An amperometric lactate biosensor based on lactate dehydrogenase immobilized onto graphene oxide nanoparticles‐modified pencil graphite electrode

A novel amperometric lactate biosensor was developed based on immobilization of lactate dehydrogenase onto graphene oxide nanoparticles‐decorated pencil graphite electrode. The enzyme electrode was characterized by scanning electron microscopy, Fourier transform infrared spectroscopy (FTIR), and cyclic voltammetry at different stages of its construction. The biosensor showed optimum response within 5 s at pH 7.3 (0.1 M sodium phosphate buffer) and 35°C, when operated at 0.7 V. The biosensor exhibited excellent sensitivity (detection limit as low as 0.1 μM), fast response time (5 s), and wider linear range (5–50 mM). Analytical recovery of added lactic acid in serum was between 95.81–97.87% and within‐batch and between‐batch coefficients of variation were 5.04 and 5.40%, respectively. There was a good correlation between serum lactate values obtained by standard colorimetric method and the present biosensor (r = 0.99). The biosensor measured lactate levels in sera of apparently healthy subjects and persons suffering from lactate acidosis and other biological materials (milk, curd, yogurt, beer, white wine, and red wine). The enzyme electrode lost 25% of its initial activity after 60 days of its regular uses, when stored dry at 4°C.     

Amperometric l-lysine determination biosensor amplified with l-lysine oxidase nanoparticles and graphene oxide nanoparticles

We describe the amplification of amperometric l-lysine biosensor using l-lysine oxidase nanoparticles (LOxNPs) and graphene oxide nanoparticles (GrONPs) immobilized onto pencil graphite electrode (PGE). LOxNPs and GrONPs were characterized by UV spectroscopy and transmission electron microscopy (TEM). The working electrode (LOxNPs/GrONPs/PGE) was studied by scanning electron microscopy (SEM) and cyclic voltammetry at different stages of its construction. The biosensor showed optimum current at 0.7 V, pH 6.5, 35 °C, a detection limit of 0.01 μM, response time as 3.95 s and a wider linear range 0.01–1000 μM. The analytical recovery of added lysine in sera was 97 %. The within assay and between batch coefficients of variation for the biosensor were 0.068 and 0.074 % respectively. The biosensor measured l-lysine levels in sera of healthy adults and human immunodeficiency virus (HIV) patients. The biosensor exhibited good correlation with standard spectrophotometric method (R² = 0.989). The biosensor lost 35 % of its original activity after its regular uses for a period of 180 days, while being stored dry at 4 °C.     

Stable mediated amperometric biosensors using a graphite electrode embedded with tetrathiafulvalene in silicone oil

A new method has been developed to incorporate the mediator, tetrathiafulvalene (TTF), to the electrode/solution interface of an amperometric biosensor. TTF was dissolved in methylphenyl polysiloxane (silicone oil) and embedded in a graphite disc electrode. The mediator was able to diffuse to the electrode surface at an electrocatalytically significant speed. The storage of TTF in the inert polysiloxane provided a long-lasting and stable mediator supply.

TTF-silicone oil electrodes with immobilized glucose oxidase, xanthine oxidase, or amino acid oxidase exhibited sensitive, fast and reproducible responses. The glucose oxidase electrode was very stable for at least 2 months when stored at 4°C. Together with flow injection analysis (FIA), the enzyme electrodes were reused for at least 500 repeated analyses during a 25 h operation without losing their initial activity.     

Development of glucose biosensor based on reconstitution of glucose oxidase onto polymeric redox mediator coated pencil graphite electrodes

In this study, a novel glucose biosensor was fabricated by reconstitutional immobilization of glucose oxidase (GOx) onto a poly(glycidyl methacrylate-co-vinylferrocene) (poly(GMA-co-VFc)) film coated pencil graphite electrode (PGE). The amperometric current response of poly(GMA-co-VFc)-GOx to glucose is linear in the concentration range between 1 and 16 mM (correlation coefficient of 0.9998) with a detection limit of 2.7 μM (S/N = 3). Experimental parameters were studied in detail and optimized, including the pH and temperature governing the analytical performance of the biosensor. The stability and reusability of the biosensor as well as its kinetic parameters have also been studied.     

