Immobilization of urease on poly(N-vinyl carbazole)/stearic acid Langmuir-Blodgett films for application to urea biosensor

Urease was immobilized in mixed monolayers of poly(N-vinyl carbazole) (PNVK) and stearic acid (SA) formed at an air-water interface. The monolayers were transferred onto indium-tin-oxide (ITO) coated glass plates using Langmuir-Blodgett (LB) film deposition technique. Urease immobilized on PNVK/SA LB films, characterized using FTIR and UV-visible spectroscopy, was found to exhibit increased stability over a wide pH (6.5-8.5) and temperature (25-50 degrees C) range. Potentiometric measurements on these urease electrodes were carried out using an ammonium ion analyzer. Two values for K(m)(app) were obtained at lower and higher concentrations of substrate urea.     

Fast and easy preparation of an amperometric glucose biosensor

Summary Electrochemical polymerization of pyrrole in aqueous KCl solution containing glucose oxidase produces adherent films at platinum electrode surface. Such coated electrodes are prepared in 20 min and can determine glucose in the range 0 to 100mm. The useful lifetime of the electrode is 85 assays. Its stability is at least 75 days under storage in PBS at 4°C.     

A polymer nanostructured Fabry-Perot interferometer based biosensor

A polymer nanostructured Fabry–Perot interferometer (FPI) based biosensor is reported. Different from a conventional FPI, the nanostructured FPI has a layer of Au-coated nanopores inside its cavity. The Au-coated nanostructure layer offers significant enhancement of optical transducing signals due to the localized surface plasmon resonance effect and also due to the significantly increased sensing surface area, which is up to at least two orders of magnitude larger than that of a conventional FPI-based biosensor. Using this technical platform, the immobilization of captures proteins (protein A) on the nanostructure layer and their binding with immunoglobulin G (IgG) has been monitored in real time, resulting in the shift of the interference fringes of the optical transducing signals. Current results show that the limit-of-detection of the biosensor should be lower than 10 pg/mL for IgG-protein A binding.     

Piezoelectric urea biosensor based on immobilization of urease onto nanoporous alumina membranes

The urease was immobilized onto nanoporous alumina membranes prepared by the two-step anodization method, and a novel piezoelectric urea sensing system with separated porous alumina/urease electrode has been developed through measuring the conductivity change of immobilized urease/urea reaction. The process of urease immobilization was optimized and the performance of the developed urea biosensor was evaluated. The obtained urea biosensor presented high-selectivity monitoring of urea, better reproducibility (S.D. = 0.02, n = 6), shorter response time (30 s), wider linear range (0.5 μM to 3 mM), lower detection limit (0.2 μM) and good long-term storage stability (with about 76% of the enzymatic activity retained after 30 days). The clinical analysis of the urea biosensor confirmed the feasibility of urea detection in urine samples.     

A nylon membrane based amperometric biosensor for polyphenol determination

A nylon membrane based amperometric biosensor employing banana fruit polyphenol oxidase (PPO) is presented for polyphenol detection. Nylon membrane was first activated and then coupled with chitosan. PPO was covalently attached to this membrane through glutaraldehyde coupling. The membrane bioconjugate was characterized by scanning electron microscopy (SEM) and Fourier Transform Infrared (FTIR) study and then mounted onto Au electrode using parafilm to construct a working electrode. Once assembled along with Ag/AgCl as reference and Pt as auxiliary electrode, the biosensor gave optimum response within 15 s at pH 7.5 and 30 °C, when polarized at +0.4 V. The response (in mA) was directly proportional to polyphenol concentration in the range 0.2–400 μM. The lower detection limit of the biosensor was 0.2 μM. The biosensor was employed for determination of polyphenols in tea, beverages and water samples. The enzyme electrode showed 25% decrease in initial activity after 150 reuses over 6 months, when stored at 4 °C.     

Immobilization of urease on vermiculite

Urease (EC 3.5.1.5) of high activity was obtained when the enzyme was immobilized on vermiculite crosslinked with 2.5% glutaraldehyde in chilled EDTA-phosphate buffer (pH 5.5). The highest activity of the immobilized enzyme was at 65°C and pH 6.5 while the optimum temperature for free urease was found to be 25°C. The thermal stability of immobilized urease was observed to be much better than that of the free urease. When stored at 4°C, urease immobilized on vermiculite retained 69 to 81% of its activity after 60 days and 61 to 75% of its original activity was retained after 4 repeated uses.     

