Application of an enzyme-based biofuel cell containing a bioelectrode modified with deoxyribonucleic acid-wrapped single-walled carbon nanotubes to serum

Enzyme-based biofuel cells (EFCs) are a form of biofuel cells (BFCs) that can utilize redox enzymes as biocatalysts. Applications of an EFC to an implantable system are evaluated under mild conditions, such as ambient temperature or neutral pH. In the present study, an EFC containing a bioelectrode modified with deoxyribonucleic acid (DNA)-wrapped single-walled carbon nanotubes (SWNTs) was applied to a serum system. The protection of immobilized glucose oxidase (GOD) using DNA-wrapped SWNTs was investigated in a trypsin environment, which can exist in a serum. GOD is immobilized by masking the active site onto the anode electrode. The anode/cathode system in the cell was composed of GOD/laccase as the biocatalysts and glucose/oxygen as the substrates in serum. The electrical properties of the anode in serum according to cyclic voltammetry (CV cycle) were improved using the DNA-wrapped SWNTs. Overall, an EFC that employed DNA-wrapped SWNTs and GOD immobilization in conjunction with protection of the active site increased the stability of GOD in serum, which enabled a high level of power production (ca. 190 μW/cm(2)) for up to 1 week.     

Mediatorless sugar/oxygen enzymatic fuel cells based on gold nanoparticle-modified electrodes

We report on the fabrication and characterisation of a gold-nanoparticle (AuNP)-based mediatorless sugar/oxygen biofuel cell (BFC) operating in neutral sugar-containing buffers and human physiological fluids, such as blood and plasma. First, Corynascus thermophilus cellobiose dehydrogenase (CtCDH) and Myrothecium verrucaria bilirubin oxidase (MvBOx), used as anodic and cathodic bioelements, respectively, were immobilised on gold electrodes modified with 20 nm AuNPs. Detailed characterisation and optimisation of a new CDH/AuNP-based bioanode were performed and the following fundamental parameters were obtained: (i) the redox potential of the haem-containing centre of the enzyme was measured to be 75 mV vs. NHE, (ii) the surface coverage of CtCDH was found to be 0.65 pmol cm(-2) corresponding to a sub-monolayer coverage of the thiol-modified AuNPs by the enzyme, (iii) a turnover number for CtCDH immobilised on thiol-modified AuNPs was calculated to be ca. 0.5 s(-1), and (iv) the maximal current densities as high as 40 μA cm(-2) were registered in sugar-containing neutral buffers. Second, both biomodified electrodes, namely the CtCDH/AuNP-based bioanode and the MvBOx/AuNP-based biocathode, were combined into a functional BFC and the designed biodevices were carefully investigated. The following characteristics of the mediator-, separator- and membrane-less, miniature BFC were obtained: in phosphate buffer; an open-circuit voltage of 0.68 V, a maximum power density of 15 μW cm(-2) at a cell voltage of 0.52 V and in human blood; an open-circuit voltage of 0.65 V, a maximum power density of 3 μW cm(-2) at a cell voltage of 0.45 V, respectively. The estimated half-lives of the biodevices were found to be >12, <8, and <2 h in a sugar-containing buffer, human plasma, and blood, respectively. The basic characteristics of mediatorless sugar/oxygen BFCs were significantly improved compared with previously designed biodevices, because of the usage of three-dimensional AuNP-modified electrodes.     

A biofuel cell harvesting energy from glucose-air and fruit juice-air

The membraneless biofuel cell (BFC) is facile prepared based on glucose oxidase and laccase as anodic and cathodic catalyst, respectively, by using 1,1'-dicarboxyferrocene as the mediators of both anode and cathode. The BFC can work by taking glucose as fuel in air-saturated solution, in which air serves as the oxidizer of the cathode. More interestingly, the fruit juice containing glucose, e.g. grape, banana or orange juice as the fuels substituting for glucose can make the BFC work. The BFC shows several advantages which have not been reported to our knowledge: (1) it is membraneless BFC which can work with same mediator on both anode and cathode; (2) fruit juice can act as fuels of BFCs substituting for usually used glucose; (3) especially, the orange juice can greatly enhance the power output rather than that of glucose, grape or banana juice. Besides, the facile and simple preparation procedure and easy accessibility of fruit juice as well as air being whenever and everywhere imply that our system has promising potential for the development and practical application of BFCs.     

