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.     

Lactate Biosensor Based on Hydrotalcite‐Like Compounds: Performances and Application to Serum Samples

A lactate biosensor based on lactate oxidase supported onto a hydrotalcite, electrochemically deposited on a platinum surface, was developed for the first time. For the best electrode configuration, a linear response up to 0.8 mM, with a limit of detection of 14 μM and a sensitivity of 91 mA M^?1 cm^?2, was obtained. The influence of some interferents due to the oxidation of hydrogen peroxide (at +0.35 V vs. SCE) was also studied. By controlling carefully the experimental conditions, the determination of lactate in a commercial serum sample in the presence of interferents was successfully accomplished.     

An L‐Lactate Amperometric Enzyme Electrode Based on L‐Lactate Oxidase Immobilized in a Laponite Gel on a Glassy Carbon Electrode. Application to Dairy Products and Red Wine

A biosensor based on the immobilization of Lactate oxidase in laponite–organosilasesquioxane films on glassy carbon electrode for the quantification of L ‐lactate in wine and dairy products is presented. The bioelectrode showed a very high sensitivity (0.33±0.01) A cm^?2 M^?1 and a short time response (10 s) for less than 1 U of enzyme. No significant interferences, including ascorbic acid, were detected. For red wine, matrix effects assigned to polyphenols and anthocyanins were observed, which ware easily overcome by sample dilution. Our L ‐lactate determinations were in good agreement with those of two standard methods.     

Minimally‐invasive Microneedle‐based Biosensor Array for Simultaneous Lactate and Glucose Monitoring in Artificial Interstitial Fluid

Here we report the first mediated pain free microneedle‐based biosensor array for the continuous and simultaneous monitoring of lactate and glucose in artificial interstitial fluid (ISF). The gold surface of the microneedles has been modified by electrodeposition of Au‐multiwalled carbon nanotubes (MWCNTs) and successively by electropolymerization of the redox mediator, methylene blue (MB). Functionalization of the Au‐MWCNTs/polyMB platform with the lactate oxidase (LOX) enzyme (working electrode 1) and with the FAD‐Glucose dehydrogenase (FADGDH) enzyme (working electrode 2) enabled the continuous monitoring of lactate and glucose in the artificial ISF. The lactate biosensor exhibited a high sensitivity (797.4±38.1 μA cm^?2 mM^?1), a good linear range (10–100 μM) with a detection limit of 3 μM. The performance of the glucose biosensor were also good with a sensitivity of 405.2±24.1 μA cm^?2 mM^?1, a linear range between 0.05 and 5 mM and a detection limit of 7 μM. The biosensor array was tested to detect the amount of lactate generated after 100 minutes of cycling exercise (12 mM) and of glucose after a normal meal for a healthy patient (10 mM). The results reveal that the new microneedles‐based biosensor array seems to be a promising tool for the development of real‐time wearable devices with a variety of sport medicine and clinical care applications.     

Design and characterization of a lactate biosensor based on immobilized lactate oxidase onto gold surfaces

The design and characterization of a lactate biosensor and its application to the determination of this analyte in wine and beer are described. The biosensor is developed through the immobilization of lactate oxidase (LOx) using two different strategies including direct adsorption and covalent binding. The characterization of the resulting lactate oxidase monolayers was performed in aqueous phosphate buffer solutions using atomic force microscopy (AFM) and quartz crystal microbalance (QCM) techniques. In presence of lactate and using hydroxymethylferrocene as a redox mediator, biosensors obtained by either direct adsorption or by covalent binding exhibit a clear electrocatalytic activity, and lactate could be determined amperometrically at 300 mV versus SSCE. Results obtained under these conditions give a linear current response versus lactate concentration up to 0.3 mM, with a detection limit of 10 μM of lactate and a sensitivity of 0.77 ± 0.08 μA mM⁻¹. Finally, biosensors were applied to the determination of lactate in wine and beer. The results obtained are in good agreement with those obtained by a well-established enzymatic-spectrophotometric assay kit.     

