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.     

Enzymatic Biosensors Based on Carbon Nanotubes Paste Electrodes

The performance of enzymatic biosensors based on the immobilization of different enzymes within a carbon nanotubes paste electrode (CNTPE) prepared by dispersion of multi‐wall carbon nanotubes (MWNT) and mineral oil is reported in this work. The strong electrocatalytic activity of carbon nanotubes towards the reduction of hydrogen peroxide and quinones and the oxidation of NADH have allowed an effective low‐potential amperometric determination of lactate, phenols, catechols and ethanol, in connection with the incorporation of lactate oxidase, polyphenol oxidase and alcohol dehydrogenase/NAD⁺, respectively, within the composite matrix. Compared to the analogous enzymatic CPEs, a great enhancement in the response is observed at the enzymatic CNTPEs. Therefore, highly sensitive lactate, phenols, catechols and alcohols biosensors without using any metal or redox mediator can be obtained with this new composite material.     

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.     

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.     

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.     

Determination of lactate in human saliva with an electrochemical enzyme probe

Electrochemical biosensors for lactate were assembled and used for the determination of lactic acid in saliva. Saliva was collected from healthy subjects and immediately screened for its lactate content. The electrochemical and biological interferences from saliva were discriminated by using a dual platinum electrode and blocking membranes. The stability, reproducibility and lifetime of the probe were studied. Lactate was measured in eight subjects in fasting conditions and after eating, showing an increase in lactate for each subject after meals. Correlation with a spectrophotometric lactate measurement is reported. Subjects before, during and after physical exercise showed consistent variations of lactate in saliva.     

New Strategy for Dehydrogenase Amperometric Biosensors Using Surfactant to Enhance the Sensitivity of Diaphorase/Ferrocene Modified Carbon Paste Electrodes for Electrocatalytic Oxidation of NADH

A carbon paste electrode (CPE) modified with diaphorase (DAP) and ferrocene (FcH) has been developed for determination of NADH at low working potential. The sensitivity and operational stability, towards the detection of the reduced form of the nicotinamide adenine dinucleotide (NADH) in flow injection analysis (FIA), were greatly improved (5 times) upon adding Tween 20 into the electrode matrix. The magnitude of the amperometric signal was dependent on DAP, FcH and surfactant loading, into the modified carbon paste electrode. A rapid and repeatable response was observed to the variation of NADH concentration in the vicinity of the electrode surface. Such advantages of the DAP/FcH/Tween 20 modified carbon paste were successfully used in the construction of L ‐lactate dehydrogenase modified electrodes. The use of this new approach can be generalized to other dehydrogenases and represents a decisive step for a versatile preparation method of amperometric biosensors.     

A microband lactate biosensor fabricated using a water-based screen-printed carbon ink

The present study demonstrated for the first time that screen-printed carbon microband electrodes fabricated from water-based ink can readily detect H₂O₂ and that the same ink, with the addition of lactate oxidase, can be used to construct microband biosensors to measure lactate. These microband devices were fabricated by a simple cutting procedure using conventional sized screen-printed carbon electrodes (SPCEs) containing the electrocatalyst cobalt phthalocyanine (CoPC). These devices were characterised with H₂O₂ using several electrochemical techniques. Cyclic voltammograms were found to be sigmoidal; a current density value of 4.2 mA cm⁻² was obtained. A scan rate study revealed that the mass transport mechanism was a mixture of radial and planar diffusion. However, a further amperometric study under quiescent and hydrodynamic conditions indicated that radial diffusion predominated. A chronoamperometric study indicated that steady-state currents were obtained with these devices for a variety of H₂O₂ concentrations and that the currents were proportional to the analyte concentration. Lactate microband biosensors were then fabricated by incorporating lactate oxidase into the water-based formulation prior to printing and then cutting as described. Voltammograms demonstrated that lactate oxidase did not compromise the integrity of the electrode for H₂O₂ detection. A potential of +400 mV was selected for a calibration study, which showed that lactate could be measured over a dynamic range of 1-10 mM which was linear up to 6 mM; a calculated lower limit of detection of 289 μM was ascertained. This study provides a platform for monitoring cell metabolism in-vitro by measuring lactate electrochemically via a microband biosensor.     

