Bilirubin Oxidase Research Articles

A-209 Application of Bilirubin Oxidase for Improvement of False Positive Error Caused by Bilirubin on Cholesterol Efflux Capacity Assay by Immobilize Liposome-Bound Gel Beads method

Abstract Background High density lipoprotein (HDL) exerts various atheroprotective functions including cholesterol efflux capacity (CEC). HDL can attenuate lipid burden at atherosclerotic lesion by CEC. Based on the HDL`s role for moderate atherosclerosis, HDL-cholesterol level which indicates the amount of HDL has been evaluated to assess the risk of cardiovascular disease (CVD). Nevertheless, many clinical studies demonstrated that HDL-cholesterol level does not always reflect the risk of CVD, and CEC which represents the quality of HDL is considered as surrogate biomarker for CVD. CEC is prospective biomarker for CVD risk assessment, however, CEC measurement encounters barriers for clinical application because of its requirements of cultured cell and radioactive substance for conventional CEC assay. To overcome these problems, we invented a novel CEC assay called immobilized liposome-bound gel beads (ILG) method. This method is cell-free assay and utilizes a fluorescence substance as cholesterol tracer. Although the ILG method is useful assay for clinical application, previous study revealed that bilirubin caused false positive error on CEC value. Hence, current ILG method might produce incorrect CEC evaluation for patients with hyperbilirubinemia and even healthy subjects within the range of biological variation of bilirubin. In this study, we improved the influence of bilirubin for ILG method by using bilirubin oxidase (BOD). Methods BOD (Asahi Kasei Corp.) was added to 10 mM Tris-HCl (pH 7.4) containing 150 mM of NaCl and 1 mM of Na2EDTA (Buffer A). Bilirubin from ‘Interference check A Plus’ (Sysmex Inc.) was added to serum (free-: 10 mg/dL, conjugated-:9.7 mg/dL), and apoB-depleted serum (BDS) was derived from the bilirubin additive serum using polyethylene glycol. After the prepared bilirubin additive BDS was incubated with BOD-containing Buffer A for 16 h, the absorbance spectrum was analyzed. In addition, the time course of bilirubin decrement was monitored during 16 h-incubation. We further measured the fluorescence intensity of bilirubin additive BDS incubated with BOD-containing Buffer A. Using the newly developed BOD-containing Buffer A for ILG method, CEC of bilirubin additive serum was evaluated. Finally, we measured CEC of 4 healthy individuals by conventional and the improved ILG method. The CEC value was standardized by fluorescence intensity of reference sample which was measured in every assay. Results BOD contained in Buffer A could convert bilirubin to biliverdin from results of absorbance spectrum. According to the time course of bilirubin decrement, both free- and conjugated-bilirubin were oxidized to biliverdin in approximately 6 h. By converting bilirubin to biliverdin, fluorescence light was weakened greatly. We confirmed that there was no significant difference of CEC values between non bilirubin-additive serum and bilirubin additive serum. Compared healthy individual CEC values measured by conventional and improved ILG method, all subjects showed significant decrease of CEC values from the improved method, and false positive error caused by bilirubin was ameliorated. Conclusion In this study, we achieved to improve the positive error caused by bilirubin on CEC assay using ILG method. This improvement would contribute to more accurate CEC assessment by ILG method and advance the clinical application of this method.

Surfactant-Free Formate/O2 Biofuel Cell with Electropolymerized Phenothiazine Derivative-Modified Enzymatic Bioanode.

A formate (HCOO-) bioanode was developed by utilizing a phenothiazine-based electropolymerized layer deposited on sucrose-derived carbon. The electrode modified with NAD-dependent formate dehydrogenase and the electropolymerized layer synergistically catalyzed the oxidation of the coenzyme (NADH) and fuel (HCOO-) to achieve efficient electron transfer. Further, the replacement of carbon nanotubes with water-dispersible sucrose-derived carbon used as the electrode base allowed the fabrication of a surfactant-free bioanode delivering a maximum current density of 1.96 mA cm-2 in the fuel solution. Finally, a separator- and surfactant-free HCOO-/O2 biofuel cell featuring the above bioanode and a gas-diffusion biocathode modified with bilirubin oxidase and 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonate) was fabricated, delivering a maximum power density of 70 μW cm-2 (at 0.24 V) and an open-circuit voltage of 0.59 V. Thus, this study demonstrates the potential of formic acid as a fuel and possibilities for the application of carbon materials in bioanodes.