Glucose biosensor based on glucose oxidase immobilized at gold nanoparticles decorated graphene-carbon nanotubes

Biopolymer pectin stabilized gold nanoparticles were prepared at graphene and multiwalled carbon nanotubes (GR-MWNTs/AuNPs) and employed for the determination of glucose. The formation of GR-MWNTs/AuNPs was confirmed by scanning electron microscopy, X-ray diffraction, UV–vis and FTIR spectroscopy methods. Glucose oxidase (GOx) was successfully immobilized on GR-MWNTs/AuNPs film and direct electron transfer of GOx was investigated. GOx exhibits highly enhanced redox peaks with formal potential of −0.40 V (vs. Ag/AgCl). The amount of electroactive GOx and electron transfer rate constant were found to be 10.5 × 10⁻¹⁰ mol cm⁻² and 3.36 s⁻¹, respectively, which were significantly larger than the previous reports. The fabricated amperometric glucose biosensor sensitively detects glucose and showed two linear ranges: (1) 10 μM – 2 mM with LOD of 4.1 μM, (2) 2 mM – 5.2 mM with LOD of 0.95 mM. The comparison of the biosensor performance with reported sensors reveals the significant improvement in overall sensor performance. Moreover, the biosensor exhibited appreciable stability, repeatability, reproducibility and practicality. The other advantages of the fabricated biosensor are simple and green fabrication approach, roughed and stable electrode surface, fast in sensing and highly reproducible.     

Disposable biosensor based on enzyme immobilized on Au-chitosan-modified indium tin oxide electrode with flow injection amperometric analysis

Indium tin oxide (ITO) electrode is used to fabricate a novel disposable biosensor combined with flow injection analysis for the rapid determination of H2O2. The biosensor is prepared by entrapping horseradish peroxidase (HRP) enzyme in colloidal gold nanoparticle-modified chitosan membrane (Au-chitosan) to modify the ITO electrode. The biosensor is characterized by scanning electron microscope, atomic force microscope, and electrochemical methods. Parameters affecting the performance of the biosensor, including concentrations of o-phenylenediamine (OPD) and pH of substrate solution, were optimized. Under the optimal experimental conditions, H2O2 could be determined in the linear calibration range from 0.01 to 0.5 mM with a correlation coefficient of 0.997 (n=8). The amperometric response of the biosensor did not show an obvious decrease after the substrates were injected continuously 34 times into the flow cell. The prepared biosensor not only is economic and disposable, due to the low-cost ITO film electrode obtained from industrial mass production, but also is capable with good detection precision, acceptable accuracy, and storage stability for the fabrication in batch.     

A novel biosensor based on activation effect of thiamine on the activity of pyruvate oxidase

A biosensor based on pyruvate oxidase (POX) enzyme was developed for the investigation of the effect of thiamine (vitamin B(1)) molecule on the activity of the enzyme. The biosensor was prepared with a chemical covalent immobilization method on the dissolved oxygen (DO) probe by using gelatin and cross-linking agent, glutaraldehyde. POX catalyzes the degradation of pyruvate to acetylphosphate, CO(2) and H(2)O(2) in the presence of phosphate and oxygen. Thiamine is an activator for POX enzyme and determination method of the biosensor was based on this effect of thiamine on the activity of the enzyme. The biosensor responses showed increases in the presence of thiamine. Increases in the biosensor responses were related to thiamine concentration. Thiamine determination is based on the assay of the differences on the biosensor responses on the oxygenmeter in the absence and the presence of thiamine. The biosensor response depend linearly on thiamine concentration between 0.025 and 0.5 microM with 2 min response time. In the optimization studies of the biosensor the most suitable enzyme amount was found as 2.5 U cm(-2) and also phosphate buffer (pH 7.0; 50 mM) and 35 degrees C were obtained as the optimum working conditions. In the characterization studies of the biosensor some parameters such as activator and interference effects of some substances on the biosensor response and reproducibility were carried out.     

An amperometric oxalate biosensor based on sorghum oxalate oxidase bound carboxylated multiwalled carbon nanotubes-polyaniline composite film

A highly sensitive, specific and rapid electrochemical oxalate biosensor was constructed by covalently immobilizing sorghum leaf oxalate oxidase on carboxylated multiwalled carbon nanotubes and conducting polymer, polyaniline nanocomposite film electrodeposited over the surface of platinum (Pt) wire using N-ethyl-N′-(3-dimethylaminopropyl) carbodiimide (EDC) and N-hydroxy succinimide (NHS) chemistry. The modified electrode was characterized by scanning electron microscopy (SEM) and Fourier transform infrared (FTIR) spectrophotometry. The optimized oxalate biosensor showed linear response range of 8.4-272 μM with correlation coefficient of 0.93 and rapid response within 5 s at a potential of 0.4 V vs Ag/AgCl. The sensitivity was approximately 0.0113 μA/μM with a detection limit of 3.0 μM. Proposed oxalate biosensor was successfully applied to human urine sample.     

