Development Of Graphene Field Effect Transistor Biosensor For Ultrasensitive Detection Of Microvesicles Released By Liver Cancer Cells

Microvesicles(MVs),which are heterogeneous membrane-bound nanovesicles(100-1000 nm)shed from the cell surface into the extracellular environment in a budding manner,have been widely regarded as promising"biofingerprint"for early diagnosis of various cancer.Quantitative detection of entire MVs population will help us understand the intracellular and intercellular communication process(including transport,secretion and receptor-mediated signal transduction,etc.)from a new perspective.Moreover,the potential regulatory mechanism of MVs formation and clearance can be explained by analyzing the overall level of MVs,which can further help us deeply understand the pathogenesis of various diseases with abnormal concentrations of MVs.High-performance detection of cancerous MVs plays a vital role in the early diagnosis of various cancers.However,high-performance detection of MVs is still technically challenging.Traditional MVs detection methods,such as nanoparticle tracking analysis(NTA),enzyme-linked immunosorbent assay(ELISA),etc.often suffer from shortcomings such as low detection sensitivity,large sample and reagent consumption,and long detection time.Hence,the above-mentioned MVs detection technologies can not meet the actual clinical application requirements.With the rapid development of modern nanotechnology,reduced graphene oxide(RGO)field effect transistor biosensors(FET)stands out among various biosensor platforms because of their superiorities,such as label-free detection,high sensitivity,good specificity,fast analysis speed,low sample and reagent consumption,low test cost,easy to miniaturize and integrate and etc.According to previously reported works,RGO-FET biosensors have been applied for highly sensitive detection of various biomolecules,such as nucleic acids,proteins,neurotransmitters,virus,bacteria and its metabolites,antibiotics and gas molecules.However,there are no reports of using RGO-FET biosensors to detect MVs.Therefore,RGO-FET biosensor is promising to be a powerful tool for the detection of MVs,and has broad clinical application prospects in future.Based on the challenges mentioned above,two functionalized RGO-FET biosensors are constructed in this paper and have been applied for highly sensitive detection of the overall level of MVs and highly sensitive,specific and label-free detection of MVs derived from HepG2 cells(HepG2-MVs),respectively.The main research works are as follows:Part I:Microvesicle detection by a reduced graphene oxide field-effect transistor biosensor based on a membrane biotinylation strategyIn this part,we have successfully fabricated a streptavidin(SA)-functionalized RGO-FET biosensor for the highly sensitive detection of the overall level of MVs based on a membrane biotinylation strategy.This FET biosensor uses RGO as the sensing nanomaterial.After the FET chip was prepared by the traditional standard semiconductor technology,the RGO-FET biosensor could be obtained by dropping-casting the RGO suspension onto the channel surface.Due to the lack of well-defined protein markers on the surface of MVs,we firstly labelled the MVs with biotin(B-MVs)by adding the biotinylated phospholipid derivatives to the cell culture medium.To detect B-MVs using RGO-FET biosensor,SA probe was modified on the chip surface by a chemical linker.Since the biotin on B-MVs could specifically bind with SA,B-MVs could be specifically captured by the chip.B-MVs are negatively charged and RGO-FET is a p-type device.Therefore,B-MVs could cause a n-doping effect to RGO,which eventually led to the change of the electrical signal(the change of the Dirac point in the transfer characteristic curves).This platform can detect B-MVs over the range from 10~5 particles/mL to 10~9 particles/mL,and the detection limit is as low as 20 particles/μL.In addition,the platform has good specificity,successfully avoiding the possible interference of other non-specific substances.Moreover,this platform has good versatility(can detect B-MVs derived from various cell lines)and anti-interference ability(can detect B-MVs in serum).All the above results proved that the SA-functionalized RGO-FET biosensor provides a new and reliable detection strategy and platform for the analysis of the overall level of MVs in biomedical research.Part II:Dual-aptamer modified graphene field-effect transistor biosensor for label-free and specific detection of hepatocellular carcinoma-derived microvesiclesIn this part,we have developed a dual-aptamer functionalized RGO-FET biosensor for the highly specific and sensitive detection of MVs derived from liver cancer cells(HepG2-MVs).First,HepG2-MVs were isolated and purified from the culture supernatant of HepG2 cells by differential centrifugation.To specifically detect HepG2-MVs using RGO-FET biosensor and improve the detection sensitivity,AuNPs were deposited onto RGO surface by chemical reduction reaction after the RGO-FET biosensor was firstly prepared.Subsequently,the thiolated TLS11a and EpCAM aptamers were modified onto the surface of AuNPs through Au-S bond.Finally,the dual-aptamer functionalized RGO-FET biosensor(AAP-GFET)was obtained.HepG2-MVs are negatively charged,and RGO-FET is a p-type device.Therefore,when HepG2-MVs are specifically captured by the aptamer probes modified on the chip surface,the negative charge on the surface of HepG2-MVs can generate n-doping effect to RGO,which eventually causes the change of electrical signals(the change of the Dirac point).This AAP-GFET biosensor can achieve highly sensitive detection of HepG2-MVs in PBS,with a detection limit as low as 84 particles/μL.It also shows good specificity,avoiding the possible interference from liver cancer-related serum proteins and MVs derived from other cell lines.In addition,the dual-aptamer-based nanosensing interface greatly enhances the sensing signals,thus effectively improving the detection sensitivity of the sensor.Using AAP-GFET biosensor to detect HepG2-MVs in clinical blood samples,we successfully distinguished hepatocellular carcinoma(HCC)patients from healthy individuals.All the above results indicate that the AAP-GFET biosensor is promising to be a powerful tool for early diagnosis of HCC.