Determination of lactate dehydrogenase in human erythrocytes by capillary electrophoresis with electrochemical detection

Capillary electrophoresis (CE) was employed to analyze lactate dehydrogenase (LDH) in human erythrocytes using an amperometric detector with a carbon fiber micro-disk bundle electrode. LDH activity was measured by determining the amount of NADH generated by LDH through a enzyme-catalyzed reaction between NAD(+) and lithium lactate. The factors influencing the enzyme-catalyzed reaction, separation and detection were examined and optimized. The following conditions were suitable for the determination of LDH: running buffer, 5.0 x 10(-2)mol/l Tris-HCl (pH 7.5); separation voltage, 20.0 kV; detection potential, 1.00 V (versus saturated calomel electrode (SCE)). The conditions of enzyme-catalyzed reaction were: reaction buffer, 5.0 x 10(-2)mol/l Tris-HCl (pH 9.3); substrates, 5.0 x 10(-2)mol/l lithium lactate and 5.0 x 10(-3)mol/l NAD(+); reaction time, 10 min. The concentration limit of detection (LOD) of the method was 0.017 U/ml at a signal-to-noise (S/N) ratio of 3, which corresponded to 1.10 x 10(-10)mol/l, and the mass LOD was 2 x 10(-20)mol. The linear dynamic range was 0.039-4.65 U/ml for the injection voltage of 5.0 kV and injection time of 10s. The relative standard deviation (R.S.D.) was 0.85% for the migration time and 1.8% for the electrophoretic peak area. The method was applied to determine LDH in human erythrocytes. The recovery of the method was between 98 and 101%.     

Development of a xylitol biosensor composed of xylitol dehydrogenase and diaphorase

In preparation for the development of a xylitol biosensor, the xylitol dehydrogenase of Candida tropicalis IFO 0618 was partially purified and characterized. The optimal pH and temperature of the xylitol dehydrogenase were pH 8.0 and 50 degrees C, respectively. Of the various alcohols tested, xylitol was the most rapidly oxidized, with sorbitol and ribitol being reduced at 65% and 58% of the xylitol rate. The enzyme was completely inactive on arabitol, xylose, glucose, glycerol, and ethanol. The enzyme's xylitol oxidation favored the use of NAD+ (7.9 U/mg) over NADP+ (0.2 U/mg) as electron acceptor, while the reverse reaction, D-xylulose reduction, favored NADPH (7.7 U/mg) over NADH (0.2 U/mg) as electron donor. The K(m) values for xylitol and NAD+ were 49.8 mM and 38.2 microM, respectively. For the generation of the xylitol biosensor, the above xylitol dehydrogenase and a diaphorase were immobilized on bromocyan-activated sephallose. The gel was then attached on a dissolved oxygen electrode. In the presence of vitamin K3, NAD+ and phosphate buffer, the biosensor recorded a linear response to xylitol concentration up to 3 mM. The reaction was stable after 15 min. When the biosensor was applied to a flow injection system, optimal operation pH and temperature were 8.0 and 30 degrees C, respectively. The strengths and limitations of the xylitol biosensor are its high affinity for NAD+, slow reaction time, narrow linear range of detection, and moderate affinity for xylitol.     

Voltammetric biosensors for the determination of formate and glucose-6-phosphate based on the measurement of dehydrogenase-generated NADH and NADPH

This paper describes the development of a modified electrode for the electrocatalytic oxidation of beta-nicotinamide adenine dinucleotide (beta-NADH) and beta-nicotinamide adenine dinucleotide phosphate (beta-NADPH) using electropolymerised 3,4-dihydroxybenzaldehyde (3,4-DHB). Two voltammetric biosensors using enzyme-immobilised membranes were constructed for the determination of formic acid and glucose-6-phosphate (G6P), respectively. The formic acid biosensor based on the combination of formate dehydrogenase (FDH)-modified membrane with 3,4-DHB-coated glassy carbon electrode is one to two orders more sensitive (LOD, 5.0x10(-5) M) than previously reported electrochemical biosensors. Similarly, lower detection limit (4.0x10(-5) M) for the measurement of G6P was achieved using glucose-6-phosphate dehydrogenase (G6PDH) in the presence of beta-NADP(+). The interference of uric acid and ascorbate was minimised by incorporating an additional membrane modified with uricase and ascorbate oxidase, respectively. The biosensing scheme developed in this study can be adopted universally with a number of dehydrogenases for the detection of different substrates.     

