| Fluorescence detection has been widely used in medical testing,food hygiene,animal and plant inspection and quarantine. It is of great significance of quantitative analysis and specific identification of nucleic acids by fluorescence detection in biomedicine. MicroRNA(miRNA) in nucleic acid is closely related to the occurrence and development of various diseases in the development of life. Besides, miRNA regulation of gene expression precedes the change of protein in the regulatory chronological order,therefore it can be used as a potential marker for early diagnosis of diseases. What’s more, it is difficult to meet the needs of the rapid and real-time detection of low abundance miRNA requirements, considering that most of the traditional miRNA detection methods should be operated by the specially trained operators with expensive detection equipments which inevitably would take long detection time and high detection costs. This study based on the characteristics of miRNA that can be expressed in advance on some diseases to mark the specific miRNA with the rolling circle amplification(RCA) detection technology of miRNA markers and detect the fluorescence intensity of the target using photoelectric detection method. By analyzing the initial concentration of miRNA, it provides a new technology for rapid and real-time detection of specific miRNA. The miRNA fluorescence detection system developed by this technique can provide a risk stratification and early warning instrument for chronic diseases such as coronary heart disease. The specific work has been done in the following aspects concering the rapid detection of fluorescence:Based on the mechanism of fluorescence generation, the number of fluorescence photons that can be generated by the structure of confocal fluorescence detection system and non-confocal fluorescence detection system is calculated through theoretical analysis. If the confocal structure is used and with the light power of the excited light is up to 1.2mW, the minimum detection sensitivity of the photodetector is not less than 2.77×10-7W, while using the non confocal structure, the minimum detection sensitivity of the photodetector is not less than 1.98×10-7W under the same light intensity. According to the theoretical calculation results, the positive incident structure in the nonconforming structure is chosen as the optical structure of the miRNA fluorescence detection equipment.According to the characteristics of miRNA fluorescence detection, the specific miRNA of coronary heart disease is amplified by technique of rolling circle amplification in the system design. The SYBR Green II fluorescent dye is used as the fluorescent marker,the corresponding light source is selected for the light-emitting diode (LED) according to the fluorescent dye parameters, and the detector is the photomultiplier tube (PMT). Through the theoretical calculation, semiconductor heating is selected as the heating method of miRNA fluorescence detection system due to the characteristics of rolling ring amplification and fluorescence detection structure. Through the optical shaping design of the excited light beam, it can meet the application requirements of the system. The sample pool was designed to facilitate the rolling circle amplification reaction and facilitate the detection of fluorescence signals,and to isolate the crosstalk between the fluorescence signals of different samples as well. Finally,data processing and human-computer interaction are completed through software design.Through the design of the corresponding validation experiments, a number of technical indicators of miRNA fluorescence detection system were obtained. The linear bias of fluorescence intensity of miRNA fluorescence detection system was no more than ±5%,the repeatability no less than 95%,and the lowest detection limit was 12 copies. The coronary heart disease marker miR-499 which was extracted from myocardial cells are amplified by RCA using miRNA fluorescence detection system.It can be seen from the test results that miRNA fluorescence detection system can meet the purpose of coronary heart disease-specific miRNA real-time detection. It was concluded that the time required for coronary heart disease-specific miRNA detection was less than half an hour by setting the rolling ring amplification reaction conditions reasonably.It can be concluded through the results of the experiment that the sensitivity of miRNA fluorescence detection equipment is similar to that of real-time fluorescence quantitative PCR,while the detection time of miRNA fluorescence detection equipment is much lower than that of real-time fluorescence quantitative PCR instrument. This provides a valuable detection technique for the rapid and immediate detection of specific miRNAs. By using the orthogonal detection structure and the overall of the designment to reduce the background noise of the equipment, improve the efficiency of fluorescence collection and realize the weak fluorescence collection,it successfully provides a miniaturized design for the biological fluorescence detection.The miRNA fluorescence detection equipment based on miRNA fluorescence detection technology has the characteristics of low cost, small size, easy to carry and operate. It can not only be used as laboratory testing equipment for the rolling ring amplification but also be used as a common device for daily detection of small and medium-sized medical institutions, It provides a new auxiliary diagnostic method for the risk stratification, early warning and disease tracking of chronic diseases such as coronary heart disease and cancer. |
