Pyrroloquinoline Quinone Glucose Dehydrogenase Research Articles

High voltage flexible glucose/O2 fully printed hydrogel-based enzymatic fuel cell

Herein we report on a novel enzymatic fuel cell (EFC) based on stencil printed electrodes modified with pyrrolo quinoline quinone glucose dehydrogenase and bilirubin oxidase, which are assembled by considering two different configurations: (i) normal assembling in liquid electrolyte and (ii) six EFCs connected in series, each one comprising both bioanode and biocathode, coupled through a hydrogel-based electrolyte in a stack-like mode similar to a Voltaic pile. After a deep electrodes characterization, they are assembled according to the first configuration obtaining an open circuit voltage (OCV) of 0.562 ± 0.002 V. Moreover, the EFC performance are substantially improved by using the second configuration (six EFCs connected in series) obtaining an OCV of 2.36 ± 0.22 V with a maximum power output of 22.9 ± 0.9 μW at a cell voltage of 1.95 V (operating in 10 mM D-glucose). This innovative approach represents a proof-of-concept towards the development of renewable power sources and could serve as a critical step in powering implantable bioelectronics, such as pacemakers.

Nanobody-guided redox and enzymatic functionalization of icosahedral virus particles for enhanced bioelectrocatalysis

Icosahedral, 30 nm diameter, grapevine fanleaf virus (GFLV) virus particles are adsorbed onto electrodes and used as nanoscaffolds for the assembly of an integrated glucose oxidizing system, comprising the enzyme pyrroloquinoline quinone-glucose dehydrogenase (PQQ-GDH) and ferrocenylated polyethylene glycol chains (Fc-PEG) as a redox co-substrate. Two different GFLV-specific nanobodies, either fused to the enzyme, or chemically conjugated to Fc-PEG, are used for the regio-selective immunodecoration of the viral particles. A comprehensive kinetic characterization of the enzymatic function of the particles, initially decorated with the enzyme alone shows that simple immobilization on the GFLV capsid has no effect on the kinetic scheme of the enzyme, nor on its catalytic activity. However, we find that co-immobilization of the enzyme and the Fc-PEG co-substrate on GFLV does induce enzymatic enhancement, by promoting cooperativity between the two subunits of the homodimeric enzyme, via “synchronization” of their redox state. A decrease in inhibition of the enzyme by its substrate (glucose) is also observed.

Polyazine nanoparticles as anchors of PQQ glucose dehydrogenase for its most efficient bioelectrocatalysis

We report on the drop-cast production of glucose biosensors based on the most efficient bioelectrocatalysis by pyrroloquinoline quinone dependent glucose dehydrogenase (PQQ GDH). To orient the enzyme upon immobilization we suggest using poly(Methylene Green) (p(MG)) nanoparticles acting as anchors. Synthesis of polymeric anchors has been carried out in course of Methylene Green electropolymerization, which allowed to tune polymer-to-monomer ratio in the drop-cast mixtures varying the monomer concentration and applying different number of potential sweep cycles. Except for elimination of electrochemical step for the electrode modification, opening the prospects for mass-production, the use of drop-cast enzyme anchors provides advantageous characteristics of the resulting biosensors. Catalytic current of PQQ GDH immobilized over p(MG) nanoparticles obtained in optimal conditions is increased only 2–2.5 times after addition of the freely diffusing mediator. Obviously, the lowest ratio of mediated-to-reagentless current points to the most efficient bioelectrocatalysis. The obtained ratio is 2.5 times lower than that for biosensors based on electropolymerized p(MG) films and practically an order of magnitude lower than that for the best reagentless sensors based on PQQ GDH immobilized over conductive nanomaterials. The achieved most efficient bioelectrocatalysis provides high sensitivity of the elaborated biosensors even at 0.0 V potential, which allows to operate them in power generation mode and control relative sweat glucose variation as a tool for non-invasive diabetes monitoring.

