L-lactate Biosensor Research Articles

More Than One Enzyme: Exploring Alternative FMN-Dependent L-Lactate Oxidases for Biosensor Development.

The α-hydroxy acid oxidoreductase (HAOx) family contains a diverse group of enzymes that can be applied in biosensors for L-lactate detection, most prominently lactate oxidase (LOx). The limited availability and a lack of diversity of L-lactate-oxidizing enzymes have currently hindered advancements in L-lactate biosensor development. Until now, the field has mostly relied on a single, commercially available enzyme, namely Aerococcus viridans L-lactate oxidase (AvLOx). In this study, we present newly discovered alternative L-lactate oxidases that exhibit a narrow substrate specificity and varied kinetic efficiencies toward L-lactate, making them suitable for integration into existing biosensor configurations. Some of these FMN-dependent L-lactate oxidases could be obtained in substantial amounts from routine E. coli expression, potentially facilitating commercial production. Using electrochemical characterization with a mediated biosensor setup, we present 7 enzymes that perform comparable or even better than commercial AvLOx. Finally, we show that their electrochemical performance is not directly correlating with their biochemical performance, making predictions of the suitability of enzymes for biosensor applications extremely difficult. Our research emphasizes the significance of expanding the enzyme toolbox of L-lactate oxidases for the development of improved L-lactate biosensors.

High-Performance Genetically Encoded Green Fluorescent Biosensors for Intracellular l-Lactate.

l-Lactate is a monocarboxylate produced during the process of cellular glycolysis and has long generally been considered a waste product. However, studies in recent decades have provided new perspectives on the physiological roles of l-lactate as a major energy substrate and a signaling molecule. To enable further investigations of the physiological roles of l-lactate, we have developed a series of high-performance (ΔF/F = 15 to 30 in vitro), intensiometric, genetically encoded green fluorescent protein (GFP)-based intracellular l-lactate biosensors with a range of affinities. We evaluated these biosensors in cultured cells and demonstrated their application in an ex vivo preparation of Drosophila brain tissue. Using these biosensors, we were able to detect glycolytic oscillations, which we analyzed and mathematically modeled.

LACCO Series: Genetically Encoded L-Lactate Biosensors

LACCO Series: Genetically Encoded L-Lactate Biosensors

Lactate biosensors for spectrally and spatially multiplexed fluorescence imaging

l-Lactate is increasingly appreciated as a key metabolite and signaling molecule in mammals. However, investigations of the inter- and intra-cellular dynamics of l-lactate are currently hampered by the limited selection and performance of l-lactate-specific genetically encoded biosensors. Here we now report a spectrally and functionally orthogonal pair of high-performance genetically encoded biosensors: a green fluorescent extracellular l-lactate biosensor, designated eLACCO2.1, and a red fluorescent intracellular l-lactate biosensor, designated R-iLACCO1. eLACCO2.1 exhibits excellent membrane localization and robust fluorescence response. To the best of our knowledge, R-iLACCO1 and its affinity variants exhibit larger fluorescence responses than any previously reported intracellular l-lactate biosensor. We demonstrate spectrally and spatially multiplexed imaging of l-lactate dynamics by coexpression of eLACCO2.1 and R-iLACCO1 in cultured cells, and in vivo imaging of extracellular and intracellular l-lactate dynamics in mice.

Flavocytochrome b2-Mediated Electroactive Nanoparticles for Developing Amperometric L-Lactate Biosensors.

L-Lactate is an indicator of food quality, so its monitoring is essential. Enzymes of L-Lactate metabolism are promising tools for this aim. We describe here some highly sensitive biosensors for L-Lactate determination which were developed using flavocytochrome b2 (Fcb2) as a bio-recognition element, and electroactive nanoparticles (NPs) for enzyme immobilization. The enzyme was isolated from cells of the thermotolerant yeast Ogataea polymorpha. The possibility of direct electron transfer from the reduced form of Fcb2 to graphite electrodes has been confirmed, and the amplification of the electrochemical communication between the immobilized Fcb2 and the electrode surface was demonstrated to be achieved using redox nanomediators, both bound and freely diffusing. The fabricated biosensors exhibited high sensitivity (up to 1436 A·M-1·m-2), fast responses, and low limits of detection. One of the most effective biosensors, which contained co-immobilized Fcb2 and the hexacyanoferrate of gold, having a sensitivity of 253 A·M-1·m-2 without freely diffusing redox mediators, was used for L-Lactate analysis in samples of yogurts. A high correlation was observed between the values of analyte content determined using the biosensor and referenced enzymatic-chemical photometric methods. The developed biosensors based on Fcb2-mediated electroactive nanoparticles can be promising for applications in laboratories of food control.

