RESUMEN

Enzyme-modified electrodes are core components of electrochemical biosensors for diagnostic and environmental analytics and have promising applications in bioelectrocatalysis. Despite huge research efforts spanning decades, design of enzyme electrodes for superior performance remains challenging. Nanoporous gold (npAu) represents advanced electrode material due to high surface-to-volume ratio, tunable porosity, and intrinsic redox activity, yet its coupling with enzyme catalysis is complex. Here, the study reports a flexible-modular approach to modify npAu with functional enzymes by combined material and protein engineering and use a tailored assortment of surface and in-solution methodologies for characterization. Self-assembled monolayer (SAM) of mercaptoethanesulfonic acid primes the npAu surface for electrostatic adsorption of the target enzyme (flavocytochrome P450 BM3; CYT102A1) that is specially equipped with a cationic protein module for directed binding to anionic surfaces. Modulation of the SAM surface charge is achieved by electrochemistry. The electrode-adsorbed enzyme retains well the activity (33%) and selectivity (complete) from in-solution. Electrochemically triggered nanoscale stirring in the internal porous network of npAu-SAM enhances speed (2.5-fold) and yield (3.0-fold) of the enzyme immobilization. Biocatalytic reaction is fueled from the electrode via regeneration of its reduced coenzyme (NADPH). Collectively, the study presents a modular design of npAu-based enzyme electrode that can support flexible bioelectrochemistry applications.

RESUMEN

The convenient and sensitive detection of metabolites is of great significance for understanding human health status and drug development. Solid-phase electrochemiluminescence (ECL) enzyme electrodes show great potential in metabolite detection based on the enzyme-catalyzed reaction product hydrogen peroxide (H2O2). Herein, a solid-phase ECL enzyme sensor was fabricated based on a confined emitter and an immobilized enzyme using electrostatic nanocage array, constructing a platform for the sensitive detection of cholesterol. The electrostatic cage nanochannel consists of a bipolar and bilayer vertically aligned mesoporous silica film (bp-VMSF). The upper layer of bp-VMSF is an amino-modified, positively charged VMSF (p-VMSF), and the lower layer is a negatively charged VMSF (n-VMSF). The most commonly used ECL probe tris(bipyridine)ruthenium(II) (Ru(bpy)32+) is fixed in n-VMSF by electrostatic adsorption from n-VMSF and electrostatic repulsion from the upper p-VMSF, generating significantly enhanced and stable ECL signals. The successful preparation of the electrostatic cage was characterized by scanning electron microscopy (SEM) and electrochemical methods. After amino groups on the outer surface of bp-VMSF were derivatized with aldehyde, cholesterol oxidase (ChOx) molecules were covalently immobilized. The successful construction of the enzyme electrode was characterized by cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). When the corresponding enzyme substrate, cholesterol, was present in the solution, the ECL signal of Ru(bpy)32+ was quenched by the enzyme-catalyzed reaction product H2O2, enabling the high-sensitivity detection of cholesterol. The linear range for detecting cholesterol was from 0.05 mM to 5.0 mM, with a limit of detection (LOD) of 1.5 µM.

Asunto(s)

RESUMEN

Assembling functional molecules on the surface of an enzyme electrode is the most basic technique for constructing a biosensor. However, precise control of electron transfer interface or electron mediator on the electrode surface remains a challenge, which is a key step that affects the stability and sensitivity of enzyme-based biosensors. In this study, we propose the use of controllable free radical polymerization to grow stable 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO) polymer as electron mediator on enzyme surface for the first time. Through scanning electron microscopy (SEM), Raman spectroscopy, electrode surface coverage measurement, static contact angle (SCA), and a series of electrochemical methods, it has been demonstrated that the TEMPO-based enzyme electrode exhibits a uniform hydrophilic morphology and stable electrochemical performance. Furthermore, the results show that the sensor demonstrates high sensitivity for detecting glucose biomolecules in artificial sweat and serum. Attributing to the quantitative and controllable radical polymerization of TEMPO redox assembled enzyme electrode surface, the as-proposed biosensor providing a use, storage, and inter-batch sensing stability, providing a vital platform for wearable/implantable biochemical sensors.