Microfluidic Electrochemical Glucose Biosensor with In Situ Enzyme Immobilization

The development and characterization of a microfluidic electrochemical glucose biosensor are presented herein. The transducer part is based on thin-film metal electrodes on a glass substrate. The biological recognition element of the biosensor is the pyrroloquinoline quinone-glucose dehydrogenase (PQQ-GdhB) enzyme, selectively in situ immobilized via microcontact printing of a mixed self-assembling monolayer (SAM) on a gold working electrode, while the microfluidic part of the device comprises microchannel and microfluidic connections formed in a polydimethylsiloxane (PDMS) elastomer. The electrode properties throughout all steps of biosensor construction and the biosensor response to glucose concentration and analyte flow rate were characterized by cyclic voltammetry and chronoamperometry. A measurement range of up to 10 mM in glucose concentration with a linear range up to 200 μM was determined. A detection limit of 30 µM in glucose concentration was obtained. Respective biosensor sensitivities of 0.79 nA/µM/mm2 and 0.61 nA/µM/mm2 were estimated with and without a flow at 20 µL/min. The developed approach of in situ enzyme immobilization can find a wide number of applications in the development of microfluidic biosensors, offering a path towards continuous and time-independent detection.

Construction of electroactive polyamine-enzyme assemblies nondependent on the electrical charge

The assembly of polyelectrolytes and biomacromolecules (proteins, DNA) has attracted great attention in several fields related to medicine, molecular biology, and clinical chemistry. Here, a quick and easy multicomponent self-assembly is used to unite two species with the same charge to form an electroactive polyamine-enzyme assembly. Phosphate ions are added as a crosslinker agent to create a stable structure. Osmium-derivatized polyallylamine is combined with horseradish peroxidase (pI = 8.6) or PQQ-glucose dehydrogenase (pI = 9.5) on graphite electrodes. The assemblies are built by layer-by-layer or one-pot deposition. In both cases, the electrocatalytical signals observed are comparable to the state-of-the-art results. The proposed construction method represents a new alternative for the assembly of polyamines with proteins independently of their charge.

Continuous long-term glucose biosensor in agitation condition for bioconversion processes

The use of a glucose biosensor in the bioconversion processes is challenging due to a long period of intensive stirring that is required to mix the heterogenous substants together. In this work, a glucose biosensor was developed to be accurate and robust for an extended period of measuring time in agitated conditions. The sensor probe was fabricated by electro-polymerization of polyaniline (PANI) nanostructures on a gold wire before putting in gold nanoparticles (AuNPs) and carrageenan (carr) solution to make an PANI-carr-AuNPs layer. Then the probe was soaked in PQQ-Glucose dehydrogenase (GDH) solution before coating with polyurethane (PU) to prevent the enzymes from leaching away from the sensor. The sensor probe showed a sensitivity of 0.85 μA/mg/ml of glucose with R2 of 0.9973. The storage stability of the probe was up to 4 days with stable signal. In agitated condition of 150 rpm, steady signals from the sensor were continuously obtained for 72 h. In sugarcane bagasse hydrolysis with speed at 150 rpm for 72 h, the biosensor continuously displayed signals which were not significantly different in glucose concentration to the results from the HPLC method. This biosensor displayed promising applications in bioconversion processes.

Differential colonization and functioning of microbial community in response to phosphate levels

Microbes play a major role in phosphate cycling and regulate its availability in various environments. The metagenomic study highlights the microbial community divergence and interplay of phosphate metabolism functional genes in response to phosphate rich (100 mgL−1), limiting (25 mgL−1), and stressed (5 mgL−1) conditions at lab-scale bioreactor. Total five core phyla were found responsive toward different phosphate (Pi) levels. However, major variations were observed in Proteobacteria and Actinobacteria with 33–81% and 5–56% relative abundance, respectively. Canonical correspondence analysis reflects the colonization of Sinorhizobium (0.8–4%), Mesorhizobium (1–4%), Rhizobium (0.5–3%) in rich condition whereas, Pseudomonas (1–2%), Rhodococcus (0.2–2%), Flavobacterium (0.2–1%) and Streptomyces (0.3–4%) colonized in limiting and stress condition. The functional profiling demonstrates that Pi limiting and stress condition subjected biomass were characterized by abundant PQQ-Glucose dehydrogenase, alkaline phosphatase, 5′-nucleotidase, and phospholipases C genes. The finding implies that the major abundant genera belonging to phosphate solubilization enriched in limiting/stressed conditions decide the functional turnover by modulating the metabolic flexibility for Pi cycling. The study gives a better insight into intrinsic ecological responsiveness mediated by microbial communities in different Pi conditions that would help to design the microbiome according to the soil phosphate condition. Furthermore, this information assists in sustainably maintaining the ecological balance by omitting excessive chemical fertilizers and eutrophication.

