Flow-based systems for rapid and high-precision enzyme kinetics studies.

Enzyme kinetics studies normally focus on the initial rate of enzymatic reaction. However, the manual operation of steps of the conventional enzyme kinetics method has some drawbacks. Errors can result from the imprecise time control and time necessary for manual changing the reaction cuvettes into and out of the detector. By using the automatic flow-based analytical systems, enzyme kinetics studies can be carried out at real-time initial rate avoiding the potential errors inherent in manual operation. Flow-based systems have been developed to provide rapid, low-volume, and high-precision analyses that effectively replace the many tedious and high volume requirements of conventional wet chemistry analyses. This article presents various arrangements of flow-based techniques and their potential use in future enzyme kinetics applications.

Sequential injection Lab-at-valve (SI-LAV) segmented flow system for kinetic study of an enzyme.

A sequential injection-Lab-at-valve (SI-LAV) segmented flow system for kinetic study of an enzyme was developed. Air segments were introduced for separation of enzyme and substrate zones and separation of the stacked zones from the carrier solution which ensure the measurement of the initial rate and minimize the dilution/dispersion effect. The open- ended mixing chamber makes it possible to use air segments in the flow system without the need for additional air segment discarding steps. The enzyme horseradish peroxidase (HRP) kinetic parameters based on initial rate was used as a model study. The operation of the system is virtually the same as that of the conventional batch-wise process. The kinetic parameters (i.e. K(m) and V(max)) of HRP obtained using the proposed system agree well with those obtained using the batch-wise process as well. The proposed system offers additional benefits of volume down scaling, improved rapidity and automatic features that does not require a skillful operator.

Exploiting green analytical procedures for acidity and iron assays employing flow analysis with simple natural reagent extracts.

Green analytical methods employing flow analysis with simple natural reagent extracts have been exploited. Various formats of flow based analysis systems including a single line FIA, a simple lab on chip with webcam camera detector, and a newly developed simple lab on chip system with reflective absorption detection and the simple extracts from some available local plants including butterfly pea flower, orchid flower, and beet root were investigated and shown to be useful as alternative self indicator reagents for acidity assay. Various tea drinks were explored to be used for chromogenic reagents in iron determination. The benefit of a flow based system, which allows standards and samples to go through the analysis process in exactly the same conditions, makes it possible to employ simple natural extracts with minimal or no pretreatment or purification. The combinations of non-synthetic natural reagents with minimal processed extracts and the low volume requirement flow based systems create some unique green chemical analyses.

Sequential injection-capillary immunoassay system for determination of sialoglycoconjugates.

An automatic immunoassay system for an assay of sialoglycoconjugates was developed based on the sequential injection technique. A cost effective plain glass capillary tube was used as a solid surface for immobilization of biomolecules via a simple physical adsorption which is adequate to tolerate the force of solution flowing through the capillary during the multi-steps immunoassay process. Immunoassay could be performed with many improvements-rapidity per sample as compared to the conventional micro-plate format (40 min vs. 5-8 h); lower cost and simpler as compared to fused silica capillary with covalent immobilization; and without problem of back pressure as compared to flow injection-bead based immunoassay. Performance of the sequential injection-capillary immunoassay was demonstrated by assay of sialoglycoconjugates level in human serum to differentiate cancer patients from healthy people.

Online assay of bone specific alkaline phosphatase with a flow injection-bead injection system.

Alkaline phosphatase (ALP) has been used as one of the biomarkers for bone resorption and liver diseases. Normally, total alkaline phosphatase is quantified along with other symptoms to determine the releasing source of the alkaline phosphatase. A semi-automated flow injection-bead injection system was proposed to conveniently and selectively assay bone alkaline phosphatase (BALP) based on its specific binding to wheat germ coated beads. Amount of BALP in serum was determined from the intensity of the yellow product produced from bound BALP on the retained beads and its substrate pNPP. The used beads were discarded and the fresh ones were introduced for the next analysis. The reaction cell was designed to be opened and closed using a computer controlled solenoid valve for a precise incubation time. The performance of the proposed system was evaluated by using it to assay BALP in human serum. The results were compared to those obtained by using a commercial ELISA kit. The system is proposed to be an easy and cost effective system for quantification of BALP as an alternative to batch wise wheat germ specific binding technique.

Exploiting flow injection system with mini-immunoaffinity chromatographic column for chondroitin sulfate proteoglycans assay.

A flow injection (FI) system with a mini-immunoaffinity chromatographic column was used to perform on-line assays of specific proteoglycans. The 300-microL mini-column contained beads coupled with monoclonal antibodies against the specific sulfation pattern of chondroitin sulfate proteoglycans, which have been reported to be a potential biomarker for cancer. The amount of these proteoglycans present was estimated indirectly from their protein content using the Bradford assay, which is an alternative to a direct carbohydrate assay. The system developed was tested by assaying for chondroitin sulfate proteoglycans in sera from patients with various cancers and comparing the results to those obtained for sera from healthy people. The results indicated that this approach could be used as a cost-effective alternative system for determining the amount of these specific biomarker proteoglycans. The column could be reused at least 90 times, with each run consisting of 200 microL of serum sample diluted twofold; an analysis rate of 30 min per run was achieved, as compared to 4 h for a batch procedure.

Simple bead injection-flow injection system for the determination of copper.

