Amperometric Transducer Research Articles

Simple and suitable immunosensor for β-lactam antibiotics analysis in real matrixes: Milk, serum, urine

The anti-penicillin G was conjugated to avidin–peroxidase and biotin to obtain immunogen and competitor which were then used to develop a competitive immunosensor assay for the detection of penicillin G and other β-lactam antibiotics, with Kaff values of the order of 108M−1. The new immunosensor appears to afford a number of advantages in terms of sensitivity, possibility of “in situ” analysis, but especially of simplicity and lower costs, compared with other existing devices, or different chemical instrumental methods reported in the literature and used for the analysis of β-lactam compounds. Satisfactory results were found in the analysis of real matrixes and good recoveries were obtained by applying the standard addition method to spiked milk, urine, serum and drug samples. The new device uses an amperometric electrode for hydrogen peroxide as transducer, the BSA–penicillin G immobilized on polymeric membrane overlapping the amperometric transducer and the peroxidase enzyme as marker. It proved to be highly sensitive, inexpensive and easily reproducible; LOD was of the order of 10−11M. Lastly, the new immunosensor displayed low selectivity versus the entire class of β-lactam antibiotics and higher selectivity toward other classes of non-β-lactam antibiotics.

Amperometric biosensor system for simultaneous determination of adenosine-5'-triphosphate and glucose.

The majority of biosensors for adenosine-5'-triphosphate (ATP) determination are based on cascades of enzymatic reactions; therefore, they are sensitive to glucose or glycerol (depending on the enzymatic system) as well as to ATP. The presence of unknown concentrations of these substances in the sample greatly complicates the determination of ATP. To overcome this disadvantage of known biosensors, we developed a biosensor system consisting of two biosensors: the first one is based on glucose oxidase and is intended for measuring glucose concentration, and the second one is based on glucose oxidase and hexokinase and is sensitive toward both glucose and ATP. Using glucose concentration measured by the first biosensor, we can analyze the total response to glucose and ATP obtained by the second biosensor. Platinum disc electrodes were used as amperometric transducers. The polyphenilenediamine membrane was deposited onto the surface of platinum electrodes to avoid the response to electroactive substances. The effect of glucose concentration on biosensor determination of ATP was studied. The reproducibility of biosensor responses to glucose and ATP during a day was tested (relative standard deviation, RSD, of responses to glucose was 3-6% and to ATP was 8-12%) as well as storage stability of the biosensors (no decrease of glucose responses and 43% drop of ATP responses during 50 days). The measurements of ATP and glucose in pharmaceutical vials (including mixtures of ATP and glucose) were carried out. It was shown that the developed biosensor system can be used for simultaneous analysis of glucose and ATP concentrations in water solutions.

STUDY OF CHARACTERISTICS OF AMEROMETRIC NICKEL TRANSDUCERS USING POTENTIOSTAT ‘PalmSens’ AND IT’S UKRAINIAN ANALOG

Characteristics of nickel amperometric transducers connected to experimental Ukrainian potentiostat and to commercial potentiostat PalmSens were studied in this work. During this, intrinsic characteristics of transducers and differences caused by connection to different potentiostats were examined. Comparison was made in several working modes. Amperometric electrodes made from nickel and biosensors for glucose determination (obtained by immobilization of glucose oxidase onto the surface of these electrodes) were connected to the potentiostats and effectiveness of measurement of hydrogen peroxide and glucose contents by both potentiostats was compared. I.e., sensor responses, signal noise, limit of detection of substances, calibration curves for determination of hydrogen peroxide and glucose, reproducibility of responses during continuous operation were compared. Also selectivity of sensors to hydrogen peroxide respectively to main interfering substances (ascorbic acid and ethanol) was checked. It was shown that nickel transducers can be a basis for creation of chemo- and biosensors. Besides, it was demonstrated that for the most parameters new Ukrainian potentiostat is not inferior to the potentiostat PalmSens and Ukrainian potentiostat can serve as a cheaper analog of PalmSens during development of biosensor test systems

