Ultrasensitive Detection Of Gastric Cancer Exosomes Using Aptamer-Based Biosensors

Gastric carcinoma is one of the most frequent gastrointestinal carcinomas.According to GLOBOCAN,gastric carcinoma is the fifth most common malignancy and the third leading cause of cancer mortality,mainly due to the advanced stage at diagnosis.Exosomes are bilayer lipid vesicles secreted by cells into the extracellular environment.Studies have shown the presence of exosomes in physiological fluids,including peripheral blood,saliva and urine.Exosomes contain a collection of essential molecules representing the communicative function of exosomes.It has been demonstrated that the transfer of molecules via exosomes is related to various diseases including gastric carcinoma,and the existence and content of exosomes could be utilized as biomarkers in the diagnosis and prognosis of gastric carcinoma.The research contents are as follows:1.The identification of gastric cancer exosome-specific aptamers.The application of exosomes in clinical practice remains challenging as most of the detection probes used in current available strategies lack specificity,which could cause high level of“biological noise”.Aptamers are short ss DNA or RNA molecules which can specifically bind to their targets.Aptamers have been commonly used as detection probes in various biosensors.The purpose of this section is to screen an appropriate detection probe.Firstly,exosomes were isolated from cell supernatant through filtration.Then several reported aptamers specific to gastric cancer cells or cancer-related protein were synthesized as candidates of the detection probe.The morphology of isolated exosomes was observed using transmission electron microscope.We also verified the exosomes based on their surface protein markers using western blot and immunogold labeling.Results showed that the Mucin 1（MUC1）aptamer could recognize target exosomes specifically and would be the ideal detection probe in the following experiment.2.Rapid fluorescence detection of gastric cancer exosomes using aptamer-graphene oxide and DNase I-based target recycling reaction.We developed a simple and fast fluorescence aptasensor in this section.Graphene oxide（GO）is a member of the graphene family,which has been widely used in biosensors.The aptasensor we constructed consisted of FAM-aptamer conjugate-assembled graphene oxide and DNase I.The addition of target exosomes could cause the dissociation of FAM-aptamer from GO.The newly formed aptamer/exosome could be readily cleaved by DNase I and the target exosomes were released,which could trigger target recycling to generate numerous free fluorophores.The fluorescence aptasensor could deliver results in 1～2 h and the detection limitation was 1.69×10⁶ exosomes/m L.3.A sensitive aptasensor based on a hemin/G-quadruplex assisted signal amplification strategy for electrochemical detection of gastric cancer exosomes.As the sensitivity and specificity of the GO-based fluorescence assay need to be improved,we developed a rolling circle amplification and hemin/G-quadruplex-assisted signal amplification strategy for the electrochemical detection of gastric cancer exosomes.The presence of target exosomes could trigger rolling circle amplification and multiple G-quadruplex units were produced.This HRP mimicking DNAzyme could catalyzes the reduction of H₂O₂ and generate electrochemical signal.The detection range of the electrochemical aptasensor was 10²～10⁶exosomes/m L and the detection limit was 9.43×10² exosomes/m L.Furthermore,test was performed by detecting healthy human plasma samples containing normal exosomes and gastric cancer exosomes and a significant difference was observed between the two groups（P<0.0001）.4.As the electrochemical assay has complicated and error-prone steps,we further developed branched rolling circle（BRCA）amplification-assisted signal amplification strategy for the fluorescence detection of gastric cancer exosomes.The designed padlock probe was cyclized after incubation with aptamer binding with target exosomes.Quantitative detection was achieved by adding SYBR Green I.The detection limit was 4.27×10⁴ exosomes/m L.At last,gastric cancer patient plasmas were tested using this aptasensor and a significant difference was observed between the two groups（P<0.0001）.