Polyphenol biosensor based on laccase immobilized onto silver nanoparticles/multiwalled carbon nanotube/polyaniline gold electrode

Laccase purified from Ganoderma sp. was immobilized covalently onto electrochemically deposited silver nanoparticles (AgNPs)/carboxylated multiwalled carbon nanotubes (cMWCNT)/polyaniline (PANI) layer on the surface of gold (Au) electrode. A polyphenol biosensor was fabricated using this enzyme electrode (laccase/AgNPs/cMWCNT/PANI/Au electrode) as the working electrode, Ag/AgCl as the reference electrode, and platinum (Pt) wire as the auxiliary electrode connected through a potentiostat. The biosensor showed optimal response at pH 5.5 (0.1 M acetate buffer) and 35 °C when operated at a scan rate of 50 mV s⁻¹. Linear range, response time, and detection limit were 0.1–500 μM, 6 s, and 0.1 μM, respectively. The sensor was employed for the determination of total phenolic content in tea, alcoholic beverages, and pharmaceutical formulations. The enzyme electrode was used 200 times over a period of 4 months when stored at 4 °C. The biosensor has an advantage over earlier enzyme sensors in that it has no leakage of enzyme during reuse and is unaffected by the external environment due to the protective PANI microenvironment.     

A novel amperometric bienzymatic biosensor based on alcohol oxidase coupled PVC reaction cell and nanomaterials modified working electrode for rapid quantification of alcohol

Abstract

A new amperometric sensor has been fabricated for sensitive and rapid quantification of ethanol. The biosensor assembly was prepared by covalently immobilizing alcohol oxidase (AOX) from Pichia pastoris onto chemically modified surface of polyvinylchloride (PVC) beaker with glutaraldehyde as a coupling agent followed by immobilization of horseradish peroxidase (HRP), silver nanoparticles (AgNPs), chitosan (CHIT), carboxylated multi-walled carbon nanotubes (c-MWCNTs) and nafion (Nf) nanocomposite onto the surface of Au electrode (working electrode). Owing to properties such as chemical inertness, light weight, weather resistance, corrosion resistance, toughness and cost-effectiveness, PVC membrane has attracted a growing interest as a support for enzyme immobilization in the development of biosensors. The amperometric biosensor displayed optimum response within 8?s at pH 7.5 and 35°C temperature. A linear response to alcohol in the range of 0.01mM–50?mM and 0.0001?µM as a minimum limit of detection was displayed by the proposed biosensor with excellent storage stability (190?days) at 4°C. The sensitivity of the sensor was found to be 155?µA mM^?1?cm^?2. A good correlation (R²?=?0.99) was found between alcohol level in commercial samples as evaluated by standard ethanol assay kit and the current biosensor which validates its performance.     

An ultrasensitive electrochemiluminescent sensor based on a pencil graphite electrode modified with CdS nanorods for detection of chlorogenic acid in honeysuckle

In this paper, a novel and ultrasensitive electrochemiluminescent sensor employing a solvothermal‐synthesized CdS nanorod‐modified pencil graphite electrode (CdS/PGE) for the determination of chlorogenic acid (CA) is fabricated. In the first step, the PGE surface is modified using CdS nanorods. In the next step, the developed electrode is used to detect CA using a electrochemiluminescent (ECL) technique, in which potassium persulfate (K₂S₂O₈) served as a co‐reactant. The possible ECL mechanism is investigated, and the influences of pH and cyclic voltammetric scanning rate on the signal response are studied. The ECL intensity decreases quantitatively in relation to the concentration of the target molecule. Under optimized conditions, the linear correlation between the quenched ECL intensity and the logarithm of CA concentration is observed in the range from 2 × 10^?9 to 8 × 10^?7 mol L^?1 with a limit of detection of 1 × 10^?9 mol L^?1. This proposed method is applied to the analysis of CA in honeysuckle flower, giving recoveries of 99‐107%. The experimental results demonstrate that this ECL sensor shows good stability and reproducibility.     

