Triglyceride determination with use of an enzyme thermistor

The application of a split-flow type of enzyme thermistor for determining triglycerides is described. The device measures the protonation heat produced when a triglyceride is passed through a column containing triacyl glycerol lipase covalently bound to controlled-pore glass beads. The time required for a determination is less than 5 min, and the calibration graph for a triolein standard is linear up to 5 mM. The procedure is used for determining triglycerides in human blood sera and is compared with a conventional spectrophotometric method.     

Flow-injection determination of sugars with immobilized enzyme reactions and chemiluminescence detection

Glucose is determined by reaction with gluocose oxidase to produce hydrogen peroxide which is quantified via a chemiluminescence reaction with luminol. Sucrose, maltose, lactose and fructose are determined by enzymatic conversion to glucose (using invertase, amyloglucosidase, lactase. and glucose isomerase, respectively) and subsequent determination of the glucose, All enzymes are immobilized on controlled-pore glass and contained in flow-through reactors. For glucose, sucrose, and maltose the linear log-log working range 0.2 μM-1 mM, with a detection limit of 0.1 μM; for lactose and fructose the linear working range is 3 μM-1 mM with a detection limit of 1 μM. Assay time is 2 min.     

Flow-injection method for the determination of serine using immobilized enzyme

A flow-injection method for the determination of serine using a mini-column containing immobilized serine dehydratase isolated and purified from rat liver is described. Ammonia produced from the enzymatic reaction is coupled with hypochlorite and phenol in an alkaline medium yields a blue product due to the indophenol anion formation, which is the basis of a spectrophotometric detection at 640 nm. The limit of detection (2×blank noise) is 0.01 mM with a sample throughput of 25 h⁻¹. Calibration graph is linear in the range 0.2–1.0 mM, with relative standard deviation 0.6–1.0%.     

Amperometric determination of cholinesterase with use of an immobilized enzyme electrode

A choline-sensitive electrode consisting of an immobilized choline oxidase layer and an oxygen electrode is described. Cholinesterase (0.5–60 I.U. l^-1) is measured by addition of acetylcholine, and detection of the choline produced. The precision is 3%, and the electrode is stable for more than 2 weeks (140 assays).     

Flow-injection determination of malate with immobilized malate dehydrogenase

Malate dehydrogenase (MDH) is immobilized chemically on controlled-pore glass and used on-line in a glass minicolumn (25×2.5 mm i.d.). Malate solution passes through the minicolumn of immobilized MDH and the NADH formed is monitored spectrophotometrically from 9 × 10-^?4 down to 7 × 10^?6 M (36 ng in 40 μl) at 50 samples h^?1.     

Flow-injection determination of l-tyrosine in serum with immobilized tyrosinase

A flow-injection method for the determination of the serum l-tyrosine is described. The method involves the conversion of tyrosine into dopaquinone by reaction of tyrosinase, followed by derivation of the dopaquinone with fluorogenic agent 1,2-diphenylethylenediamine. Serum was deproteinized with tungstic acid. Sample solution was injected into a reactor (50 x 4 mm i.d.) packed with glass beads on which tyrosinase was immobilized. The fluorescence was detected at 480 nm (excitation at 350 nm). The calibration graph was linear for 5 x 10(-7)-2 x 10(-4)Ml-tyrosine; the detection limit was 2 x 10(-7)M.     

Flow-injection amperometric determination of oxalate with immobilized oxalate oxidase

Oxalate is immobilized on controlled-pore glass and is used on-line in a glass minicolumn (2.5×25 mm). The hydrogen peroxide formed is detected amperometrically. Oxalate (6×10^?6?9×10^?4 M) is determined in a flowing stream of pH 3.5 citrate (or succinate) buffer. As little as 20 ng (in 40 μl; 5.7×10^?6 M) of oxalate can be detected. Copper inhibition can be removed either by adding EDTA to the carrier stream or incorporating a chelating-resin minicolumn into the flow system prior to the enzyme column.     

Flow-injection spectrophotometric determination of acetyl-coenzyme A with immobilized phosphotransacetylase

Phosphotransacetylase (PTA) is immobilized on AF-Tresyl TOYOPEARL 650 gel and used on-line in a stainless-steel column (10 × 4 mm i.d.). CoA-SH liberated enzymatically from acetyl-CoA is reacted with Ellman's reagent [5,5′-dithiobis(2-nitrobenzoic acid)] in the carrier stream. The calibration graph for acetyl-CoA is linear from 4 × 10^?6 to 4 × 10^?4 M and the detection limits is 8 × 10^?7 M. The immobilized enzyme can be employed for over 4 months without any significant decrease in activity. The enzyme retains its activity in methanol even though the initial rate of reaction is decreased.     

