Continuous-flow Assay Research Articles

Influence of flow regime on the concentration of cyanide producing anaerobic process inhibition

A study was conducted to analyze the effects of cyanide on methanogenic bacterial populations in both continuous and batch reactors. The appropriate toxicity assays were divided into two parts, i.e. 1) batch assays, and 2) continuous flow assays, using a 6.5-litre up-stream anaerobic sludge blanket reactor (UASB). A synthetic substrate was used as feed for both assays using 99% sucrose as a carbon source, and potassium cyanide (KCN) as a free cyanide source. In the first part, the treatments with the lowest and highest percentages of Specific Methanogenic Activity (SMA %) were 3 and 10 mg CN − /l. A 0.96 mg/l concentration of cyanide caused methanogenic activity inhibition by 50% during the exposure stage. During the recovery stage, Methanogenic Activity of treatments had a considerable increase compared to the results from direct cyanide exposure, except for treatments of 7 and 10 mg CN − /l. The concentration of cyanide that inhibits Specific Methanogenic Activity by 50% was 6.1 mg CN − /l during this stage. In the second part, the reactor was continuously fed with a synthetic substrate (COD≈ 1500 mg/l). After the anaerobic sludge became acclimated, cyanide concentrations from 0.5 and 130 mg CN − /l were progressively added. The hydraulic retention time remained constant at 12 hours throughout the entire experiment. Results indicated that while partial inhibition occurred for concentrations of 3, 20 and 100 mg CN − /l, severe inhibition occurred for 130 mg CN − /l.

A methyl-accepting protein involved in multiple-sugar chemotaxis by Cellulomonas gelida.

Tethered-cell and capillary assays indicated that L-methionine is required by Cellulomonas gelida for its normal cell motility pattern and chemotaxis and that S-adenosylmethionine is involved in sugar chemotaxis by this cellulolytic bacterium. In addition, in vivo methylation assays showed that several proteins were methylated in the absence of protein synthesis. The incorporated methyl groups were alkali sensitive. Of special interest was the observation that the methylation level of a 51,000-Mr protein increased two- to fivefold upon addition of various sugar attractants and decreased after the removal of the attractants. The increase was less pronounced in mutants defective in sugar chemotaxis and appeared to be specifically involved with sugar chemotaxis. Furthermore, cell fractionation and in vitro methylation assays demonstrated that the 51,000-Mr protein is located in the cytoplasmic membrane. These results suggest that a specific methyl-accepting chemotaxis protein is involved in multiple-sugar chemotaxis by C gelida. During chemotaxis, the changes of methylesterase activity in C gelida cells were similar to those in Escherichia coli RP437 cells, as determined by a continuous-flow assay for methanol evolution. Thus, the mechanism of methyl-accepting chemotaxis protein-mediated chemotaxis of the gram-positive C. gelida appears to be similar to that of the gram-negative E. coli rather than to that of other gram-positive bacteria, such as Bacillus subtilis.

Continuous-flow assay with immobilized enzymes for determining of inorganic phosphate in serum

An automated method for the determination of inorganic phosphate based on flow-injection analysis and on the use of immobilized enzymes is reported. The method features a linear range between 0.1 and 20 mumol/L with a CV < 2.1% and 3.4% for the within-run and between-run studies, respectively, and a sampling throughput of 40 h-1. The sensitivity of the method makes a 1:250 dilution of the serum samples feasible, thus making undetectable the interferences from analytes commonly present in serum. The method shows an excellent correlation with conventional automated analyzers based on the same enzymatic reaction (Hitachi, r = 0.988) but with the catalyst in solution, and with the Kodak Ektachem method (r = 0.974) based on the use of dry reagents and formation of the phosphomolybdo heteropolyacid.

Continuous flow assay of ammonia in plasma using immobilized enzymes

A simple, convenient and rapid enzymatic method for the determination of ammonia in plasma based on the immobilization of glutamate dehydrogenase and on a continuous flow system is proposed. The fixed-time measuring principle allows shortening of the assay time without loss of sensitivity, so a sampling frequency of 40 h −1 can be achieved in a linear range of 5–500 μM with a coefficient of variation of less than 2%. The method correlates well with the results of the Kodak Ektachem analyzer and can also successfully be compared with other automated batch methods which it surpasses in rapidity, simplicity and lower cost of the assay.

