Enzyme-based Biosensors Research Articles

Sequential detection of dopamine and L-DOPA by a 2,3-dopa-dioxygenase from Streptomyces sclerotialus

A variety of enzyme-based colorimetric biosensors have been developed for clinical practice; however, these methods will only become cost-effective when they are able to process multiple samples with a high degree of sensitivity. In this study, a novel heat-stable enzyme, 2,3-dopa-dioxygenase from the thermophilic bacterium Streptomyces sclerotialus (SsDDO), was used in the development of a protein- and cell-based biosensor for the detection of L-DOPA for the first time. SsDDO catalyzes the oxidative cleavage of L-DOPA forms linear semialdehyde (AHMS) and cyclizes to a 3-carboxy-3-hydroxyallylidene-3,4-dihydropyrrole-2-carboxylic acid (CHAPCA). We next derivatized CHAPCA by reacting with 3-aminobenzoic acid (MABA) to yield a red-fluorescent pigment. Overall, the detection of L-DOPA via the red fluorescent signal can be completed in only 30 min. We also developed a sequential analysis method to detect the coexistence of dopamine and L-DOPA with a high degree of sensitivity using the dual-fluorescent signals to monitor the therapy of patients with Parkinson's disease treated with L-DOPA. The robustness and applicability of the system were further validated in serum. In addition, paper microfluidics modified with chitosan was applied for fast and cost-effective analysis of dopamine and L-DOPA in the mixed solutions.

An amperometric glucose biosensor based on electrostatic force induced layer-by-layer GOD/chitosan/pyrite on a glassy carbon electrode.

Pyrite (PR), as a representative sulfide mineral, possesses the advantages of abundancy, thermodynamic stability, non-toxicity and semi-conductivity. In this study, an amperometric glucose biosensor (GOD/CS/PR/GCE) based on layer-by-layer of glucose oxidase (GOD), chitosan (CS) and pyrite (PR) on a glassy carbon electrode (GCE) was fabricated through electrostatic force. In this research, PR suspension prepared in phosphate buffer (pH 5.5) was first immobilized on the GCE surface, which exhibits a negative charge. Then, positively charged CS was adsorbed on the PR/GCE by electrostatic force. Finally, negatively charged GOD was further modified on the CS/PR/GCE surface through electrostatic force again. The surface morphology and adsorbance mechanism were supported by field emission scanning electron microscopy, quartz crystal microbalance with dissipation and atomic force microscope. The step-by-step procedure gives both strong adhesion ability and good bioelectrocatalytic activity of GOD on the CS- and PR-modified electrode surface. The linear range of this GOD/CS/PR/GCE biosensor was achieved from 0.5 to 60mM with the linear regression equation of y = 0.897x - 0.3016 (R2 = 0.9996) and a limit of detection value of 50µM. This approach of using pyrite and chitosan as physically modified GOD to serve as electrostatic glues could be useful for designing better enzyme-based biosensors for a wide variety of practical applications.

Rational Design of ZIF-8 for Constructing Luminescent Biosensors with Glucose Oxidase and AIE-Type Gold Nanoclusters.

The development of modern technologies has acclimatized biosensors to complicated applicable scenarios with integrated properties as a whole instead of the pursuit of a single-point breakthrough. Here, we targeted a few concerns in the development of enzyme-based biosensors, including stability, analyte enrichment, and signal transduction, and developed a general biosensing model utilizing enzymes, aggregation-induced emission (AIE) luminogens, and stimuli-responsive framework materials as the units. We propose such proof-of-concept of glucose biosensors by coencapsulating glucose oxidase and AIE-type gold nanoclusters into acid-sensitive zeolite imidazolate framework (ZIF)-8 nanocrystals. The acid-activated degradation of ZIF-8 bridges the molecular signals produced by the enzyme-catalytic reaction of glucose and the photon signals generated by ZIF-8-induced AIE effects of gold nanoclusters, resulting in the "turn-off" model nanoprobes for glucose detection with high selectivity. After embedding the nanoprobes into hollow-out tapes, the formed paper biosensors can conveniently detect glucose with the help of a smartphone.

Fabrication of a New, Low-Cost, and Environment-Friendly Laccase-Based Biosensor by Electrospray Immobilization with Unprecedented Reuse and Storage Performances.

