Amperometric Lactate Biosensor Research Articles

Electrochemical detection of lactate produced by foodborne presumptive lactic acid bacteria

The detection of lactate is an important indicator of the freshness, stability, and storage stability of products as well as the degree of fermentation in the food industry. In addition, it can be used as a diagnostic tool in patients' healthcare since it is known that the lactate level in blood increases in some pathological conditions. Thus, the determination of lactate level plays an important role in not only the food industry but also in health fields. As a result, biosensor technologies, which are quick, cheap, and easy to use, have become important for lactate detection. In the current study, amperometric lactate biosensors based on lactate oxidase immobilization (with Nafion 5% wt) were designed and the limit of detection, linear range, and sensitivity values were determined to be 31μM, 50-350μM, and 0.04μAμM-1cm-2, respectively. Then, it was used for the measurement of lactic acid that produced by six different and morphologically identified presumptive lactic acid bacteria (LAB) which are isolated from different naturally fermented cheese samples. The biosensors were then used to successfully perform lactate measurements within 3min for each sample, even though a few of them were out of the limit of detection. Thus, electrochemical biosensors should be used as an alternative and quick solutions for the measurement of lactate metabolites rather than the traditional methods which require long working hours. This is the first study to use a biosensor to measure lactate produced by foodborne LAB in a real sample.

BIOSSENSORES DE SUOR PARA MONITORAMENTO ESPORTIVO

ABSTRACT Introduction: Biometric sweat monitoring is an important tool for optimizing sports training. The possibility of developing a wearable amperometric lactate biosensor using a screen-printed carbon electrode (SPCE) impregnated by Ag nanoparticles (Ag NPs) for sweat determination in sports monitoring is questioned. Objective: To develop a sensor with GCE substrate coated on Ag NPs/SPCE. Methods: FESEM and XRD analysis were used for the morphological and structural characterizations of Ag and SPCE NPs, respectively. Results: FESEM, EDS, and XRD revealed that Ag NPs were uniformly dispersed in SPCE. The electrochemical biosensor responded linearly to lactate in phosphate buffer solutions, with detection and sensitivity limits of 1.2 µM and 14.2 mAcm-2 mM-1, respectively. Conclusion: The results suggest that Ag NPs/SPCE can be used to continuously monitor lactate levels in sweat as a practical and reliable biosensor for use. Level of evidence II; Therapeutic studies - investigation of treatment outcomes.

Non-invasive monitoring of interstitial fluid lactate through an epidermal iontophoretic device

The development of a non-invasive sensing technology that allows collection of interstitial fluid (ISF) lactate and its subsequent analysis without exertion requirement, could enable lactate monitoring from rested individuals. Here, we describe a wearable, soft epidermal adhesive patch that integrates a reverse iontophoretic (RI) system, and an amperometric lactate biosensor placed on the anodic electrode with a porous hydrogel reservoir, for simultaneous ISF lactate extraction and quantification via electrochemical sensing, respectively. The iontophoretic system includes agarose hydrogels for preventing skin electrocution, while a porous polyvinyl alcohol-based hydrogel facilitates the effective transport of lactate from skin to the biosensor. The flexible skin-worn device tested on healthy individuals at rest showed rapid lactate collection from the ISF after 10 min of reverse iontophoresis with no evidence of discomfort or irritation to the skin. Detailed characterization of the enzymatic biosensor before and during on-body trials along with relevant control experiments confirmed the efficient extraction and selective detection of ISF lactate. Such an epidermal technology represents the first demonstration of an all-in-one platform that integrates non-invasive collection and subsequent analysis of lactate from iontophoretically extracted ISF toward point-of-care operation.