Flow-injection determination of branched-chain L-amino acids with immobilized leucine dehydrogenase

L-Leucine, L-isoleucine and L-valine are determined by passage through a column of the enzyme immobilized on polystyrene beads, at pH 11.0. The NADH produced is monitored fluorimetrically. The detection limit is 0.1 nmol of L-leucine.     

Flow-injection system for the fluorimetric determination of fructose with an immobilized mannitol dehydrogenase reactor

Immobilized mannitol dehydrogenase is used for the determination of D-fructose in a flow-injection system. The enzyme is immobilized on poly(vinyl alcohol) beads. The oxidation of NADH occurs simultaneously and the disappearance of NADH is measured fluorimetrically. The response is linearly related to fructose concentration in the range 6–600 μM; 30 samples per hour can be analysed. The immobilized enzyme retains over 80% of its initial activity after repetitive use for 2 months.     

Flow-injection determination of l-tyrosine in serum with an immobilized tyrosinase reactor and fluorescence detection

Tyrosinase is immobilized on controlled-pore glass beads and packed into a stainless-steel column (5 cm × 4 mm i.d.). Serum is deproteinized with tungstate and sulphuric acid. The carrier stream is 0.3 M phosphate buffer (pH 7.2), and is mixed with 5 M potassium hydroxide after the enzyme reactor. The fluorescent dihydroxyindole formed is detected at 490 nm (excitation at 375 nm). The calibration graph is linear for 1 × 10^?7 ?1 × 10^?4 M tyrosine; the detection limit is 2 × 10^?8 M.     

Flow-injection determination of paraoxon by inhibition of immobilized acetylcholinesterase

Two flow-injection methods (continuous-flow and stopped-flow) are proposed for the determination of paraoxon, applying the dual-injection technique and spectrophotometric detection. They are based on the inhibition of the acetylcholinesterase-catalysed hydrolysis of α-naphthyl acetate and subsequent reaction of the α-naphthol produced with p-nitrobenzenediazonium fluoroborate. For the continuous-flow system the calibration graph was linear from 5 × 10^?7 to 1.5 × 10^?5 M, the relative standard deviation (r.s.d.) (n=6) for an 8 × 10^?6 M standard was 1.4%, the limit of detection (3σ) was 4 × 10^?7 M and the sample throughput was ca. 60 h^?1. For the stopped-flow system the linear range was from 1 × 10^?8 to 4 × 10^?7 M, the r.s.d. for a 2.5 × 10^?7 M standard was 0.9%, the limit of detection was 8 × 10^?9 M and the sample throughput was 30 h^?1.     

Flow-injection analysis for l-glutamate using immobilized l-glutamate oxidase: comparison of an enzyme reactor and enzyme electrode

An enzyme electrode and enzyme based on immobilized l-glutamate oxidase are used for the determination of l-glutamate in a flow-injection system. The hydrogen peroxide produced is monitored amperometrically. The enzyme reactor system surpasses the enzyme electrode system with regard to sensitivity and analytical speed. For both systems, the peak current is linearly related to the l-glutamate concentration in the range 5 × 10^?6-1 × 10^?3 M. l-Glutamate in seasoning can be determined very selectively with < 0.7% r.s.d.     

Flow-injection determination of starch and total carbohydrate with an immobilized glucoamylase reactor and pulsed amperometric detection

Starch and total carbohydrate are determined by using a flow-injection system comprised of an immobilized glucoamylase reactor followed by pulsed amperometric detection of the glucose produced. Glucoamylase, immobilized onto porous silica and packed into a short stainless steel column, is capable of nearly quantitative (98%) conversion of the starch to glucose. The sensitivity of pulsed amperoemtric detection for soluble starch is increased 26-fold by first passing the starch through the immobilized glucoamylase reactor. This system is successfully used to determine total carbohydrate in beer samples. The method is simple, rapid and sensitive for starch.     

Flow-injection determination of adenosine and inosine in blood plasma with immobilized enzyme columns connected in series and fluorimetric detection

A rapid and sensitive flow-injection method is described for the enzymatic determination of adenosine and inosine in human blood plama. Teflon columns prepared by packing adenosine deaminase. purine nucleoside phosphorylase, xanthine oxidase, uricase and horseradish peroxidase immobilized chemically on controlled-pore glass beads are connected in series in that order in the flow line. Hydrogen peroxide formed in the enzymatic conversion of adenosine and inosine is measured fluorimetrically after reaction with 3-(p-hydroxyphenyl)propionic acid. Linear calibrations were obtained for 0.5–500 pmol of adenosine or inosine in hte 20 μl sample injected. Necessary deproteination routines are outlined.     