Smart biocathodic double signal amplification design empowered self-powered bioplatform ultrasensitive detection of tumor biomarker

MicroRNA-21 (miRNA-21) is widely expressed in various organs and tissues of mammals, including the heart, lungs, brain, pancreas, small intestine, colon, breast, and kidneys, showing high sensitivity and specificity in the diagnosis of multiple types of tumors. It can serve as a new clinical early diagnosis and prognostic biomarker for tumors. This study proposes a self-powered bioplatform, which employs a biological cathode double signal amplification design to construct a high-performance enzyme biofuel cell for the ultra-sensitive detection of tumor biomarker miRNA-21. The biocathodic double signal mechanism involves the catalyzed hairpin self-assembly introduction signal I bilirubin oxidase and a tetrahedral DNA nanostructure that produces a double-helical DNA chain to adsorb signal II electron acceptor [Ru(NH3)6]3+. This approach not only improves the stability of the sensing system, but also synergistically enhances the open circuit voltage. The developed assay exhibits a wide linear range from 0.1 fM to 1000 pM, with a low detection limit of 0.04 fM (S/N = 3). This study provides a strategy for designing biosensors with improved sensitivity and potential application in medical diagnosis field.

High electrolyte concentration effect on enzymatic oxygen reduction

The nature, the composition and the concentration of electrolytes is essential for electrocatalysis involving redox enzymes. Here, we discuss the effect of various electrolyte compositions with increasing ionic strengths on the stability and activity towards O2 reduction of the bilirubin oxidase from Myrothecium verrucaria (Mv BOD). Different salts, Na2SO4, (NH4)2SO4, NaCl, NaClO4, added to a phosphate buffer (PB) were evaluated with concentrations ranging from 100 mM up to 1.7 M. On functionalized carbon nanotube-modified electrodes, it was shown that the catalytic current progressively decreased with increasing salt concentrations. The process was reversible suggesting it was not related to enzyme leakage. The enzyme was then immobilized on gold electrodes modified by self-assembling of thiols. When the enzyme was simply adsorbed, the catalytic current decreased in a reversible way, thus behaving similarly as on carbon nanotubes. Enzyme mobility at the interface induced by a modification in the interactions between the protein and the electrode upon salt addition may account for this behavior. When the enzyme was covalently attached, the catalytic current increased. Enzyme compaction is proposed to be at the origin of such catalytic current increase because of shorter distances between the first copper site electron acceptor and the electrode.

Improvement in bilirubin influence on cholesterol efflux capacity evaluation using the immobilized liposome-bound gel beads method.

High-density lipoprotein (HDL) has a cholesterol efflux capacity (CEC) that protects against atherosclerosis. Recently, we developed an assay for CEC evaluation, named the immobilized liposome-bound gel beads (ILG) method, which is a highly accurate, simple, and safe method for CEC evaluation because it uses liposomes and BODIPY-labeled cholesterol instead of cultured cells and radioactive substances, respectively. Although the ILG method can be implemented in clinical settings, our previous study revealed that bilirubin causes a positive error in the CEC value. Therefore, in the present study, we attempted to improve the influence of bilirubin levels on the ILG method. To investigate why bilirubin caused a positive error in CEC values when using the ILG method, 3D fluorescence spectra of BODIPY-labeled cholesterol and bilirubin were measured. To avoid the fluorescence emitted by bilirubin, CEC was measured using the ILG method with shifting of excitation wavelength for BODIPY-labeled cholesterol quantification. In addition, we used bilirubin oxidase to oxidize bilirubin during the incubation time of the ILG method to weaken bilirubin fluorescence. We found that bilirubin emitted fluorescence at the measurement setting of the ILG method. By shifting the excitation wavelength, the positive error caused by bilirubin was improved by approximately 70%. Furthermore, by utilizing bilirubin oxidase, the false-high values of CEC were improved by approximately 80%. Bilirubin interferes with CEC assay using BODIPY-cholesterol, but we successfully improved the influence of bilirubin on CEC evaluation using the ILG method. These improvements will promote the clinical application of the ILG method.