Diamond nanoparticles as a way to improve electron transfer in sol–gel l-lactate biosensing platforms

In the present work, we have included for the first time diamond nanoparticles (DNPs) in a sol–gel matrix derived from (3-mercaptopropyl)-trimethoxysilane (MPTS) in order to improve electron transfer in a lactate oxidase (LOx) based electrochemical biosensing platform. Firstly, an exhaustive AFM study, including topographical, surface potential (KFM) and capacitance gradient (CG) measurements, of each step involved in the biosensing platform development was performed. The platform is based on gold electrodes (Au) modified with the sol–gel matrix (Au/MPTS) in which diamond nanoparticles (Au/MPTS/DNPs) and lactate oxidase (Au/MPTS/DNPs/LOx) have been included. For the sake of comparison, we have also characterized a gold electrode directly modified with DNPs (Au/DNPs). Secondly, the electrochemical behavior of a redox mediator (hydroxymethyl-ferrocene, HMF) was evaluated at the platforms mentioned above. The response of Au/MPTS/DNPs/LOx towards lactate was obtained. A linear concentration range from 0.053 mM to 1.6 mM, a sensitivity of 2.6 μA mM⁻¹ and a detection limit of 16 μM were obtained. These analytical properties are comparable to other biosensors, presenting also as advantages that DNPs are inexpensive, environment-friendly and easy-handled nanomaterials. Finally, the developed biosensor was applied for lactate determination in wine samples.     

A Biofuel Cell Based on Biocatalytic Reactions of Lactate on Both Anode and Cathode Electrodes – Extracting Electrical Power from Human Sweat

Electrodes composed of carbon fibers were modified with graphene nano‐sheets in order to increase their surface area and facilitate electrochemical reactions. Electrocatalytic species, such as Meldola's blue (MB) and hemin were immobilized on the graphene surface due to their π‐π stacking and then used for electrocatalytic oxidation of NADH and reduction of H₂O₂, respectively. Further modification of these electrodes with enzymes producing NADH and H₂O₂ in situ (lactate dehydrogenase, LDH, and lactate oxidase, LOx, respectively), allowed assembling of a biofuel cell operating in the presence of lactate, oxygen and NAD⁺. The cathode of the biofuel cell required lactate and O₂ for its operation, while the anode operated in the presence of lactate and NAD⁺. Notably, both bioelectrocatalytic electrodes operated in the presence of lactate, one producing H₂O₂ in the reaction catalyzed by LOx in the presence of O₂, second producing NADH in the reaction catalyzed by LDH in the presence of NAD⁺. Both reactions were performed in the biofuel cell without separation of the cathodic and anodic solutions and with no need of a membrane. The biofuel cell was tested in solutions mimicking human sweat and then in real human sweat samples, demonstrating substantial power release being able to activate electronic devices.     

Determination of Phenolic Compounds Based on Co‐Immobilization of Methylene Blue and HRP on Multi‐Wall Carbon Nanotubes

This work evaluated an amperometric biosensor based on multi‐wall carbon nanotubes (MWCNT), chemically modified with methylene blue (Met) and horseradish peroxidase (HRP), for detection of phenolic compounds. The dependences of the biosensor response due to the enzyme immobilization procedure, HRP amounts, pH and working potential were investigated. The amperometric response for catechol using the proposed biosensor showed a very wide linear response range (1 to 150 μmol L^?1), good sensitivity (50 nA cm^?2 μmol^?1 L), excellent operational stability (after 300 determinations the response remained at 97%) and very good storage stability (lifetime>3 months). Based on all these characteristics, it is possible to affirm that the material is promising for phenol detection due to its good electrochemical response and enzyme stabilization. The biosensor response for various phenolic compounds was investigated.     

Amperometric Lactate Biosensor Based on Carbon Paste Electrode Modified with Benzo[c]cinnoline and Multiwalled Carbon Nanotubes

Amperometric lactate biosensor based on a carbon paste electrode modified with benzo[c]cinnoline and multiwalled carbon nanotubes is reported. Incorporation of benzo[c]cinnoline acting as a mediator and multiwalled carbon nanotubes providing a conduction pathway to accelerate electron transfer due to their excellent conductivity into carbon paste matrix resulted in a high performance lactate biosensor. The resulting biosensor exhibited a fast response, high selectivity, good repeatability and storage stability. Under the optimal conditions, the enzyme electrode showed the detection limit of 7.0×10^?8 M with a linear range of 2.0×10^?7 M–1.1×10^?4 M. The usefulness of the biosensor was demonstrated in serum samples.     

