Bilirubin Oxidase Research Articles

A photoelectrochemical sensor for highly sensitive detection of H2O2 based on [Fcmim][N(CN)2]@Nafion® film modified GaN through a parallel catalysis strategy

A photoelectrochemical (PEC) sensing system for H2O2 detection had been proposed based on parallel catalytic method in the presence of a ferrocene derivative. 3,3-bis(1-ferrocene methyleneimidazole-3-) boron dihydrodicyanamide ([Fcmim][N(CN)2]) was synthesized and immobilized on the surface of a GaN photoanode by the Nafion® film. A circulating H2O2-O2-H2O2 system was constructed using [Fcmim][N(CN)2] as bifunctional catalyst. In the cyclic process, H2O2 is catalyzed by [Fcmim][N(CN)2] to produce O2, which is then reduced to H2O2 by photogenerated electrons of GaN. The reduction process of O2 is also catalyzed by the same ferrocene catalyst. The regenerated H2O2 continues to be decomposed, causing O2 and H2O2 to fall into a photoelectrochemical cycle of H2O2-O2-H2O2. The decrease of the photocurrent by O2 was markedly amplified because the H2O2 and O2 were periodically regenerated. On the other hand, the diffusion rate of dissolved O2 through the Nafion® film is lower than the photoelectrochemical reduction rate of O2 on the GaN photoanode, thus the interference of dissolved O2 in the solvent could be eliminated during repeated on/off cycles of illumination. In the range of 4.00 nM - 0.720 μM and the limit of detection was 1.23 nM (3 S/N), PEC signal is linearly correlated with the logarithm of H2O2 concentration under optimized conditions. This method has a wide application prospect in environmental analysis and food safety analysis. For example, bilirubin can be detected using Nafion® in combination with bilirubin oxidase and [Fcmim][N(CN)2].

Polydopamine mediator for glucose oxidation reaction and its use for membraneless enzymatic biofuel cells

Polydopamine (PDA) is used as a biocompatible mediator for fabricating glucose oxidase (GOx) based bioanode of enzymatic biofuel cells (EBFCs). Through the autoxidation and polymerization of dopamine monomer, spherical PDA particles are well synthesized, and their chemical and optical characteristics are verified. Although the durability of bioanode including CNT and PDA (CNT/PDA/GOx/Nafion) is poor, when catalyst including polyethyleneimine (PEI) upper layer (CNT/PDA/GOx/PEI/Nafion) is fabricated, CNT/PDA/GOx/PEI/Nafion shows excellent durability. This indicates PEI plays a critical role in suppressing the leaching out of PDA and GOx molecules. By the role of PEI, the anodic current density of CNT/PDA/GOx/PEI/Nafion (142.6 ± 1.98 μA cm−2 at 0.6 V (vs. Ag/AgCl)) is 17.3 times better than that of CNT/PDA/GOx/Nafion. When maximum power density (MPD) of EBFC using CNT/PDA/GOx/PEI/Nafion is measured, that of EBFC including membrane and platinum-based air–cathode is 552.8 ± 5.4 µW cm−2, and that of membraneless EBFC including bilirubin oxidase biocathode is 135.2 ± 5.45 μW cm−2, which is better than that of other reported EBFCs studies. This confirms that the suggested new catalyst is a viable option as bioanode for EBFC.

Inhibition of direct-electron-transfer-type bioelectrocatalysis of bilirubin oxidase by silver ions.

In enzyme-based biosensors, Ag+ eluted from the reference electrode inhibits the enzyme activity. Herein, to suppress the inhibition of bilirubin oxidase (BOD) by Ag+, kinetic analysis was used to examine the effect of Ag+ on the activity of BOD. It was confirmed that the addition of Ag+ decreased the bioelectrocatalytic activity of BOD. Atomic absorption spectroscopy (AAS) suggested that Ag+ was attached to BOD. Moreover, the changes in the visible absorption spectra after Ag+ addition showed that Ag+ was bound to the type I Cu sites in BOD. During oxygen reduction by BOD, the direct-electron-transfer-type bioelectrocatalytic current decreased after Ag+ was added. The decay of the catalytic current was evaluated using kinetic analysis (assuming a pseudo-first-order reaction). Based on the analysis, the inhibition of BOD was suppressed when the Ag+ concentration was below 0.1µM. Referring to the solubility product of AgCl, Cl- at a concentration of 1mM suppressed the inhibition of the enzymatic activity by 95%.

