Pentacyanoammineferrate-Based Non-Enzymatic Electrochemical Biosensing Platform for Selective Uric Acid Measurement.

The electrochemical-based detection of uric acid (UA) is widely used for diagnostic purposes. However, various interfering species such as ascorbic acid, dopamine, and glucose can affect electrochemical signals, and hence there is an outstanding need to develop improved sensing platforms to detect UA with high selectivity. Herein, we report a pentagonal mediator-based non-enzymatic electrochemical biosensing platform to selectively measure UA in the presence of interfering species. The working electrode was fabricated by electrodepositing polymerized 1-vinylimidazole (PVI), which has an imidazole ligand, onto indium tin oxide (ITO), and then conjugating nickel ions to the PVI-coated ITO electrode. Electrode performance was characterized by cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) measurements and integrated together with pentacyanoammineferrate, which can bind to the amine groups of UA and function as an electron transferring mediator. The experimental results showed a wide linear range of UA concentration-dependent responses and the multi-potential step (MPS) technique facilitated selective detection of UA in the presence of physiologically relevant interfering species. Altogether, these findings support that pentacyanoammineferrate-based non-enzymatic electrodes are suitable biosensing platforms for the selective measurement of UA, and such approaches could potentially be extended to other bioanalytes as well.

Stretchable Fiber Biofuel Cell by Rewrapping Multiwalled Carbon Nanotube Sheets.

The fiber-type biofuel cell is attractive as an implantable energy source because the fiber can modify various structures and the wound can be stitched like a suture. In addition, in daily life, the biofuel cell is forced by human motion, and stretchability is a critical requirement for real applications. Therefore, we introduce a new type of highly stretchable, stable, soft fiber biofuel cell with microdiameter dimensions as an energy harvester. The completed biofuel cell operated well in fluids similar to human fluids, such as 20 mM phosphate-buffered 0.14 M NaCl solution (39.5 mW/cm2) and human serum (36.6 µW/cm2). The fiber-type biofuel cell can be reversibly stretched up to 100% in tensile direction while producing sustainable electrical power. In addition, the unique rewrapping structure, which traps the enzyme between multiwalled carbon nanotube sheets, enormously enhanced the stability of the biofuel cell when the biofuel cell was repeatedly stretched (the power density retention increased from 63 to 99%) and operated in human serum (the power density retention increased from 29 to 86%). The fiber can be easily woven into various structures, such as McKibben braid yarn, and scaled up by series and parallel connections.

A Simple Interfacial Platform for Homogeneous Electrochemical Immunoassays Using a Poly(Vinylimidazole)-Modified Electrode.

In this study, a homogeneous method featuring simple, one-step detection was developed to analyze hippuric acid (HA), a major metabolite of toluene. High sensitivity was achieved with the facile immobilization of poly(vinylimidazole) (PVI) on an indium tin oxide (ITO) electrode. Using a previously developed approach, pentacyanoferrate was coordinated with pyridyl-N ligands, and the redox-active Fe(II/III) centers were bound to Ni(II) ions on the electrode via electrostatic cyanide bridges. The detection was accomplished by the competitive binding of free HA and pentacyanoferrate-(4-aminomethylpyridine-hippuric acid) (Fe-HA, the electron transfer mediator) to the HA antibody on the Ni(II) ions-modified PVI-ITO (Ni-PVI-ITO) electrode. The electrical and physicochemical characterization of the electrode was carried out by cyclic voltammetry, differential pulse voltammetry, field emission scanning electron microscopy, and X-ray photoelectron spectroscopy. At low mediator concentrations, the electrical signals were proportional to the HA concentration between 0.1 µg/mL and 1.0 mg/mL. The same method may be extended to other small organic molecules.

Disposable Non-Enzymatic Glucose Sensors Using Screen-Printed Nickel/Carbon Composites on Indium Tin Oxide Electrodes.

Disposable screen-printed nickel/carbon composites on indium tin oxide (ITO) electrodes (DSPNCE) were developed for the detection of glucose without enzymes. The DSPNCE were prepared by screen-printing the ITO substrate with a 50 wt% nickel/carbon composite, followed by curing at 400 °C for 30 min. The redox couple of Ni(OH)2/NiOOH was deposited on the surface of the electrodes via cyclic voltammetry (CV), scanning from 0-1.5 V for 30 cycles in 0.1 M NaOH solution. The DSPNCE were characterized by field-emission scanning electron microscopy (FE-SEM), X-ray photoelectron spectroscopy (XPS), and electrochemical methods. The resulting electrical currents, measured by CV and chronoamperometry at 0.65 V vs. Ag/AgCl, showed a good linear response with glucose concentrations from 1.0-10 mM. Also, the prepared electrodes showed no interference from common physiologic interferents such as uric acid (UA) or ascorbic acid (AA). Therefore, this approach allowed the development of a simple, disposable glucose biosensor.