Hierarchical Structure of Gold and Carbon Electrode for Bilirubin Oxidase-Biocathode.

Biofuel cells (BFCs) with enzymatic electrocatalysts have attracted significant attention, especially as power sources for wearable and implantable devices; however, the applications of BFCs are limited owing to the limited O2 supply. This can be addressed by using air-diffusion-type bilirubin oxidase (BOD) cathodes, and thus the further development of the hierarchical structure of porous electrodes with highly effective specific surface areas is critical. In this study, a porous layer of gold is deposited over magnesium-oxide-templated carbon (MgOC) to form BOD-based biocathodes for the oxygen reduction reaction (ORR). Porous gold structures are constructed via electrochemical deposition of gold via dynamic hydrogen bubble templating (DHBT). Hydrogen bubbles used as a template and controlled by the Coulomb number yield a porous gold structure during the electrochemical deposition process. The current density of the ORR catalyzed by BOD without a redox mediator on the gold-modified MgOC electrode was 1.3 times higher than that of the ORR on the MgOC electrode. Furthermore, the gold-deposited electrodes were modified with aromatic thiols containing negatively charged functional groups to improve the orientation of BOD on the electrode surface to facilitate efficient electron transfer at the heterogeneous surface, thereby achieving an ORR current of 12 mA cm-2 at pH 5 and 25 °C. These results suggest that DHBT is an efficient method for the fabrication of nanostructured electrodes that promote direct electron transfer with oxidoreductase enzymes.

Influence of distal glycan mimics on direct electron transfer performance for bilirubin oxidase bioelectrocatalysts

Bilirubin oxidase (BOD) is a bioelectrocatalyst that reduces dioxygen (O2) to water and is capable of direct electron transfer (DET)-type bioelectrocatalysis via its electrode-active site (T1 Cu). BOD from Myrothecium verrucaria (mBOD) has been widely studied and has strong DET activity. mBOD contains two N-linked glycans (N-glycans) with N472 and N482 binding sites distal to T1 Cu. We previously reported that different N-glycan compositions affect the enzymatic orientation on the electrode by using recombinant BOD expressed in Pichia pastoris and the deglycosylation method. However, the individual function of the two N-glycans and the effects of N-glycan composition (size, structure, and non-reducing termini) on DET-type reactions are still unclear. In this study, we utilize maleimide-functionalized polyethylene glycol (MAL-PEG) as an N-glycan mimic to evaluate the aforementioned effects. Site-specific enzyme-PEG crosslinking was carried out by specific binding of maleimide to Cys residues. Recombinant BOD expressed in Escherichia coli (eBOD), which does not have a glycosylation system, was used as a benchmark to evaluate the effect. Site-directed mutagenesis of Asn residue (N472 or N482) into Cys residue is utilized to realize site-specific glycan mimic modification to the original binding site.

A disposable enzymatic biofuel cell for glucose sensing via short-circuit current

It is essential to construct a biofuel cell-based sensor and develop an effective strategy to detect glucose without any potentiostat circuitry in order to create a simple and miniaturized device. In this report, an enzymatic biofuel cell (EBFC) is fabricated by the facile design of an anode and cathode on a screen-printed carbon electrode (SPCE). To construct the anode, thionine and flavin adenine dinucleotide-dependent glucose dehydrogenase (FAD-GDH) are covalently immobilized via a crosslinker to make a cross-linked redox network. As a cathode, the Pt-free oxygen reduction carbon catalyst is employed alternative to the commonly used bilirubin oxidase. We proposed the importance of EBFC-based sensors through the connection of anode and cathode; they can identify a short-circuit current by means of applied zero external voltage, thereby capable of glucose detection without under the operation of the potentiostat. The result shows that the EBFC-based sensor could be able to detect based on a short-circuit current with a wide range of glucose concentrations from 0.28 to 30 mM. Further, an EBFC is employed as a one-compartment model energy harvester with a maximum power density of (36 ± 3) μW cm− 2 in sample volume 5 μL. In addition, the constructed EBFC-based sensor demonstrates that the physiological range of ascorbic acid and uric acid shows no significant effect on the short-circuit current generation. Moreover, this EBFC can be used as a sensor in artificial plasma without losing its performance and thereby used as a disposable test strip in real blood sample analysis.

