The Application And Fundamental Research Of Nanotechnology For Self-assembly Of Hemoglobin And Electrochemical Sensors

Nowadays, nano–science and nanotechnology has caught more and more attentionby scientists all over the world. It has been discovered that materials in the nano-sizescale display size–dependent optical, electronic, magnetic and chemical properties. Inaddition, nanomaterials have special chemical and physical properties that differ greatlyfrom the bulk substances.Biomacromolecules including proteins and enzymes are the primary groups of life.They are involved in metabolism and other important physiological processes which arecharacteristics of electron transter between their oxidation and reduction states. In asense, to study the process of life is to investigate the electron transfer in essence.Therefore, electrochemical method has a special advantage for the study of electrontransfer process of proteins.Direct electrochemistry of redox proteins and enzymes has aroused great interest inbiological and bioelectrochemical fields. Studies on direct electrochemistry of redoxproteins and enzymes can be used to extract essential physicochemical data concerningthe kinetics and energetics of proteins’redox reactions, providing mechanistic studies ofelectron exchange among proteins in biological systems. Moreover, direct electrontransfer between immobilized proteins and the underlying electode can establish afoundation for fabricating new kinds of mediator-free biosensors, biofuel cells,bio-reactors etc. Therefore, it is very important to look for more effective method torealize the direct electron transfer of proteins and enzymes. Constructing the superiorperformance of biosensors to meet the biomedical, environmental testing and industrialquick analysis needs, will become the trend of the development of this field.The nanomaterials can enhance the active surface available for protein binding greatly over the geometrical area, and maintain the proteins’ physiological activitywithout detectable denaturation. Nanomaterials have unique physical and chemicalproperties such as high surface area, high surface free energy and excellentbiocompatibility. They can activate the electrode surface, penetrate to the internal andthe electrical activity center of protein, so as to shorten the distance between the proteinactivity center and the electrode surface. Therefore, they can help to speed up theelectron transfer directly and keep the biological activity of protein well.Research on the mechanism of electrochemistry and electrocatalysis for protein willhelp us to obtain a series of high sensitivity and high selectivity of biological sensorsand molecular devices. Also, it will provide valuable detection means and reveal thephysiological functions for some biomolecules for the life science.Modified electrodes based on nanomaterials combined with high surface area andgood electrocatalytic abilities that can largely improve electrical responses and thedetection sensitivity. Today, many wonderful nanostructured materials have beenapplied in electroanalytical chemistry and some important progresses in these topicshave recently achieved. Therefore, this dissertation research focuses on developingmodified electrodes based on nanostructrued materials, which is one of most activeareas in electroanalytical chemisty of redox proteins. We are adhering to an organiccombination of electoanalytical chemistry, nanotechnology and redox proteinschemistry. The details are given as follows:1. Electrochemical biosensing of hemoglobin by direct electrochemistry onsilver nanoparticles–chitosan FilmThis paper reports on the fabrication of hemoglobin （Hb） silver nanoparticles（AgNPs） chitosan film on the glassy carbon electrode and its application onelectrochemical biosensing. AgNPs could greatly enhance the electron–transferreactivity of Hb as a bridge. In the phosphate buffer solution with pH value of7.0, Hbshowed a pair of well–defined redox peaks with the formal potential （E0’） of0.33V （vs.SCE）. The immobilized Hb in the film maintained its biological activity, showing asurface controlled process with the heterogeneous electron transfer rate constant （ks） of1.83s1and displayed the same features of a peroxidase in the electrocatalytic reduction of oxygen and hydrogen peroxide （H₂O₂）. The linear range for the determination ofH₂O₂was from0.75μmol/L to0.22mmol/L with a detection limit of0.50μmol/L（S/N=3）. Such a simple assemble method could offer a promising platform for furtherstudy on the direct electrochemistry of other redox proteins and the development of thethird-generation electrochemical biosensors.2. Direct electrochemical behavior of hemoglobin at surface of Au@Fe₃O₄magnetic nanoparticlesNanoparticles （NPs） consisting of an Fe₃O₄core and a thin gold shell （referred to asAu@Fe₃O₄NPs） were self–assembled on the surface of a glassy carbon electrodemodified with ethylenediamine. Following adsorption of hemoglobin, its interactionwith the NPs was studied by UV–vis spectroscopy （UV–vis）, electrochemicalimpedance spectroscopy（EIS）, and cyclic voltammetry（CV）. Stable and well–definedredox peaks were observed at about–0.35V and–0.13V in pH6.0buffer. The modifiedelectrode was used as a mediator–free sensor for H₂O₂, with a linear range from3.4μmol/L to4.0mmol/L of H₂O₂, and with a0.67μmol/L detection limit （S/N=3）. Theapparent Michaelis-Menten constant is2.3mmol/L.3. Nanostructured biosensors built by Layer–by–Layer electrostatic assemblyof hemoglobin and Pt@Fe₃O₄nanocompositeIn this paper, we reported the