Whole cell-enzyme hybrid amperometric biosensor for direct determination of organophosphorous nerve agents with p-nitrophenyl substituent

In this paper, we reported the construction of a hybrid biosensor for direct, highly selective, sensitive, and rapid quantitative determination of organophosphate pesticides with p-nitrophenyl substituent using purified organophosphorus hydrolase (OPH) for the initial hydrolysis and Arthrobacter sp. JS443 for subsequent p-nitrophenol oxidation. The biocatalytic layer was prepared by co-immobilizing Arthrobacter sp. JS443 and OPH on a carbon paste electrode. OPH catalyzed the hydrolysis of organophosphorus pesticides with p-nitrophenyl substituent such as paraoxon and methyl parathion to release p-nitrophenol that was oxidized by the enzymatic machinery of Arthrobacter sp. JS443 to carbon dioxide through electroactive intermediates 4-nitrocatechol and 1,2,4-benzenetriol. The oxidization current of the intermediates was measured and correlated to the concentration of organophosphates. The best sensitivity and response time were obtained using a sensor constructed with 0.06 mg dry weight of cell and 965 IU of OPH operating at 400 mV applied potential (vs. Ag/AgCl reference) in 50 mM citrate-phosphate pH 7.5 buffer at room temperature. Using these conditions, the biosensor measured as low as 2.8 ppb (10 nM) of paraoxon and 5.3 ppb (20 nM) of methyl parathion without interference from phenolic compounds, carbamate pesticides, triazine herbicides, and organophosphate pesticides that do not have the p-nitrophenyl substituent. The biosensor had excellent operational life-time stability with no decrease in response for more than 40 repeated uses over a 12-h period when stored at room temperature, while its storage life was approximately 2 days when stored in the operating buffer at 4 degrees C.     

Amperometric microbial biosensor for direct determination of organophosphate pesticides using recombinant microorganism with surface expressed organophosphorus hydrolase.

An amperometric microbial biosensor for the direct measurement of organophosphate nerve agents is described. The sensor is based on a carbon paste electrode containing genetically engineered cells expressing organophosphorus hydrolase (OPH) on the cell surface. OPH catalyzes the hydrolysis of organophosphorus pesticides with p-nitrophenyl substituent such as paraoxon, parathion and methyl parathion to p-nitrophenol. The later is detected anodically at the carbon transducer with the oxidation current being proportional to the nerve-agent concentration. The sensor sensitivity was optimized with respect to the buffer pH and loading of cells immobilized using paraoxon as substrate. The best sensitivity was obtained using a sensor constructed with 10 mg of wet cell weight per 100 mg of carbon paste and operating in pH 8.5 buffer. Using these conditions, the biosensor was used to measure as low as 0.2 microM paraoxon and 1 microM methyl parathion with very good sensitivity, excellent selectivity and reproducibility. The microbial biosensor had excellent storage stability, retaining 100% of its original activity when stored at 4 degrees C for up to 45 days.     

A hydroquinone biosensor using modified core-shell magnetic nanoparticles supported on carbon paste electrode

A hydroquinone biosensor was developed and used to determine hydroquinone concentration in compost extracts based on the immobilization of laccase on the surface of modified magnetic core-shell (Fe(3)O(4)-SiO2) nanoparticles. Laccase was covalently immobilized on the magnetic nanoparticles by glutaraldehyde, which was modified with amino groups on its surface. The obtained magnetic bio-nanoparticles were attached to the surface of carbon paste electrode with the aid of a permanent magnet to determine hydroquinone. A good microenvironment for retaining the bioactivity of laccase was provided by the immobilization matrix. The linear range for hydroquinone determination was 1 x 10(-7) to 1.375 x 10(-4)M, with a detection limit of 1.5 x 10(-8)M. The current reached 95% of the steady-state current within about 60s. Hydroquinone concentration in compost extracts was determined by laccase biosensor and HPLC, the results of the two methods were approximately the same.     

Effect of carbon starvation on p-nitrophenol degradation by a Moraxella strain in buffer and river water