Bioluminescence-based fibre-optic sensor with entrapped co-reactant: an approach for designing a self-contained biosensor

The possibility of designing a self-contained fibre-optic biosensor, i.e., that can be used without renewing reagents in the probe, was investigated. A probe specific for NADH involves immobilized bioluminescent enzymes which require the presence of two co-reactants [a flavin substrate (FMN) and an aldehyde] to catalyse light emission. The FMN was non-covalently immobilized in a synthetic film and was internally released in the vicinity of the bound enzymes, at the sensing tip of the bioprobe. Release of FMN was achieved from the two different matrices tested: a collagen film and a poly(vinyl alcohol) (PVA) network. Continuous-flow assays of NADH could be performed over a linear dynamic range from 10 pmol to 1 nmol. A PVA matrix appears to be a promising support for designing a self-contained biosensor, as 30–35 reliable measurements (R.S.D.=5%) could be achieved without a decrease in the sensor signal, compared with only 10–15 assays with a collagen film.

A continuous flow immunoassay for rapid and sensitive detection of small molecules

An immunosensor operating in continuous flow and capable of detecting low molecular weight antigens is described. The approach differs from previously described continuous flow assays by not requiring incubation steps or the introduction of additional reagents following the loading of the sample into the system. Detection of the antigen is rapid, occurring within 3 min in the system described. The assay is based on the binding of labeled antigen to an immobilized antibody, with subsequent displacement of the labeled antigen when antigen is present in the buffer flow. Signal detection occurs downstream of the antigen recognition event. In this study, the hapten 2,4-dinitrophenol DNP as (DNP)-lysine was used as model antigen. To generate a labeled antigen, DNP was coupled to the terminal amino group of insulin A chain (tetra S-sulfonate form) which provides two tyrosine residues for the introduction of an 125I-label (DNP-Ins- 125I) or three carboxyl groups for the attachment of three fluorescein residues (DNP-Ins-Fl). The radiolabeled antigen was used to establish assay conditions. Subsequently, fluorescein was substituted for the radioisotope label in order to develop an assay independent of the restrictions associated with isotopes. Using this flow immunoassay, we were able to detect DNP-lysine down to a detection limit of 143 nM (29 pmol/200 μl) using DNP-Ins- 125I or DNP-Ins-Fl as labeled antigen. The density of immobilized antibody and the flow rate were identified to be critical parameters for the sensitivity of the assay.

Nodule structure and nitrogenase activity ofCoriaria arborea in response to varying pO2

When excised root nodules ofCoriaria arborea are assayed for nitrogenase activity at various pO2 they show a broad optimum between 20 and 40 kPa O2, with some evidence for adaptation. Continuous flow assays of nodulated root systems of intact plants indicate that Coriaria shows an acetylene induced decline in nitrogenase activity. When root systems were subject to step changes in pO2 nitrogenase activity responded with a steep decline followed by a slower rise in activity both at lower and higher than ambient pO2. Thus Coriaria nodules are able to adapt rapidly to oxygen levels well above and well below ambient. Measurement of nodule diffusion resistance showed that the adaptation is accompanied by rapid increase in resistance at above ambient pO2 and decrease in resistance at below ambient pO2. Plants grown with root systems at pO2 from 5–40 kPa O2 did not differ in growth or nodulation. The anatomy of Coriaria nodules shows they have a dense periderm which encircles the nodule and also closely invests the infected zone. The periderm is both thicker and more heavily suberised in nodules grown at high pO2 than at low pO2. Vacuum infiltration of India ink indicates that oxygen diffusion is entirely through the lenticel and via a small gap adjacent to the stele.

The determination of plasma oxalate concentrations using an enzyme/bioluminescent assay. 2. Co-immobilisation of bioluminescent enzymes and studies of in vitro oxalogenesis

An inexpensive, continuous flow assay for the determination of oxalate in plasma is described. The assay is based on the bioluminescent determination of NADH, a product of the degradation of oxalate by oxalate decarboxylase and formate dehydrogenase, using bioluminescent enzymes immobilized on cyanogen bromide-activated sepharose. The detection limit of the assay is 0.8 μmol/l. Intra-batch CV values of 5.2 and 3.8% were obtained at oxalate concentrations of 18 and 60 μmol/l. Recovery of added oxalate averaged 100.7%. Plasma oxalate ranged from < 0.8 to2 μmol/l in 14 healthy subjects, and from 6 to 134 μmol/l in 125 patients with renal disease treated by continuous ambulatory peritoneal dialysis. Ascorbic and dehydroascorbic acid did not directly interfere in the assay. In vitro oxalogenesis was observed in blood from 12 healthy subjects, but only after samples had stood at room temperature for more than 6 h. No significant oxalate generation occurred in blood from 24 patients with impaired renal function, even after standing at room temperature for 24 h. Oxalate generation was inhibited by the addition of oxalate to plasma, but the addition of urea and creatinine was without effect.

Continuous-flow bioluminescent determination of ATP in platelets using firefly luciferase immobilized on epoxy methacrylate.

