The Use of Poly(Vinyl Alcohol) to Cross‐link Penicillinase for the Fabrication of a Penicillin Potentiometric Biosensor

Chemically cross‐linked PVA films are permeable matrices for the fabrication of biosensors. PVA provides an attractive immobilisation method as it preserves the enzymatic activity. Penicillinase (P’nase) was cross‐linked with poly(vinyl alcohol) (PVA) and bovine serum albumin (BSA). The optimum conditions for the of BSA‐PVA‐P’nase film were: 2.5 % w/v PVA, 0.006 % w/v BSA, 2.4 mM penicillin (Pen) and 16 U/mL P’nase. The minimum detectable concentration was 1.7 µM. The linear concentration range obtained for the BSA‐PVA film was 7.5–283 µM. The BSA‐PVA P’nase biosensor detected penicillin in amoxycillin with an average percentage recovery of 97±12 %. Higher penicillin concentrations (10–20 ppm) were detected more successfully than lower concentrations (≤5 ppm). These results indicate that further work is required to enable the successful detection of lower penicillin concentrations such as 5 ppm.     

Fabrication of a Single Layer and Bilayer Potentiometric Biosensors for Penicillin by Galvanostatic Entrapment of Penicillinase into Polypyrrole Films

A single layer and bilayer potentiometric biosensors for the detection of penicillin have been developed. The favourable conditions that were established for the polypyrrole‐penicillinase ((PPy‐P’nase) single layer biosensor were 0.03 M pyrrole, 50 U/mL P’nase, 0.01 M penicillin, applied current density of 0.9 mA/cm² and a polymerisation time of 40 s. The optimum conditions for the formation of the outer layer of the bilayer were: (a) 0.1 M Py, 19 U/mL P’nase, 0.01 M pen, current density of 0.9 mA/cm² and a polymerisation time of 40 s. The minimum detectable penicillin concentration with the bilayer potentiometric biosensor was 0.3 µM and the linear concentration range was 7.5–146 µM. The average percentage recovery of penicillin that was found in amoxycillin 500 mg was 113±24 %. The determination of penicillin in milk was fraught with problems of non‐specific binding of penicillin to the milk.     

A Novel Bioelectrochemical Sensor Based on Immobilized Urease on the Surface of Nickel Oxide Nanoparticle and Polypyrrole Composite Modified Pt Electrode

Nickel oxide nanoparticle (NiO?NP) and polypyrrole (PPy) composite were deposited on a Pt electrode for fabrication of a urea biosensor. To develop the sensor, a thin film of PPy?NiO composite was deposited on a Pt substrate that serves as a matrix for the immobilization of enzyme. Urease was immobilized on the surface of Pt/PPy?NiO by a physical adsorption. The response of the fabricated electrode (Pt/PPy?NiO/Urs) towards urea was analyzed by chronoamperometry and cyclic voltammetry (CV) techniques. Electrochemical response of the bio‐electrode was significantly enhanced. This is due to electron transfer between Ni²⁺ and Ni³⁺ as the electro‐catalytic group and the reaction between polypyrrole and the urease‐liberated ammonium. The fabricated electrode showed reliable and demonstrated perfectly linear response (0.7–26.7 mM of urea concentration, R²= 0.993), with high sensitivity (0.153 mA mM^?1 cm^?2), low detection of limit (1.6 μM), long stability (10 weeks), and low response time (～5 s). The developed biosensor was highly selective and obtained data were repeatable and reproduced using PPy‐NiO composite loaded with immobilized urease as urea biosensors.     