Paper-type membraneless enzymatic biofuel cells using a new biocathode consisting of flexible buckypaper electrode and bilirubin oxidase based catalyst modified by electrografting

A new biocathode consisting of bilirubin oxidase (BOD), 4-aminophthalic acid (4-APA) and buckypaper (BP) electrode is suggested for enzymatic biofuel cells (EBFCs). Here, to enhance the electron transfer rate of BOD, electrografting method is imported. When this method and 4-APA are utilized, BP surface has negative charges by the polarity arrangement of 4-APA, forming BP/4-APA structure. With the modification of surface charge, T1 active site within BODs having positive charges are strongly bonded with the negative charges of BP/4-APA in neutral pH condition, while the orientation of T1 site is well aligned by electrostatic interaction. This treatment increases both reactivities of cathodic reaction and EBFC performance. To prove that, the performance of membraneless paper-type EBFCs using the new flexible BP/4-APA/BOD biocathode is investigated. According to the evaluations, their maximum power density and open circuit voltage are 92.025 ± 0.892 μW cm−2 and 0.612 ± 0.002 V with injection of 10 mM glucose which is a condition of living body fluid. Such performances are far better than those of other membraneless EBFCs and are large enough to be utilized as a power source of implantable devices for the human body.

Glucose Oxidase-like Rhodium Single-Atom Nanozymes: A Mimic Platform for Biometabolism and Electrometabolism of Glucose Oxidation at Neutral pH

Rhodium single-atom nanozymes (Rh SANs), mimicking the function of natural glucose oxidase (GOD) for both biometabolism and electrometabolism of glucose oxidation, were designed in this work. Here, Rh SANs were proved to have the same biocatalytic mechanism as natural GOD, as well as a similar electrocatalytic path, namely two-electron electrooxidation from glucose to gluconic acid. On this basis, by coupling a bilirubin oxidase (BOD) biocathode, the Rh SANs-based glucose/O2 enzymatic biofuel cell (EBFC) was developed, and a maximum power density of 135.0 ± 3.0 μW cm–2 was obtained, which was nearly 2-fold enhanced over that of natural GOD-based EBFC. In addition to gathering fuels from soft drinks available in daily life, this EBFC could be designed as a novel, low-cost, and miniaturized bio-energy device. With an ingenious catalyst and flexible configuration, the device not only accelerates the development of nanozymes but paves avenues for powering portable and wearable devices.

Self-powered logically operated fluorescent detection of hepatitis B virus (HBV)

In this study, a novel sensing strategy based on double sensing/actuating pathway is demonstrated, being capable to trigger the DNA-based AND gate for the sensitive and selective detection of hepatitis B virus DNA (HBV-DNA). Such an approach encompasses an enzymatic machinery logically operated using the variation of physiologically relevant biomarkers for liver dysfunctions. Alanine aminotransferase (ALT) and lactate dehydrogenase (LDH) are used as inputs of an AND gate generating an output signal, namely lactate. In particular, lactate is oxidized back to pyruvate at the anodic electrode by lactate oxidase connected in mediated electron transfer through ferrocene moieties (creating an amplifying recycling mechanism). The anodic electrode is further connected with a Myrothecium verrucaria bilirubin oxidase (MvBOx) based biocathode modified with SiO2 nanoparticles (SiO2NPs) functionalized with phenyl boronic acid and trigonelline, triggering the release of quenching DNA (qDNA) upon local pH change at the electrode surface (notably, modified SiONPs gets negatively recharged upon local pH gradient releasing negatively charged DNA). Next, the released qDNA labeled with BHQ2 and detecting DNA (dDNA, labeled with FAM) are detecting HBV-DNA. The proposed biosensor can discriminate between the absence and presence of HBV-DNA setting the threshold at 0.05 fM in model buffer solutions and 1 fM in human serum. This enzymatic/DNA logic network can be of particular interest for future biomedical applications (e.g., early detection of liver cancer disease etc.). In the future development this technology could be easily integrated with a smartphone camera, allowing more user-friendly applications.