An enhanced L-lactate biosensor based on nanohybrid of chitosan, iron-nanoparticles and carboxylated multiwalled carbon nanotubes

An enhanced biosensor was developed for the determination of blood lactate in lacto-acidosis patients. The biosensor employed a nanohybrid composed of chitosan/iron oxide nanoparticles and carboxylated multiwalled carbon nanotubes (CHIT/Fe3O4NPs/c-MWCNTs), electrodeposited onto an Au electrode, followed by covalent immobilization of L-lactate oxidase (LOx) onto this nano-hybrid. The biosensor (LOx/CHIT/Fe3O4NPs/c-MWCNTs/AuE) exhibited notable improvements in its analytical characteristics such as a rapid response time (4s), a lower detection limit of 0.15 μM and a wider linear range of 1–3000 μM of L-lactic acid. Additionally, it displayed enhanced reproducibility and an extended shelf life of 100 days. The biosensor was employed to measure the concentration of L-lactate in the plasma of both apparently healthy individuals and lacto-acidosis patients. The results showed that the L-lactate concentrations ranged from 112 ± 1.24 to 183 ± 29.15 μmol/L in apparently healthy individuals, whereas it ranged from 2236 ± 33.29 to 4949 ± 72.39 μmol/L in lacto-acidosis patients, which is significantly higher than in apparently healthy individuals. Thus, the integration of the CHIT/Fe3O4NPs/c-MWCNTs hybrid film in the biosensor led to the enhanced analytical performance of the biosensor.

A Selective Fluorescent l-Lactate Biosensor Based on an l-Lactate-Specific Transcription Regulator and Förster Resonance Energy Transfer.

Selective detection of l-lactate levels in foods, clinical, and bacterial fermentation samples has drawn intensive attention. Many fluorescent biosensors based on non-stereoselective recognition elements have been developed for lactate detection. Herein, the allosteric transcription factor STLldR from Salmonella enterica serovar Typhimurium LT2 was identified to be stereo-selectively respond to l-lactate. Then, STLldR was combined with Förster resonance energy transfer (FRET) to construct a fluorescent l-lactate biosensor FILLac. FILLac was further optimized by truncating the N- and C-terminal amino acids of STLldR between cyan and yellow fluorescent proteins. The optimized biosensor FILLac10N0C exhibited a maximum emission ratio change (ΔRmax) of 33.47 ± 1.91%, an apparent dissociation constant (Kd) of 6.33 ± 0.79 μM, and a limit of detection of 0.68 μM. FILLac10N0C was applied in 96-well microplates to detect l-lactate in bacterial fermentation samples and commercial foods such as Jiaosu and yogurt. The quantitation results of FILLac10N0C exhibited good agreement with that of a commercial l-lactate biosensor SBA-40D bioanalyzer. Thus, the biosensor FILLac10N0C compatible with high-throughput detection may be a potential choice for quantitation of l-lactate in different biological samples.

Flavin Mononucleotide-Dependent l-Lactate Dehydrogenases: Expanding the Toolbox of Enzymes for l-Lactate Biosensors

The development of L-lactate biosensors has been hampered in recent years by the lack of availability and knowledge about a wider range and diversity of L-lactate-oxidizing enzymes that can be used as bioelements in these sensors. For decades, L-lactate oxidase of Aerococcus viridans (AvLOx) has been used almost exclusively in the field of L-lactate biosensor development and has achieved somewhat like a monopoly status as a biocatalyst for these applications. Studies on other L-lactate-oxidizing enzymes are sparse and are often missing biochemical data. In this work, we made use of the vast amount of sequence information that is currently available on protein databases to investigate the naturally occurring diversity of L-lactate-utilizing enzymes of the flavin mononucleotide (FMN)-dependent α-hydroxy acid oxidoreductase (HAOx) family. We identified the HAOx sequence space specific for L-lactate oxidation and additionally discovered a not-yet described class of soluble and FMN-dependent L-lactate dehydrogenases, which are promising for the construction of second-generation biosensors or other biotechnological applications. Our work paves the way for new studies on α-hydroxy acid biosensors and proves that there is more to the HAOx family than AvLOx.

Amperometric L-Lactate Biosensor Based upon a Gold Nanoparticles/Reduced Graphene Oxide/Polyallylamine Hydrochloride Modified Screen-Printed Graphite Electrode

This work describes a novel L-lactate biosensor based on the immobilization of L-lactate dehydrogenase enzyme on the screen-printed electrode modified with a ternary composite based on gold nanoparticles, electrochemically-reduced graphene oxide, and poly (allylamine hydrochloride). The enzyme was stabilized by crosslinking with glutaraldehyde. Applied working potential, pH and NAD+ concentration were optimized. The biosensor reports a specific sensitivity of 1.08 µA/mM·cm2 in a range up to 3 mM L-lactic acid with a detection limit of 1 µM. The operational and long-term stability as well as good selectivity allowed the L-lactic acid measurement in dairy products and wine samples.

