Research On Ultrasensitive Detection Of Cathepsin B Activity By Multiple Cycle Signal Amplification Method

Cysteine cathepsin is a protease located in lysosomal vesicles,the main function is to mediate protein hydrolysis.As one of the important members of cathepsin family,Cathepsin B is the only protease with both endopeptidase and exopeptidase activity.It can identify peptide bonds at specific sites of amino acids,inactivate the cell-adhesion protein,activate other proteases(e.g.laminins),degrade the extra-cellular matrix,and release the metastatic cells from solid tumors,achieving the invasion and metastasis of cancer cells.Cathepsin B is closely related to many diseases,such as arthritis,cardiovascular disease,lung cancer and colon cancer.Alteration of Cathepsin B expression pattern,imbalance of proteolytic activity and change of localization can disrupt the homeostasis of normal biological tissues or cells,participate key steps of the tumor disease process,drive cancer progression in the tumor microenvironment.Thus,cathepsin B is becoming a promising tumor biomarker whose concentration and distribution can be used to guide the diagnosis and analysis of cancer.Cathepsin B detection methods mainly include affinity assays and activity assays,but these assays are usually require specific antibodies,and have problems such as time-consuming,poor sensitivity and large sample consumption.Therefore,sensitive and accurate detection of cathepsin B is critical for early diagnosis and therapy of cancer.Using nanomaterials such as peptide-DNA conjugates and magnetic beads,taking advantage of multiple cycle signal amplification technology,we developed a simple fluorescence method for detecting cathepsin B activity.We designed two DNA templates with multiple domains for DNA strand extension and digestion to convert cathepsin B activity into measurable fluorescent signals by binding ribonuclease H assisted amplification.The method is simple and easy to operate,short time-consuming and does not involve any thermal cycle.The method has the following characteristics: First,the method can completely eliminate the steric hindrance effect in the process of nucleic acid amplification by translating the primer DNA containing amino acid residues into the new primer DNA without amino acid residues.Second,the amplification of cyclic signal induced by multiple strand displacement reactions.Third,RNase H-driven cyclic digestion of signal probes further amplifies the fluorescence signal.This method can sensitively detect cathepsin B activity with an extremely low detection limit of 8.1 pg/mL and a large dynamic range of 4 orders of magnitude from 0.01 to 100 ng/m L.This method can measure cathepsin B activity in the human cervical carcinoma cell line(He La cells)at the single-cell level,and can be further applied for the screening of cathepsin B inhibitors.