Label-free Electrochemical Aptasensor For The Detection Of Carcinoebryonic Antigen

Electrochemical biosensor has been widely concerned by the analytical researchers,because it has many advantages,such as simple instrument,easy to carry,fast response and high sensitivity.The classical electrochemical biosensor mainly uses antigen/antibody DNA or enzyme as biometric molecules.The preparation of antigen/antibody is difficult,the procedure is cumbersome,the period is long,the enzyme is inactivated easily,and it is difficult to preserve.These shortcomings limit the development of electrochemical biosensors to some extent.Aptamers have attracted a considerable attention due to their ability to bind target protein with high affinity and specificity and have many advantages over traditional biological recognition molecules.These properties of aptamers make aptamers ideal candidates as recognition elements in a wide range of bioassays for drugs and proteins.In recent years,nanomaterials have been widely used in the construction of electrochemical biosensors,because of their large specific surface area,high catalytic activity and special physicochemical properties.If the combination of nanpartical and aptamer,it may bring a new opportunity for the electrochemical biosensor.In this paper,the homemade carbon electrode and commercial gold electrode were used as the basic electrode,and the multi-walled carbon nanotubes and gold nanoparticles were used as the electrode modification materials.A new electrochemical method for the detection of small drug moleculeand free-labeled electrochemical aptasensors for detection of carcinoembryonic antigen(CEA)were constructed.The thesis mainly includes the following parts:Multi-walled carbon nanotubes modified screen-printed carbon two-array electrode was used to detect folic acid.A stable integrated carbon two electrodes array,including two carbon working electrodes,a carbon counter electrode and a thick-film Ag/AgCI reference electrode,was fabricated by mask technique of polyethylene sticky film and the screen printing technique.MWCNTs were used as modifying materials,folic acid as a model molecule,a simple electrochemical method for the detection of folic acid with high sensitivity was constructed.The result showed that MWCNTs modified carbon two electrodes array had excellent electrochemical performance,the relative average deviation of the peak current of K3[Fe(CN)6]on two working electrodes was 1.8%.At the MWCNTs modified carbon two electrodes array,folic acid had an obviously catalytic effect,and the oxidation peak current were greatly enhanced compared with carbon two electrodes array.Under the optimized conditions,the oxidation peak currents were linear over ranges from 3.0 μmol/L to 100 μmol/L,for folic acid with a detection limit of 1.77 μmol/L(S/N=3).With the standard addition method,the reliability of the method was tested by commercially available folic acid tablets,with a recovery rate of 90.0%～108.7%.Free-labeled impedance aptasensor for the determination of carcinoembryonic antigen(CEA).A integrated thick film carbon working electrode,a large thick film carbon counter electrode and Ag/AgCI reference electrode was fabricated on FR-4 glass fiber substrate by the screen printing technology.Gold nanoparticles were deposited on the surface of carbon working electrode using the cyclic voltammetry,and then,the CEA aptamers were immobilized on the surface of carbon working electrode modified by gold nanoaprtical using thiolated self-assembling.The interaction between aptamer and the target CEA was monitored by the the change in interfacial electron transfer resistance using[Fe(CN)6]3-/4-as probe.When the target CEA interacted with aptamer,the interfacial electron transfer resistance decreased due to the interference of bound CEA with the transfer of[Fe(CN)6]3-/4-.The experimental results showed:when the concentration of CEA was 0.5 ng/mL to 13.0 ng/mL,there was a good linear relationship between electrochemical aptasensor and CEA.The Linear equation was as follow:△Ret=40.7972c(ng/mL)+762.7832,R=0.9960.The detection limit was 0.094 ng/mL(S/N=3).Free-labeled impedance aptasensor for the determination of carcinoembryonic antigen(CEA)based on target-induced aptamer displacement.Using CEA as the research model,[Fe(CN)6]3-/4-.as electrochemical probe,an impedance aptasensor was developed for the detection of CEA based on the target induced aptamer displacement.The Aptamer-DNA duplex with sulfhydryl group was obtained by the hybridizion of the CEA aptamer and the partial complementary DNA with sulfhydryl group.And then,the aptasensor was structured for the determination of CEA by the self-assembling thiolated of the aptamer-DNA duplex on the surface of gold electrode.To measure CEA,the change in interfacial electron transfer resistance of the aptasensor using a redox couple of[Fe(CN)6]3-/4-as the probe was monitored.The introduction of target CEA induced the displacement of the aptamer from the DNA-aptamer duplex on the gold electrode into the solution,decreasing the electron transfer resistance of the aptasensor.The experimental results showed that the change of interfacial electron transfer resistance △Ret was linear in the range of 0.8 ng/mL-10.4 ng/mL for CEA detection.The detection limit was 0.15 ng/mL(S/N=3).The results also showed that the aptasensor with short complementary DNA chain and aptamer can obtain higher sensitivity.