Controllable assembly of three-dimensional porous graphene-Au dual aerogels and its application for high-efficient bioelectrocatalytic O2 reduction

Aerogels derived from the colloidal nanoparticles featured with hierarchical interconnected pore-rich networks guarantee their great potentials in various applications. Herein, the controllable assembly of three-dimensional aerogels based on Au nanoparticles (Au NPs) and reduced graphene oxide (rGO) nanosheets as building blocks via a bottom-up approach have been systematically clarified. The difference of building blocks and their assembly sequence were crucially to the final aerogel morphologies and electrochemical properties. Specifically, the highly porous graphene-gold dual aerogels (rGO-Au DAGs) with interconnected rGO nanosheets and Au nanowires showed high conductivity, large surface area and good biocompatibility. Thus, it was employed as an excellent matrix to immobilize enzyme for high-efficient bioelectrocatalysis. Taking bilirubin oxidase as an example, a more positive on-set potential (0.60 V) and a larger catalytic current density (0.77 mA cm−2@0.40 V) than those of other rGO-Au assemblies were achieved for direct bioelectrocatalytic O2 reduction. This study will provide an efficient strategy for unique dual-structural aerogels design and shed light to develop new functional materials for bioelectrocatalytic applications such as biosensors and biofuel cells.

Microenergy generation and dioxygen sensing by bilirubin oxidase immobilized on a nanostructured carbon paper transducer

Biological systems can be envisaged as energy-sustainable solutions for both microenergy production to power small electronics and sensing purposes. In the present work, an enzymatic bioelectrode was characterized as biocathode in the fabrication of a biobattery and as biosensor for dioxygen detection. Carbon fibre paper was selected as transducer given its unique tailoring and electrocatalytic characteristics, being modified with carbon nanotubes for subsequent attachment of bilirubin oxidase enzyme. The immobilization process was based on a simple but effective method using the crosslinker 1-pyrenebutanoic acid N-hydroxysuccinimide ester, which creates stable bonds by maintaining a good response (88%) for over 34 days. The biobattery, that consisted of a zinc anode coupled with the developed biocathode, produced an open circuit potential of 1.69 V and maximum power of 40 μW, being capable of powering a digital watch for over 2 h. When applied as a dioxygen biosensor, the biocathode revealed high sensitivity of 606 ± 22 μA mM−1 cm−2 and low limit of detection of 1.3 ± 0.2 μM in chronoamperometric measurements at +0.4 V. The application of bilirubin oxidase in the fabrication of biobatteries is scarcely studied even given its advantages over other dioxygen reduction enzymes. Likewise, the use of the remarkable features of CP in dioxygen biosensing is yet to be explored. Therefore, this work stands as an interesting opportunity in the development of relatively simple and affordable bioelectrodes viably intended for energy and biosensing applications.

Enhancing long-term stability of bio-photoelectrochemical cell by defect engineering of a WO3-x photoanode

Bio-photoelectrochemical cells (BPECs) can further expand the use of conventional biofuel cells for renewable energy, but the poor stability of the photoelectrode still hinders their practical application. Herein, a BPEC capable of long-term operating in a fuel-free model is fabricated by WO3-x photoanode with oxygen vacancy (Ov) and bilirubin oxidase catalyzed biocathode. The construction of Ov on the WO3 surface significantly suppresses the dissolution of W species into the electrolyte, and improves the charge separation efficiency and the reaction kinetics during the photoelectrochemical oxygen evolution process, thus enhancing the stability and power output performance of the BPEC. As a result, the assembled BPEC can output an open circuit voltage of 0.81 V and deliver a maximum output power of up to 283 μW cm−2. Impressively, the BPECs maintain 97% of their original power after 36000 s of consecutive discharge under an enclosed environment. This fuel-free BPEC based on a robust WO3-x photoanode shows excellent promise for accurate application.