We demonstrate the extended application of a simple bead injection-flow injection system with a modified simple colorimetric detection unit for the determination of low amount of Cu2+ in samples of different matrices (water and supplement tablet samples) with minimal sample pretreatment by employing ammonium pyrrolidinedithiocarbamate (APDC) reagent.

Novel approach for mono-segmented flow micro-titration with sequential injection using a lab-on-valve system: a model study for the assay of acidity in fruit juices.

A new concept for micro-titration using a "lab-on-valve"(LOV) system with sequential injection of mono-segmented flow is proposed. The performance of the system was demonstrated by the assay of acidity in fruit juices which is based on acid-base neutralization. A standard/sample solution containing citric acid, indicator, sodium hydroxide, were sandwiched between air segments and were aspirated in microliter volumes through a selection valve into a holding coil. The acid, indicator, and base were mixed by flow reversal. After removing air segments, the solution was pushed to the detector for monitoring of the change in absorbance of the indicator color, which depended on the concentration of the remaining base. With LOV, microliter volumes of the solution can be detected without dispersion of the color zone. A calibration graph (plot of absorbance vs. acidity value) in the range of 0.2-1.2% (w/v) as citric acid was established. Sample throughput of 30 sample h(-1) and good reproducibility (RSD = 1.2%, n= 11 for 0.6% acidity) were achieved. The procedure has been applied to determine acidity in fruit juices.

Review on screening and analysis techniques for hemoglobin variants and thalassemia.

Thalassemia involves gene mutation that causes the production of an insufficient amount of normal structure globin chains while Hb variant involves gene mutation that causes the change in type or number of amino acid of the globin chain. It has been reported that some 200 million people worldwide had hemoglobinopathies of some sort. Attempts to develop effective and economical techniques for screening and analysis of thalassemia and Hb variants have become very important. In this review, we report the different techniques available, ranging from initial screening to extensive analysis, comparing advantages and disadvantages. Some indirect studies related to thalassemia indication and treatment follow-up are also included. We hope that information on these various techniques would be useful for some scientists who are working on development of a new technique or improving the existing ones.

Sequential injection-ELISA based system for online determination of hyaluronan.

Sequential injection-bead-based immunoassay system has been developed. The main purpose is to make immunoassay process more automated by manipulating the precise delivery of micro-volumes of reagents and the precise timing of incubation and washing steps with a computer program that controls the bi-directional syringe pump. The manifold was designed with the aims of reducing back pressure from beads that act as solid surfaces for immobilization of the target substance, reducing dispersion and dilution of the reagent during incubation, and maximizing signal while minimizing incubation time. This was done by introducing air segment to separate the reagent zone from the carrier stream and by using a suitable sensitive detector which, in this case, was an amperometer. In this study, hyaluronan (HA) was used as a target analyte because of its clinical significance as a potential biomarker for liver, bone and cancer diseases. Amount of hyaluronan was determined using competitive enzyme linked immuno sorbent assay (ELISA) based technique where immobilized HA and HA in solution compete to bind with a fixed amount of biotinylated HA-binding proteins (b-HABPs). Upon separation of the two phases, anti-biotin conjugated with enzyme and a suitable substrate were introduced to follow the binding reaction of the immobilized HA and b-HABPs whose degree of binding is indirectly proportional to the amount of HA in solution. A calibration curve was constructed from a series of concentrations of HA standards. Lowest detectable concentration was found to be 1 ng/mL with the dynamic working range of 1-5000 ng/mL and R.S.D. of intra-assay (n=7) and inter-assay (n=3) of various HA concentrations were 4-10% and 9-12%, respectively. Used beads could be reused by washing with 2M guanidine. Total analysis time for this automatic assay was about 30 min as compared to the 5-8h used in conventional batch well ELISA. The system could be applied to assay HA in human serum.

Exploiting guava leaf extract as an alternative natural reagent for flow injection determination of iron.

Guava leaf extract is utilized as an alternative natural reagent for quantification of iron. The flow injection technique enables the use of the extract in acetate buffer solution without the need of further purification. Some properties of the extract such as its stability and ability to form a colored complex with iron were studied. The proposed system is an environmentally friendly method for determination of iron with less toxic chemical wastes.

A simple flow injection-reduced volume column system for hemoglobin typing.

A flow injection (FI)-reduced volume column system was developed for hemoglobin (Hb) typing to be used as an initial screening method for thalassemia. The column was packed with 140 microl diethylaminoethyl (DEAE)-Sephadex A-50 ion exchange beads. Hb can be separated using Tris-HCl buffer solution with pH gradient 8.5-6.5 and then monitored spectrophotometrically at 415 nm. The hemolysate of 40 blood samples from packed red cells were screened for thalassemia by determining the amount of HbA(2) and HbE present. The proposed system was able to predict positive test results from those samples with beta, E-trait and EE homozygous thalassemia, Hb types that were independently identified following the conventional method at the hospital laboratory. Advantages of the proposed system over the conventional column technique include low amount of reagents and blood sample needed, short analysis time and low cost. Each analysis required only 80 microl of 50 times diluted packed cells, which is equivalent to 1.6 microl undiluted packed cells, and it can be completed in only 35 min. This simple FI-reduced volume column system was demonstrated to be an economic alternative system for Hb typing to initially screen some types of thalassemia such as beta-trait, E-trait and EE-homozygous which are commonly found in Thailand.