Search Results (24)

A biosensor for the determination of glucose, lactate, ethanol and starch in beverages has been developed using enzymes immobilized by a redox-active gel on a screen-printed electrode. A significant improvement proposed for multichannel biosensors, overcoming stability and sensitivity issues by covalently binding phenazine mediators to a biocompatible protein hydrogel, enhancing the packaging of the enzyme. Glucose oxidase (GOx), alcohol oxidase (AOx) and lactate oxidase (LOx) were used as biological materials, as well as a mixture of GOx with γ-amylase (Am). Redox gels were synthesized from bovine serum albumin (BSA) and phenazine derivatives. It was shown that a neutral red-based redox gel combined with single-walled carbon nanotubes is more promising than other substrates for enzyme immobilization. The lower limit of quantification for glucose, ethanol, lactate and starch using these systems is 0.035 mM, 2.3 mM, 15 mM and 2 mg/L, respectively. Biosensors were used to analyze the content of these substances in alcoholic, kvass and fermentation mass. Statistical analysis of the results showed that the values of glucose, ethanol, lactic acid and starch determined using biosensors and obtained by reference methods differ insignificantly. A set of biosensors developed on the basis of specifically selected enzymes is effective for controlling biotechnological processes and can be used as an alternative to classical analytical methods. Full article

Artificial enzymes or nanozymes (NZs) are gaining significant attention in biotechnology due to their stability and cost-effectiveness. NZs can offer several advantages over natural enzymes, such as enhanced stability under harsh conditions, longer shelf life, and reduced production costs. The booming interest in NZs is likely to continue as their potential applications expand. In our previous studies, we reported the “green” synthesis of copper hexacyanoferrate (gCuHCF) using the oxidoreductase flavocytochrome b₂ (Fcb₂). Organic–inorganic micro-nanoparticles were characterized in detail, including their structure, composition, catalytic activity, and electron-mediator properties. An SEM analysis revealed that gCuHCF possesses a flower-like structure well-suited for concentrating and stabilizing Fcb₂. As an effective peroxidase (PO) mimic, gCuHCF has been successfully employed for H₂O₂ detection in amperometric sensors and in several oxidase-based biosensors. In the current study, we demonstrated the uniqueness of gCuHCF that lies in its multifunctionality, serving as a PO mimic, a chemosensor for ammonium ions, a biosensor for L-lactate, and exhibiting perovskite-like properties. This exceptional ability of gCuHCF to enhance fluorescence under blue light irradiation is being reported for the first time. Using gCuHCF as a PO-like NZ, novel oxidase-based sensors were developed, including an optical biosensor for L-arginine analysis and electrochemical biosensors for methanol and glycerol determination. Thus, gCuHCF, synthesized via Fcb₂, presents a promising platform for the development of amperometric and optical biosensors, bioreactors, biofuel cells, solar cells, and other advanced devices. The innovative approach of utilizing biocatalysts for nanoparticle synthesis highlights a groundbreaking direction in materials science and biotechnology. Full article

We report the development of amperometric biosensors (ABSs) employing flavocytochrome b₂ (Fcb₂) coupled with nanoparticles (NPs) of noble metals on graphite electrode (GE) surfaces. Each NPs/GE configuration was evaluated for its ability to decompose hydrogen peroxide (H₂O₂), mimicking peroxidase (PO) activity. The most effective nanoPO (nPO) was selected for developing ABSs targeting L-lactate. Consequently, several Fcb₂/nPO-based ABSs with enhanced sensitivity to L-lactate were developed, demonstrating mediated ET between Fcb₂ and the GE surface. The positive effect of noble metal NPs on Fcb₂-based sensor sensitivity may be explained by the synergy between their dual roles as both PO mimetics and electron transfer mediators. Furthermore, our findings provide preliminary data that may prompt a re-evaluation of the mechanism of L-lactate oxidation in Fcb₂-mediated catalysis. Previously, it was believed that L-lactate oxidation via Fcb₂ catalysis did not produce H₂O₂, unlike catalysis via L-lactate oxidase. Our initial research revealed that the inclusion of nPO in Fcb₂-based ABSs significantly increased their sensitivity. Employing other PO mimetics in ABSs for L-lactate yielded similar results, reinforcing our hypothesis that trace amounts of H₂O₂ may be generated as a transient intermediate in this reaction. The presence of nPO enhances the L-lactate oxidation rate through H₂O₂ utilization, leading to signal amplification and heightened bioelectrode sensitivity. The proposed ABSs have been successfully tested on blood serum and fermented food samples, showing their promise for L-lactate monitoring in medicine and the food industry. Full article