The fabrication of enzyme-based biosensors has received much attention for their selectivity and sensitivity. In particular, laccase-based biosensors have attracted a lot of interest for their capacity to detect highly toxic molecules in the environment, becoming essential tools in the fields of white biotechnology and green chemistry. The manufacturing of a new, metal-free, laccase-based biosensor with unprecedented reuse and storage capabilities has been achieved in this work through the application of the electrospray deposition (ESD) methodology as the enzyme immobilization technique. Electrospray ionization (ESI) has been used for ambient soft-landing of laccase enzymes on a carbon substrate, employing sustainable chemistry. This study shows how the ESD technique can be successfully exploited for the fabrication of a new promising environment-friendly electrochemical amperometric laccase-based biosensor, with storage capability up to two months without any particular care and reuse performance up to 63 measurements on the same electrode just prepared and 20 measurements on the one-year-old electrode subjected to redeposition. The laccase-based biosensor has been tested for catechol detection in the linear range 2–100 μM, with a limit of detection of 1.7 μM, without interference from chrome, cadmium, arsenic, and zinc and without any memory effects.

Towards Multi-Analyte Detection with Field-Effect Capacitors Modified with Tobacco Mosaic Virus Bioparticles as Enzyme Nanocarriers.

Utilizing an appropriate enzyme immobilization strategy is crucial for designing enzyme-based biosensors. Plant virus-like particles represent ideal nanoscaffolds for an extremely dense and precise immobilization of enzymes, due to their regular shape, high surface-to-volume ratio and high density of surface binding sites. In the present work, tobacco mosaic virus (TMV) particles were applied for the co-immobilization of penicillinase and urease onto the gate surface of a field-effect electrolyte-insulator-semiconductor capacitor (EISCAP) with a p-Si-SiO2-Ta2O5 layer structure for the sequential detection of penicillin and urea. The TMV-assisted bi-enzyme EISCAP biosensor exhibited a high urea and penicillin sensitivity of 54 and 85 mV/dec, respectively, in the concentration range of 0.1–3 mM. For comparison, the characteristics of single-enzyme EISCAP biosensors modified with TMV particles immobilized with either penicillinase or urease were also investigated. The surface morphology of the TMV-modified Ta2O5-gate was analyzed by scanning electron microscopy. Additionally, the bi-enzyme EISCAP was applied to mimic an XOR (Exclusive OR) enzyme logic gate.

Development of amperometric enzyme‐based biosensor to evaluate the adulteration in virgin coconut oil

Virgin Coconut oil (VCO), owing to its functional properties (important in COVID-19), is costly and, therefore, susceptible to adulteration with other cheaper oils like coconut oil. An enzyme-based biosensor confirmative test of VCO was constructed by co-immobilizing enzymes onto a glassy carbon electrode. The performance of the biosensor was optimized at a potential of +0.5 V with 45 mg gelatin, 30 mg BSA coupled with 2.5% glutaraldehyde at pH 7.0 with an incubation time of 1 hr. Adulterated samples of VCO with coconut oil (CO) were analyzed. The concentration of diglyceride (DG) was estimated from the empirical relation, which showed a linear increase with the increase in adulteration. The developed biosensor was validated using high-performance liquid chromatography (HPLC) methods using a paired sample t test at a 5% significance level. The biosensor could detect adulteration in VCO with CO above 20% within 3–5 s and can be reused for 25 days. Practical applications Adulteration of edible oils is a massive issue in today's market. Virgin coconut oil due to its functional and antiviral properties becomes important for human health. This study revealed that the developed biosensor could be an effective method for adulteration detection in virgin coconut oil with coconut oil.

A FRET Approach to Detect Paraoxon among Organophosphate Pesticides Using a Fluorescent Biosensor.

The development of faster, sensitive and real-time methods for detecting organophosphate (OP) pesticides is of utmost priority in the in situ monitoring of these widespread compounds. Research on enzyme-based biosensors is increasing, and a promising candidate as a bioreceptor is the thermostable enzyme esterase-2 from Alicyclobacillus acidocaldarius (EST2), with a lipase-like Ser–His–Asp catalytic triad with a high affinity for OPs. This study aimed to evaluate the applicability of Förster resonance energy transfer (FRET) as a sensitive and reliable method to quantify OPs at environmentally relevant concentrations. For this purpose, the previously developed IAEDANS-labelled EST2-S35C mutant was used, in which tryptophan and IAEDANS fluorophores are the donor and the acceptor, respectively. Fluorometric measurements showed linearity with increased EST2-S35C concentrations. No significant interference was observed in the FRET measurements due to changes in the pH of the medium or the addition of other organic components (glucose, ascorbic acid or yeast extract). Fluorescence quenching due to the presence of paraoxon was observed at concentrations as low as 2 nM, which are considered harmful for the ecosystem. These results pave the way for further experiments encompassing more complex matrices.

