Bienzymatic Biosensor Research Articles

Thread-Based Bienzymatic Biosensor for Linoleic Acid Detection.

The concentration of nonesterified fatty acids (NEFAs) in biological media is associated with metabolic and cardiovascular disorders (e.g., diabetes, cancer, and cystic fibrosis) and in food products is indicative of their quality. Therefore, the early identification of NEFAs is crucial for both medical diagnosis and food quality assessment. However, the development of a portable and scalable sensor capable of detecting these compounds at a low cost presents challenges due to their considerable chemical and physical stability. This research endeavors to illustrate the viability of detecting linoleic acid using a chemiresistive bienzymatic sensor constructed with cotton thread. The sensor's design incorporates the conductive polymer poly(3,4-ethylenedioxythiophene):polystyrenesulfonate (PEDOT:PSS) within the thread, alongside the enzymes horseradish peroxidase (HRP) and lipoxygenase (LOX). By implementing this technology, a sensitive detection range spanning from 161 nM to 16.1 μM is achieved when the PEDOT:PSS/HRP/LOX system is integrated into a single thread. The sensor exhibits exceptional selectivity toward linoleic acid, owing to the specific enzymatic reaction between LOX and linoleic acid. This selectivity is upheld even in the presence of other unsaturated fatty acids. This system can be used for future designs with the capability to detect polyunsaturated fatty acids and other intricate biomolecules.

Multi-walled carbon nanotubes functionalized with a new Schiff base containing phenylboronic acid residues: applicationtothe development of a bienzymatic glucose biosensor using a response surface methodology approach.

Aninnovative supramolecular architectureis reportedfor bienzymatic glucose biosensing based on the use of a nanohybrid made of multi-walled carbon nanotubes (MWCNTs) non-covalently functionalized with a Schiff base modified with two phenylboronic acid residues (SB-dBA) as platform for the site-specific immobilization of the glycoproteins glucose oxidase (GOx) and horseradish peroxidase (HRP). The analytical signal was obtained from amperometric experiments at - 0.050V in the presence of 5.0 × 10-4M hydroquinone as redox mediator. The concentration of GOx and HRP and the interaction time between the enzymes and the nanohybrid MWCNT-SB-dBA deposited at glassy carbon electrodes (GCEs) were optimized through a central composite design (CCD)/response surface methodology (RSM). The optimal concentrations of GOx and HRP were 3.0mgmL-1 and 1.50mgmL-1, respectively, while the optimum interaction time was 3.0min. The bienzymatic biosensor presented a sensitivity of (24 ± 2) × 102 µA dL mg-1 ((44 ± 4) × 102 µA M-1), a linear range between 0.06mg dL-1 and 21.6mg dL-1 (3.1µM-1.2mM) (R2 = 0.9991),and detection and quantification limits of 0.02mg dL-1 (1.0µM) and 0.06mg dL-1 (3.1µM), respectively. The reproducibility for five sensors prepared with the same MWCNT-SB-dBA nanohybrid was 6.3%, while the reproducibility for sensors prepared with five different nanohybrids and five electrodes each was 7.9%. The GCE/MWCNT-SB-dBA/GOx-HRP was successfully used for the quantification of glucose in artificial human urine and commercial human serum samples.

Acetylcholinesterase and butyrylcholinesterase co-immobilized on acopper containing Prussian blue modified electrode for the broad screening of insecticides.

We have developed a bienzymatic biosensor that contains acetylcholinesterase together with butyrylcholinesterase co-immobilized on the same electrode modified with a stabilized copper containing Prussian blue electrodeposited on electrodes coated with 4-aminothiophenol monolayer using diazonium chemistry and copper nanoparticles for improved sensitivity. There are organophosphorus and carbamate neurotoxic insecticides that inhibit only one of the two enzymes, e.g., pirimicarb inhibits butyrylcholinesterase at much lower concentrations than acetylcholinesterase while methomyl inhibits only acetylcholinesterase. Our system is simple and in a single measurement provides a sensitive signal for insecticides' presence based on the inhibition of the enzyme with the highest affinity for each toxic compound. The limits of detection are 50ng/mL pirimicarb for thebienzymatic biosensor in comparison with 400ng/mL pirimicarb for theacetylcholinesterase biosensor and 6ng/mL methomyl for thebienzymatic biosensor, while inhibition is obtained forthe butyrylcholinesterase biosensor at 700ng/mL.