METHOD OF TESTING AND OPTIMIZATION OF AMPEROMETRIC TRANSDUCERS

Method of testing and optimization of amperometric transducers were proposed in this work. This method must essentially facilitate preliminary testing of transducers during development of amperometric biosensors and it will allow more effectively use these transducers for creation of biosensors. Proposed method includes the sequence of necessary operations, that researcher has to make during development of each enzyme amperometric biosensors based on registration of peroxide hydrogen oxidation without mediators. The sequence of necessary operations includes study of main working characteristics, optimization of these characteristics with the purpose of following effective usage of transducers as a part of biosensor.

P-HEMA Membrane Fabrication Applying Nanogold for Biochemical Sensor

A p-HEMA (poly-hydroxy ethyl methacrylic acid) membrane using alcohol oxidase deposited on a carbon screen printed electrode (SPE) as working electrode for amperometric transducer has been built for biochemical sensor. Acetophenon derivate has been applied to generate polymeric cross linking while ferrocene mediator has been added to wire the electrochemical reaction. The UV photocuring attached the membrane polymer onto the carbon SPE. This study has obtained that nanogold application enhanced the formaldehyde biosensor performance. Optimum result using cyclic voltametric (CV) mode in amperometric assay has been found by applying 1.6% acetophenon, 1% ferrocene, 7.7u alcohol oxidase and 0.2% nanogold in phosphate buffer of pH 7.2 at 500s UV photocure.

Development of a Disposable Gold Electrodes-Based Sensor for Electrochemical Measurements of cDNA Hybridization

This work deals with the development of a disposable electrochemical biosensor for the specific detection of short DNA sequences. The sensor is an amperometric transducer with three planar electrodes, comprising a working, a counter and a pseudo-reference electrode, all made of a gold layer over a polycarbonate substrate. For the development of the genosensor, the working electrode was modified using thiol-tethered 33-mer DNA probe by chemisorptions, in a concentration range from 0.1 μM to 5 μM. Immobilization of ssDNA on gold surface was monitored with electrochemical impedance spectroscopy (EIS) and differential pulse voltammetry (DPV) in Fe(CN)64−/13− and Ruthenium(II)/(III) solutions. The time dependence of ssDNA probe immobilization was also studied. The hybridization detection is then compared with EIS and DPV measurements.

A biosensor based on the self-entrapment of glucose oxidase within biomimetic silica nanoparticles induced by a fusion enzyme

We constructed a fusion protein (GOx–R5) consisting of R5 (a polypeptide component of silaffin) and glucose oxidase (GOx) that was expressed in Pichia pastoris. Silaffin proteins are responsible for the formation of a silica-based cell matrix of diatoms, and synthetic variants of the R5 protein can perform silicification in vitro [1]. GOx secreted by P. pastoris was self-immobilized (biosilicification) in a pH 5 citric buffer using 0.1 M tetramethoxysilane as a silica source. This self-entrapment property of GOx–R5 was used to immobilize GOx on a graphite rod electrode. An electric cell designed as a biosensor was prepared to monitor the glucose concentrations. The electric cell consisted of an Ag/AgCl reference electrode, a platinum counter electrode, and a working electrode modified with poly(neutral red) (PNR)/GOx/Nafion. Glucose oxidase was immobilized by fused protein on poly(neutral red) and covered by Nafion to protect diffusion to the solution. The morphology of the resulting composite PNR/GOx/Nafion material was analyzed by scanning electron microscopy (SEM). This amperometric transducer was characterized electrochemically using cyclic voltammetry and amperometry in the presence of glucose. An image produced by scanning electron microscopy supported the formation of a PNR/GOx complex and the current was increased to 1.58 μA cm −1 by adding 1 mM glucose at an applied potential of −0.5 V. The current was detected by way of PNR-reduced hydrogen peroxide, a product of the glucose oxidation by GOx. The detection limit was 0.67 mM (S/N = 3). The biosensor containing the graphite rod/PNR/GOx/Nafion detected glucose at various concentrations in mixed samples, which contained interfering molecules. In this study, we report the first expression of R5 fused to glucose oxidase in eukaryotic cells and demonstrate an application of self-entrapped GOx to a glucose biosensor.