Asunto(s)

RESUMEN

The accomplishment of concurrent interenzyme chain reaction and direct electric communication in a multienzyme-electrode is challenging since the required condition of multienzymatic binding conformation is quite complex. In this study, an enzyme cascade-induced bioelectrocatalytic system has been constructed using solid binding peptide (SBP) as a molecular binder that coimmobilizes the invertase (INV) and flavin adenine dinucleotide (FAD)-dependent glucose dehydrogenase gamma-alpha complex (GDHγα) cascade system on a single electrode surface. The SBP-fused enzyme cascade was strategically designed to induce diverse relative orientations of coupling enzymes while enabling efficient direct electron transfer (DET) at the FAD cofactor of GDHγα and the electrode interface. The interenzyme relative orientation was found to determine the intermediate delivery route and affect overall chain reaction efficiency. Moreover, interfacial DET between the fusion GDHγα and the electrode was altered by the binding conformation of the coimmobilized enzyme and fusion INVs. Collectively, this work emphasizes the importance of interenzyme orientation when incorporating enzymatic cascade in an electrocatalytic system and demonstrates the efficacy of SBP fusion technology as a generic tool for developing cascade-induced direct bioelectrocatalytic systems. The proposed approach is applicable to enzyme cascade-based bioelectronics such as biofuel cells, biosensors, and bioeletrosynthetic systems utilizing or producing complex biomolecules.

Asunto(s)

RESUMEN

Viruses have unique coat proteins that are genetically modifiable. Their surface can serve as a nano-template on which electroactive molecules are immobilized. In this study, we report filamentous bacteriophage as a backbone to which redox mediators are covalently and densely tethered, constructing redox nanowire, i.e. an electron conducting biomaterial. The highly ordered coat proteins of a filamentous bacteriophage provide flexible and biocompatible platform to constitute a biohybrid redox nanowire. Incorporating bacteriophage and redox molecules form an entangled assembly of nanowires enabling facile electron transfer. Electron transfer among the molecular mediators in the entangled assembly originates apparent electron diffusion of which the electron transfer rate is comparable to that observed in conventional redox polymers. Programming peptide terminals suggests further enhancement in electron mediation by increasing redox species mobility. In addition, the redox nanowire film functions as a favorable matrix for enzyme encapsulation. The stability of the enzymes entrapped in this unique matrix is substantially improved.

Asunto(s)

RESUMEN

Real-time sweat monitoring is vital for athletes in order to reflect their physical conditions, quantify their exercise loads, and evaluate their training results. Therefore, a multi-modal sweat sensing system with a patch-relay-host topology was developed, which consisted of a wireless sensor patch, a wireless data relay, and a host controller. The wireless sensor patch can monitor the lactate, glucose, K+, and Na+ concentrations in real-time. The data is forwarded via a wireless data relay through Near Field Communication (NFC) and Bluetooth Low Energy (BLE) technology and it is finally available on the host controller. Meanwhile, existing enzyme sensors in sweat-based wearable sports monitoring systems have limited sensitivities. To improve their sensitivities, this paper proposes a dual enzyme sensing optimization strategy and demonstrates Laser-Induced Graphene (LIG)-based sweat sensors decorated with Single-Walled Carbon Nanotubes (SWCNT). Manufacturing an entire LIG array takes less than one minute and costs about 0.11 yuan in materials, making it suitable for mass production. The in vitro test result showed sensitivities of 0.53 µA/mM and 3.9 µA/mM for lactate and glucose sensing, and 32.5 mV/decade and 33.2 mV/decade for K+ and Na+ sensing, respectively. To demonstrate the ability to characterize personal physical fitness, an ex vivo sweat analysis test was also performed. Overall, the high-sensitivity lactate enzyme sensor based on SWCNT/LIG can meet the requirements of sweat-based wearable sports monitoring systems.