Nanoarchitectonics with NADPH Catalyst and Quantum Dots Copper Sulfide on Titanium Dioxide Nano-sheets Electrode for Electrochemical Biosensing of Sorbitol Detection.

Sorbitol accumulation in the tissue is known to cause diabetic complications. Nanotechnology-enabled biosensor methods have high sensitivity, selectivity, and more rapid detection of an analytic for sorbitol which is used as a biomarker of diabetic complications. The biosensor used aldose reductase from serum blood to oxidize the NADPH by the enzymatic reaction and reduce glucose to sorbitol. Biosensors can be developed for diagnostic testing. Developing a simple, sensitive, and rapid method for sorbitol detection is significant for efficient monitoring of diabetic complications like neuropathy at the initial stages. This project synthesized quantum dots of copper sulfide (CuS QDs) to fabricate an Electrochemical sensor for the detection of sorbitol by the UV-irradiation technique. The crystal structure of CuS QDs was characterized using X-ray diffraction (XRD), which confirmed the synthesized sample's hexagonal shape. The structure of the manufactured product was examined using energy-dispersive X-ray spectroscopy (EDX), and the result revealed just copper (Cu) and sulfide (S) elements, indicating that the synthetic material was pure. The morphology, optical properties, and particle size were investigated by scanning electron microscope (SEM), photoluminescence spectroscopy (PL), and transmission electron spectroscopy (TEM), respectively. The particle sizes of the CuS QDs were found to range between 5.4 to 9.1 nm. The CuS QDs will be dedicated to the conventional methods to synthesize the modified electrode functionalized with NADPH and covered with CuS QD (Ti-TiO2/CuS/NADPH) demonstrated switchable interfacial properties. The electrochemical process was characterized by cyclic voltammetry (CV). The developed sensor was successfully tested to detect sorbitol in human serum samples. The high catalytic activity and the redox behavior of CuS QD make it an efficient matrix for the realization of sorbitol. These results indicate that CuS QD is a suitable candidate material for developing enzyme-based sorbitol biosensors.

Molecular understanding of acetylcholinesterase adsorption on functionalized carbon nanotubes for enzymatic biosensors.

The immobilization of acetylcholinesterase on different nanomaterials has been widely used in the field of amperometric organophosphorus pesticide (OP) biosensors. However, the molecular adsorption mechanism of acetylcholinesterase on a nanomaterial's surface is still unclear. In this work, multiscale simulations were utilized to study the adsorption behavior of acetylcholinesterase from Torpedo californica (TcAChE) on amino-functionalized carbon nanotube (CNT) (NH2-CNT), carboxyl-functionalized CNT (COOH-CNT) and pristine CNT surfaces. The simulation results show that the active center and enzyme substrate tunnel of TcAChE are both close to and oriented toward the surface when adsorbed on the positively charged NH2-CNT, which is beneficial to the direct electron transfer (DET) and accessibility of the substrate molecule. Meanwhile, the NH2-CNT can also reduce the tunnel cost of the enzyme substrate of TcAChE, thereby further accelerating the transfer rate of the substrate from the surface or solution to the active center. However, for the cases of TcAChE adsorbed on COOH-CNT and pristine CNT, the active center and substrate tunnel are far away from the surface and face toward the solution, which is disadvantageous for the DET and transportation of enzyme substrate. These results indicate that NH2-CNT is more suitable for the immobilization of TcAChE. This work provides a better molecular understanding of the adsorption mechanism of TcAChE on functionalized CNT, and also provides theoretical guidance for the ordered immobilization of TcAChE and the design, development and improvement of TcAChE-OPs biosensors based on functionalized carbon nanomaterials.

A Dual Electrode Biosensor for Glucose and Lactate Measurement in Normal and Prolonged Obese Mice Using Single Drop of Whole Blood.

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.

