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.     

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.     

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.     

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.     

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.     

Reagentless d-sorbitol biosensor based on d-sorbitol dehydrogenase immobilized in a sol–gel carbon nanotubes–poly(methylene green) composite

A reagentless d-sorbitol biosensor based on NAD-dependent d-sorbitol dehydrogenase (DSDH) immobilized in a sol–gel carbon nanotubes–poly(methylene green) composite has been developed. It was prepared by durably immobilizing the NAD⁺ cofactor with DSDH in a sol–gel thin film on the surface of carbon nanotubes functionalized with poly(methylene green). This device enables selective determination of d-sorbitol at 0.2 V with a sensitivity of 8.7?μA?mmol^?1?L?cm^?2 and a detection limit of 0.11 mmol?L^?1. Moreover, this biosensor has excellent operational stability upon continuous use in hydrodynamic conditions.

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.     

Emissive Synthetic Cofactors: A Highly Responsive NAD+ Analogue Reveals Biomolecular Recognition Features

Apart from its vital function as a redox cofactor, nicotinamide adenine dinucleotide ( NAD⁺ ) has emerged as a crucial substrate for NAD⁺ -consuming enzymes, including poly(ADP-ribosyl)transferase 1 (PARP1) and CD38/CD157. Their association with severe diseases, such as cancer, Alzheimer's disease, and depressions, necessitates the development of new analytical tools based on traceable NAD⁺ surrogates. Here, the synthesis, photophysics and biochemical utilization of an emissive, thieno[3,4-d]pyrimidine-based NAD⁺ surrogate, termed N^thAD⁺ , are described. Its preparation was accomplished by enzymatic conversion of synthetic ^th ATP by nicotinamide mononucleotide adenylyltransferase 1 (NMNAT1). The new NAD⁺ analogue possesses useful photophysical features including redshifted absorption and emission maxima as well as a relatively high quantum yield. Serving as a versatile substrate, N^thAD⁺ was reduced by alcohol dehydrogenase (ADH) to N^thADH and afforded ^thADP-ribose ( ^th ADPr ) upon hydrolysis by NAD⁺ -nucleosidase (NADase). Furthermore, N^thAD⁺ was engaged in cholera toxin A (CTA)-catalyzed mono(^thADP-ribosyl)ation, but was found incapable in promoting PARP1-mediated poly(^thADP-ribosyl)ation. Due to its high photophysical responsiveness, N^thAD⁺ is suited for spectroscopic real-time monitoring. Intriguingly, and as an N7-lacking NAD⁺ surrogate, the thieno-based cofactor showed reduced compatibility (i.e., functional similarity compared to native NAD⁺ ) relative to its isothiazolo-based analogue. The distinct tolerance, displayed by diverse NAD⁺ producing and consuming enzymes, suggests unique biological recognition features and dependency on the purine N7 moiety, which is found to be of importance, if not essential, for PARP1-mediated reactions.     

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.     

Electroenzymatic reactions with sorbitol dehydrogenase on gold electrodes

Sorbitol dehydrogenase (SDH) originating from recombinant Escherichia coli cells is immobilized on gold electrodes. First of all, (4-carboxy-2,5,7-trinitrofluorenyliden)malon-nitrile (CTFM) is adsorbed on the surface as mediator. In a second step, the cofactor β-nicotinamide adenine dinucleotide (NAD⁺) is immobilized on the gold electrode. Due to the formation of a complex between the mediator and the cofactor, the electron transfer rate can be enhanced by adding calcium ions to the buffer. The immobilization of NAD⁺ and SDH on the surface has been achieved by cross-linking with the glutaraldehyde/bovine serum albumin system. The successful biofunctionalization is monitored by cyclic voltammetry.Paper presented at the “Jahrestagung der Fachgruppe Angewandte Electrochemie der Gesellschaft Deutscher Chemiker, Düsseldorf, 11.-14.09.2005”.     

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.     

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.     

