Enzyme Electrode Research Articles

Electrochemical impedance spectroscopy: Simultaneous detection of different diffusion behaviors as seen in finite element method simulations of mediator-type enzyme electrodes

Herein, a different interpretation of electrochemical impedance spectra with a double semicircle and a straight line obtained for systems involving diffusion processes is proposed. Simulations of electrochemical impedance spectra of mediator-type enzyme electrodes by using the finite element method revealed that under certain conditions, diffusion can be a combination of apparent semi-infinite and finite length, with the diffusion type differing depending on the position on the electrode or the diffusion direction. The Nyquist plot of a system with one charge transfer and the mixed-type diffusion shows a semicircle for the charge transfer, a second semicircle for finite length diffusion (short Warburg impedance), and a straight line for semi-infinite diffusion (Warburg impedance). In mediator-type enzyme electrodes with a free-diffusing enzyme and a mediator, such mixed-type diffusion can be observed at medium substrate concentrations when the reaction plane is close to the electrode at the edges and farther away in the middle of the electrode. These simulation results may help (bio-)electrochemists to more accurately interpret impedance spectra and gain better understanding of the phenomena occurring at mediator-type enzyme electrodes and other cases involving diffusion.

Nanocellulose Paper Semiconductor with a 3D Network Structure and Its Nano-Micro-Macro Trans-Scale Design.

Semiconducting nanomaterials with 3D network structures exhibit various fascinating properties such as electrical conduction, high permeability, and large surface areas, which are beneficial for adsorption, separation, and sensing applications. However, research on these materials is substantially restricted by the limited trans-scalability of their structural design and tunability of electrical conductivity. To overcome this challenge, a pyrolyzed cellulose nanofiber paper (CNP) semiconductor with a 3D network structure is proposed. Its nano–micro–macro trans-scale structural design is achieved by a combination of iodine-mediated morphology-retaining pyrolysis with spatially controlled drying of a cellulose nanofiber dispersion and paper-crafting techniques, such as microembossing, origami, and kirigami. The electrical conduction of this semiconductor is widely and systematically tuned, via the temperature-controlled progressive pyrolysis of CNP, from insulating (1012 Ω cm) to quasimetallic (10–2 Ω cm), which considerably exceeds that attained in other previously reported nanomaterials with 3D networks. The pyrolyzed CNP semiconductor provides not only the tailorable functionality for applications ranging from water-vapor-selective sensors to enzymatic biofuel cell electrodes but also the designability of macroscopic device configurations for stretchable and wearable applications. This study provides a pathway to realize structurally and functionally designable semiconducting nanomaterials and all-nanocellulose semiconducting technology for diverse electronics.

Control of carbon monoxide dehydrogenase orientation by site-specific immobilization enables direct electrical contact between enzyme cofactor and solid surface

Controlling the orientation of redox enzymes on electrode surfaces is essential in the development of direct electron transfer (DET)-based bioelectrocatalytic systems. The electron transfer (ET) distance varies according to the enzyme orientation when immobilized on an electrode surface, which influences the interfacial ET rate. We report control of the orientation of carbon monoxide dehydrogenase (CODH) as a model enzyme through the fusion of gold-binding peptide (gbp) at either the N- or the C-terminus, and at both termini to strengthen the binding interactions between the fusion enzyme and the gold surface. Key factors influenced by the gbp fusion site are described. Collectively, our data show that control of the CODH orientation on an electrode surface is achieved through the presence of dual tethering sites, which maintains the enzyme cofactor within a DET-available distance (<14 Å), thereby promoting DET at the enzyme–electrode interface.

Construct α-FeOOH-Reduced Graphene Oxide Aerogel as a Carrier for Glucose Oxidase Electrode.