Microelectronic amperometric sensors for biochemical research

The problems of choice of electrode systems for amperometric type multibiosensors were considered. The design of planar amperometric electrode chips for multisensory electrochemical cell was developed. The manufacturing technique of disposable electrode chips for amperometric transducers of biosensory systems was simplified and its cost was reduced. The portable biosensor measurement system which includes manufactured amperometric transducers was described and the results of its testing were provided

Further development of lactoferrin immunosensor (part III)

The last year we fabricated some new immunosensors for the analysis of lactoferrin protein. In the present research the immunological and analytical characteristics of the immunosensor method have been extensively investigated. The study was therefore extended to cover the ability of the analyte and the corresponding antibody to produce the immunocomplex. A rough estimation of the K aff value was obtained at the midpoint of the Langmuir curve, where K aff = 1/IC 50. The K aff value was found to be of the order of 10 6 M −1. In addition we attempted in the present study to reduce the excessively long time required for each measurement, due to the fact that, in the previous researches the measurement procedure used was the classic “competition” ELISA type, employing an ad hoc enzymatic marker. One possible way of reducing measurement time was found to be to perform a kind of completely innovative “direct” measurement developed by us, which still uses an enzymatic marker and an amperometric transducer, as in the previous competition method.

A Mediated Glucose Biosensor Incorporated with Reverse Iontophoresis Function for Noninvasive Glucose Monitoring

The objective of this study was to develop a ferrocene mediated glucose biosensor for reverse iontophoresis. An amperometric ferrocene mediated glucose biosensor based on a three electrodes planar configuration was constructed using screen printing technique. Different combinations of glucose oxidase and ferrocene loading were drop coated onto the surface of the amperometric transducer. The amperometric transducer was characterized electrochemically using cyclic voltammetry and its electrochemical characteristics (DeltaE(p) = 70 mV, I(pa)/I(pc) = 0.89) were found close to an ideal amperometric transducer. The biosensor on the detection of glucose at 200 mV (vs. Ag/AgCl) showed a linear response range (0-4 mM). The response time of the biosensor was about 10 s. Finally, the biosensor was used together with reverse iontophoresis technique. By the use of an actual model for evaluation, an excellent linear relationship (r(2) = 0.99) was found between the glucose concentration of the actual model and the biosensor current response. In conclusion, a ferrocene mediated glucose biosensor incorporated with reverse iontophoresis function was developed.

A Novel Amperometric Transducer Electrode with Iridium‐Niobium Binary Alloys

AbstractWe have discovered that an Ir‐23Nb binary alloy more effectively oxidizes hydrogen peroxide than Ir, Ir‐13Nb, Ir‐17Nb, Ir‐30Nb, Ir‐43Nb, Ir‐62Nb, or Nb. The oxidation capability was determined via cyclic voltammogram measurements of pH‐buffer and hydrogen peroxide. We ascertained that applying a 0.7 V potential produces hydrogen peroxide currents of 9.2 μA/mm2 Ir‐23Nb, 5.3 μA/mm2 Ir, 5.1 μA/mm2 Ir‐17Nb, 3.7 μA/mm2 Ir‐13Nb, 2.0 μA/mm2 Ir‐30Nb, 1.5 μA/mm2 Ir‐43Nb, 0.6 μA/mm2 Ir‐62Nb, and 0.13 μA/mm2 Nb. These results indicate that the effective oxidation of Ir‐23Nb for hydrogen peroxide might be due to its fcc+L12 two‐phase structure and that Ir‐23Nb can be used as an amperometric transducer material.