Amperometric assay for the ketone body 3-hydroxybutyrate

A stable dry-strip electrochemical sensor for the direct measurement of 3-hydroxybutyrate in blood is described. The sensor utilizes the electrocatalytic oxidation of enzymically generated NADH by the redox mediator 4-methyl-o-quinone. The enzyme 3-hydroxybutyrate dehydrogenase, cofactor NAD⁺ and 4-methyl-o-quinone were incorporated into single-use disposable strip electrodes.     

Construction of L‐Lysine Sensor by Layer‐by‐Layer Adsorption of L‐Lysine 6‐Dehydrogenase and Ferrocene‐Labeled High Molecular Weight Coenzyme Derivative on Gold Electrode

A ferrocene‐labeled high molecular weight coenzyme derivative (PEI‐Fc‐NAD) and a thermostable NAD‐dependent L ‐lysine 6‐dehydrogenase (LysDH) from thermophile Geobacillus stearothermophilus were used to fabricate a reagentless L ‐lysine sensor. Both LysDH and PEI‐Fc‐NAD were immobilized on the surface of a gold electrode by consecutive layer‐by‐layer adsorption (LBL) technique. By the simple LBL method, the reagentless L ‐lysine sensor, with co‐immobilization of the mediator, coenzyme, and enzyme was obtained, which exhibited current response to L ‐lysine without the addition of native coenzyme to the analysis system. The amperometric response of the sensor was dependent on the applied potential, bilayer number of PEI‐Fc‐NAD/LysDH, and substrate concentration. A linear current response, proportional to L ‐lysine concentration in the range of 1–120 mM was observed. The response of the sensor to L ‐lysine was decreased by 30% from the original activity after one month storage.     

Catalytic electrooxidation of NADH for dehydrogenase amperometric biosensors

The developments in the techniques of NADH catalytic oxidation relevant for incorporation in amperometric biosensors with dehydrogenase enzymes are reviewed with special emphasis in the years following 1990. The review stresses the direct electro-catalytic methods of NAD⁺ recycling as opposed to enzymatic regeneration of the coenzyme. These developments are viewed and evaluated from a mechanistic perspective of recycling of NADH to enzymatically active NAD⁺, and from the point of view of development of technologically useful reagentless dehydrogenase biosensors. An effort is made to propose a method for the standardization of evaluation of new mediating and direct coenzyme recycling schemes. A perspective is given for the requirements that have to be met for successful biosensor development incorporating dehydrogenase enzymes that open the analytical possibilities to a number of new analytes. The intrinsic limitations of the system are finally discussed and a view of the future of the field is presented.     

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.     

NADP+-Specific Isocitrate Dehydrogenase from Oleaginous Yeast Yarrowia lipolytica CLIB122: Biochemical Characterization and Coenzyme Sites Evaluation

NADP⁺-dependent isocitrate dehydrogenase from Yarrowia lipolytica CLIB122 (YlIDP) was overexpressed and purified. The molecular mass of YlIDP was estimated to be about 81.3 kDa, suggesting its homodimeric structure in solution. YlIDP was divalent cation dependent and Mg²⁺ was found to be the most favorable cofactor. The purified recombinant YlIDP displayed maximal activity at 55 °C and its optimal pH for catalysis was found to be around 8.5. Heat inactivation studies revealed that the recombinant YlIDP was stable below 45 °C, but its activity dropped quickly above this temperature. YlIDP was absolutely dependent on NADP⁺ and no NAD-dependent activity could be detected. The K _m values displayed for NADP⁺ and isocitrate were 59 and 31 μM (Mg²⁺), 120 μM and 58 μM (Mn²⁺), respectively. Mutant enzymes were constructed to tentatively alter the coenzyme specificity of YlIDP. The K _m values for NADP⁺ of R322D mutant was 2,410 μM, being about 41-fold higher than that of wild type enzyme. NAD⁺-dependent activity was detected for R322D mutant and the K _m and k _cat values for NAD⁺ were 47,000 μM and 0.38 s^?1, respectively. Although the R322D mutant showed low activity with NAD⁺, it revealed the feasibility of engineering an eukaryotic IDP to a NAD⁺-dependent one.     

