Ultrasensitive Detection Of Exosomal MiRNA With A PMO-Graphene Quantum Dots Functionalized Field Effect Transistor Biosensor

Tumor disease has long been a problem that plagues human health and is the second leading cause of death worldwide.If tumors can be diagnosed at an early stage and early detection and treatment can be achieved,the mortality rate of tumors patients can be effectively reduced.In recent years,exosomes have become a hot research topic because of their important role in tumorigenesis,development and metastasis.Exosomes carry a variety of molecules with biologically active functions,especially exosomal mi RNAs,which are inextricably linked to intercellular communication.Therefore,the levels of exosomal mi RNAs（Exo-mi Rs）are highly correlated with disease progression and tumor malignancy.In addition,Exo-mi Rs secreted by donor cells can target recipient cells and directly regulate gene expression.Compared with plasma mi RNAs,Exo-mi Rs are more advantageous as biomarkers.Firstly,Exo-mi Rs are protected from degradation in circulation by the phospholipid bilayer in exosomes.Secondly,being protected by exosomes,Exo-mi Rs remain biologically active even under multiple freeze-thaw cycles or extreme p H conditions.Although Exo-mi Rs are emerging as biomarkers for malignancies,their highly sensitive detection remains a challenge due to their short length,high homology and low abundance.With the rapid development of modern nanotechnology,field-effect transistor（FET）biosensors,as a label-free detection tool,are among the most promising biosensors in recent years.However,the current functionalization method of FET mainly depends on immobilization of probe molecules on the channel surface,and such functionalization method may limit the detection sensitivity of FET biosensors.To improve the detection sensitivity,the nanoparticle-based functionalization method can enable more probe molecules to be anchored on the surface.This functionalization method can achieve higher hybridization efficiency compared to planar surfaces due to its three-dimensional structure.Graphene quantum dots（GQDs）are a new zero-dimensional member of the carbon family.GQDs were first isolated and characterized in 2004-2006.In terms of size,GQDs are graphene sheets with lateral dimensions less than 10 nm.Due to their extremely small size and easy modification,they are widely used for the attachment of various biological probes.Compared to DNA probes,Phosphorodiamidate morpholino oligomer（PMO）developed by AVI pharmacy in the United States in 1997,which are DNA molecular analogs and can bind to RNA and DNA single strands through base complementary pairing.Due to structural changes that PMO does not carry charge.Therefore,PMO has good specificity and stability when hybridizing with DNA and RNA.Here,we report a GQDs-functionalized reduced graphene oxide（RGO）FET biosensor that can detect Exo-mi Rs in the blood of breast cancer patients with ultra-sensitive sensitivity.GODs were incubated with PMO to obtain GQDs-PMO hybrid.After RGO-FET preparation,polylysine（PLL）was assembled onto the RGO surface.Subsequently,the GQDs-PMO complexes were immobilized on the chip surface to construct the GPPR-FET biosensor.The PMO probe was hybridized with Exo-mi Rs for sensitive and specific detection of Exo-mi Rs.The work was divided into two parts as follows.Part Ⅰ: Fabrication of GPPR-FET BiosensorFirstly,PMO-GQDs hybrid is obtained by incubating GODs with amine-modified PMO.After the RGO FET is fabricated,polylysine（PLL）is assembled onto the RGO surface,allowing the functionalized GQDs-PMO hybrid to be immobilized on the chip surface via amide formation.We optimized various conditions for the preparation of GQDs-PMO complexes and deposition of PLL on the RGO surface.Meanwhile,the interference resistance and stability of the GPPR-FET biosensor were investigated.Part Ⅱ: GPPR-FET biosensor detection of exosomal mi RNAFirstly,the sensitivity of GPPR-FET biosensor was investigated.By adding different concentrations of mi RNA21 to the surface of GPPR-FET biosensor,the detection of mi RNA can be realized by detecting the changes of Dirac point before and after PMO-mi RNA hybridization.The results showed that the sensor can have a detection limit as low as 85 a M,which is more sensitive than those reported by other sensing methods.In addition,GPPR-FET biosensor was able to distinguish between complementary mi RNAs,non-complementary mi RNAs and single base mismatched mi RNAs,showing a high specificity.Then,GPPR-FET biosensor was used to detect MCF-7 cell derived exosomal mi RNA21 and MCF-10 A cell derived exosomal mi RNA21,respectively.It was found that the content of mi RNA21 from MCF-7 cells was significantly higher than that from MCF-10 A cells.The results of GPPR-FET biosensor were compared with those of gold standard q RT-PCR,and the results were found to be in good agreement.Finally,GPPR-FET biosensor was used to detect clinical samples.It was demonstrated that the biosensor can distinguish between normal population and breast cancer patients by detecting exosome mi RNA21 in patients’ plasma.