University of North Florida
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Stuart Chalk, Ph.D.
Department of Chemistry
University of North Florida
Phone: 1-904-620-1938
Fax: 1-904-620-3535
Email: schalk@unf.edu
Website: @unf

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Applied Biochemistry and Biotechnology

  • Publisher: Springer
  • FAD Code: ABAB
  • CODEN: ABIBDL
  • ISSN: 0273-2289
  • Abbreviation: Appl. Biochem. Biotechnol.
  • DOI Prefix: 10.1385/ABAB
  • Language: English
  • Comments: Fulltext from 1976 V1

Citations 11

"A Sequential Enzymatic Microreactor System For Ethanol Detection Of Gasohol Mixtures"
Appl. Biochem. Biotechnol. 2005 Volume 121, Issue 1-3 Pages 361-371
Eliana M. Alhadeff; Andréa M. Salgado; Nei Pereira; Belkis Valdman

Abstract: A sequential enzymatic double microreactor system with dilution line was developed for quantifying ethanol from gasohol mixtures, using a colorimetric detection method, as a new proposal to the single µreactor system used in previous work. Alcohol oxidase (AOD) and horseradish peroxidase (HRP) immobilized on glass beads, one in each microreactor, were used with phenol and 4-aminophenazone and the red-colored product was detected with a spectrophotometer at 555 nm. Good results were obtained with the immobilization technique used for both AOD and HRP enzymes, with best retention efficiencies of 95.3 ± 2.3% and 63.2 ± 7.0%, respectively. The two microreactors were used to analyze extracted ethanol from gasohol blends in the range 1-30% v/v (10.0-238.9 g ethanol/L), with and without an on-line dilution sampling line. A calibration curve was obtained in the range 0.0034-0.087 g ethanol/L working with the on-line dilution integrated to the biosensor-FIA system proposed. The diluted sample concentrations were also determined by gas chromatography (GC) and high-pressure liquid chromatography (HPLC) methods and the results compared with the proposed sequential system measurements. The effect of the number of analysis performed with the same system was also investigated. Copyright © 2005 by Humana Press Inc. All rights of any nature whatsoever reserved.

"Noncompetitive Enzyme Immunoassay For α-Fetoprotein Using Flow Injection Chemiluminescence"
Appl. Biochem. Biotechnol. 2004 Volume 117, Issue 2 Pages 93-102
Lin, Jiehua; Yan, Feng; Ju, Huangxian

Abstract: A novel, direct noncompetitive flow injection enzyme immunoassay for ?-fetoprotein (AFP) was developed by enhanced chemiluminescence detection. The method was based on off-line incubation of AFP and horseradish peroxidase (HRP)-labeled anti-AFP, and then trapping of the unbound enzyme conjugate by an immunoaffinity column filled with AFP-modified Sepharose. The immunocomplex formed in incubation passed through the column and then was directly detected by a post-column chemiluminescence technique. The optimal conditions for the immunoassay procedure and chemiluminescence detection were established. At a 1:10 dilution of enzyme conjugate solution, the linear range for chemiluminescence detection of AFP was from 2.0 to 75 ng/mL with a correlation coefficient of 0.993 and a coefficient of variation of 2.67% at 30 ng/mL. The detection limit was 0.5 ng/mL. This method was flexible, sensitive, and rapid. The immunoaffinity column of 200 µL could be repeatedly used 100 times without a single decrease. The whole assay time including the preincubation step was only 30 min for one sample.

"Immobilized Enzyme Studies In A Microscale Bioreactor"
Appl. Biochem. Biotechnol. 2004 Volume 113, Issue 1-3 Pages 261-272
Jones, Francis; Forrest, Scott; Palmer, Jim; Lu, Zonghuan; Elmore, John; Elmore, Bill B.

Abstract: Novel microreactors with immobilized enzymes were fabricated using both silicon and polymer-based microfabrication techniques. The effectiveness of these reactors was examined along with their behavior over time. Urease enzyme was successfully incorporated into microchannels of a polymeric matrix of polydimethylsiloxane and through layer-bylayer self-assembly techniques onto silicon. The fabricated microchannels had cross-sectional dimensions ranging from tens to hundreds of micrometers in width and height. The experimental results for continuous-flow microreactors are reported for the conversion of urea to ammonia by urease enzyme. Urea conversions of >90% were observed.