A promising α-FeOOH-reduced graphene oxide aerogel (FeOOH-GA) has been prepared for the assembly of an enzyme electrode. The α-FeOOH-reduced graphene oxide aerogel was characterized by X-ray powder diffraction, field emission scanning electron microscopy, transmission electron microscopy, Fourier transform infrared spectroscopy, Raman, energy-dispersive X-ray spectroscopy, and X-ray photoelectron spectroscopy. The results reveal that graphene oxide is reduced by Fe2+ ion and α-FeOOH nanorods anchored on the reduced graphene oxide sheet through the Fe-O-C bond. Analyses using scanning electron microscopy and the Brunauer–Emmett–Teller method show that FeOOH-GA displays a various and interconnected pore structure. The FeOOH-GA was used as a support material on the glass carbon electrode (GCE) for glucose oxidase (GOD). Electrochemistry properties and bioelectrocatalytic activities of Nafion/GOD/FeOOH-GA/GCE were achieved from cyclic voltammetry and electrochemical impedance spectroscopy. The results show that Nafion/GOD/FeOOH-GA/GCE maintains outstanding catalytic activity and electrochemical properties. The FeOOH-GA could immobilize GOD through the hydrophobicity of the reduced graphene oxide and hydroxide radical of α-FeOOH. Appropriate α-FeOOH and diversified pore structure are beneficial for electron transfer, enzyme electrode storage, and interfacial electron transfer rate. All results indicated that the α-FeOOH-reduced graphene oxide aerogel as a carrier could effectively immobilize the tested enzyme.

Numerical Modeling and Experiment of a Thin-Film Enzyme Electrode with an Enzyme Adsorption Experiment to Design High-Current-Density Biofuel Cells

Numerical Modeling and Experiment of a Thin-Film Enzyme Electrode with an Enzyme Adsorption Experiment to Design High-Current-Density Biofuel Cells

Lactose Biosensor Development and Deployment in Dairy Product Analysis

Herein, development of an enzymatic biosensor for rapid quantitation of the disaccharide lactose in dairy samples (whey permeates and milk protein isolates (MPI)) is presented. Biosensor fabrication involved a chitosan/enzyme/crosslinker configuration with enzymes glucose oxidase and β-galactosidase at platinum and glassy carbon electrodes. Solution phase mediation was used to lower the operating potential (Eapp = 0.3 V vs Ag/AgCl), minimising any contribution from endogenous background electroactive species. Potential sweep and potentiostatic experiments realised analytical data for the lactose sensor with linear range 5.83 × 10−3 to 1.65 × 10−2 M, sensitivity 9.41 × 10−4 C cm−2 mM−1 and LOD of 1.38 mM. Scanning electrochemical microscopy realised surface characterisation of the enzyme layers with approach curves and redox competition mode imaging achieved over the active enzyme bilayer. Both glucose and lactose sensing was realised in whey permeate sample measuring 23.7 mM lactose, correcting for free glucose contributing signals, with 92.2% correlation with results obtained from the sample certificate of analysis. Solution phase mediation at glassy carbon enzyme electrodes resulted in lactose quantitation in milk protein isolates, measuring 1.16 mM for the MPI 1 (low lactose) and 1.54 mM for the MPI 10 (standard) sample with validation by HPLC analysis.

Designing a Highly Stable Enzyme–Graphene Oxide Biohybrid as a Sensitive Biorecognition Module for Biosensor Fabrication with Superior Performance and Stability

Biosensors are developed for monitoring signal molecules in a simple and rapid way, but their performance and stability are mainly limited by unstable biorecognition elements. Herein, we used a predesigned bioinorganic catalytic composite (nOPH10@GO) as a sensitive biological material to fabricate a high-performance organophosphorus hydrolase (OPH)-based biosensor. At first, the stability of OPH was enhanced through the in situ radical polymerization to generate an OPH nanocapsule (nOPH10), which was then assembled on graphene oxide (GO) to form a bioinorganic hybrid (nOPH10@GO). We confirmed the successful formation of a polymer shell on an enzyme surface and subsequently investigated its protective effects using the thermal treatment and organic solvent test. Compared to native OPH, nOPH10 exhibited enhanced enzymatic performance and stability, which could be also observed on the proposed electrode (nOPH10@GO/GCE). Specifically, nOPH10@GO/GCE can work steadily in the presence of different external stimuli and exhibited improved selectivity and stability. More importantly, owing to the enhanced catalytic activity of nOPH10@GO, the fabricated biosensor presented a wider linear range (0.2–60 μM), a lower limit of detection (0.013 μM), and higher repeatability and reproducibility in comparison with the naked enzyme electrode (OPH@GO/GCE). Our results highlighted the importance of the rational predesign of biorecognition materials in the development of a high-performance biosensing platform.