Heterogeneous electrochemical immunoassay of hippuric acid on the electrodeposited organic films.

By directly coordinating hippuric acid (HA) to the ferrate (Fe) as an electron transfer mediator, we synthesized a Fe-HA complex, which shows a good electrochemical signal and thus enables the electrochemical immunoanalysis for HA. We electrodeposited organic films containing imidazole groups on the electrode surface and then bonded Ni ion (positive charge) to induce immobilization of Fe-HA (negative charge) through the electrostatic interaction. The heterogeneous competitive immunoassay system relies on the interaction between immobilized Fe-HA antigen conjugate and free HA antigen to its antibody (anti-HA). The electric signal becomes weaker due to the hindered electron transfer reaction when a large-sized HA antibody is bound onto the Fe-HA. However, in the presence of HA, the electric signal increases because free HA competitively reacts with the HA antibody prior to actual reaction and thus prevents the HA antibody from interacting with Fe-HA at the electrode surface. This competition reaction enabled an electrochemical quantitative analysis of HA concentration with a detection limit of 0.5 µg mL(-1), and thus allowed us to develop a simple and rapid electrochemical immunosensor.

Homogeneous electrochemical detection of hippuric acid in urine based on the osmium-antigen conjugate.

A homogeneous electrochemical immunoassay is based on the interaction of osmium-antigen conjugate with its antibody. The novelty presented herein is the direct conjugation of the osmium complex to a small antigen and the application of the quantitative analysis of the antigen and its antibody as the electrical signal for homogeneous immunoassay. The small antigen chosen is hippuric acid (HA), a major urinary metabolite in toluene-exposed humans. As a redox mediator, [Os(4,4'-dimethoxy-2,2'-bipyridine)2(4-aminomethylpyridine-HA)Cl](+/2+) (Os-HA antigen) has been synthesized and characterized on screen-printed carbon electrodes. The synthesized Os-HA antigen shows reversible redox peaks at E(½)=0.056 V versus Ag/AgCl. The homogeneous competitive immunoassay relies on the interaction between Os-HA antigen conjugate and free antigen to its antibody, which can generate electrical signals linearly proportional to the free antigen monitored by cyclic voltammetry and differential pulse voltammetry in the range of 10 µg mL(-1) to 5.12 mg mL(-1). The cutoff concentration of HA in urine samples is 2.0 mg mL(-1), so the method can be used to develop a HA immunosensor. Moreover, the proposed homogeneous electrochemical immunoassay method can be applied to detect low concentrations of small antigens found in the healthcare area.

Development of a Glucose Sensor Based on Glucose Dehydrogenase Using Polydopamine-Functionalized Nanotubes.

The electrochemical-based detection of glucose is widely used for diagnostic purposes and is mediated by enzyme-mediated signal transduction mechanisms. For such applications, recent attention has focused on utilizing the oxygen-insensitive glucose dehydrogenase (GDH) enzyme in place of the glucose oxidase (GOx) enzyme, which is sensitive to oxygen levels. Currently used Ru-based redox mediators mainly work with GOx, while Ru(dmo-bpy)2Cl2 has been proposed as a promising mediator that works with GDH. However, there remains an outstanding need to improve Ru(dmo-bpy)2Cl2 attachment to electrode surfaces. Herein, we report the use of polydopamine-functionalized multi-walled carbon nanotubes (PDA-MWCNTs) to effectively attach Ru(dmo-bpy)2Cl2 and GDH onto screen-printed carbon electrodes (SPCEs) without requiring a cross-linker. PDA-MWCNTs were characterized by Fourier transform infrared (FT-IR) spectroscopy, Raman spectroscopy, and thermal gravimetric analysis (TGA), while the fabrication and optimization of Ru(dmo-bpy)2Cl2/PDA-MWCNT/SPCEs were characterized by cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) measurements. The experimental results demonstrate a wide linear range of glucose-concentration-dependent responses and the multi-potential step (MPS) technique facilitated the selective detection of glucose in the presence of physiologically relevant interfering species, as well as in biological fluids (e.g., serum). The ease of device fabrication and high detection performance demonstrate a viable pathway to develop glucose sensors based on the GDH enzyme and Ru(dmo-bpy)2Cl2 redox mediator and the sensing strategy is potentially extendable to other bioanalytes as well.