Glucose/O2 biofuel cell based on enzymes, redox mediators, and multiple-walled carbon nanotubes deposited by AC-electrophoresis then stabilized by electropolymerized polypyrrole

In this study, we developed an automated strategy to manufacture an enzyme BFC powered by glucose/O(2). The bioanode consists of GOx enzyme and PQQ redox mediator adsorbed over night on MWCNTs then deposited by means of AC-electrophoresis at 30 Hz and 160 V(p-p) and, finally stabilized by electropolymerized polypyrrole. The biocathode is constructed from LAc enzyme and ABTS redox mediator adsorbed over night on MWCNTs, then electrophoretically deposited under AC-electric field at 30 Hz and 160 V(p-p) and, finally stabilized by electrodeposited polypyrrole. The BFC was studied under air in phosphate buffer solution pH 7.4 containing 10 mM glucose and in human serum with 5 mM glucose addition at the physiological temperature of 37°C. Under these conditions, the maximum power density reaches 1.1 μW · mm(-2) at a cell voltage of 0.167 V in buffer solution and 0.69 μW · mm(-2) at cell voltage of 0.151 V in human serum. Such automated BFCs have a great potential to be optimized, miniaturized to micro and nanoscale devices suitable for in vivo studies.     

High-performance amperometric biosensors and biofuel cell based on chitosan-strengthened cast thin films of chemically synthesized catecholamine polymers with glucose oxidase effectively entrapped

Rapid oxidation of dopamine (DA) or L-noradrenaline (NA) by K(3)Fe(CN)(6) yields poly(DA) (PDA(C)) or poly(NA) (PNA(C)) with glucose oxidase (GOx) effectively entrapped, and such an enzyme-entrapped catecholamine polymer is cast on an Au electrode followed by chitosan (CS) strengthening for biosensing and fabrication of a biofuel cell (BFC). The optimized glucose biosensor of CS/PDA(C)-GOx/Au displays an extremely high sensitivity up to 135 μA mM(-1) cm(-2), a very low limit of detection of 0.07 μM, a response time of <3 s, good suppression of interferents, striking thermostability (lifetime of 3 weeks at 60°C and over 2 months at 30°C), and high resistance to urea denaturation. The biosensor also works well in the second generation biosensing mode with p-benzoquinone (BQ) or ferrocene monocarboxylic acid (Fc) as an artificial mediator, with greatly broadened linear detection ranges (2.0 μM-48.0 mM for BQ and 2.0 μM-16.0 mM for Fc) and up to mA cm(-2)-scale glucose-saturated current density. The good permeability of artificial mediators across the enzyme film enables the quantification of the surface concentration of immobilized GOx on the basis of a reported kinetic model, and UV-Vis spectrophotometry is used to measure the enzymatic activity, revealing high enzymatic activity/load at CS/PDA(C)-GOx/Au. A BFC is also successfully fabricated with a bioanode of CS/PDA(C)-GOx/Au in phosphate buffer solution containing 100 mM glucose and 4.0 mM BQ and a carbon cathode in Nafion-membrane-isolated acidic KMnO(4), and its maximum power density of 1.62 mW cm(-2) is superior to those of most BFC hitherto reported.     

Development of nanostructured bioanodes containing dendrimers and dehydrogenases enzymes for application in ethanol biofuel cells

This paper describes the use of the electrostatic layer-by-layer (LbL) technique for the preparation of bioanodes with potential application in ethanol/O(2) biofuel cells. More specifically, the LbL technique was employed for immobilization of dehydrogenase enzymes and polyamidoamine (PAMAM) dendrimers onto carbon paper support. Both mono (anchoring only the enzyme alcohol dehydrogenase, ADH) and bi-enzymatic (anchoring both ADH and aldehyde dehydrogenase, AldDH) systems were tested. The amount of ADH deposited onto the Toray? paper was 95 ng cm(-2) per bilayer. Kinetic studies revealed that the LbL technique enables better control of enzyme disposition on the bioanode, as compared with the results obtained with the bioanodes prepared by the passive adsorption technique. The power density values achieved for the mono-enzymatic system as a function of the enzyme load ranged from 0.02 to 0.063 mW cm(-2) for the bioanode containing 36 ADH bilayers. The bioanodes containing a gas diffusion layer (GDL) displayed enhanced performance, but their mechanical stability must be improved. The bi-enzymatic system generated a power density of 0.12 mW cm(-2). In conclusion, the LbL technique is a very attractive approach for enzyme immobilization onto carbon platform, since it enables strict control of enzyme disposition on the bioanode surface with very low enzyme consumption.     

