Amperometric Ethanol Biosensors Based on Chitosan‐NAD+‐Alcohol Dehydrogenase Films

The reagentless and oxygen‐independent biosensors for ethanol were developed based on the covalent immobilization of alcohol dehydrogenase (ADH) and its cofactor nicotinamide adenine dinucleotide (NAD⁺) on chitosan (CHIT) chains. The CHIT‐NAD⁺‐ADH structures were adsorbed onto carbon nanotubes (CNT) in order to provide a signal transduction based on the recycling of redox states of NAD cofactor at CNT (detection limit, 8–30 µM ethanol; dynamic range up to 20 mM). The CHIT‐NAD⁺‐dehydrogenase/CNT hybrid material represents a general approach to the development of dehydrogenases‐based electrochemical biosensors. Interestingly, the CHIT‐NAD⁺ solutions preserved their enzymatic activity even after five years of storage at 4 °C.     

Discovery of an NAD+ analogue with enhanced specificity for PARP1

Among various protein posttranslational modifiers, poly-ADP-ribose polymerase 1 (PARP1) is a key player for regulating numerous cellular processes and events through enzymatic attachments of target proteins with ADP-ribose units donated by nicotinamide adenine dinucleotide (NAD⁺). Human PARP1 is involved in the pathogenesis and progression of many diseases. PARP1 inhibitors have received approvals for cancer treatment. Despite these successes, our understanding about PARP1 remains limited, partially due to the presence of various ADP-ribosylation reactions catalyzed by other PARPs and their overlapped cellular functions. Here we report a synthetic NAD⁺ featuring an adenosyl 3′-azido substitution. Acting as an ADP-ribose donor with high activity and specificity for human PARP1, this compound enables labelling and profiling of possible protein substrates of endogenous PARP1. It provides a unique and valuable tool for studying PARP1 in biology and pathology and may shed light on the development of PARP isoform-specific modulators.

An analogue of nicotinamide adenine dinucleotide (NAD⁺) featuring an azido group at 3′-OH of adenosine moiety is found to possess high specificity for human PARP1-catalyzed protein poly-ADP-ribosylation.     

Emissive Synthetic Cofactors: Enzymatic Interconversions of tzA Analogues of ATP,NAD+, NADH,NADP+, and NADPH

A series of enzymatic transformations, which generate visibly emissive isofunctional cofactors based on an isothiazolo[4,3‐d]pyrimidine analogue of adenosine ( ^tz A ), was developed. Nicotinamide adenylyl transferase condenses nicotinamide mononucleotide and ^tz ATP to yield N^tzAD⁺ , which can be enzymatically phosphorylated by NAD⁺ kinase and ATP or ^tz ATP to the corresponding N^tzADP⁺ . The latter can be engaged in NADP‐specific coupled enzymatic transformations involving conversion to N^tzADPH by glucose‐6‐phosphate dehydrogenase and reoxidation to N^tzADP⁺ by glutathione reductase. The N^tzADP⁺ / N^tzADPH cycle can be monitored in real time by fluorescence spectroscopy.     

Emissive Synthetic Cofactors: Enzymatic Interconversions of tzA Analogues of ATP,NAD+, NADH,NADP+, and NADPH

A series of enzymatic transformations, which generate visibly emissive isofunctional cofactors based on an isothiazolo[4,3‐d]pyrimidine analogue of adenosine ( ^tz A ), was developed. Nicotinamide adenylyl transferase condenses nicotinamide mononucleotide and ^tz ATP to yield N^tzAD⁺ , which can be enzymatically phosphorylated by NAD⁺ kinase and ATP or ^tz ATP to the corresponding N^tzADP⁺ . The latter can be engaged in NADP‐specific coupled enzymatic transformations involving conversion to N^tzADPH by glucose‐6‐phosphate dehydrogenase and reoxidation to N^tzADP⁺ by glutathione reductase. The N^tzADP⁺ / N^tzADPH cycle can be monitored in real time by fluorescence spectroscopy.     

