Fabrication And Performance Of Stimuli–responsive Enzymatic Electrode And Its Biofuel Cell

Enzymatic biofuel cell(EBFC)is a new type of fuel cell that utilizes enzymes as catalysts for fuels to converse the chemical energy to electricity.The EBFCs have attracted much attention in the area of life science and clinical medicine,due to their advantages such as mild operating conditions,good biocompatibility and ability to acquire energy from biowastes,thus can be applied as green powers for micro devices.If their output behavior correlates with the physiological conditions,the EBFCs would be potentially working in vivo with logic response to automatically switch on and off,which is significant for manufacturing implanted sensing-actuating devices.However,before fabricating a well-performanced stimuli-responsive EBFC,we should first solve the basic problems of general EBFC:a)the catalytic active sites are generally embedded in a thick isolated protein shell,making electrons hard to transmit from these sites to electrode surface;b)the specific 3-dimentional protein structure of enzyme is highly environmentally sensitive in which the deformation is unfavorable;c)the weak interactions between enzymes and electrodes leading to a leakage of enzymes on the electrodes.All these problems could significantly impair the EBFC electrochemical output performance and stability.Furthermore,for the stimuli-responsive polymers coated enzymatic electrodes,the enzymes loading,surface charges,and substrate diffusion are all dependent on the structure and composition of the polymer at electrodes,which eventually affect the final conductivity,catalysis and response activity at electrode,indicating that the accurate control of polymer on the surface of electrode is critical to improving the EBFC performance.In this thesis,the conductive nanohybrid enzymatic electrodes were initially prepared to improve the enzyme loading and electron transfer at electrode,and constructed a well-performance EBFC.Subsequently,the stimuli responsive polymer was introduced onto electrode surface to investigate the effect of polymer structure on electrochemical ’on/off’behavior,as well as the interaction of polymer brushes and enzyme molecules.Through the regulation and control of polymer structure and chemistry,the conductivity and enzymes loading were optimized.On this basis,the interaction between different polymer brushes and oligonucleotides,as well as the effect on genetic delivery efficiency were discussed.The main research aspects are as follows:1.To solve the problems to enzyme immobilization and electron transfer on electrode,a facile route to prepare chemical reduced graphene-silver nanoparticles hybrid(AgNPs@CR-GO)was designed involving in β-cyclodextrin(β-CD)as reducing and stabilizing agent.The close-packed AgNPs structure was used as a conductive matrix to adsorb enzyme and facilitate the electron transfer between immobilized enzyme and electrode.It was demonstrated by electrochemical testing that the electrode with close-packed AgNPs provided high GOx loading(Γ=4.80×10-10 mol cm-2)and fast electron transfer rate(ks=5.76 s-1).By employing GOx based-electrode as anode and laccase based-electrode as cathode,the assembled enzymatic biofuel cell exhibited a maximum power density of 77.44 μW cm-2 and an open-circuit voltage of 0.705 V.2.To further improve the enzyme loading and electron transfer on the nanomaterial-based electrode,and enhance the performance of enzymatic electrode and EBFC,we investigated a strategy to simultaneously improve the two aspects by assistance of the cationic surfactant,stearyltrimethylammonium bromide(STAB).STAB could firmly adsorb a substantial number of enzymes via electrostatic interaction in a favorable orientation on the conductive nanomaterial surface for electron transfer.On the other hand,STAB acts as a dispersant and stabilizer for traditionally conductive nanomaterials(reduced graphene oxide,carbon nanotubes,and gold nanoparticles)to guarantee their unique properties and form a well-conductive network.Electrochemical measurements demonstrated that enzymatic electrodes based on the nanohybrid possessed fast electron transfer rate(ks=8.36 s-1),a large quantity of immobilized enzymes(2.03×10-10 mol cm-2),and good activity toward glucose oxidation or oxygen reduction.The glucose biosensor performed linear response range of 0.01-11.71 mM,detection limit of 3.84×10-3 mM,and sensitivity of 10.42 μA mM-1 cm-2,while the glucose/O2 