Motility enhancement of human spermatozoa using electrical stimulation in the nano-Ampere range with enzymatic biofuel cells.

Sperm motility is a crucial factor for normal fertilisation that is partly supported by mitochondrial activity. Enzymatic biofuel cells (EBFCs) generate electric currents by an electron grade from anodic to cathodic electrodes in a culture media. We demonstrate that electrical stimulation by EBFC at the nano-Ampere range enhances sperm motility that can potentially allow the development of a new therapeutic tool for male infertility, including poor motility. EBFC was set up with three different electrical currents (112 nA/cm2 and 250 nA/cm2) at two different times (1 h, 2 h). Each sample was evaluated for its motility by computer-assisted sperm analyses and sperm viability testing. In the expanded study, we used the optimal electrical current of the EBFC system to treat asthenozoospermia and sperm with 0% motility. Results showed that optimal electrical stimulation schemes with EBFCs enhanced sperm motility by 30-40% compared with controls. Activated spermatozoa led to tyrosine phosphorylation in the tail area of the sperm following the electrical stimulation in the nano-Ampere range. However, the electrically stimulated group did not exhibit increased acrosomal reaction rates compared with the control group. In cases related to asthenozoospermia, 40% of motility was recovered following the electrical stimulation at the nano-Ampere range. However, motility is not recovered in sperm with 0% motility. In conclusion, we found that sperm motility was enhanced by exposure to electrical currents in the nano-Ampere range induced by optimal EBFCs. Electrical stimulation enhanced the motility of the sperm though tyrosine phosphorylation in spermatozoa. Therefore, our results show that electrical currents in the nano-Ampere range can be potentially applied to male infertility therapy as enhancers of sperm motility in assisted reproductive technology.

Two-Ply Carbon Nanotube Fiber-Typed Enzymatic Biofuel Cell Implanted in Mice.

Implantable devices have emerged as a promising industry. It is inevitable that these devices will require a power source to operate in vivo. Thus, to power implantable medical devices, biofuel cells (BFCs) that generate electricity using glucose without an external power supply have been considered. Although implantable BFCs have been developed for application in vivo, they are limited by their bulky electrodes and low power density. In the present study, we attempted to apply to living mice an implantable enzymatic BFC (EBFC) that was previously reported to be a high-power EBFC comprising carbon nanotube yarn electrodes. To improve their mechanical properties and for convenient implantation, the electrodes were coated with Nafion and twisted into a micro-sized, two-ply, one-body system. When the two-ply EBFC system was implanted in the abdominal cavity of mice, it provided a high-power density of 0.3 mW/cm2. The two-ply EBFC system was injected through a needle using a syringe without surgery and the inflammatory response in vivo initially induced by the injection of the EBFC system was attenuated after 7 days, indicating the biocompatibility of the system in vivo.

Microfluidic chip-based electrochemical immunoassay for hippuric acid.

Urinary hippuric acid (HA), of molecular weight 180 Da, is one of the major metabolites in toluene-exposed humans and is a major biological indicator. Simple and ubiquitous monitoring of exposure to toluene is very important in occupational health care, and a microfluidic chip-based electrochemical immunoassay for rapid and quantitative detection of HA in human urine is proposed in this paper. The system employs a conjugate of ferrocene (Fc) and hippuric acid (HA). The competition between hippuric acid (HA) and the ferrocene-hippuric acid complex (Fc-Lys-HA) to bind with a HA antibody coated onto polybeads generated electrical signals proportional to the HA concentration in the range of 0-40 mg mL(-1). All the complicated HA detection processes were integrated on the single microfluidic platform. The quantitative advantages of our HA detection chip are as follows: (1) the total chip size was reduced to 3.0 x 2.0 x 0.5 cm and is small enough to be portable, (2) the assay time took 1 min, and is shorter than that of conventional electrochemical HA immunoassay systems (about 20 min) and (3) 40 microL of the sample solution was enough to detect HA in the range of 0-40 mg mL(-1), which is enough range to be used for the point-of-care system. In addition, we suggest the improved chip-based HA assay method by the combination of electrochemical and enzymatic amplification processes for the detection of greater electrical signals. The sensitivity of the combined method was increased about three times compared to that of the non-enzymatic process.