L-Lactate is an important bioanalyte in the food industry, biotechnology, and human healthcare. In this work, we report the development of a new L-lactate electrochemical biosensor based on the use of multiwalled carbon nanotubes non-covalently functionalized with avidin (MWCNT-Av) deposited at glassy carbon electrodes (GCEs) as anchoring sites for the bioaffinity-based immobilization of a new recombinant biotinylated lactate oxidase (bLOx) produced in Escherichia coli through in vivo biotinylation. The specific binding of MWCNT-Av to bLOx was characterized by amperometry, surface plasmon resonance (SPR), and electrochemical impedance spectroscopy (EIS). The amperometric detection of L-lactate was performed at −0.100 V, with a linear range between 100 and 700 µM, a detection limit of 33 µM, and a quantification limit of 100 µM. The proposed biosensor (GCE/MWCNT-Av/bLOx) showed a reproducibility of 6.0% and it was successfully used for determining L-lactate in food and enriched serum samples. Full article

Lactate serves as a crucial biomarker that indicates sepsis assessment in critically ill patients. A rapid, accurate, and portable analytical device for lactate detection is required. This work developed a stepwise polyurethane–polyaniline–m-phenylenediamine via a layer-by-layer based electrochemical biosensor, using a screen-printed gold electrode for lactate determination in blood samples. The developed lactate biosensor was electrochemically fabricated with layers of m-phenylenediamine, polyaniline, a crosslinking of a small amount of lactate oxidase via glutaraldehyde, and polyurethane as an outer membrane. The lactate determination using amperometry revealed the biosensor’s performance with a wide linear range of 0.20–5.0 mmol L⁻¹, a sensitivity of 12.17 ± 0.02 µA·mmol⁻¹·L·cm⁻², and a detection limit of 7.9 µmol L⁻¹. The developed biosensor exhibited a fast response time of 5 s, high selectivity, excellent long-term storage stability over 10 weeks, and good reproducibility with 3.74% RSD. Additionally, the determination of lactate in human blood plasma using the developed lactate biosensor was examined. The results were in agreement with the enzymatic colorimetric gold standard method (p > 0.05). Our developed biosensor provides efficiency, reliability, and is a great potential tool for advancing lactate point-of-care testing applications in the early diagnosis of sepsis. Full article

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. Full article

The electrochemical enzyme sensors based on direct electron transfer (DET)-type oxidoreductase-based enzymes are ideal for continuous and in vivo monitoring. However, the number and types of DET-type oxidoreductases are limited. The aim of this research is the development of a versatile method to create a DET-type oxidoreductase complex based on the SpyCatcher/SpyTag technique by preparing SpyCatcher-fused heme c and SpyTag-fused non-DET-type oxidoreductases, and by the in vitro formation of DET-type oxidoreductase complexes. A heme c containing an electron transfer protein derived from Rhizobium radiobacter (CYTc) was selected to prepare SpyCatcher-fused heme c. Three non-DET-type oxidoreductases were selected as candidates for the SpyTag-fused enzyme: fungi-derived flavin adenine dinucleotide (FAD)-dependent glucose dehydrogenase (GDH), an engineered FAD-dependent d-amino acid oxidase (DAAOx), and an engineered FMN-dependent l-lactate oxidase (LOx). CYTc-SpyCatcher (CYTc-SC) and SpyTag-Enzymes (ST-GDH, ST-DAAOx, ST-LOx) were prepared as soluble molecules while maintaining their redox properties and catalytic activities, respectively. CYTc-SC/ST-Enzyme complexes were formed by mixing CYTc-SpyCatcher and SpyTag-Enzymes, and the complexes retained their original enzymatic activity. Remarkably, the heme domain served as an electron acceptor from complexed enzymes by intramolecular electron transfer; consequently, all constructed CYTc-SC/ST-Enzyme complexes showed DET ability to the electrode, demonstrating the versatility of this method. Full article