An amperometric biosensor based on laccase immobilized onto MnO2NPs/cMWCNT/PANI modified Au electrode

A method is described for construction of an amperometric biosensor for detection of phenolic compounds based on covalent immobilization of laccase (Lac) onto manganese dioxide nanoparticles (MnO(2)NPs) decorated carboxylated multiwalled carbon nanotubes (cMWCNTs)/PANI composite electrodeposited onto a gold (Au) electrode through N-ethyl-N'-(3-dimethylaminopropyl) carbodiimide (EDC) and N-hydroxy succinimide (NHS) chemistry. The modified electrode was characterized by scanning electron microscopy (SEM), cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). The biosensor showed optimum response at pH 5.5 (0.1M sodium acetate buffer) and 35°C, when operated at 0.3 V vs. Ag/AgCl. Linear range, response time, detection limit were 0.1-10 μM (lower concentration range) and 10-500 μM (higher concentration range), 4s and 0.04 μM, respectively. Biosensor measured total phenolic content in tea leaves extract. The enzyme electrode was used 150 times over a period of 5 months.     

Amperometric cholesterol biosensor based on in situ reconstituted cholesterol oxidase on an immobilized monolayer of flavin adenine dinucleotide cofactor

A new amperometric biosensor for determining cholesterol based on deflavination of the enzyme cholesterol oxidase (ChOx) and subsequent reconstitution of the apo-protein with a complexed flavin adenine dinucleotide (FAD) monolayer is described. The charge transfer mediator pyrroquinoline quinone (PQQ) was covalently bound to a cystamine self-assembled monolayer (SAM) on an Au electrode. Boronic acid (BA) was then bound to PQQ using the carbodiimide procedure, and the BA ligand was complexed to the FAD molecules on which the apo-ChOx was subsequently reconstituted. The effective release of the FAD from the enzyme and the successful reconstitution were verified using molecular fluorescence and cyclic voltammetry. The optimal orientation of FAD toward the PQQ mediator and the distances between FAD and PQQ and between PQQ and electrode enhance the charge transfer, very high sensitivity (about 2,500 nAmM(-1)cm(-2)) being obtained for cholesterol determination. The biosensor is selective toward electroactive interferents (ascorbic acid and uric acid) and was tested in reference serum samples, demonstrating excellent accuracy (relative errors below 3% in all cases). The biosensor activity can be successfully regenerated in a simple process by successive reconstitution with batches of recently prepared apo-ChOx on the same immobilized Au/SAM-PQQ-BA-FAD monolayer (it was tested five times); the lifetime of the biosensor is about 45-60 days.     

An amperometric uric acid biosensor based on multiwalled carbon nanotube-gold nanoparticle composite

An amperometric uric acid biosensor was fabricated by immobilizing uricase (EC 1.7.3.3) onto gold nanoparticle (AuNP)/multiwalled carbon nanotube (MWCNT) layer deposited on Au electrode via carbodiimide linkage. Determination of uric acid was performed by oxidation of enzymically generated H₂O₂ at 0.4 V. The sensor showed optimal response within 7 s at 40 °C in 50 mM Tris–HCl buffer (pH 7.5). The linear working range of the biosensor was 0.01–0.8 mM. The limit of detection (LOD) was 0.01 mM. The sensor measured uric acid levels in serum of healthy individuals and persons suffering from gout. The analytical recoveries of the added uric acid, 10 and 20 mg L^–1, were 98.0% and 96.5%, respectively. Within- and between-batch coefficients of variation were less than 5.6% and less than 4.7%, respectively. A good correlation (r = 0.998) was obtained between serum uric acid values by the standard enzymic colorimetric method and the current method. A number of serum substances had practically no interference. The sensor was used in more than 200 assays and had a storage life of 120 days at 4 °C.     

An amperometric biosensor based on horseradish peroxidase immobilized onto maize tassel-multi-walled carbon nanotubes modified glassy carbon electrode for determination of heavy metal ions in aqueous solution

A biosensor for trace metal ions based on horseradish peroxidase (HRP) immobilized on maize tassel-multiwalled carbon nanotube (MT-MWCNT) through electrostatic interactions is described herein. The biosensor was characterized using Fourier transform infrared (FTIR), UV–vis spectrometry, voltammetric and amperometric methods. The FTIR and UV–vis results inferred that HRP was not denatured during its immobilization on MT-MWCNT composite. The biosensing principle was based on the determination of the cathodic responses of the immobilized HRP to H₂O₂, before and after incubation in trace metal standard solutions. Under optimum conditions, the inhibition rates of trace metals were proportional to their concentrations in the range of 0.092–0.55 mg L⁻¹, 0.068–2 mg L⁻¹ for Pb²⁺ and Cu²⁺ respectively. The limits of detection were 2.5 μg L⁻¹ for Pb²⁺ and 4.2 μg L⁻¹ for Cu²⁺. Representative Dixon and Cornish-Bowden plots were used to deduce the mode of inhibition induced by the trace metal ions. The inhibition was reversible and mixed for both metal ions. Furthermore, the biosensor showed good stability, selectivity, repeatability and reproducibility.     

