The Electrochemical Detection Of Heavy Metal Ions And Its Application In Immune Analysis

Objective:By chemical deposition method,we constructed novel nano-gap electrodes,so that the electrode gap shortened from micron to nanometer.Nano gap biosensor based on thymine mercury thymine?T-Hg-T?coordination chemistry structure,which has specific identification sensitive interface for detection of heavy metal ions Hg²⁺,was prepared.Metal ion functionalized immune complexes as signal marker,gold nanoparticles decorated with chitosan coated multi-wall carbon nanotube?CHIT-Au-MWCNTs?as sensitive interface,electrochemical immune sensor was prepared,to quantitative detect tumor markers alpha fetal protein?AFP?.Methods:1.About 16 nm gold nanoparticles?Au NPs?in size are successfully prepared,through the simple hydrothermal synthesis method.By chemical deposition method the gold nanoparticles were fixed on the interdigitated array microelectrode?IDAM?with chemical bonding,then through the in situ growth of gold nanoparticles to regulate the size of the gap.Gold nanoparticles were evenly distributed in interdigitated array microelectrode gap from 2.5 micron to nanometer level wide.A novel nano gap electrode?NGE?was constructed and the surface of the electrode and the gap between the electrodes was characterized by scanning electron microscopy?SEM?.Chooseing a suitable rich thymine?T?and the 5 'end sulfhydryl?-SH?,3' end amino group?-NH2?oligonucleotide nucleic acid as probe,Ferrocenecarboxylic acid?Fc?was immobilized on the 3 'end of the nucleic acid probe by EDC/NHS condensation reaction.The?Fc?labeled nucleic acid probes were immobilized on the nano gap electrode?NGE?surface by self assembled of Au NPs at the end of the 5 'end group?-SH?.A nano gap biosensor was prepared for specific detection Hg²⁺ ion.2.Alpha fetal protein?AFP?is an important tumor marker,which can be used for differential diagnosis,disease monitoring and therapeutic evaluation of liver cancer.In this study,we use multi walled carbon nanotubes?MWCNT?,chitosan?CHIT?and the Chloride four water?HAu Cl4·4H2O?to build a sensitive interface.First antibody?Ab1?was fixed on the sensitive interface by the Au-N bonding and the residual non-binding active site was blocked with bovine serum albumin?BSA?to fabricate immune-sensor successfully.Then by one pot synthesis methods amino functionalized magnetic nanoparticles?PAN@Fe₃O₄?were synthetized.which fixed second antibody?Ab2?as a carrier.Metal ion functionalized immune complexes were prepared by using metal ion Hg²⁺ as signal marker,which can be used for detection of AFP.Scanning electron microscopy?SEM?,cyclic voltammetry?CV?were used to characterize the whole construction process of the immune sensor,differential pulse voltammetry?DPV?was used to detect the response signal of AFP on the sensor.Results:1.Uniform particle size about 16 nm gold nanoparticles were prepared and deposited on the surface of the IDAM to built the appropriate nanometer pitch NGE.Gold nanoparticles size and NGE gap size were characterized by scanning electron microscopy?SEM?.The self-assembly process of the nucleic acid probe on the surface of the NGE was characterized by electrochemical method,cyclic voltammetry?CV?and electrochemical impedance spectroscopy?EIS?.The results of the sensor for Hg²⁺ detection:with different concentration of Hg²⁺ ions?1 p M¹5 p M?from low concentration to high concentration continuously combined to the surface of the NGE,the response current of the electrode is gradually decreased,and the electrochemical impedance increases with the increase of Hg²⁺ concentration,concentration?C?and the absolute value of the electron transfer impedance??Ret?showed a good linear relationship,Z/105?=4.81+0.249C/p M?R²=0.989?.The detection results show that the biosensor has high sensitivity and good selectivity for the Hg²⁺ ion response.2.We have been synthesized chitosan-gold-multi walled carbon nanotubes?CHITAu NPs-MWCNTs?compound material that modified glassy carbon electrode to construct sensitive interface.Through the synthesis of immune complexes(Hg²⁺-PAN@ Fe₃O₄-Ab2),which consist of amino functionalized magnetic nanoparticles as the second anti carrier and heavy metal as the signal markers,to construct based on heavy metal signal marker of electrochemical immune sensor application in tumor mark detection of AFP.Electrochemical method cyclic voltammetry?CV?was used to characterize the immune sensor and the steps of electrochemical detection.After optimized conditions,the response current of the immune sensor with AFP concentration in the concentration range was 10pg/m L-2.5ng/m L and 2.5ng/m L-60ng/m L have a good linear relationship,Correlation equations are ?I=1.50+0.61 CAFP R²=0.976,?I=2.51+0.071 CAFP R²=0.976.In the actual sample analysis with ELLSA method at the same time detection of 6 samples,the detection results of the two methods of the relative error is less than 5.83%.Conclusion:1.Nano gap biosensor based on thymine mercury thymine?T-Hg-T?coordination chemistry structure,which has specific identification sensitive interface for detection of heavy metal ions Hg²⁺,was prepared,and it has successful application in the analysis of the application of serum samples.2.Metal ion functionalized immune complexes(Hg²⁺-PAN@Fe₃O₄-Ab2)as signal marker,gold nanoparticles decorated with chitosan coated multi-wall carbon nanotube?CHIT-Au-MWCNTs?as sensitive interface,electrochemical immune sensor was prepared,to quantitative detect tumor markers alpha fetal protein?AFP?..The immune sensor showed good sensitivity for detection of AFP,through the analysis of the actual samples with the ELLSA method,the detection results of our constructed immune sensor can meet some trace concentrations of tumor markers AFP detection.