Covalent Immobilization of Dehydrogenases on Carbon Felt for Reusable Anodes with Effective Electrochemical Cofactor Regeneration

This study presents the immobilization with aldehyde groups (glyoxyl carbon felt) of alcohol dehydrogenase (ADH) and formate dehydrogenase (FDH) on carbon‐felt‐based electrodes. The compatibility of the immobilization method with the electrochemical application was studied with the ADH bioelectrode. The electrochemical regeneration process of nicotinamide adenine dinucleotide in its oxidized form (NAD⁺), on a carbon felt surface, has been deeply studied with tests performed at different electrical potentials. By applying a potential of 0.4 V versus Ag/AgCl electrode, a good compromise between NAD⁺ regeneration and energy consumption was observed. The effectiveness of the regeneration of NAD⁺ was confirmed by electrochemical oxidation of ethanol catalyzed by ADH in the presence of NADH, which is the no active form of the cofactor for this reaction. Good reusability was observed by using ADH immobilized on glyoxyl functionalized carbon felt with a residual activity higher than 60 % after 3 batches.     

Electrical communication between electrode and dehydrogenase by a ferrocene-labeled high molecular-weight cofactor derivative: application to a reagentless biosensor

A ferrocene-labeled high molecular weight cofactor derivative (PEI-Fc-NAD) was prepared by attaching both ferrocene and nicotinamide adenine dinucleotide (NAD⁺) to a water soluble polyelectrolyte, polyethylenimine (PEI). Approximately 9.8% and 2.9% of all the primary amino groups of PEI were coupled with ferrocene and the bioactive cofactor, respectively. The cyclic voltammograms of PEI-Fc-NAD exhibited a one-electron transfer process, and the difference between the anodic and cathodic peak potential was found to be 80 mV. The PEI-Fc-NAD was used together with NAD-dependent dehydrogenase to construct a reagentless biosensor. An NAD-dependent alcohol dehydrogenase (ADH) was selected as the model enzyme, and both ADH and PEI-Fc-NAD were retained onto the sensing area of gold electrodes by a dialysis membrane or immobilized by layer-by-layer adsorption method. In both cases, the modified electrodes showed current response to ethanol without the addition of native NAD⁺ to the system, which suggested that the electrical communication between ADH and the electrode was achieved through PEI-Fc-NAD. In summary, PEI-Fc-NAD provides a new way for immobilization of mediator and cofactor, and exhibits the potential as a platform for constructing reagentless NAD-dependent dehydrogenase biosensors.     

Recycling of NAD+ crosslinked to albumin or hemoglobin immobilized with multienzyme systems in artificial cells

A soluble immobilized NAD⁺ was prepared by creating a peptide binding between bovine serum albumin and N⁶-[(6-aminohexyl)]carbamoyl-methyl] nicotinamide adenine dinucleotide. When immobilized within semipermeable microcapsules with alcohol dehydrodgenase (EC 1.1.1.1) and malic dehydrogenase (EC 1.1.1.37) the albumin-NAD⁺ exhibited high cofactor recycling rates. NAD⁺ can also be similarly crosslinked to hemoglobin.     

Effect of Enzyme and Cofactor Immobilization on the Response of Ethanol Oxidation in Zirconium Phosphate Modified Biosensors

Two different self‐contained ethanol amperometric biosensors incorporating layered [Ru(phend)₂bpy]²⁺‐intercalated zirconium phosphate (ZrP) as the mediator as well as yeast‐alcohol dehydrogenase (y‐ADH) and its cofactor nicotinamide adenine dinucleotide (NAD⁺) were constructed to improve upon a design previously reported where only this mediator was immobilized in the surface of a modified electrode. In the first biosensor, a [Ru(phend)₂bpy]²⁺‐intercalated ZrP modified carbon paste electrode (CPE) was improved by immobilizing in its surface both y‐ADH and NAD⁺ using quaternized Nafion membrane. In the second biosensor, a glassy carbon electrode was modified with [Ru(phend)₂bpy]²⁺‐intercalated ZrP, y‐ADH, and NAD⁺ using Nafion as the holding matrix. Calibration plots for ethanol sensing were constructed in the presence and absence of ZrP. In the absence of ZrP in the surface of the modified glassy carbon electrode, leaching of ADH was observed as detected by UV‐vis spectrophotometry. Ethanol sensing was also tested in the presence and absence of ascorbate to measure the selectivity of the sensor for ethanol. These two ethanol biosensors were compared to a previously reported one where the y‐ADH and the NAD⁺ were in solution, not immobilized.     

