Dyer Narinesingh

Polypyrrole-hydrogel composites for the construction of clinically important biosensors

The present study reports on the use of p(2-hydroxyethyl methacrylate) (pHEMA) in which polypyrrole and various oxidoreductase enzymes were physically entrapped to function as a viable matrix for the construction of clinically important amperometric biosensors. Glucose oxidase, cholesterol oxidase and galactose oxidase biosensors were constructed. Electrode-supported hydrogel films were prepared by UV polymerization of the HEMA component (containing the dissolved enzyme) followed immediately by electrochemical polymerization (+0.7V vs. Ag/AgCl) of the pyrrole component within the interstitial spaces of the pre-formed hydrogel network. The optimized glucose oxidase biosensor displayed a wide linear glucose response range (5.0 x 10(-5) to 2.0 x 10(-2) M), a detection limit (3S(y/x)/sensitivity) of 25 microM and a response time of 35-40 s. The analytical recovery of glucose in serum samples ranged from 98 to 102% with mean coefficients of variation of 4.4% (within-day analyses) and 5.1% (day-to-day analyses). All three sensors displayed good stabilities when stored desiccated in the absence of buffer (>9 months).

Kosmotropes enhance the yield of antibody purified by affinity chromatography using immobilized bacterial immunoglobulin binding proteins.

Abstract The yield of antibody purified using affinity chromatography on immobilized Protein A or Protein G was increased up to 5‐fold (500%) by including kosmotropic salts in the binding buffer. The binding buffer is used to equilibrate the affinity column before applying a sample to the column and also to dilute the sample prior to loading onto the affinity column to optimize conditions for a maximal binding of antibodies to affinity gels. In this study, the kosmotropic salts that were effective in greatly increasing antibody binding to Protein A included both inorganic and organic salts of ammonium; sodium; or potassium sulfate, phosphate, polycarboxylates; for example, succinate, citrate, isocitrate, N‐(2‐hydroxyethylene diamine triacetate (HEDTA), ethylene diamine tetraacetate (EDTA), and ethylene glycol‐O,O′‐bis(2‐aminoethyl)‐N,N,N′N′‐tetra acetate(EGTA). On an equal‐molar basis, the greater the number of carboxylic groups within the polycarboxylate molecule, the greater the increase in the yield of the purified antibody that was observed. The data show that kosmotropes can be used as effective additives to enhance the binding of immunoglobulins to Protein A or Protein G gels with a resultant increase in the yield of the purified antibodies. Thus, it appears that strongly hydrated anions (citrate, sulfate, and phosphate) and weakly hydrated cations (ammonium, potassium) increase the yield of antibody purified on either Protein A or Protein G affinity gels.

Interferent Suppression Using a Novel Polypyrrole-Containing Hydrogel in Amperometric Enzyme Biosensors

Amperometric biosensors for three clinically important analytes; glucose, cholesterol and galactose were prepared using a novel polymer composite material consisting of a poly(hydroxyethylmethacrylate) [p(HEMA)] hydrogel intimately combined with polypyrrole (PPy), the appropriate enzyme and fabricated on platinum electrodes. These biosensors were evaluated for their sensitivity to two very common electrooxidizable interferents, ascorbic acid and L-cysteine. The composite polymer films showed effective suppression of these interferents in a serum matrix with a deviation of <5% of the biosensor response being observed at ascorbic acid and L-cysteine concentrations that were twice as high as the normal physiological levels found in serum (10 mg L−1 and 4 mg L−1 respectively). In contrast, biosensor films containing an external layer of peroxidase or Nafion were found to be less effective than the p(HEMA)/PPy composite films in screening these two interferents. The sieving properties of the crosslinked hydrogel and the anion exchange properties of the cationic polypyrrole together with its “dopant” anion effectively inhibit transport of these anion interferents to the electrode.