Asunto(s)

RESUMEN

This study successfully created a portable acetylcholinesterase sensor on a printed hybrid electrode capable of detecting chlorpyrifos in the field. While a screen-printed electrode was chosen herein to enable a single-use and portable platform for the in-field application, the hybrid material was incorporated to ensure ultrasensitive detection at lower electrode potentials. The hybrid ink of gold nanoparticles (AuNPs) decorated on graphene (GP) sheets in poly(3,4-ethylenedioxythiophene):poly(styrenesulfonate) (PEDOT:PSS) was synthesized through a simple completely-green one-pot process. The subsequent characterization was carried out via transmission electron microscopy (TEM), X-ray diffraction (XRD), scanning electron microscopy (SEM), and Fourier-transform infrared spectroscopy (FTIR). The synergy resulting from the greater surface area and enhanced transfer of electrons combined with high levels of electrocatalytic activity and superb conductivity offered by GP, AuNP, and PEDOT:PSS allows the sensor to exhibit ultrasensitive chlorpyrifos detection at the relatively low detection limit of 0.07 nM. The sensor demonstrated in this study also exhibits good reproducibility, desirable stability, and a successful application for the real sample with satisfactory recovery results of around 106 %, indicating its potential for use as a tool in the analysis of pesticides.

Asunto(s)

RESUMEN

The demand for the ubiquitous detection of gases in complex environments is driving the design of highly specific gas sensors for the development of the Internet of Things, such as indoor air quality testing, human exhaled disease detection, monitoring gas emissions, etc. The interaction between analytes and bioreceptors can described as a "lock-and-key", in which the specific catalysis between enzymes and gas molecules provides a new paradigm for the construction of high-sensitivity and -specificity gas sensors. The electrochemical method has been widely used in gas detection and in the design and construction of enzyme-based electrochemical gas sensors, in which the specificity of an enzyme to a substrate is determined by a specific functional domain or recognition interface, which is the active site of the enzyme that can specifically catalyze the gas reaction, and the electrode-solution interface, where the chemical reaction occurs, respectively. As a result, the engineering design of the enzyme electrode interface is crucial in the process of designing and constructing enzyme-based electrochemical gas sensors. In this review, we summarize the design of enzyme-based electrochemical gas sensors. We particularly focus on the main concepts of enzyme electrodes and the selection and design of materials, as well as the immobilization of enzymes and construction methods. Furthermore, we discuss the fundamental factors that affect electron transfer at the enzyme electrode interface for electrochemical gas sensors and the challenges and opportunities related to the design and construction of these sensors.

Asunto(s)

RESUMEN

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.

RESUMEN

Enzyme electrode biosensor blood glucose detector is a new rapid blood glucose measurement method. There is no corresponding product standard and industry standard reference at home and abroad, and there is a lack of corresponding reference experience in performance research and clinical evaluation. This study discusses the safety and effectiveness of enzyme electrode biosensor blood glucose detector from the perspective of technical evaluation of medical devices. It is expected to provide some reference for medical device registration technical reviewers and registration application enterprises.

Asunto(s)

RESUMEN

A biosensor based on the release of the enzyme substrate from its structure was developed for the inhibitive detection of benzoic acid. A polyurethane support comprising two perforated microcapsules (800 µm in diameter) filled with methylene blue as a model compound and covered with a conductive deposit of multiwalled carbon nanotubes, continuously released this stored dye for 24 h. An increase in methylene blue concentration of 0.5-0.75 µmol L-1 h-1 and 1.5-2 µmol L-1 h-1, in the presence and absence of the multiwalled carbon nanotube coating, respectively, was demonstrated by UV-vis spectroscopy in a 2 mL UV cuvette. The same configuration with microcapsules filled with catechol was modified by a laponite clay coating containing tyrosinase enzyme. The resulting biosensor exhibits a constant cathodic current at -0.155 V vs AgCl/Ag, due to the reduction of the ortho-quinone produced enzymatically from the released catechol. The detection of benzoic acid was recorded from the decrease in cathodic current due to its inhibiting action on the tyrosinase activity. Reagentless biosensors based on different deposited quantity of tyrosinase (100, 200, 400 and 600 µg) were investigated for the detection of catechol and applied to the detection of benzoic acid as inhibitor. The best performance was obtained with the 400 µg-based configuration, namely a detection limit of 0.4 µmol L-1 and a sensitivity of 228 mA L mol-1. After the inhibition process, the biosensors recover 97-100% of their activity towards catechol, confirming a reversible inhibition by benzoic acid.