Firefly luciferase was immobilized on epoxy methacrylate beads and used for a continuous-flow assay of ATP extracted from platelets. The immobilized luciferase had a half-life of 3 days at 25 degrees C; there was a 25% recovery of luciferase activity upon immobilization, and ca 50 reactors were made from 1 mg of commercial enzyme. The sensitivity of the assay was 0.3 pmol of ATP, and the response was linear between 1 and 500 pmol of ATP. The ATP content of platelets obtained with the present method correlated well with those obtained using soluble luciferase.

Lactulose interferes in the alkaline picrate assay for creatinine.

Errors of more than 10 mg/L in measured serum creatinine concentrations were encountered for an azotemic patient who was given lactulose orally. The magnitude of the apparent error fluctuated with the dose of lactulose. Additions of lactulose to serum produced a linear increase in the creatinine measured by each of two automated methods that involve use of the alkaline picrate (Jaffé) reaction. A lactulose concentration of 100 g/L produced positive interferences of 30 and 65 mg/L in kinetic (Beckman Astra) and continuous-flow (Technicon SMAC) assays, respectively, but caused no problem in an enzymatic assay for creatinine. The results of creatinine assays must be interpreted with caution in patients treated with lactulose.

14] Continuous-flow assays with nylon tube-immobilized bioluminescent enzymes

Publisher Summary Nylon tube-immobilized bioluminescent enzymes make it possible to specifically assay NAD(P)H and bile acids at picomole levels. The precision of the method, as well as its correlation with other methods, such as radioimmunoassay, enzyme immunoassay, and high-performance liquid chromatography, is satisfactory. The adopted continuous-flow system is simple, requires only minor modifications of a commercial detector, and allows analyzing of about 20–30 samples per hour. Unlike Sepharose columns, nylon reactors present no problems with packing or disruption of the gel matrix, nor bacterial growth, which markedly enhances background light level. This, together with its handiness, makes the nylon tube a very suitable enzyme support or continuous-flow analysis, in spite of the relatively low activity recovery of immobilized enzymes. Up to 500–700 bile acid samples are analyzed with use of only a few milligrams of the enzymes, and therefore this bioluminescent method appears highly competitive with other methods, such as radioimmunoassay, enzyme immunoassay, high-performance liquid chromatography, and fluorometry where radioactive materials, separation steps, sample manipulation, or expensive equipment are needed. Potentially, a variety of other NAD(P)H-generating metabolites could be analyzed using the bioluminescent reactor coupled with a proper immobilized dehydrogenase.

Bioluminescent continuous-flow assay of adenosine 5'-triphosphate using firefly luciferase immobilized on nylon tubes.

Bioluminescent continuous-flow assay of adenosine 5'-triphosphate using firefly luciferase immobilized on nylon tubes.

Measurement of plasma oxalate in healthy subjects and in patients with chronic renal failure using immobilised oxalate oxidase

A continuous flow assay using immobilised oxalate oxidase was used to measure the level of oxalate in plasma ultrafiltrate obtained from healthy subjects and from patients with chronic renal failure. The levels of oxalate in plasma from normal subjects ranged from 1.3–3.1 μmol/l (mean 2.03; sd = 0.52) with fe0males showing a higher ( p < 0.05) level (mean 2.25 μmol/l) than males (mean 1.87 μmol/l). The mean oxalate/creatinine clearance ratio in fourteen healthy subjects was greater than unity, thus indicating a net tubular secretion of oxalate. At physiological pH, l-ascorbate was converted to oxalate in whole blood following venepuncture, in plasma and in plasma ultrafiltrate. Reduction of the spontaneous generation of oxalate in the samples prior to analysis was achieved by acidification and treatment with sodium nitrite. A linear correlation ( r = 0.92; p < 0.001) was found between plasma oxalate and plasma creatinine in patients with chronic renal failure.

Preincubation of serum aspartate aminotransferase with pyridoxal 5'-phosphate in the SMAC: comparison with revised DuPont aca method and recommended IFCC method.

The method for continuous-flow assay of aspartate aminotransferase with the Technicon SMAC was modified to include preincubation of the serum enzyme with pyridoxal 5'-phosphate, to be consistent with the recommendations of IFCC and the Standards Committee of AACC. Preliminary estimates of the imprecision of the modified method on SMAC gave day-to-day standard deviations of 5.3 U/L at mean of 48 U/L (n = 66) and 6.2 U/L at 155 U/L (n = 61). Added bilirubin, sodium pyruvate, ascorbic acid, and endogenous lipids did not interfere. Comparison of results for 50 samples by this method with those by the manual IFCC method gave y = 1.1113x - 0.3 U/L, Sy/x = 4.4 U/L, and r = 0.997. Similar data are presented for the revised AST method for the DuPont aca discrete analyzer. Clinical data show that AST activities increase by as much as 200% when the serum is preincubated with pyridoxal 5'-phosphate.