Lactate detection sensors for food, clinical and biological applications: a review

l-lactate is a major analyte in the food industry, bioprocess engineering, sports medicine and clinical care unit. l-lactate detection by standard analytical techniques is expensive, time-consuming and difficult for in situ studies. Therefore, selective l-lactate biosensors are developed to overcome the current challenges of food industries and biological sectors. High-throughput nanobiosensors appear promising for on-site detection of lactate in industrial- and laboratory-scale applications. Here, we review principles of lactate sensing techniques and trends in transduction approaches. We discuss nanomaterials, merits and demerits of enzyme-based lactate analysis and commercial testing kits. We present also non-enzymatic nanobiosensors.

Enhanced Performance of Reagent-Less Carbon Nanodots Based Enzyme Electrochemical Biosensors.

This work reports on the advantages of using carbon nanodots (CNDs) in the development of reagent-less oxidoreductase-based biosensors. Biosensor responses are based on the detection of H2O2, generated in the enzymatic reaction, at 0.4 V. A simple and fast method, consisting of direct adsorption of the bioconjugate, formed by mixing lactate oxidase, glucose oxidase, or uricase with CNDs, is employed to develop the nanostructured biosensors. Peripherical amide groups enriched CNDs are prepared from ethyleneglycol bis-(2-aminoethyl ether)-N,N,N′,N′-tetraacetic acid and tris(hydroxymethyl)aminomethane, and used as precursors. The bioconjugate formed between lactate oxidase and CNDs was chosen as a case study to determine the analytical parameters of the resulting L-lactate biosensor. A linear concentration range of 3.0 to 500 µM, a sensitivity of 4.98 × 10−3 µA·µM−1, and a detection limit of 0.9 µM were obtained for the L-lactate biosensing platform. The reproducibility of the biosensor was found to be 8.6%. The biosensor was applied to the L-lactate quantification in a commercial human serum sample. The standard addition method was employed. L-lactate concentration in the serum extract of 0.9 ± 0.3 mM (n = 3) was calculated. The result agrees well with the one obtained in 0.9 ± 0.2 mM, using a commercial spectrophotometric enzymatic kit.

Noninvasive Sweat-Lactate Biosensor Emplsoying a Hydrogel-Based Touch Pad

This study is the first report demonstrating proof-of-concept for a hydrogel-based touch sensor pad used for the non-invasive extraction and detection of sweat components. The sensor device was composed of an electrochemical L-lactate biosensor covered with an agarose gel in a phosphate buffer saline. When human skin contacts the agarose gel, L-lactate in sweat was continuously extracted into the gel, followed by in-situ potentiometric detection without controlled conditions. This novel type of sweat sensor is expected to enable the simple, non-invasive daily periodic monitoring of sweat biomarkers for advanced personal healthcare methods in the future.

An L-lactate Biosensor Based on Printed Organic Inverter Circuitry and with a Tunable Detection Limit

An L-lactate Biosensor Based on Printed Organic Inverter Circuitry and with a Tunable Detection Limit

L-lactate Biosensor Based on LOx/ SiO2@ZrONPs/CHIT/Au

L-lactate Biosensor Based on LOx/ SiO2@ZrONPs/CHIT/Au

Minimizing the effects of oxygen interference on l-lactate sensors by a single amino acid mutation in Aerococcus viridansl-lactate oxidase

l-lactate biosensors employing l-lactate oxidase (LOx) have been developed mainly to measure l-lactate concentration for clinical diagnostics, sports medicine, and the food industry. Some l-lactate biosensors employ artificial electron mediators, but these can negatively impact the detection of l-lactate by competing with the primary electron acceptor: molecular oxygen. In this paper, a strategic approach to engineering an AvLOx that minimizes the effects of oxygen interference on sensor strips was reported. First, we predicted an oxygen access pathway in Aerococcus viridans LOx (AvLOx) based on its crystal structure. This was subsequently blocked by a bulky amino acid substitution. The resulting Ala96Leu mutant showed a drastic reduction in oxidase activity using molecular oxygen as the electron acceptor and a small increase in dehydrogenase activity employing an artificial electron acceptor. Secondly, the Ala96Leu mutant was immobilized on a screen-printed carbon electrode using glutaraldehyde cross-linking method. Amperometric analysis was performed with potassium ferricyanide as an electron mediator under argon or atmospheric conditions. Under argon condition, the response current increased linearly from 0.05 to 0.5mM l-lactate for both wild-type and Ala96Leu. However, under atmospheric conditions, the response of wild-type AvLOx electrode was suppressed by 9–12% due to oxygen interference. The Ala96Leu mutant maintained 56–69% of the response current at the same l-lactate level and minimized the relative bias error to −19% from −49% of wild-type. This study provided significant insight into the enzymatic reaction mechanism of AvLOx and presented a novel approach to minimize oxygen interference in sensor applications, which will enable accurate detection of l-lactate concentrations.

