Pierre Bouvrette

Developments and applications of biosensors in food analysis

The food industry needs suitable analytical methods for process and quality control; that is, methods that are rapid, reliable, specific and cost-effective in their provision of information about physical and chemical characteristics of food. Apart from a few important analytes, such as sugars, alcohols, amino acids, flavours and sweeteners, food applications mainly focus on the determination of contaminants. However, very few biosensors play a prominent role in food processing or quality control. Considerable effort must be made to develop biosensors that are inexpensive, reliable, and robust enough to operate under realistic conditions.

Raman-based detection of bacteria using silver nanoparticles conjugated with antibodies

Surface enhanced Raman scattering (SERS) has been used to detect bacteria captured by polyclonal antibodies sorbed onto protein-A-modified silver nanoparticles. The selectivity and discrimination of the technique were assured by using a specific antibody to the model bacterium, Escherichia coli. As the SERS enhancement mechanism depends upon the metal surface proximity, 8 nm was considered as the optimum distance between the bacterium and the nanoparticle surface. Spectral reproducibility was verified using Principal Components Analysis to differentiate the clusters corresponding to the biomolecules and/or bacteria sorbed onto nanoparticles. Compared to the normal Raman spectrum, the SERS technique resulted in an intensity enhancement of over 20-fold.

Amperometric biosensor for diamine using diamine oxidase purified from porcine kidney

Diamine oxidase was purified over 2300-fold from porcine kidney to a specific activity of 1 U mg−1. The final preparation exhibited a single 103 kDa protein band and contained relatively large amounts of Asx and Glx acidic residues. When stored at −80°C, soluble enzyme retained its original catalytic activity for at least five months. The optimal pH and temperature of the enzyme immobilized by intramolecular cross-linking via glutaraldehyde 3ctivation and deposited onto a preactivated nylon membrane were 7.4 and 60°C with cadaverine as substrate. The apparent Km″ values (Michaelis-Menten constants) of immobilized diamine oxidase with histamine, putrescine, and cadaverine as substrates were estimated to be 0.27, 3.2, and 0.64 mm, respectively. Artificial mediators such as ferrocene derivatives, 2,6-dichlorophenolindophenol, potassium ferricyanide and 4-animodiphenylamine were not observed to facilitate electron transfer from the reduced enzyme to the electrode. The biosensor using the immobilized diamine oxidase and a platinum working electrode (poised at +700 mV vs AgAgCl for determination of hydrogen peroxide released from the enzymatic oxidation) was linear up to 6 mm histamine, cadaverine, or putrescine with a lower detection limit of 25 μm. Each analysis could be performed in 3 min including washing and time for the current to return to baseline. The enzyme membranes were stable at 5°C for at least two months and could be used for more than 60 repeated analyses without significant loss of sensitivity.

A coupled enzymatic assay for salicylate and acetylsalicylate using salicylate hydroxylase and tyrosinase

Abstract Salicylate hydroxylase was used together with tyrosinase in a coupled enzymatic assay for determining salicylate and acetylsalicylate. In the presence of NADH and dioxygen, salicylate hydroxylase catalyzed the hydroxylation and decarboxylation of salicylate to catechol, which was then oxidized by tyrosinase to o -quinone. When NADH was used in excess, the resulting o -quinone product was recycled to its catechol form since o -quinone can oxidize NADH to produce NAD + . Consequently, a cycle was established in which several NADH molecules were utilized by each catechol, which was easily followed by monitoring the rate of absorbance decrease at 340 nm. In comparison to its non-amplified counterpart, the recycling of catechol and o -quinone improved the detection limit of the spectrophotometric assay about ninety-fold. The method developed is a rapid, simple spectrophotometric assay with a linear response up to 1.4 μM salicylate and detection limit of 6.5 nM. The recycling assay was applied to determine salicylate in plasma and urine samples and the results obtained agreed well with the established Trinder method.

Derivatization of resin acids with a fluorescent label for cyclodextrin-modified electrophoretic separation

Abstract A derivatization reaction for resin acids (RAs), commonly found in untreated pulp mill effluents, was performed and evaluated in the separation and sensitive detection of the resin acids by cyclodextrin-modified capillary electrophoresis. The procedure involved the reaction of 4-bromomethyl-7-methoxycoumarin, a fluorescent label, with the carboxyl group of the resin acid in the presence of potassium carbonate to form an ester. Both MS data and HPLC analysis equipped with UV or scanning fluorescence revealed that the derivatization was free from side products and very quantitative. The derivatized esters were very stable and fluoresced optimally at 325 nm, excitation and 400 nm emission, an ideal condition for capillary electrophoresis equipped with He–Cd laser-induced fluorescence. Cyclodextrin-modified capillary electrophoresis using a mixture of negatively charged sulfobutylether-β-CD (SBCD) and neutral methyl-β-CD (MECD) was then optimized for separation and detection of the derivatized RAs. Separation at pH 4.5 and +15 kV applied potential using 42.5 mM sulfobutylether–β-CD and 12.5 mM methyl-β-CD in 50 mM sodium acetate has allowed the achievement of baseline separation of eight very closely related derivatized resin acids. With laser-induced fluorescence, the extrapolated concentration limit of detection (3σ) of the resin acid esters was about 10–20 μg/l, based on capillary electrophoresis analysis of a standard solution containing 500 μg/l of each derivative.

