Immobilization Of Lactate Oxidase Research Articles

Sweat lactate biosensor based on lactate oxidase immobilized with flower-like NiCo2O4 and carbon nanotubes

We design a portable and non-invasive lactate biosensor that can conveniently monitor lactate levels in real human sweat. The proposed biosensor is constructed through the immobilization of lactate oxidase (LOx) and horseradish peroxidase (HRP) on flower-like NiCo2O4 microspheres coupled with single-walled carbon nanotubes (SWCNTs). The synergistic effect between NiCo2O4 microspheres and SWCNTs can increase the conductivity and the active surface area of the electrode. Using ferrocene–methanol (FcMeOH) as the electron transfer mediator in buffer, the developed biosensor can detect lactate in a wide linear range of 0.1 mM to 30 mM with a detection limit of 39.9 µM under optimal conditions. Additionally, the biosensor exhibits excellent selectivity and stability. As proof of concept, the biosensor was applied to monitor lactate level in real human sweat samples, and the results were in good agreement with those obtained by high-performance liquid chromatography (HPLC) analysis. The lactate biosensor is cost effective and can be as a non-invasive alternative to blood analysis, which is suitable for rapid analysis of lactate levels in the fields of fitness, health monitoring and clinical diagnosis.

Mill Scale-Derived Magnetite Nanoparticles: A Novel Substrate for Lactate Oxidase-Based Biosensors.

Recycling and revalorization of waste are currently essential for sustainable growth. Mill scale, a waste product from steel production industries, which contains high levels of iron and minimal impurities, is proposed in this study as the source to synthesize magnetite nanoparticles (Fe3O4) for an enhancement of a lactate biosensor range. The synthesized Fe3O4 nanoparticles were coated with polydopamine (PDA) to prevent aggregation and degradation, creating a stable platform for immobilizing lactate oxidase enzyme (LOx) on their surfaces. The characterization of the Fe3O4@PDA material was carried out using transmission electron microscopy (TEM), dynamic light scattering (DLS), and measurement of the polydispersity index (PdI). The Fe3O4@PDA-LOx material was then deposited on a screen-printed carbon electrode modified with Prussian blue (SPCE-PB) for lactate detection. The biosensor exhibited a broad, dual linear concentration-response range, one from 0.1 to 4.62 mM with a limit of detection of 0.32 mM and sensitivity of 1.54 μAmM-1cm-2, and another one from 4.62 to 149.21 mM with a limit of detection of 6.31 mM and sensitivity of 0.08 μAmM-1cm-2. The dual-range concentration response of the biosensor makes it an ideal tool for lactate determination in various applications, including sports medicine, clinical diagnosis, and industrial bioprocessing.

Lactate biosensing based on covalent immobilization of lactate oxidase onto chevron-like graphene nanoribbons via diazotization-coupling reaction

We have designed and prepared an electrochemical biosensor for lactate determination. Through a diazotation process, the enzyme lactate oxidase (LOx) is anchored onto chevron-like graphene nanoribbons (GNR), previously synthesized by a solution-based chemical route, and used as modifiers of glassy carbon electrodes. In a first step, we have performed the grafting of a 4-carboxyphenyl film, by electrochemical reduction of the corresponding 4-carboxyphenyl diazonium salt, on the GNR-modified electrode surface. In this way, the carboxylic groups are exposed to the solution, enabling the covalent immobilization of the enzyme through the formation of an amide bond between these carboxylic groups and the amine groups of the enzyme. The biosensor design was optimized through the morphological and electrochemical characterization of each construction step by atomic force microscopy, scanning electron microscopy, cyclic voltammetry and electrochemical impedance spectroscopy.The cyclic voltammetric response of the biosensor in a solution of hydroxymethylferrocene in presence of l-lactate evidenced a clear electrocatalytic effect powered by the specific design of the biosensing platform with LOx covalently attached to the GNR layer. From the calibration procedures employed for l-lactate determination, a linear concentration range of 3.4 · 10−5– 2.8 · 10−4 M and a detection limit of 11 μM were obtained, with relative errors and relative standard deviations less than 6.0% and 8.4%, respectively. The applicability of the biosensor was tested by determining lactate in apple juices, leading to results that are in good agreement with those obtained with a well-established enzymatic spectrophotometric assay kit.

