| Nucleic acids, with high diversity and specificity, could be targeted as the biomarker for identifying and detecting different biological species, individual organisms, or even single genes. Nature journal pointed out that WHO developed a list of general characteristics that make a diagnostic test appropriate for resource-limited sites, known as ASSURED. According to this criteria, significant efforts have been poured to solve the technical difficulties in nucleic acid testing, including simplifying the operating procedure, improving the detection performance and efficiency, and lowering the user threshold. In order to overcome deficiencies and problems of current methods for qualitative and quantitative nucleic acid testing, especially for point-of-care testing, we integrated microfluidics together with nucleic acid isothermal amplification technique, and developed an Rotate & React chip and a centrifugal spiral chip, respectively, for qualitative and quantitative visual detection of pathogenic nucleic acid based on loop-mediated isothermal amplification (LAMP). Our approach offers novel nucleic acid testing platform for various applications including disease diagnosis, biochemical analysis, environmental monitoring, forensic identification and food safety control.Firstly, we established a semiclosed Rotate & React microfluidic chip platform which was successfully applied in visual LAMP assay. By hydrophobic modification on the surfaces of the chip glasses, multiple liquid samples could be introduced and parallel mixing reaction could be conducted on the chip by one-step rotational slipping operation, without leakage or cross-contamination. Fluorescence quenching reactions were carried out on the chip to further evaluate the method and nearly 3 orders of magnitude in fluorescein concentration were quenched linearly. The isothermal control on the chip was improved by a custom-built peltier TEC heater which could start the LAMP reaction within 5 min. Different quenching efficiencies of fluorescent indicator for LAMP assay were tested using copper ion, silver nano-particles and sodium hydroxide, respectively, and we optimized the best conditions for visual LAMP detection on chip, which achieve a 4 to 5-fold positive/negative fluorescent signal ratio while reagent consumption was downscaled to 0.7?L per chamber. Besides, we investigated the pros and cons that would cause carryover contamination in LAMP, and determined the dU-degradable reagent setup and standard operation process for a robust and safe LAMP assay on chip.Secondly, qualitative LAMP detection was attempted for five common digestive pathogens including Bacillus cereus, Escherichia coli, Salmonella enterica, Vibrio fluvial is and Vibrio parahaemolyticus based on the visual LAMP detection method established in Chapter 2. By carrying out a series of parallel LAMP assays on chip, the best working concentrations for major ingredients including inner primers, dNTPs, Bst DNA polymerase, betaine and magnesium ion, were screened. Under such reagents setup, the visual LAMP detection on chip for Vibrio fluvialis was realized, and a detection limit of about 7.2 copies/?L was obtained while good specificity of the self-designed primers was confirmed and verified. Then we further attempted simultaneous visual LAMP detection of multiple pathogens on the Rotate & React chip and the results were validated in comparison with off-chip references, which showed good consistency.Thirdly, a mathematical model for digital isothermal amplification quantitative method was derived and built on the theories of poisson statistics and chemometrics. Validity and reliability of maximum likelihood estimation were proved and demonstrated in theory by a MATLAB coding Monte Carlo simulation program. Then the technical parameters including precision, accuracy and dynamic range, were calculated for 1200-pex and 12000-plex digital quantitative assays respectively. Moreover we compared the pros and cons between digital assay and the Ct-method real-time assay by theoretical calculation, and the quantification resolutions were deduced and analyzed. Based on previous work, a new centrifugal digital spiral chip was designed and fabricated to further test the performance of sample partitioning, temperature maintenance and evaporation control. Calcein was loaded into the spiral chip and fluorescence image showed that the volume and intensity of the partitioned chambers were evenly distributed, with coefficients of variation equal to 2.27% and 0.676%, respectively. This chip facilitates fluorescence detection by naked eye and portable optical sensors, which could sharply cut down the cost of quantitative assays for nucleic acid.Finally, by integrating centrifugal spiral chip together with visual isothermal amplification technique, the microbial loads of Vibrio fluvialis were quantitatively measured. We screened and optimized the reaction temperature, polymerase activity and reaction duration and evaluated the influence of imaging conditions on the quantification results. Further experiments indicated that this chip has the capability of quantifying pathogenic genomic nucleic acid samples that spanned over 4 orders of magnitude in concentration, and the results were in good consistency with real-time assays. In comparison with the qualitative assay established previously, the detection limit was further lowered to 8.7×10-2 copies/?L, and the response time was shortened to 40 min with a competitively low cost for each single assay. By incorporating with our custom-built peltier TEC heater and portable optical sensors, the developed digital assay could get free of dependence on real-time fluorescence detectors and thermal cyclers, satisfying the urgent need for applications in point-of-care service. |
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