Talanta 85 (2011) 804–808 Contents lists available at ScienceDirect Talanta journal homepage: www.elsevier.com/locate/talanta Short communication Sequential injection Lab-at-valve (SI-LAV) segmented flow system for kinetic study of an enzyme Kraingkrai Ponhong, Supaporn Kradtap Hartwell, Kate Grudpan ∗ Department of Chemistry and Center for Innovation in Chemistry, Faculty of Science, Chiang Mai University, Chiang Mai 50200 Thailand, and Center of Excellence for Innovation in Analytical Science and Technology, Chiang Mai University, Chiang Mai 50200, Thailand article info Article history: Received 5 January 2011 Received in revised form 26 March 2011 Accepted 30 March 2011 Available online 8 April 2011 Keywords: Sequential injection Lab-at-valve Segmented flow system Kinetic study Enzyme Horseradish peroxidase Down scaling Automation abstract 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. © 2011 Elsevier B.V. All rights reserved. 1. Introduction Kinetic studies of enzymes have been carried out for various objectives. Comparison of various enzymes for a certain task, com- parison of the efficiencies of the same enzymes extracted from various sources, and quantification of analyte via kinetic approach of a fixed amount of enzyme label and substrate are just some examples. In any study related to enzyme kinetics, the critical point is to develop a reliable initial velocity enzyme assay procedure where real time progression of enzyme-substrate reaction can be recorded [1]. Conventional spectrophotometric batch-wise proce- dure requires a rather high volume (i.e. ∼2–3 mL) of reagents and sample [2] which is not suitable for expensive and rare enzymes. Availability of a micro-cuvette helps in reducing the volume of liq- uid needed for spectrophotometric measurement. However, the main disadvantage in the manual operation is the imprecision in initial detection time of the repeated measurements of several cuvettes containing different substrate concentrations [3], which is due to the manual mixing and placement of the cuvettes in the detector. ∗ Corresponding author. E-mail address: kate@chiangmai.ac.th (K. Grudpan). Flow injection (FI) technique, which has been used for automa- tion of various wet chemical/biochemical analyses, has also been adapted for enzyme kinetic studies in both solution and immobi- lized phases [4–9]. The important benefits of the FI system include the ability to automate the chemical analysis using low volume of reagent and sample with various possible manifold arrangements [10] that can also be coupled online with other analysis techniques (i.e. LC and microdialysis) for extended studies [11]. FI technique can also be operated in stopped flow mode for continuous recording of reaction progress over time [12]. However, while the detection can be done precisely in terms of measurement time, there is impre- cision in the measurement of initial rate due to dead time between point of mixing and point of detection [13] and unknown concen- tration of enzyme and product owing to dispersion, diffusion and incomplete mixing in the flow line [14]. These can yield incorrect enzyme kinetic parameters such as K m and V max . The micro-fluidic/micro-chip technology where small volume reagents/samples are incorporated with chemical/biochemical reactions in tiny micro-channels [15–18] helps minimize the dilu- tion/dispersion effect. However, their relatively higher cost makes them not suitable for general use. The micro-sequential injection with Lab-on-valve (LOV) and a micro-reactor was also reported [19]. Its small volume micro-channels and on-reactor detection should help to minimize dilution due to dispersion, although the dilution might not yet be completely eliminated. 0039-9140/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.talanta.2011.03.086