Bacterial cellulose-based electrochemical sensing platform: A smart material for miniaturized biosensors

Substantial scientific and technological progresses have prompted the development of point of care testing for health monitoring. However, most of the commercially available devices rely on invasive analysis based on blood samples. In this scenario, wearable sensing platforms have arisen as a promising non-invasive analytical tool. To fully exploit their capabilities, challenges must be overcome regarding good mechanical flexibility of wet devices upon bending and stretching movements, preserving the device integrity over the skin. Herein, we successfully developed an electrochemical biosensor made on bacterial cellulose (BC) substrate, and as a proof of concept, we demonstrated the detection of lactate in artificial sweat by immobilizing lactate oxidase (LOx) directly on BC substrate instead of on electrode surface. BC is a smart biocompatible material used as wound dressing with remarkable mechanical properties due to its nanometric fibers. The significant advantages of the proposed substrate for biosensors include biocompatibility, mass production of screen-printed electrodes (SPE) on BC, and superior mechanical resistance in comparison with vegetal cellulose even when hydrated. The electrochemical sensing platform was designed using a carbon-based working electrode modified with Prussian blue nanocubes, an efficient electron mediator for hydrogen peroxide, and LOx immobilized directly onto BC surface. The fabricated biosensor exhibited excellent amperometric response to lactate in the range of 1.0–24.0 mmol L−1 in artificial sweat with detection limit of 1.31 mmol L−1, and quantification limit of 4.38 mmol L−1. The proposed sensing platform based on BC substrate paves the way to wearable devices with superior mechanical resistance and biocompatibility.

Wearable Tape-Based Smart Biosensing Systems for Lactate and Glucose

The development of new bioelectronic platforms could provide exciting new opportunities for clinical analysis, environmental monitoring, and defense. Wearable adhesive tapes could adhere to skin to be in close contact with sweat that consists of many healthcare-related proteins, small molecules, and ions, and thus could function as unique platforms for the sensor construction. Here, we show for the first time the construction of wearable adhesive tape-based smart biosensing systems, and their determinations of lactate and glucose. The sensor is built by printing carbon graphite and Ag/AgCl inks on an adhesive tape to function as the working, reference, and counter electrodes, followed by the immobilization of lactate oxidase or glucose oxidase on the working electrode. The sensor shows a sensitive and broad detection range from micromolar to millimolar, and a quick measuring time of within 5 min for the determination of lactate or glucose. It is expected that these results could open up exciting new avenues for the development of novel wearable sensors, and toward advanced sweat-sensor applications.

A nanocomposite prepared from platinum particles, polyaniline and a Ti3C2 MXene for amperometric sensing of hydrogen peroxide and lactate.

A nanocomposite consistingof platinum particles, polyaniline and Ti3C2 MXene (Pt/PANI/MXene) was used tomodify a screen-printed carbon electrode (SPCE) to obtain sensors for hydrogen peroxide and lactate. This nanocomposite was characterized by scanning electron microscopy (SEM), energy dispersive X-ray spectroscopy (EDX), transmission electron microscopy (TEM) and X-ray powder diffraction (XRD) to determine the physical morphologies and the nanocomposite elements. The modified electrode exhibited the improved current response towards hydrogen peroxide (H2O2) compared with an unmodified electrode and provided a low detection limit of 1.0μM. When lactate oxidase was immobilized on the modified electrode, the electrode responded to lactate via the H2O2 generated in the enzymatic reaction. The lactate assaywas performed by amperometry at a constant potential of +0.3V (vs. Ag/AgCl). The linear range was found to be from0.005 to 5.0mM with a detection limit of 5.0μM for lactate. Ultimately, this biosensor was used for the determination of lactate in milk samples with high stability and reliability. Graphical abstractSchematic representation of a novel platinum particles/polyaniline/MXene nanocomposite (Pt/PANI/MXene) for screen-printed carbon electrode (SPCE) modification to enhance the specific surface area for immobilization of lactate oxidase (LOx) and use as enzymatic biosensor for lactate determination in milk sample.