Lactate Biosensor Based on the Adsorption of Polyelectrolyte Stabilized Lactate Oxidase into Porous Conductive Carbon

 In this work the development a lactate biosensor is illustrated. Lactate oxidase is stabilized with the cationic polyelectrolyte diethylaminoethyl-dextran, and the resulting enzyme-polyelectrolyte complexes are physically absorbed into a highly porous and conductive carbon electrode for the construction of the biosensor. The amount of diethylaminoethyl-dextran used is optimized with respect to the sensor sensitivity and stability. Optimum results obtained with enzyme solution containing 0.5% w/v diethylaminoethyl-dextran and 200 U/ml lactate oxidase. The resulting biosensors present increased operational (over 240 hours of continuous polarization) and storage stability (more than 5 months), while the reproducibility was calculated to be better than 5.0% RSD.     

Sensitive lactate determination based on acclimated mixed bacteria and palygorskite co-modified oxygen electrode

A sensitive bacteria biosensor was prepared for the detection of trace lactate. The sensitive bioelement, Lactobacillus bulgaricus and Streptococcus thermophilus mixed cultrue, and palygorskite, a perfect matrix for bacteria, was co-immobilized on the surface of oxygen electrode. The biosensor possesses fine selective specificity, good sensitivity and longer operational life time, which were due to the mutual help relationship of symbiotic bacteria and 240 days acclimation with lactate as the carbon source. Hydrodynamic amperometry, an advanced electrochemical method, is suitable for on-line monitoring the concentration change of dissolved oxygen that is closely accompanied with the metabolism of lactate. Electrochemical data show that the current is very sensitive to the changes of the concentration of lactate. The response current was linear with lactic acid concentration in the range from 0 to 300 μmol L^? 1, where the response time is no more than 240 s (R = 0.9952), and the sensitivity was 1.87 mA mol^? 1 L. Experiments show the biosensor is also very useful for long time on-line monitoring of lactate, such as fermentation progress.     

Biosensor for H2O2 Response Based on Horseradish Peroxidase: Effect of Different Mediators Adsorbed on Silica Gel Modified with Niobium Oxide

Reagentless biosensors sensitive to hydrogen peroxide have been developed and compared. These biosensors are comprised of a carbon paste electrode modified with horseradish peroxidase (HRP) and one phenothiazine (methylene blue), one phenoxazine (meldola's blue) or one phenazine (phenazine methosulfate) dye adsorbed on silica gel modified with niobium oxide (SN). The enzyme was immobilized onto the graphite powder by cross‐linking with glutaraldehyde and mixing with one of the electron transfer mediators (dyes) adsorbed on SN. The amperometric response was based on the electrocatalytic properties of the dye to mediate electrons, which were generated in the enzymatic reaction of hydrogen peroxide under catalysis of HRP. The dependence on the biosensor response in terms of pH, buffer, HRP amounts and applied potential was investigated. The best results were found with a biosensor containing methylene blue dye showing an excellent operational stability (around 92% of the activity was maintained after 300 determinations). The proposed biosensor also presented good sensitivity (32.87 nA cm^{?2 μ}mol^?1 L) allowing hydrogen peroxide quantification at levels down to 0.52×10^?6 mol L^?1 an optimum response at pH 6.8 and at a potential of ?50 mV (vs. SCE) and showing a wide linear response range (from 1 to 700 μmol L^?1 for hydrogen peroxide).     