Effects of Interactions between Speek or Nafion Ionomers and Bilirubin Oxidase on O2 Enzymatic Reduction

Effects of Interactions between Speek or Nafion Ionomers and Bilirubin Oxidase on O2 Enzymatic Reduction

Superior performance of a graphdiyne self-powered biosensor with exonuclease III-assisted signal amplification for sensitive detection of microRNAs.

Graphdiyne (GDY) is an sp and sp2 co-hydrocarbon allotrope whose particular structure endows it with many fascinating properties, including abundant chemical bonds, high conjugation, natural pores, high carrier mobility, high conductivity and stability, etc. In this work, two-dimensional graphdiyne is prepared as an electrode substrate material coupling with an exonuclease III-assisted amplification strategy to construct a superior-performance self-powered biosensor based on enzymatic biofuel cells for highly sensitive detection of the tumour marker miRNA-21. Glucose oxidase (GOD) is first immobilized on the GDY/AuNP composite to prepare a bioconjugate. GDY/AuNP modified carbon cloth is used as an enzyme biofuel cell electrode, which is then modified with bilirubin oxidase as a biocathode. The bioconjugate binds to GOD through specific binding to the bioanode. When miRNA-21 is present, specific recognition by exonuclease III in the system results in cleavage of the capture probe, and miRNA-21 is recovered and involved in the cycle. The target miRNA-21 then causes corresponding changes in the open-circuit voltage of the self-powered system. Based on this, a sensitive detection method was constructed, within the scope from 0.1 fM to 0.1 nM with a shallow detection limit of 55.2 aM (S/N = 3). The new approach triumphantly has been used to detect miRNA-21 in serum, which provides a compelling new way for early diagnosis of related cancers.

Dual-feature photobioanodes based on nanoimprint lithography for photoelectric biosupercapacitors

Direct transformation of solar energy into electrical energy by means of biological photosynthesis is considered as an attractive option for sustainable electrical energy production. Thylakoid membranes, the site of photosynthesis, are regarded as a promising biological material for the development of photoelectric biodevices, which produce electrical power consuming only light energy as oxygen evolves at photobioanode upon irradiation and biocathode converts it back to water. Therefore, in this work dual-feature photobioanode based on nanoimprinted gold substrates modified with thylakoids in combination with a capacitive part made of a planar gold substrate coated with a conductive polymer was designed and evaluated, providing open-circuit potential of −0.21 V vs. Ag|AgCl|KClsat and a capacitance of ca. 60 F m−2 both at ambient light and artificial illumination of 400 W m−2. Combination of thylakoid based dual-feature photobioanode with bilirubin oxidase modified transparent and capacitive indium tin oxide biocathode resulted in a photoelectric biosupercapacitor with remarkable characteristics at ambient light, viz. an open-circuit voltage as high as 0.74 V, which was stable upon charge-discharge cycles during ca. 2 h.

Integration of a capacitor to a 3-D DNA walker and a biofuel cell-based self-powered system for ultrasensitive bioassays of microRNAs.

A self-powered microRNA biosensor with triple signal amplification systems was assembled through the integration of three-dimensional DNA walkers, enzymatic biofuel cells and a capacitor. The DNA walker is designed from an enzyme-free target triggered catalytic hairpin assembly of modified gold nanoparticles. When triggered by the target microRNA, the DNA walker will move along the catalytic hairpin track, resulting in a payload release of glucose oxidase. The enzymatic biofuel cell contains the glucose oxidase bioanode and a bilirubin oxidase biocathode that bring a dramatic open circuit voltage to realize the self-powered bioassays of microRNA. A capacitor is further coupled with the enzymatic biofuel cell to further amplify the electrochemical signal, and the sensitivity increases 28.82 times through optimizing the matching capacitor. Based on this design, the present biosensor shows high performance, especially for detection limit and sensitivity. Furthermore, the present biosensor was successfully applied for serum samples, directly demonstrating its good application in clinical biomedicine and disease diagnosis.