Construction of an enzyme-based all-fiber SPR biosensor for detection of enantiomers

Chiral analysis of amino acids (AAs) is of great importance in medical science due to the distinctive effect of AA isomers on human health. Although various chiral recognition techniques have been developed, the quantitative chiral recognition of low-level AA isomers remains challenging. Here, we combined the fiber optic SPR with an enzyme-substrate recognition mechanism to construct a direct-assay-type chiral AA biosensor. As a proof-of-concept attempt, a recently discovered Rasamsonia emersonii D-amino acid oxidase (ReDAAO) with a wide substrate spectrum and high stability was immobilized on the graphene oxide and gold nanorods composites (GO-AuNRs), using both EDC/NHS coupling and the gold-binding peptide (GBP) method. Such a biosensor can distinguish two AA isomers at the same concentration. It achieved specific detection of D-amino acids (D-AAs) with a linear range from 5x10-4 mM to 30 mM. Furthermore, it showed good resistance to enantiomeric interference. When the percentage of D-AA increases in the isomer mixture, a good linear relationship between the D/(D + L)-AA ratio and SPR spectral shift was obtained. This unique combination of the enzyme, nanocomposite, and SPR taps into the rich reservoir of proteins for chiral receptors. It lays the foundation for protein-based chiral recognition of other clinically important small molecules in future biosensor designs.

Nanometer-Thick Mn:NiO and Co:NiO Films for High Performance Nonenzymatic Biosensors

People with diabetes require regular blood sugar level monitoring, using commercial enzyme-based biosensors. There is a considerable need to develop biosensors with nonenzymatic electrodes to elimi...

MXene-based electrochemical sensors for detection of environmental pollutants: A comprehensive review

Since the discovery of MXenes at Drexel University in the United States in 2011, there has been extensive research regarding various applications of MXenes including environmental remediation. MXenes with a general formula of Mn+1XnTx are a class of two-dimensional (2D) transition metal carbides, carbonitrides, and nitrides with unique chemical and physical characteristics as nanomaterials. MXenes feature characteristics such as high conductivity, hydrophobicity, and large specific surface areas that are attracting attention from researchers in many fields including environmental water engineering such as desalination and wastewater treatment as well as designing and building efficient sensors to detect hazardous pollutants in water. In this study, we review recent developments in MXene-based nanocomposites for electrochemical (bio) sensing with a particular focus on the detection of hazardous pollutants, such as organic components, pesticides, nitrite, and heavy metals. Integration of these 2D materials in electrochemical enzyme-based and affinity-based biosensors for environmental pollutants is also discussed. In addition, a summary of the key challenges and future remarks are presented. Although this field is relatively new, future research on biosensors of MXene-based nanocomposites need to exploit the remarkable properties of these 2D materials.

Cytochrome P450 2B6 Amperometric Biosensor for Continuous Monitoring of Propofol

Despite the growing evidence of improved patient outcomes and of a substantially reduced environmental impact of propofol-based total intravenous anesthesia when compared to volatile-based techniques, the vast majority of general anesthetics still use volatile agents for the maintenance phase. A significant reason for this is the lack of suitable point-of-care, real-time blood propofol measurement techniques. Here we present an enzyme-based electrochemical biosensor for the detection of propofol. Deactivated yeast cells expressing the enzyme cytochrome P450 2B6 are immobilized, alongside gold nanoparticles, within a chitosan film upon the surface of a screen printed electrode. In the presence of the cofactor NADPH, the enzyme converts propofol to a quinone/quinol redox pair that can be detected using simple electrochemistry. This approach avoids the issue of electrode fouling that commonly renders electrochemical propofol sensors impractical. The sensor has a limit of detection of 67 ± 7 ng/ml and a sensitivity of 4.2 ± 0.2 nA/ <inline-formula xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink"> <tex-math notation="LaTeX">$\mu \text{g}$ </tex-math></inline-formula> /ml/mm <inline-formula xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink"> <tex-math notation="LaTeX">$^{\mathbf {2}}$ </tex-math></inline-formula> . It has been successfully demonstrated in a serum-like solution and has shown a linear response across the therapeutic range of propofol (1 – <inline-formula xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink"> <tex-math notation="LaTeX">$10~\mu \text{g}$ </tex-math></inline-formula> /ml). Additionally, the sensor has shown good specificity with regards to potential interfering compounds.

Enzyme (Single and Multiple) and Nanozyme Biosensors: Recent Developments and Their Novel Applications in the Water-Food-Health Nexus.