Advanced electrocatalytic materials based biosensors for cancer cell detection – A review

Herein, we have highlighted the latest developments on biosensors for cancer cell detection. Electrochemical (EC) biosensors offer several advantages such as high sensitivity, selectivity, rapid analysis, portability, low-cost, etc. Generally, biosensors could be classified into other basic categories such as immunosensors, aptasensors, cytosensors, electrochemiluminescence (ECL), and photo-electrochemical (PEC) sensors. The significance of the EC biosensors is that they could detect several biomolecules in human body including cholesterol, glucose, lactate, uric acid, DNA, blood ketones, hemoglobin, and others. Recently, various EC biosensors have been developed by using electrocatalytic materials such as silver sulfide (Ag₂S), black phosphene (BPene), hexagonal carbon nitrogen tube (HCNT), carbon dots (CDs)/cobalt oxy-hydroxide (CoOOH), cuprous oxide (Cu₂O), polymer dots (PDs), manganese oxide (MnO₂), graphene derivatives, and gold nanoparticles (Au-NPs). In some cases, these newly developed biosensors could be able to detect cancer cells with a limit of detection (LOD) of 1 cell/mL. In addition, many remaining challenges have to be addressed and validated by testing more real samples and confirm that these EC biosensors are more accurate and reliable to measure cancer cells in the blood and salivary samples.     

Novel electron transfer mediators based on dichloroindophenol derivatives for lactate oxidase

Novel indophenol derivatives were synthesized and characterized electrochemically with respect to their abilities to act as electron-transfer mediators for lactate oxidase. These compounds showed suitable redox potentials and high second-order rate constants, k_med, in solution for electron-transfer from the reduced enzyme. A lactate sensor using these derivatives demonstrated high sensitivity and good substrate selectivity. This sensor could also achieve excellent durability which retained more than 70% residual activity and good linearity in the range from 0 to 16 mM lactate concentration even after 10 days.     

Application of screen-printed microband biosensors incorporated with cells to monitor metabolic effects of potential environmental toxins

Microband biosensors were fabricated from a screen-printed water-based carbon ink containing cobalt phthalocyanine redox mediator and glucose oxidase or lactate oxidase enzyme. The microbiosensors were characterised for their ability to monitor ferrocyanide and H₂O₂ in phosphate buffer solution: sigmoidal cyclic voltammograms, high current density values and steady-state amperometric responses confirmed the existence of radial-diffusion-limiting microelectrode behaviour. The lactate microband biosensors were then used, in conjunction with a screen-printed Ag/AgCl reference and platinum counter electrode, to monitor lactate levels in culture medium, with a linear range of 0.5–5 mM, sensitivity of 20 nA.mM^?1, and dynamic range up to >9 mM. The lactate microband biosensors could operate continuously in culture medium over extended times (up to 24 h) at 37 °C. These biosensors were then applied to detect changes in lactate release from cultured cells in response to toxic challenge: m-dinitrobenzene (500 μM) caused a reduction in lactate production by high-passage number HepG2 single cells; D-galactosamine (20 mM) induced release of lactate by HepG2 spheroid cultures. This novel use of microband biosensors in cell culture has the potential for further application in toxicity monitoring, in both environmental and pharmaceutical areas.     

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.     

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.     

Development of a Carbon Paste Electrode for Lactate Detection Based on Meldola’s Blue Adsorbed on Silica Gel Modified with Niobium Oxide and Lactate Oxidase

A reagentless amperometric biosensor sensitive to lactate was developed. The sensor employs a carbon paste electrode modified with lactate oxidase (LOx) and Meldola’s Blue (MB) adsorbed on silica gel coated with niobium oxide. The dependence on the biosensor response was investigated in terms of pH, supporting electrolyte, ionic strength, lactate oxidase (LOx) amounts and applied potential. The biosensor showed an excellent operational stability (96 % of the activity was maintained after 150 determinations) and storage stability (allowing measurements for more than 1.5 months, when stored in a refrigerator). The proposed biosensor also presented good sensitivity allowing lactate quantification at levels down to 6.5×10^?7 mol L^?1. Moreover, the biosensor showed a good linear response range (from 0.1 to 5.0 mmol L^?1 for lactate). Lactate analysis in biological samples such as blood was also performed. The precision of the data obtained by the proposed biosensor showed reliable results for real complex matrices.     

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.     

