Dual Antibody-functionalized Nanochannel Biosensor For Sensitive Detection Of Exosomes

In recent years,cancer has become one of the leading causes of human death in the world.Statistics show that 90% of cancer-related deaths are caused by cancer metastasis.Therefore,early diagnosis,early treatment,real-time monitoring,and prevention of cancer metastasis are the keys to increasing the survival rate of cancer patients.Biomarkers are important for the early diagnosis of major diseases such as cancer and heart disease.Currently,the most commonly used cancer-related biomarkers in the clinic are proteins/antigens.For example,prostate specific antigen(PSA),alpha-fetoprotein(AFP),carcinoembryonic antigen(CEA),etc.Exosomes are another powerful class of biomarkers that are rapidly drawing attention and are considered the future of biomarkers in medicine.Exosomes are discoid vesicles(30-150 nm)that are released from multivesicular bodies to the extracellular matrix via the endolysosomal pathway.Exosomes carry a great deal of genetic information from their parent cells,including transmembrane and cytoplasmic proteins,mRNA,DNA and micro RNA.As such,they act as messengers,whose indications and changes are associated with disease,and in particular have an important role in changing the state of cancer-related physiopathology.And almost all cell types can secrete exosomes and can enter various body fluids,including blood,urine,breast milk,bile,ascites,tears and saliva,with high stability.In particular,it has recently been reported that exosomal concentrations in the circulation of cancer patients are significantly increased.As a result,exosome is now recognized as a promising biomarker for early cancer diagnosis.At present,although there are many research reports on the purification detection of exosomes,the quantification of exosomes is still challenging.Traditional methods for quantitative analysis of exosomes include(1)flow cytometry,which can provide high-throughput detection,but because of weak light scattering,small vesicles(<200 nm)are often missed;New counting method of(2)nanoparticle tracking analysis(NTA)or(3)dynamic light scattering(DLS),which can provide more accurate particle counting,103 times higher than those reported by flow cytometry,but with a limited detection concentration range;Conventional molecular assays such as(4)western blot and(5)enzyme-linked immunoassay require a large number of samples,which cannot be widely used in clinical research and detection,especially continuous analysis of limited samples in large patient groups or biological repositories.Therefore,the analysis and detection of exosomes in the conventional clinical environment is still a difficult task,mainly due to the lack of a sufficiently sensitive and rapid assay platform.Nowadays,low-cost,simple and portable biosensor detection technology is developing rapidly.Nanochannel biosensor is a new type of biosensor developed in recent years.Its physical structure is stable,the surface is easy to be chemically modified,the measurement results are simple and fast,and it has high sensitivity.The principle is that the inner wall of the nanochannel is modified by a functional molecule,which has a recognition site for the target.The binding between the functional probe and the target causes changes in the effective pore size,internal surface charge,and wettability of the internal wall,resulting in changes in the ion current.Therefore,by measuring the current-voltage(I-V)curve before and after the target is added,target detection can be achieved.The nanochannel sensing platform provides a fixed three-dimensional environment for the interaction between functional molecules and analytes at the nanoscale,which rapidly and frequently generates the kinetic effects of transporting reactants in the nanochannel.These characteristics make the detection of nanochannel biosensors simple,fast and highly sensitive,and are widely used for sensing detection of various indicators of clinical medical and health environment samples.Its application range includes gas,various protein molecules,nucleic acid molecules,cells and so on.Based on this research background,this paper constructs a dual antibody-functionalized nanochannel biosensor and applies it to the highly sensitive and rapid detection of exosomes.The work is mainly divided into the following two parts:Part Ⅰ: Construction of a dual antibody-functionalized nanochannel biosensorIn this work,we selected a porous anodized aluminum(PAA)membrane as the substrate to build the sensing device.First,the PAA membrane was acidified to activate a large number of hydroxyl groups on the channel surface;then 3-aminopropyltriethoxysilane(APTES)and glutaraldehyde(GA)were sequentially modified to the surface of the nanochannel,and finally the dual antibody(CD63 antibody and Ep CAM antibody)was modified on the surface of the channel by Schiff reaction.To verify the successful construction of the sensor,a series of characterization experiments were performed,such as electrical tests,contact angle(CA)and X-ray photoelectron spectroscopy(XPS)analysis,and fluorescent antibody experiments.The above experimental results show that APTES,GA and diabody have been successfully modified on the outer surface of the nanochannel and the structure is stable.The constructed dual-antibody-functionalized nanochannel biosensor can be applied to the next exosome detection.Part Ⅱ: Ultrasensitive detection of exosomes based on dual antibody-functionalized nanochannel biosensorsIn this stage of work,we applied the previously constructed dual antibody functionalized nanochannel sensor to the highly sensitive and rapid detection of exosomes.First,exosomes are selectively captured on the surface of nanochannel membranes by recognition of antigens and antibodies carried on the surface of the exosomal membrane.Because the particle size of the exosomes is much larger than the pore diameter of the nanochannel,the exosomes will cover the surface of the nanochannel membrane and cannot enter the nanochannel interior.Then,the entire process can be monitored by recording the I-V curve to achieve accurate and sensitive detection of exosomes.The results of characterization experiments using transmission electron microscopy(TEM),nanoparticle tracking analysis(NTA),and Western blotting(WB)showed that exosomes were successfully extracted from cell culture supernatants.And electrical experiments and scanning electron microscope(SEM)results proved that exosomes were successfully captured on the surface of nanochannels.In this work,we selected two antibodies to capture and detect exosomes at the same time.Compared with the detection ratio of such antibodies,the detection efficiency of the dual antibody-functionalized nanochannel sensor has been improved by almost two times.This method can effectively detect exosomes with a concentration range of 3.3×104 to 3.3×109 particles/m L,and its detection limit is as low as 7.3×103particles/m L.Finally,we tested and analyzed exosomes extracted from the plasma of 5 patients with liver cancer,13 patients,and 10 healthy individuals to prove the clinical application of the method.The results show that the plasma exosomes content of cancer patients is generally higher than that of healthy individuals,and this method is helpful for the early screening of clinical cancer.