Supramolecular complex based on MWNTs/Boltorn H40 provides fast response to a Sandwich-type amperometric lactate biosensor

Most sandwich-type biosensors can be used several times, since their long-time stability generally ranges from weeks to several months. However, the response-time of this kind of sensors is typically slow because reagents and products have to diffuse through filtering membranes and the enzymatic matrix. In this manuscript, multiwalled carbon nanotubes (MWNTs) have been used for wiring the enzymatic matrix to the electrode surface. To achieve this, the hyperbranched polymer Boltorn H40 (BH40) has been self-assembled at the surface of MWNTs. After the assembling, the suspension of BH40-MWNTs became stable and its functionality was tested by replacing mucin in the enzymatic matrix of a previously reported sandwich-type lactate biosensor. The inclusion of BH40-MWNTs in the biosensor: increased the diffusion coefficient of soluble species by decreasing the elastic properties of the enzymatic matrix, kept the characteristics of the microenvironment where the enzyme is stored, and wired the enzymatic matrix through a 13μm thick polycarbonate membrane to the electrode surface. It is important to understand that the superstructure of BH40-MWNTs does not increase the active surface of the electrode. Instead of this, it is a conductive 3D network that connects the enzymatic matrix to the electrode. As a result, the response-time of this novel sandwich-type lactate biosensor is much shorter than that of conventional biosensors because the mediator of the enzyme (oxygen) can be regenerated at the enzymatic matrix and it does not have to diffuse to the electrode surface.

Construction of an Amperometric Lactate Biosensor Based on Immobilization of Lactate Dehydrogenase Nanoparticles onto Pencil Graphite Electrode

Nanoparticles (NPs) of commercial lactate dehydrogenase from rabbit muscle were prepared, characterized and immobilized covalently onto pencil graphite (PG) electrode to fabricate an improved amperometric lactate biosensor (LDHNPs/PGE). The biosensor showed an optimum response within 2.5s at pH 7.0, temperature 35°C, at an applied potential of -0.4V. The wide linear response was produced by biosensor with in a concentration range of lactic acid (0.001 μM to 45 mM) and current (mA) under optimal conditions. The LDHNPs/PGE electrode showed high sensitivity (2.45 ± 2.0 μA cm−2 μM−1); a lower detection limit (0.001 μM) and good correlation coefficient (R2 = 0.99) with a standard enzymic colorimetric method. The evaluation study of biosensor offered a good analytical recovery of 98.01%, when lactic acid concentration was added in the sera sample. In addition, within and between batches coefficients of variation for working electrode were detected as 0.03% and 0.04%, respectively. The biosensor was applied for the detection of lactic acid in the serum of apparently healthy subject and patients affected from cardiogenic shocks. There was a 10% loss in initial activity of biosensor after its regular use over a time period of 180 days, while being stored at 4°C.

Bendable Electro-chemical Lactate Sensor Printed with Silver Nano-particles.

Here we report a flexible amperometric lactate biosensor using silver nanoparticle based conductive electrode. Mechanically bendable cross-serpentine-shaped silver electrode is generated on flexible substrate for the mechanical durability such as bending. The biosensor is designed and fabricated by modifying silver electrode with lactate oxidase immobilized by bovine serum albumin. The in-sensor pseudo Ag/AgCl reference electrode is fabricated by chloridization of silver electrode, which evinced its long-term potential stability against a standard commercial Ag/AgCl reference electrode. The amperometric response of the sensor shows linear dependence with lactate concentration of 1~25 mM/L. Anionic selectivity is achieved by using drop-casted Nafion coated on silver electrode against anionic interferences such as ascorbate. This non-invasive electrochemical lactate sensor also demonstrates excellent resiliency against mechanical deformation and temperature fluctuation which leads the possibility of using it on human epidermis for continuous measurement of lactate from sweat. Near field communication based wireless data transmission is demonstrated to reflect a practical approach of the sensor to measure lactate concentration portably using human perspiration.