Amperometric Biosensor for Evaluation of Competitive Cholinesterase Inhibition by the Reactivator HI‐6

Abstract

Amperometric biosensors containing enzymes butyrylcholinesterase or acetylcholinesterase were prepared. The biosensors were employed for studying of cholinesterase reactivator: HI‐6. Competitions between HI‐6 and acetylthiocholine as enzyme substrate were used for determination of IC₅₀ value. Biosensors with butyrylcholinesterase from human serum determined IC₅₀ as (1.00±0.02)×10^?6 M; the biosensor with acetylcholinesterase from human erythrocytes performance provided IC₅₀ (3.31±0.13)×10^?6 M, the one with human recombinant acetylcholinesterase (2.00±0.06)×10^?6 M and finally biosensor with acetylcholinesterase from electric eel (6.17±0.17)×10^?6 M when 5 mM acetylthiocholine as substrate was used. We are encouraged to consider presented biosensors as a very useful for evaluation of newly prepared cholinesterase reactivators.     

Potentiometric Biosensor System Based on Recombinant Urease and Creatinine Deiminase for Urea and Creatinine Determination in Blood Dialysate and Serum

A biosensor system for simultaneous determination of creatinine and urea in blood serum and dialysate samples was developed. It consisted of creatinine and urea biosensors based on a potentiometric transducers with two identical pH‐sensitive field‐effect transistors. In creatinine biosensor, creatinine deiminase immobilized via photopolymerization in PVA/SbQ polymer on one transistor served as a biorecognition element, while bovine serum albumin in PVA/SbQ polymer placed on the second transistor was used for reference. The urea biosensor was created in the same way but recombinant urease was used instead of creatinine deiminase. The linear ranges of creatinine and urea measurement were 0.02–2 mM and 0.5–15 mM, correspondingly, which allowed simultaneous determination of the metabolites. Response time of the biosensor system was 2–3 min; RSD of responses did not exceeded 5 %. The biosensors demonstrated absence of non‐selective response towards components of blood dialysate and serum. Urea and creatinine concentrations were determined in 20 samples of blood dialysate and serum. The results correlated well with traditional methods of analysis. Creatinine and urea biosensors were stable during five months of storage (during this time the responses decreased by about 10 %). The proposed biosensor system can be effectively used for analysis of serum samples and for hemodialysis control.     

Fabrication of a bilayer potentiometric phosphate biosensor by cross-link immobilization with bovine serum albumin and glutaraldehyde

Chemical cross-linking of purine nucleoside phosphorylase (PNP) and xanthine oxidase (XOD) with glutaraldehyde (GLA) and bovine serum albumin (BSA) has been used to fabricate a stable and reliable bilayer potentiometric phosphate biosensor. The bilayer arrangement consists of an inner BSA-GLA layer and an outer BSA-GLA-PNP-XOD layer. The inclusion of the inner BSA-GLA layer improves the adhesion of the outer BSA-GLA-PNP-XOD layer and ensures stability of the phosphate biosensor. Established optimum conditions for immobilization of the enzymes in the outer layer and for reliable potentiometric measurement were 4.5% v/v GLA, 6.8% w/v BSA, XOD:PNP mole ratio of 1:8, and a film drying time of 30 min. As little as 20 μM of phosphate can be detected with the BSA-GLA/BSA-GLA-XOD-PNP bilayer biosensor with a linear concentration range between 40 and 120 μM. The biosensor was very stable for 21 days, achieving a good reproducibility with a rsd of only 5.7% and, even after more than a month, the change in the initial potential value was only 10%.     

Pure Organic Phase Phenol Biosensor Based on Tyrosinase Entrapped in a Vapor Deposited Titania Sol‐Gel Membrane

A novel amperometric biosensor was constructed for the determination of phenols in pure organic phase. This biosensor was fabricated by immobilizing tyrosinase in a titania sol‐gel membrane which was obtained with a vapor deposition method. This method was facile and avoided the calcination step needed in conventional titania sol‐gel process. The titania sol‐gel membrane could effectively retain the essential water layer around the enzyme molecule needed for maintaining its activity in organic phase. The experimental parameters such as solvent and operating potential were optimized. At ?100 mV this biosensor showed a good amperometric response to phenols in pure chloroform without any mediator and rehydration of the enzyme. For catechol determination the sensor exhibited a fast response of less than 5 seconds. The sensitivity of different phenols was as follows: catechol > phenol > p‐cresol. Additionally, the apparent Michaelis‐Menten constants of the encapsulated tyrosinase to catechol, phenol and p‐cresol were found to be 0.15±0.003, 0.17±0.008 and 0.21±0.004 mM, respectively. The biosensor had also good reproducibility and stability. This work provided a promising platform for the construction of pure organic phase biosensors and the determination of substrates with poor water solubility.     