This study examines the effect of carbon starvation on the ability of a Moraxella sp. strain to degrade p-nitrophenol (PNP). Carbon starvation for 24 h decreased the induction time for p-nitrophenol degradation by the bacterium in a minimal salt medium from 6 to 1 h but it did not completely eliminate the induction time. Moraxella cells with 2-day carbon starvation had an induction time of 3 h and the induction time of the 3-day starved cells was 6 h. A 100% increase in density of the non-starved cells did not affect the induction time for p-nitrophenol degradation by the bacterium, indicating that the initial increase in cell density of the carbon-starved culture did not cause the faster onset of p-nitrophenol degradation. However, the initial uptake of p-nitrophenol of the 1-day carbon-starved Moraxella cells was 3-fold higher than the non-starved cells. A green fluorescent protein gene (gfp)-labelled Moraxella (M6 strain) was constructed to examine the survival of and p-nitrophenol degradation by the bacterium in non-sterile river water samples. Similar p-nitrophenol degradation behaviour was observed in the river water samples inoculated with the M6 cells. The time needed for complete degradation of p-nitrophenol by the non-starved M6 was 19-27 and 33 h in samples spiked with 80, 200 and 360 microM p-nitrophenol, respectively. However, the 1-day carbon-starved inocula required about 16 h to degrade the p-nitrophenol completely regardless of its concentration in the water samples. Survival of the carbon-starved and non-starved M6 was not significantly different from each other in the river water regardless of the p-nitrophenol concentration. In the absence of p-nitrophenol, the inoculum density decreased continuously. At 200 and 360 microM p-nitrophenol, the cell densities of M6 increased in the first two days of incubation and declined steadily afterward.     

Simultaneous degradation of organophosphorus pesticides and p-nitrophenol by a genetically engineered Moraxella sp. with surface-expressed organophosphorus hydrolase.

Moraxella sp., a native soil organism that grows on p-nitrophenol (PNP), was genetically engineered for the simultaneous degradation of organophosphorus (OP) pesticides and p-nitrophenol (PNP). The truncated ice nucleation protein (INPNC) anchor was used to target the pesticide-hydrolyzing enzyme, organophosphorus hydrolase (OPH), onto the surface of Moraxella sp., alleviating the potential substrate uptake limitation. A shuttle vector, pPNCO33, coding for INPNC-OPH was constructed and the translocation, surface display, and functionality of OPH were demonstrated in both E. coli and Moraxella sp. However, whole cell activity was 70-fold higher in Moraxella sp. than E. coli. The resulting Moraxella sp. degraded organophosphates as well as PNP rapidly, all within 10 h. The initial hydrolysis rate was 0.6 micromol/h/mg dry weight, 1.5 micromol/h/mg dry weight, and 9.0 micromol/h/mg dry weight for methyl parathion, parathion, and paraoxon, respectively. The possibility of rapidly degrading OP pesticides and their byproducts should open up new opportunities for improved remediation of OP nerve agents in the future.     

Development of amperometric horseradish peroxidase based biosensors for clozapine and for the screening of thiol compounds

Two amperometric biosensors with immobilized horseradish peroxidase (HRP) were developed for the investigation of the clozapine drug oxidation and for thiols screening based on biosensor signal inhibition. The HRP was retained either in magnetized nanoporous silica microparticles (MMPs) or in a carbon paste (CP). The latter served for the carbon paste electrode while the MMPs were attracted in close proximity of a magnetized carbon electrode. The potential use of these configurations for drug oxidation and inhibition studies was illustrated by the enzymatic oxidation of clozapine (CLZ) in the presence of hydrogen peroxide. The biosensor signal corresponded to the electro-reduction of CLZ oxidation products namely a nitrenium ion (CLZox) generated by the enzyme HRP. Several thiols reactive towards CLZox were investigated and the biosensor signal inhibition (IC(50)) was comparatively determined. A protective effect of the nanoporous silica and carbon paste matrices towards HRP inactivation was inferred by comparing the biosensor inhibition results with those obtained with the free enzyme in solution.     

Bienzymatic amperometric biosensor for choline based on mediator thionine in situ electropolymerized within a carbon paste electrode

An amperometric enzyme biosensor for the determination of choline utilizing two enzymes, choline oxidase (CHOD) and horseradish peroxidase (HRP), is described. The biosensor consisted of CHOD cross-linked onto a HRP-immobilized carbon paste electrode. The biosensor was prepared by in situ electropolymerization of poly(thionine) within a carbon paste containing the enzyme HRP and thionine monomer and then CHOD was immobilized by using chitosan film through cross-linking with glutaraldehyde. The in situ electrogenerated poly(thionine) displays excellent electron transform efficiency between the enzyme HRP and the electrode surface, and the polymer enables improvement in enzyme immobilization within the paste. Several parameters such as the amount of thionine and enzyme, the applied potential, the pH, etc. have been studied. Amperometric detection of choline was realized at an applied potential of -0.2V vs saturated calomel electrode in 1/15M phosphate buffer solution (pH 7.4) with a linear response range between 5.0 x 10(-6) and 6.0 x 10(-4)M choline and a response time of 15s. When applied to the analysis of phosphatidylcholine in serum samples, a 0.997 correlation was obtained between the biosensor results and those obtained by a hospital method.     