"Development And Application Of An Integrated System For Monitoring Ethanol Content Of Fuels"
Appl. Biochem. Biotechnol. 2004 Volume 113, Issue 1-3 Pages 125-136
Alhadeff, Eliana M.; Salgado, Andrea M.; Pereira, Nei; Valdman, Belkis

Abstract: An automated flow injection analysis (FIA) system for quantifying ethanol was developed using alcohol oxidase, horseradish peroxidase, 4-amino-phenazone, and phenol. A colorimetric detection method was developed using two different methods of analysis, with free and immobilized enzymes. The system with free enzymes permitted analysis of standard ethanol solution in a range of 0.05-1.0 g of ethanol/L without external dilution, a sampling frequency of 15 analyzes/h, and relative SD of 3.5%. A new system was designed consisting of a microreactor with a 0.91 mL internal volume filled with alcohol oxidase immobilized on glass beads and an addition of free peroxidase, adapted in an FIA line, for continued reuse. This integrated biosensor-FIA system is being used for quality control of biofuels, gasohol, and hydrated ethanol. The FIA system integrated with the microreactor showed a calibration curve in the range of 0.05-1.5 g of ethanol/L, and good results were obtained compared with the ethanol content measured by high-performance liquid chromatography and gas chromatography standard methods.

"Development Of Novel Microscale System As Immobilized Enzyme Bioreactor"
Appl. Biochem. Biotechnol. 2002 Volume 98, Issue 1-9 Pages 627-640
Jones, Francis; Lu, Zonghuan; Elmore, Bill B.

Abstract: This study involves a novel method for immobilized enzyme catalysis. The focus of the work was to design and construct a microscale bioreactor using microfabrication techniques traditionally employed within the semiconductor industry. Enzymes have been immobilized on the microreactor walls by incorporating them directly into the wall material. Fabricated microchannels have cross-sectional dimensions on the order of hundreds of micrometers, constructed using polydimethylsiloxane cast on silicon/ SU-8 molds. The resulting ratio of high surface area to volume creates an efficient, continuous-flow reaction system. Transverse features also containing enzymes were molded directly into the channels.

"Quaternized Wood As Sorbent For Hexavalent Chromium"
Appl. Biochem. Biotechnol. 2001 Volume 90, Issue 1 Pages 75-87
Low, Kun-She; Lee, Chnoong-Kheng; Lee, Chun-Yuan

Abstract: The potential of quaternized wood (QW) chips in removing hexavalent chromium from synthetic solution and chrome waste under both batch and continuous-flow conditions was investigated. Sorption was found to be dependent on pH, metal concentration, and temperature. QW chips provide higher sorption capacity and wider pH range compared with untreated wood chips. The equilibrium data could be fitted into the Langmuir isotherm model, and maximum sorption capacities were calculated to be 27.03 and 25.77 mg/g in synthetic chromate solution and chrome waste, respectively. The presence of sulfate in high concentration appeared to suppress the uptake of chromium by QW chips. Column studies showed that bed depth influenced the breakthrough time greatly whereas flow rate of influent had little effect on its sorption on the column.
Activated carbon Adsorption

"Amperometric Biosensors Based On Microflow Injection System"
Appl. Biochem. Biotechnol. 2000 Volume 89, Issue 2-3 Pages 217-230
Eshkenazi, Inna; Sacks, Vered; Neufeld, Tova; Rishpon, Judith

Abstract: Novel electrochemical cells based on a microflow system combined with amperometric enzyme electrodes were developed and served for quantitative determination of various compounds, such as organophosphates and lactose. The resulting biosensors are selective and efficient owing to immobilization of the sensing elements on the electrodes. The sensors are easy to operate, and the procedures are rapid, accurate, reproducible, and inexpensive, requiring neither special skills and training nor complicated instrumentation. The use of a microflow cell ensures the continuous flux of a new substrate, thus preventing the accumulation or adsorption of products to the electrode. Miniaturization of the sensor has two main advantages: (1) it is easy to carry and therefore can be used outdoors as well, and (2) it allows working with low volumes of compounds and reagents, which is highly important when dealing with hazardous compounds.

"Biosensor For Determination Of Glucose And Sucrose In Fruit Juices By Flow Injection Analysis"
Appl. Biochem. Biotechnol. 2000 Volume 89, Issue 2-3 Pages 171-181
Guémas, Yann; Boujtita, Mohamed; El Murr, Nabil

Abstract: Glucose and sucrose were measured with an amperometric method by using the flow injection analysis technique. A carbon paste electrode with a renewable surface containing glucose oxidase, horseradish peroxidase, and ferrocene was used in combination with the soluble enzymes invertase and mutarotase. The Effect of invertase, mutarotase, and ascorbic acid on the electrode response was examined. Glucose and sucrose concentrations were determined with <3% errors. The proposed method for glucose and sucrose measurements was validated in real samples of fruit juices. The results were also compared with those obtained with the ultraviolet method.
Glucose

"Fiberoptic Biosensors Based On Chemiluminescent Reactions"
Appl. Biochem. Biotechnol. 2000 Volume 89, Issue 2-3 Pages 107-115
Marquette, Christophe A.; Degiuli, Agn&eagrave;s; Blum, Lo&iuml;c J.