Theoretical and Numerical Analysis of Nonlinear Processes in Amperometric Enzyme Electrodes with Cyclic Substrate Conversion

A theoretical model of amperometric enzyme electrodes has been developed in which chemical amplification occurs in a single enzyme membrane via cyclic substrate conversion. The system is based on non-stationary diffusion equations with a nonlinear factor related to the Michaelis–Menten kinetics of the enzymatic reaction. By solving the nonlinear equations using the AGM technique, simple analytical expressions of concentration substrate, product, and amperometric current response are derived. Further, biosensor sensitivity, resistance, and gain are obtained from the current. MATLAB programming was used to carry out the digital simulation. The analytical results are validated with the numerical results. The effect of substrate concentration, maximum enzymatic rate, and membrane thickness on biosensor response was evaluated.

Electrochemical Impedance Simulation of Porous (Enzyme) Electrodes with Variously Shaped Pores Using 3-Dimensional Finite Element Method

Electrochemical Impedance Simulation of Porous (Enzyme) Electrodes with Variously Shaped Pores Using 3-Dimensional Finite Element Method

Electrochemical determination of (S)-7-hydroxywarfarin for analysis of CYP2C9 catalytic activity

An electrochemical method for the quantitative determination of (S)-7-hydroxywarfarin as a metabolite of the vitamin K antagonist warfarin has been developed to analyze the catalytic activity of cytochrome P450 2C9 (CYP2C9). The electrochemical properties of (S)-7-hydroxywarfarin were investigated using screen-printed carbon electrodes. We have shown by cyclic voltammetry that irreversible (S)-7-hydroxywarfarin electrochemical oxidation, in contrast to (S)-warfarin, can be registered by an oxidation peak at approximately 0.6 V (vs. Ag pseudo-reference electrode). A linear dependence of the oxidation peak amplitude in the range of 0.57–0.61 V on (S)-7-hydroxywarfarin concentration (in the range of 0.1–1 μM) was shown by the square-wave voltammetry method. The limit of (S)-7-hydroxywarfarin detection and the sensitivity were calculated as 0.091 μM and 0.0075 A/M, respectively. Using an electrochemical system based on recombinant human CYP2C9 immobilized on screen-printed carbon electrodes modified by didodecyldimethylammonium bromide, we have shown the possibility of (S)-7-hydroxywarfarin electrochemical quantification without separation of the enzymatic system component. The (S)-7-hydroxywarfarin quantitative electrochemical determination was used to calculate the kinetic parameters of CYP2C9 immobilized on electrode toward (S)-warfarin: the values of the maximal reaction rate (Vmax) and the Michaelis constant (KM) were calculated as 0.1 ± 0.002 min−1 and 3.03 ± 0.38 μM, respectively. The usage of an enzymatic CYP2C9-containing electrode as a catalyst and an indicator electrode as a sensor for the formation of metabolite allows to effectively register the kinetic parameters of the process. Such a two-electrode system can be applied to assess drug-drug interactions.

Controlling electrocatalytic, photoelectrocatalytic, and load release processes using soft material-modified electrodes