Highly stable enzyme precipitate coatings and their electrochemical applications

This paper describes highly stable enzyme precipitate coatings (EPCs) on electrospun polymer nanofibers and carbon nanotubes (CNTs), and their potential applications in the development of highly sensitive biosensors and high-powered biofuel cells. EPCs of glucose oxidase (GOx) were prepared by precipitating GOx molecules in the presence of ammonium sulfate, then cross-linking the precipitated GOx aggregates on covalently attached enzyme molecules on the surface of nanomaterials. EPCs-GOx not only improved enzyme loading, but also retained high enzyme stability. For example, EPC-GOx on CNTs showed a 50 times higher activity per unit weight of CNTs than the conventional approach of covalent attachment, and its initial activity was maintained with negligible loss for 200 days. EPC-GOx on CNTs was entrapped by Nafion to prepare enzyme electrodes for glucose sensors and biofuel cells. The EPC-GOx electrode showed a higher sensitivity and a lower detection limit than an electrode prepared with covalently attached GOx (CA-GOx). The CA-GOx electrode showed an 80% drop in sensitivity after thermal treatment at 50°C for 4 h, while the EPC-GOx electrode maintained its high sensitivity with negligible decrease under the same conditions. The use of EPC-GOx as the anode of a biofuel cell improved the power density, which was also stable even after thermal treatment of the enzyme anode at 50°C. The excellent stability of the EPC-GOx electrode together with its high current output create new potential for the practical applications of enzyme-based glucose sensors and biofuel cells.     

High-performance bioanode based on the composite of CNTs-immobilized mediator and silk film-immobilized glucose oxidase for glucose/O2 biofuel cells

A high-performance bioanode based on the composite of carbon nanotubes (CNTs)-immobilized mediator and silk film (SF)-immobilized glucose oxidase (GOD) was developed for glucose/O(2) biofuel cell (BFC). Ferrocenecarboxaldehyde (Fc) was used as the mediator and covalently immobilized on the ethylenediamine (EDA)-functionalized CNTs (CNTs-EDA). GOD was cross-linked on the SF with glutaraldehyde (GA) as the cross-linking agent. The resulting electrode (CNTs-Fc/SF-GOD/glassy carbon (GC) electrode) exhibited good catalytic activity towards glucose oxidation and excellent stability. For the assembled glucose/O(2) BFC with the CNTs-Fc/SF-GOD/GC electrode as the bioanode and a commercial E-TEK Pt/C modified GC electrode as the cathode, the open circuit potential is 0.48 V and the maximum power density of 50.70 μW cm(-2) can be achieved at 0.15 V.     

Enzyme precipitate coatings of lipase on polymer nanofibers

Lipase (LP) was immobilized on electrospun and ethanol-dispersed polystyrene–poly(styrene-co-maleic anhydride) (PS–PSMA) nanofibers (EtOH-NF) in the form of enzyme precipitate coatings (EPCs). LP precipitate coatings (EPCs-LP) were prepared in a three-step process, consisting of covalent attachment, LP precipitation, and crosslinking of precipitated LPs onto the covalently attached LPs via glutaraldehyde treatment. The LP precipitation was performed by adding various concentrations of ammonium sulfate (20–50%, w/v). EPCs-LP improved the LP activity and stability when compared to covalently attached LPs (CA-LP) and the enzyme coatings of LPs (EC-LP) without the LP precipitation. For example, the use of 40% (w/v) ammonium sulfate resulted in EPC40-LP with the highest activity, which was 4.0 and 3.6 times higher than those of CA-LP and EC-LP, respectively. After 165-day incubation under rigorous shaking at 200 rpm, the residual activities of EPC50-LP were 0.5 μM/min mg of EtOH-NF, representing 113 and 75 times higher than those of CA-LP and EC-LP, respectively. When LP was partially purified via a simple ammonium sulfate precipitation and dialysis, both activities and stabilities of EC-LP and EPC-LP could be marginally improved. It is anticipated that the improved LP activity and stability in the form of EPCs would allow for their potential applications in various bioconversion processes such as biodiesel production and ibuprofen resolution.     