Mutation of Tyr-218 to Phe in <Emphasis Type="Italic">Thermoanaerobacter ethanolicus</Emphasis> Secondary Alcohol Dehydrogenase: Effects on Bioelectronic Interface Performance

Bioelectronic interfaces that facilitate electron transfer between the electrode and a dehydrogenase enzyme have potential applications in biosensors, biocatalytic reactors, and biological fuel cells. The secondary alcohol dehydrogenase (2° ADH) from Thermoanaerobacter ethanolicus is especially well suited for the development of such bioelectronic interfaces because of its thermostability and facile production and purification. However, the natural cofactor for the enzyme, β-nicotinamide adenine dinucleotide phosphate (NADP⁺), is more expensive and less stable than β-nicotinamide adenine dinucleotide (NAD⁺). PCR-based, site-directed mutagenesis was performed on 2° ADH in an attempt to adjust the cofactor specificity toward NAD⁺ by mutating Tyr²¹⁸ to Phe (Y218F 2° ADH). This mutation increased the K _m(app) for NADP⁺ 200-fold while decreasing the K _m(app) for NAD⁺ 2.5-fold. The mutant enzyme was incorporated into a bioelectronic interface that established electrical communication between the enzyme, the NAD⁺, the electron mediator toluidine blue O (TBO), and a gold electrode. Cyclic voltammetry, impedance spectroscopy, gas chromatography, mass spectrometry, constant potential amperometry, and chronoamperometry were used to characterize the mutant and wild-type enzyme incorporated in the bioelectronic interface. The Y218F 2° ADH exhibited a fourfold increase in the turnover ratio compared to the wild type in the presence of NAD⁺. The electrochemical and kinetic measurements support the prediction that the Rossmann fold of the enzyme binds to the phosphate moiety of the cofactor. During the 45 min of continuous operation, NAD⁺ was electrically recycled 6.7 × 10⁴ times, suggesting that the Y218F 2° ADH-modified bioelectronic interface is stable.     

411 - Role of adsorption in the electrochemical behaviour of nicotinamide adenine dinucleotide at a gold electrode

The role of adsorption in the electrochemical reduction of nicotinamide adenine dinucleotide (NAD⁺) at a gold electrode was examined by cyclic voltammetry and the measurement of specular reflectivity of the electrode⋎solution interface. Reduction of the pyridine ring in NAD⁺ takes place at a less negative potential compared with its compounds such as nicotinamide mononucleotide (NMN⁺) and nicotinamide. All of these compounds are found to be adsorbed on the electrode surface around the point of zero charge from the reflectance data. NAD⁺ remains adsorbed on the electrode surface until the potential scanning attains its reduction potential, whereas NMN⁺ and nicotinamide are desorbed at potentials far prior to reduction. A possible model of the adsorbed NAD⁺ was proposed to explain how the adsorption of NAD⁺ serves to make its reduction easier than NMN⁺ and nicotinamide.     

An enzymatic-fluorimetric method for monitoring of ethanol in ambient air

A method is described for the continuous monitoring of ethanol in ambient air. The system consists of a scrubber coil for enrichment of the analyte from air in an aqueous solution and a directly connected fluorescence detector. Because of using a reagent solution containing alcohol dehydrogenase (ADH) and nicotinamide adenine dinucleotide (NAD⁺) for absorption, ethanol can react directly with ADH and NAD⁺ during air sampling, producing NADH, which can be measured by fluorescence detection. The influence of reagent concentrations, gas flow rate and scrubber solution flow rate on the performance of the instrument was tested. Possible ozone interferences can be avoided by placing a KI coated filter in front of the scrubber inlet. The response time of the system was found to be 2.3 min and the detection limit about 1 ppb_V. The applicability of the developed method was demonstrated during a field campaign in Brazil.     

Coenzyme Regeneration in Hexanol Oxidation Catalyzed by Alcohol Dehydrogenase

The enzymatic ways of coenzyme regeneration include the addition of a second enzyme to the system or the addition of the co-substrate. In the present study, both methods of enzymatic coenzyme (NAD⁺) regeneration were studied and compared in the reaction of hexanol oxidation catalyzed by alcohol dehydrogenase (ADH). As a source of ADH, commercial isolated enzyme and the whole baker??s yeast cells were used. First, coenzyme regeneration was employed in the reaction of acetaldehyde reduction catalyzed by the same enzyme that catalyzed the main reaction, and then NAD⁺ regeneration was applied in the reaction of pyruvate reduction catalyzed by l-lactate dehydrogenase (l-LDH). Hexanal was obtained as the product of hexanol oxidation catalyzed by isolated ADH while hexaonic acid was detected as a product of the same reaction catalyzed by baker??s yeast cells. All of the used biocatalysts were kinetically characterized. The mass reactions were described by the mathematical models. All models were validated in the batch reactor. One hundred percent hexanol conversion was obtained using permeabilized yeast cells using both methods of cofactor regeneration. By using isolated enzyme ADH, the higher conversion was achieved in a system with cofactor regeneration catalyzed by l-LDH.     