Thermal Annealing‐Enhanced Bioelectrocatalysis in Membraneless Glucose/Oxygen Biofuel Cell based on Hydrophilic Carbon Fibers

AbstractThe electrode material is a pivotal component in achieving efficient electron transfer in an enzymatic biofuel cell (EBFC). Herein, novel hydrophilic carbon fibers (HCFs) with a helical structure are synthesized through chemical vapor deposition and are used as an electrode material for the immobilization of biocatalysts to fabricate bioelectrodes, where glucose dehydrogenase, serving as the anodic enzyme, is co‐immobilized with 1,4‐naphthoquinone on the HCFs for the catalysis of the glucose oxidation reaction (GOR), and bilirubin oxidase, as the cathodic enzyme, is co‐immobilized with protoporphyrin IX for the catalysis of the oxygen reduction reaction (ORR). It is found that thermal annealing the HCFs improves the bioelectrocatalytic performances of the as‐fabricated bioanode for the GOR and biocathode for the ORR, owing to the enhanced electron transfer kinetics. Subsequently, a membraneless glucose/O2 EBFC assembled by using the as‐synthesized bioelectrodes is able to generate a high open circuit voltage of 0.8 V and a maximum power density of 0.7 mW cm−2 at 0.44 V with good operational stability. The practicability in fabricating membraneless EBFCs makes the synthesized HCFs promising in miniature bioenergy devices.

Photo-stimulated self-powered electrochemical system for DNA release

The DNA-release activated by light in the self-powered bioelectronic system has been studied. The system was composed of two connected electrodes: one photo-active electrode was modified with photosynthetic thylakoid membranes and another electrode was modified with O2-reducing bilirubin oxidase and nanoparticles functionalized with molecules changing their charge upon pH variation. When the photo-electrode was illuminated, it produced current resulting in O2 reduction and local pH change at the releasing electrode. The interfacial charge was changed from positive, when negatively charged DNA was loaded, to negative, when the DNA was repulsed and released.

Multiscale modelling of diffusion and enzymatic reaction in porous electrodes in Direct Electron Transfer mode

This work is dedicated to a multi-scale modelling of coupled diffusion and reaction in a porous micro-electrode operating in the Direct Electron Transfer mode. The pore-scale physico-electrochemical unsteady model is developed considering the oxygen reduction, catalyzed by an enzyme coating the pores of the electrode, coupled to the diffusion of oxygen and mass balance of enzymes. This model is formally upscaled to obtain an original closed unsteady macroscopic model operating at the electrode scale, together with the associated closure providing the effective diffusivity tensor. A validation of this model is carried out from a comparison with the solution of the initial 3D pore-scale governing equations considering the bilirubin oxydase as the catalyst. The relevance and accuracy of the macroscale model are proved allowing a considerable simulation speedup. It is further employed to successfully predict experimental voltammetry results obtained with porous gold electrodes functionnalized with a bilirubin oxidase mutant (BOD S362C). This model represents a breakthrough by providing an operational simple way of understanding and further optimizing porous electrodes functioning in DET mode.

Bile Duct Ligation Upregulates Expression and Function of L-Amino Acid Transporter 1 at Blood-Brain Barrier of Rats via Activation of Aryl Hydrocarbon Receptor by Bilirubin.

Liver failure is associated with increased levels of brain aromatic amino acids (AAAs), whose transport across the blood–brain barrier (BBB) is mainly mediated by L-amino acid transporter 1 (LAT1). We aimed to investigate whether liver failure induced by bile duct ligation (BDL) increases levels of brain AAAs by affecting the expression and function of LAT1. The LAT1 function was assessed using the brain distribution of gabapentin. It was found that BDL significantly increased levels of gabapentin, phenylalanine, and tryptophan in the cortex, hippocampus, and striatum of rats, and upregulated the expression of total LAT1 protein in hippocampus and striatum as well as cortex membrane LAT1 protein. HCMEC/D3 served as in vitro BBB model, and the data showed that both the serum of BDL rats and bilirubin induced LAT1 expression and function, while bilirubin oxidase almost abolished the upregulation of LAT1 protein by bilirubin and the serum of BDL rats. The enhanced function and expression of LAT1 were also observed in the hippocampus and striatum of hyperbilirubinemia rats. Both aryl hydrocarbon receptor (AhR) antagonist α-naphthoflavone and AhR silencing obviously attenuated the upregulation of LAT1 protein by bilirubin or omeprazole. This study provides the first evidence that BDL upregulates LAT1 at the rat BBB, attributed to the activation of AhR by the increased plasma bilirubin. The results highlight the mechanisms causing BDL-increased levels of brain AAAs and their physiological significance.