Fabrication of a highly sensitive electrochemiluminescence lactate biosensor using ZnO nanoparticles decorated multiwalled carbon nanotubes

ZnO nanoparticles (nanoZnO) were decorated on multiwalled carbon nanotubes (MWCNTs) and then the prepared nano-hybrids, nanoZnO-MWCNTs, were immobilized on the surface of a glassy carbon electrode (GCE) to fabricate nanoZnO-MWCNTs modified GCE. The prepared electrode, GCE/nanoZnO-MWCNTs, showed excellent electrocatalytic activity towards luminol electrochemiluminescence (ECL) reaction. The electrode was then further modified with lactate oxidase and Nafion to fabricate a highly sensitive ECL lactate biosensor. Two linear dynamic ranges of 0.01-10 μmol L⁻¹ and 10-200 μmol L⁻¹ were obtained for lactate with the correlation coefficient better than 0.9996. The detection limit (S/N = 3) was 4 nmol L⁻¹ lactate. The relative standard deviation for repetitive measurements (n = 6) of 10 μmol L⁻¹ lactate was 1.5%. The fabrication reproducibility for five biosensors prepared and used in different days was 7.4%. The proposed ECL lactate biosensor was used for determination of lactate in human blood plasma samples with satisfactory results.     

Evaluation of Polycation-Stabilized Lactate Oxidase in a Silica Sol–Gel as a Biosensor Platform

 Whereas glucose oxidase and related proteins are encapsulated readily in silica sol–gels, α-hydroxy enzymes such as lactate oxidase (LOx), are reported to be damaged by electrostatic interaction with these matrices. Based on a previous report, poly(ethyleneimine), PEI, was evaluated as a protecting compound under conditions suited to analytical measurements. With LOx and PEI co-encapsulated in a silica sol–gel, the enzyme retained 62% of its initial activity after 20 days. In the absence of PEI, activity was lost during the processing. Batch analytical measurements with enzyme-doped sol–gel yielded a linear response over the range 0.5–2.0 mM lactate and a detection limit of 0.03 mM lactate. Both simple incorporation of LOx in a silica sol–gel and an alternative protection method, blocking the ion-exchange sites on silica with La(III), failed. These negative results supported the hypothesis that the efficacy of PEI was related to its formation of a protective sheath around the enzyme. Author for correspondence. E-mail: coxja@muohio.edu Received July 29, 2002; accepted December 15, 2002 Published online May 19, 2003     

Novel Multiplexed Biosensor System for the Determination of Lactate and Pyruvate in Blood Serum

Multiplexing is one of the main current trends in biosensors, especially important for clinical diagnosis. However, simultaneous determination of several substances in one sample is often difficult due to different performance and working conditions of separate biosensors. This work was aimed at the development of a multiplexed biosensor system for the determination of lactate and pyruvate concentrations in liquid samples (i. e., blood serum). The system consisted of two amperometric biosensors based on lactate oxidase and pyruvate oxidase, which worked simultaneously in a single measuring cell. Conditions for the biosensor system work were selected. Linear range of lactate determination was 5–1000 μM, pyruvate – 10–5000 μM. Steady‐state response time was 30 s and 50 s for the lactate and pyruvate biosensors, respectively. After 2 weeks of storage biosensor responses decreased to 95 % (lactate biosensor) or 82 % (pyruvate biosensor) of the initial value. A scheme of analysis of the concentrations of lactate and pyruvate in human blood serum was proposed. The lactate and pyruvate concentrations as well as their ratio in human blood serum samples were determined and compared with the control method. The proposed biosensor system is suitable for the rapid detection of lactate, pyruvate and their ratio and can be used for clinical diagnosis, e. g., evaluation of the reasons of lactic acidosis and prognosis of patient's recovery.     

流动注入式乳酸生物传感器

研制了一种测定L－乳酸的生物传感器,将乳酸氧化酶（LOD）通过共价键固定在尼龙钢上制备乳酸氧化酶膜,将制得的酶膜固定在氧电极上构成乳酸生物传感器;将透析膜放在氧化酶膜上产生对L－乳酸扩散高度限制来改变该生物传感器的响应;酶膜机械强度高,在氧电极上反复装卸而不损坏,所构成的乳酸生物传感器的校正曲线的乳酸定量上限达5mmol/L,响应时间小于30s;初步血样测试的结果显示该乳酸生物传感器用于临床血乳酸的测定具有可行性。     