Bienzyme Based Biosensing Platform Using Functionalized Carbon Nanotubes

This work presents a novel approach to design an amperometric transducer capable of detecting glucose and hydrogen peroxide. The biosensor is based on a bienzyme-channelling configuration, employing the enzymes glucose oxidase and horseradish peroxidase, which were immobilized with Toluidine blue (TB) functionalized multiwalled carbon nanotubes (MWNTs). TB was covalently immobilized with carboxylic acid groups of the carbon nanotubes via carbodiimide reaction. The MWNT/TB/GOD/HRP/Nf nanobiocomposite was prepared by mixing the GOx and HRP solution with TB functionalized CNTs followed by mixing homogeneously with Nafion. The MWNTiTB/GOD/HRP/Nf nanobiocomposite was characterized by scanning electron microscopy. The performance of the MWNT/TB/GOD/HRP/Nf nanobiocomposite modified electrode was examined by electrochemical impedance spectroscopy and cyclic voltammetry. Linear calibrations were achieved upto 1.4 x 10(-7)-1.6 x 10(-3) M for glucose and 6.8 x 10(-8)-1.7 x 10(-3) M for H2O2. A remarkable feature of this bienzyme electrode is the possibility to detect glucose and H2O2 at very low applied potentials where the noise level and interferences from other electro-active compounds are minimal.

Urea amperometric biosensors based on a multifunctional bipolymeric layer: Comparing enzyme immobilization methods

This paper outlines the results obtained with biosensors designed for urea amperometric detection. The incorporation of urease into a bipolymeric substrate consisting of poly(pyrrole) and poly(5-amino-1-naphthol) was performed through four different approaches: direct adsorption, entrapment in cellulose acetate layer, cross-linking with glutaraldehyde, and also covalent attachment to the polymeric matrix. Poly(pyrrole) acts as amperometric transducer in these biosensors, while poly(5-amino-1-naphthol) drastically reduces the interference signal of agents such as ascorbic and uric acids. The biosensors containing urease covalently attached to the substrate provided interesting results in terms of sensitivity towards urea (0.50 μA cm −2 mmol −1 L), lifetime (20 days) and short response times, due to the enzyme immobilization method used. All biosensors analyzed showed also a wide linear concentration range (up to 100 mmol L −1) and low detection limits (0.22–0.58 mmol L −1).

Fabrication of bienzyme nanobiocomposite electrode using functionalized carbon nanotubes for biosensing applications

Mediated biosensors consisting of an oxidase and peroxidase (PO x) have attracted increasing attention because of their wider applicability. This work presents a novel approach to fabricate nanobiocomposite bienzymatic biosensor based on functionalized multiwalled carbon nanotubes (MWNTs) with the aim of evaluating their ability as sensing elements in amperometric transducers. Electrochemical behavior of the bienzymatic nanobiocomposite biosensor is investigated by Faradaic impedance spectroscopy and cyclic voltammetry. The results indicate that glucose oxidase (GOD) and horseradish peroxidase (HRP) are strongly adsorbed on the surface of the thionin (TH) functionalized MWNTs and demonstrate a facile electron transfer between immobilized GOD/HRP and the electrode via the functionalized MWNTs in a Nafion film. The functionalized carbon nanotubes act as molecular wires to allow efficient electron transfer between the underlying electrode and the redox centres of enzymes through TH. Linear ranges for these electrodes are from 10 nM to 10 mM for glucose and 17 nM to 56 mM for hydrogen peroxide with the detection limit of 3 and 6 nM, respectively. A remarkable feature of the bienzyme electrode is the possibility to determine glucose and hydrogen peroxide at a very low applied potential where the noise level and interferences from other electroactive compounds are minimal. Performance of the biosensor is evaluated with respect to response time, detection limit, selectivity, temperature and pH as well as operating and storage stability.