Asunto(s)

RESUMEN

Wearable devices that generate power using sweat have garnered much attention in the field of skin electronics. These devices require high performance with a small volume and low production rate of sweat by living organisms. Here we demonstrate a high-power biofuel cell bracelet based on the lactate in human sweat. The biofuel cell was developed by using a lactate oxidase/osmium-based mediator/carbon nanotube fiber for lactate oxidation and a bilirubin oxidase/carbon nanotube fiber for oxygen reduction; the fibers were woven into a hydrophilic supportive textile for sweat storage. The storage textile was sandwiched between a hydrophobic textile for sweat absorption from the skin and a hydrophilic textile for water evaporation to improve sweat collection. The performance of the layered cell was 74 µW at 0.39 V in 20 mM artificial sweat lactate, and its performance was maintained at over 80% for 12 h. Furthermore, we demonstrated a series-connection between anode/cathode fibers by tying them up to wrap the bracelet-type biofuel cell on the wrist. The booster six-cell bracelet generated power at 2.0 V that is sufficient for operating digital wrist watches.

Asunto(s)

RESUMEN

Biodevices in which biomolecules such as enzymes and antibodies are immobilized on the surface of electrode materials are capable of converting chemical energy into electrical energy, and are expected to contribute to solving energy problems and developing medical measurements especially as biobatteries and biosensors. Device performance depends on the interface formed between the biomolecule layer and electrode material, and the interface is required to simultaneously achieve a highly efficient enzymatic reaction and electron transfer. However, when enzymes were immobilized on a material surface, the enzymes undergoes a structural change due to the interaction between the enzyme and the electrode surface, making it difficult to maximize the function of the enzyme molecule on the material surface. In this study, we postulate that the structural change of the enzyme would be reduced and the electrochemical performance improved by making the contact area between the enzyme and the electrode extremely small and adsorbing it as a point. Therefore, we aimed to develop a high-power biodevice that retains enzyme structure and activity by interposing gold nanoparticles (AuNPs) between the enzyme and the electrode. The enzymatic and electrochemical properties of pyrroloquinoline quinone-dependent glucose dehydrogenase adsorbed on AuNPs of 5-40 nm diameter were investigated. We found that the characteristics differed among the particles, and the enzyme adsorbed on 20 nm AuNPs showed the best electrochemical characteristics.

Asunto(s)

RESUMEN

Fungi-derived flavin adenine dinucleotide (FAD)-dependent glucose dehydrogenases (FADGDHs) are the most popular and advanced enzymes for SMBG sensors because of their high substrate specificity toward glucose and oxygen insensitivity. However, this type of FADGDH hardly shows direct electron transfer (DET) ability. In this study, we developed a new DET-type FADGDH by harboring Cytochrome b562 (cyt b562) derived from Escherichia coli as the electron transfer domain. The structural genes encoding fusion enzymes composed of cyt b562 at either the N- or C-terminus of fungal FADGDH, (cyt b562-GDH or GDH-cyt b562), were constructed, recombinantly expressed, and characteristics of the fusion proteins were investigated. Both constructed fusion enzymes were successfully expressed in E. coli, as the soluble and GDH active proteins, showing cyt b562 specific redox properties. Thusconstructed fusion proteins showed internal electron transfer between FAD in FADGDH and fused cyt b562. Consequently, both cyt b562-GDH and GDH-cyt b562 showed DET abilities toward electrode. Interestingly, cyt b562-GDH showed much rapid internal electron transfer and higher DET ability than GDH-cyt b562. Thus, we demonstrated the construction and production of a new DET-type FADGDH using E.coli as the host cells, which is advantageous for future industrial application and further engineering.