An amperometric glucose biosensor based on poly(o-aminophenol) and Prussian blue films at platinum electrode

Prussian blue (PB), as a good catalyst for the reduction of hydrogen peroxide, has been combined with nonconducting poly(o-aminophenol) (POAP) film to assemble glucose biosensor. Compared with PB-modified enzymatic biosensor, the biosensor based on glucose oxidase immobilized in POAP film at PB-modified electrode shows much improved stability (78% remains after 30 days) in neutral medium. Additionally, the biosensor, at an applied potential of 0.0 V, exhibits other good characteristics, such as relative low detection limit (0.01 mM), short response time (within 5s), large current density (0.28 mA/cm2), high sensitivity (24 mAM(-1)cm(-2)), and good antiinterferent ability. The apparent activation energy of enzyme-catalyzed reaction and apparent Michaelis-Menten constant are 34.2 KJmol(-1) and 10.5 mM, respectively. In addition, effects of temperature, applied potential used in the determination, pH value of the detection solution, and electroactive interferents on the amperometric response of the sensor were investigated and are discussed.     

An amperometric biosensor for determining amino acids using a bienzymatic system containing amino acid oxidase and protease

An amperometric biosensor for rapid determination of the concentration of l-amino acids has been developed using l-amino acid oxidase (l-AAO) immobilized by gel entrapment with poly(carbamoyl) sulfonate hydrogel. The broad substrate range of l-AAO allows this biosensor to be flexible in application. The artificial sweetener, aspartame, was determined by coupling l-AAO with pronase.     

A novel glucose biosensor based on the immobilization of glucose oxidase onto gold nanoparticles-modified Pb nanowires

A novel glucose biosensor was developed, based on the immobilization of glucose oxidase (GOD) with cross-linking in the matrix of bovine serum albumin (BSA) on a Pt electrode, which was modified with gold nanoparticles decorated Pb nanowires (GNPs-Pb NWs). Pb nanowires (Pb NWs) were synthesized by an l-cysteine-assisted self-assembly route, and then gold nanoparticles (GNPs) were attached onto the nanowire surface through –SH–Au specific interaction. The morphological characterization of GNPs-Pb NWs was examined by transmission electron microscopy (TEM). Cyclic voltammetry and chronoamperometry were used to study and to optimize the electrochemical performance of the resulting biosensor. The synergistic effect of Pb NWs and GNPs made the biosensor exhibit excellent electrocatalytic activity and good response performance to glucose. The effects of pH and applied potential on the amperometric response of the biosensor have been systemically studied. In pH 7.0, the biosensor showed the sensitivity of 135.5 μA mM⁻¹ cm⁻², the detection limit of 2 μM (S/N = 3), and the response time <5 s with a linear range of 5–2200 μM. Furthermore, the biosensor exhibits good reproducibility, long-term stability and relative good anti-interference.     

Immobilization of glucose oxidase and lactate dehydrogenase onto magnetic nanoparticles for bioprocess monitoring system

Glucose oxidase (GOD) and lactate dehydrogenase (LDH) were immobilized onto magnetic nanoparticles, viz. Fe₃O₄, via carbodiimide and glutaraldehyde. The immobilization efficiency was largely dependent upon the immobilization time and concentration of glutaraldehyde. The magnetic nanoparticles had a mean diameter of 9.3 nm and were superparamagnetic. The immobilization of GOD and LDH on the nanoparticles slightly decreased their saturation magnetization. However, the FT-IR spectra showed that GOD and LDH were immobilized onto the nanoparticles by different binding mechanisms, the reason for which was not well explained. The optimum pH values of the immobilized GOD and LDH were changed to 8 and 10, respectively. The free and immobilized enzyme kinetic parameters (K_m and V_max) were determined by Michaelis-Menten enzyme kinetics. The Km values for free and immobilized GOD were 0.168 and 0.324 mM, respectively, while those for free and immobilized LDH were 0.19 and 0.163 mM for NAD, and 2.976 and 4.785 mM for lactate, respectively. High operational stability was observed, with more than 80% of the initial enzyme activity being retained for the immobilized GOD up to 12 h and for the immobilized LDH up to 24 h. The immobilized GOD was applied to a sequential injection analysis system for the application of bioprocess monitoring.