Amperometric IgG Immunosensor using a Tyrosinase‐Colloidal Gold‐Graphite‐Teflon Biosensor as a Transducer

An IgG immunosensor using a colloidal gold‐Tyrosinase‐graphite‐Teflon composite biosensor as an amperometric transducer is reported. Protein A was used to immobilize the antibody on the biosensor surface and a sandwich‐type configuration using alkaline phosphatase (AP) labeled anti‐IgG was employed. Phenyl phosphate was used as the AP‐substrate, and the enzyme reaction product, phenol, was catalytically oxidized by tyrosinase to the o‐quinone, which is subsequently reduced at −0.1 V at the biocomposite electrode. Variables such as the concentration of phenyl phosphate, the amount of antibody attached to the electrode surface, the immersion time into a 2% BSA solution and the incubation time into IgG, protein A and AP conjugate solutions, were optimized. Electrochemical impedance spectroscopy was used to monitor all the steps involved in the preparation of the immunosensor. A linear calibration graph for IgG was obtained between 5 and 100 ng ml−1 IgG, with a slope value of 11.8 nA ng−1 ml, and a detection limit of 2.6 ng ml−1. These analytical characteristics are competitive with other IgG electrochemical immunosensor designs. The developed anti‐IgG (Tyr‐Aucoll‐graphite‐Teflon) immunosensor was applied to IgG determination in a spiked serum sample with a recovery of 103±6% for a 10 ng ml−1 concentration level.

Immunosensor for the determination of Staphylococcus aureus using a tyrosinase–mercaptopropionic acid modified electrode as an amperometric transducer

An amperometric immunosensor for the quantification of Staphylococcus aureus based on the coimmobilization of rabbit immunoglobulin G (RbIgG) and tyrosinase on a mercaptopropionic acid self-assembled monolayer modified gold electrode is reported. A competitive mode in which protein-A-bearing S. aureus cells and antiRbIgG labeled with alkaline phosphatase (AP) compete for the binding sites of immobilized RbIgG was used. Monitoring of the affinity reaction was carried out by the amperometric detection at -0.15 V of phenol generated in the enzyme reaction with AP, at the tyrosinase-modified electrode through the electrochemical reduction of the o-quinone formed. Optimization of the working variables, such as the immunosensor composition and incubation times, the applied potential, the working pH and the concentration of phenyl phosphate used as the AP substrate, was carried out. Under the optimized conditions, both the repeatability of the measurements and the reproducibility of the responses obtained with different immunosensors yielded relative standard deviation values for the steady-state current lower than 10%. The immunosensor showed a dynamic range from 4.4x10(5) to 1.8x10(7) S. aureus cells mL(-1), with a detection limit of 1.7x10(5) cells mL(-1). The limit of detection was remarkably improved by subjecting S. aureus cells to wall lysis by heat treatment. The value obtained was 2.3x10(3) cells mL(-1), which is adequate for the monitoring of S. aureus contamination levels in some foodstuffs. As an application, milk samples spiked with bacteria at the 4.8x10(3) cells mL(-1) level were analyzed.

Nanostructured progesterone immunosensor using a tyrosinase–colloidal gold–graphite–Teflon biosensor as amperometric transducer

A novel progesterone immunosensor using a colloidal gold–graphite–Teflon–tyrosinase composite biosensor as amperometric transducer is reported. A sequential competitive configuration between the analyte and progesterone labelled with alkaline phosphatase (AP) was used. Phenyl phosphate was employed as the AP-substrate and the enzyme reaction product, phenol, was oxidized by tyrosinase to o-quinone, which is subsequently reduced at −0.1 V at the biocomposite electrode. Variables such as the concentration of phenyl phosphate, the amount of antibody attached to the electrode surface, immersion time in a 2% BSA solution, working pH and incubation times in progesterone and AP conjugate were optimized. A linear calibration graph for progesterone was obtained between 0 and 40 ng mL −1 with a slope value of −82.3 nA ng −1 mL, and a detection limit of 0.43 ng mL −1. The time needed to reach the steady-state current from the addition of phenyl phosphate was 30–40 s. These analytical characteristics improve substantially those reported for other progesterone immunosensors. A lifetime of 14 days with no need to apply any regeneration procedure was also achieved. The usefulness of the immunosensor was evaluated by determining progesterone in milk samples spiked with the analyte at 5.0 and 1.5 ng mL −1 concentration levels. Following a very simple procedure, involving only sample dilution, mean recoveries ( n = 7) of 98 ± 3% and 99 ± 3%, respectively, were obtained.