An acetylcholinesterase biosensor for determination of low concentrations of Paraoxon and Dichlorvos

The characterization of an economic and ease-to-use carbon paste acetylcholinesterase (AChE) based biosensor to determine the concentration of pesticides Paraoxon and Dichlorvos is discussed. AChE hydrolyses acetylthiocholine (ATCh) in thiocoline (TC) and acetic acid (AA). When AChE is immobilized into a paste carbon working electrode kept at +410 mV vs. Ag/AgCl electrode, the enzyme reaction rate using acetylthiocholine chloride (ATCl) as substrate is monitored as a current intensity. Because Paraoxon and Dichlorvos inhibit the AChE reaction, the decrease of the current intensity, at fixed ATCl concentration, is a measure of their concentration. Linear calibration curves for Paraoxon and Dichlorvos determination have been obtained. The detection limits resulted to be 0.86 ppb and 4.2 ppb for Paraoxon and Dichlorvos, respectively, while the extension of the linear range was up 23 ppb for the former pesticide and up to 33 ppb for the latter. Because the inhibited enzyme can be reactivated when immediately treated with an oxime, the biosensor reactivation has been studied when 1,1'-trimethylene bis 4-formylpyridinium bromide dioxime (TMB-4) and pyridine 2-aldoxime methiodide (2-PAM) were used. TMB-4 resulted more effective. The comparison with the behavior of similar AChE based biosensors is also presented.     

Biodegradation of p-nitrophenol via 1,2,4-benzenetriol by an Arthrobacter sp.

The degradation of p-nitrophenol (PNP) by Moraxella and Pseudomonas spp. involves an initial monooxygenase-catalyzed removal of the nitro group. The resultant hydroquinone is subject to ring fission catalyzed by a dioxygenase enzyme. We have isolated a strain of an Arthrobacter sp., JS443, capable of degrading PNP with stoichiometric release of nitrite. During induction of the enzymes required for growth on PNP, 1,2,4-benzenetriol was identified as an intermediate by gas chromatography-mass spectroscopy (GC-MS) and radiotracer studies. 1,2,4-Benzenetriol was converted to maleylacetic acid, which was further degraded by the beta-ketoadipate pathway. Conversion of PNP to 1,2,4-benzenetriol is catalyzed by a monooxygenase system in strain JS443 through the formation of 4-nitrocatechol, 4-nitroresorcinol, or both. Our results clearly indicate the existence of an alternative pathway for the biodegradation of PNP.     

Immobilization of metallothionein to carbon paste electrode surface via anti-MT antibodies and its use for biosensing of silver

In this paper, heavy metal biosensor based on immobilization of metallothionein (MT) to the surface of carbon paste electrode (CPE) via anti-MT-antibodies is reported. First, the evaluation of MT electroactivity was done. The attention was focused on the capturing of MT to the CPE surface. Antibodies incorporated and mixed into carbon paste were stable; even after two weeks the observed changes in signal height were lower than 5%. Further, the interaction of MT with polyclonal chicken antibodies incorporated in carbon paste electrode was determined by square-wave voltammetry. In the voltammogram, two signals--labelled as cys(MT) and W(a)--were observed. The cys(MT) corresponded to -SH moieties of MT and W(a) corresponded to tryptophan residues of chicken antibodies. Time of interaction (300 s) and MT concentration (125 μg/ml) were optimized to suggest a silver(I) ions biosensor. Biosensor (CPE modified with anti-MT antibody) prepared under the optimized conditions was then used for silver(I) ions detection. The detection limit (3 S/N) for silver(I) ions was estimated as 0.5 nM. The proposed biosensor was tested by detection spiking of silver(I) ions in various water samples (from very pure distilled water to rainwater). Recoveries varied from 74 to 104%.     

Studies on the electrochemical performance of glucose biosensor based on ferrocene encapsulated ORMOSIL and glucose oxidase modified graphite paste electrode