An amperometric lactate biosensor based on lactate dehydrogenase immobilized onto graphene oxide nanoparticles‐modified pencil graphite electrode

A novel amperometric lactate biosensor was developed based on immobilization of lactate dehydrogenase onto graphene oxide nanoparticles‐decorated pencil graphite electrode. The enzyme electrode was characterized by scanning electron microscopy, Fourier transform infrared spectroscopy (FTIR), and cyclic voltammetry at different stages of its construction. The biosensor showed optimum response within 5 s at pH 7.3 (0.1 M sodium phosphate buffer) and 35°C, when operated at 0.7 V. The biosensor exhibited excellent sensitivity (detection limit as low as 0.1 μM), fast response time (5 s), and wider linear range (5–50 mM). Analytical recovery of added lactic acid in serum was between 95.81–97.87% and within‐batch and between‐batch coefficients of variation were 5.04 and 5.40%, respectively. There was a good correlation between serum lactate values obtained by standard colorimetric method and the present biosensor (r = 0.99). The biosensor measured lactate levels in sera of apparently healthy subjects and persons suffering from lactate acidosis and other biological materials (milk, curd, yogurt, beer, white wine, and red wine). The enzyme electrode lost 25% of its initial activity after 60 days of its regular uses, when stored dry at 4°C.

Layer by layer construction of ascorbate interference-free amperometric lactate biosensors with lactate oxidase, ascorbate oxidase, and ceria nanoparticles

We report on the design of an ascorbate interference-free lactate biosensor that takes advantage of the redox properties of ceria (CeO2) nanoparticles that make this material suitable to be used in electrochemical biosensors operating in low oxygen conditions. First, ceria-free lactate biosensors were fabricated using a positively charged polymer, polyethylenimine (PEI), for the immobilization of lactate oxidase (LOx) and ascorbate oxidase (AOx) on Pt electrodes. When AOx was not immobilized on the electrode, the sensor was interfered by ascorbate. Immobilization of AOx on the electrode surface, however, resulted in a loss of interference by ascorbate due to the depletion of oxygen in the enzyme layer. To address this challenge, we exploited the oxygen storage capacity of ceria nanoparticles. Their introduction into the enzyme layer enabled the fabrication of lactate biosensors that are not interfered by ascorbate and yet can be operated under low oxygen conditions. The Pt/CeO2–3(PEI/LOx)-3(PEI/AOx) biosensors, if operated at a working voltage of 0.6 V (vs. Ag/AgCl) display a wide linear range (20 μM to 1 mM), a low detection limit (0.3 μM) and a sensitivity of 172.9 ± 11.7 μA∙mM−1∙cm−2. Evidently, the layer-by-layer configuration eliminates interferences by common species such as ascorbic acid, uric acid, and glucose. The practical applicability of the sensors was evaluated by detecting lactate in human serum.

Novel CeO2–CuO-decorated enzymatic lactate biosensors operating in low oxygen environments

The detection of the lactate level in blood plays a key role in diagnosis of some pathological conditions including cardiogenic or endotoxic shocks, respiratory failure, liver disease, systemic disorders, renal failure, and tissue hypoxia. Here, we described for the first time the use of a novel mixed metal oxide solution system to address the oxygen dependence challenge of first generation amperometric lactate biosensors. The biosensors were constructed using ceria-copper oxide (CeO2–CuO) mixed metal oxide nanoparticles for lactate oxidase immobilization and as electrode material. The oxygen storage capacity (OSC, 492 μmol-O2/g) of these metal oxides has the potential to reduce the oxygen dependency, and thus eliminate false results originated from the fluctuations in the oxygen concentration. In an effort to compare the performance of our novel sensor design, ceria nanoparticle decorated lactate sensors were also constructed. The enzymatic activity of the sensors were tested in oxygen-rich and oxygen-lean solutions. Our results showed that the OSC of the electrode material has a big influence on the activity of the biosensors in oxygen-lean environments. While the CeO2 containing biosensor showed an almost 21% decrease in the sensitivity in a O2-depleted solution, the CeO2–CuO containing electrode, with a higher OSC value, experienced no drop in sensitivity when moving from oxygen-rich to oxygen-lean conditions. The CeO2–CuO decorated sensor showed a high sensitivity (89.3 ± 4 μA mM−1 cm−2), a wide linear range up to 0.6 mM, and a low limit of detection of 3.3 μM. The analytical response of the CeO2–CuO decorated sensors was studied by detecting lactate in human serum with good selectivity and reliability. The results revealed that CeO2–CuO containing sensors are promising candidates for continuous lactate detection.

