Research On Hemoglobin Enzyme-like Biosensor

Hemoglobin?Hb?contains the porphyrin ring and Fe???electroactive center,it would have an enzyme-like activity.H₂O_2,O_2,chloropropanols would be electrochemically reduced by Hb biocatalysis.In turn,based on Hb enzyme-like property,it might be determined electrochemically,rather than the enzyme substance.A heme group consists of an Fe???ion which is the electron active center,held in the porphyrin ring.Because the heme group is embedded deeply by the peptide chains,the electron transfer is very slow,and the direct redox of Hb on the electrode is difficult.A Hb enzyme-like biosensor based on Hb immobilized with magnetic molecularly imprinted nanoparticles?MMIPs NPs?or adsorbed by chitosan?CS?under an alkaline medium on a magnetic electrode was constructed for direct electrochemical determination of Hb,3-chloro-1,2-propandiol?3-MCPD?and hydrogen peroxide?H₂O₂?.The main contents of the thesis were as follows:1.A biosensor for determination of 3-MCPD was developed based on the electrochemically catalytic reduction by Hb.Hb was concentrated and immobilized with MMIPs NPs.The surface-imprinted technique was used to prepare MMIPs NPs,using Fe₃O₄@SiO₂ NPs as the carrier,Hb as the template molecule,and tetraethyl orthosilicate?TEOS?as the imprinted polymer monomer.The MMIPs NPs had a core-shell structure,with magnetic Fe₃O₄ NPs as the core and SiO₂ as the shell.MMIPs NPs were modified on the surface of a magnetic electrode to constitute the Hb enzyme-like sensor.The biosensor could catalyze the electrochemical reduction of 3-MCPD,and a direct electrochemical determination of 3-MCPD could be achieved for the first time.The biosensor,where Hb immobilized with MMIPs NPs had high electrochemical response for 3-MCPD,increased by37.5%compared with Hb immobilized with magnetic non-imprinted nanoparticle?MNIPs NPs?.The biosensor increased the response by 19.56%again under an external magnetic field.The reduction peak current had a linear relationship with 3-MCPD concentration in the range of 1.0-160 mg·L^-11 with a detection limit?S/N=3?of 0.25 mg·L^-1.The biosensor was successfully used for the detection of 3-MCPD in soy sauce samples with recoveries between97.4%and 99.6%.2.MMIPs NPs were immobilized with CS on the surface of a magnetic electrode to constitute Hb enzyme-like biosensor to catalyze the reduction of hydrogen peroxide?H₂O₂?.Compared with MNIPs NPs,the biosensor enhanced the response by 14.3%.Notably,an introduction of a magnetic field made the biosensor be more sensitive owing to the paramagnetism of MNIPs NPs,Hb and O₂ molecules.The reduction current of H₂O₂ on Hb/MMIPs NPs modified magnetic glassy carbon electrode increased by 60.0%.Under the optimum conditions,the linear detection range of H₂O₂ was 25-200?mol·L^-1with the detection limit of 3?mol·L^-1?S/N=3?,which showed that Hb enzyme-like biosensor had a good catalytic performance for H₂O₂.3.A simple biosensor based on a magnetic glassy carbon electrode?MGCE?modified with CS was developed for directly electrochemical determination of Hb.Hb,as an enzyme-like catalyst,was adsorbed and immobilized by CS membrane on the MGCE to electrochemically catalyze the reduction of dissolved oxygen.In turn,Hb could be determined in terms of the reduction peak current.The biosensor,where Hb adsorbed under an alkaline condition had high electrochemical response,increased by 49.37%compared with Hb adsorption under the neutral conditions.In addition,the biosensor increased the response by13.77%under an external magnetic field,which was ascribed to the paramagnetism of Hb and O₂.All the experimental parameters,such as pH,temperature of the electrolyte solution and ionic strength,which influenced on the Hb enzyme-like activity and saturation of dissolved oxygen were optimized.Differential pulse voltammetry?DPV?showed that the reduction peak current had log-linear relationship with Hb concentration.The determination range for Hb was 0.05-50?g·mL^-1with the detection limit of 0.01?g·mL^-1?S/N=3?.Hb in human blood samples was determined by DPV,the determination results were in good accordance with the reference values from typical clinical analysis.4.A facile and highly sensitive biosensor for determination of H₂O₂ was developed based on the electrochemically catalytic reduction of H₂O₂ by Hb.Hb was enriched and immobilized by CS membrane on the surface of a magnetic electrode to constitute Hb enzyme-like sensor.The biosensor could catalyze the electrochemical reduction of H₂O₂,and an ultrasensitive determination of H₂O₂ could be achieved.The biosensor had a higher response when Hb was adsorbed by CS under an alkaline medium,almost twice as high as under the neutral condition.Under an external magnetic field,the response of the biosensor increased by 16.2%owing to Hb paramagnetism.The effect of pH on Hb adsorbed by CS,as well as the effect of an external magnetic field on Hb configuration were investigated with UV-Vis spectroscopic studies.The reduction peak current had log-linear relationships with H₂O₂ concentration in the range of 5-250?mol·L^-11 and 0.01-1?mol·L^-1,respectively.The detection limit?S/N=3?was as low as 0.003?mol·L^-1.The biosensor was successfully applied to the determination of H₂O₂ in milk samples and the disinfectant solution with recoveries of 96.3%-105.4%and 95.3%-107.7%,respectively.