The electrochemical performance of a new glucose biosensor is reported. The glucose biosensor is developed using glucose oxidase (GOD) and ferrocene encapsulated palladium (Pd)-linked organically modified sol-gel glass (ORMOSIL) material incorporated within graphite paste electrode. The ORMOSIL material incorporated within graphite paste electrode behaves as an excellent electrocatalyst for the oxidation of enzymatically reduced GOD. The electrochemical behavior of new glucose biosensor has been examined by cyclic volammetry and amperometric measurements. The bioelectrocatalysis of ORMOSIL embedded within graphite paste as a function of storage time and varying concentration of ORMOSIL is reported. The initial amperometric response on glucose sensing is recorded to be 145 microA at 15% (w/w) concentration of the ORMOSIL which is decreased to 20 microA at 5% of the same keeping GOD concentration constant. The variation of electrochemical behavior of the ORMOSIL embedded within graphite paste as a function of time has also been studied based on cyclic voltammetry. The voltammograms showing the reversible electrochemistry of ORMOSIL encapsulated ferrocene is changed into a plateau shape as a function of time, however, the electrocatalytic behavior is still retained. The practical usability of new glucose sensor has been compared with earlier developed glucose sensor. The sensitivity, response time and linearity of the new glucose biosensor are found to be excellent over earlier reported glucose biosensor. The amperometric response, calibration curve and practical applications of new glucose sensor are reported.     

Development of an enzyme-modified carbon paste electrode for determining inhibitors of lipoxygenase

In this study, a 15-lipoxygenase-modified carbon paste electrode (15-LOX-MCPE) was developed in connection with the help of voltammetry, which can be used as an assay system for screening drugs with inhibiting lipoxygenase (LOX) activity. The influence of different experimental conditions (LOX loading of carbon paste, pH, type of buffer system etc.) was investigated in order to optimise the biosensing device. The best composition of the biosensor is 30% paraffin oil, 68% graphite powder and 2% LOX. The optimised voltammetric measurement medium is S?rensen/NaOH (0.1M, pH 9.0) using linoleic acid as a substrate. Under these conditions the hydroperoxy linoleic acid is formed, which can be oxidised at a potential of +0.9 V versus Ag/AgCl/3M KCl. The applicability of the LOX biosensor as assay of lipoxygenase inhibitors was successfully tested with nordihydroguaiaretic acid, zileuton and fenleuton, which are well known inhibitors of LOX.     

Xanthine biosensor based on XO/AuNP/PtNP/MWCNT hybrid nanocomposite modified GCPE

A new xanthine (X) biosensors based on a hybrid nanocomposite containing multi-walled carbon nanotubes (MWCNT), Pt nanoparticles (PtNP) and gold nanoparticle (AuNP) was presented. X biosensor was fabricated by dropping AuNP/PtNP/MWCNT onto xanthine oxidase (XO) modified glassy carbon paste electrode (GCPE). Resulted XO/AuNP/PtNP/MWCNT/GCPE biosensor showed two linearity between 2.0 and 50 µM and 0.25 and 6.0 mM for X. RSD value was calculated as 2.46 (n = 5). Finally, the biosensor was applied to the X detection in synthetic serum samples and good recovery value was obtained.     

Microbial biosensor for detection of methyl parathion using screen printed carbon electrode and cyclic voltammetry

Whole cells of recombinant Escherichia coli were immobilized on the screen printed carbon electrode (SPCE) using glutaraldehyde. Recombinant E. coli was having high periplasmic expression of organophosphorus hydrolase enzyme, which hydrolyzes the methyl parathion into two products, p-nitrophenol and dimethyl thiophosphoric acid. Cells immobilized SPCE was studied under SEM. Cells immobilized SPCE was associated with cyclic voltammetry and cyclic voltammograms were recorded before and after hydrolysis of methyl parathion. Detection was calibrated based on the relationship between the changes in the current observed at +0.1 V potential, because of redox behavior of the hydrolyzed product p-nitrophenol. As concentration of methyl parathion was increased the oxidation current also increased. Only 20 μl volume of the sample was required for analysis. Detection range of biosensor was calibrated between 2 and 80 μM of methyl parathion from the linear range of calibration plot. A single immobilized SPCE was reused for 32 reactions with retention of 80% of its initial enzyme activity.     

Amperometric determination of choline released from rat submandibular gland acinar cells using a choline oxidase biosensor

A choline (CHO) biosensor based on the determination of H(2)O(2) generated at the electrode surface by the enzyme choline oxidase (CHOx) was developed. The biosensor consisted of CHOx retained onto a horseradish peroxidase (HRP) immobilized solid carbon paste electrode (sCPE). The HRPsCPE contained the molecule phenothiazine as redox mediator and CHOx was physically retained on the electrode surface using a dialysis membrane. Several parameters have been studied such as, mediator amount, influence of applied potential, etc. The CHO measurements were performed in 0.1 M phosphate buffer, pH 7.4. Amperometric detection of CHO was realized at an applied potential of 0.0 mV vs Ag/AgCl. The response is linear over the concentration range 5.0x10(-7)-7.0x10(-5) M, with a detection limit of 1.0x10(-7) M. This biosensor was used to detect choline released from phosphatidylcholine (PC) by phospholipase D (PLD) in isolated rat salivary gland cells stimulated by a purinergic agonist (ATP).     