• Hemin/G-quadruplex hydrogel-modified electrode reveals triggered stiffness and switchable electrocatalysis. • Enzyme-loaded pH-responsive hydrogel-modified electrode show electroswitchable stiffness and load release functions. • An integrated photosystem I/glucose oxidase-modified electrode act as photoelectrochemical cell. Soft materials, such as hydrogels, polymers or biomaterials associated with electrodes provide interfaces revealing electrocatalytic, photoelectrocatalytic and controlled release of loads functions. These will be addressed by three examples demonstrated by our laboratory, including: (i) The assembly of a stimuli-responsive nucleic acid-based hydrogel on an electrode support, consisting of a hydrogel matrix crosslinked by duplex nucleic acid bridges and K + -ion-stabilized G-quadruplex bridges. By cyclic and reversible formation and dissociation of the G-quadruplexes by K + -ions and crown ether (CE), the switchable stiffness properties of the hydrogel are demonstrated. The integration of hemin into the G-quadruplex bridging units on electrocatalytic interface for the electrocatalyzed reduction of H 2 O 2 is introduced. In the presence of K + -ions/crown ether the switchable electrocatalytic functions of the electrode are demonstrated. (ii) A pH-responsive nucleic acid-based hydrogel matrix is immobilized on an electrode surface. The hydrogel interface is crosslinked by permanent nucleic acid bridges and pH-responsive crosslinking units. At acidic pH values, the pH responsive bridges are unlocked via generating i-motif structures, leading to an hydrogel interface of lower stiffness. Immobilization of ferrocene-modified glucose oxidase (GOx) in the hydrogel yields an electrically contacted enzyme electrode that stimulates the bioelectrocatalyzed oxidation of glucose and the acidification of the hydrogel. By incorporating a load into the hydrogel, the electrocatalyzed oxidation of glucose and the accompanying acidification of the hydrogel lead to a hydrogel of lower stiffness, allowing the controlled release of the load. (iii) A supramolecular, electrochemically contacted, photoresponsive matrix consisting of native photosystem I (PS I) interlinked to glucose oxidase (GOx) by an electrically contacting redox polymer consisting of polyvinyl imidazole Os 2+/3+ (bipyridine) 2 Cl complex is introduced. The organized assembly acts as a photobioelectrochemical fuel cell where photoinduced electron transfer from PS I to the electrode stimulates the bioelectrocatalyzed oxidation of glucose. The system demonstrates the light-induced oxidation of the glucose-fuel and the concomitant generation of electrical power.

An improved amperometric sarcosine biosensor based on graphene nanoribbon/chitosan nanocomposite for detection of prostate cancer

We describe herein an improved amperometric biosensor for detection of sarcosine, a potential biomarker for prostate cancer. The biosensor is based on covalent immobilization of sarcosine oxidase (SOx) onto nanocomposite of chitosan (CHIT) and graphene nanoribbons (GNRs) electrodeposited onto Au electrode. The GNRs were studied by transmission electron microscopy and UV spectroscopy. The working electrode (SOx/CHIT/GNRs/AuE) exhibited maximum current at a potential of 0.1V against Ag/AgCl, generated from electrochemical oxidation of H2O2, from sarcosine by immobilized SOx. The biosensor showed optimum response i.e. current (mA) within 2s at pH 7.3 and 35 ​°C. There was a linearity between current (mA) and sarcosine concentration in a wider range 0.001–100 ​μM with a minimum detection limit of 0.001 ​μM and a high sensitivity of 277.5 ​μA/μM/cm. Analytical recoveries of added sarcosine in sera were 97.35%, within and between-batch coefficients of variation were 1.08% and 1.40% respectively. A good correlation (R2 ​= ​0.99) was obtained between sera sarcosine values, as measured by the standard immuno kit method and present biosensor. The biosensor measured sarcosine levels in sera of prostate cancer patients, which was significantly higher than in apparently healthy persons. The enzyme electrode lost 20% of its initial activity during 180days, when stored dry at 4°C.

Transient potentiometry based d-serine sensor using engineered d-amino acid oxidase showing quasi-direct electron transfer property

d-Serine biosensing has been extensively reported based on enzyme sensors using flavin adenine dinucleotide (FAD) -dependent d-amino acid oxidase (DAAOx), based on the monitoring of hydrogen peroxide generated by the enzymatic reaction, which is affected by dissolved oxygen concentration in the measurement environment in in vivo use. Here we report a novel sensing principle for d-serine, transient potentiometry based d-serine sensor using engineered DAAOx showing quasi-direct electron transfer (DET) property. DAAOx Gly52Val mutant, revealed to possess dye-mediated dehydrogenase activity using artificial synthetic electron acceptors, while its oxidase activity was negligible. The enzyme was immobilized on electrode and was modified with amine-reactive phenazine ethosulfate, resulted an enzyme electrode showing quasi-DET type response. Although OCP based monitoring took more than several minutes to obtain steady state OCP value, the time dependent OCP change monitoring, transient potentiometry, provided rapid and sensitive sensor signals. While dOCP/dt based monitoring was suitable for sensing with longer than 5 s time resolution with d-serine concentration range between 0.5 mM and 5 mM, dOCP/d t based monitoring is suitable for d-serine monitoring with much shorter time resolution (less than 1 s) with high sensitivity with wider dynamic range (20 μM–30 mM). The maximum dOCP/d t was −39.2 ± 2.0 mV/s1/2, the Km(app) was 1.9 mM, and the lower limit of detection was 20 μM. In addition, d-serine monitoring was also possible in the artificial cerebrospinal fluid. The transient potentiometry based sensing reported in this study will be further utilized to realize miniaturized, continuous, real-time, in vivo sensor for d-serine monitoring.