Passive fuel delivery and efficient anoxic condition in anode improve performance of methanol biofuel cell

The operational stability, power efficiency, and technical simplicity of enzymatic biofuel cells are the focus area of research for practical applications of this green energy-generating device. Here, we report some critical findings on these issues on a methanol-fuelled pure biofuel cell fabricated by using alcohol oxidase and bilirubin oxidase as anodic and cathodic catalysts, respectively. The cell was fabricated with a new design strategy comprising efficient anoxic condition in the anodic chamber, adequate airflow to the cathode for enhancing oxygen reduction reactions, and a passive fuel pumping facility to the anode. A magnetic nanoparticle-based bio-nanocomposite matrix on the carbon-cloth electrode offered as a biocompatible enzyme immobilization matrix for harvesting electrons in the cell through the direct electron transfer mechanism as validated by cyclic voltammetry. Six units of the cells, when connected in a series, the device's potential increased to 4.3-fold (3.1 V) and rested at a stable state under a load with a half-life of ∼ 372 days and a coulombic efficiency of 60%. This high operational stability has been attributed to the efficient anoxic setup in the anodic chamber that supported the stability of alcohol oxidase, the activity of which was intact even after 49 days of the operation. This work also demonstrated that the prolonged interaction of molecular oxygen with the oxidase drastically inactivates it without affecting the structural integrity of the enzyme protein. This enzymatic fuel cell with improved design and functions is a step forward for achieving practical application as a standalone power supply to small-scale devices.

A self-powered biosensor for glucose detection using modified pencil graphite electrodes as transducers

Biofuel cells are an interesting way of microenergy production that can be advantageously channeled for biosensing applications. In this study, a biofuel cell was developed and applied as a self-powered biosensor for glucose detection. Glucose oxidase was initially tested for the bioanode and, since no evidence of direct electron transfer was observed in the absence of the co-substrate, it was not further considered. The enzymes Pyrroloquinoline quinone-dependent glucose dehydrogenase (PQQGDH) and Bilirubin oxidase (BOx) were immobilized on Pencil graphite electrodes (PGE), previously nanostructured with MWCNT, serving as bioanode and biocathode, respectively. The bifunctional crosslinker 1-pyrenebutanoic acid succinimidyl ester (PBSE) was used as a simple but efficient tethering agent, enabling the establishment of direct electron transfer events. Cyclic and linear sweep voltammetry, and amperometry were employed for individual characterization of bioanode and biocathode as biosensors for the respective enzymatic substrates. A mean sensitivity of 77.7 ± 5.9 μA cm−2 mM−1 was obtained for glucose with a Limit of detection (LOD) of 4.0 ± 2.2 μM, whereas in the detection of O2, the sensitivity and LOD achieved were 336 ± 22 μA cm−2 mM−1 and 3.2 ± 1.0 μM, respectively. Finally, the PQQGDH bioanode and BOx biocathode were conjugated into a biofuel cell and further characterized as a self-powered biosensor for glucose, exhibiting a linear range up to 1 mM, an excellent sensitivity of 8.08 μW cm−2 mM−1, a low LOD of 0.084 mM and long stability (94% of the original response after 12 days). Therefore, PGE transducers and PBSE are valuable strategies for the design of simple and efficient self-powered biosensors.

A Fluorescence-Based Quantitative Analysis for Total Bilirubin in Blood and Urine.

Bilirubin is a catabolic product of heme metabolism that circulates in the bloodstream in its unconjugated or glucuronide-conjugated form. Because the accumulation of bilirubin in the blood is a common symptom of liver diseases, its measurement in plasma (serum) is important for the diagnosis of these diseases. We developed a method to assess total bilirubin levels in serum and urine, using the fluorescent protein UnaG and β-glucuronidase. Our results indicate good correlation in serum total bilirubin levels between UnaG and the conventional bilirubin oxidase (BOD) methods. We found low levels of conjugated and unconjugated bilirubin in the urine of healthy subject individuals. Urinary bilirubin levels were elevated in patients with liver or bile duct diseases. A simple spot test of bilirubin using serum and urine showed a strong signal in patients with liver diseases. The proposed method to assess bilirubin levels in serum and urine will contribute to the accurate diagnosis of health conditions such as jaundice, anemia, and liver disease.

