Electrochemiluminescent Sensing For Caspase 3 And Electrochemical Sensing For DNA

Electrochemiluminescence(ECL)is a combination of electrochemical techniques and chemiluminescence.Compared to the traditional photoluminescence,it has no need of excitation light source,samll background,high sensitivity,good reproducibility and selectivity and it has unique advantages in the field of life and chemical analysis.ECL analysis has been widely applied in immunoassay,nucleic acid hybridization analysis,cell analysis and determination of other biochemical substances,etc.In this thesis,we explore the application of ECL analysis technology in protein analysis by developing the ECL sensor platform.At the same time,we use the nucleic acid amplification technology to construct an ultrasensitive DNA sensor through electrochemical analysis methods.The main contents are as follows:1.Electrochemiluminescent sensing for caspase 3 activity and inhibition Caspase 3 is one of the most frequently activated cysteine proteases during the apoptosis process,which has been identified as a well-established cellular marker of apoptosis.In this study,a novel electrochemiluminescence(ECL)approach for sensitive determination of caspase 3 activity and inhibition based on Ru(bpy)32+-doped silica and tri-n-propylamine(TPA)ECL system.A glassy carbon electrode(GCE)was modified with nano-materials composed of Au nanoparticles(AuNPs),poly(dimethyl diallyl ammonium chloride)(PDDA),and carbon nanotubes(CNTs)to construct the sensor platform.The biotinylated DEVD-peptide(biotin-Gly-Asp-Gly-Asp-Glu-Val-Asp-Gly-Cys)was immobilized on the surface of the GCE via the strong bonding between AuNPs and the thiol group.Then the streptavidin-modified Ru(bpy)32+-doped silica(Ru@SiO2)was modified on the sensor via the specific interaction between biotin and streptavidin.Caspase 3 could specifically recognize and cleave the N-terminus of DEVD,which led to the loss of the biotin label and the decrease of ECL intensity.The ECL intensity is related to the activity of caspase 3.Using this proposed new method,a novel electrochemiluminescent sensing platform for sensitive determination of caspase 3 activity and inhibition was developed.2.Ultrasensitive electrochemical detection of BCR/ABL fusion gene fragment based on circular strand replacement polymerization coupling with cascade hybridization reactionThe molecular beacons(MB)which could hybridiz with target DNA was immobilized on the electrode through the Au-thiol interaction.After target DNA was added to open up the loop,assist-DNA1 could attach on the stem part of the MB.In the presence of Klenow Fragment(3'?5' exo-),DNA polymerization occurred.The lengthening new strand could release the target DNA,which would trigger new polymerization cycle,resulting in the multiplication of assist-DNAl on the electrode.Then assist-DNA2 probes hybridized with complementary regions on each of two assist-DNA1 probe to create long concatamers structure containing abundant amino-group.QDs could be tagged on the assist-DNA2 easily via the classic EDC/NHS coupling reaction.After dissolved by HNO3,we could detection the signal of Cd2+ using the anodic stripping voltammetry.The dynamic range of the sensor was from 10-14 mol·L-1 to 10-10 mol·L-1,and the detection limit was 2×10-15 mol·L-1.The biosensor could discriminate perfectly matched target DNA from single-base mismatch DNA and thus might have a promising future in early diagnosis and prognosis of chronic myelogenous leukaemia.Additionally,the assay could be used for detecting other DNA chain by changing bases of the capture MB probe conveniently.