A self-powered amperometric lactate biosensor based on lactate oxidase immobilized in dimethylferrocene-modified LPEI.

Lactate is an important biomarker due to its excessive production by the body during anerobic metabolism. Existing methods for electrochemical lactate detection require the use of an external power source to supply a positive potential to the working electrode of a given device. Herein we describe a self-powered amperometric lactate biosensor that utilizes a dimethylferrocene-modified linear poly(ethylenimine) (FcMe2-LPEI) hydrogel to simultaneously immobilize and mediate electron transfer from lactate oxidase (LOx) at the anode and a previously described enzymatic cathode. Operating as a half-cell, the FcMe2-LPEI electrode material generates a jmax of 1.51 ± 0.13 mAcm(-2) with a KM of 1.6 ± 0.1 mM and a sensitivity of 400 ± 20 μAcm(-2)mM(-1) while operating with an applied potential of 0.3 V vs. SCE. When coupled with an enzymatic biocathode, the self-powered biosensor has a detection range between 0mM and 5mM lactate with a sensitivity of 45 ± 6 μAcm(-2)mM(-1). Additionally, the FcMe2-LPEI/LOx-based self-powered sensor is capable of generating a power density of 122 ± 5 μWcm(-2) with a current density of 657 ± 17 μAcm(-2) and an open circuit potential of 0.57 ± 0.01 V, which is sufficient to act as a supplemental power source for additional small electronic devices.

Non-Invasive Sensor for Quantitative Determination of Lactate in Sweat

Lactic acid is a key biomarker of anaerobic respiration and descriptor of an individual’s health state. Currently, lactate monitoring is performed though continuous invasive blood sampling and sample processing which is inconvenient and unpractical when trying to obtain real time measurements of an active individual. Non-invasive sensing methods are needed for monitoring human performance in sports, military and health care fields. Lactate is found in various bodily fluids including sweat and a correlation between blood and sweat lactate concentration has been determined (1). Hence, monitoring sweat lactate levels is a good noninvasive alternative to blood sampling methods. Here we report the development, characterization and optimization of an amperometric lactate sensor based on a lactate dehydrogenase and carbon nanotube chitosan electrode coupling system previously developed by our group (3). Due to the high overpotential of NADH oxidation, the electrocatalyst, polymethylene green (PMG) was electrochemically deposited onto the surface of multi-walled carbon nanotube (CNT) material called Bucky paper for NAD⁺ regeneration. Lactate dehydrogenase was immobilized onto the Bucky paper-PMG electrode with a chitosan-based carbon nanotubes (CNT) mixture. The bioelectrodes were tested in a standard three-electrode polycarbonate cell hardware consisting of enzymatic working electrode, Ag/AgCl reference and platinum wire counter electrode and operated in a chronoamperometric regime. Sweat composition varies between individuals and different parts of the human body resulting in variations in pH, salt concentration (related to conductivity,) and lactate levels before and after exercise with rates of release decreasing over time. In order to address some of these issues, calibration curves at various buffer pHs and buffer concentrations were created (Fig 1). The variation of pH of solutions from 5 to 7 showed an increase in bioelectrode response. The slope of the current/lactate concentration linear dependence was strongly influenced by the solution pH with low pH leading to decreased sensitivity most likely as a result of influenced enzyme activity. A similar trend was observed when the concentration of the electrolyte was varied from 0.01 to 0.245 M. The slope of the calibration curve was strongly influenced by the solution conductivity with low values leading to decreased reproducibility and sensitivity. In all cases the electrode response was linear with lactate concentration allowing one to tailor the system based on individual needs. An artificial sweat was prepared, composed of NaCl, urea, pH 6.5, glucose, NAD⁺, and having a conductivity of 16.6 mS/cm. As it was expected the initial tests of the LDH-electrode with the artificial sweat showed lower sensitivity most likely due to low conductivity of the solution, the generated current form the lactate oxidation followed linear dependence from lactate concentration (data not shown). The strong dependence of the slope of the sensor calibration curve from the electrolyte conductivity and pH can be minimized by buffering the sweat, which in real conditions is performed by impregnation of the sensor sweat collector with a buffer salts. A prototype of a patch lactate sensor was developed composed of a LDH-working electrode, a Ag/AgCl reference electrode and carbon yarn counter electrode. The three electrodes were placed on a medical adhesive and covered with a bandage, preventing a contact between electrodes and the skin and at the same time adsorbing and collecting the sweat. The designed patch sensor displayed an increase in current density with increasing lactate concentration with high sensitivity. To avoid artificial introduction of NAD+ in the sweat, a method for NAD+immobilization of the electrode surface will be implemented along with encapsulation of the enzyme into silica gel matrix, which improve the linearity of the response and prolong the life -time of the sensor. The proposed amperometric enzyme electrode coupling approach along with optimization experiments provides the opportunity to monitor noninvasively and in real-time sweat lactate concentrations. (1) Sakharov, D.A. et al., 2010. Relationship between lactate concentrations in active muscle sweat and whole blood. Bull. Exp. Biol. Med.. 150 (1) 83-5. (2) Nikolaus, N., and Strehlitz B., 2008. Review. Amperometric lactate biosensors and their application in (sports) medicine, for life quality and wellbeing. Microchim Acta. 160, 15-55. (3). Narvaez Villarrubia C.W. et al., 2011. Biofuel cell anodes integrating NAD⁺-dependent enzymes and multiwalled carbón nanotube papers. ACS Appl. Mater Interfaces. 2011, 3(7), p. 2402-9 (7) Figure 1

