Development Of UV-Vis-Integrated Microfluidic System For Concentration Detection Of Nucleic Acid

Digital polymerase chain reaction(dPCR)enables the absolute quantification of nucleic acid molecules,which is widely used in gene cloning,DNA sequencing and disease diagnosis.The accuracy of dPCR detection depends not only on the number of reaction units,but also on the concentration of molecules in each reaction unit.The optimal concentration for obtaining the highest accuracy of dPCR is about 1.59 molecules in each reaction unit.Hence it is necessary to quantify the concentration of nucleic acid sample before running dPCR,and then the sample can be diluted into the optimal concentration.Microfluidics refer to the miniaturized devices and experimental methodologies of manipulating small liquid volume in interdisciplinary research,such as biology,chemistry,biomedicine,and other engineering fields,with versatile applications.In this dissertation,the detection of nucleic acid concentration based on UV-Vis and microfluidic technologies with low sample consumption,wide detection range and high integration is realized on the premise of ensuring detection sensitivity and repeatability.The major work in this dissertation is described as follows:1.A microfluidic chip to detect the nucleic acid concentration was designed and processed.The chip was fabricated by 3D printed mold and casting.Meandering channel was designed for temporary storage of sample solution.The concentration detection in the chip is based on UV-Vis.Polydimethylsiloxane(PDMS),used as the material for chip replication,features transparence in UV-Vis light.Three optical path lengths,0.2 mm,1 mm,and 10 mm were patterned innovatively in the microfluidic chip to determine the nucleic acid concentration.2.A setup capable of reliably fixing the microfluidic chip and precisely switching the optical path lengths was designed and manufactured.The setup mainly consists of three parts: a clamper to fix the microfluidic chip,a cross slide to move the optical fiber,and an electronic circuit to control the sliding.The clamper plays an important role in supporting and fixing the microfluidic chip stably on the setup.The coupling optical fiber is innovatively separated from the microfluidic chip in the setup,which is fixed on the cross slide.With the precise control of the electronic circuit hardware,the cross slide carries the coupling optical fiber to move among the three detection regions with different optical path lengths,so that the absorbance of UV-Vis light could be obtained through a micro fiber spectrometer.3.A user interface for the detection operation,and real-time data acquisition,analysis and visualization was designed.The user interface software was implemented based on MATLAB GUI.With the acquired raw data from spectrometer,the sample concentration was calculated based on Lambert-Beer law.In conclusion,we demonstrated that the microfluidic system based on UV-Vis was capable of the quantitative detection of nucleic acid concentration with the wide dynamic range of 1.5 ～ 3500 ng/μL,the high sensitivity of 0.9 ng/μL and the low coefficient of variation(CV)of less than 10%.Potentially,the microfluidic system could be integrated into a d PCR instrument as a functional module for quantifying the concentration of nucleic acid sample before running PCR.