Photosystem II as a chemiluminescence-induced photosensitizer for photoelectrochemical biofuel cell-type biosensing system

Herein, photosystem II (PSII), extracted from spinach, is used for the first time as an efficient and green sensitizer for a photobioanode in a photoelectrochemical glucose biofuel cell (PBFC) setup. The concept is based on the formation of hemin-catalyzed luminol chemiluminescence (CL) after the enzymatic oxidation of glucose and the simultaneous production of hydrogen peroxide by glucose oxidase. The photosynthetic enzyme PSII, combined with an osmium polymer serving as mediator and photosensitizer, is immobilized and wired on microporous carbonaceous material (MC) for the chemiluminescence-induced oxidation of water to O2 at the photobioanode (GCE|MC|Os polymer|PSII). Also, bilirubin oxidase immobilized on multiwalled carbon nanotubes (MWCNTs) coated electrode (GCE|MWCNT|BOx) serves as a biocathode. The photoelectrochemical biofuel cell (PBFC) is applied to a biosensor model system to validate the appropriateness of such a bioanode operating in a self-powered mode. Os redox polymer attached to MCs provides abundant PSII immobilization and a reliable electron transfer pathway. The well-matching energy levels of photosensitive entities reduce recombination phenomena while MC enhances the charge collection. Substantial photocatalytic water oxidation was observed under CL due to the well-matched CL emission and PSII absorption. The electrode is rationally designed to gain the maximum luminol CL power for the photobioanode. The open circuit potential of PBFC linearly increased with the CL power intensity and, in turn, glucose concentrations in the range of 0–6 mmol L−1. The PBFC yielded an OCP of 0.531 V in 30 mmolL−1 glucose. The study may open a new horizon to the green and pioneering PEC biosensing realm.

Fabrication and characterization of glucose-oxidase-trehalase electrode based on nanomaterial-coated carbon paper.

Multienzyme systems are essential for utilizing di-, oligo-, and polysaccharides as fuels in enzymatic fuel cells effectively. However, the transfer of electrons generated by one enzymatic reaction in a multienzyme cascade at the electrode may be impeded by other enzymes, potentially hindering the overall efficiency. In this study, carbon paper was first modified by incorporating single-walled carbon nanotubes (SWCNTs) and gold nanoparticles (AuNPs) sequentially. Subsequently, glucose oxidase (GOx) and a trehalase-gelatin mixture were immobilized separately on the nanostructured carbon paper via layer-by-layer adsorption to mitigate the electron transfer hindrance caused by trehalase. The anode was first fabricated by immobilizing GOx and trehalase on the modified carbon paper, and the cathode was then fabricated by immobilizing bilirubin oxidase on the nanostructured electrode. The SWCNTs and AuNPs were distributed adequately on the electrode surface, which improved the electrode performance, as demonstrated by electrochemical and morphological analyses. An enzymatic fuel cell was assembled and tested using trehalose as the fuel, and a maximum power density of 23 μW cm-2 was obtained at a discharge current density of 60 μA cm-2. The anode exhibited remarkable reusability and stability.

Nanostructured electrodes based on multiwalled carbon nanotube/glyconanoparticles for the specific immobilization of bilirubin oxidase: Application to the electrocatalytic O2 reduction

Here we describe the design and the characterization of novel electrode materials consisting of multi-walled carbon nanotubes coated with glyconanoparticles (GNPs) functionalized with anthraquinone sulfonate. The resulting modified electrodes were characterized by scanning electron microscopy and cyclic voltammetry. Their electrochemical behavior reveals a stable pH-dependent redox signal characteristic of anthraquinone sulfonate. Immobilization of bilirubin oxidase on these three-dimensional electrodes leads to the electroenzymatic reduction of O2 to water with an onset potential of 0.5 V/SCE (saturated calomel electrode). A catalytic cathodic current of 174 µA (0.88 mA cm−2) at 0.1 V/SCE, demonstrates that glyconanoparticles modified by anthraquinone sulfonate were able to interact and orientate bilirubin oxidase by electrostatic interactions.

Hollow-Channel Paper Analytical Devices Supported Biofuel Cell-Based Self-Powered Molecularly Imprinted Polymer Sensor for Pesticide Detection.