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 FILLac_10N0C exhibited a maximum emission ratio change (ΔR_max) of 33.47 ± 1.91%, an apparent dissociation constant (K_d) of 6.33 ± 0.79 μM, and a limit of detection of 0.68 μM. FILLac_10N0C 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 FILLac_10N0C exhibited good agreement with that of a commercial l-lactate biosensor SBA-40D bioanalyzer. Thus, the biosensor FILLac_10N0C compatible with high-throughput detection may be a potential choice for quantitation of l-lactate in different biological samples. Full article

Precision analysis of the key biological metabolites such as L-lactate has great practical importance for many technological processes in food technology, including beverage production. Here we describe a new, highly selective, and sensitive biosensor for accurate L-lactate assay based on a combination of peroxidase-mimetic nanozymes with microbial lactate oxidase (LOx) immobilized onto the surface of a graphite-rod electrode (GE). The peroxidase-like nanozymes were synthesized using the debris of carbon microfibers (CFs) functionalized with hemin (H) and modified with gold nanoparticles (AuNPs) or platinum microparticles (PtMPs). The nanozyme formed with PtMPs as well as corresponding bioelectrodes based on it (LOx-CF-H-PtMPs/GE) is characterized by preferable catalytic and operational characteristics, so it was selected for the analysis of L-lactate content in real samples of grape must and red wine. The results of the L-lactate analysis obtained by the developed biosensors are highly correlated with a very selective spectrophotometric approach used as a reference. The developed biosensor, due to its high selectivity and sensitivity, is very prospective not only for the beverage industry and food technology, but also for clinical diagnostics and medicine, as well as in other applications where the accurate analysis of L-lactate is highly important. Full article

Aflatoxin M1 (AFM1) is one of the most widespread aflatoxins that can be present in the milk of lactating mammals. It can cause carcinogenicity, mutagenesis, teratogenesis, genotoxicity and immunosuppression. The WHO recommends reducing the AFM1 concentration in food products, so the European Commission has set a maximum allowable limit of 0.05 µg L⁻¹ in milk and its products. Thus, there is a need to develop new methodologies to satisfy the demand for reliable, cost-effective, robust and sensitive AFM1 routine controls. In the present work, a competitive phosphorescent immunosensor for AFM1 quantification in milk, based on antibody–antigen recognition and Mn:ZnS quantum dots (d-QDs) as photoluminescent labels, has been developed. Two different assay strategies based on the use of d-QDs as labels of secondary antibodies (direct assay), or of a derivative species of the antigen AFM1-Bovine Serum Albumin (indirect assay) were compared in terms of analytical performance for AFM1 quantification. The best analytical results were obtained with the immunoassay format that uses d-QDs as tags of secondary antibodies (direct assay), and said design was finally selected. The selected immunosensor provided a detection limit for AFM1 quantification of only 0.002 µg L⁻¹, which greatly satisfied the maximum tolerable limit of AFM1 in milk of 0.05 µg L⁻¹. The accuracy, calculated as recovery of AFM1 in fortified skimmed milk samples, ranged from 81 to 90%, with relative standard deviations from 3% to 14%. These results bring to light the good performance of such phosphorescent biosensors as simple and fast alternatives to conventional chromatographic analytical methods. Full article

Understanding the levels of glucose (G) and lactate (L) in blood can help us regulate various chronic health conditions such as obesity. In this paper, we introduced an enzyme-based electrochemical biosensor adopting glucose oxidase and lactate oxidase on two working screen-printed carbon electrodes (SPCEs) to sequentially determine glucose and lactate concentrations in a single drop (~30 µL) of whole blood. We developed a diet-induced obesity (DIO) mouse model for 28 weeks and monitored the changes in blood glucose and lactate levels. A linear calibration curve for glucose and lactate concentrations in ranges from 0.5 to 35 mM and 0.5 to 25 mM was obtained with R-values of 0.99 and 0.97, respectively. A drastic increase in blood glucose and a small but significant increase in blood lactate were seen only in prolonged obese cases. The ratio of lactate concentration to glucose concentration (L/G) was calculated as the mouse’s gained weight. The results demonstrated that an L/G value of 0.59 could be used as a criterion to differentiate between normal and obesity conditions. With L/G and weight gain, we constructed a diagnostic plot that could categorize normal and obese health conditions into four different zones. The proposed dual electrode biosensor for glucose and lactate in mouse whole blood showed good stability, selectivity, sensitivity, and efficiency. Thus, we believe that this dual electrode biosensor and the diagnostic plot could be used as a sensitive analytical tool for diagnosing glucose and lactate biomarkers in clinics and for monitoring obesity. Full article