Insertional Profiling of Pyrroloquinoline Quinone Glucose Dehydrogenase for Two-Component Biosensor Engineering

Colorectal cancer (CRC) is the third most common cancer worldwide and the second leading cause of cancer death. Currently, it is both expensive and invasive to screen for CRC. Developing a potential screening method that is more affordable and less invasive than endoscopy and biopsy would be beneficial to vulnerable communities. This method should also be able to diagnose and monitor the development and progression of GI cancers in real-time. A sensor that is capable of sending out an electrical signal in the presence of a cancer biomarker would alleviate the physical and economic costs of endoscopy while offering a significant amount of information about the gastrointestinal environment. Pyrroloquinoline quinone (PQQ) glucose dehydrogenase (PQQ-GDH), a redox enzyme, is capable of using glucose as a substrate for electron transfer. Engineering PQQ-GDH by insertion of a ligand-binding domain that is specific to a cancer biomarker would create an allosteric enzymatic switch that only produces current in the presence of the biomarker. To begin building this platform, I am creating a comprehensive insertion library to identify functionally important residues and domains of the PQQ-GDH. From there, I will profile which locations are capable of small primary, secondary, and/or tertiary structure disruptions. After identifying these regions, I will then create two-component protein switches capable of detecting cancer biomarkers. Creating an oxidoreductase switch that controls current transfer in the presence of gastrointestinal cancer biomarkers will advance the development of a novel, portable bio-recognition device capable of real-time, continuous sensing.

Exploring Performance Parameters of Artificial Allosteric Protein Switches

Biological information processing networks rely on allosteric protein switches that dynamically interconvert biological signals. Construction of their artificial analogues is a central goal of synthetic biology and bioengineering. Receptor domain insertion is one of the leading methods for constructing chimeric protein switches. Here we present an in vitro expression-based platform for the analysis of chimeric protein libraries for which traditional cell survival or cytometric high throughput assays are not applicable. We utilise this platform to screen a focused library of chimeras between PQQ-glucose dehydrogenase and calmodulin. Using this approach, we identified 50 chimeras (approximately 23% of the library) that were activated by calmodulin-binding peptides. We analysed performance parameters of the active chimeras and demonstrated that their dynamic range and response times are anticorrelated, pointing to the existence of an inherent thermodynamic trade-off. We show that the structure of the ligand peptide affects both the response and activation kinetics of the biosensors suggesting that the structure of a ligand:receptor complex can influence the chimera’s activation pathway. In order to understand the extent of structural changes in the reporter protein induced by the receptor domains, we have analysed one of the chimeric molecules by CD spectroscopy and hydrogen–deuterium exchange mass spectrometry. We concluded that subtle ligand-induced changes in the receptor domain propagated into the GDH domain and affected residues important for substrate and cofactor binding. Finally, we used one of the identified chimeras to construct a two-component rapamycin biosensor and demonstrated that core switch optimisation translated into improved biosensor performance.

Nanostructured Interface Loaded with Chimeric Enzymes for Fluorimetric Quantification of Cyclosporine A and FK506.