Lactate biosensor and energy supply for the enzyme molecule

A biosensor of lactate has been constructed, made, and tested. The lactate biosensor uses the lactate dehydrogenase molecules from muscle. The lactate biosensor works according to the simplest scheme. An immobilized lactate dehydrogenase molecule binds a L-lactate molecule in the absence of the coenzyme NAD+. Then the L-lactate molecule is oxidized by the electric field of a metal electrode of the biosensor to generate an electron. The transfer of this electron between the immobilized lactate dehydrogenase molecule and the metal electrode of the biosensor is carried out without a redox mediator molecule. A new mechanism for the energy supply of the enzyme molecule is proposed to explain this effect. The new mechanism is based on the electric dipole-dipole interactions occurring in the enzyme molecule and surrounding water and on the thermal energy of this water.     

Internal supply of coenzyme to an amperometric glucose biosensor based on a chemically modified electrode

A biosensor for glucose using glucose dehydrogenase immobilized on a chemically modified graphite electrode was supplied with coenzyme, nicotinamide adenine dinucleotide (NAD+), through pores in the material. A graphite rod was hollowed out, leaving 0.3 mm at the end contacting the solution, filled with 10 mM NAD+ and pressurized. The response factor was 40% of that obtained when 2 mM NAD+ was mixed with the sample solution in a flow system. The coenzyme consumption was 11 microliters h-1 representing a 500-fold saving compared to supply through the bulk solution. The biosensor had a linear calibration curve from the detection limit, 1 microM, to 2 mM glucose and a repeatability of 0.3%. The graphite electrode was modified by adsorption of a bis-(benzophenoxazinyl)-terephthaloyl derivative in order to be able to oxidize NADH at 0 mV versus Ag/AgCl, 0.1 M KCl.     

Bioluminescent flow sensors: L-lactate dehydrogenase activity determination in serum

The catalytic activity of serum L -lactate dehydrogenase (LDH), was determined by monitoring the NADH produced by LDH with bacterial bioluminescent enzymes immobilized on a nylon coil. The LDH reaction of L -lactate with NAD took place in a flow-through mixing coil that preceded the bioluminescent detector coil. The response was linear from 1 to 5000 U/l at 37°C and from 3 to 2000 U/l at 25°C. The intra- and inter-assay reproducibility (CV%) were less than 10% and recovery range was 92% to 110%. The results agreed well with those obtained with a spectrophotometric method.     

Low potential detection of NADH based on Fe₃O₄ nanoparticles/multiwalled carbon nanotubes composite: fabrication of integrated dehydrogenase-based lactate biosensor

Fe(3)O(4) magnetic nanoparticles were in situ loaded on the surface of multiwalled carbon nanotubes (MWCNTs) by a simple coprecipitation procedure. The resulting Fe(3)O(4)/MWCNTs nanocomposite brings new capabilities for electrochemical sensing by combining the advantages of Fe(3)O(4) magnetic nanoparticles and MWCNTs. It was found that Fe(3)O(4) has redox properties similar to those of frequently used mediators used for electron transfer between NADH and electrode. The cyclic voltammetric results indicated the ability of Fe(3)O(4)/MWCNTs modified GC electrode to catalyze the oxidation of NADH at a very low potential (0.0 mV vs. Ag/AgCl) and subsequently, a substantial decrease in the overpotential by about 650 mV compared with the bare GC electrode. The catalytic oxidation current allows the stable and selective amperometric detection of NADH at an applied potential of 0.0 mV (Ag/AgCl) with a detection limit of 0.3 μM and linear response up to 300 μM. This modified electrode can be used as an efficient transducer in the design of biosensors based on coupled dehydrogenase enzymes. Lactate dehydrogenase (LDH) and NAD(+) were subsequently immobilized onto the Fe(3)O(4)/MWCNTs nanocomposite film by covalent bond formation between the amine groups of enzyme or NAD(+) and the carboxylic acid groups of the Fe(3)O(4)/MWCNT film. Differential pulse voltammetric detection of lactate on Fe(3)O(4)/MWCNT/LDH/NAD(+) modified GC electrode gives linear responses over the concentration range of 50-500 μM with the detection limit of 5 μM and sensitivity of 7.67 μA mM(-1). Furthermore, the applicability of the sensor for the analysis of lactate concentration in human serum samples has been successfully demonstrated.     

