Peptide Nucleic Acid-Functionalized Nanochannels Biosensor For Highly Sensitive Detection Of Tumor Exosomal MicroRNA

Early tumor detection is the key to improve the cure rate,and tumor marker detection is one of the favorable methods for early tumor detection,so it is of great significance to search for appropriate tumor markers and carry out monitoring for early detection and prognosis evaluation of tumors.Studies have found that highly stable miRNAs in human serum or plasma can regulate oncogene expression,tumor proliferation,angiogenesis and metastasis,which can act as tumor suppressors and promoters,and are regarded as a new generation of tumor markers.Since exosomal miRNAs are protected by the lipid membrane of vesicles,they can avoid the degradation of RNase in the blood,and have higher abundance and stability than free miRNAs in blood.Therefore,exosomal miRNAs can be regarded as an ideal molecular marker for early diagnosis of cancer.Therefore,the construction of highly specific and sensitive exosomal miRNAs sensors is of great significance for the early diagnosis of cancer.In recent years,nano-biosensors have been widely used in the detection of disease-related molecular markers due to their high specificity,fast analysis speed,and easy operation.Nanochannel biosensor is a new type of biosensor developed in recent years.Because the size of the nanochannel is controlled in the nanoscale range,the channel has a high specific surface area,which is conducive to highefficiency functional modification of the channel surface.Nano confinement effect increases the probability of interaction between the detection substrate and the ligand in a narrow nano space.The inner diameter of the channel and the modified functional groups can be controlled according to needs,so that it has high specificity,sensitivity and rapidity for detecting substrates.Therefore,biomimetic nanochannels have attracted much attention in the fields of analysis and detection of biomolecules.Based on this research background,this paper constructed a peptide nucleic acid(PNA)functionalized nanochannels biosensor to detect tumor exosomal miRNAs.Since PNA is electrically neutral,the stability and specificity of binding to miRNA are greatly improved,showing good affinity and stability.Through the change of the surface charge of the nanochannel before and after hybridization,the sensitive,specific and rapid detection of tumor exosomal miRNAs can be realized.Due to the high specific surface area in the nanochannel,the nano confinement effect and the use of electrically neutral PNA,the hybridization efficiency of PNA and miRNA is improved,which is expected to solve the outstanding problems of current tumor exosomal miRNAs detection,such as low recognition efficiency,unsatisfactory specificity,and operation time.The work is divided into the following two parts:Part Ⅰ: Construction of PNA functionalized nanochannels biosensor and miRNA detectionFirst,Polyethylene terephthalate(PET)conical nanochannels were prepared by asymmetric chemical etching.The probe PNA is modified on the surface of the channels by covalent bonding,and a series of characterization experiments prove the successful construction of PNA-functionalized nanochannels biosensor.The sensor was further applied to detect miRNA in PBS buffer,which could not only highly distinguish target miRNA-10 b from single-base mismatches and noncomplementary sequences,but also has a detection limit as low as 75 aM.In conclusion,the PNA functionalized nanochannels biosensor has high specificity and sensitivity for miRNA detection,and can be further used to detect tumor exosomal miRNAs.Part Ⅱ: PNA functionalized nanochannels biosensor for the detection of tumor exosomal miRNAThe PNA functionalized nanochannels biosensor was used to detect total RNA extracted from exosomes derived from pancreatic cancer cells and normal pancreatic cells.The electrical signal response of miRNA-10 b derived from pancreatic cancer cells was four times higher than that of miRNA-10 b derived from normal pancreatic cells.After statistical analysis,the signal difference between the two was significant(P<0.001),The results indicate that this biosensor could effectively distinguish pancreatic cancer tumor-derived exosomes from normal control group,and the detection results show good consistency with those of qRT-PCR method.Finally,the sensor was used to detect exosomal miRNA-10 b in clinical plasma samples,and it was found that the content of exosomal miRNA-10 b in the plasma of cancer patients is generally higher than that of healthy individuals,proving that this method is expected to be applied to early clinical cancer screening.