| Microdrop digital PCR detection technology has received widespread attention due to its advantages of absolute quantification,high sensitivity,and high accuracy.Compared with foreign countries,domestic research on this technology has made slow progress.Most detector products have problems such as fewer detection channels,low detection efficiency,and large volume.In this thesis,a multiple fluorescence microdroplet digital PCR detection system is designed to address the above issues.Focusing on the two key technologies used in the detection system,microfluidic technology and flow fluorescence detection technology,the flow channel model and optical system are simulated,and the circuit processing and peak value extraction of fluorescence signals are studied.Firstly,microfluidic technology is used to separate droplets in the flow channel for subsequent detection.A three-dimensional cross flow channel model is designed in Solid Works software,analyzing the relationship between droplet spacing and two-phase flow velocity,searching for the range of the ratio of sample flow to sheath flow velocity,and achieving optimal fluid focus so that droplets pass through the detection area one by one under the sheath flow wrapping;Secondly,a four channel fluorescence detection micro drop digital PCR optical detection system was designed around the flow fluorescence detection technology,and an X-prism was added to the system to compress the optical path.Through simulation and stray light analysis of the optical path of the detection system,it is verified that the light energy utilization rate of the system reaches over 93% and the stray light interference is small.The results show that the detection efficiency of this optical system is 4 times higher than that of traditional single channel systems,and its volume is also significantly reduced;Finally,a fluorescence signal acquisition and processing module is designed.PMT collects weak fluorescence signals and converts them into electrical signals,which are then amplified,filtered,and converted into digital signals through a shift and phase difference circuit,filter and amplification circuit,and analog-to-digital conversion circuit.After that,the analog signals are transmitted to the host computer for processing.The principle and process of extracting fluorescence signal peaks are emphatically analyzed,and the effective fluorescence signal peaks are successfully extracted.Based on the above simulation analysis,a dual detection channel micro droplet digital PCR detection system was built,and an injection pump and a flow pump were selected to drive the microfluidic liquid circuit.Three samples were used for the detection experiment,and the results showed that the proportion of target molecules in the detected sample and the proportion of target molecules in the original sample had a small difference.The experiment proved the feasibility of the multiple fluorescence microdroplet digital PCR detection system.The above research has laid a solid foundation for the development and optimization of multicolor digital PCR detectors in the future,providing innovative technical references. |