Enzyme Deposition by Polydimethylsiloxane Stamping for Biosensor Fabrication

High‐performance biosensors were fabricated by efficiently transferring enzyme onto Pt electrode surfaces using a polydimethylsiloxane (PDMS) stamp. Polypyrrole and Nafion were coated first on the electrode surface to act as permselective films for exclusion of both anionic and cationic electrooxidizable interfering compounds. A chitosan film then was electrochemically deposited to serve as an adhesive layer for enzyme immobilization. Glucose oxidase (GOx) was selected as a model enzyme for construction of a glucose biosensor, and a mixture of GOx and bovine serum albumin was stamped onto the chitosan‐coated surface and subsequently crosslinked using glutaraldehyde vapor. For the optimized fabrication process, the biosensor exhibited excellent performance characteristics including a linear range up to 2 mM with sensitivity of 29.4±1.3 μA mM⁻¹ cm⁻² and detection limit of 4.3±1.7 μM (S/N=3) as well as a rapid response time of ∼2 s. In comparison to those previously described, this glucose biosensor exhibits an excellent combination of high sensitivity, low detection limit, rapid response time, and good selectivity. Thus, these results support the use of PDMS stamping as an effective enzyme deposition method for electroenzymatic biosensor fabrication, which may prove especially useful for the deposition of enzyme at selected sites on microelectrode array microprobes of the kind used for neuroscience research in vivo .     

Glucose Biosensor Based on the Fabrication of Glucose Oxidase in the Bio-Inspired Polydopamine-Gold Nanoparticle Composite Film

A highly efficient enzyme immobilization method has been developed for electrochemical biosensors using polydopamine films with gold nanoparticles (AuNPs) embedded. This simple enzyme fabrication method can be performed in very mild conditions and stored in a long time with high bioactivity. The fabricated amperometric glucose biosensor exhibited a high and reproducible sensitivity, wide linear dynamic range and low limit of detection (LOD) (0.1 μmol·L^?1). A low value of 1.5 mmol·L^?1 for the apparent Michaelis‐Menten constant K^app_M was obtained. The high sensitivity, wide linear range, good reproducibility and stability make this biosensor a promising candidate for portable amperometric glucose biosensor.     

Methyl Parathion Degrading Enzyme‐based Nano‐hybrid Biosensor for Enhanced Methyl Parathion Recognition

Enzyme‐based electrochemical biosensors with sufficient sensing specificity are useful analytical tools for detection of biologically important substances in complicated systems. Here, we present the design of a nano‐hybrid biosensor for the specific and sensitive detection of methyl parathion (MP). The nano‐hybrid sensing film was prepared via the formation of Au nanoparticals (AuNPs) on silica nanoparticles (SiNPs), mixing with multiwall carbon nanotube (MWNTs) and subsequent immobilization of methyl parathion degrading enzyme (MPD). The fabrication procedure was characterized by scanning electron images, linear scan voltammetry and electrochemical impedance spectroscopy. The combined MPD exhibited high affinity to it substrate and thus a selective, sensitive, fast and cheap method for determination of MP, quantitatively was proposed. A significant synergistic effect of nano‐hybrid on the biosensor performance was observed in biosensing MP. The square wave voltammetric responses displayed well defined peaks, linearly proportional to the concentration of MP in the range from 0.001 to 5.0 μg/mL with a detection limit of 0.3 ng/mL. The proposed biosensor also showed good precision and reproducibility, acceptable stability and accuracy in garlic samples analysis. It provided a platform for the simple and fast construction of biosensors with good performance for the determination of enzyme‐specific electroactive species.     