A screening method for trace mercury analysis using flow injection with urease inhibition and fluorescence detection

Abstract A flow-injection screening method based on urease inhibition is described for the determination of trace amounts of the heavy metal ions, mercury and silver. The method is based on the inhibitory effect that these ions have on the enzyme urease which catalyzes the conversion of urea to carbon dioxide and ammonia. The liberated ammonia is monitored fluorimetrically using o -phthalaldehyde as the fluorophore (λ ex = 340 nm, λ em = 455 nm). A detection limit of 2 ppb can be achieved. Comparison of this flow injection method with that of the standard cold vapour atomic absorption technique for mercury determination gave a standard deviation of 0.02 and a coefficient of variation of 2%. Also one-way ANOVA shows no significant difference between these two methods.

DESIGN AND CHARACTERIZATION OF A GALACTOSE BIOSENSOR USING A NOVEL POLYPYRROLE-HYDROGEL COMPOSITE MEMBRANE

ABSTRACT A rapid, two-step method for constructing galactose biosensors by entrapment of galactose oxidase within a polymeric composite has been developed. The composite material is formed as an interpenetrating network of polypyrrole grown within a UV cross-linked poly(2-hydroxyethyl methacrylate) [p(HEMA)] hydrogel. The optimized galactose biosensor exhibited a linear response range from 5.0 × 10−5 to 1.0 × 10−2 M and detection limit of 25 µM toward galactose. The response time of the biosensor was 70 s. The analytical recovery of galactose in serum samples ranged from 97 to 105% with mean coefficients of variation of 3.8% (within-day analyses) and 4.4% (day-to-day analyses). The biosensor was effective in screening up to twice the physiological levels of ascorbate, urate and acetaminophen interferents and retained 70% of initial enzyme activity after 9 months when stored desiccated in the absence of buffer at 4°C.

Cytochrome P4502D6 (CYP2D6) Bioelectrode for Fluoxetine

Abstract A bioelectrode system consisting of polyaniline (PAn)‐doped glassy carbon electrode (GCE) and cytochrome P4502D6 (CYP2D6) enzyme solution was used for the amperometric study of in vitro fluoxetine biotransformation. The PAn film was potentiostatically grown at +700 mV vs. Ag/AgCl (20°C) on a 0.071 cm2 GCE and used for cyclic voltammetric (CV) measurements in phosphate buffer solution (0.1 M, pH 7.5, 0.1 M KCl) of the enzyme. The response of the CYP2D6 bioelectrode to fluoxetine was consistent with uncompetitive substrate inhibition kinetics. Apparent Michaelis–Menten constant K′ m of the CYP2D6 electrode was 3.7 µmol/L, which is within the intra‐hepatic fluoxetine concentration between 2 and 7 µmol/L. Thus PAn‐mediated electrochemistry can be used to observe the monooxygenation reaction of CYP2D6. K′ m is the apparent Michaelis–Menten constant and K′ i is the apparent substrate inhibition constant.

COMPOSITE HYDROGELS CONTAINING POLYPYRROLE AS SUPPORT MEMBRANES FOR AMPEROMETRIC ENZYME BIOSENSORS

Conducting polymers and redox hydrogels are shown to be attractive materials for biocompatible electrodes in amperometric biosensors. We have combined electrically conducting polypyrrole (PPy) with crosslinked poly(2-hydroxyethylmethacrylate) (p-HEMA) to produce a novel composite hydrogel membrane. The high water content of these materials provides a biocompatible environment for the long-term immobilization of enzymes and a more favorable medium for the rapid movement of charge neutralizing ions. Electrode-supported composite films were prepared by UV polymerization of the hydrogel component (containing dissolved enzyme) followed immediately by electrochemical polymerization (+0.7V vs. Ag/AgCl) of the pyrrole component within the interstitial spaces of the pre-formed hydrogel network. Typical monomer compositions consisted of HEMA:TEGDA:pyrrole in an 85:10:05 vol%. (TEGDA = tetraethyleneglycol diacrylate). An optimized glucose biosensor displayed a wide linear response range of 5.0 × 10−5 to 2.0 × 10−2 M, a detection limit (3Sy/x/sensitivity) of 25 μM and a response time of 35–40 seconds. The analytical recovery of glucose in serum samples ranged from 98 to 102% with mean coefficients of variation of 4.4% (within-day analyses) and 5.1% (day-to-day analyses). The optimized cholesterol and galactose biosensors also displayed wide linear response ranges (5.0 × 10−4 – 1.5 × 10−2M and 1.0 × 10−4 – 1.0 × 10−2M, respectively) towards their respective substrates. All three biosensors retained > 70% of initial activity after 9 months when stored desiccated in the absence of buffer. An attractive feature with all the biosensors was their ability to effectively screen the endogenous interferents ascorbic acid, uric acid, L-cysteine and acetaminophen. This characteristic, coupled with the high biocompatibility of the polymeric hydrogel composites make these mate rials potential candidates for in-vivo biosensors.