biofuel cell exhibited maximum power density of 121.87μW cm-2 and open-circuit voltage of 0.663 V.Both of the devices showed better performances than those of devices without STAB or conductive nanomaterials in this work.3.To obtain a stable light-switchable electrode,we used Langmuir-Schaefer technique to control the arrangement and aggregation of azobenzene-containing polymer molecules on the ITO electrode surface decorated with gold nanoparticles.The polymer formed a compact,complete thin film on the electrode surface and offered reversible and stable switching performance.The conductivity and hydrophilicity of the electrode changed under UV/visible light due to the photoisomerization of the azobenzene moieties in the polymer film,influencing electron transfer and mass transport at the electrode.The electrochemical characterization demonstrated that the electrode exhibited reversibly switchable electrochemical behavior.In its active state,the as-prepared electrode possessed efficient electrocatalytic capability towards uric acid oxidation with a maximum anodic current density 0.97 mA cm-2.The uric acid/air fuel cell assembled from the photo-triggered anode and a Pt/C-modified cathode operated with an open circuit voltage of 0.12 V and a maximum power density of 41.33μW cm-2.The cell exhibited reversible switching performance and high stability:after one month the power output was 94.2%of the original maximum value.4.Based on the non-enzymatic switchable electrode and high-loading enzymatic electrode,to fabricate a switchable bioelectrode loading with large amount of enzyme and performing excellent responsiveness towards the environment conditions,we prepared enzyme-embedded poly(2-(dimethylamino)ethyl methacrylate-N-isopropylacrylamide)(PDMAEMA-PNIPAm)block copolymer brush using "grafting from" method on gold electrode via surface-initiated atom transfer radical polymerization.The bottom PDMAEMA brush offered pH-responsiveness and adsorbed enzyme by electrostatic interaction,while the top PNIPAm layer provided temperature-responsiveness and prevent the leakage of enzyme from the electrode surface and provided an anti-fouling surface for electrode.We varied the grafting density,thickness,cross-linking degree and the chemistry of the block copolymer brushes,and studied their impact to enzyme loading,electron transfer,mass diffusion and the responsiveness on electrode.The electrochemical characterization demonstrated that the brush with grafting density of 0.332 chains per nm2,thickness of 15 nm for each block allowed a better enzyme permeation and adsorption,and well controlled the responsiveness process on electrode based on good electron transfer.The switchable bioelectrode possessed good glucose sensing performance,in which the linear response range is 0.05-8 mM,detection limit is 0.02 mM,and sensitivity of 4.63 μA mM-1 cm-2.The switching property is reversible and stable.After several switching cycles,the catalytic current of glucose remained around 89.0±0.6%of the original current.5.Based on the study correlating to the interaction between polymer brushes and enzyme,to systematically investigate the interaction between cationic polymer brushes and oligonucleotides,we use the Surface Plasmon Resonance technique and developed a kinetic model of brush binding and infiltration.We identify the striking impact that brush grafting density and thickness have on oligonucleotide kinetics of infiltration,binding affinity,and maximum loading.Surprisingly,double-stranded RNA molecules are found to load at significantly higher levels compared to DNA molecules of identical sequence(apart from uracils/thymines).Furthermore,analysis of the kinetics of adsorption of these oligonucleotides indicates that the stoichiometry of binding(ratio of amine versus phosphate residues)is close to parity for the uptake of 20 bp double-stranded RNA.Finally,nanoparticles were designed to be used as gene transfection vectors and to quantify if the brush grafting density and thickness significantly impact transfection efficiencies in a small interfering RNA knockdown assay.Therefore,this study demonstrates the rational design of polymer brush-based nanoparticle vectors for efficient delivery of oligonucleotides.The model developed will allow to uncover how the refinement of the physicochemical and structural properties of polymer brushes enable the tuning of RNA binding and allow the systematic study of cationic vectors efficiency for RNA delivery.