An amperometric lactate biosensor using H2O2 reduction via a Prussian Blue impregnated poly(ethyleneimine) surface on screen printed carbon electrodes to detect anastomotic leak and sepsis

Lactate detection by biosensors is of increasing importance in clinical applications to provide rapid, point of care, serial measurements for early diagnosis of disorders such as intra-abdominal sepsis to reduce morbidity and mortality. We present a method for the detection and quantification of lactate using pre-impregnated Prussian Blue screen printed carbon electrodes and polyethyleneimine polymer. The resulting amperometric lactate biosensor exhibited a reproducible response to lactate. Data was validated using cyclic voltammetry, and key parameters for sensor fabrication were systematically evaluated. The biosensor showed little response to possible interferents, including ascorbic acid. Biosensor results using clinical peritoneal drain fluid samples demonstrated significant concordance with a commercial lactate assay (rs=0.891, P=0.001). We therefore present a robust and sensitive lactate biosensor using a simple adsorption based methodology which has the capacity to be a potentially valuable diagnostic test for intra-abdominal sepsis and anastomotic leak.

Amperometric lactate biosensor for flow injection analysis based on a screen-printed carbon electrode containing Meldola's Blue-Reinecke salt, coated with lactate dehydrogenase and NAD +

A biosensor for the measurement of lactate in serum has been developed, which is based on a screen-printed carbon electrode, modified with Meldola's Blue-Reinecke Salt (MBRS-SPCE), coated with the enzyme lactate dehydrogenase NAD + dependent (from Porcine heart), and NAD +. A cellulose acetate layer was deposited on the top of the device to act as a permselective membrane. The biosensor was incorporated into a commercially available, thin-layer, amperometric flow cell operated at a potential of only +0.05 V vs. Ag/AgCl. The mobile phase consisted of 0.2 M phosphate buffer pH 10 containing 0.1 M potassium chloride solution; a flow rate of 0.8 ml min −1 was used throughout the investigation. The biosensor response was linear over the range 0.55–10 mM lactate; the former represents the detection limit. The precision of the system was determined by carrying out 10 repeat injections of 10 mM l(+)lactic acid standard; the calculated coefficient of variation was 4.28%. It was demonstrated that this biosensor system could be applied to the direct measurement of lactate in serum without pre-treatment; therefore, this would allow high throughput-analysis, at low cost, for this clinically important analyte.

