1. Department of Chemistry, School of Physics and Chemistry, Gwangju Institute of Science and Technology (GIST), 261 Cheomdan-gwagiro, Gwangju 500-712, Republic of Korea;
2. Department of Electrical Engineering, University of California, Los Angeles, California 90095, United States;
3. Department of Physiology Kyungpook National University School of Medicine, Daegu 41944, Republic of Korea;
4. Mmonitor Incorporation, Daegu 41914, Republic of Korea;
5. Department of Bionanotechnology, Gachon University, Sungnam 13120, Republic of Korea;
6. Department of Nanobiotechnology, Korea University of Science and Technology (UST), 305-350 Republic Korea;
7. Hazards Monitoring Bionano Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), 305-806 Republic of Korea.
* These authors contributed equally to this work.
Paper-based diagnostic devices have many advantages as a one of the multiple diagnostic test platforms for point-of-care (POC) testing because they have simplicity, portability, and cost-effectiveness. However, despite high sensitivity and specificity of nucleic acid testing (NAT), the development of NAT based on a paper platform has not progressed as much as the others because various specific conditions for nucleic acid amplification reactions such as pH, buffer components, and temperature, inhibitions from technical differences of paper-based device. Here, we propose a paper-based device for performing loop-mediated isothermal amplification (LAMP) with real-time simultaneous detection of multiple DNA targets. We determined the optimal chemical components to enable dry conditions for the LAMP reaction without lyophilization or other techniques. We also devised the simple paper device structure by sequentially stacking functional layers, and employed a newly discovered property of hydroxynaphthol blue fluorescence to analyze real-time LAMP signals in the paper device. This proposed platform allowed analysis of three different meningitis DNA samples in a single device with single-step operation. This LAMP-based multiple diagnostic device has potential for real-time analysis with quantitative detection of 102-105 copies of genomic DNA. Furthermore, we propose the transformation of DNA amplification devices to a simple and affordable paper system approach with great potential for realizing a paper-based NAT system for POC testing.
Keywords: loop-mediated isothermal amplification, paper, biosensor, molecular diagnosis, nucleic acid testing, point-of-care.