The non-specific inhibition of enzymes by environmental pollutants: a study of a model system towards the development of electrochemical biosensor arrays

Previous research has shown that lactate dehydrogenase (LDH) was competitively inhibited by pentachlorophenol (PCP) and a modified assay produced a detection limit of 1 μM (270 μg l⁻¹). This work used spectrophotometric rate-determination but in order to move towards biosensor development the selected detection method was electrochemical. The linkage of LDH to lactate oxidase (LOD) provided the electroactive species, hydrogen peroxide. This could be monitored using a screen-printed carbon electrode (SPCE) incorporating the mediator, cobalt phthalocyanine, at a potential of +300 mV (vs. Ag/AgCl). A linked LDH/LOD system was optimised with respect to inhibition by PCP. It was found that the SPCE support material, PVC, acted to reduce inhibition, possibly by combining with PCP. A cellulose acetate membrane removed this effect. Inhibition of the system was greatest at enzyme activities of 5 U ml⁻¹ LDH and 0.8 U ml⁻¹ LOD in reactions containing 246 μM pyruvate and 7.5 μM NADPH. PCP detection limits were an EC₁₀ of 800 nM (213 μg l⁻¹) and a minimum inhibition detectable (MID) limit of 650 nM (173 μg l⁻¹). The inclusion of a third enzyme, glucose dehydrogenase (GDH), provided cofactor recycling to enable low concentrations of NADPH to be incorporated within the assay. NADPH was reduced from 7.5 to 2 μM. PCP detection limits were obtained for an assay containing 5 U ml⁻¹ LDH, 0.8 U ml⁻¹ LOD and 0.1 U ml⁻¹ GDH with 246 μM pyruvate, 400 mM glucose and 2 μM NADPH. The EC₁₀ limit was 150 nM (39.9 μg l⁻¹) and the MID was 100 nM (26.6 μg l⁻¹). The design of the inhibition assays discussed has significance as a model for other enzymes and moves forward the possibility of an electrochemical biosensor array for pollution monitoring.     

Evaluation of different strategies for the development of amperometric biosensors for L-lactate

Two strategies were investigated for the development of lactate biosensors based on sol-gel matrixes and polysulfone composite films, both containing L-lactate dehydrogenase (LDH). Firstly, reagentless disposable screen-printed electrodes (SPE's) with Meldola's Blue (MB) and the cofactor NAD(+) inside a sol-gel matrix were prepared. These showed relatively low sensitivities (260 microA/M). Secondly, mediator-modified-polysulfone-graphite composite films deposited over both cylindrical epoxy-graphite and SPE's. These electrodes showed enhanced performance characteristics: improved sensitivity (80 mA/M), detection limit (0.87 microM) and reproducibility (2%). Reagentless electrodes, incorporating NAD(+) in the polysulfone film, had a decreased sensitivity, although better than that achieved by the sol-gel electrodes. While sol-gel electrodes showed a linear range between 1.25 x 10(-4) and 2.48 x 10(-3)M, the epoxy-graphite composite electrodes based on polysulfone composite films allowed the detection of lactate at a linear range of lower concentrations from 1 x 10(-6) to 1.2 x 10(-5)M. Finally, the performance of the LDH-MB-polysulfone-composite film-based SPE's in a flow system was studied. Short response times were obtained (t<30s). Furthermore, repeatability and reproducibility values were notably improved, especially when working with electrodes covered with a polyamide layer prepared with N-(2-aminoethyl)-piperazine.     

