| Protease is an indispensable biological macromolecule in life activities.It can process specific substrates with high selectivity,restriction and efficiency,which can lead to irreversible changes in protein translation level,and then affect important biological processes such as embryonic development,neuron growth,cell cycle regulation and so on.The change of protease structure and expression pattern is the basis of many human pathological processes,which is of great significance to the study of disease pathogenesis.Although immunoassay based on antigen-antibody interaction can sensitively detect the mass concentration of protease,the protease precursor(proenzyme)can cause false positive signal.Moreover,the analysis methods based on protease hydrolysis substrate polypeptide lack effective signal amplification process,and thus their sensitivity cannot fully meet the requirements of clinical biochemical analysis.Therefore,it is important to develop an efficient signal conversion amplification strategy to realize highly sensitive protease activity analysis.In this paper,taking the T7 RNA polymerase-lysozyme fusion protein as the module,we constructed a proteolysis-activated transcription-based transducer element,which can transform and amplify the protease activity signal to nucleic acid signal out,and further established a new protease biosensor method to realize ultra-sensitive analysis of protease activity.Specific work is as follows:1.Construction of proteolysis-activated transcription-based signal transduction amplifiers for the analysis of protease activity.T7 lysozyme can inhibit the transcription activity of T7 RNA polymerase(T7 RNAP),while a protease can split the linkage peptide between them,and restore the activity of T7 RNAP,accompanied by the generation of large amounts of RNA through transcription.The output RNA was designed as a G-quadruplex sequence,which was combined with a small molecule dye to generate fluorescence,and thus the activity of the protease can be reflected the intensity of the fluorescence signal.Using thrombin as the protease model,a limit of detection of 0.33 pM was obtained,which is lower than that of many existing detection methods.This method can not only realize the transformation from protein signal to nucleic acid signal,but also improve the sensitivity of target detection by using the amplification strategy of transcription,which provides a favorable tool for the detection of protease and lays a foundation for the follow-up research.2.We constructed a novel colorimetric method for the detection of tumor marker MMP-2 based on the proteolysis-activated transcription.On the basis of above research,we replaced the linkage peptide in the fusion protein with the specific substrate peptide of MMP-2,and constructed a proteolysis-activated transcription-based transducer for MMP-2.The output RNA was designed as the linker to induced the cross-link between DNA-functionalized AuNPs,and the aggregation of the AuNPs generate a colorimetric signal.The quantitative analysis of MMP-2 was carried out based on the changes of the color or the absorption spectra of AuNPs,and a limit of detection of 17 pM was obtained.3.On the basis of the proteolysis-activated transcription,we constructed a paper-based sensor that enables fast,visual detection of MMP-2.The colorimetric reaction in former study was moved from EP tubes to the glass fiber,and the quantitative analysis was carried out via the smartphone with a limit of detection of 17 pM was obtained for MMP-2.The paper-based sensor was further applied to analyze the expression level of MMP-2 in different cells,and the results of which are agreed well with that of the standard method,indicating the potential application of the proposed sensor in clinical diagnosis. |
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