Advances in protein engineering resulted in increased efforts to create protein biosensors that can replace instrumentation-heavy analytical and diagnostic methods. Sensitivity, amenability to multiplexing, and manufacturability remain to be among the key issues preventing broad utilization of protein biosensors. Here, we attempt to address these by constructing arrays utilizing protein biosensors based on the artificial allosteric variant of PQQ-glucose dehydrogenase (GDH). We demonstrated that the silica nanoparticle-immobilized GDH protein could be deposited on fiberglass sheets without loss of activity. The particle-associated GDH activity could be monitored using changes in the fluorescence of the commonly used electron mediator phenazine methosulfate. The constructed biosensor arrays of macrocyclic immunosuppressant drugs cyclosporine A and FK-506 displayed very low background and a remarkable dynamic range exceeding 300-fold that resulted in a limit of detection of 2 pM for both analytes. This enabled us to quantify both drugs in human blood, serum, urine, and saliva. The arrays could be stored in dry form and quantitatively imaged using a smartphone camera, demonstrating the method's suitability for field and point-of-care applications. The developed approach provides a generalizable platform for biosensor array development that is compatible with inexpensive and potentially scalable manufacturing.

Circular Permutated PQQ‐Glucose Dehydrogenase as an Ultrasensitive Electrochemical Biosensor

AbstractProtein biosensors play an increasingly important role as reporters for research and clinical applications. Here we present an approach for the construction of fully integrated but modular electrochemical biosensors based on the principal component of glucose monitors PQQ‐glucose dehydrogenase (PQQ‐GDH). We designed allosterically regulated circular permutated variants of PQQ‐GDH that show large (>10‐fold) changes in enzymatic activity following intramolecular scaffolding of the newly generated N‐ and C termini by ligand binding domain/ligand complexes. The developed biosensors demonstrated sub‐nanomolar affinities for small molecules and proteins in colorimetric and electrochemical assays. For instance, the concentration of Cyclosporine A could be measured in 1 μL of undiluted blood with the same accuracy as the leading diagnostic technique that uses 50 times more sample. We further used this biosensor to construct highly porous gold bioelectrodes capable of robustly detecting concentrations of Cyclosporine A as low as 20 pM and retained functionality in samples containing at least 60 % human serum.

Circular Permutated PQQ-Glucose Dehydrogenase as an Ultrasensitive Electrochemical Biosensor.

Protein biosensors play an increasingly important role as reporters for research and clinical applications. Here we present an approach for the construction of fully integrated but modular electrochemical biosensors based on the principal component of glucose monitors PQQ-glucose dehydrogenase (PQQ-GDH). We designed allosterically regulated circular permutated variants of PQQ-GDH that show large (>10-fold) changes in enzymatic activity following intramolecular scaffolding of the newly generated N- and C termini by ligand binding domain/ligand complexes. The developed biosensors demonstrated sub-nanomolar affinities for small molecules and proteins in colorimetric and electrochemical assays. For instance, the concentration of Cyclosporine A could be measured in 1 μL of undiluted blood with the same accuracy as the leading diagnostic technique that uses 50 times more sample. We further used this biosensor to construct highly porous gold bioelectrodes capable of robustly detecting concentrations of Cyclosporine A as low as 20 pM and retained functionality in samples containing at least 60 % human serum.

Anchoring PQQ-Glucose Dehydrogenase with Electropolymerized Azines for the Most Efficient Bioelectrocatalysis.

Catalytic current of pyrroloquinoline quinone (PQQ)-glucose dehydrogenase (PQQ-GDH) immobilized over electropolymerized methylene green (MG) is increased only five times after the addition of the freely diffusing mediator. This value, being an efficiency criterion for bioelectrocatalysis, is several (three to six) times lower than that for the best reagentless glucose electrodes reported for this enzyme. Thermodynamics of the polyMG|PQQ-GDH electrode is determined by the enzyme-catalyzed reaction pointing to the direct bioelectrocatalysis. PQQ-GDH immobilized over polyMG displays the current plateau region from 0.0 to 0.2 V in the presence of glucose; at 0.00 V, being the optimal potential for biosensing applications, the catalytic current of the polyMG|PQQ-GDH electrode is 700-fold higher than that for the enzyme on a blank electrode. Successful glucose detection in human sweat by means of the corresponding enzyme electrode confirms that the reported bioelectrocatalytic system is attractive for advanced biosensors, as well as for biofuel cells.