Fabrication of Miniaturised Screen‐printed Glucose Biosensors,Using a Water‐based Ink,and the Evaluation of their Electrochemical Behaviour

This paper describes a simple, convenient approach to the fabrication of microband electrodes and microband biosensors based on screen printing technology. These devices were printed in a three‐electrode configuration on one strip; a silver/silver chloride electrode and carbon counter electrode served as reference and counter electrodes respectively. The working electrodes were fabricated by screen‐printing a water‐based carbon ink containing cobalt phthalocyanine for hydrogen peroxide detection. These were converted into a glucose microband biosensor by the addition of glucose oxidase into the carbon ink. In this paper, we discuss the fabrication and application of glucose microband electrodes for the determination of glucose in cell media. The dimensions (100–400 microns) of the microband electrodes result in radial diffusion, which results in steady state responses in the absence of stirring. The microband biosensors were investigated in cell media containing different concentrations of glucose using chronoamperometry. The device shows linearity for glucose determination in the range 0.5 mM to 2.5 mM in cell media. The screen‐printed microband biosensor design holds promise as a generic platform for future applications in cell toxicity studies.     

Disposable Horseradish Peroxidase Biosensors for the Selective Determination of Tyramine

This work reports the development of horseradish peroxidase based biosensors using screen‐printed carbon electrodes for the determination of tyramine (tyr). A novel procedure based on the insertion of the enzyme in the screen‐printing process (SPC_HRPEs) has been compared with the cross‐linked immobilization into the carbon working electrode (HRP/SPCEs). Both biosensors were characterized obtaining good capability of detection (2.1±0.2 and 0.2±0.01 µM for SPC_HRPEs and HRP/SPCEs, respectively). The reproducibility was 3.4 % and 6.8 % for SPC_HRPEs and HRP/SPCEs, respectively. The repeatability was 2.2 % and 7.1 % for SPC_HRPEs and HRP/SPCEs, respectively. The specificity towards different biogenic amines was analyzed. The developed biosensors were applied to the determination of tyr content in cheese samples.     

Galvanostatic Entrapment of Sulfite Oxidase into Ultrathin Polypyrrole Films for Improved Amperometric Biosensing of Sulfite

The entrapment of sulfite oxidase (SOx) into ultrathin polypyrrole (PPy) films of 27–135 nm thickness has been successfully used for amperometric biosensing of sulfite with considerably improved performance. Optimum galvanostatic entrapment was accomplished in an electrolyte‐free solution which contained 0.1 M pyrrole and 5 U/mL of SOx with a polymerization period of 120 seconds and an applied current density of 0.2 mA cm^?2. Evidence of the incorporation and retention of SOx in the ultrathin PPy film was obtained by scanning electron microscopy, cyclic voltammetry and amperometric measurements. Entrapment of the enzyme in a 54 nm thick PPy‐SOx film gave optimum amperometric response for sulfite and enabled the detection of as little as 0.9 μM of sulfite with a linear concentration range of 0.9 to 400 μM. The successful application of the biosensor to the determination of sulfite in beer and wine samples is reported. Comparison with a spectrophotometric method indicates that the biosensor was more superior for the determination of sulfite in red wine.     

Encapsulation of tyrosinase within liposome bioreactors for developing an amperometric phenolic compounds biosensor

A novel tyrosinase (Tyr) biosensor based on liposome bioreactor and chitosan (CS) nano-composite has been developed for the detection of phenolic compounds. Liposome-based bioreactors were prepared by encapsulating the enzyme Tyr in l-α-phosphatidylcholine liposome resulting in spherical bioreactor with a mean diameter of 8.5?±?1.25 μm. The encapsulation efficiency and drug loading content of the Tyr-loaded liposome-based bioreactors were about 46.35?±?0.85 and 41.15?±?0.95 %, respectively. Porins were embedded into the lipid membrane, allowing for the free substrate transport, but not that of the enzyme due to size limitations. The glassy carbon electrode (GCE) was alternately immersed in CS and Tyr liposome bioreactor (TLB) to assemble bilayer films [(CS/TLB)/GCE]. The presence of Tyr in the biosensor was confirmed by scanning electron microscopy, cyclic voltammetry, and electrochemical measurements. The results indicated that the biosensor was applied to detect phenol with a broad linear range from 0.25 nM to 25 μM, the detection limit was brought down to 0.091 nM. The apparent Michaelis–Menten constant, K _m, for the enzymatic reaction was 34.78 μM. The novel biosensor exhibits good repeatability and stability. Such new biosensor based on encapsulation of Tyr within liposome bioreactors shows great promise for rapid, simple, and cost-effective analysis of phenolic contaminants in environmental samples. The proposed strategy can be extended for the development of other enzyme-based biosensors.     