482—Red-ox transformations of NAD+ model compounds

The electrochemical reduction of four Nl-substituted nicotinamides, and the properties of their electrochemically generated dimers, have been examined. Bearing in mind previously reported data for dimers of NAD⁺, NMN⁺. Nl-methylnicotinamide and the parent nicotinamide, it is shown that the nature of the Nl-substituent exerts a marked effect on the stability of the dimers and their conversion productsAll dimers undergo photo-oxidation to the parent monomers with quantum yields of the order of 0.1. The rate of oxidation by molecular oxygen and by an enzyme extract from mung beans, is much more rapid for dimers of N₁-subtituted nicotinamide then for dimers of NAD⁺ and NMN⁺,The lability of the dimer of the phosphate ester of N₁-hydroxyethylnicotinamide was particularly marked, due in this case to phosphate-catalysed, so-called acid hydration which, for this derivative, is so rapid that it occurs during electrolysis at pH 9.5. The phosphate group also influences the electrode process for reduction of the nicotinamide moiety on wave I, leading to a pH-dependence of U_1/2. Esterification of the secondary phosphate hydroxyl liquidates both effects.The ¹H NMR spectrum of the, NMN dimer indicates that it consists of several stereoisomers of the 4-4′ linked dimer The foregoing data are discussed in relation to the choice of suitable simpler model analogues for NAD⁺     

Multi‐walled Carbon Nanotubes Non‐covalently Functionalized with Polyarginine: A New Alternative for the Construction of Reagentless NAD+/Dehydrogenase‐based Ethanol Biosensor

This work reports for the first time the development of a reagentless enzymatic amperometric biosensor for ethanol based on the use of a glassy carbon electrode (GCE) modified with multi‐walled carbon nanotubes (MWCNTs) non‐covalently functionalized with polyarginine (Polyarg) as platform for the robust immobilization of alcohol dehydrogenase (ADH) and NAD⁺. The new strategy allows to obtain an integrated GCE/MWCNTs‐Polyarg/NAD⁺‐ADH ethanol biosensor with important advantages compared to the existing ethanol biosensors: avoids the external addition of the cofactor for each measurement, ensures a fast and sensitive quantification of ethanol due to the intimate interaction of the components, and allows the detection at considerably lower potentials due to the catalytic activity of the carbon nanostructures. These unique properties have made possible a very efficient ethanol quantification with a sensitivity of (1487±6) μA M^?1, detection limit of 0.65 μM, response time of 8 s, and reproducibility of 5.5 % with a very successful application for the quantification of ethanol in different commercial beverages.     

Equilibrium (NAD+/NADH) potential on poly(Neutral Red) modified electrode

The electrochemical regeneration of nicotinamide adenine dinucleotide (NAD⁺/NADH) has been one of the central subjects of bioelectrochemistry during past three decades. We report on the unique chemical electrocatalyst for NAD⁺/NADH regeneration based on electropolymerized Neutral Red. Using poly(Neutral Red) modified electrodes, the reversible polarographic waves of nicotinamide adenine dinucleotide reduction–oxidation and the equilibrium (NAD⁺/NADH) potential were observed. This was impossible using all known catalytic and mediator systems. The unique poly(Neutral Red) based electrocatalyst allowed us to determine the standard (NAD⁺/NADH) potential more precisely (E^′≅0.59 V SCE, pH 6.0).     