The effect of physico-chemically immobilized methylene blue and neutral red on the anode of microbial fuel cell

A fast and cost effective immobilization of electron carriers, methylene blue (MB) and neutral red (NR) by pH shift was proposed to improve bioanodic performance. The adsorption of mediators onto the carbon cloth anode was verified using cyclic voltammogram (CV) and the effect of the immobilized mediators on acclimation, power density, and acetate removal of MFCs was investigated. A peak power density of P _max(MB) = 11.3 W/m³ was achieved over days 110 ∼ 120, as compared to P _max(Control) = 5.4 W/m³ and P _max(NR) = 3.1 W/m³ for the treated anode after 15 sequential fed-batch operations. The VFA removal rates however were similar for all MFC systems, ranging from 82 to 87%. It could be suggested that the increase in power density for the MB treated electrode resulted from an enhanced electron transport from exo-electrogenic bacteria. MB may also have a selective effect on the bacterial community during the start-up stage, increasing the voltage production and acetate removal from day 1 to 16. However, MFC with NR treated anode produced an initial voltage under 100 mV, with lower coulombic efficiency (CE). NR exhibited less favourable mediator molecule binding to the electrode surface, when subject to pH driven physico-chemical immobilization.     

Enhanced Growth of Endothelial Precursor Cells on PCG-Matrix Facilitates Accelerated,Fibrosis-Free,Wound Healing: A Diabetic Mouse Model

Diabetes mellitus (DM)-induced endothelial progenitor cell (EPC) dysfunction causes impaired wound healing, which can be rescued by delivery of large numbers of ‘normal’ EPCs onto such wounds. The principal challenges herein are (a) the high number of EPCs required and (b) their sustained delivery onto the wounds. Most of the currently available scaffolds either serve as passive devices for cellular delivery or allow adherence and proliferation, but not both. This clearly indicates that matrices possessing both attributes are ‘the need of the day’ for efficient healing of diabetic wounds. Therefore, we developed a system that not only allows selective enrichment and expansion of EPCs, but also efficiently delivers them onto the wounds. Murine bone marrow-derived mononuclear cells (MNCs) were seeded onto a PolyCaprolactone-Gelatin (PCG) nano-fiber matrix that offers a combined advantage of strength, biocompatibility wettability; and cultured them in EGM2 to allow EPC growth. The efficacy of the PCG matrix in supporting the EPC growth and delivery was assessed by various in vitro parameters. Its efficacy in diabetic wound healing was assessed by a topical application of the PCG-EPCs onto diabetic wounds. The PCG matrix promoted a high-level attachment of EPCs and enhanced their growth, colony formation, and proliferation without compromising their viability as compared to Poly L-lactic acid (PLLA) and Vitronectin (VN), the matrix and non-matrix controls respectively. The PCG-matrix also allowed a sustained chemotactic migration of EPCs in vitro. The matrix-effected sustained delivery of EPCs onto the diabetic wounds resulted in an enhanced fibrosis-free wound healing as compared to the controls. Our data, thus, highlight the novel therapeutic potential of PCG-EPCs as a combined ‘growth and delivery system’ to achieve an accelerated fibrosis-free healing of dermal lesions, including diabetic wounds.     

Immobilization of lipase by ultrafiltration and cross-linking onto the polysulfone membrane surface

In this study, a biphasic enzymatic membrane reactor was made by immobilizing Candida Rugosa lipase onto the dense surface of polysulfone ultrafiltration membrane by filtration and then cross-linking with glutaraldehyde solution. The reactor was further applied for the hydrolysis of olive oil, the performance of which was evaluated in respect of apparent reaction rate based on the amount of fatty acids extracted into the aqueous phase per minute and per membrane surface. It was found that the ultrafiltration and cross-linking process greatly improved the reaction rate per unit membrane area and the enzyme lifetime. The highest reaction rate reached 0.089 micromol FFA/min cm2 when the enzyme loading density was 0.098 mg/cm2. The results also indicated that the performance of lipase immobilized on the membrane surface was superior to that immobilized in the pores, and the apparent reaction rate and stability of immobilized lipases were improved greatly after cross-linking. It suggested that immobilization of enzymes by filtration and then cross-linking the enzymes onto the membrane surface is a simple and convenient way to prepare a high-activity immobilized enzyme membrane.     