The use of sensors in critical areas for human development such as water, food, and health has increased in recent decades. When the sensor uses biological recognition, it is known as a biosensor. Nowadays, the development of biosensors has been increased due to the need for reliable, fast, and sensitive techniques for the detection of multiple analytes. In recent years, with the advancement in nanotechnology within biocatalysis, enzyme-based biosensors have been emerging as reliable, sensitive, and selectively tools. A wide variety of enzyme biosensors has been developed by detecting multiple analytes. In this way, together with technological advances in areas such as biotechnology and materials sciences, different modalities of biosensors have been developed, such as bi-enzymatic biosensors and nanozyme biosensors. Furthermore, the use of more than one enzyme within the same detection system leads to bi-enzymatic biosensors or multi-enzyme sensors. The development and synthesis of new materials with enzyme-like properties have been growing, giving rise to nanozymes, considered a promising tool in the biosensor field due to their multiple advantages. In this review, general views and a comparison describing the advantages and disadvantages of each enzyme-based biosensor modality, their possible trends and the principal reported applications will be presented.

Development of Mlra Enzyme-Based Electrochemical Biosensor for the Direct Detection of MC-LR

Microcystins are potent hepatotoxins and carcinogens produced by cyanobacteria (blue-green algae). In particular, MC-LR, one of the most toxic microcystins, tends to accumulate and pollute water reservoirs around the world, exhibiting extraordinary environmental stability. Microcystinase (MlrA) is a key enzyme in the detoxification pathway of MC-LR expressed by aerobic gram-negative bacteria. MlrA hydrolyzes a peptide bond within the cyclic MC-LR structure thus generating a nearly benign linear product.The substrate-specificity and stability of MlrA makes it an ideal candidate for an enzyme-based electrochemical biosensor. In this study, the enzyme kinetics of partially purified, heterologously expressed recombinant Mlr-A was elucidated. Polypyrrole-modified screen-printed electrodes were constructed and MlrA was incorporated via covalent immobilization. The resulting enzyme-modified chips were used to study the electrochemical properties of MC-LR and MlrA. Within a small potential window, both MC-LR substrate and MlrA enzyme are electrochemically inert. However, it was shown that enzymatic hydrolysis of MC-LR significantly affected the capacitive current measured overtime. These concentration-dependent changes in non-faradic current were observed even at relatively low MC-LR concentration of 10mM/L, and provide a proof-of-concept for the use of MlrA in an enzyme-based electrochemical biosensor. This study may pave the way towards designing biosensors for environmental diagnostics that enable both monitoring and remediation. Figure 1

Facile Fabrication of Stable Enzyme-Based Colorimetric Glucose Biosensor on Cotton Using Polymer Entrapment

Easy and rapid analyte detection can be democratised using fabric-based colorimetric sensor, which can be readily incorporated into everyday clothing or accessories, and digitally imaged by non-expert users using pervasive smartphones. However, fabricating colorimetric arrays, particularly enzyme-based sensors, on fabrics is nontrivial because enzyme activity cannot be guaranteed upon solid-state immobilisation on textiles, and because textiles present a highly-textured surface that could cause interference during digital imaging and reduce the limit of detection of known sensing formulations. Here, an enzyme-based multilayer sensor is fabricated on selected fabrics via physical adsorption and drop-casting, and utilised for proof-of-concept colorimetric detection of glucose on textiles. Substrates like cotton, cotton lycra, and filter paper are compared for colour uniformity and potential interference during digital imaging. Colour development on cotton achieved optimal homogeneity and reproducibility. The entrapment of enzymes with polyvinyl alcohol is demonstrated to enhance linear dynamic range, water fastness, and storage stability under different conditions. An image-processing algorithm is developed in OpenCV to automatically detect the colour spot and aid in quantitative analysis of glucose concentration. The present study suggests a facile strategy to fabricate a robust biosensor on textile that can achieve noninvasive monitoring of glucose in human biological fluid.