IMPROVEMENT OF THE ANALYTICAL CHARACTERISTICS OF OXIDASE-BASED ELECTROCHEMICAL BIOSENSORS BY ADDING AN ADDITIONAL ENZYME – CATALASE TO THE BIOSELECTIVE MEMBRANE

In this study, the additional enzyme catalase is added to the bioselective membrane of the monoenzyme biosensor to improve the analytical characteristics of the biosensor. In this case, catalase is used to increase the saturate the biomembrane with oxygen, and, accordingly, facilitate the passage of the reaction catalyzed by enzymes of the oxidase class. A standard monoenzyme conductometric biosensor based on glucose oxidase for glucose determination and a new bi-enzyme biosensor based on glucose oxidase and catalase were compared. The optimal method of coimmobilization of enzymes on the surface of the conductometric transducer was investigated, as well as the dependence of the biosensor performance on the concentration of catalase in the bioselective membrane. The stability of the biosensor preparation procedure and the reproducibility of its results were verified. The analytical characteristics of the proposed bi-enzyme biosensor are compared with the monoenzyme biosensor. It is shown that the addition of catalase to the bioselective element of the biosensor as an additional enzyme significantly improves the analytical characteristics of the biosensor, including sensitivity, reproducibility of measurement results, and linear and dynamic ranges of operation. The developed method could be used in future to improve the parameters of other oxidase-based biosensors.

TECHNIQUE FOR IMPROVING THE ANALYTICAL CHARACTERISTICS OF BIOSENSORS BASEDON ENZYMES OF THE OXIDASE SUBCLASS

Aim. In this study, the possibility of improving the analytical performance of a monoenzyme biosensor based on the main oxidase pathway by adding catalase to bioselective membranes was increased. Catalase helps strengthen the acid biomembrane, which facilitates the reaction that catalyzes glucose oxidase (GOD). Methods. A standard glucose oxidase conductometric biosensor for increasing glucose was tested and a new bienzyme biosensor with glucose oxidase and catalase was created. To create bioselective membranes, enzymes were immobilized on the electrode surface for additional covalent formation of molecules in glutaraldehyde vapor. Results. An optimal method for stabilizing enzymes and slowing down the biosensor operation depending on the catalase concentration was proposed. The stability of the biosensor preparation procedures and the recognition of the results were assessed. The presented analytical characteristics of a bienzyme biosensor with a monoenzyme based on GOD showed that the addition of catalase provided high efficiency, output of results and linear dynamism of the secret range operation. Conclusion. The proposed method can be used to improve other oxidase-based biosensors.

Bienzyme photoelectrochemical biosensor for the sensitive determination of phosphatidylcholine in vegetable oil

A bienzyme photoelectrochemical (PEC) biosensor was explored for the sensitive determination of phosphatidylcholine (PC) in vegetable oil based on phospholipase D (PLD) and choline oxidase (ChOx) immobilized on an indium tin oxide (ITO) electrode. The bienzyme electrode is based on the modification with SnO2 nanoparticles (NPs) (SnO2 NPs) and polythionine (PTh). SnO2 NPs were obtained by using a hydrothermal method. Electropolymerization was performed to load the PTh layer on the ITO/SnO2 NPs electrode surface. PLD and ChOx were immobilized on the surface of modified ITO electrode using embedding and cross-linking methods. The prepared ITO/SnO2 NPs/PTh/ChOx/PLD electrode was finally applied as bienzyme PEC biosensor for PC detection. Under optimized conditions, a good linear relationship was obtained in the range of 0.01 mmol/L to 5 mmol/L with a detection limit of 0.002 mmol/L (S/N = 3). The proposed PEC biosensor exhibited remarkable reproducibility, selectivity, and stability. The prepared bienzyme biosensor was used to detect PC in soybean oil by acceptable recoveries. The results were consistent with those of liquid chromatography, confirming the suitability of this PEC sensing strategy for the rapid and sensitive determination of PC in vegetable oil.