Self-powered molecule release systems activated with chemical signals processed through reconfigurable Implication or Inhibition Boolean logic gates

The Implication (IMPLY) and Inhibition (INHIB) Boolean logic gates were realized using switchable chimeric pyrroloquinoline quinone-dependent glucose dehydrogenase (PQQ-GDH-Clamp) containing a fused affinity clamp unit recognizing a signal-peptide. The second component of the logic gate was the wild-type PQQ-glucose dehydrogenase working cooperatively with the PQQ-GDH-Clamp enzyme. The IMPLY and INHIB gates were realized using the same enzyme composition activated with differently defined input signals, thus representing reconfigurable logic systems. The logic gates were first tested while operating in a solution with optical analysis of the output signals. Then, the enzymes were immobilized on a buckypaper electrode for electrochemical transduction of the output signals. The switchable modified electrodes mimicking the IMPLY or INHIB logic gates were integrated with an oxygen-reducing electrode modified with bilirubin oxidase to operate as a biofuel cell activated/inhibited by various input signal combinations processed either by IMPLY or INHIB logic gates. The switchable biofuel cell was used as a self-powered device triggering molecule release function controlled by the logically processed molecule signals.

Coenzyme Coupling Boosts Charge Transport through Single Bioactive Enzyme Junctions.

SummaryOxidation of formate to CO2 is catalyzed via the donation of electrons from formate dehydrogenase (FDH) to nicotinamide adenine dinucleotide (NAD+), and thus the charge transport characteristics of FDH become essential but remain unexplored. Here, we investigated the charge transport through single-enzyme junctions of FDH using the scanning tunneling microscope break junction technique (STM-BJ). We found that the coupling of NAD+ with FDH boosts the charge transport by ∼2,100%, and the single-enzyme conductance highly correlates with the enzyme activity. The combined flicker noise analysis demonstrated the switching of the coenzyme-mediated charge transport pathway and supported by the significantly reduced HOMO-LUMO gap from calculations. Site-specific mutagenesis analysis demonstrated that FDH-NAD+ stably combined own higher bioactivity and boosts charge transport, and the coupling has been optimized via the natural selection. Our work provides evidence of hydrogen bond coupling in bioactivity but also bridges the charge transport through single-enzyme junctions and enzyme activities.

Special Collection on Bioelectrochemistry

Special Collection on Bioelectrochemistry

Pyrroloquinoline quinone glucose dehydrogenase adopted in thermometric analysis for enhancement of glucose determination

A broad measurement range of glucose is often required in clinical analysis, especially for diabetic patients where glucose levels can be very high. Pyrroloquinoline quinone glucose dehydrogenase (PQQGDH) has previously been used in electrochemical quantification of glucose with an extended linear range. However, in real sample determination of glucose, interferences from electroactive substances in blood are unavoidable. Calorimetric biosensors, e.g., the Enzyme Thermistor, are insensitive to either directly electroactive or optical interferences often present in real clinical samples. This paper describes a novel analytical strategy where the intrinsic advantages of PQQGDH are combined with the Enzyme Thermistor as biosensor using calorimetric detection as general measurement principle. When compared with the most frequently used enzyme glucose oxidase, PQQGDH has a higher catalytic efficiency and is insensitive to the availability of oxygen. The use of calorimetry in this context resulted in a broad linear range of glucose measurements, from 0.009 to 100 mM, an excellent specificity and insignificant side effects of compounds present in blood at high concentrations, such as lactate and urea.

Ca2+-Switchable Glucose Dehydrogenase Associated with Electrochemical/Electronic Interfaces: Applications to Signal-Controlled Power Production and Biomolecular Release.