Separation of resin acids using cyclodextrin‐modified capillary electrophoresis

A cyclodextrin‐modified capillary electrophoretic method has been developed for the analysis of eleven common resin acids using a pH 4.5, 20 mM sodium acetate buffer containing 10% acetonitrile, 20 mM methyl‐β‐cyclodextrin (MECD) and 30 mM sulfobutylether‐β‐cyclodextrin (SBCD) as buffer modifiers. At pH below their pKa (< 5.7—6.4) the resin acids were virtually unionized and insoluble; however, they formed water‐soluble inclusion complexes with MECD (20 mM) or SBCD (30 mM) even at pH 4.5. The analytes were separated in 25 min and, with the exception of two pairs, 12‐ or 14‐chlorodehydroabietic/12,14‐dichlorodehydroabietic acid and dehydroabietic/palustric acid, the remaining resin acids were baseline‐separated. Analysis time was significantly shortened (< 12 min) at pH 9.25 using 30 mM SBCD and 20 mM MECD in 20 mM sodium borate. Resin acids were baseline‐separated with the exception of two pairs, pimaric/ sandaracopimaric acid and 12‐ or 14‐chlorodehydroabietic/abietic acid. The addition of 7.5% methanol to the running buffer resolved the abietic acid peak. Both HPLC and micellar capillary electrokinetic chromatography using 20 mM deoxycholic acid, 10% acetonitrile in 20 mM sodium borate, pH 9.25, failed to resolve the resin acids. The simple capillary electrophoretic method developed would be useful for the rapid separation and characterization of several important resin acids in pulp mill effluents and other contaminated samples.

Rapid detection of microorganisms with nanoparticles and electron microscopy

Rapid detection of microorganisms is highly desirable. A procedure has been developed based on interactions between gold nanoparticles and proteins of microorganisms (Escherichia coli, Rhodococcus rhodochrous, and Candida sp.) followed by scanning electron microscopy (SEM). The nanoparticle‐cell interaction was confirmed by ultraviolet resonance Raman spectroscopy (UVRS) in the SEM focus. Cell suspensions in a buffer were interacted with gold nanoparticles (<10 nm in diameter) prepared from tetrachloroauric acid and sodium borohydride. Possible interference of elevated salt concentrations was eliminated by dialysis in deionized water. Small (10 μL) aliquots of cell‐nanoparticle suspensions were dried on a silicon wafer and photographed under an SEM. Characteristic bacterial or yeast cell images in the micrographs indicated the actual presence of microorganisms in the suspension examined. This was further confirmed by UV resonance Raman spectroscopy. Microsc. Res. Tech., 2008.

Isolation, purification, and further characterization of an L-phenylalanine oxidase fromMorganella morganii

A l-amino acid oxidase was isolated, purified, and characterized fromMorganella morganii 53187, a bacterium formerly known asProteus morganii. The synthesis of the enzyme by this bacterial strain was growth-associated and decreased sharply when the culture just reached the stationary phase. Based on this finding, the preparation of spheroplast by lysozyme-ethylene-diaminetetra-acetic acid (EDTA) disruption was carried out using the cells harvested during the exponential growth phase. Among several detergents tested, at the detergent-to-protein ratio of 2.5, 3-[(3-cholamidopropyl)dimethylammonio]-l-propane-sulfonate (CHAPS) was very effective in solubilizing most of the enzyme attached to the membranes while still preserving the activity of the solubilized enzyme. The resulting enzyme solution was then purified by hydrophobic interaction chromatography, followed by ion exchange chromatography and gel permeation.The enzyme was purified 19-fold with an overall recovery yield of 12%, corresponding to a specific activity of 252.2 U/mg protein. The selectivity of the purified enzyme toward l-amino acids was pH-dependent. At pH 6.35, the enzyme was very specific to l-leucine, whereas the selectivity for l-phenylalanine could be improved at pH 7.4. The enzyme exhibited a wide optimum temperature range 35–43‡C and exhibited 1, l−dimethylferricinium reductase capability in the presence of l-phenylalanine.

Use of δ-(α-aminoadipoyl) chromogenic amides in screening for aminoadipoyl amidohydrolases

Abstract The synthesis of δ-(α-aminoadipoyl) aromatic amides and their use in screening for enzymes able to cleave δ-(α-aminoadipoyl) residues off the synthetic amides and cephalosporin C are described. A number of commercially available proteases and peptidases were not active with δ-(α-aminoadipoyl) chromogenic amides. Also, most tested microbial strains known to produce acylases did not hydrolyze these compounds. Only one microbial strain, Xanthomonas maltophila , had an appreciable activity toward the racemic form of chromogenic substrates. Activity measured in crude extracts from Xanthomonas cells indicated that this bacterium produces predominantly l -specific aminoadipoyl amidohydrolase and γ-glutamyl hydrolase. A low level of cephalosporin C and glutaryl-cephalosporin acylase activities was also found.