Detection of NAD+-dependent alcohol dehydrogenase activities in YR-1 strain of Mucor circinelloides, a potential bioremediator of petroleum contaminated soils

Different soluble NAD⁺-dependent alcohol dehydrogenase (ADH) isozymes were detected in cell-free homogenates from aerobically grown mycelia of YR-1 strain of Mucor circinelloides isolated from petroleumcontaminated soil samples. Depending on the carbon source present in the growth media, multiple NAD⁺-dependent ADHs were detected when hexadecane or decane was used as the sole carbon source in the culture media. ADH activities from aerobically or anaerobically grown mycelium or yeast cells, respectively, were detected when growth medium with glucose added was the sole carbon source; the enzyme activity exhibited optimum pH for the oxidation of different alcohols (methanol, ethanol, and hexadecanol) similar to that of the corresponding aldehyde (≈7.0). Zymogram analysis conducted with partially purified fractions of extracts from aerobic mycelium or anaerobic yeast cells of the YR-1 strain grown in glucose as the sole carbon source indicated the presence of a single NAD⁺-dependent ADH enzyme in each case, and the activity level was higher in the yeast cells. ADH enzyme from mycelium grown in different carbon sources showed high activity using ethanol as substrate, although higher activity was displayed when the cells were grown in hexadecane as the sole carbon source. Zymogram analysis with these extracts showed that this particular strain of M. circinelloides has four different isozymes with ADH activity and, interestingly, one of them, ADH4, was identified also as phenanthrene-diol-dehydrogenase, an enzyme that possibly participates in the aromatic hydrocarbon biodegradation pathway.     

Amperometric Biosensor for Lactate Based on Meldola's Blue Adsorbed on Silica Gel Modified with Niobium Oxide

A reagentless amperometric biosensor sensitive to lactate was developed. This sensor comprises a carbon paste electrode modified with lactate dehydrogenase (LDH), nicotinamide adenine dinucleotide (NAD⁺) cofactor and Meldola's blue (MB) adsorbed on silica gel coated with niobium oxide. The amperometric response was based on the electrocatalytic properties of MB to oxidize NADH, which was generated in the enzymatic reaction of lactate with NAD⁺ under catalysis of LDH. The dependence on the biosensor response was investigated in terms of pH, supporting electrolyte, ionic strength, LDH and NAD⁺ amounts and applied potential. The biosensor showed an excellent operational stability (95% of the activity was maintained after 250 determinations) and storage stability (allowing measurements for over than 2.5 months, when stored in a refrigerator). The proposed biosensor also presented good sensitivity allowing lactate quantification at levels down to 6.5×10^?6 mol L^?1. Moreover, the biosensor showed a wide linear response range (from 0.1 to 14 mmol L^?1 for lactate). These favorable characteristics allowed its application for direct measurements of lactate in biological samples such as blood. The precision of the data obtained by the proposed biosensor show reliable results for real complex matrices.     

Preparation of an Electrode Modified with an Electropolymerized Film as a Mediator of NADH Oxidation and Its Application in an Ethanol/O2 Biofuel Cell

This paper reports a novel mediator for the oxidation of β‐nicotinamide adenine dinucleotide (NAD⁺/NADH), an electropolymeric film (pAPRu) of [Ru(NH₂‐phen)₃]²⁺. A pAPRu‐modified electrode was prepared via electropolymerization and exhibited catalytic activity toward the electrochemical oxidation of NADH due to the imine moieties of pAPRu. The electrochemical oxidation of ethanol was observed using an alcohol dehydrogenase (ADH)‐immobilized electrode. A compartmentless ethanol/O₂ biofuel cell composed of an ADH anode and a bilirubin oxidase cathode was constructed. The maximum current density and the maximum power density of the biofuel cell were 190 µA cm^?2 and 31 µW cm^?2 (at 0.29 V), respectively.     

Entrapment of co-enzymically active NAD+ polymers in fibres

A new functionalized NAD⁺ derivative, nicotinamide 6-(2-hydroxy-3-carboxypropylamino)purine dinucleotide (NAD⁺—But), synthesized in these laboratories, has been purified by ion-exchange chromatography and subsequently crystallized as a pure product. Its co-enzymatic activity has been determined with several dehydrogenases, values ranging from 80% to 40% relative to NAD⁺ being found.Soluble and insoluble high-molecular-weight NAD⁺ derivatives, obtained by coupling NAD⁺—But to several polymers, exhibit high percentages of co-enzymatic activity.Water-soluble high-molecular-weight NAD⁺ derivatives have been entrapped in cellulose triacetate fibres together with lactic and alanine dehydrogenases. The immobilized enzymic system was stable under operating conditions and catalyzed the synthesis of L-alanine from lactic acid and ammonia without requiring any co-enzyme in the external reaction mixture.