| As key regulatory factors in cells,micro RNAs(mi RNAs)affect many important biological processesses,such as growth and development of the human body.Recent studies have shown that mi RNAs also play a key role in the generation and development of tumors and other major diseases.At the same time,mi RNAs released into the human circulatory system can also become potential markers for early tumor diagnosis.However,due to the cumbersome operation,the limited sensitivity,and the dependence on the complex instruments,the application of mi RNAs-based diagnostic techniques in the developing areas is hindered.Therefore,under the premise of ensuring sensitivity and specificity,the establishment of a simple,easy-to-use,low-cost mi RNAs detection technology will provide the technical foundation for popularizing early tumor screening and improving the level of medical services in resource limited areas in our country.In this thesis,by using protein self-labeling technology we designed and constructed a fragmented luciferase-DNA chimera probe with high signal-to-noise ratio base.By combining the as-designed probes with the rolling circle amplification(RCA)system,the smart phone was used to achieve high-sensitivity detection of lung cancer mi RNAs markers in human peripheral blood.The following works have been done:(1)By fusing the SNAP tag to the fragmented Nano Luciferase(Nluc),the covalent cross-linking of the protein fragments and single-stranded DNA(ss DNA)is achieved on the basis of 1:1 stoichiometric reaction.The fragmented luciferase-DNA chimera can produce specific signal output to the DNA template through the base pairing.The signal-to-noise ratio of this system is 175.3.(2)By combining the chimera probes with the specially designed RCA system,the ultra-sensitive detection of the lung cancer marker mi R-21 was achieved by using a microplate reader,with a detection limit of 23.4 a M.Compared with the unamplified detection system,the sensitivity is improved by 7 orders of magnitude.In addition,compared with the existing nucleic acid bioluminescence detection method,its sensitivity is improved by at least 5 orders of magnitude.(3)In order to verify the applicability of the above method in point-of-care(POC)settings,we further used the smart phone as a portable detection equipment to evaluate the analytical performance of this method.Studies have shown that the detection limit of mi R-21 is 11.2 a M,which is consistent with the results of the microplate reader.At the same time,smartphone-based detection can accurately distinguish single-base mutations of mi R-21 targets.(4)In order to further verify the versatility of the above methods in the detection of different mi RNAs,we further analyzed the lung cancer marker mi R-148 b and the commonly used external reference target cel-mi R-39.The results showed that the detection limits of the above two targets were 15.9 a M and 16.3 a M,respectively,and they had single-base specificity.Therefore,this method is applicable to the detection of different mi RNAs.(5)Finally,we verified the applicability of this method in clinical diagnosis.By analyzing the serum samples of the healthy group and the lung cancer group(12samples each),the differences in the expression of mi R-21 and mi R-148 b in the above two samples can be detected,and the results are consistent with real-time fluorescent quantitative polymerase chain reaction(q RT-PCR)experiments.Therefore,this method has high analytical performance,and demonstrates the advantages of low instrument cost and simple operation,thus providing a new tool for mi RNA-based clinical diagnosis. |
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