Microbiosensor for Salicylate Based on Modified Carbon Fibre

ABSTRACT

A microbiosensor is proposed for a quick and easy amperometric determination of salicylate. The methodology involves the use of the enzyme salicylate hydroxylase (SH) to convert salicylate to catechol, which is then oxidised at the carbon fibre electrode. The covalent immobilisation of the enzyme onto a carbon fibre electrode via carbodiimide results in an amperometric biosensor with high sensitivity, low detection limit and good stability. The response of the biosensor is linearly proportional to the salicylate concentration between 1.0 10^?7 and 2.0 10^?6 mol L^?1, at an applied potential of 300 mV vs SCE, with a response time of 3.5 s and a detection limit of 3.3 10^?8 mol L^?1. The relative standard deviation for the determination was 4.1% for n=10. The biosensor was applied to determine salicylate in urine and pharmaceutical samples and compared to the reference method with a good correlation.     

Lactate biosensor based on human erythrocytes

An amperometric lactate biosensor based on human erythrocytes is described. The erythrocyte suspension is retained near the platinum electrode by means of a semipermeable membrane. The response is based on lactate dehydrogenase activity in the erythrocytes and uses the oxidation of NADH by hexacyanoferrate(III) and amperometric detection of the resulting hexacyanoferrate(II). The limit of detection is 2.8 × 10^?5 mol l^?1, and the response is linear up to 1 mmol l^?1 lactate in the analyzed solution (11 mmol l^?1 in a blood sample). The response time is 7 min, and the useful lifetime is 2 weeks. The response is influenced only by reducing substances (uric acid) and malic acid. The effect of uric acid is readily compensated, and there is insufficient malic acid in blood to affect the results.     

Amperometric enzyme electrodes of glucose and lactate based on poly(diallyldimethylammonium)-alginate-metal ion-enzyme biocomposites

Sodium alginate (AlgNa) and poly(diallyldimethylammonium chloride) (PDDA) were mixed to obtain an interpenetrating polymer composite via electrostatic interaction and then cast on an Au electrode surface, followed by incorporation of metal ions (e.g. Fe³⁺ or Ca²⁺, to form AlgFe or AlgCa hydrogel) and glucose oxidase (GOx) (or lactate oxidase (LOx)), to prepare amperometric enzyme electrodes. The interactions of PDDA, Alg, and Fe³⁺ are studied by visual inspection as well as microscopic and electrochemical methods. Under optimized conditions, the PDDA-AlgFe-enzyme/Au and PDDA-AlgCa-enzyme/Au electrodes can give good analytical performance (e.g. nM-scale limit of detection of glucose or lactate, and sensitivities > 50 μA cm⁻² mM⁻¹) in the first-generation biosensing mode, which are better than the reported analogs using typical polysaccharide biopolymers as enzyme-immobilization matrices. The enzyme electrodes also worked well in the second-generation biosensing mode in the coexistence of p-benzoquione or ferrocene monocarboxylic acid artificial mediator. Biofuel cells (BFCs) with the enzyme electrodes as the bioanodes and glucose (or lactate) as the biofuel were also fabricated with satisfactory results. The proposed protocols for preparation of high performance Alg-based biocomposites may find wide applications in bioanalysis.     

一种基于壳聚糖固定葡萄糖氧化酶的葡萄糖生物传感器的研究

本文选用生物相容性好的壳聚糖作为基体材料，使其与戊二醛交联成网状结构包埋葡萄糖氧化酶制成电化学传感器。这种壳聚糖膜不仅可以减小葡萄糖氧化酶的流失，而且能为酶提供了适宜的微环境。用红外光谱、紫外光谱及透射电镜对膜的形态和性质进行了表征。实验结果表明该传感器具有很快的响应速度，很好的稳定性和重现性，能选择性地催化葡萄糖并测定其浓度。该传感器的制备方法简单，成本低，于冰箱中放置两周信号保持在90％以上，对葡萄糖测量的线性范围为1×10^-5 - 3.4×10^-3mol•L^-1，当信噪比为3:1时检测限为5×10^-6mol•L^-1。