Three-dimensional catalysis and the efficient bioelectrocatalysis beyond surface chemistry

The exceptional catalysis for the oxygen reduction reaction (ORR) of bilirubin oxidase (BOD) encouraged us to reevaluate the existing catalytic models. Experimental evidence of the electron-transfer reaction’s dependence on the three-dimensional structure of BOD is demonstrated by operando X-ray absorption spectroscopy (XAS) combined with Quantum Mechanics/Molecular Mechanics (QM/MM). We suggest that if a catalytic reaction occurs in a three-dimensional environment (3D-catalysis), the energies involved in the transition states during the electron transfer cannot be explained by adsorption kinetics. In contrast, there is the formation of a three-dimensional complex (TDC). Our data suggest influence of the metallic cofactor electronic structure as well as the spatial disposition of its surrounding ligands on the electron transfer that promotes the biocatalysis. We propose that investigating enzymatic redox processes involving a dynamic three-dimensional enzymatic structure can provide further insights as how heterogeneous catalysis can work beyond single atoms and surface chemistry.

Separate immobilization of glucose oxidase and trehalase, and optimization of enzyme-carbon nanotube layers for the anode of enzymatic fuel cells utilizing trehalose

Insect cyborgs can be used as search robots in disaster areas and as environmental monitoring robots. In this study, we aimed to develop an anode for compact enzymatic fuel cells (EFCs) utilizing trehalose to power electronic devices implanted in such insects. Conventional trehalose-EFCs are fabricated by co-immobilization of glucose oxidase (GOx) and trehalase (TREH) on the surface of the anode. Here, we showed that current generation can be enhanced by separate immobilization of GOx and TREH using agarose. The effects of GOx-redox mediator layers, TREH loading, and the incorporation of single-walled carbon nanotubes (SWCNTs) on the performance of GOx-TREH anodes were investigated. The optimal arrangement of GOx-redox mediator layers with or without SWCNTs was determined. The highest oxidation current density (494 μA/cm2) was obtained at an electrode composed of three GOx-redox mediator layers and three GOx-redox mediator-SWCNTs layers, and agarose containing TREH. The bilirubin oxidase (BOD)-redox mediator layers and SWCNTs also influenced the performance of cathode. The highest reduction current intensity was obtained for the cathode with six BOD-redox mediator-SWCNTs layers. EFCs with the optimized anode and cathode showed a power density of ca. 15 μW/cm2 at a cell voltage of 0.3 V under a discharge current density of 50 μA/cm2.

Self-Powered Diaper Sensor with Wireless Transmitter Powered by Paper-Based Biofuel Cell with Urine Glucose as Fuel.

A self-driven sensor that can detect urine and urine sugar and can be mounted on diapers is desirable to reduce the burden of long-term care. In this study, we created a paper-based glucose biofuel cell that can be mounted on diapers to detect urine sugar. Electrodes for biofuel cells were produced by printing MgO-templated porous carbon on which poly(glycidyl methacrylate) was modified using graft polymerization. A new bioanode was prepared through covalently modifying flavin-adenine-dinucleotide-dependent glucose dehydrogenase and azure A with pendant glycidyl groups of poly(glycidyl methacrylate). We prepared a cathode with covalently bonded bilirubin oxidase. Covalent bonding of enzymes and mediators to both the bioanode and biocathode suppressed elution and improved stability. The biofuel cell could achieve a maximum output density of 0.12 mW cm–2, and by combining it with a wireless transmission device, the concentration of glucose sensed from the transmission frequency was in the range of 0–10 mM. The sensitivity of the sensor was estimated at 0.0030 ± 0.0002 Hz mmol–1 dm3. This device is expected to be a new urine-sugar detection device, composed only of organic materials with a low environmental load and it can be useful for detecting postprandial hyperglycemia.