A novel urea sensitive biosensor with extended dynamic range based on recombinant urease and ISFETs

A novel urea biosensor based on immobilised recombinant urease as sensitive element and ion sensitive field effect transistor as transducer was developed. Recombinant urease from E. coli with an increased Km was photoimmobilised in PVA/SbQ (poly(vinyl alcohol) containing styrylpyridinium) membrane and has demonstrated quite good performance as biosensitive element. Enzymatic field effect transistors based on such a bioselective element were studied in model buffer solutions. This biosensor demonstrated an extended dynamic range up to 80 mM, a quite good reproducibility (standard deviation of the sensor responses was approximately 2.5%, n= 20 for urea concentration 10 mM) and a high stability. Such characteristics fit with the analytical requirements needed for urea control in plasma and liquids used during renal dialysis.     

Immobilization of urease on gelatin — poly (HEMA) copolymer preparation and characterization

Urease was immobilized onto gelatin-poly (HEMA) copolymer by covalent linkage. Maximum amount of urease was immobilized onto the support at a pH of 8.5. The optimal pH of the immobilized urease was similar to that of free urease; the optimal temperature showed an increase of 10 °C over the free enzyme. The stability of the immobilized urease for a range of pH, temperature and shelf life was greater than the corresponding values for the free enzyme. The same result was obtained for k _m also.Grateful acknowledgement is made to CSIR, Govt. of India for the research associateship conferred on Dr. M. Chellapandian which helped the progress of this piece of research investigation.     

Simultaneous topographic and amperometric membrane mapping using an AFM probe integrated biosensor

The investigation of the plasma membrane with intercorrelated multiparameter techniques is a prerequisite for understanding its function. Presented here, is a simultaneous electrochemical and topographic study of the cell membrane using a miniaturized amperometric enzymatic biosensor. The fabrication of this biosensor is also reported. The biosensor combines a scanning force microscopy (AFM) gold-coated cantilever and an enzymatic transducer layer of peroxidases (PODs). When these enzymes are brought in contact with the substrate, the specific redox reaction produces an electric current. The intensity of this current is detected simultaneously with the surface imaging. For sensor characterization, hydroquinone-2-carboxylic acid (HQ) is selected as an intrinsic source of H(2)O(2). HQ has been electrochemically regenerated by the reduction of antraquinone-2-carboxylic acid (AQ). The biosensor reaches the steady state value of the current intensity in 1 ± 0.2s.     

Development of potentiometric urea biosensor based on urease immobilized in PVA-PAA composite matrix for estimation of blood urea nitrogen (BUN)

A urea biosensor was developed using the urease entrapped in polyvinyl alcohol (PVA) and polyacrylamide (PAA) composite polymer membrane. The membrane was prepared on the cheesecloth support by gamma-irradiation induced free radical polymerization. The performance of the biosensor was monitored using a flow-through cell, where the membrane was kept in conjugation with the ammonia selective electrode and urea was added as substrate in phosphate buffer medium. The ammonia produced as a result of enzymatic reaction was monitored potentiometrically. The potential of the system was amplified using an electronic circuit incorporating operational amplifiers. Automated data acquisition was carried by connecting the output to a 12-bit analog to digital converter card. The sensor working range was 1–1000 mM urea with a response time of 120 s. The enzyme membranes could be reused 8 times with more than 90% accuracy. The biosensor was tested for blood urea nitrogen (BUN) estimation in clinical serum samples. The biosensor showed good correlation with commercial Infinity™ BUN reagent method using a clinical chemistry autoanalyzer. The membranes could be preserved in phosphate buffer containing dithiothreitol, β-mercaptoethanol and glycerol for a period of two months without significant loss of enzyme activity.     

Lipid membrane immobilized horseradish peroxidase biosensor for amperometric determination of hydrogen peroxide

Stable films of didodecyldimethylammonium bromide (DDAB, a synthetic lipid) and horseradish peroxidase (HRP) were made by casting the mixture of the aqueous vesicle of DDAB and HRP onto the glassy carbon (GC) electrode. The direct electron transfer between electrode and HRP immobilized in lipid film has been demonstrated. The lipid films were used to supply a biological environment resembling biomembrane on the surface of the electrode. A pair of redox peaks attributed to the direct redox reaction of HRP were observed in the phosphate buffer solution (pH 5.5). The cathodic peak current increased dramatically while anodic peak decreased by addition of small amount H(2)O(2). The pH effect on amperometric response to H(2)O(2) was studied. The biosensor also exhibited fast response (5 s), good stability and reproducibility.     