Development and Optimization of an Amperometric Bi-enzyme Biosensor for Aspartame Determination

An amperometric bi-enzyme biosensor based on α-chymotrypsin (CHY) and alcohol oxidase (AOX) was developed for detection of aspartame in a flow injection analysis (FIA) system. The CHY and AOX were separately immobilized on bead supports using an adsorption technique. The cleavage of aspartame was catalyzed by CHY to produce methanol that was converted by AOX to formaldehyde and hydrogen peroxide. The formed hydrogen peroxide was detected amperometrically at a platinum electrode. The biosensor performance was optimized with respect to enzyme immobilization and operating conditions. The most suitable conditions for enzyme immobilization were 250 U/mL CHY and 100 U/mL AOX with 60 min immobilization time. The optimal conditions for operating the developed aspartame biosensor were a flow rate of 0.5 mL/min and pH 8.0 at an applied potential of +0.70 V versus Ag/AgCl. Under the optimal conditions, the developed biosensor showed a linear response over the aspartame concentration range 0.15–1.0 mM (coefficient of determination (R2) = 0.9941) with a sensitivity of 5.52 μA/mM·cm2 and a detection limit of 0.10 mM. The developed biosensor was successfully applied in the determination of aspartame in commercial samples.

Amperometric bienzymatic biosensor in flow injection analysis system for determination of aspartame in foods.

An amperometric bienzymatic biosensor was developed for the determination of aspartame in a flow injection analysis (FIA) system, consisting of two enzyme reactor columns packed with immobilized α-chymotrypsin (CHY) and alcohol oxidase (AOX) beads and a hydrogen peroxide electrode, connected in series. The CHY and AOX were separately immobilized on glutaraldehyde (GA)-activated beads through covalent bonding. The biosensor fabrication and operational conditions were optimized. The optimal fabrication conditions were: 2% GA with 120min activation time; and 250 U/mL CHY and 100 U/mL AOX, with 180min enzyme immobilization time. The optimal operational conditions were a flow rate of 0.5mL/min and pH 8.0 at room temperature. The developed biosensor showed linearity over the aspartame concentration range 0.01-1.2mM, with a detection limit of 0.005mM. The developed biosensor was satisfactorily applied for detecting aspartame in beverage samples without any excessive pretreatments.

An origami paper-based electrochemical biosensing platform for quality control of agri-food waste in the valorization strategy

The increasing demand for food and the need for a sustainability vision in the agri-food sector have boosted novel approaches for food management, enhancing the valorization of wastes and by-products belonging to the food industry. Herein, we present a novel paper-based origami device to assess the amount of both glucosinolate and glucose in a food waste product belonging to Brassicaceae plants, to evaluate the quality value and the correct management of waste samples. The device has been designed as an origami paper-based platform constituted of two paper-based biosensors to work synergistically in a multiplexed detection. In detail, a monoenzymatic biosensor and a bienzymatic biosensor were configured for the detection of glucose and glucosinolates, respectively, using filter paper pads preloaded with glucose oxidase and/or myrosinase. To complete the paper-based platform, the enzyme-preloaded pads were combined with office paper-based electrodes modified with Carbon black/Prussian Blue nanoparticles for the measurement of enzymatic by-product at a low applied potential (i.e., 0 V versus Ag/AgCl). Overall, this paper-based platform measured glucose and glucosinolate (i.e., sinigrin) with a linear range up to 2.5 and 1.5 mM, and detection limits of 0.05 and 0.07 mM, respectively. The repeatability corresponded to an RSD% equal to 5% by testing 10 mM of glucose, and 10% by testing 1 mM of sinigrin. The accuracy of the developed multiplex device was evaluated by recovery studies at two different levels of sinigrin, i.e., 0.25 and 0.5 mM, obtaining recoveries values equal to (111 ± 3) % and (86 ± 1) %, respectively. The multiplex detection of both glucose and glucosinolate in Brassicaceae samples evaluates the quality values of the waste sample, ensuring the quality of the re-used food product waste by using an eco-designed analytical tool. The combination of paper-based devices for quality control of food waste with the re-use of these food products represents a sustainable approach that perfectly matches sustainable agrifood practices as well as the overall approach of the circular economy.Graphical abstract