Thermal self-regulatory smart biosensor based on horseradish peroxidase-immobilized phase-change microcapsules for enhancing detection of hazardous substances

Conventional enzyme-based biosensors are highly sensitive to operation temperature due to a strong temperature dependency of biocatalytic activity. Aiming to enhance the biosensing detection of hazardous substances at high ambient temperatures, we focused on the design and construction of a thermal self-regulatory smart biosensor through an innovative combination of phase change material (PCM) and bioelectrocatalytic material. A bioelectrocatalytic phase-change microcapsule system was first fabricated by microencapsulating n-eicosane as a PCM core in the TiO2 shell and then depositing polypyrrole (PPy) as an electroactive coating layer on the surface of TiO2 shell, followed by immobilizing horseradish peroxidase on the surface of PPy coating layer through physical adsorption. The resultant microcapsules exhibit a regular spherical morphology and layer-by-layer core–shell microstructure with the desired chemical compositions. The microcapsules not only exhibit a good thermal management ability to perform effective temperature regulation under a latent-heat capacity of approximately 115 J/g, but also reveal high thermal impact resistance and good thermal cycle stability for the long-term thermal management application in biosensors. A working electrode was modified with the microcapsules obtained above and then used to construct an electrochemical biosensing system imparted with a thermal self-regulation capability. With a high sensitivity of 5.571 µA·L·µmol−1·cm−2 and a low detection limit of 5.384 µmol/L at 55 °C, the resultant smart biosensor exhibits a better determination ability to detect catechol as a model hazardous substance at high operation temperatures than conventional biosensors thanks to the in-situ thermal management derived from its n-eicosane core. This study provides a new approach for development of thermal self-regulatory smart biosensors with an enhanced identification capability to detect hazardous substances over a wide range of temperatures.

Thermal self-regulatory intelligent biosensor based on carbon-nanotubes-decorated phase-change microcapsules for enhancement of glucose detection

Enzyme-based biosensors are sensitive to temperature due to their strong temperature dependency of catalytic activity. Aiming at enhancing biosensing detection for glucose assay over a wide range of applicable temperatures, we designed a thermal self-regulatory intelligent biosensor through an innovative integration of phase change material (PCM) and bioelectrocatalytic substances. An electroactive phase-change microcapsule system was firstly fabricated by microencapsulating n-docosane as a PCM core in the SiO2 shell, followed by depositing polydopamine along with carbon nanotubes as an electroactive layer on the surface of SiO2 shell. The resultant microcapsules showed a regularly spherical morphology and well-defined core-shell microstructure. They also exhibited a satisfactory latent heat capacity of around 137 J/g for implementing temperature regulation with a good working stability. An electrochemical biosensing system was constructed with the resultant electroactive microcapsules together with glucose oxidase as a redox enzyme, achieving a thermal self-regulation capability to enhance the biosensing detection of glucose under in-situ thermal management at higher temperatures. With a high sensitivity of 5.95 μA⋅mM−1⋅cm−2 and a lower detection limit of 13.11 μM at 60 °C, the intelligent biosensor developed by this study demonstrated a superior determination capability and better detection performance toward glucose than conventional biosensors in a high temperature region thanks to effective regulation of microenvironment temperature in the electrode system. This study provides a promising strategy for the development of thermal self-regulatory smart biosensors with an enhanced identification ability to detect various chemical substances over a wide range of applicable temperatures.

Graphene for the Building of Electroanalytical Enzyme-Based Biosensors. Application to the Inhibitory Detection of Emerging Pollutants.

Graphene and its derivates offer a wide range of possibilities in the electroanalysis field, mainly owing to their biocompatibility, low-cost, and easy tuning. This work reports the development of an enzymatic biosensor using reduced graphene oxide (RGO) as a key nanomaterial for the detection of contaminants of emerging concern (CECs). RGO was obtained from the electrochemical reduction of graphene oxide (GO), an intermediate previously synthesized in the laboratory by a wet chemistry top-down approach. The extensive characterization of this material was carried out to evaluate its proper inclusion in the biosensor arrangement. The results demonstrated the presence of GO or RGO and their correct integration on the sensor surface. The detection of CECs was carried out by modifying the graphene platform with a laccase enzyme, turning the sensor into a more selective and sensitive device. Laccase was linked covalently to RGO using the remaining carboxylic groups of the reduction step and the carbodiimide reaction. After the calibration and characterization of the biosensor versus catechol, a standard laccase substrate, EDTA and benzoic acid were detected satisfactorily as inhibiting agents of the enzyme catalysis obtaining inhibition constants for EDTA and benzoic acid of 25 and 17 mmol·L−1, respectively, and a maximum inhibition percentage of the 25% for the EDTA and 60% for the benzoic acid.