Amperometric biosensor based on a site-specific immobilization of acetylcholinesterase via affinity bonds on a nanostructured polymer membrane with integrated multiwall carbon nanotubes

Acetylcholinesterase (AChE) was immobilized on chemically modified poly-(acrylonitrile-methyl-methacrylate-sodium vinylsulfonate) membranes in accordance with three different methods, the first of which involved random enzyme immobilization via glutaraldehyde, the second one—site-specific enzyme immobilization via glutaraldehyde and Concanavalin A (Con A) and the third method—modified site-specific enzyme immobilization via glutaraldehyde in the presence of a mixture of multiwall carbon nanotubes and albumin (MWCNs + BSA), glutaraldehyde and Con A. Preliminary tests for the activity of immobilized AChE were carried out using these three methods. The third method was selected as the most efficient one for the immobilization of AChE and the prepared enzyme carriers were used for the construction of amperometric biosensors for the detection of acetylthiocholine (ATCh).A five level three factorial central composite design was chosen to determine the optimal conditions for the enzyme immobilization with three critical variables: concentration of enzyme, Concanavalin A and MWCNs. The design illustrated that the optimum values of the factors influencing the amperometric current were C_E: 70 U mL⁻¹; C_{Con A}: 1.5 mg mL⁻¹ and C_MWCN: 11 mg mL⁻¹, with an amperometric current 0.418 μA. The basic amperometric characteristics of the constructed biosensor were investigated. A calibration plot was obtained for a series of ATCh concentrations ranging from 5 to 400 μM. A linear interval was detected along the calibration curve from 5 to 200 μM. The correlation coefficient for this concentration range was 0.995. The biosensor sensitivity was calculated to be 0.065 μA μM⁻¹ cm⁻². The detection limit with regard to ATCh was calculated to be 0.34 μM. The potential application of the biosensor for detection and quantification of organophosphate pesticides was investigated as well. It was tested against sample solutions of Paraoxon. The biosensor detection limit was determined to be 1.39 × 10⁻¹² g L⁻¹ of Paraoxon, as well as the interval (10⁻¹¹ to 10⁻⁸ g L⁻¹) within which the biosensor response was linearly dependant on the Paraoxon concentration. Finally the storage stability of the enzyme carrier was traced for a period of 120 days. After 30-day storage the sensor retained 76% of its initial current response, after 60 days—68% and after 120 days—61%.     

Development of urea biosensor using non-covalent complexes of urease with aldehyde derivative of PEG and analysis on serum samples

Abstract

Non-covalent complexes of urease/polyethylene glycol (PEG)-aldehyde were synthesized using regular molar ratios of urease and PEG-aldehyde at room temperature. The physical properties of the non-covalent complexes were analyzed in order to investigate the impact of coupling ratio, temperature, pH, storage stability, and thermal stability. Urease activity was analyzed by UV–Vis spectrophotometer at 630?nm. The results showed that the strongest thermal resistance was obtained using n_U/n_PEG:1/1 (mg/mL) complex within all molar ratios tested. The enzymatic activity of n_U/n_PEG:1/1 complex doubled the activity of the free enzyme. Therefore, this complex was chosen to be used in the analyses. When coupled with PEG-aldehyde, urease exhibited improved activity between pH 4.0–9.0 and the optimum pH was found to be 7.0. The thermal inactivation results of the complex demonstrated that higher activity remained (40%) when compared with the free enzyme (10%) at 60?°C. The storage stability of the non-covalent complex was 4 weeks which was greater than the storage stability of the free enzyme. A kinetic model was suggested in order to reveal the mechanism of enzymatic conversion. Potentiometric urea biosensor was prepared using two different membranes: carboxylated poly vinyl chloride (PVC) and palmitic acid containing PVC. The potentiometric responses of both sensors were tested against pH and temperature and the best results were obtained at pH 7.0 and 20–30?°C. Also, selectivity of the suggested biosensors toward Na⁺, Li⁺ Ca²⁺, and K⁺ ions was evaluated and the reproducibility responses of the urea biosensors were measured with acceptable results.     