Optical analysis of lactate dehydrogenase and glucose by CdTe quantum dots and their dual simultaneous detection

Biomolecules detection by size-controlled quantum dots (QDs) was promising in developing clinic diagnostic techniques. In this work, a novel bioanalytical platform was developed to detect the activity of nicotinamide adenine dinucleotide (NAD) dependent enzyme, lactate dehydrogenase (LDH), and the concentration of glucose by the changes of fluorescence intensities of the QDs based on the electron transfer between QDs and sensitive biomolecules. The fluorescence intensities of the QDs was firstly quenched by NAD and then intensified with increasing amounts of the LDH because of the consumption of the NAD by the biocatalyzed reaction. Also the glucose led to the decline of fluorescence due to the formation of hydrogen peroxide (H(2)O(2)) which was the product of the glucose reacting with the glucose oxidase (GOD). The linear calibration plots of the activity of LDH and glucose were obtained from 250 to 6000 U/L and 1.67 to 6.67 mM, respectively. The detection system was also successfully applied to detect LDH and glucose in human serum samples. This analysis process was very convenient and simple because the QDs need not to be modified by any organic or biological molecules before they were used into the system. Moreover, the established method had great potential in detection of the physiological level of some biomolecules in clinical diagnostics of various diseases.     

Dehydrogenase based reagentless biosensor for monitoring phenylketonuria

Phenylketonuria (PKU) is a disease characterized by an inability to metabolize the amino acid l-phenylalanine. The resulting buildup leads to brain damage and ultimately mental retardation in children if their phenylalanine intake is not carefully controlled. The National Institutes of Health recently suggested that people with PKU monitor their phenylalanine levels throughout their life and be put on a low phenylalanine diet. As an alternative approach to analysis using blood, this paper describes the first reagentless dehydrogenase based sensor for the determination of phenylalanine in human urine. The clinical range of phenylalanine in human urine is 20-60mM for people with PKU. Although most clinical analysis is performed using blood, urine was chosen due to its high concentrations of phenylalanine in phenylketonurics, as well as its simple, safe, and painless collection. The sensor is comprised of a carbon paste electrode with nicotinamide adenine dinucleotide (NAD(+)), phenylalanine dehydrogenase (PDH), uricase, and an electron mediator, 3,4-dihydroxybenzaldehyde (3,4-DHB), all mixed into the paste. The electron mediator reacts with the electrode surface to produce two redox species, which catalytically oxidize NADH. The behavior of the electron mediator mixed into a carbon paste electrode has not been previously investigated. Cyclic voltammetry was used to characterize the sensor's response to NADH, and with the addition of PDH and NAD(+) to the paste, its response to phenylalanine in human urine. The limit of detection for phenylalanine is 0.5mM (S/N=3).     

Development of a glucose-6-phosphate biosensor based on coimmobilized p-hydroxybenzoate hydroxylase and glucose-6-phosphate dehydrogenase

This work reports the development of an amperometric glucose-6-phosphate biosensor by coimmobilizing p-hydroxybenzoate hydroxylase (HBH) and glucose-6-phosphate dehydrogenase (G6PDH) on a screen-printed electrode. The principle of the determination scheme is as follows: G6PDH catalyzes the specific dehydrogenation of glucose-6-phosphate by consuming NADP(+). The product, NADPH, initiates the irreversible the hydroxylation of p-hydroxybenzoate by HBH in the presence of oxygen to produce 3,4-dihydroxybenzoate, which results in a detectable signal due to its oxidation at the working electrode. The sensor shows a broad linear detection range between 2 microM and 1000 microM with a low detection limit of 1.2 microM. Also, it has a fast measuring time which can achieve 95% of the maximum current response in 20s after the addition of a given concentration of glucose-6-phosphate with a short recovery time (2 min).     