Asunto(s)

RESUMEN

Carbon-based nanoelectrodes fabricated by means of pyrolysis of an alkane precursor gas purged through a glass capillary and subsequently etched with HF were modified with redox polymer/enzyme films for the detection of glucose at the single-cell level. Glucose oxidase (GOx) was immobilized and electrically wired by means of an Os-complex-modified redox polymer in a sequential dip coating process. For the synthesis of the redox polymer matrix, a poly(1-vinylimidazole-co-acrylamide)-based backbone was used that was first modified with the electron transfer mediator [Os(bpy)2Cl]+ (bpy = 2,2'-bipyridine) followed by the conversion of the amide groups within the acrylamide monomer into hydrazide groups in a polymer-analogue reaction. The hydrazide groups react readily with bifunctional epoxide-based crosslinkers ensuring high film stability. Insertion of the nanometre-sized polymer/enzyme modified electrodes into adherently growing single NG108-15 cells resulted in a positive current response correlating with the intracellular glucose concentration. Moreover, the nanosensors showed a stable current output without significant loss in performance after intracellular measurements.

Asunto(s)

RESUMEN

Stability of glucose-oxidising enzyme electrodes is affected by substances in physiological solutions, hampering deployment as long-term implantable biosensors or fuel cells. The performance of Nafion over-coated enzyme electrodes, consisting of multiwalled carbon nanotubes and flavin adenine dinucleotide-dependent glucose dehydrogenase (FADGDH) or glucose oxidase (GOx) crosslinked with osmium-complex based redox polymer, was compared to uncoated electrodes in presence of uric acid and artificial plasma. Nafion over-coating resulted in lower glucose oxidation current densities compared to no over-coating. The highest initial current density for Nafion over-coated electrodes in artificial plasma in 100 mM glucose was 8.0 ± 2.0 mA cm-2 for GOx electrodes with 0.5% w/v Nafion coating. These electrodes retained 83% of initial current after 12 h continuous operation in artificial plasma while similarly prepared FADGDH electrodes retained 58% signal. This is compared to retention of only 73% or 31% observed for GOx or FADGDH electrodes in artificial plasma with no Nafion membrane. Enzyme electrodes over-coated with Nafion maintain improved signal stability when tested continuously in the presence of uric acid, identified as being the main contributing substance to FADGDH enzyme electrode instability, showing promise for application to continuous use glucose-oxidising enzyme electrodes.

Asunto(s)

RESUMEN

Enzymatic electrolysis cell (EEC) has advantages over microbial electrolysis cell (MEC) due to the needless of microbe inoculation and high-efficiency of enzymatic reaction. In this study, an EEC was first applied to achieve the effective degradation of halogenated organic pollutants and dichloromethane (CH2Cl2) was utilized as a model pollutant. The results indicate that the degradation efficiency of CH2Cl2 after 2 hr reaction in the EEC was almost 100%, which was significantly higher than that with enzyme (51.1%) or current (19.0%). The current induced the continuous regeneration of reduced glutathione (GSH), thus CH2Cl2 was degraded under the catalysis of GSH-dependent dehalogenase through stepwise dechlorination, and successively formed monochloromethane (CH3Cl) and methane (CH4). The kinetic result shows that with a current of 15 mA, the maximum specific degradation rate of CH2Cl2 (3.77 × 10-3hr-1) was increased by 5.7 times. The optimum condition for CH2Cl2 dechlorination was also obtained with pH, current and temperature of 7.0, 15 mA and 35°C, respectively. Importantly, this study helps to understand the behavior of enzymes and the fate of halogenated organic pollutants with EEC, providing a possible treatment technology for halogenated organic pollutants.