Flow-injection determination of 3-hydroxybutyrate in serum with an immobilized 3-hydroxybutyrate dehydrogenase reactor and fluorescence detection

A flow-injection system with an immobilized enzyme reactor is proposed for the determination of 3-hydroxybutyrate. 3-Hydroxybutyrate dehydrogenase is immobilized on aminated poly(vinyl alcohol) beads and packed into a stainless-steel column (4 cm x 4 mm I.D.). Serum is diluted and filtered. Sample solution (20 mul) is injected into the carrier stream [4mM NAD(+) in glycine buffer (pH 9.3)]. The NADH formed is detected at 465 nm (excitation at 340 nm). The calibration graph is linear for 0.7-500muM 3-hydroxybutyrate; the detection limit is 0.5muM.     

Flow-injection determination of thyroxine using immobilized enzyme with tris(2,2'-bipyridyl)ruthenium(III) chemiluminescence detection.

A flow-injection method is reported for the determination of thyroxine based on its enhancement effect on the tris(2,2'-bipyridyl)ruthenium(III) chemiluminescence reaction in the presence of NADH using immobilized alcohol dehydrogenase purified from baker yeast. The limit of detection (3 sigma blank) was 5.0 x 10(-8) M with a sample throughput of 80 h(-1). The calibration graph was linear over the range 0.5 - 10 x 10(-7) M (r2= 0.9988) with the relative standard deviation in the range 1.4 - 3.5% (n = 4). The effect of common excipients used in pharmaceutical preparations, some organic compounds and metal ions was studied. The method was applied to pharmaceutical thyroxine tablets, and the obtained results were not significantly different from the amount quoted.     

Flow-injection determination of 3-hdroxybutyrate in serum with an immobilized 3-hydroxybutyrate dehydrogenase reactor and chemiluminescence detection

A flow-injection system for the determination of 3-hydroxybutyrate in serum is described. 3-Hydroxybutyrate dehydrogenase is immobilized on poly(vinyl alcohol) beads and incorporated in a flow-injection system. 1-Methoxy-5-methylphenazinium methylsulphate reacts with enzymatically generated NADH to give H₂O₂, which is detected chemiluminometrically with the reaction of luminol and hexacynoferrate(III). Serum is diluted and filtered through an ultrafiltration membrane. The system responds linearly to injected samples (80 μl) in the concentration range 0.5–300 μM; the detection limit is 0.1 μM. The within-day relative standard deviation (n = 90) for 58 μM 3-hydroxybutyrate in serum is 0.8%. The maximum throughout is 20 samples per hour. The immobilized enzyme is stable for at least 1 month.     

Flow-injection determination of L-histidine with an immobilized histidine oxidase from Brevibacillus borstelensis KAIT-B-022 and chemiluminescence detection.

A chemiluminometric flow injection analytical system for the quantitation of L-histidine is described. Histidine oxidase (EC 1.4.3.-) from Brevibacillus borstelensis KAIT-B-022 was immobilized on tresylated poly(vinyl alcohol) beads and packed into a stainless-steel column. The hydrogen peroxide produced was detected chemiluminometrically by a flowthrough sensor containing immobilized peroxidase (EC 1.1 1.1.7). The maximum sample throughput was 10 h(-1). The calibration graph was linear from 0.05 to 5 mM; the detection limit (signal to noise ratio = 3) was 0.01 mM. The activity of immobilized histidine oxidase reduced to 65% of the initial value after 350 injections. The system was applied to the determination of L-histidine in fish meat, such as salmon, tunny, bonito, and mackerel.     

Application of an enzyme thermistor for the determination of glucose in complex fermentation media

An enzyme thermistor was used for on-line glucose determination during cultivation of Cephalosporium acremonium for a period of 160 h. The complex medium consisted of 100 g 1^?1 peanut meal. Automatic control and data registration were achieved by interfacing a process computer with the thermistor unit. Problem caused by long-term application are discussed. On-line and off-line data obtained with the enzyme thermistor are compared with the results obtained with a commercial glucose analyzer. The results of these experiments show that an enzyme thermistor can be used to determine glucose concentration in complex fermentation media under real cultivation conditions for about 60 h.