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.

Nanozymes “Artificial Peroxidase”: Enzyme Oxidase Mixtures for Single‐Step Fabrication of Advanced Electrochemical Biosensors

AbstractWe report the single‐step fabrication of advanced electrochemical biosensors upon drop‐casting of “artificial peroxidase”, that is, oxidase‐containing mixtures. Compared to bienzyme biosensors, we substitute peroxidase with more active and stable nanozymes based on catalytically synthesized Prussian Blue. The electroactivity of nanozymes in the sensing membrane was facilitated with carbon black nanoparticles, which resulted in record sensitivity of the H2O2 transducer exceeding 1.1 A M−1 ⋅ cm−2. Both glucose and lactate oxidases were co‐immobilized with nanozymes in alkoxysilane or perfluorosulfonated ionomer membranes. The proposed drop‐casting approach results in biosensors that are advantageous over those conventionally produced upon layer‐by‐layer immobilization in terms of i) almost an order of magnitude increased sensitivity, ii) unsurpassed biosensor‐to‐transducer sensitivity ratio reaching the value of 0.3, and iii) three times extended operational lifetime.

Potentiometric sensing of histamine using immobilized enzymes on layered double hydroxides.

Diamine oxydase and peroxidase have been co-immobilized onto layered double hydroxide (LDH) thin films for the development of real-time histamine biosensors. The chosen LDH materials are Mg2AlCO3, Mg4FeCl and Ca2AlCl. Prepared bi-enzymatic hybrid nanomaterials are capable of detecting histamine through the electrochemical oxidation of H2O2 and are used as the sensitive membrane for potentiometric microelectrode. Histamine biosensors developed in this work have fast response of less than 20s, are sensitive and selective, with a large dynamic range of 10-8-10-3M and a limit of detection of less than 10-8M. The detection limit of the developed bi-enzymatic biosensors is relatively higher than those corresponding with gas and liquid chromatography, which are still considered as the reference methods. Finally, the reproducibility, the specificity and the storage stability of the biosensors were studied.

Preparation of acetylcholine biosensor for the diagnosis of Alzheimer's disease

We report herein the design of a novel biosensor sensing strategy for sensitive detection of acetylcholine based on PAMAM-Sal dendrimer. PAMAM-Sal, salicylaldehyde and PAMAM dendrimer have been synthesized by means of condensation. It has been determined that PAMAM-Sal dendrimer was formed by the formation of Schiff base with FT-IR, 1H NMR and UV spectra. In addition, the structure has been supported by elemental analysis. Later, a bienzymatic biosensor system has been developed. The bienzymatic biosensor system with acetylcholine esterase (AChE) and choline oxidase (ChO) was prepared with carbon paste electrode modified with PAMAM-Sal for determination of the amount of acetylcholine. Acetylcholine esterase and choline oxidase enzymes were immobilized onto modified carbon paste electrode by cross-linking with glutaraldehyde. Determination of acetylcholine was carried out by the oxidation of enzymatically produced H2O2 at +0.4 V vs. Ag/AgCl. The linear working range for acetylcholine determination of biosensor was identified. The effects of pH and temperature on the response of the biosensor were examined. Reusability and storage stability of the biosensor were determined. Interference effects of interferants which might be in biologic media on the response of the biosensor were also studied.