Thermostable amperometric lactate biosensor with Clostridium thermocelluml-LDH for the measurement of blood lactate

The gene for Clostridium thermocellum l-lactate dehydrogenase enzyme was cloned into pGEX-4T-2 purification vector to supply a source for a thermostable enzyme in order to produce a stable lactate biosensor working at relatively high temperatures. The purified thermostable enzyme ( t-LDH) was then immobilized on a gold electrode via polymerization of polygluteraldehyde and pyrrol resulting in a conductive co-polymer. t-LDH working electrode ( t-LDHE) was used for determination of lactate in CHES buffer. Amperometric response of the produced electrodes was measured as a function of lactate concentration, at a fixed bias voltage of 200 mV in a three-electrode system. The linear range and sensitivity of the biosensor was investigated at various temperatures in the range of 25–60 °C. The sensitivity t-LDHE increased with increasing the temperature and reached its highest value at 60 °C. The calculated value was nearly 70 times higher as compared to the sensitivity value of the same electrode tested at 25 °C. The sensing parameters of t-LDHE were compared with the electrodes produced by commercially available rabbit muscle LDH ( m-LDH). The sensitivity of t-LDHE was nearly 8 times higher than that of m-LDHE. t-LDHE was found to retain its activity for a week incubation at refrigerator (+5 °C), while m-LDHE lost its activity in this period. t-LDHE was also tested in the presence of human blood serum. The results showed that the current increased with increasing concentrations of lactate in the human blood serum and the biosensor is more sensitive to serum lactate as well as the commercial lactate dissolved in serum as compared to the commercial lactate dissolved in CHES buffer.

Development of Amperometric Lactate Biosensor Modified with Pt-black Nanoparticles for Rapid Assay

For point-of-care determination, a disposable biosensor for the analysis of L-lactate in athletes' serum samples was developed and optimized. The planar electrochemical biosensor was fabricated with gold thin-film two-electrode system and was modified with platinum-black nanoparticles and ferricyanide mediator. Platinum-black nanoparticles were deposited on the electrodes to improve the sensitivity and stability. Lactate oxidase (LOD, E.C.1.1.3.2) was immobilized on working electrodes with ferricyanide as mediator to improve the electron transfer ability by lowering the work potential to +0.2 V. The optimizations of the deposition of platinum-black nanoparticles, concentration of LOD, and concentration of ferricyanide in this article have resulted in the development of a lactate electrode with a wide linear range of 1–20 mM lactate, a high sensitivity of 1.43 µA mM−1, a fast detection time of 50 s, and a coefficient variation (CV) of 0.0549. The activity of biosensor held above 90% after one year storage at room temperature. The biosensor was successfully applied to the determination of L-lactate in serum samples without dilution. This disposable biosensor combined with portable meter is promising for rapid determination of point-of-care lactate.

A lactate biosensor based on lactate dehydrogenase/nictotinamide adenine dinucleotide (oxidized form) immobilized on a conducting polymer/multiwall carbon nanotube composite film

An amperometric lactate biosensor was developed based on a conducting polymer, poly-5,2′-5′,2′′-terthiophene-3′-carboxylic acid (pTTCA), and multiwall carbon nanotube (MWNT) composite on a gold electrode. Lactate dehydrogenase (LDH) and the oxidized form of nicotinamide adenine dinucleotide (NAD +) were subsequently immobilized onto the pTTCA/MWNT composite film. The modified electrode was characterized by quartz crystal microbalance (QCM), scanning electron microscopy (SEM), and electrochemical experiments. The detection signal was amplified by the pTTCA/MWNT assembly onto which a sufficient amount of enzyme was immobilized and stabilized by the covalent bond formation between the amine groups of enzyme and the carboxylic acid groups of the pTTCA/MWNT film. Experimental parameters affecting the sensor responses, such as applied potential, pH, and temperature, were assessed and optimized. Analytical performances and dynamic ranges of the sensor were determined, and the results showed that the sensitivity, stability, and reproducibility of the sensor improved significantly using pTTCA/MWNT composite film. The calibration plot was linear ( r 2 = 0.9995) over the range of 5 to 90 μM. The sensitivity was approximately 0.0106 μA/μM, with a detection limit of 1 μM, based on a signal/noise ratio of 3. The applicability of the sensor for the analysis of l-lactate concentration in commercial milk and human serum samples was demonstrated successfully.