Herein, a paper-based glucose/air biofuel cell (BFC) was constructed and implemented for self-powered pesticide detection. Our developed paper-based chip relies on a hollow-channel to transport fluids rather than capillarity, which reduces analysis times as well as physical absorption. The gold nanoparticles (Au NPs) and carbon nanotubes (CNTs) were adapted to modify the paper fibers to fabricate the flexible conductive paper anode/cathode electrode (Au-PAE/CNT-PCE). Molecularly imprinted polymers (MIPs) using 2,4-dichlorophenoxyacetic acid (2,4-D) as a template were synthesized on Au-PAE for signal control. In the cathode, bilirubin oxidase (BOD) was used for the oxygen reduction reaction. Based on a competitive reaction between 2,4-D and glucose-oxidase-labeled 2,4-D (GOx-2,4-D), the amount of GOx immobilized on the bioanode can be simply tailored, thus a signal-off self-powered sensing platform was achieved for 2,4-D determination. Meanwhile, the coupling of the paper supercapacitor (PS) with the paper-based chip provides a simple route for signal amplification. Combined with a portable digital multi-meter detector, the amplified signal can be sensitively readout. Through rational design of the paper analytical device, the combination of BFC and PS provides a new prototype for constructing a low-cost, simple, portable, and sensitive self-powered biosensor lab-on-paper, which could be easily expanded in the field of clinical analysis and drug delivery.

All-carbon sandwich-type self-powered biosensor for ultrasensitive detection of femtomolar miRNA-141

The latest research shows that the expression level of microRNA-141 can predict the number of prostate cancer cells in the human body and has become an important biomarker. In this paper, an all-carbon sandwich self-powered biosensor based on graphene and carbon cloth is constructed for the highly sensitive detection of the prostate tumor marker miRNA-141. First, gold nanoparticles modified carbon cloth is applied for substrate electrode, and bilirubin oxidase is then immobilized on it to prepare the biocathode of the biofuel cell. Then, aptamer 1 is immobilized on gold nanoparticles-modified carbon cloth as the electrode substrate. The bioconjugate is prepared by immobilizing the aptamer 2-glucose oxidase complex on gold nanoparticles/graphene. In the biofuel cell-based self-powered sensing system, when the target microRNA-141 is present, it undergoes complementary base pairing with aptamer 1 and aptamer 2, and the bioconjugates are immobilized on the anode to form the sandwich structure. The enzyme on the anode undergoes an oxidation reaction to catalyze the reduction of oxygen, and the electrochemical respond of the system increases significantly. The results show that the concentration of microRNA-141 is proportional to the open-circuit voltage value ranging from 0.0001 to 1000 pmol/L with a detection limit of 50 amol/L (S/N = 3). The method has high sensitivity and excellent selectivity and can be applied to sensitively detect tumor marker microRNA-141 in biological matrix.

Local pH Modulation during Electro-Enzymatic O2 Reduction: Characterization of the Influence of Ionic Strength by In Situ Fluorescence Microscopy.

Understanding how environmental factors affect the bioelectrode efficiency and stability is of uttermost importance to develop high-performance bioelectrochemical devices. By coupling fluorescence confocal microscopy in situ to electrochemistry, this work focuses on the influence of the ionic strength on electro-enzymatic catalysis. In this context, the 4 e-/4 H+ reduction of O2 into water by the bilirubin oxidase from Myrothecium verrucaria (MvBOD) is considered as a model. The effects of salt concentration on the enzyme activity and stability were probed by enzymatic assays performed in homogeneous catalysis conditions and monitored by UV-vis absorption spectroscopy. They were also investigated in heterogeneous catalysis conditions by electrochemical measurements with MvBOD immobilized at a graphite microelectrode. We demonstrate that the catalytic activity and stability of the enzyme both in solution and in the immobilized state at the bioelectrode were conserved with an electrolyte concentration of up to 0.5 M, both in a buffered and a non-buffered electrolyte. Relying on this, we used fluorescence confocal laser scanning microscopy coupled in situ to electrochemistry to explore the local pH of the electrolyte at the vicinity of the electrode surface at various ionic strengths and for several overpotentials. 3D proton depletion profiles generated by the interfacial electro-enzymatic reaction were recorded in the presence of a pH-sensitive fluorophore. These concentration profiles were shown to contract with increasing ionic strength, thus highlighting the need for a minimal electrolyte concentration to ensure availability of charged substrates at the electrode surface during electro-enzymatic experiments.