Investigation and evaluation of a method for determination of ethanol with the SIRE Biosensor P100, using alcohol dehydrogenase as recognition element

A new method for rapid determination of ethanol was developed, using alcohol dehydrogenase as recognition element for the SIRE (sensors based on injection of the recognition element) Biosensor, which is an amperometric biosensor. The method was simple, fast, accurate, specific and cost-effective. The recognition element solution used was stable at least for 24 h in room temperature, and at least one month when lyophilised. The optimal potential versus the silver wire electrode, the optimal pH of the buffer and the optimal temperature of the water bath was determined to be +950 mV, 8.1 and 308 K, respectively. The optimal concentrations of alcohol dehydrogenase, BSA and NAD(+) were determined to be 200 U/ml, 20 mg/ml and 15 mM, respectively. The total analysis time was between 50 s and 4 min per analysis, depending on the concentration range. The linear range was 0-12.5 mM. The detection limit was less than 0.1 mM. The repeatability (%R.S.D.) was 3-5% (n=10). The reproducibility was 5-8% (n=5). Methanol gave no signal at all, but higher alcohols, such as propanol, pentanol and hexanol, gave significant signals, decreasing with increasing length of the carbon chain. The price for one measurement was calculated to be 0.052 euro. The results from measurements with the biosensor were compared to those from an established analysis kit for ethanol. The results correlated well (R(2)=0.9874). The concentration of ethanol in different alcoholic beverages was investigated and correlated well with the concentrations given by the manufacturers.     

Direct electrochemistry of lactate dehydrogenase immobilized on silica sol-gel modified gold electrode and its application

The direct electrochemistry of lactate dehydrogenase (LDH) immobilized in silica sol-gel film on gold electrode was investigated, and an obvious cathodic peak at about -200 mV (versus SCE) was found for the first time. The LDH-modified electrode showed a surface controlled irreversible electrode process involving a one electron transfer reaction with the charge-transfer coefficient (alpha) of 0.79 and the apparent heterogeneous electron transfer rate constant (K(s)) of 3.2 s(-1). The activated voltammetric response and decreased charge-transfer resistance of Ru(NH(3))(6)(2+/3+) on the LDH-modified electrode provided further evidence. The surface morphologies of silica sol-gel and the LDH embedded in silica sol-gel film were characterized by SEM. A potential application of the LDH-modified electrode as a biosensor for determination of lactic acid was also investigated. The calibration range of lactic acid was from 2.0 x 10(-6) to 3.0 x 10(-5) mol L(-1) and the detection limit was 8.0 x 10(-7) mol L(-1) at a signal-to-noise ratio of 3. Finally, the effect of environmental pollutant resorcinol on the direct electrochemical behavior of LDH was studied. The experimental results of voltammetry indicated that the conformation of LDH molecule was altered by the interaction between LDH and resorcinol. The modified electrode can be applied as a biomarker to study the pollution effect in the environment.     

Kinetic analysis of duck epsilon-crystallin, a lens structural protein with lactate dehydrogenase activity.

Biochemical characterization and kinetic analysis of epsilon-crystallin from the lenses of common ducks were undertaken to elucidate the enzyme mechanism of this unique crystallin with lactate dehydrogenase (LDH) activity. Despite the structural similarities between epsilon-crystallin and chicken heart LDH, differences in charge and kinetic properties were revealed by isoenzyme electrophoresis and kinetic studies. Bi-substrate kinetic analysis examined by initial-velocity and product-inhibition studies suggested a compulsory ordered Bi Bi sequential mechanism with NADH as the leading substrate followed by pyruvate. The products were released in the order L-lactate and NAD+. The catalysed reaction is shown to have a higher rate in the formation of L-lactate and NAD+. Substrate inhibition was observed at high concentrations of pyruvate and L-lactate for the forward and reverse reactions respectively. The substrate inhibition was presumably due to the formation of epsilon-crystallin-NAD(+)-pyruvate or epsilon-crystallin-NADH-L-lactate abortive ternary complexes, as suggested by the product-inhibition studies. The significance and the interrelationship of duck epsilon-crystallin with other well-known LDHs are discussed with special regard to its role as a structural protein with some enzymic function in lens metabolism.     