‘Artificial peroxidase’ nanozyme – enzyme based lactate biosensor

In contrast to bienzyme biosensors, we propose the nanozyme-enzyme based ones substituting the enzyme peroxidase with the more active and stable nanoparticles “artificial peroxidase”. The use of catalytically synthesized Prussian Blue based nanozymes simplifies assembling of hydrogen peroxide transducer providing its higher sensitivity. For immobilization of lactate oxidase the composite alkoxysilane – perfluorosulfonated ionomer (PFSI) membranes are proposed achieving the significantly improved operating stability. The resulting nanozyme-enzyme lactate biosensor displays twice higher sensitivity (>0.2 A M−1 cm−2) compared to the Prussian Blue film based one. Nanozymes “artificial peroxidase” are expected to find wide use in elaboration of oxidase based biosensors.

Amperometric lactate nanobiosensor based on reduced graphene oxide, carbon nanotube and gold nanoparticle nanocomposite.

A sensitive amperometric method is reported for the determination of lactate. A platinum electrode was modified with a composite prepared from reduced graphene oxide (rGO), carbon nanotubes (CNTs) and gold nanoparticles. The composite was synthesized by the in-situ reduction of gold(III) ions on the GO-CNT hybrid. Triple composite components showed synergistic effects on the enzyme loading, electrocatalytic activity and electron transfer between receptor and electrode surface. The amperometric lactate sensor was obtained by immobilization of lactate oxidase (LOx) on the modified electrode. LOx catalyzes the conversion of lactate into pyruvate and hydrogen peroxide. The generated hydrogen peroxide is simultaneously involved in the oxidation reaction, which is associated with the electron production. These electrons act as amperometric signal generators. At the low potential of 0.2V, the nanobiosensor shows a relatively wide linear analytical range (i.e., 0.05-100mM of lactate) with high electrochemical sensitivity (35.3μA mM-1 cm-2) and limit of detection of 2.3μM. The sensor is stable, repeatable and reproducible. It is a highly sensitive tool for the detection of lactate in biological samples under both normoxic and hypoxic conditions. Graphical abstract Schematic representation of an electrochemical biosensor for sensitive detection of lactate in biological and food samples based on reduced graphene oxide-carbon nanotube-gold nanocomposite, as a surface modifier, and lactate oxidase, as a bioreceptor.

Selective determination of lactic acid in dry-fermented sausages using a sensor based on immobilized lactate oxidase

ABSTRACTSelective determination of lactic acid in dry-fermented sausages is an indicator of quality and its presence prevents the growth of pathogenic bacteria. The analysis of lactic acid represents a high cost for the relevant food industries. The use of an enzymatic sensor would allow to reduce the time and cost of this analysis. An enzymatic sensor employing lactate oxidase (LacOx) with the immobilized enzyme system in combination with an oxygen electrode was optimized to determine the lactic acid content in dry-fermented sausages. In this study using LacOx from Pediococcus sp., a voltage of −600 mV, low volumes of reaction and the amperometric signal obtained due to the oxygen depletion (consumed oxygen) during the lactic acid oxidation was recorded at 15 s in the immobilized enzyme sensor so that the reaction rates (slope) were related to the lactic acid content. A positive linear relationship between the consumed oxygen as a function of time (mg O2/L*s−1) and the lactic acid concentration in the range of 250–600 μM, with a coefficient ofR2 = 0.9936 for the immobilized enzyme system, was determined. The immobilized enzyme sensor showed a high specificity KM = 0.865 and sensitivity of 0.25 mM and was stable enough to allow the reutilization of the membranes up to 20 times without loss of activity, where 90% of its initial activity remained after 45 days. The analysis of lactic acid with the immobilized enzyme system in dry-fermented sausages revealed very good agreement with the determination performed through standard HPLC methodology using the same linear range in both methods, which validated the use of this sensor as an alternative technique to evaluate cured meat quality.

Wearable Carbon Nanotube-Based Biosensors on Gloves for Lactate.