L-Dopa Synthesis on Conducting Polymers

With regards to the synthesis of L-Dopa (l-3,4-dihydroxy phenylalanine) two types of biosensors were designed by immobilizing tyrosinase on conducting polymers; polypyrrole (PPy) and poly(3,4-ethylenedioxythiophene) (PEDOT). PPy and PEDOT were synthesized electrochemically and tyrosinase immobilized by entrapment during electropolymerization. The kinetic parameters of the designed biosensors, maximum reaction rate of the enzyme (V_max) and Michaelis Menten constant (K_m) were determined. V_max were found as 0.013 for PPy matrix and 0.041 μ mol/min.electrode for PEDOT matrix. K_m values were determined as 3.7 and 5.2mM for PPy and PEDOT matrices respectively. Calibration curves for enzyme activity vs. substrate concentration were drawn for the range of 0.8 to 2.5 mM L-Tyrosine. Optimum temperature and pH, operational and shelf life stabilities of immobilized enzyme were also examined.     

Characterization and Potential Applications of Immobilized Glucose Oxidase and Polyphenol Oxidase

Pyrrole functionalized polystyrene (PStPy) was copolymerized with pyrrole to obtain a conducting copolymer, P(PStPy‐co‐Py) which is used as the immobilization matrix. Glucose oxidase and polyphenol oxidase enzymes were immobilized via the entrapment method by electrochemical polymerization. Enzyme electrodes were prepared by electrolysis at a constant potential using sodium dodecyl sulfate (SDS) as the supporting electrolyte during the copolymerization of PStPy with pyrrole. Maximum reaction rates (V_max) and enzyme affinities (Michaelis‐Menten constants, K_m) were determined for the enzyme entrapped both in polypyrrole (PPy) and P(PStPy‐co‐Py) matrices. Optimizations of enzyme electrodes were done by examining the effects of temperature and pH on enzymes' activities along with the shelf life and operational stability investigations. Glucose oxidase enzyme electrodes were used for human serum analysis and glucose determination in two brands of orange juices. Polyphenol oxidase enzyme electrodes were used for the determination of phenolics in red wines of Turkey.     

A New Enzyme Immobilization Technique Based on Thionine‐Bovine Serum Albumin Conjugate and Gold Colloidal Nanoparticles for Reagentless Amperometric Biosensor Applications

A novel enzyme immobilization technique based on thionine‐bovine serum albumin conjugate (Th‐BSA) and gold colloidal nanoparticles (nano‐Au) was developed. Thionine was covalently bound onto the BSA film with glutaraldehyde(GA) as cross‐linker to achieve Th‐BSA conjugate. The free amino groups of thionine were then used to attach nano‐Au for the immobilization of horseradish peroxidase (HRP). Such nano‐Au/Th‐BSA matrix shows a favorable microenvironment for retaining the native activity of the immobilized HRP and thionine immobilized in this way can effectively shuttle electrons between the electrode and the enzyme. The proposed biosensor displays excellent catalytic activity and rapid response for H₂O₂. The linear range for the determination of H₂O₂ is from 4.9×10^?7 to 1.6×10^?3 M with a detection limit of 2.1×10^?7 M at 3σ and a Michaelies‐Menten constant K value of 0.023 mM.     