Microorganism-immobilized carbon nanoparticle anode for microbial fuel cells based on direct electron transfer

A fast and convenient bacterial immobilization method was proposed as an attempt to improve the anode efficiency of a microbial fuel cell, in which bacteria were entrapped into carbon nanoparticle matrix. The direct electron transfer from the entrapped bacterial cells to the anode was verified using cyclic voltammogram (CV). Using the immobilized bioanode, the start-up time of the MFC was greatly reduced. Meanwhile, the maximum power density of 1,947 mW m⁻² with the modified anode was much higher than that with the biofilm-based carbon cloth anode (1,479 mW m⁻²). Impedance measurements suggested that performance improvement resulted from the decrease in charge transfer and diffusion resistances. The results demonstrated that bacteria immobilization using carbon nanoparticle matrix was a simple and efficient approach for improving the anodes performances in MFCs.     

Chronic administration of NMU into the paraventricular nucleus stimulates the HPA axis but does not influence food intake or body weight

C-reactive protein (CRP), a predictor of future cardiovascular diseases, has been reported to damage the vascular wall by inducing endothelial dysfunction and inflammation. This proatherogenic CRP was speculated to have a role in attenuating angiogenic functions of human endothelial progenitor cells (EPCs), possibly impairing vascular regeneration and increasing cardiovascular vulnerability to ischemic injury. Herein, we investigated the direct effect of CRP on angiogenic activity and gene expression in human EPCs. Incubation of EPCs with human recombinant CRP significantly inhibited EPC migration in response to vascular endothelial growth factor, possibly by decreasing the expression of endothelial nitric oxide synthase and subsequent nitric oxide production. In addition, CRP-treated EPCs showed the reduced adhesiveness onto an endothelial cell monolayer. When assayed for the gene expression of arteriogenic chemo-cytokines, CRP substantially decreased their expression levels in EPC, in part due to the upregulation of suppressors of cytokine signaling proteins. These results suggest that CRP directly attenuates the angiogenic and possibly arteriogenic functions of EPCs. This CRP-induced EPC dysfunction may impair the vascular regenerative capacity of EPCs, thereby leading to increased risk of cardiovascular diseases.     

Screen-printed electrodes modified with glucose oxidase immobilized in hybrid organosilicon sol-gel matrix

A new polymer composition was developed based on hybrid sol-gel material for the immobilization of enzymes on the surface of screen-printed carbon electrodes modified with Prussian blue. The existing and developed methods for the glucose oxidase (GO) immobilization are compared. Highly stable bioelectrodes were shown to be obtained by enzyme immobilization on a hybrid composition consisting of solgel/poly vinyl alcohol (PVA) (up to 60 sequential chains) and agar gel (up to 45 sequential chains). The range of glucose concentrations detected during enzyme immobilization in a hybrid sol-gel/PVA composition or agar gel without dilution of a sample was 1.0–5.9 μM and 3.6–6.3 μM, respectively. An analysis of wine products was conducted. The results obtained using the proposed biosensors were shown to differ insignificantly from those obtained by high-performance liquid chromatography (the correlation coefficient was 0.9998).     

Biomimetic CO₂ sequestration using purified carbonic anhydrase from indigenous bacterial strains immobilized on biopolymeric materials

The present study deals with immobilization of purified CA and whole cell of Pseudomonas fragi, Micrococcus lylae, and Micrococcus luteus 2 on different biopolymer matrices. Highest enzyme immobilization was achieved with P. fragi CA (89%) on chitosan-KOH beads, while maximum cell immobilization was achieved with M. lylae (75%) on chitosan-NH(4)OH beads. A maximum increase of 1.08-1.18 fold stability between 35 and 55°C was observed for M. lylae immobilized CA. The storage stability was improved by 2.02 folds after immobilization. FTIR spectra confirmed the adsorption of CA on chitosan-KOH beads following hydrophilic interactions. Calcium carbonate precipitation was achieved using chitosan-KOH immobilized P. fragi CA. More than 2 fold increase in sequestration potential was observed for immobilized system as compared to free enzyme. XRD spectra revealed calcite as the dominant phase in biomimetically produced calcium carbonate.     