Developing a simple and direct approach for interfacing a sensor and a target analyte is of great interest for fields such as medical diagnosis, threat detection, food quality control, and environmental monitoring. Gloves provide a unique interface for sensing applications. Here, we show for the first time the development of wearable carbon nanotube (CNT)-based amperometric biosensors painted onto gloves as a new sensing platform, used here for the determination of lactate. Three sensor types were studied, configured as: two CNT electrodes; one CNT electrode, and an Ag/AgCl electrode, and two CNT electrodes and an Ag/AgCl electrode. The sensors are constructed by painting the electrodes using CNT or Ag/AgCl inks. By immobilizing lactate oxidase onto the CNT-based working electrodes, the sensors show sensitive detections of lactate. Comparison of sensor performance shows that a combination of CNT and Ag/AgCl is necessary for highly sensitive detection. We anticipate that these findings could open exciting avenues for fundamental studies of wearable bioelectronics, as well as practical applications in fields such as healthcare and defense.

A Wearable Amperometric Biosensor on a Cotton Fabric for Lactate

The development of new bioelectronic sensors could enable significant advances in clinical analysis, environmental monitoring, and defense. Cotton fabric is a wearable material, which can contact the skin closely. Here, we demonstrate for the first time the development of a wearable amperometric biosensor on a cotton fabric for the detection of lactate. The sensor is constructed by printing carbon graphite ink and Ag/AgCl ink on a cotton fabric as the working, reference, and counter electrode. Via the immobilization of lactate oxidase, the sensor shows a sensitive detection of lactate with a detection range of 0.05–1.5 mM and a rapid measuring time of around 5 min. We anticipate that these results could open exciting opportunities for fundamental studies and practical applications of wearable bioelectronics in areas ranging from healthcare to defense.

Graphene Supported Platinum Nanoparticles Modified Electrode and Its Enzymatic Biosensing for Lactic Acid

Lactic acid is a metabolic product of human physiological activity. It is of great importance in the diagnosis and treatment of diseases and scientific sports management. It is necessary to build a lactate biosensor that can realize rapid and accurate detection of lactic acid. Graphene supported platinum nanoparticles (GPNs) were prepared by the redox reaction between graphene oxide, chloroplatinic acid and ethylene glycol. Pt nanoparticles were highly dispersed on the graphene, and the surface platinum content of the prepared GPNs was as high as 28%. The GPNs showed excellent electrochemical performance compared to graphene modified electrodes or platinum modified electrodes. An enzymatic lactic acid biosensor (ELB) was prepared by immobilizing lactate oxidase (LOx) on the GPNs modified glassy carbon electrode. The response current of the sensor at +0.4 V in the presence of lactic acid showed a wide linear relationship in the range of 0–1.0 mM, and the limit of detection (LOD) was calculated to be 8.0 μM according to S/N = 3. The ELB was free from common interfering species, and exhibited excellent stability. From the 1st day to the 15th day, the response signal lost less than 2%.

Sonochemically Synthesized ZnO Nanostructure-Based L-Lactate Enzymatic Sensors on Flexible Substrates

ABSTRACTWe report on highly sensitive and flexible L-lactate enzymatic sensors. The sensing materials of this biosensor, two-dimensional (2D) zinc oxide nanoflakes (ZnO NFs), are synthesized on flexible gold(Au)-coated polyethylene terephthalate (PET) substrate using one step sonochemical approach for non-invasive lactate monitoring in human perspiration. ZnO NFs show high isoelectric points (IEP) and biocompatibility. Taking advantage of these unique properties, we immobilized Lactate oxidase (LOx) on the synthesized ZnO NFs. PET/Au/ZnO NFs sensors demonstrated detection of lactate in the range of 10 pM-10 µM for the electrode area of 0.5×0.5 cm2. The sensitivity of this linker free lactate sensor was found 2.23μA/M /cm2 and shows 4 times better response than conventional Au electrode with linker.

Reagent-Less and Robust Biosensor for Direct Determination of Lactate in Food Samples.