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.

Disposable L-lactate biosensor based on a screen-printed carbon electrode enhanced by graphene

In this work, an amperometric L-lactate biosensor based on a graphene-modified screen-printed carbon electrode (SPCE) was constructed. First, the electrocatalytic performance of the SPCE modified with graphene by a one-step electrodeposition process (OerGO/SPCE) was investigated. The cyclic voltammogram of OerGO/SPCE, which showed a well-defined redox peak, had a smaller peak potential separation than that of SPCE, revealing the improvement in electron transfer speed brought about by modifying with graphene. Next, lactate oxidase and potassium ferricyanide were dropped on the OerGO/SPCE to construct a graphene-modified L-lactate biosensor (LOD/K3[Fe(CN)6]/OerGO/SPCE). The proposed biosensor, with a detection limit of 60 μM, had a high sensitivity (42.42 μA mM−1 cm−2) when working at a low working potential (0.15 V). The linear range was 0.5 mM–15 mM, covering the detecting range of L-lactate in clinical applications. The L-lactate biosensor had a short response time (10 s) and required only 10 μl of the sample. This L-lactate sensor modified with electrodeposited graphene had a larger sensitivity than that based on the bare SPCE. Thus, our low-cost and disposable L-lactate biosensor enhanced by graphene can perform as an attractive electrochemical device that can be manufactured for point-of-care testing (POCT) devices and be employed in POCT applications.

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.

Polyoxometalate@magnetic graphene as versatile immobilization matrix of Ru(bpy)32+ for sensitive magneto-controlled electrochemiluminescence sensor and its application in biosensing

We demonstrated here the exploration of polyoxometalate (POM) coated magnetic Fe3O4/reduced graphene oxide (POM@mrGO) composite as the versatile immobilization matrix for the electrochemiluminescence (ECL) agent Ru(bpy)32+. The effective modification of Ru(bpy)32+/POM@mrGO hybrid simply involved using magnetic electrode showed 10-fold ECL intensity increase than that observed for Ru(bpy)32+/Nafion@mrGO to the same concentration of nicotinamide adenine dinucleotide (NADH), which is largely due to POM׳s good electrocatalytic activity towards NADH oxidation. These findings allowed the stable and ultrasensitive ECL detection of NADH as low as 0.1nM. The good stability and high sensitivity of the magneto-controlled ECL sensor enabled us to explore the feasibility of applying the sensing platform to fabricating the ECL biosensors in which the NADH was produced from the dehydrogenase-based enzymatic reaction in the presence of NAD+ cofactor. With l-lactate dehydrogenase as a model, a l-lactate biosensor was successfully constructed where we showed that the ECL intensity of the biosensor increased with the increasing l-lactate concentration. Excellent performance of the presented biosensor has been achieved including a wide linear range extended from 5.0×10−9M to 5.0×10−4M and an extremely low detection limit of 0.4nM. Such sensing strategy combines enzymatic selectivity with simple sensor preparation can be used as a new and biocompatible platform for dehydrogenase-based ECL biosensing.

Amperometric l-lactate biosensor based on screen-printed carbon electrode containing cobalt phthalocyanine, coated with lactate oxidase-mesoporous silica conjugate layer

A novel amperometric biosensor for the measurement of l-lactate has been developed. The device comprises a screen-printed carbon electrode containing cobalt phthalocyanine (CoPC-SPCE), coated with lactate oxidase (LOD) that is immobilized in mesoporous silica (FSM8.0) using a polymer matrix of denatured polyvinyl alcohol; a Nafion layer on the electrode surface acts as a barrier to interferents. The sampling unit attached to the SPCE requires only a small sample volume of 100 μL for each measurement. The measurement of l-lactate is based on the signal produced by hydrogen peroxide, the product of the enzymatic reaction. The behavior of the biosensor, LOD-FSM8.0/Naf/CoPC-SPCE, was examined in terms of pH, applied potential, sensitivity and operational range, selectivity, and storage stability. The sensor showed an optimum response at a pH of 7.4 and an applied potential of +450 mV. The determination range and the response time for l-lactate were 18.3 μM to 1.5 mM and approximately 90 s, respectively. In addition, the sensor exhibited high selectivity for l-lactate and was quite stable in storage, showing no noticeable change in its initial response after being stored for over 9 months. These results indicate that our method provides a simple, cost-effective, high-performance biosensor for l-lactate.