Attempting to remove the substrate inhibition ofL-lactate dehydrogenase fromBacillus stearothermophilus by site-directed mutagenesis

L-lactate dehydrogenase (LDH) catalyzes the interconversion of an oxoacid (pyruvate) and hydroxy-acid (lactate) using the NADH/NAD⁺ pair as a redox cofactor. The enzyme has a commercial significance, as it can be used to produce chiral building blocks for the synthesis of key pharmaceuticals and agrochemicals. However, the substrate inhibition which is due to an abortive NAD⁺-pyruvate complex reducing the steady state concentration of functional LDH limits its use in industry. This substrate inhibition can be overcome by weaking the binding of NAD⁺. The conserved aspartic acid residue at position 38 was replaced by the longer basic arginine side chain (D38R) using PCR based overlap extension mutagenesis technique in the hope of weakening NAD⁺-binding. The mutant gene was overexpressed in theEscherichia coli high-expression vector pKK223-3 in JM105 cells; then, the mutant protein was produced. Comparing the effect of substrate inhibition in the arginine-38 mutant with wild-type, substrate inhibition is decreased threefold.     

Sensing the Lactic Acid in Probiotic Yogurts Using an L-Lactate Biosensor Coupled with a Microdialysis Fiber Inserted in a Flow Analysis System

An amperometric biosensor for the determination of L-lactic acid in probiotic yogurts has been assembled using L-lactate dehydrogenase (EC 1.1.1.27, LDH) entrapped in 1% (v/v) neutralized Nafion® solution deposited on Variamine blue redox mediator modified screen-printed electrodes. The Variamine blue was previously covalently linked to oxidized single-walled carbon nanotubes and used for modifying screen-printed electrodes. The electrochemical cell, containing the L-lactate biosensor operating at an applied working potential of +200 mV vs. Ag|AgCl, was coupled with a microdialysis fiber and connected with a flow system, thus obtaining a microdialysis based sampling experimental set-up. Various analytical parameters, such as the cofactor concentration (2 mM, NAD⁺), the flow rate (10.5 μL/min), the applied working potential (+200 mV vs. Ag|AgCl), the working buffer (50 mM phosphate buffer +0.1 M KCl), and pH (7.5), were optimized in batch amperometric experiments. The dynamic linear working range was comprised between 2·10^?4 and 1·10^?3 M. The proposed biosensor was challenged with real samples of yogurt, properly diluted in working buffer, and the performances of the L-lactate biosensor were compared with a commercially available kit for the determination of L-lactic acid in foodstuffs from R-Biopharm GmbH, Germany, showing a good agreement.     

Alcohol,lactate and glutamate sensors based on oxidoreductases with regeneration of nicotinamide adenine dinucleotide

Flowthrough enzyme electrodes are reported for determinations of alcohol, lactate and glutamate. Oxidoreductases mixed with immobilized NAD⁺ cofactor are held between a suitable platinum electrode and a semipermeable membrane. The coenzyme is readily regenerated either directly by electrochemical oxidation or by using phenazine methosulphate (PMS⁺) as intermediate. Continuous flow conditions are used. The sensitivity obtained with the alcohol dehydrogenase electrode was 50, 620 or 810 nA mol^-1 of ethanol, respectively, when regeneration was done electrochemically or with 0.1 or 0.5 mM PMS⁺. The sensitivities for the lactate and glutamate sensors in the presence of 0.5 mM PMS⁺, were 14 and 50 nA mmol^-1 for D,L-lactate and L-glutamate, respectively. The calibration curves were linear for concentrations up to 0.5, 1.5 and 100 mM of glutamate, lactate and ethanol, respectively. The sensitivity of the alcohol and lactate sensors decreased by 50–55% within 60 h and that of the glutamate sensor within 6 h.