Immobilization of hemoglobin on zirconium dioxide nanoparticles for preparation of a novel hydrogen peroxide biosensor

Direct electrochemistry and thermal stability of hemoglobin (Hb) immobilized on a nanometer-sized zirconium dioxide (ZrO2) modified pyrolytic graphite (PG) electrode were studied. The immobilized Hb displayed a couple of stable and well-defined redox peaks with an electron transfer rate constant of (7.90 +/- 0.93)s(-1) and a formal potential of -0.361 V (-0.12 V versus NHE) in 0.1M pH 7.0 PBS. Both nanometer-sized ZrO2 and dimethyl sulfoxide (DMSO) could accelerate the electron transfer between Hb and the electrode. Spectroscopy analysis of the Hb/ZrO2/DMSO film showed that the immobilized Hb could retain its natural structure. This modified electrode showed a high thermal stability up to 74 degrees C and an electrocatalytic activity to the reduction of hydrogen peroxide (H2O2) without the aid of an electron mediator. The electrocatalytic response showed a linear dependence on the H2O2 concentration ranging from 1.5 to 30.2 microM with a detection limit of 0.14 microM at 3sigma. The apparent Michaelis-Menten constant KMapp for H2O2 sensor was estimated to be (0.31 +/- 0.02) mM, showing a high affinity.     

Facile preparation of amperometric laccase biosensor with multifunction based on the matrix of carbon nanotubes-chitosan composite

The carbon nanotubes–chitosan (CNTs–CS) composite provides a suitable biosensing matrix due to its good conductivity, high stability, and good biocompatibility. Enzymes can be firmly incorporated into the matrix without the aid of other cross-linking reagents. The composite is easy to form insoluble film in solution above pH 6.3. Based on this, a facilely fabricated amperometric biosensor by entrapping laccase into the CNTs–CS composite film has been developed. At pH 6.0, the fungi laccase incorporated into the composite film remains better catalytic activity than that dissolved in solution. The system is in favor of the accessibility of substrate to the active site of laccase, thus the affinity to substrates is improved greatly, such as 2,2′-azino-bis-(3-ethylbenzthiazoline-6-sulfonic acid) diammonium salt (ABTS), catechol, and O₂ with K_m values of 19.86 μM, 9.43 μM, and 3.22 mM, respectively. The major advantages of the as-prepared biosensor are: detecting different substrates (ABTS, catechol, and O₂), possessing high affinity and sensitivity, durable long-term stability, and facile preparation procedure. On the other hand, the system can be applied in fabrication of biofuel cells as the cathodic catalysts based on its good electrocatalysis for oxygen reduction. It can be extended to immobilize other enzymes and biomolecules, which will greatly facilitate the development of biosensors, biofuel cells, and other bioelectrochemical devices.     

Immobilizing Pt nanoparticles and chitosan hybrid film on polyaniline naofibers membrane for an amperometric hydrogen peroxide biosensor

A convenient and effective way for fabricating amperometric hydrogen peroxide (H₂O₂) biosensor was designed in this paper. First, the polyaniline (PANI) nanofibers membrane with good conductance and high surface area was electropolymerized on a gold electrode surface. Then, Pt nanoparticle (PtNP) was electrochemically deposited on the PANI nanofibers membrane. Finally, the hybrid film of gold nanoparticle, chitosan, and horseradish peroxidase (HRP) was cast onto the modified electrode to form a stable biofunctional film, which was also employed as a protective layer to PtNP. The proposed biosensor exhibited a rapid response to H₂O₂ with the linear range from 7.0 × 10⁻⁶ to 1.4 × 10⁻² M and a detection limit of 2.8 × 10⁻⁶ M (S/N = 3). The sensitivity of 558 μA mM⁻¹ cm⁻² was obtained. The Michaelis–Menten constant, K_\textM^\textapp K_{\text{M}}^{\text{app}} value was 1.90 mM suggesting a high affinity. Moreover, it displayed a good reproducibility and long-term stability.     

Interference elimination of an amperometric glucose biosensor using poly(hydroxyethyl methacrylate) membrane

A permselective membrane fabricated from photo‐cross‐linked poly(hydroxyethyl methacrylate) (pHEMA) was studied as a potential selective membrane that can eliminate electrochemical interferences commonly faced by a hydrogen peroxide‐based biosensor. The quantitative selection of the permselective membrane was based on the permeabilities of hydrogen peroxide and acetaminophen (AC). AC was used as a model of the interfering substance due to its neutral nature. pHEMA membrane with the cross‐linking ratio of 0.043 was found to achieve a selectivity of hydrogen peroxide over AC of 10, while maintaining an acceptable degree of hydrogen peroxide response. A two‐layer glucose biosensor model consisting of glucose oxidase entrapped within a freeze‐thawed poly(vinyl alcohol) matrix and the cross‐linked pHEMA membrane was challenged with AC, ascorbic acid and uric acid. 0.2 mM AC and 0.2 mM ascorbic acid were completely eliminated. However, 0.2 mM uric acid could not be completely eliminated and still gave a bias of approximately 6.6% relative to 5 mM glucose. The results showed that cross‐linked pHEMA was quite promising as an interference eliminating inner membrane.