Flexible and Stretchable Enzymatic Biofuel Cell with High Performance Enabled by Textile Electrodes and Polymer Hydrogel Electrolyte.

Biofuel cells with good biocompatibility are promising to be used as the power source for flexible and wearable bioelectronics. We here report a type of highly flexible and stretchable biofuel cells, which are enabled by textile electrodes of graphene/carbon nanotubes (G/CNTs) composite and polymer hydrogel electrolyte. The CNT array covalently grown from a graphene layer not only can be served as a conducting substrate to immobilize enzyme molecules but also can provide efficient charge transport channels between the enzyme and graphene electrode. As a result, the developed biofuel cells deliver a high open-circuit voltage of 0.65 V and output power density of 64.2 μW cm-2, which are much higher than previously reported results. Benefiting from the unique textile structure of electrodes and the polymer hydrogel electrolyte, the biofuel cells exhibit high retention of power density after 400 bending cycles and even stretched to a high strain of 60%.

Spider nest shaped multi-scale three-dimensional enzymatic electrodes for glucose/oxygen biofuel cells

A spider nest shaped multi-scale three-dimensional substrate consisting of reduced graphene oxide (RGO) and nickel foam is fabricated for enzymatic electrodes and biofuel cells. According to the excellent conductivity and large electroactive surface area of this special structure, the enzymatic electrodes show large enzymatic loading density, low electron transfer resistance and high electrocatalytic efficiency. The strong forces in the spider nest shaped structure and the excellent enzymatic embedding method ensure the stability of the glucose oxidase bioanodes and laccase biocathodes. The Michaelis–Menten constant value for the bioanodes to glucose is calculated to be 2.24 mM, which is close to the Michaelis–Menten constant for free glucose oxidase, implying a remarkably high enzymatic activity. Employed the obtained bioelectrodes with great properties, the relative glucose/oxygen biofuel cell has an open-circuit voltage of 0.70 V, with a high output power performance, and a maximum output power of 7.05 ± 0.05 mW cm−2, owe to the high enzyme loading and low electron transfer resistance of the electrode based on the spider nest shaped structure. After 60 days of periodic storage experiments, the performance of the biofuel cell still maintained 84.2%, showing a good long-term stability.

Thermoplastic Electrode (TPE)‐based Enzymatic Glucose Sensor Using Polycaprolactone‐graphite Composites

AbstractCarbon composite electrodes are frequently used for enzymatic electrodes but suffer from poor electrocatalytic activity and/or are single use. Our group has recently developed thermoplastic electrodes (TPEs) that use a thermopolymer binder. The electrodes can be easily molded without extensive use of organic solvents enabling catalysts and enzymes to be added directly to the electrode material. The TPE material is bulk‐modified with cobalt phthalocyanine and glucose oxidase, resulting in a robust sensor that is stable for over 100 minutes. These sensors can be molded into intricate shapes and sanded for surface renewal, allowing the sensors to be continuously reused.

Conducting Polymer Based Enzyme Electrodes Fabricated by Invertase and Polyphenol Oxidase

Novel carbon fiber enzyme electrodes were constructed and evaluated. Fabrication of electrodes was performed via electrochemical deposition of a conducting matrice composed of polythiophene and polypyrrole (PTh–PPy) onto carbon fiber substrates. The enzyme was entrapped into the matrix during electropolymerization. Resultant biosensors represented higher kinetic parameters, Vmax and Km, in comparison to PPy matrice, which are 2.471 ± 0.150 mol min1- electrode1- and 30.60 ± 5.30 mM for invertase, 0.056 ± 0.012 mol min1- electrode1- and 842.00 ± 37.50 mM for polyphenol oxidase respectively. Optimum pH and temperature of the immobilized enzyme within PTh–PPy composite indicates that this matrice provides a more protective environment. The detection limit (LOD) of polyphenols was obtained as 0.037 mg mL1-. Polyphenol oxidase enzyme electrodes were proved to be used for the determination of polyphenolic substances in real samples and the results were confirmed by the Folin-Ciocalteau method

