Construction Of Elelctrochemical Aptasensor Based On Split-Aptamer For The Detection Of Tumor Exosomes

Exosomes are nanovesicles with a diameter of 40 to 160 nm secreted by cells.These vesicles are widespread in the supernatant of cell culture and various body fluids.Exosomes have phospholipid bilayer membranes and carry a variety of biological molecules from parental cells,and play an important role in the process of information exchange between cells.At present,a number of studies have shown that exosomes are related to the process of tumor proliferation and metastasis,and has become a new biomarker for cancer screening,diagnosis and prognostic treatment.However,due to the small size of exosomes and the low content of exosomes in biological body fluids at the early stage of cancer,it is still a challenge to achieve highly sensitive and selective detection of exosomes.Therefore,it is of great significance to develop efficient methods for the detection of exosomes.Split-aptamer originates from one intact aptamer sequence that is artificially divided into two or more fragments,which can induce aggregation and form recognition configuration in the presence of the target.They are more flexible and easier to design for sensors due to their short sequences.Therefore,this paper designed a series of electrochemical biosensors for the detection of tumor exosomes based on the split-aptamer binding DNA nanostructures and nucleic acid amplification technology.The details are as follows:(1)Split-aptamer Mediated Regenerable Temperature-sensitive Electrochemical Biosensor for the Detection of Tumour ExosomesIn view of the poor reproducibility and stability of electrochemical aptasensor caused by the modification difference between electrodes,we constructed a regeneable electrochemical biosensor based on the temperature-sensitive split-aptamer for the detection of exosomes.In the prescence of the target,the split-aptamer fragment had recognition function and could form sandwich structure at 4℃.The signal probe was connected to the electrode to generate signal.At higher temperature,the split-aptamer losed recognition ability and the sandwich structure was broken,leading the signal probe left the electrode and the signal disappeared.The sensor could detect tumor exosomes sensitively and specifically with a detection limit(LOD)of 1.5 × 106 particles/mL.The electrode could be regenerated quickly and easily by washing with PBS at 37℃ for 30 s without any additives and maintained good performance.In this experiment,the regeneration strategy was used to reduce the modification difference between different electrodes and improve the reproducibility and stability of electrochemical biosensors.(2)An electrochemical aptasensor based on DNA tetrahedron and HCR reaction for highly sensitive detection of tumor exosomesTraditional electrochemical sensors usually modify the single-strand DNA probe on the gold electrode surface.However,due to electrostatic repulsion and the reaction between nitrogen atoms in the base and gold electrode and other factors,the singlestranded DNA probes tend to intertwine with each other when assembled on the electrode surface,resulting in non-specific adsorption,which seriously affects the target recognition of the probe and limits the sensitivity of the sensor.Therefore,in the electrochemical aptasensor proposed in the previous chapter,DNA tetrahedron and HCR nucleic acid amplification technology were introduced to improve the sensitivity of the sensor.The three vertices of the DNA tetrahedron(TDN)were fixed to the surface of the gold electrode by gold-sulfur bonds,and a sequence extending from the other vertex could be complementary to the partial sequence of probe a,thus fixing probe a to the surface of the electrode.In the presence of target e,probes a and b formed a sandwich structure with exosomes,and the split Trigger approached and triggered HCR reaction to generate long DNA double strand which could load with more signal molecules to realize signal generation and amplification.The introduction of DNA tetrahedron and HCR reaction greatly improved the sensitivity of the sensor,and the detection limit(LOD)was as low as 1.27 × 103 particles/mL,realizing the high sensitivity and specificity of exosome detection.(3)A label-free electrochemical biosensor based on silver clusters synthesized in situ using DNA as template for the detection of tumor exosomesAt present,most electrochemical sensors need to modify small molecules or enzymes on the recognition element,which is relatively high cost.Therefore,we constructed a label-free electrochemical biosensor using silver clusters synthesized in situ using DNA as templates as signals for the detection of tumor exosomes.The detection of exosomes was first converted into DNA detection by magnetic beads.Probes modified on magnetic beads could capture exosomes and be complementary to a DNA sequence(B12).In the presence of target,the probe configuration changed,causing B12 to drop from the magnetic beads into the solution.Next,the B12 was separated by magnetic separation and detected by the electrode.B12 could not only partially complement the C sequence modified on the electrode,but also connected the double strand formed by Ha and Hb to the electrode.Ha and Hb contain C-rich sequences and provide templates for AgNCs generation.The AgNCs generated in situ could generate signals on the electrode surface and realized the detection of B12,so as to realize the sensitive detection of exosomes.The detection limit(LOD)was 3.12 × 103 particles/mL.This sensor reliazed highly sensitive and selective detection of exosomes without labeling.