Lactic acid is a relevant analyte in the food industry, since it affects the flavor, freshness, and storage quality of several products, such as milk and dairy products, juices, or wines. It is the product of lactose or malo-lactic fermentation. In this work, we developed a lactate biosensor based on the immobilization of lactate oxidase (LOx) onto N,N′-Bis(3,4-dihydroxybenzylidene) -1,2-diaminobenzene Schiff base tetradentate ligand-modified gold nanoparticles (3,4DHS–AuNPs) deposited onto screen-printed carbon electrodes, which exhibit a potent electrocatalytic effect towards hydrogen peroxide oxidation/reduction. 3,4DHS–AuNPs were synthesized within a unique reaction step, in which 3,4DHS acts as reducing/capping/modifier agent for the generation of stable colloidal suspensions of Schiff base ligand–AuNPs assemblies of controlled size. The ligand—in addition to its reduction action—provides a robust coating to gold nanoparticles and a catalytic function. Lactate oxidase (LOx) catalyzes the conversion of l-lactate to pyruvate in the presence of oxygen, producing hydrogen peroxide, which is catalytically oxidized at 3,4DHS–AuNPs modified screen-printed carbon electrodes at +0.2 V. The measured electrocatalytic current is directly proportional to the concentration of peroxide, which is related to the amount of lactate present in the sample. The developed biosensor shows a detection limit of 2.6 μM lactate and a sensitivity of 5.1 ± 0.1 μA·mM−1. The utility of the device has been demonstrated by the determination of the lactate content in different matrixes (white wine, beer, and yogurt). The obtained results compare well to those obtained using a standard enzymatic-spectrophotometric assay kit.

An improved amperometric L-lactate biosensor based on covalent immobilization of microbial lactate oxidase onto carboxylated multiwalled carbon nanotubes/copper nanoparticles/polyaniline modified pencil graphite electrode.

An improved amperometric l-lactate biosensor was constructed based on covalent immobilization of lactate oxidase (LOx) from Pediococcus species onto carboxylated multiwalled carbon nanotubes (cMWCNT)/copper nanoparticles (CuNPs)/polyaniline (PANI) hybrid film electrodeposited on the surface of a pencil graphite electrode (PGE). The enzyme electrode was characterized by cyclic voltammetry (CV), scanning electron microscopy (SEM), Fourier transform infrared (FTIR) spectroscopy and electrochemical impedance spectroscopy (EIS), while CuNPs synthesized by chemical reduction method, were characterized by transmission electron microscopy (TEM), UV spectrascopy and X-ray diffraction (XRD). The biosensor showed maximum response within 5s at pH 8.0 in 0.05M sodium phosphate buffer and 37°C, when operated at 20mVs-1. The biosensor had a detection limit of 0.25μM with a wide working range between 1μM-2500μM. The biosensor was employed for measurement of l-lactic acid level in plasma of apparently healthy and diseased persons. Analytical recovery of added lactic acid in plasma was 95.5%. Within- and between-batch coefficients of variations were 6.24% and 4.19% respectively. There was a good correlation (R2=0.97) between plasma lactate values as measured by standard enzymatic spectrophotometric method and the present biosensor. The working enzyme electrode was used 180 times over a period of 140 days, when stored 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.

Covalent Immobilization of Lactate Oxidase onto Zirconia Coated Silica Nanoparticles/Chitosan Hybrid Film for Amperometric Determination of Lactate

An improved amperometric L-lactate biosensor was constructed based on covalent immobilization of lactate oxidase (LOx) onto zirconia coated silica nanoparticles (SiO2@ZrONPs)/chitosan (CHIT) hybrid film electrodeposited on the surface of a gold electrode (AuE). The enzyme electrode was characterized by cyclic voltammetry (CV), scanning electron microscopy (SEM), Fourier transform infrared (FTIR) spectroscopy and electrochemical impedance spectroscopy (EIS), while SiO2@ZrONPs were synthesized by chemical reduction method and characterized by transmission electron microscopy (TEM), UV spectroscopy and X-ray diffraction (XRD). The biosensor showed an optimal response within 3s at pH 7.5 in 0.05M sodium phosphate buffer and 30°C, when operated at 20 mVs-1. The biosensor had a low detection limit of 0.2 nM with a wide working / linear range between 0.1 – 4000 μM. The biosensor was employed for measurement of L-lactic acid level in plasma of apparently healthy and diseased persons. Analytical recovery of added lactic acid (5.0 mM and 10.0 mM)) in plasma was 99% and 96.6% respectively. Within- and between-batch coefficients of variations were 1.79% and 2.89% respectively. There was a good correlation (R2=0.99) between plasma lactate values as measured by standard enzymatic spectrophotometric method and the present biosensor. The enzyme electrode was used 160 times over a period of 120 days, when stored dry at 4°C.