Lignosulfonate-assisted synthesis of platinum nanoparticles deposited on multi-walled carbon nanotubes for biosensing of glucose

It is presented in this work that lignosulfonate (LS) can be preferentially adsorbed on the surface of multi-walled carbon nanotubes (MWCNT) giving rise to the functional platform for platinum nanoparticles (NPt) deposition. The novel MWCNT/LS/NPt hybrid material has been characterized by X-ray diffraction (XRD), Fourier transformation infrared spectroscopy (FT-IR), X-ray photoelectron spectroscopy (XPS). The morphology of the MWCNT/LS/NPt electrodes has been investigated by atomic force microscopy (AFM). The electrochemical studies of MWCNT/LS/NPt hybrid material revealed strong electrocatalytic properties towards hydrogen peroxide. In addition, the effects of lignosulfonate amount adsorbed at the MWCNT on the voltammetric response of the hydrogen peroxide were discussed and used to select the optimal and effective conditions for the synthesis of the electrode material. An amperometric biosensor for glucose was developed based on the covalent linkage of glucose oxidase (GOx) at the MWCNT/LS/NPt. The enzyme electrode exhibited excellent electrocatalytic activity and rapid response for glucose. The linear range of the glucose determination was 50–1400 µM and LOD was quantified as 15.67 µM.

Bioelectrochemistry in Undergraduate Curriculum and Research

Electrochemistry has a wide range of uses and applications, but undergraduate students do not appreciate this when the topic is first introduced in General Chemistry. Introducing more electrochemical methods in later chemistry courses can encourage interest in the field. In the chemistry department at Lebanon Valley College, the students first encounter electrochemistry through the typical introduction of redox reactions, the Nernst equation, and galvanic cells. The topic is expanded in Analytical Chemistry for third years during which the students learn about and perform electroanalytical methods, including potentiometric analysis, coulometry, amperometry, and voltammetry. Students who choose the advanced elective Chemical Sensors and Biosensors learn about electrochemical sensors as one unit of the course. This unit also includes a section on bioelectrochemical sensors. Students develop a hydrogen peroxide biosensor and use amperometry to evaluate its performance, giving them a better understanding of concepts like limit of detection, sensitivity, and dynamic linear range.In addition to the chemistry curriculum, students can choose to do electrochemical research in the department. Recent projects include fabricating and evaluating enzyme electrodes for a wide range of biologically relevant molecules, designing self-powered biosensors, and designing a biophotovoltaic device for solar energy conversion with plants. We also currently have a research project with the focus of developing an electrosynthesis experiment to be added to the organic lab sequence. Throughout these research projects, students gain experience with several electrochemical methods, analysis of electrochemical data, and general lab skills. They are also required to present their work, either in posters or oral presentations, a skill that will prepare them for their careers after college. Overall this combination of curriculum and research leads to more students being interested in electrochemistry. They learn that these methods can be used for every area of chemistry, not just analytical. It has lead to an increase in the number of graduating students who continue working in the field, whether in industry or academia.

Исследование влияния физико-химических факторов на активность работы модифицированных ферментных электродов

To modify graphite electrodes with a conductive enzyme polymer matrix, the method of drip application of a liquid polymer solution of various compositions was used: polyvinylpyrrolidone (40%), chitosan (0.1%), glutaric dialdehyde (0.1%), glucoxidase and peroxidase in a ratio of 2:5. To assess the effect of the immobilization time (x1), the pH of immobilization (x2) and the enzyme/carrier ratio (x3) on the activity of the enzyme electrodes, a three-factor and three-level Box-Benken and RSM design was used. This model was able to adequately predict the results of immobilization within the range of variables used. The most favorable conditions and the largest number of molecules of the enzyme complex are in the electrochemically active state when they are immobilized on PVP using modifying agents chitosan and glutaraldehyde. The results demonstrate that the productivity of the enzymatic biofuel element is directly proportional to the activity of the immobilized GOX/HRP complex, since in this case the glucose oxidation reaction can proceed more efficiently.