A sensitive NADH and glucose biosensor tuned by visible light based on thionine bridged carbon nanotubes and gold nanoparticles multilayer

A NADH and glucose biosensor based on thionine cross-linked multiwalled carbon nanotubes (MWNTs) and Au nanoparticles (Au NPs) multilayer functionalized indium-doped tin oxide (ITO) electrode were presented in this paper. The effect of light irradiation on the enhancement of bioelectrocatalytic processes of the biocatalytic systems by the photovoltaic effect was investigated. This bioelectrode exhibited excellent catalytic activity of the oxidation towards dihydronicotinamide adenine dinucleotide (NADH). Most interesting, the performance of this NADH sensor could be tuned by the visible light. When the biosensor was performed in the dark, the anodic current increased linearly with NADH concentration over the range from 0.5 to 237 microM with detection limit 0.1 microM and sensitivity 17 nA microM(-1). The sensitivity became 115 nA microM(-1) with detection limit 0.05 microM with the light irradiation. Compared with the reaction in dark, the sensitivity increased around 7 folds while the detection limit decreased 2 folds. The glucose biosensor also exhibited the same behavior. The linear range was from 10 microM to 2.56 mM with the sensitivity of 7.8 microAmM(-1) and detection limit 5.0 microM in the dark. After the light irradiation, the linear range was from 1 microM to 3.25 mM with the sensitivity of 18.5 microA mM(-1) and detection limit 0.7 microM. It indicated a potential to provide an operational access to develop new kinds of photocontrolled dehydrogenase enzyme-based bioelectronics.     

An amperometric bi-enzyme sensor for determination of formate using cofactor regeneration

A biosensor for detection of formate at submicromolar concentrations has been developed by co-immobilizing formate dehydrogenase (FDH, E.C. 1.2.1.2), salicylate hydroxylase (SHL, E.C. 1.14.13.1) and NAD(+) linked to polyethylene glycol (PEG-NAD(+)) in a poly(vinyl alcohol) (PVA) matrix in front of a Clark-electrode. The principle of the bi-enzyme scheme is as follows: formate dehydrogenase converts formate into carbon dioxide using PEG-NAD(+). Corresponding PEG-NADH produced is then oxidized to PEG-NAD(+) by salicylate hydroxylase using sodium salicylate and oxygen. The oxygen consumption is monitored with the Clark-electrode. The advantages of this biosensor approach are the effective re-oxidation of PEG-NADH, and the entrapment of PEG-NAD(+) resulting in avoiding the addition of expensive cofactor to the working medium for each measurement. This bi-enzyme sensor has achieved a linear range of 1-300 microM and a detection limit of 1.98 x 10(-7) M for formate (S/N=3), with the response time of 4 min. The working stability is limited to 7 days due to the inactivation of the enzymes. Only sodium salicylate was needed in milli-molar amounts.     

Durable cofactor immobilization in sol-gel bio-composite thin films for reagentless biosensors and bioreactors using dehydrogenases

A new strategy directed to the durable immobilization of NAD(+)/NADH cofactors has been tested, along with a suitable redox mediator (ferrocene), in biocompatible sol-gel matrices encapsulating a bi-enzymatic system (a dehydrogenase and a diaphorase, this latter being useful to the safe regeneration of the cofactor), which were deposited as thin films onto glassy carbon electrode surfaces. It involves the chemical attachment of NAD(+) to the silica matrix using glycidoxypropylsilane in the course of the sol-gel process (in smooth chemical conditions). This approach based on chemical bonding of the cofactor (which was checked by infrared spectroscopy) led to good performances in terms of long-term stability of the electrochemical response. The possibility to integrate all components (proteins, cofactor, mediator) in the sol-gel layer in an active and durable form gave rise to reagentless devices with extended operational stability (i.e. high amperometric response maintained for more than 12h of continuous use under constant potential, whereas the signal completely vanished within the first few minutes of working with non-covalently bonded NAD(+)). To confirm the wide applicability of the proposed approach, the same strategy has been applied to the elaboration of biosensors for D-sorbitol, D-glucose and L-lactate with using D-sorbitol dehydrogenase, D-glucose dehydrogenase and L-lactate dehydrogenase respectively. The analytical characteristics of the glucose sensors are given and compared to previous approaches described in the literature for the elaboration of reagentless biosensors.     