Author Correction: Performance of a glucose-reactive enzyme-based biofuel cell system for biomedical applications.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Performance of a glucose-reactive enzyme-based biofuel cell system for biomedical applications.

A glucose-reactive enzyme-based biofuel cell system (EBFC) was recently introduced in the scientific community for biomedical applications, such as implantable artificial organs and biosensors for drug delivery. Upon direct contact with tissues or organs, an implanted EBFC can exert effects that damage or stimulate intact tissue due to its byproducts or generated electrical cues, which have not been investigated in detail. Here, we perform a fundamental cell culture study using a glucose dehydrogenase (GDH) as an anode enzyme and bilirubin oxidase (BOD) as a cathode enzyme. The fabricated EBFC had power densities of 15.26 to 38.33 nW/cm2 depending on the enzyme concentration in media supplemented with 25 mM glucose. Despite the low power density, the GDH-based EBFC showed increases in cell viability (~150%) and cell migration (~90%) with a relatively low inflammatory response. However, glucose oxidase (GOD), which has been used as an EBFC anode enzyme, revealed extreme cytotoxicity (~10%) due to the lethal concentration of H2O2 byproducts (~1500 µM). Therefore, with its cytocompatibility and cell-stimulating effects, the GDH-based EBFC is considered a promising implantable tool for generating electricity for biomedical applications. Finally, the GDH-based EBFC can be used for introducing electricity during cell culture and the fabrication of organs on a chip and a power source for implantable devices such as biosensors, biopatches, and artificial organs.

Ultrasonic synthesis and characterization of poly(acrylamide)-co-poly(vinylimidazole)@MWCNTs composite for use as an electrochemical material.

Applying a nanocomposite to increase the conductivity of an electrode can facilitate electrochemical analysis. In this regard, multi-walled carbon nanotubes (MWCNTs) evenly dispersed in hydrophilic solution can play an important role in electrochemical bio-sensing due to their unique properties, such as their high electrical conductivity and ability to conjugate with hydrophilic enzymes. Herein, we report the simple ultrasonic synthesis of a highly dispersible, enzyme-binding nanocomposite, poly(acrylamide)-co-poly(vinyl imidazole) (7:1 mol ratio)-MWCNTs (PAA-PVI@MWCNTs). This material, having a zeta potential of 36.6 ±â€¯0.53 mV, was applied as a film to an electrode surface and stably bound with glucose oxidase to transfer an electron between the enzyme and electrode in the presence of glucose. The PAA-PVI@MWCNTs composite, which was readily dispersed in deionized water, can be used as a biocompatible material for applications such as bio-sensing, point-of-care testing (POCT), and other health care functions.

Immunochromatographic analysis of hippuric acid in urine.

Toluene, a clear, colorless liquid with a distinctive smell, is the most commonly used industrial organic solvent. The adverse effects of chronic toluene exposure have been reported. The abuse of volatile substances is practiced mainly by adolescents and young adults. Chronic toluene abuse causes permanent changes in brain structure correlated with brain dysfunction; therefore, it is important to monitor the level of toluene exposure to prevent neurological damages. In this study, immunochromatographic analysis was performed to measure a level of the exposed toluene easily and accurately in urine. Inhaled toluene is metabolized to hippuric acid (HA) in the liver and secreted in urine. Therefore, the monoclonal antibodies against HA were generated and characterized by indirect competitive ELISA. The sensitivity was then monitored in order to adjust the cutoff concentration to 2 mg of HA/mL of urine. Using these monoclonal antibodies as raw materials, the immunochromatographic device was manufactured with the lateral flow system. The clinical studies were performed with suspected users' urine samples, and the results were confirmed by high-performance liquid chromatography.

Electrical stimulation by enzymatic biofuel cell to promote proliferation, migration and differentiation of muscle precursor cells.

Electrical stimulation is a very important biophysical cue for skeletal muscle maintenance and myotube formation. The absence of electrical signals from motor neurons causes denervated muscles to atrophy. Herein, we investigate for the first time the utility of an enzymatic biofuel cell (EBFC) as a promising means for mimicking native electrical stimulation. EBFC was set up using two different enzymes: one was glucose oxidase (GOX) used for the generation of anodic current followed by the oxidation of glucose; the other was Bilirubin oxidase (BOD) for the generation of cathodic current followed by the reduction of oxygen. We studied the behaviors of muscle precursor cells (MPCs) in terms of proliferation, migration and differentiation under different electrical conditions. The EBFC electrical stimulations significantly increased cell proliferation and migration. Furthermore, the electrical stimulations promoted the differentiation of cells into myotube formation based on expressions at the gene and protein levels. The EBFC set up, with its free forms adjustable to any implant design, was subsequently applied to the nanofiber scaffolding system. The MPCs were demonstrated to be stimulated in a similar manner as the 2D culture conditions, suggesting potential applications of the EBFC system for muscle repair and regeneration.

