Electrochemical Signal Amplification And Detection Of Prostate Tumor Exosomes Based On Aptamer Recognition

A liquid biopsy,also known as fluid biopsy or fluid phase biopsy,is the sampling and analysis of non-solid biological tissue,primarily blood.Comparing with the traditional biopsy,the advantage of liquid biopsy is that it can provide a resource for noninvasive diagnosis,which reduce the risk of biopsy.In addition,liquid biopsy can be done more frequently which can better track tumors and mutations over a duration of time.Currently,the tests for liquid biopsy include circulating tumor cells(CTCs),circulating tumor DNA(ctDNA)fragments and exosomes(carrying a variety of proteins,lipids,DNA and RNA related to cell original).Exosomes are more abundant and more likely to be enriched when compares to CTCs.Secretion of vesicles can efficiently protect the nucleic acids and avoid the problem of ctDNA easily degradation in blood.Therefore,the detection of exosomes has broad prospects in clinical application.Exosomes,the diameter of 30-150 nm,are nanoscale extracellular lipid bilayer vesicles of endocytic origin.They can convey functional information to neighboring cells or distant cells,thus promoting intercellular communication.Due to the high abundance and good stability of exosomes in body fluids,especially in the blood,they are being evaluated as a reliable source of circulating biomarkers for cancer.Exosomes carried the tumor-specific proteins and miRNA are successfully used as biomarkers for several diseases,such as ovarian cancer,breast cancer,prostate cancer and colon cancer.At present,the detection of exosomes is based on the antibodies or aptamers specific recognition of proteins.Aptamers are oligonucleotide molecules consisted of DNA or RNA and exhibit excellent stability and selectivity when comparing with antibodies.In addition,aptamers are less susceptible to environmental factors such as pH and temperature.Herein,we constructed a biosensor through using the specific binding between aptamer and antigen expressed on the membrane of exosomes to detect the tumor-derived exosomes.The purpose of this project is to establish a non-invasive cancer detection method that provides a theoretical basis for early diagnosis and treatment.We have established an electrochemical biosensor based on AuNPs-aptamer signal amplification to detect prostate cancer cell-derived exosomes.In this work,the probe DNA was modified on the gold electrode for specific recognition with the four-transmembrane protein CD63 expressed on the exosome membrane,which allowing the exosomes can be immobilized on the gold electrode.AuNPs-aptamer was gold nanoparticle modified CD63 aptamers on the surface.On the one hand,AuNPs-aptamer was used for the specific identification of exosomes.On the other hand,it carries a large amount of DNA and the DNA phosphate backbone is negatively charged,thus can adsorb more of the active substance,RuHeX,to enhance the signal.The aptasensor shows a detection limit of 3.568×104 particle/mL,with the linear range from 9.15×104 particle/mL to 3.25x107 particle/mL.Many tumor-derived exosomes would carry the four-transmembrane protein CD63.Therefore,the biosensor has universal applicability that has an important guiding significance for the early diagnosis of disease.We constructed a method that basing on the HCR for amplified the electrochemical signal to detect the exosomes of prostate cancer.Exosomes secreted by the parental prostate cells will be carry a large number of PSMA,which is a specific protein of prostate cancer cells.We design two hairpins that can trigger the HCR in the presence of initiating strand(One end of the CD63 aptamer is another sequence that can initiate the hybridization of the two hairpins).In the presence of targets,the PSMA aptamer modified on the electrode that can capture the exosomes through specific recognition.The initiator,on the one hand,was used to specific recognized the CD63 protein expressed on the exosomes and on the other hand,it was employed to trigger the HCR.The self-assembly nanostructure could insert more RuHeX into the nicked double helixes,which can efficiently generate an amplified electrochemical signal.