Development of Biosensor for Catechol Using Electrosynthesized Poly(3‐methylthiophene) and Incorporation of LAC Simultaneously

Simultaneous electropolymerization of 3‐methylthiophene and incorporation of Laccase (LAC) was carried out in the presence of propylene carbonate as a medium by amperometric method. This enzyme modified electrode was used for the sensing of polyphenol. Catechol is taken as a model compound for the study. UV‐Vis spectral studies suggest no denaturation of LAC in presence of propylene carbonate. The SEM studies reveal the surface morphology and incorporation of LAC in P3MT with agglomerated flaky masses are observed in with and without enzyme micrographs. The cyclic voltammograms were recorded for 0.01 mM catechol on plain glassy carbon, polymer and enzyme incorporated electrodes at pH 6.0 and scan rate 50 mV s^?1. The fabricated electrochemical biosensor was used for the determination of catechol in aqueous solution by Differential Pulse Voltammetry (DPV) technique. The concentration linear range of 8×10^?8 to 1.4×10^?5 M a value of Michealis? Menten constant K_m=7.67 µmol dm^?3 and activation energy is 32.75 kJ mol^?1. It retains 83 % of the original activity after 60 days which is much higher than that of other biosensors. The developed biosensor was used to quantify catechol in the determination in real samples.     

Composite Multienzyme Amperometric Biosensors for an Improved Detection of Phenolic Compounds

A biosensor design, in which glucose oxidase and peroxidase are coimmobilized by simple physical inclusion into the bulk of graphite‐Teflon pellets, is reported for the detection of phenolic compounds. This design allows the “in situ” generation of the H₂O₂ needed for the enzyme reaction with the phenolic compounds, which avoids several problems detected in the performance of single peroxidase biosensors as a consequence of the presence of a high H₂O₂ concentration. So, a much lower surface fouling was found at the GOD‐HRP biosensor in comparison with a graphite‐Teflon‐HRP electrode, suggesting that the controlled generation of H₂O₂ makes more difficult the formation of polymers from the enzyme reaction products. The construction of trienzyme biosensors, in which GOD, HRP and tyrosinase were coimmobilized into the graphite‐Teflon matrix is also reported, and their performance was compared with that of GOD‐HRP bienzyme electrodes. The practical applicability of the composite multienzyme amperometric biosensors was evaluated by the estimation of the phenolic compounds content in waste waters from a refinery, and the results were compared with those obtained by using a colorimetric official method based on the reaction with 4‐aminoantipyrine.     

Disposable Amperometric Biosensor Based on Poly‐L‐lysine and Fe3O4 NPs‐chitosan Composite for the Detection of Tyramine in Cheese

An amperometric tyramine biosensor based on poly‐L‐lysine (PLL) and Fe₃O₄ nanoparticles (Fe₃O₄NP) modified screen printed carbon electrode (SPCE) was developed. PLL was formed on the SPCE by the electropolymerization of L‐lysine. Subsequently, Fe₃O₄NP suspension prepared in chitosan (CH) solution was casted onto the PLL/SPCE. Tyrosinase (Ty) enzyme was immobilized onto the modified Fe₃O₄?CH/PLL/SPCE and the electrode was coated with Nafion to fabricate the Ty/Fe₃O₄?CH/PLL/SPCE. Different techniques including scanning electron microscopy, chronoamperometry (i–t curve), cyclic voltammetry and electrochemical impedance spectroscopy were utilized to study the fabrication processes, electrochemical characteristics and performance parameters of the biosensor. The analytical performance of the tyramine biosensor was evaluated with respect to linear range, sensitivity, limit of detection, repeatability and reproducibility. The response of the biosensor to tyramine was linear between 4.9×10^?7–6.3×10^?5 M with a detection limit of 7.5×10^?8 M and sensitivity of 71.36 μA mM^?1 (595 μA mM^?1 cm^?2). The application of the developed biosensor for the determination of tyramine was successfully tested in cheese sample and mean analytical recovery of added tyramine in cheese extract was calculated as 101.2±2.1 %. The presented tyramine biosensor is a promising approach for tyramine analysis in real samples due to its high sensitivity, rapid response and easy fabrication.