Development Of Fluorescent Detection System For Point-of-care Testing Instrument Of Nucleic Acid

Nucleic acid testing plays an important role in the prevention and diagnosis of infectious diseases,for example,since the outbreak of the novel coronavirus(SARSCoV-2),nucleic acid testing results have become one of the important bases for detecting and isolating infected individuals to limit the further expansion of the outbreak.However,due to the complex operation of traditional nucleic acid testing techniques and the susceptibility of samples to contamination causing false positives,the efficiency of nucleic acid testing is low,the waiting time for issuing report results is long,and it cannot be applied to primary care units on a large scale.In order to solve the limitation,our research group has developed and designed a rapid nucleic acid detection instrument based on microfluidics(iNAT)which is used for point-of-care testing.It can achieve "sample input,answer output".And it can work at anytime and anywhere.This article aims to develop a real-time fluorescent detection system that can be used in iNAT.The research includes the design of the optical module,the hardware circuit design of control system,fluorescence signal processing and analysis,and experimental validation:(1)Based on the structural characteristics of iNAT and the demand of miniaturized instrument use,the design and construction of multi-channel optical detection module was completed,mainly including excitation optical path,emission optical path,multichannel integration and switching device.The light-emitting diode was used as the excitation light source to provide stable excitation light;the photodiode was used as the photodetector to realize reliable photoelectric conversion;the narrow-band filters with different cutoff wavelengths were used as the filter element to reduce stray light interference.In order to improve the coupling between the optical path,the instrument amplification module and chip,use plano-convex lenses and an optical fiber with linear light spots as light transmission elements.As for the overall optical path structure,an orthogonal optical path structure was adopted to reduce the interference of excitation light to fluorescence acquisition.In addition,in order to achieve multi-channel integration and switching,a multi-channel integration and switching device was designed and fabricated by 3D printing technology,which has realized the assembly and integration of multiple optical components and completed the detection of fluorescence intensity in different wavelengths.(2)Based on the real-time fluorescence quantitative detection process and requirements of the polymerase chain reaction process,the hardware circuit was designed and implemented to complete the control and data acquisition processing of the detection system.The main sub-circuits include power supply module circuit,LED constant current drive circuit,communication control circuit,stepper motor drive circuit,and the signal conditioning and sampling circuits.(3)In order to complete the quantitative analysis of nucleic acid detection,a fluorescence signal processing and analysis algorithm was established.This article used the five-point moving average filter and the Savitzky-Golay filter algorithm to smooth the curve.And we proposed a calculation method of Ct value.The method of linear fitting and the calculation and comparison of linearity R2 is used to distinguish the data of exponential period and linear period.Finally,the Ct value(Cycle threshold)of the PCR amplification curve is solved by the method of solving the intersection of the threshold and the fitted straight line of the data of linear period.(4)The multi-channel real-time fluorescence detection system was integrated into the prototype instrument for testing.Concentration gradient detection was performed with three different channel fluorescent dyes of FAM,HEX,and ROX(excitation light bands are 450-490nm,510-530nm,570-590nm).The limits of detection were:0.39μg/ml for the FAM channel,0.06μg/ml for the HEX channel,which were consistent with those of commercial fluorimeters.And the two channels of the system have better linearity.For the ROX channel,the limit of detection of the system is 0.31 μg/ml,which has a certain gap compared with the commercial fluorimeter.And the linearity also has a gap.Further research and optimization are needed for the follow up.At the same time,10 consecutive repetitive tests were performed on different gradient fluorescent dyes.The coefficients variation(CV)of the test results were less than 1%,which means the system has good detection repeatability.We took Cytomegalovirus(CMV)samples as the object,used FAM,HEX and ROX fluorescent probes to label the samples and then performed gradient sample dilution to detect the nucleic acid.The linearity of the Ct values obtained from the three channels was more than 0.98,which was good.At the same time,the repeatability test of multi-channel real-time fluorescent PCR was completed.The experimental results showed that the C V values of the test results were FAM:1.8%;HEX:2.9%;ROX:2.5%,which all met the national pharmaceutical industry standard of no more than 3%,indicating that the repeatability was good.In general,fluorescent detection system designed and implemented in this paper can successfully achieve multi-channel nucleic acid quantitative detection.For the pointof-care testing of nucleic acid,the detection results are consistent with the current commercial laboratory instruments.