A biosensor based on immobilization of lactate oxidase in a PB-CTAB film for FIA determination of lactate in beer samples

An amperometric lactate biosensor with lactate oxidase immobilized into a Prussian Blue (PB) modified electrode was fabricated. The advantage of using cetyltrimethylammonium bromide (CTAB) in the electrodeposition step of PB films onto glassy carbon surfaces was confirmed taking into account both the stability and sensitivity of the measurements. The biosensor was used in the development of a FIA amperometric method for the determination of lactate. Under optimal operating conditions (pH = 6.9, E = -0.1 V), the linear response of the method was extended up to 0.28 µmol L-1 lactate with a limit of detection of 0.84 mmol L-1. The repeatability of the method for injections of a 0.28 mmol L-1 lactate solution was 2.2 % (n = 18). The usefulness of the method was demonstrated by determining lactate in beer samples and the results were in good agreement with those obtained by using a reference spectrophotometric enzyme method.

Amperometric biosensor for lactate analysis in wine and must during fermentation

A lactate oxidase-based amperometric biosensor is designed for lactate determination. Two methods of lactate oxidase immobilization on the surface of commercial SensLab platinum printing electrodes are compared. The sensor with lactate oxidase immobilized by physical adsorption in Resydrol polymer is shown to have both narrower dynamic range (0.004–0.5 mМ lactate) and higher sensitivity (320 nA/mM) as compared with that immobilized in poly(3,4-ethylenedioxythiophene) by electrochemical polymerization (0.05–1.6 mM and 60 nA/mM respectively). The operational stability of the biosensors developed is studied; the immobilization method is shown to be of no influence. The lactate content in wine and in wine material during fermentation is analyzed. The data obtained by amperometric lactate biosensor correlated with those of standard chromatography. The biosensor developed can be used in food industry for control and optimization of process of wine fermentation as well as for control of wine quality.

Amperometric lactate biosensors and their application in (sports) medicine, for life quality and wellbeing

The article reviews the use of electrochemical biosensors for detecting lactate, a key metabolite of the anaerobic glycolytic pathway. This compound plays an important role in (sports) medicine, in the nutritional sector, in food quality control and touches environmental concerns. Amperometric biosensors offer a sensitive and selective means to monitor organic analytes like lactate. A detailed study on different aspects of amperometric lactate biosensor preparation is described: the main configuration aspects are compiled regarding electrode materials, biorecognition elements, immobilization methods, mediators and cofactors as well as fields of application. Comparative studies are conducted correlating different configuration aspects and performance of the resulting biosensors. This review contains 214 references from the years 1974 to 2007.

Permeabilized cells of flavocytochrome b2 over-producing recombinant yeast Hansenula polymorpha as biological recognition element in amperometric lactate biosensors

A l-lactate-selective microbial biosensor was developed using permeabilized cells of gene-engineered thermotolerant methylotrophic yeast Hansenula polymorpha, over-producing l-lactate:cytochrome c-oxidoreductase (EC 1.1.2.3, flavocytochrome b 2, FC b 2). The construction of FC b 2-producers by over-expression of the gene CYB2 H. polymorpha encoding FC b 2 is described. The HpCYB2 gene under the control of the strong H. polymorpha alcohol oxidase promoter in the frame of a plasmid for multicopy integration was transformed to the recipient strain H. polymorpha C-105 ( gcr1 catX) impaired in glucose repression and devoid of catalase activity. The permeabilized cells were either immobilized on the graphite working electrode by physical entrapment of the cell suspension by means of a dialysis membrane or by integration of the cells in an electrochemically generated layer using a cathodic electrodeposition polymer. Phenazine methosulphate was used as a free-diffusing redox mediator. It was assumed that the mediator reacts with mitochondrial FC b 2 after entering the cells in the presence of l-lactate. The biosensor based on recombinant yeast cells exhibited a higher K M app value and hence expanded linear range toward l-lactate as compared to a similar sensor based on the initial cells of H. polymorpha C-105.