Development Of LAMP Microfluidics For Point-of-care Detection Of Biomolecules

This work creatively combined the loop-mediated isothermal amplification (LAMP) and microfluidic technology. We developed various LAMP microfluidic chips, including singleplex/multiplex format (sμLAMP), qualitative/quantitative format (mμLAMP) and integrated format (iμLAMP). These formats of LAMP chips were successfully used to the rapid and accurate diagnosis of pseudorabies virus (PRV), influenza virus (Flu), mycobacterium tuberculosis (MTB) and so forth. We made use of software Vector NTI to determine three specific gene markers of DNA-binding protein (DBP) gene, hemagglutinin gene and Gyr B gene for PRV, Flu and MTB, respectively, while LAMP primers, targeting at these gene markers, were successfully designed by Primer Explorer V3software and microfluidic chips in this study were designed and fabricated by MEMS technology and soft-lithography method.1) Singleplex quatitative/quanlitative LAMP chip (sμLAMP) was developed based on the merits of the biocompatibility, gas tightness, and light transmissibility of the PDMS in this part of work. The whole volume of the amplification chamber is5μL, while the length of the chamber is10mm, width:0.6mm and depth:0.8mm. The assay is straightforward in manipulation and successfully applied in the rapid diagnosis of pesudorabies. It requires a sample volume of0.4μL and is complete within1h. The sensitivity of the assay is comparable to standard methods, where10fg of target DNA could be detected under isothermal conditions (63℃). A real-time quantitative μLAMP assay using absorbance detection was achieved by integrating transmissible light-based optical fibers within the chip, and the standarnd curve displayed high correlation between target concentration and time-to-positive value (TTP) with the coefficient of0.9894.2) Multiplex quatitative/quanlitative LAMP chip (mμLAMP) was developed in this part of the work. This format of LAMP chip was designed to have multiple microchambers and each was connected to the corresponding thin microchannel via a dimension gradient bridge. Thin microchannels with low-mass-transfer coefficient were critical to forbid the cross-talk of probes among different microchambers. This mμLAMP assay with the ability of analyzing multiple genes qualitatively and quantitatively is highly specific, operationally simple and cost/time-effective with the detection limit of less than10copies/μL in2microlitre quantities of sample within0.5h. A real-time quantitative mμLAMP assay using absorbance detection was achieved by integrating reflectable light-based optical fibers within the chip. We obtained standard curves with correlation coefficients of0.9984,0.9897and0.9962for flu A, seasonal H1N1and pandemic H1N1, respectively.3) Single/multiplex integrated micro-LAMP chip (iμLAMP) was developed in this part of work. We successfully integrated the nucleic acid extraction, amplification and signal read-out in this format of chip based on the elasticity merit of the PDMS material, achieving the simple-to-answer effect. This iμLAMP was successfully applied to be used in the diagnosis of MTB and differention of bacteria within45min to60min via naked eye read-out with the10μL sample volume demanded.These micro-LAMP chips, including sμLAMP, mμLAMP and iμLAMP established in this work was very simple-fabricated, low-cost, small sample volume-demanded, and with high specificity, sensitivity and rapidity, which was very suitable for the point-of-care detection of biomolecules. These micro-LAMP chips could also be applied in detecting other important pathogens, holding much significance in developing point-of-care diagnostic assays to combat various epidemics or genetic diseases, especially in some resource-limited countries.