Development and comparison of biosensors for in-vivo applications

Electrochemical biosensors have been of increasing interest, especially those developed to be directly applied in diagnostic areas, such as neuroscience. We have been interested in developing a range of biosensors for monitoring glucose, lactate, pyruvate, and glutamate in order to study on-line both brain function in the laboratory and to monitor brain health in neurointensive care. For a biosensor to function effectively in these situations, it has to combine the following characteristics: quick response and high sensitivity, good reproducibility and adequate stability. In this study we compared the performance of a number of different amperometric biosensors strategies. These included ferrocene mediation of immobilised enzymes (system A), a redox hydrogel based system (system B), and a conducting polymer approach using polyaniline (system C). All assays were operated as flow-injection systems with upstream immobilised enzyme beds if necessary. When calibrated for H2O2 systems A and B reacted quickly enough to give quantitative conversion up to 0.2 mM. Above this concentration the response was limited by horseradish peroxidase enzyme kinetics and eventually enzyme loading. System C showed a restricted H2O2 response. When calibrated for glucose (by use of immobilised glucose oxidase) system B exhibited the highest sensitivity but its analytical range was restricted because the system became limited by H2O2 response. System A had low sensitivity for analyte compared to H2O2 and system B, but a greater useful range. Problems of mediator cycling between the immobilised enzymes are discussed. System C gave an excellent linear range but sensitivity was limited by background noise. Stability and reproducibility of the systems are also described. In conclusion, from this study the ferrocene system proved to be overall most useful and has now been used in the first dual on-line monitoring of glucose and lactate in patients in neurointensive care.     

Gamma-irradiation immobilization of lactate oxidase in poly(vinyl alcohol) on platinized graphite electrodes

A novel method for enzyme immobilization in a polymer matrix was examined with lactate oxidase (LOD) to make a sensor for lactate. Poly(vinyl alcohol) (PVAL) and LOD were applied in layers on platinized graphite electrodes and cross-linked by exposure to a ⁶⁰Co gamma radiation source. When the sensor is dipped in lactate solution, the product of the enzymatic reaction, hydrogen peroxide, is detected at +300 mV vs. Ag/AgCl. The LOD-PVAL lactate sensor exhibits a fast response (10–50 s), a linear range between 26 μM and 1.7 mM, a detection limit of 13 μM and a sensitivity of 2.94 μA mmol^?1. The sensitivity and the linearity of the electrode were improved considerably by bubbling oxygen continuously through the lactate solution. Optimum response to lactate was obtained with a radiation dose of 3–10 Mrad. LOD was found to be active in the presence of the polymer under radiation doses as high as 40 Mrad. Repeated use of the sensors under various conditions showed a stable and reproducible response to lactate for over 80 days.     

基于丙酮酸/还原型辅酶I/乳酸脱氢酶/氧化型辅酶I/乳酸正逆反应同时测定丙酮酸/乳酸的酶荧光毛细管分析

利用丙酮酸(PA)/还原型辅酶I(NADH)/乳酸脱氢酶(LDH)/氧化型辅酶I(NAD+)/乳酸(LA)荧光反应体系的正逆反应,建立了一种可直接用于临床检验、能同时测定血清中微量PA/LA的酶荧光毛细管分析法.本方法可在常规荧光光度计上,用普通玻璃毛细管同时实现了PA/LA的高灵敏分析,每次分析试剂和样品的用量仅9 ...     

Detection Mechanism of Metallized Carbon Epoxy Oxidase Enzyme Based Sensors

The coenzyme FAD has been identified to play an important role in the detection mechanism of oxidase enzyme based biosensors. Incorporating FAD into the carbon composite improved sensitivity to H₂O₂ consequently increasing sensitivity to the respective analyte. The amount of active enzyme also increased thus enhancing the overall performance of the sensors. Polycarboxybetaine (PCB) has been used as a biocompatible membrane coating. The PCB coated sensors gave reproducible calibrations in protein solutions, which has been shown to be a valid protocol for testing biocompatibility. The importance of reporting selectivity in a manner which indicates the “fitness of purpose” of biosensors has been discussed.     

Microscopic and voltammetric characterization of bioanalytical platforms based on lactate oxidase

A microscopic and voltammetric characterization of lactate oxidase- (LOx-) based bioanalytical platforms for biosensor applications is presented. In this context, emphasis is placed on amperometric biosensors based on LOx that have been immobilized by direct absorption on carbon surfaces, in particular, glassy carbon (GC) and highly ordered pyrolytic graphite (HOPG). The immobilized LOx layers have been characterized using atomic force microscopy (AFM) under liquid conditions and cyclic voltammetry. In addition, spatially resolved mapping of enzymatic activity has been carried out using scanning electrochemical microscopy (SECM). In the presence of lactate with hydroxymethylferrocene (HMF) as a redox mediator in solution, biosensors obtained by direct adsorption of LOx onto GC electrodes exhibited a clear electrocatalytic activity, and lactate could be determined amperometrically at 300 mV versus SSCE. The proposed biosensor also exhibits good operating performance in terms of linearity, detection limit, and lifetime.