A sensitive amperometric detection of neurotransmitter acetylcholine using carbon dot-modified carbon paste electrode.

Acetylcholine is a neurotransmitter, which is located at the intersections of the nerve and muscles in the lymph nodes of the internal organs motor systems and in various parts of the central nervous system. A decrease of acetylcholine in brain is associated with Alzheimer's disease. That is why it is an important agent for this disease. In this study, a bienzymatic biosensor system with acetylcholine esterase and choline oxidase was prepared with carbon paste electrode modified with carbon nano Dot-(3-Aminopropyl) triethoxysilane (CDs-APTES) for determination of the amount of acetylcholine. Acetylcholine esterase and choline oxidase enzymes were immobilized onto a modified carbon paste electrode by cross-linking with glutaraldehyde. Determination of acetylcholine was carried out by the oxidation of enzymatically produced H2 O2 at 0.4 V versus Ag/AgCl. The effect of temperature, pH, and substrate concentration on the acetylcholine response of the prepared biosensor was investigated. In addition, the optimum CDs-APTES amount, the linear operating range of the biosensor, and the interference effect were also investigated.

‘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.

Photoelectrochemical platform for glucose sensing based on g-C3N4/ZnIn2S4 composites coupled with bi-enzyme cascade catalytic in-situ precipitation

A unique bi-enzyme photoelectrochemical (PEC) biosensor is developed for selective and sensitive detection of glucose based on bi-enzyme cascade catalytic in-situ precipitation for signal amplification. In this design, the composites of ZnIn2S4 and graphitic carbon nitride (g-C3N4/ZnIn2S4) with better photoelectrochemical properties are used as the photoactive material. Subsequently, horseradish peroxidase (HRP) and glucose oxidase (GOx) are anchored on the modified electrode surface. In the presence of 4-chloro-1-naphthol (4-CN) and glucose, GOx catalyzes the oxidation of glucose to produce H2O2, which oxidizes 4-CN to generate a precipitate in the presence of HRP. The precipitate on the electrode surface can dramatically reduce the photocurrent signal. In the light of the signal magnification process, the bi-enzyme biosensor presents a “signal-off” sensing mode for glucose detection, which displays a dynamic detection range of 1–10000 μM with a low limit of detection of 0.28 μM. Furthermore, the constructed PEC bi-enzyme biosensor exhibits a tremendous potential for quantitative detection of glucose in practical applications.

Nanoarchitectures based on multi-walled carbon nanotubes non-covalently functionalized with Concanavalin A: A new building-block with supramolecular recognition properties for the development of electrochemical biosensors

We propose an innovative nanoarchitecture for the development of electrochemical biosensors based on the non-covalent functionalization of multi-walled carbon nanotubes (MWCNTs) with the lectin Concanavalin A (ConA) and the site-specific supramolecular binding of glycobiomolecules. As proof-of-concept, we propose the use of two glycoenzymes, glucose oxidase (GOx) and horseradish peroxidase (HRP), for building mono and bienzymatic glucose biosensors. The selected conditions for the preparation of the dispersion were 1.5 mg MWCNTs in 1.0 mL of 2.0 mg mL−1 ConA sonicated for 5.0 min with sonicator probe. The monoenzymatic glucose biosensor was prepared by casting GCE with the MWCNTs-ConA dispersion (GCE/MWCNTs-ConA) followed by the interaction with GOx (GCE/MWCNTs-ConA/GOx), while the bienzymatic one was obtained by interaction of GCE/MWCNTs-ConA with GOx + HRP (GCE/MWCNTs-ConA/GOx-HRP). The best analytical performance was obtained with the bienzymatic biosensor from the amperometric response at -0.050 V in the presence of 1.0 × 10-4 M hydroquinone. The sensitivity was (2.22 ± 0.03) μA mM−1 (which was 5.2 times higher than the one obtained with the monoenzymatic biosensor) and a detection limit of 0.31 μM. The reproducibility was 5.4% and the biosensor was challenged with human blood serum showing an excellent correlation with the values reported by the laboratory.