Ultrasensitive detection of cortisol with enzyme fragment complementation technology using functionalized nanowire

Cortisol is a member of the glucocorticoid hormone family and a key metabolic regulator. Increased intracellular cortisol levels have been implicated in type 2 diabetes, obesity, and metabolic syndrome. Cortisol is an important bio-marker of stress and its detection is also important in sports medicine. However, rapid methods for sensitive detection of cortisol are limited. Functionalized gold nanowires were used to enhance the sensitivity and selectivity of cortisol detection. Gold nanowires are used to improve the electron transfer between the electrodes. Moreover, the large surface to volume ratio, small diffusion time and high electrical conductivity and their aligned nature will enhance the sensitivity and detection limit of the biosensor several fold. The biosensor was fabricated using, aligned gold (Au) nanowires to behave as the working electrode, platinum deposited on a silicon chip to function as the counter electrode, and silver/silver chloride as reference electrode. The gold nanowires were coupled with cortisol antibodies using covalent linkage chemistry and a fixed amount of 3alpha-hydroxysteroid dehydrogenase was introduced into the reaction cell during each measurement to convert (reduce) ketosteroid into hydroxyl steroid. Furthermore, the micro-fluidic, micro-fluid part of the sensor was fabricated using micro-electro-mechanical system (MEMS) technology to have better control on liquid flow over Au nanowires to minimize the signal to noise ratio. The biosensor was characterized using SEM, AFM and FTIR technique. The response curve of the biosensor was found to be linear in the range of 10-80 microM of cortisol. Moreover, the presence of hydrocortisone is sensitively detected in the range of 5-30 microM. It is concluded that the functionalized gold nanowires with micro-fluidic device using enzyme fragment complementation technology can provide an easy and sensitive assay for cortisol detection in serum and other biological fluids.     

Deep oxidation of glucose in enzymatic fuel cells through a synthetic enzymatic pathway containing a cascade of two thermostable dehydrogenases

A sensitive glutamate biosensor is prepared based on glutamate dehydrogenase/vertically aligned carbon nanotubes (GLDH, VACNTs). Vertically aligned carbon nanotubes were grown on a silicon substrate by direct current plasma enhanced chemical vapor deposition (DC-PECVD) method. The electrochemical behavior of the synthesized VACNTs was investigated by cyclic voltammetry and electrochemical impedance spectroscopic methods. Glutamate dehydrogenase covalently attached on tip of VACNTs. The electrochemical performance of the electrode for detection of glutamate was investigated by cyclic and differential pulse voltammetry. Differential pulse voltammetric determinations of glutamate are performed in mediator-less condition and also, in the presence of 1 and 5 μM thionine as electron mediator. The linear calibration curve of the concentration of glutamate versus peak current is investigated in a wide range of 0.1-500 μM. The mediator-less biosensor has a low detection limit of 57 nM and two linear ranges of 0.1-20 μM with a sensitivity of 0.976 mA mM(-1) cm(-2) and 20-300 μM with a sensitivity of 0.182 mA mM(-1) cm(-2). In the presence of 1 μM thionine as an electron mediator, the prepared biosensor shows a low detection limit of 68 nM and two linear ranges of 0.1-20 with a calibration sensitivity of 1.17 mA mM(-1) cm(-2) and 20-500 μM with a sensitivity of 0.153 mA mM(-1) cm(-2). The effects of the other biological compounds on the voltammetric behavior of the prepared biosensor and its response stability are investigated. The results are demonstrated that the GLDH/VACNTs electrode even without electron mediator is a suitable basic electrode for detection of glutamate.