Abstract: The chemiluminescence of luminol in the presence of H2O2 has been exploited to develop fiberoptic biosensors associated with flow injection analysis systems. A chlorophenol sensor was developed based on the ability of certain halophenols to enhance the peroxidase-catalyzed luminol chemiluminescence. Horseradish peroxidase immobilized on a collagen membrane was used. Ten chlorophenols have been tested with this chemiluminescent-based sensor. The lower detection limit was obtained with 4-chloro-3-methylphenol and was equal to 0.01 µM. Electrochemiluminescent-based fiberoptic biosensors for glucose and lactate were also developed using glucose oxidase or lactate oxidase immobilized on polyamide membranes. In the presence of oxidase-generated H2O2, the light emission was triggered electrochemically by means of a glassy carbon electrode polarized at +425 mV vs a platinum pseudo-reference electrode. The detection limits for glucose and lactate were 150 and 60 pmol, respectively, and the dynamic ranges were linear from 150 Fmol to 600 nmol and from 60 pmol to 60 nmol, respectively.

"Direct And Mediated Electron Transfer Catalyzed By Anionic Tobacco Peroxidase - Effect Of Calcium Ions"
Appl. Biochem. Biotechnol. 2000 Volume 88, Issue 1-3 Pages 321-333
Munteanu, Florentina-Daniela; Gorton, Lo; Lindgren, Annika; Ruzgas, Tautgirdas; Emn&eacute;us, Jenny; Cs&ouml;regi, Elisabeth; Gazaryan, Irina G; Ouporov, Igor V; Mareeva, Elena A; Lagrimini, L Mark

Abstract: The properties of anionic tobacco peroxidase (TOP) adsorbed on graphite electrode have been studied in direct and mediated electron transfer in a wall-jet flow injection system. The percentage of tobacco peroxidase molecules active in direct electron transfer is about 83%, which is higher than that for horseradish peroxidase (40-50%). This observation is explained in terms of the lower degree of glycosylation of TOP compared with horseradish peroxidase and, therefore, a reduced interference from the oligosaccharide chains with direct electron transfer. Calcium ions cause an 11% drop in the reaction rate constant toward hydrogen peroxide. The detection limit of calcium chloride has been estimated as 5 mM. The results obtained by means of bioelectrochemistry, stopped-flow kinetics, and structural modeling provide evidence for the interaction between calcium cations and negatively charged residues at the distal domain (Glu-141, heme propionates, Asp-79, Asp-80) blocking the active site. The observation that both soluble and immobilized enzyme undergo conformational changes resulting in the blockade of the active site indicates that the immobilized enzyme preserves conformational flexibility. An even stronger suppressing effect of calcium ions on the rate constant for mediated electron transfer was observed. Ln the case of direct electron transfer, this could mean that there is no direct contact between the electrode and the active site of TOP. The electrons are shuttled from the active site to the surface of the electrode through electron transfer pathways in the protein globule that are sensitive to protein conformational changes.

"Kinetic Studies And Analytical Application Of Cholesterol Oxidase And Peroxidase Immobilized To Synthetic Polymer"
Appl. Biochem. Biotechnol. 2000 Volume 87, Issue 2 Pages 141-151
Yotova, Lyubov K.; Ivanov, Ivan P.

Abstract: A method for individual and simultaneous covalent immobilization of cholesterol oxidase and peroxidase to copolymer of acrylonitrile with acrylamide is described. The effect of immobilization on the catalytic properties of the covalently bound enzymes was studied. The immobilized enzymes showed no change in pH optima and an increase in temperature optima, activation energy, and K-m, compared to data received from experiments with soluble enzymes. A small glass column packed with immobilized multienzyme complex was used to develop a method for manual determination of cholesterol in foodstuffs (e.g., in mayonnaise Olinease). The method was characterized by high analytical precision (coefficient of variation = 2.67%). The results show high correlation with those obtained by the Kageyama method (r = 0.986). The method is economical (the enzyme-carrier conjugate may be used more than 300 times), precise, easy to perform, and less time-consuming than the manual methods utilizing soluble enzymes. The established manual method can be proposed for cholesterol determination in foodstuffs.