Study Of Tumor Biomarkers Detection Based On Porous Anodized Aluminum Film Nanochannel

Nowadays,tumor is one of main diseases that endanger human health.It’s an essential device for cancer patients to early detection and treatment in order to improve survival rate and prognosis.However,the detection methods of related tumor markers,at present,still exist many disadvantages,such as low sensitivity,cumbersome operation,expensive cost and so on.In connection with that,it’s urgent to explore and design high-efficient,sensitive,simple and rapid detection method of tumor markers.Nanopores or nanochannels have attracted a wide range of interest in field of biosensing and chemical analysis.The structure characteristic of nannochannels can embody geometrical changes of molecules by the transformation of ionic current in order that it can make an increasingly crucial role in the sensitive detection of diseases’ markers and early diagnosis of diaseases.Porous anodized aluminum film nanochannel possesses plenty of advantages in aspects of biochemical analysis and biosnesors,such as adjustable diameter of nanopores,favorable nanochannel array,feasible functionalization on the surface of nanopores,commercial availability and so on.In connection with that,this thesis utilizes PAA film nanochannel as a carrier in the detection of tumor markers.Firstly,amino group in APTES solution is connected on the surface and in the interior of nanochannel by noncovalent modification.Secondly,DNA with aldehyde group can react with amino group by covalent reaction.The strand with signal molecules is further modified on the surface of PAA film to form a compact blocking layer which affects the spatial and static hindrance reflected on the difference of electrochemical signals in order to achieve the purpose of rapid,sensitive and efficient detection of tumor markers.The specific contents are as follows:1.Generation of DNA nanoflowers in channels and new method for detection of miRNA.The electrochemical method has the performance of simple operation,high sensitivity,low cost and visualized signal output.In this part,we ultra-sensitively detect miRNA,a kind of tumor marker,based on the generated DNA flowers in the PAA nanochannel.Firstly,a proDNA complementary with microRNA-21 is modified in the interior of PAA channel.When the miRNA-21 exists in the nanochannel,it can facilitate pre-added DNA template with primer to trigger rolling circulation reaction(RCA),which generates DNA flowers.The nanoflowers,to a great extent,increase the spatial hindrance of the channel.Meanwhile,the negative charge of PAA’s phosphate group also contributes to static hindrance.The two elements can apparently decreases the anode current produced by K3[Fe(CN)6]which flows from the upper surface to the platinum working electrode and furthermore amplified electrochemical signal comes into being.Further research shows that by the signal changes of K3[Fe(CN)6]on the electrochemical work station,the reduced anode current represents linear correlation with the level of miRNA.The detection range of miRNA is 10-1000 fM and the limit of detection is 4.53 fM.This work achieve the simple,sensitive and specific detection of miRNA.Therefore,it has a broadly potential application prospect in the future clinical diagnosis.2.A new method of detection of MMP-2 based on the cleavage of collagen-DNA polymer on the channel surfaceIn this work,designed DNA strand with aldehyde group is firstly modified on the surface of PAA and then has a hybrid modification with its complementary strand.Thereafter,the PAA film is put in the collagen-like peptide solution,where the peptide can combine with double strand DNA by electrostatic interaction and hydrogen bond to form collagen-DNA polymer.The collagen can be used as a substrate for MMP-2.We fabricate a biosensor for sensitive detection of MMP-2 based on PAA film.In this regard,when the collagen is cleaved by MMP-2,the spatial hindrance of PAA film surface greatly decreases resulting in enhanced electrochemical response,which can be utilized to detect MMP-2.The detection range of miRNA is 10-20000 pg/μL and the limit of detection is 2.5 pg/μL.The method is more convenient and efficient and doesn’t require complex and intact protein.The peptide can substitute substrate to achieve the sensitive and high-efficient analysis of MMP-2 which has an excellent promising in biosensor field.