A survey of the reactivity of in vitro diagnostic bilirubin reagents developed in Japan using artificially prepared bilirubin materials: A comparison of synthetic delta, unconjugated, and taurine-conjugated bilirubin.

In vitro diagnostic bilirubin reagents based on oxidation with bilirubin oxidase or vanadic acid for total and direct-reacting bilirubin are widely used in Japan; however, their reactivity to unconjugated and conjugated bilirubin and delta bilirubin has not been completely disclosed by manufacturers. We used artificially prepared bilirubin materials to investigate the reactivity with four in vitro diagnostic bilirubin reagents. Porcine unconjugated bilirubin solution, chemically synthesized ditaurobilirubin solution, and chemically synthesized delta bilirubin solution were used as surrogates of naturally occurring unconjugated bilirubin, conjugated bilirubin, and delta bilirubin, respectively. The total bilirubin and direct-reacting bilirubin concentrations were measured by three bilirubin oxidase methods and one vanadic acid method, and the observed concentrations were compared with those obtained by the diazo-based reference measurement procedure. The unconjugated bilirubin and delta bilirubin concentrations were similar when any of the four in vitro diagnostic bilirubin reagents were used during total bilirubin measurement. This was consistent with reference measurement procedure and exhibited a converged inter-method variation. Compared with reference measurement procedure, significantly low ditaurobilirubin concentrations were observed by the in vitro diagnostic bilirubin reagents despite the converged inter-method variation. In delta bilirubin measurement, some reagents reacted doubtfully with unconjugated bilirubin, while showed lower ditaurobilirubin concentrations than its corresponding total bilirubin concentration. Reactivity with delta bilirubin was different for each method including reference measurement procedure. Some reagents were developed to react less with delta bilirubin and others to strongly react with delta bilirubin. We revealed the reactivity of IVD-TB and IVD-DB reagents to artificially prepared bilirubin materials, and their consistency with reference measurement procedure. The delta bilirubin data results vary depending on the reagents used.

Impact of Surface Hydrophilicity of Gas-Diffusion-Type Biocathodes on Their Oxygen Reduction Ability for Biofuel Cells

The surface hydrophilicity of a gas-diffusion-type biocathode, in which bilirubin oxidase was immobilized, was regulated by varying the polymer binder hygroscopicity and UV-ozone treatment time of carbon composite electrodes. The surface hydrophilicity was found to influence its oxygen reduction activity by affecting the penetration of the electrolyte and O2 gas into the electrode layer. The oxygen reduction activity was maximized at an intermediate surface hydrophilicity because of the trade-off between enzyme utilization and oxygen diffusion. A glucose/O2 biofuel cell was fabricated by combining the optimal biocathode with a suitable bioanode fabricated with carboxylic acid-functionalized carbon nanotubes. We demonstrated that the glucose/O2 gas-diffusion-type biofuel cell exhibited a maximum current density of 3.0 mA cm−2 and a maximum power density of 0.66 mW cm−2.

Construction and characterization of a functional chimeric laccase from metagenomes suitable as a biocatalyst

Screening of gene-specific amplicons from metagenomes (S-GAM) is an efficient technique for the isolation of homologous genes from metagenomes. Using the S-GAM approach, we targeted multi-copper oxidase (MCO) genes including laccase and bilirubin oxidase (BOX) in soil and compost metagenomes, and successfully isolated novel MCO core regions. These core enzyme genes shared approximately 70% identity with that of the putative MCO from Micromonospora sp. MP36. According to the principle of S-GAM, the N- and C-terminal regions of the deduced products of the mature gene come from the known parent gene, which should be homologous and compatible with the target gene. We constructed two different MCO hybrid genes using Bacillus subtilis BOX and Micromonospora sp. MP36 MCO, to give Bs-mg-mco and Mic-mg-mco, respectively. The constructed chimeric MCO genes were fused with the maltose-binding protein (MBP) gene at the N-terminus for expression in Escherichia coli cells. We found that MBP-Mic-mg-MCO/Mic-mg-MCO possessed the characteristic properties of laccase, although MBP-Bs-mg-MCO had no activity. This novel laccase (Mic-mg-MCO) demonstrated unique substrate specificity, sufficient activity at neutral pH, and high thermal stability, which are suitable properties for its use as a laccase biocatalyst.