Recombinant Microdochium nivale carbohydrate oxidase and its application in an amperometric glucose sensor

Biosensors containing recombinant carbohydrate oxidase from Microdochium nivale (rMnO) were developed by means of either chemically modified carbon paste or graphite electrode. 1-(N,N-dimethylamine)-4-(4-morpholine)benzene (AMB) and 1,1'-dimethylferrocene (DMFc) have been used as mediators. The biosensors showed a linear calibration graph up to 18 mM of glucose when operated at 0.04-0.36 V versus a saturated calomel electrode. Almost no change was detected in the sensitivity of the biosensors at pH 7.2-8.1. The biosensors responded to other aldoses in the D-configuration, however, maximal sensitivity of the biosensor was towards D-glucose. The biosensor did not response to polyhydroxylic compounds such as D-mannitol, D-sorbitol and inositol. The advantages of the biosensors based on rMnO in comparison to Aspergillus niger glucose oxidase is a wider linear range, low sensitivity to oxygen and (in some cases) broad specificity.     

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 electrochemical biosensor based on horseradish peroxidase immobilized on Ag-nanoparticles/poly(l-arginine) modified carbon paste electrode toward the determination of pyrogallol/hydroquinone

A novel electrochemical biosensor for the determination of pyrogallol (PG) and hydroquinone (HQ) has been constructed based on the poly l-arginine (poly(l-Arg))/carbon paste electrode (CPE) immobilized with horseradish peroxidase (HRP) and silver nanoparticles (AgNPs) through the silica sol–gel (SiSG) entrapment. The electrochemical properties of the biosensor were characterized by employing the electrochemical techniques. The proposed biosensor showed a high sensitivity and fast response toward the determination of PG and HQ around 0.18 V. Under the optimized conditions, the anodic peak current of PG and HQ was linear with the concentration range of 8 μM to 30 × 10^?5 M and 1–150 μM. The limit of detection (LOD) and limit of quantification (LOQ) were found to be 6.2 μM, 20 μM for PG and 0.57 μM, 1.92 μM for HQ respectively. The electrochemical impedance spectroscopy (EIS) studies have confirmed that the occurrence of electron transfer at HRP-SiSG/AgNPs/poly(l-Arg)/CPE was faster. Moreover the stability, reproducibility and repeatability of the biosensor were also studied. The proposed biosensor was successfully applied for the determination of PG and HQ in real samples and the results were found to be satisfactory.     

Development of a carbon nanotube paste electrode osmium polymer-mediated biosensor for determination of glucose in alcoholic beverages

This paper describes a new amperometric biosensor for glucose monitoring. The biosensor is based on the activity of glucose dehydrogenase (GDH) and diaphorase (DI) co-immobilized with NAD(+) into a carbon nanotube paste (CNTP) electrode modified with an osmium functionalized polymer. This mediator was demonstrated to shuttle the electron transfer between the immobilized diaphorase and the CNTP electrode, thus, showing a good electrocatalytic activity towards NADH oxidation at potentials around +0.2V versus Ag|AgCl, where interfering reactions are less prone to occur. The biosensor exhibits a detection limit of 10 micromol L(-1), linearity up to 8 x 10(-4) mol L(-1), a sensitivity of 13.4 microA cm(-2)mmol(-1)L(-1), a good reproducibility (R.S.D. 2.1%, n=6) and a stability of about 1 week when stored dry at 4 degrees C. Finally, the proposed biosensor was applied for the determination of glucose in different samples of sweet wine and validated with a commercial spectrophotometric enzymatic kit.     

Properties of modified amperometric biosensors based on methanol dehydrogenase and cells Methylobacterium nodulans

The properties of amperometric biosensors based on methanol dehydrogenase (MDH) Methylobacterium nodulans, cells, and the ferrocene-modified carbon paste electrode were investigated. It was shown that the addition of hydroxyapatite (HA) to a carbon paste increased the sensitivity and operating stability of MDH biosensors. The linear range of the electrode was 0.0135–0.5 and 0.032–1.5 mM for methanol and formaldehyde, respectively. The detection limit of methanol and formaldehyde was 4.5 and 11.0 μM, respectively. The loss of activity of the electrode within 10 days of storage in the presence of 2.0 mM KCN did not exceed 12%. Cyanide (10 mM) completely inhibited the sensor responses to formaldehyde (1.0 mM), which allowed for the selective determination of methanol in the presence of formaldehyde. The biosensor based on cells exhibited lower stability and sensitivity toward methanol and formaldehyde; the sensitivity coefficients were 980 and 21 nA/mM, respectively.