Monocrotophos detection with a bienzyme biosensor based on ionic-liquid-modified carbon nanotubes.

Acetylcholinesterase (AChE) biosensor technology is widely applied in the detection of organophosphate pesticides in agricultural production via the inhibition of AChE activity by organophosphates. However, the AChE electrode has some drawbacks, such as low stability and high overpotential. Combining the advantages of multiwalled carbon nanotubes (MWCNTs) and ionic liquids, we constructed a novel bienzyme electrode [Cl/iron porphyrin (FePP)-modified MWCNTs/AChE/glassy carbon electrode], which included AChE and mimetic oxidase FePP. In this electrode, FePP is covalently bound to the AChE carrier via ionic liquid for increased electrode sensitivity and stability. Under optimal conditions, this novel biosensor has a monocrotophos detection limit of 3.2 × 10-11 mol/L and good recovery of 89-104%. After 5 weeks of storage at 4 °C, the oxidation current was 97.8% of its original value. The biosensor has high stability and sensitivity for monocrotophos detection and is a promising device for monitoring food safety. Graphical abstract The complete synthesis process of Cl/FePP-MWCNTs/AChE/GCE.

Sampling and multiplexing in lab-on-paper bioelectroanalytical devices for glucose determination

In this work, we present a multiplexed (eight simultaneous measurements) paper-based electrochemical device developed in a very simple way and using low-cost materials, such as paper, carbon ink and multifunctional connector headers. Meanwhile, we have also combined the paper-based electrochemical platform with a glass-fiber strip in order to integrate easily a sampling step. Both approaches, simultaneous measuring and sampling, have been applied to the determination of glucose using bienzymatic biosensors. They are fabricated by adsorbing the mixture of enzymes (glucose oxidase and horseradish peroxidase), as well as the ferrocyanide, mediator of the electron transfer, on the paper-based electrode. After drying, the measuring solution (containing either glucose standards or samples) is added and the eight corresponding chronoamperograms are recorded. In the case of the microfluidic approach for sampling purposes, the glass-fiber pad (sampler) is immersed in a container with the solution, which flows by capillarity until it reaches the working electrode. The integration of one more step of the analytical process advances towards real and useful lab-on-a-chip devices. With these designs, a linear range comprised between 0.5 and 15 mM was achieved for glucose determination, with an excellent precision. If the sampler is employed, it is not necessary to use micropipettes and, nevertheless, precise measurements are obtained. The RSD of the slopes obtained for different calibrations performed in different days, with different arrays of electrochemical cells and different solutions is ca. 1%. Accurate results are obtained in the determination of glucose in real samples (orange fruit and cola beverages).

Ester flavorants detection in foods with a bienzymatic biosensor based on a stable Prussian blue-copper electrodeposited on carbon paper electrode

A stable and reproducible layer of Prussian blue (PB) modified with copper was electrodeposited on carbon paper electrodes for the multiple detection of ester flavorants with a bienzymatic biosensor. Carbon fiber composite paper was investigated as high-surface, low-cost substrate for biosensor development. The pre-activation of the electrode surface by cyclic voltammetry was necessary to improve the electrochemical properties before the electrochemical deposition of Prussian blue-copper film (PB-Cu). The stability and the reproducibility of the obtained PB-Cu carbon paper electrode was demonstrated at pH 7.4, optimum for biosensor development. The developed biosensor is based on the immobilization of two enzymes (carboxyl esterase and alcohol oxidase) by cross-linking with glutaraldehyde onto PB-Cu carbon paper electrode. A mixture of key aroma ester compounds (methyl butyrate, ethyl butyrate, methyl cinnamate and ethyl cinnamate) was detected in several food samples with low interferences.