AMP-activated protein kinase rescues the angiogenic functions of endothelial progenitor cells via manganese superoxide dismutase induction in type 1 diabetes

Endothelial progenitor cells (EPCs) play an essential role in angiogenesis but are functionally impaired in diabetes. We recently reported that decreased expression of manganese superoxide dismutase (MnSOD) critically contributes to diabetic EPC dysfunction. AMP-activated protein kinase (AMPK) activation has been shown to induce MnSOD and suppress hyperglycemia-induced mitochondrial ROS production in endothelial cells. However, whether AMPK protects EPCs from oxidative stress in diabetes is unknown. We tested the hypothesis that AMPK activation rescues impaired EPC functions through MnSOD induction in type 1 diabetes. Bone marrow-derived EPCs from adult male streptozotocin-induced diabetic mice and normal controls were used. AMPK activity was decreased in diabetic EPCs, indicated by reduced AMPK and acetyl-CoA carboxylase phosphorylation. AMPK activation by treating diabetic EPCs with its selective agonist AICAR rescued their in vitro functions, including Matrigel tube formation, adhesion, and migration. Furthermore, AICAR restored the decreased MnSOD protein and enzymatic activity and suppressed the mitochondrial superoxide level in diabetic EPCs, indicated by MitoSOX flow cytometry. These beneficial effects of AICAR on MnSOD and EPC functions were significantly attenuated by silencing MnSOD or AMPK antagonist compound C pretreatment. Finally, the expression of protein phosphatase 2A, a key enzyme for AMPK dephosphorylation and inactivation, was increased in diabetic EPCs, and its inhibition by siRNA or okadaic acid reversed the deficient AMPK activation and MnSOD level in diabetic EPCs. These findings demonstrate for the first time that AMPK activation rescues impaired EPC functions and suppresses mitochondrial superoxide by inducing MnSOD in type 1 diabetes.     

Photoautotrophic cathodic oxygen reduction catalyzed by a green alga,Chlamydomonas reinhardtii

Biofuel cells (BFCs) use enzymes and microbial cells to produce energy from bioavailable substrates and treat various wastewaters, and cathodic oxygen reduction is a key factor governing the efficiency of BFCs. In this study, we demonstrated that a green alga, Chlamydomonas reinhardtii, could directly mediate oxygen reduction. Cyclic voltammogram analysis revealed that the C. reinhardtii biofilm formed on a solid electrode was responsible for oxygen reduction without dosing of electron mediator. Furthermore, 4‐electron oxygen reduction pathway was found in this self‐sustained, light‐responded BFC. The results of this study could expand our understanding and viewpoints of biocathode catalysis, which is essential for novel catalyst design and development for BFCs. Biotechnol. Bioeng. 2013; 110: 173–179. © 2012 Wiley Periodicals, Inc.     

The potential of statin and stromal cell-derived factor-1 to promote angiogenesis

Angiogenesis requires the mobilization of progenitor cells from the bone marrow (BM) and homing of progenitor cells to ischemic tissue. The cholesterol lowering drug Statins can stimulate angiogenesis via mobilization of BM derived endothelial progenitor cells (EPCs), promoting EPC migration, and inhibiting EPC apoptosis. The chemokine stromal cell-derived factor-1 (SDF-1) augments EPC chemotaxis, facilitates EPC incorporation into the neovasculature. The combined use of a statin to mobilize EPCs and local over-expression of SDF-1 to augment EPC homing to ischemic muscle resulted in superior angiogenesis versus use of either agent alone. Their effects are through augmenting EPC mobilization, incorporation, proliferation, migration, and tube formation while inhibiting EPC apoptosis. Statin and SDF-1 therefore display synergism in promoting neovascularization by improving reperfusion of ischemic muscle, increasing progenitor cell presentation and capillary density in ischemic muscle, and diminishing apoptosis. These results suggest that the combination of statin and SDF-1 may be a new therapeutic strategy in the treatment of limb ischemia.