Urease coupled to poly(vinyl alcohol) activated by 2-fluoro-1-methylpyridinium salt: preparation of a urease potentiometric electrode and application to the determination of urea in serum

Abstract A urease electrode is described for the determination of urea in serum samples. The electrode was prepared by chemically immobilizing urease to 2-fluoro-1-methylpyridinium toluene-4-sulphonate-activated poly(vinyl alcohol) and physically adsorbing the resulting conjugate directly on to the hydrophobic membrane of an ammonia gas-sensing electrode. Linear calibration was obtained over the range 10−4–10−1 M urea. The recovery varied between 97 and 102%. The within-day relative standard deviation (R.S.D.) was 1.2–2.7% and the day-to-day R.S.D. was 2.9–4.5%. There was only a 14% decrease in electrode activity over a period of 28 days. Up to 30 serum samples can be analysed per hour.

Immobilization of xanthine oxidase and its use in the quantitation of hypoxanthine in fish muscle tissue extracts using a flow injection method

Fish muscle extracts (Scomberomorus— brasiliensis- carite) were analyzed for their hypoxanthine content using a flow injection system incorporating an immobilized xanthine oxidase bioreactor. The xanthine oxidase was immobilized under mild conditions to a 2-fluoro-1-methylpyridinium Fractogel support. The uric acid produced from the oxidation of hypoxanthine by the immobilized xanthine oxidase at pH 7.0 and 35‡C was monitored at 290 ran. Hypoxanthine concentrations as low as 4.4 Μmol/L can be detected. Up to 30 samples per hour can be analyzed at a flow rate of 1 mL/min, using 150 ΜL sample volumes and a bioreactor dimension of 1.0 cm x 2.0 mm id. Recovery yields were between 92 and 99%. Both within day and between day precisions gave CVs < 5.00% (n = 30). Good correlation (r = 0.998) is obtained when 78 fish samples were analyzed for their hypoxanthine content both by this FI method and a reference HPLC method.

Flow injection analysis of lactose using covalently immobilized β-galactosidase, mutarotase, and glucose oxidase/peroxidase on a 2-fluoro-1-methylpyridinium salt-activated fractogel support

Milk samples were analyzed for their lactose content using flow injection analysis and incorporating immobilized beta-galactosidase or beta-galactosidase/mutarotase and glucose oxidase/peroxidase bioreactors. These enzymes were immobilized, under mild conditions, on to a 2-fluoro-1-methylpyridinium salt-activated Fractogel support. The use of a phosphate buffer (0.15 M) was found to facilitate the rapid mutarotation of alpha-D-glucose and hence could obviate the need for the more expensive mutarotase. The chromogenic agents of choice for monitoring the reaction were 3-methyl-2-benzothiazolinone hydrazone and 3-dimethylaminobenzoic acid. Linearity was observed over the concentration range 16-160 micrograms/ml using lactose standards (r = 0.996). Between 30 and 40 milk samples/h can be analyzed. Comparisons are made with existing HPLC and alkaline methylamine methods for a range of milk matrices. The FIA method consistently gives the lowest standard deviations and coefficient of variation for the various milk matrices analyzed.