Highly Specific And Sensitive Detection Of Let-7a Via Exponential Rolling Circle Amplification And Chemiluminescence Instantaneous Derivatization Technology

An exponential rolling circle amplification (RCA) and chemiluminescence instantaneous derivatization technology are employed for the highly specific and sensitive detection of let-7a. Only in the presence of target let-7a, the padlock probe can be ligated and subsequent isothermal RCA process is initiated by DNA polymerase. Following that, long RCA products are restriction digested into many new "target DNA sequences", further initiating numerous new extension processes in a polymerase/nicking amplification mode. Thus, huge numbers of streptavidin aptamers are generated and then accumulated on the magnetic beads for the generation of chemiluminescence signal. With the employment of instantaneous derivatization reaction between the 3,4,5 trimethoxylphenylglyoxal and guanine nucleobases on the streptavidin aptamer backbone, a detection limit of 5 amol was obtained with a dynamic range that spanned 5 orders of magnitude. Moreover, excellent specificity is achieved by mutating single base on the fully complementary padlock probe and this approach could be easily applied to the detection of let-7a in human lung cells. Upon modification, the approach presented herein could be easily extended to detect other types of miRNA and DNA and even proteins, which enables the developed method to be a universal sensing platform for a variety of target analytes.