using of Pt@Fe₃O₄nanoparticles for introduction intohemoglobin （Hb） multilayer film assemblies, constructed by the layer–by–layer （LBL）deposition technique. The synthesized Pt@Fe₃O₄particles are characterized by X–rayphotoelectron spectroscopy （XPS）. UV–vis spectroscopy, electrochemical impedancespectroscopy, cyclic voltammetry and atomic force microscopy （AFM） were used tomonitor the film growth and demonstrated that the {Hb/Pt@Fe₃O₄}nmultilayer filmswere formed in a progressive and uniform manner but the electroactivity of the filmswas only extended to a few bilayers. CVs of {Hb/Pt@Fe₃O₄}nfilms showed a pair ofwell–defined and nearly reversible peaks, characteristic of the protein hemeFe（III）/Fe（II） redox couples. Additionally, the adsorbed Hb on the film kept highcatalytic activity toward H₂O₂and nitrite. The linear range of the determination were from0.125μM to0.16mM for H₂O₂and1.5μM to0.12mM for nitrite, with thedetection limits of0.03μM and0.29μM respectively （S/N=3）. Additionally, thebiosensor also exhibited good reproducibility, high selectivity, and long–term stability.These results demonstrate that the developed strategy making use of the advantages ofPt@Fe₃O₄and LBL assembly is ideal for the direct electrochemistry of the redoxenzymes and the construction of the sensitive and stable mediator–free biosensors.4. The pH–sensitive switchable behavior based on the layer–by–layer films ofhemoglobin and Ag nanoparticlesA switchable layer–by–layer assembly film was constructed by alternateadsorption of negatively charged Ag nanoparticles （AgNPs） and positively chargedhemoglobin （Hb） with appropriate pH value. The {Hb/AgNPs}nfilms showed strongpH–sensitive on–off responses toward the electroactive probe Fe（CN）63-. The{Hb/AgNPs}4film is active （“On”） to the negatively charged probe, Fe（CN）63-, at pH3.0, but inactive （“Off”） to the probe at pH9.0. The switching behavior is fullyreversible by simply changing the pH value of the solution and can be applied forpH–controlled reversible electrochemical reduction of H₂O₂catalyzed by Hb. ThepH–sensitive behavior maybe due to the electrostatic interaction between the films andthe probe. The modified electrode with the pH–controlled switchable redox activitywould possibly become a “smart” interface for the construction of specificelectrochemical biosensors with a signal–controlled activity.5. Electrocatalytical oxidation of nitrite and its determination based onAu@Fe₃O₄nanoparticlesA promising electrochemical nitrite sensor was fabricated by immobilizingAu@Fe₃O₄nanoparticles on the surface of L–cysteine modified glassy carbon electrode,which was characterized by scanning electron microscopy （SEM）, XPS, electrochemicalimpedance spectroscopy and cyclic voltammetry. The proposed sensor exhibitedexcellent electrocatalytic activity toward nitrite oxidation. The kinetic parameters of theelectrode reaction process were calculated,（1–α）nawas0.38and the heterogeneouselectron transfer coefficient （k） was0.13cm s^-1. The detection conditions such as supporting electrolyte and pH value were optimized. Under the optimized conditions,the linear range for the determination of nitrite was3.6×10^-6to1.0×10^-2mol/L with adetection limit of8.2×10^-7mol/L （S/N=3）. Moreover, the as-prepared electrodedisplayed good stability, repeatability and selectivity for promising practicalapplications.6. Chitosan–Fe₃O₄nanocomposite based electrochemical sensors for thedetermination of bisphenol AThis paper reports the application of chitosan–Fe₃O₄（CS–Fe₃O₄） nanocompositemodified glassy carbon electrodes for the amperometric determination of bisphenol A（BPA）. We observed that the CS–Fe₃O₄nanocomposite could remarkably enhance thecurrent response and decrease its oxidation overpotential in the electrochemicaldetection. Experimental parameters, such as the amount of the CS–Fe₃O₄, theaccumulation potential and time, the pH value of buffer solution etc. were optimized.Under the optimized conditions, the oxidation peak current was proportional to BPAconcentration in the range between5.0×10^-8and3.0×10^-5mol/L with the correlationcoefficient of0.9992and the limit of detection of8.0×10^-9mol/L （S/N=3）. Theproposed sensors were successfully employed to determine BPA in real plastic productsand the recoveries were between92.0%–106.2%. This strategy might open moreopportunities for the electrochemical determination of BPA in practical applications.Additionally, the leaching studies of BPA on incubation time using the as-preparedmodified electrode were successfully carried out.7. Enhanced sensing of diethylstilbestrol based on graphene withelectrodeposited chitosan film modified electrodeAn electrochemical sensor based on graphene oxide （GO） with electrodepositedchitosan （CS） film modified glassy carbon electrode for voltammetric determination ofdiethylstilbestrol （DES） is presented. The electrochemical behavior of DES at themodified electrode was investigated by cyclic voltammetry. The oxidation peak currentof DES increased dramatically at the GO–CS/GCE compared with the bare GCE. Themodified electrode was used to electrochemically detect DES and showed excellent electrocatalytic activity for the oxidation of DES under the optimized conditions. Usingdifferential pulse voltammetric technique, the calibration curve for DES determinationwas obtained in the range of1.5×10^-8–3.0×10^-5mol/L and the detection limit is3.0×10^-9mol/L. Using this method, DES in the tablet sample was measured. The resultsshow that this voltammetric method is reliable for the practice determination of DES.