Deviations from Michaelis-Menten kinetics in continuous flow assays: Consideration of unireactant cases

Apparent deviations from Michaelis-Menten kinetics that may appear when flow assays are used are discussed. Simulated Lineweaver-Burk plots are presented which show distortions due to product inhibition, reaction reversal, and the absence of initial velocity conditions. By employing an integrated Michaelis-Menten equation the above complications can be properly handled, allowing the true V m and K m values to be determined. Enzyme reaction velocities determined by use of a flow technique are prone to the above-mentioned complications, but they can be readily analyzed using an integrated Michaelis-Menten equation.

Enzymatic vs. Jaffé (continuous-flow) assay of creatinine in serum.

Enzymatic vs. Jaffé (continuous-flow) assay of creatinine in serum.

Analytical micro-preparative electrophoresis: Quantitation of phosphoglucose isomerase isoenzymes

A simple micro-preparative electrophoresis apparatus is described using a thin vertical slab gel and continuous elution through a narrow horizontal slot. Phosphoglucose isomerase (PGI) isoenzymes in lysates of 0.2 to 100 μl of erythrocytes were separated using this system and their activities determined using an on-line, air-segmented, continuous flow assay. Ninety-five percent recovery of the separated isoenzymes after electrophoresis was achieved and the factors influencing recovery of PGI and hemoglobin were investigated. Thus, quantitative recovery has been achieved without any loss of resolution. Up to 29 successive separations of PGI isoenzymes have been carried out on a single gel.

Enzymic fluorometric continuous-flow assays for blood glucose, lactate, pyruvate, alanine, glycerol, and 3-hydroxybutyrate.

We describe enzymic fluorometric methods of automated analysis for glucose, lactate, pyruvate, 3-hydroxybutyrate, glycerol, and alanine in perchloric acid extracts of blood. Unmodified Technicon AutoAnalyzer II apparatus is used. The usual concentrations of all these metabolites can be measured in as little as 0.1 ml of blood from a fasting subject. Within-batch and between-batch coefficients of variation ranged from 0.4 to 4.4% for all metabolites except 3-hydroxybutyrate, for which CV's were higher for low concentrations. Analytical recovery of added metabolites ranged from 92 to 98%. Glucose, lactate, alanine, and 3-hydroxybutyrate are stable in perchloric acid extracts for at least 13 days at room temperature, and a year at -20 degrees C; pyruvate shows a 6--8% loss after 3 days and 52% by one year at -20 degrees C; glycerol concentrations were stable at -20 degrees C for at least 13 days. Blank fluorescence is found in perchloric acid extracts of blood, necessitating blank runs for pyruvate, 3-hydroxybutyrate, glycerol, and alanine. The systems are simple to use, relatively inexpensive to operate, and are recommended for any laboratory with high throughput of samples.

Continuous-flow analysis for glucose, triglycerides, and ATP with immobilized enzymes in tubular form.

Small-bore ("Autozyme") tubes with immobilized enzymes at the inner wall have been developed and studied for application in the Technicon "SMAC" high-speed continuous-flow biochemical analyzer. Tubes coated with glucose oxidase (D-glucose:oxygen oxidoreductase, EC 1.1.3.4) have been prepared for the assay of glucose, with colorimetric assay of the hydrogen peroxide produced; tubes coated with glycerol kinase (ATP:glycerol phosphotransferase, EC 2.7.1.30) for the enzymatic assay of triglycerides; tubes coated with hexokinase (ATP:D-hexose-6-phosphotransferase, EC 2.7.1.1) and glucose-6-phosphate dehydrogenase (D-glucose-6-phosphate:NAD+ oxidoreductase EC 1.1.1.49) for the measurement of ATP, an intermediate product in assays for creatine kinase. With use of 10-15 cm lengths of Autozyme tube and SMAC hydraulics (150 samples per hour), assay sensitivity and carryover were similar to values for the corresponding free-enzyme methods. These immobilized enzymes were sufficiently stable for one to eight weeks of continuous use before replacemnt. We conclude that suitable bound-enzyme tubes can replace either single or multiple free-enzyme reagents in many continuous-flow assays at high sampling rates.

An automated continuous-flow assay for serum sorbitol dehydrogenase activity and its use in experimental liver damage

An automated continuous-flow assay to monitor sorbitol dehydrogenase activity (EC 1.1.1.14) in serum has been developed. The amount of serum required is 240 μl and 50 samples can be analyzed per hour. The method features an automatic blank substraction and a linear range from 0 to 500 mU/ml. In order to validate the method, sera from control rats and rats preexposed to carbon tetrachloride were analyzed and compared to results obtained using a manual reaction-rate assay. The resulting correlation coefficient was 0.99.