Organophosphorus and carbamate pesticide analysis using an inhibition tyrosinase organic phase enzyme sensor; comparison by butyrylcholinesterase + choline oxidase opee and application to natural waters

Recent research performed in our laboratory (using a butyrylcholinesterase + choline oxidase enzyme electrode) suggested the validity of the biosensor approach using enzyme inhibition OPEEs (i.e. enzyme electrodes working in organic phase) in the case of organophosphorus and carbamate pesticides, which are poorly soluble in aqueous solutions. Since these pesticides are generally much more soluble in chloroform than in water, the present research aimed at analysing this class of pesticides using a tyrosinase inhibition OPEE operating in water-saturated chloroform medium. The tyrosinase biosensor was assembled using an oxygen amperometric transducer coupled to the tyrosinase enzyme, immobilized in kappa-carrageenan gel. Lastly a detailed comparison between the inhibition monoenzymatic tyrosinase and inhibition bienzymatic (butyrylcholinesterase + choline oxidase) OPEEs was performed and discussed in this work.

Biosensor for direct determination of fenitrothion and EPN using recombinant Pseudomonas putida JS444 with surface-expressed organophosphorous hydrolase. 2. Modified carbon paste electrode

A whole cell-based amperometric biosensor for highly selective, sensitive, rapid, and cost-effective determination of the organophosphate pesticides fenitrothion and ethyl p-nitrophenol thio-benzene phosphonate (EPN) is discussed. The biosensor comprised genetically engineered p-nitrophenol (PNP)-degrading bacteria Pseudomonas putida JS444 anchoring and displaying organophosphorous hydrolase (OPH) on its cell surface as biological sensing element and carbon paste electrode as the amperometric transducer. Surface-expressed OPH catalyzed the hydrolysis of organophosphorous pesticides such as fenitrothion and EPN to release PNP and 3-methyl-4- nitrophenol, respectively, which were subsequently degraded by the enzymatic machinery of P. putida JS444 through electrochemically active intermediates to the TCA cycle. The electro-oxidization current of the intermediates was measured and correlated to the concentration of organophosphates. Operating at optimum conditions, 0.086 mg dry wt of cell operating at 600 mV of applied potential (vs Ag/AgCl reference) in 50 mM citrate phosphate buffer, pH 7.5, with 50 muM CoCl2 at room temperature, the biosensor measured as low as 1.4 ppb of fenitrothion and 1.6 ppb of EPN. There was no interference from phenolic compounds, carbamate pesticides, triazine herbicides, or organophosphate pesticides without nitrophenyl substituent. The service life of the biosensor and the applicability to lake water were also demonstrated.

Amperometric biosensor for ethanol detection based on alcohol oxidase immobilised within electrochemically deposited Resydrol film

A new biosensor based on amperometric transducer and alcohol oxidase immobilised in Resydrol polymer for ethanol detection has been developed. The optimal composition and immobilisation conditions for creating active membrane based on alcohol oxidase have been determined. Electrochemical deposition of the polymer film has been achieved by applying the potentiostatic pulse profile consisting of 20 consecutive pulses of + 1900 mV for 0.3 s and − 300 mV for 5 s. The biosensors developed show good analytical characteristics such as reproducibility, operational and storage stability. The minimal detection limit was 3.5 × 10 − 2 (% v/v) of ethanol. The biosensor developed has been shown to be a potential for ethanol detection in real alcoholic beverages.