not considered a health issue, the breakdown of food chain traceability in globalised food supply could pose a major ethical problem. Adulteration with porcine meat in
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food, in particular, is a serious religious concern in many sects such as Judaism, Hinduism and Islam6,7. Another vexing problem in the food industry is the contamination of food by pathogens that could cause illnesses in humans upon consumption, such as food poisoning by Escherichia coli and Bacillus cereus. According to WHO, contamination of food by pathogens results in up to 420,000 deaths every year8. The development of low cost, rapid point-of-care (POC) is critical for infectious disease management in food adulteration and contamination in both developed and developing countries. Various classes of paper microchip devices and technologies have been developed which opened new paradigm to develop disposable biosensor devices in a broad range of medical, environmental and food industry applications9,10. Paper is biocompatible with DNA, RNA, proteins and various clinical samples, favouring its use in diagnostics of biological samples. Moreover, paper is biodegradable and combustible, which aids in disposal and elimination after use in experimental procedures and analysis. Additionally, paper allows for passive liquid transport, a property that is exploited in a multitude of paper-based microfluidic devices reported previously9,11-14. Colorimetric sensing modality does not require any bulky components and have been rendered extensively in developing low cost POC bio-sensing tools. Such platforms are constructed by forming micro-channels through paper substrate in order to restrict the flow of fluid. These can be formed by introducing hydrophilic channels separated by hydrophobic barriers utilising wax printing to form a specific pattern of hydrophilic and hydrophobic15. These technologies integrate various sensing modalities to detect and quantify nucleic acids such as fluorescence resonance energy transfer (FRET)16, electrochemical17-19, electrical sensing20,21. However, they mainly rely on costly and bulky instruments. Colorimetric assays for nucleic acid detection are qualitative and does not provide potentially copy number of nucleic acids in the sample22-26.Yet,
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the necessity for low cost and simple colorimetric microchip to quantify nucleic acid is highly required.
Colorimetric signals can be obtained by comparing differences in color and its
intensity. Generally, nucleic acids are detected utilising fluorescent probes or pH sensitive dyes. However, these dyes are semi quantitative and cannot be applied for quantification of nucleic acids. Crystal Violet (CV), alternatively, was used as an indicator of the presence of DNA. CV is a triphenylmethane dye containing a p-quinoid group that acts as a chromophore. It is generally used for bacterial classification in Gram staining and staining of DNA in agarose gels, among others, as CV has been shown to have high affinity for dsDNA, as opposed to ssDNA or RNA27,28. In aqueous solution, CV is violet in color. However, upon addition of substituents, such as sodium sulphite (Na2SO3), CV is converted into leuco crystal violet (LCV), which is colourless. However, LCV is chemically unstable and required excess amount of sodium sulphite in order to be stable. When dsDNA is added, CV binds with DNA due to electrostatic interaction of the negatively-charged phosphate group of DNA and the positive-charged quinoid of CV as well as interacting with major groove of dsDNA to form a CV-dsDNA complex. The stability of the CV-dsDNA complex prevented decolouration of the mixture, and change the colour of the dye back to the original violet colour (Figure 1)27. Consequently, the color change from colorless (of LCV) to violet (of CV) can be used as a colorimetric sensing modality of the dsDNA. Though, polymerase chain reaction (PCR) is the most commonly used method for gene amplification, it involves with thermal cycling steps. Contrary, isothermal amplification eliminated thermal cycling steps enhancing amplification. Loop-mediated isothermal amplification (LAMP), a promising and robust isothermal amplification and it has been developed to reduce the relatively long experimental time of the PCR reaction and the use of thermal cyclers to accommodate the alternating temperature steps of the PCR protocol29,30.
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Moreover, amplification of nucleic acid products is highly efficient (up to 106-109-fold) and fast, therefore analysis can be performed easier and quicker31. The application of highly efficient and rapid LAMP has been demonstrated in various means, such as gene sequencing32, pathogen detection33-36 and meat species identification37,38. Herein, we present wax-printed paper-substrate as a platform for the colorimetric detection and quantification of dsDNA using crystal violet in meat species (porcine meat) and microbial pathogen (Bacillus subtilis) identification. The assay is relatively low-cost, sensitive, rapid, quantitative and straightforward with tremendous potential to be used at the point-of-need for detection of DNA. We have constructed a simple colorimetric DNA detection assay on paper substrate. A round wax chamber in yellow was designed and wax printed onto chromatographic paper, at the centre of which a reaction zone or sample zone was created (Figure 1A). The colorimetric detection was based on the color change observed with crystal violet (CV) dye. Upon addition of sodium sulphite to CV, the original purple color of the dye turned colorless, at which point the compound was called leuco crystal violet (LCV), and was spotted onto paper substrate within the reaction zone. In the presence of specific DNA sequence amplified by LAMP reaction, the colorless LCV returned to the original purple color and was detected visually (Figure 1B). The yellow color of the wax chamber on white chromatographic paper was employed in order to aid in the observation of the most significant color change (Figure 1C). Using this method, we have successfully identified the cytochrome B gene against the gDNA of Sus scrofa (porcine) and rpoB gene against the gDNA of Bacillus subtilis, respectively. Results Colorimetric reaction optimization
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We first optimized the reaction condition of CV solution under various pH conditions to determine proper pH and CV concentration (Figure 2). Various concentration range between 1.5 nmol to 4.5 nmol of CV and wide pH range from 6- 9.5 of CV solution was initially tested (Figure 2A-D), as well as the ratio of CV: sodium sulfite was also tested within a range between 1:0.5 to 1:2.5 (Figure 2E&F). In addition, the most significant color change was observed at 4.5 nmol which was significantly different from lower concentration of 3.5 nmol (Figure 2A&B, p