Asunto(s)

RESUMEN

A novel and highly stable biomimetic oxidase sensor system was designed for catehol detection. FePP used as biomimetic horseradish peroxidase (HRP) was immobilized onto modified multi-walled carbon nanotubes (MWCNTs). Functional groups such as -OH, -NH2 and -COOH were introduced onto the surface of MWCNTs to provide biomimetic microenvironment for iron porphyrins (FePP). Stable biomimetic enzyme electrode has been developed to detect catechol as a simple, economical and efficient method. At optimal condition, the detection limit of OH-MWCNTs/FePP/Nafion was 3.754 × 10- 6 M. After stored at - 4 °C for 35 days, the oxidation current value still maintained 98.3% of initial activity. In repetitive nature test, relative standard deviation (RSD) of oxidation current remained within 1.0% after ten consecutive measurements in the same concentration of catechol solution, while most of reported oxidase sensor was within 2.0% under the same condition.

Asunto(s)

RESUMEN

We present the plasmonic fiber based optical glucometer. A thin gold layer is coated on clad-free core of multimode optical fiber along 3 cm length to excite surface plasmons at 632.8 nm wavelength. Glucose oxidase is immobilized on the metal surface for glucose sensing. The effective surface refractive index increases by gluconic acid and hydrogen peroxide that are generated upon glucose injection, leading to plasmonic condition change with a consequence of optical power change at the fiber output. We obtain limit of detection of glucose concentration of 6.75 mg/dL, indicating higher sensitivity than the wavelength interrogating SPR glucometer that uses a spectrometer of 1nm spectral resolution. The coefficient of variation is 8.6% at a glucose concentration of 80 mg/dL at room temperature. We also examine the effects of ambient temperature variations from -10 °C to 40 °C on the performance of the presented sensor and compared them with those on commercially available glucometers that are based on enzyme electrodes. We find that the presented fiber sensor produced standard deviation of 12.1 mg/dL at a glucose concentration of 80 mg/dL under such varying temperature, which is, even without additional temperature correction function, comparable to the commercialized ones.

RESUMEN

Enzymes are highly specific and selective due to their precise, intricate three-dimensional catalytic- structure. Electron transfer in enzymes normally occurs through an active-metal centers or tunneling events that are highly insulated by the surrounding globular protein structure. In case of electrochemically active enzymes/proteins, the distance between the redox-active cofactor and the electrode surface plays key role during direct communication. Therefore, the long electron-tunneling distance can be overcome by introducing mobile redox mediators such as nanostructures specially nanowires which can diffuse into and out of the enzyme active site, ferrying reducing or oxidizing equivalents with them. Therefore, nanowire-conjugated enzymes have gained great interest in the development of biosensor devices and other electrocatalytic-biological applications. Herein we present a comprehensive review about the electrochemical enzyme-based sensor using nanowires. Over the past decade, nanowires were investigated as a versatile platform for various applications including sensors and biosensors because of their high aspect ratio and a high surface-to-volume ratio. This review aimed to summarize some of the recent developments in the enzyme based sensor research that have been achieved with various metallic and non-metallic one-dimensional nanostructure i.e. nanowires. Due to low or no toxicity and biocompatibility, enzymes conjugated with nanowires are still highly specific, sensitive and biologically active. This review demonstrates the potential usability of nanowired-enzymes for the bioanalytical applications. The review includes various types of nanowires, mode of the enzyme integration or immobilization methodologies, probe modification, biosensor fabrication and real or spiked sample testing. Biosensor parameters such as linear range and sensitivity, selectivity and detection limit of reported sensors were also considered herein. We also introduce some of the new nanowire materials which have not yet been used for biosensing or biosensor application. The limitations, challenges and prospects for the use of nanowired-enzymes in electrochemical and other real-time sensing systems as well as fabrication technologies are also discussed in this review.

Asunto(s)