An artificial Ca2+-regulated PQQ glucose dehydrogenase (PQQ-GDH) enzyme was electrically connected to conducting electrodes and semiconductor interfaces. Direct electron transfer from the enzyme to the conducting electrode support was stimulated by the addition of Ca2+ cations resulting in reversible enzyme activation. A signal-switchable biofuel cell and biomolecular release have been realized using the Ca2+-activated enzyme immobilized on conducting electrodes. Interfacing the signal-switchable enzyme with a semiconductor chip allowed electronic read out of the enzyme ON-OFF states. The developed approach based on the signal-regulated PQQ-GDH enables numerous bioelectrochemical/bioelectronic applications of the developed systems in signal-activated biosensors and biofuel cells, as well as in biomolecular computing/logic systems.

Highly Selective and Sensitive Self-Powered Glucose Sensor Based on Capacitor Circuit

Enzymatic glucose biosensors are being developed to incorporate nanoscale materials with the biological recognition elements to assist in the rapid and sensitive detection of glucose. Here we present a highly sensitive and selective glucose sensor based on capacitor circuit that is capable of selectively sensing glucose while simultaneously powering a small microelectronic device. Multi-walled carbon nanotubes (MWCNTs) is chemically modified with pyrroloquinoline quinone glucose dehydrogenase (PQQ-GDH) and bilirubin oxidase (BOD) at anode and cathode, respectively, in the biofuel cell arrangement. The input voltage (as low as 0.25 V) from the biofuel cell is converted to a stepped-up power and charged to the capacitor to the voltage of 1.8 V. The frequency of the charge/discharge cycle of the capacitor corresponded to the oxidation of glucose. The biofuel cell structure-based glucose sensor synergizes the advantages of both the glucose biosensor and biofuel cell. In addition, this glucose sensor favored a very high selectivity towards glucose in the presence of competing and non-competing analytes. It exhibited unprecedented sensitivity of 37.66 Hz/mM.cm2 and a linear range of 1 to 20 mM. This innovative self-powered glucose sensor opens new doors for implementation of biofuel cells and capacitor circuits for medical diagnosis and powering therapeutic devices.

Determination of Dehydrogenase Activities Involved in D-Glucose Oxidation in Gluconobacter and Acetobacter Strains.

Acetic acid bacteria (AAB) are known for rapid and incomplete oxidation of an extensively variety of alcohols and carbohydrates, resulting in the accumulation of organic acids as the final products. These oxidative fermentations in AAB are catalyzed by PQQ- or FAD- dependent membrane-bound dehydrogenases. In the present study, the enzyme activity of the membrane-bound dehydrogenases [membrane-bound PQQ-glucose dehydrogenase (mGDH), D-gluconate dehydrogenase (GADH) and membrane-bound glycerol dehydrogenase (GLDH)] involved in the oxidation of D-glucose and D-gluconic acid (GA) was determined in six strains of three different species of AAB (three natural and three type strains). Moreover, the effect of these activities on the production of related metabolites [GA, 2-keto-D-gluconic acid (2KGA) and 5-keto-D-gluconic acid (5KGA)] was analyzed. The natural strains belonging to Gluconobacter showed a high mGDH activity and low activity in GADH and GLDH, whereas the Acetobacter malorum strain presented low activity in the three enzymes. Nevertheless, no correlation was observed between the activity of these enzymes and the concentration of the corresponding metabolites. In fact, all the tested strains were able to oxidize D-glucose to GA, being maximal at the late exponential phase of the AAB growth (24 h), which coincided with D-glucose exhaustion and the maximum mGDH activity. Instead, only some of the tested strains were capable of producing 2KGA and/or 5KGA. In the case of Gluconobacter oxydans strains, no 2KGA production was detected which is related to the absence of GADH activity after 24 h, while in the remaining strains, detection of GADH activity after 24 h resulted in a high accumulation of 2KGA. Therefore, it is possible to choose the best strain depending on the desired product composition. Moreover, the sequences of these genes were used to construct phylogenetic trees. According to the sequence of gcd, gene coding for mGDH, Acetobacter and Komagataeibacter were phylogenetically more closely related each other than with Gluconobacter.