Enzyme Functionalized Gold Nanoparticles for the Enhanced Electrochemical Detection of Lactate

Lactate is a key molecule in the anaerobic carbohydrate metabolism ­of glucose in muscles. It is primarily considered as the biomarker for tissue oxidative stress. Heavy workout, heart failure, liver disease, drug toxicity and different cancer types causes excess production of lactate in sweat and blood. Therefore the measurement of lactate is important for clinical emergencies, sports and in general medicine. Even though there are reports in literature about lactate sensing, gold nanoparticles functionalized with the enzyme lactate oxidase using disulphide crosslinkers shows promising catalytic activity. In this work, a ultrasensitive biosensor was fabricated by covalently immobilizing lactate oxidase on self-assembled monolayer (SAM) on the gold nanoparticles modified screen printed electrodes (SPE). The sensing mechanism involves the detection of hydrogen peroxide produced during the enzymatic oxidation of lactate. Scanning electron microcopy and cyclic voltammetry were used to characterize the fabricated biosensor. The results show that SAM on uniformly deposited gold nanoparticles provides a favorable microenvironment to retain the bioactivity of enzyme towards lactate detection. The proposed lactate biosensor exhibited good analytical performances including high sensitivity and selectivity with satisfactory stability to electrochemical determination of lactate.

Enzymatic lactic acid sensing by In-doped ZnO nanowires functionalized AlGaAs/GaAs high electron mobility transistor

Indium (In) doped zinc oxide (ZnO) nanowires-gated AlGaAs/GaAs high electron mobility transistor (HEMT) was demonstrated for the detection of lactic acid. The In-doped ZnO nanowires were synthesized via chemical vapor deposition (CVD) method. Such In-doped ZnO nanowires offered an effective surface area with high surface area-to-volume ratio as well as a favorable environment for the immobilization of lactate oxidase (LOX) on the HEMT gate area. The In-doped ZnO nanowires have better conductivity than pure ZnO nanowires and retained the electroactivity of enzymes. Due to the novel structure of the Si-doped GaAs cap layer, the drain–source current of the AlGaAs/GaAs HEMT sensor showed a rapid response when lactic acid solutions at various concentrations were introduced to the gate area of the HEMT. The fabricated sensor exhibited a wide detection range from 3pM to 3mM and a low detection limit of 3pM. The result indicated that a portable, fast response and high sensitivity lactic acid detector can be realized by AlGaAs/GaAs HEMT sensor.

A comparative study of different protein immobilization methods for the construction of an efficient nano-structured lactate oxidase-SWCNT-biosensor

We constructed lactate biosensors by immobilization of lactate oxidase (LOx) onto a single-walled carbon nanotube (SWCNT) electrode. The first step of the sensor construction was the immobilization of oxidized SWCNT onto a platinum electrode modified with 4-aminothiophenol (4-ATP). Two enzyme immobilization methods were used to construct the biosensors, i.e., covalent immobilization using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) and physical adsorption. Atomic force microscopy (AFM) experiments confirmed the immobilization of SWCNT during the biosensor construction and X-ray photoelectron spectroscopy (XPS) experiments confirmed covalent immobilization of LOx onto the SWCNT in the first method. The biosensor based on covalent enzyme immobilization showed a sensitivity of 5.8μA/mM, a linearity up to 0.12mM of l-lactate, and a detection limit of 4.0μM. The biosensor based on protein adsorption displayed a sensitivity of 9.4μA/mM, retaining linearity up to 0.18mM of l-lactate with a detection limit of 3.0μM. The difference in the biosensor response can be attributed to protein conformational or dynamical changes during covalent immobilization. The stability of the biosensors was tested at different temperatures and after different storage periods. The thermostability of the biosensors after incubation at 60°C demonstrated that the biosensor with covalently immobilized LOx retained a higher response compared with the adsorbed protein. Long-term stability experiments show a better residual activity of 40% for the covalently immobilized protein compared to 20% of residual activity for the adsorbed protein after 25d storage. Covalent protein immobilization was superior compared to adsorption in preserving biosensor functionality over extended time period.