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. Full article

The pH drop in the hindgut of the horse is caused by lactic acid-producing bacteria which are abundant when a horse’s feeding regime is excessively carbohydrate rich. This drop in pH below six causes hindgut acidosis and may lead to laminitis. Lactic acid-producing bacteria Streptococcus equinus and Mitsuokella jalaludinii have been found to produce high amounts of L-lactate and D-lactate, respectively. Early detection of increased levels of these bacteria could allow the horse owner to tailor the horse’s diet to avoid hindgut acidosis and subsequent laminitis. Therefore, 16s ribosomal ribonucleic acid (rRNA) sequences were identified and modified to obtain target single stranded deoxyribonucleic acid (DNA) from these bacteria. Complementary single stranded DNAs were designed from the modified target sequences to form capture probes. Binding between capture probe and target single stranded deoxyribonucleic acid (ssDNA) in solution has been studied by gel electrophoresis. Among pairs of different capture probes and target single stranded DNA, hybridization of Streptococcus equinus capture probe 1 (SECP1) and Streptococcus equinus target 1 (SET1) was portrayed as gel electrophoresis. Adsorptive stripping voltammetry was utilized to study the binding of thiol modified SECP1 over gold on glass substrates and these studies showed a consistent binding signal of thiol modified SECP1 and their hybridization with SET1 over the gold working electrode. Cyclic voltammetry and electrochemical impedance spectroscopy were employed to examine the binding of thiol modified SECP1 on the gold working electrode and hybridization of thiol modified SECP1 with the target single stranded DNA. Both demonstrated the gold working electrode surface was modified with a capture probe layer and hybridization of the thiol bound ssDNA probe with target DNA was indicated. Therefore, the proposed electrochemical biosensor has the potential to be used for the detection of the non-synthetic bacterial DNA target responsible for equine hindgut acidosis. Full article

Lactate is an important organic molecule that is produced in excess during anaerobic metabolism when oxygen is absent in the human organism. The concentration of this substance in the body can be related to several medical conditions, such as hemorrhage, respiratory failure, and ischemia. Herein, we describe a graphene-based lactate biosensor to detect the concentrations of L-lactic acid in different fluids (buffer solution and plasma). The active surface (graphene) of the device was functionalized with lactate dehydrogenase enzyme using different substances (Nafion, chitosan, and glutaraldehyde) to guarantee stability and increase selectivity. The devices presented linear responses for the concentration ranges tested in the different fluids. An interference study was performed using ascorbic acid, uric acid, and glucose, and there was a minimum variation in the Dirac point voltage during detection of lactate in any of the samples. The stability of the devices was verified at up to 50 days while kept in a dry box at room temperature, and device operation was stable until 12 days. This study demonstrated graphene performance to monitor L-lactic acid production in human samples, indicating that this material can be implemented in more simple and low-cost devices, such as flexible sensors, for point-of-care applications. Full article

Lactate is a metabolite biomarker of tissue oxygenation and it can be used in medicine to evaluate a pathology or in sport activities to evaluate physical performance. Lactate level assessment is also important for food industry. This acid is found in food and beverages and the concentration level can be correlated with the freshness, stability and quality of several products. In this work, we present a smartphone-based enzymatic biosensor utilizing the unique colorimetric properties of the poly(aniline-co-anthranilic acid) (p(ANI-co-AA)) composite film coupled with lactate oxidase–horseradish peroxidase (LOx–HRP) enzymes. The enzymes are immobilized on the composite polymer film by adsorption and they catalyze a reversible redox color change of the host polymer from green to blue in the presence of l-lactate as the substrate. A smartphone was applied as color detector, for image acquisition and data handling. The free-of-charge Color Grab® application for Android OS was used to enable an easy and clear display of the sensor’s response, indicating remarkable changes in the optical features. The results were confirmed by spectrophotometric measurements. The developed colorimetric enzymatic biosensors were studied and optimized in relation to different experimental parameters. Moreover, the colorimetric enzymatic biosensor was applied to real matrices analysis. It has been shown by these studies that the colorimetric biosensors are promising as quick and simple tests for handheld analysis in various fields. Full article