High-power biofuel cell textiles from woven biscrolled carbon nanotube yarns.

Biofuel cells that generate electricity from glucose in blood are promising for powering implantable biomedical devices. Immobilizing interconnected enzyme and redox mediator in a highly conducting, porous electrode maximizes their interaction with the electrolyte and minimizes diffusion distances for fuel and oxidant, thereby enhancing power density. Here we report that our separator-free carbon nanotube yarn biofuel cells provide an open-circuit voltage of 0.70 V, and a maximum areal power density of 2.18 mW cm(-2) that is three times higher than for previous carbon nanotube yarn biofuel cells. Biofuel cell operation in human serum provides high areal power output, as well as markedly increased lifetime (83% remained after 24 h), compared with previous unprotected biofuel cells. Our biscrolled yarn biofuel cells are woven into textiles having the mechanical robustness needed for implantation for glucose energy harvesting.

An oxygen cathode operating in a physiological solution.

We report the electroreduction of O(2) to water under physiological conditions (pH 7.4, 0.15 M NaCl, 37.5 degrees C) at a current density of 5 mA cm(-2) and at a potential only 0.18 V reducing versus that of the reversible O(2)/H(2)O electrode at pH 7.4. The immobilized electrocatalyst enabling the reduction is the electrostatic adduct of bilirubin oxidase from Myrothecium verrucaria, a polyanion at pH >4.1, and the polycationic redox copolymer of polyacrylamide and poly (N-vinylimidazole) complexed with [Os (4,4'-dichloro-2,2'-bipyridine)(2)Cl](+/2+), cross-linked on carbon cloth. The current density of the rotating electrodes was O(2) transport limited up to 8.8 mA cm(-2); their kinetic limit was reached at 9.1 mA cm(-2). The operational life of the electrodes depended on their angular velocity, which defined not only the current density but also the mechanical shear stress stripping the electrocatalyst. When the electrodes were rotated at 300 rpm and were poised at -256 mV versus the potential of the reversible O(2)/H(2)O electrode, their 2.4 mA cm(-2) initial current density decreased to 1.3 mA cm(-2) after 6 days of continuous operation at 37.5 degrees C.

Bilirubin oxidase label for an enzyme-linked affinity assay with O2 as substrate in a neutral pH NACL solution.

Laccase, a copper enzyme catalyzing the four-electron reduction of O(2) to water, has been shown by others to be a useful label in enzyme-linked immunoassays, in which the substrate is ambient O(2) instead of an added chemical, such as hydrogen peroxide, or a phosphate ester of a phenol. Laccase-catalyzed O(2) reduction is, however, inhibited by halides, which complex the enzyme's copper ions. Replacement of laccase by bilirubin oxidase, a copper enzyme retaining its maximal activity at high chloride concentrations and at pH 7.2, allows enzyme-amplified affinity assays with O(2) as the substrate in neutral-pH chloride solutions, exemplified here by the assay of DNA, the duplexes of which are unstable at low ionic strength but are stable in strong NaCl solutions.

Enzyme-amplified amperometric detection of 3000 copies of DNA in a 10-microL droplet at 0.5 fM concentration.

We reported earlier the detection of a 38-base DNA strand at 20 pM concentration by an enzyme-amplified sandwich-type amperometric assay. The assay utilized a carbon electrode on which a redox polymer, comprising a DNA capture sequence, was electrodeposited. When present in the tested solution, part of the probed sequence hybridized with the capture probe. Hybridization of its remaining part with a horseradish peroxidase (HRP)-labeled sequence resulted in the flow of an H2O2 electroreduction current, the redox polymer wired HRP forming an electrocatalyst. Here we report a > 10(4)-fold improvement in the detection limit of the assay. DNA was detected at 0.5 fM concentration when the earlier used 3.6-mm-diameter carbon electrode was replaced by a 10-microm-diameter microelectrode. The radial diffusion of electrons through the film on the microelectrode allowed the electrodeposition of a thicker film of the redox polymer, an increase in the loading of the capture sequence, and increased the collection efficiency of the electron vacancies originating in the electroreduced H2O2. When the volume probed by the microelectrode was 10 microL, as few as 3000 copies of DNA were detected.