MicroRNA Assay Based On Catalytic Hairpin Assembly And Rare Earth Element Labeled Probes

Micro RNAs(miRNAs)are a category of short non-coding RNA molecules which play essential roles in regulating gene expression.Correspondingly,the aberrant expression of miRNAs is commonly related to the development of various diseases including cancers and cardiovascular diseases.Hence,miRNA is considered as an ideal biomarker for early diagnosis,and advanced methods for its qualification and quantification are highly demanded.However,the traditional miRNA detection methods are often complicated and suffer from poor sensitivity or high cost.Herein,an isothermal and enzyme-free miRNA assay based on catalytic hairpin assembly(CHA)amplification coupled with inductively coupled plasma mass spectrometry(ICP-MS)detection is proposed.A highly sensitive analysis of miRNA has been achieved through the efficient amplification method and the stable mass signal output.In chapter one,traditional miRNA detection methods are introduced first.Then,combined with signal output forms,some commonly used amplification methods for nucleic acid are summarized.Based on these backgrounds,the main idea and significance of this work are outlined.The preparation process of the rare earth element labeled DNA probe is shown in chapter two,and the characterization data of the labeled probes are also presented.The bifunctional chelating agent 1,4,7,10-tetraazacyclododecane-1,4,7-tris-acetic acid-10-maleimidoethylacetamide(mma-DOTA)was employed to link the DNA probe and the rare earth element.Meanwhile,the use of gel filtration chromatography makes the purification process convenient.The labeled DNA probes were verified by high-resolution quadrupole time-of-flight mass spectrometry(QTOF-MS),ICP-MS,and Nano Drop micro nucleic acid spectrophotometer.The positive results indicate that the proposed method can be used to prepare different DNA probes that are labeled with different element tags.Also,these labeled probes may be used in different nucleic acid assays.In chapter three,the feasibility of the CHA amplification procedure was demonstrated by native polyacrylamide gel electrophoresis(PAGE)first,and the amplification parameters including temperature and time were optimized.Under the optimized conditions,a series of criterion miRNA-141 solutions with different concentrations were analyzed by the proposed method.According to the calibration curve,the limit of detection(LOD)of miRNA-141 was 88 fmol with a linear range0.1 ～ 2 pmol.Compared with fluorescence mediated CHA method and ICP-MS mediated basic sandwich method,the current strategy displayed the highest sensitivity and the lowest LOD.The specificity test implied that the proposed method is capable of distinguishing the target miRNA from its family members.Ultimately,the developed method was also applied to analyze miRNA-141 and miRNA-21 spiked in 10% bovine serum samples simultaneously.The good recovery demonstrated its multiple detecting capability and practical applicability.The obtained results are summarized in chapter four and some new prospects are put forward.As an isothermal and enzyme-free miRNA detection method,the combination of CHA amplification and ICP-MS detection is convenient and reliable.It possesses many advantages such as low detection limit,high sensitivity,and high throughput.If more distinct rare earth elements are employed,the advantage of simultaneous multi-element analysis by ICP-MS can be further improved.Furthermore,the proposed miRNA assay has the potential to become a high-throughput method and brings convenience to clinical diagnosis.