Available on line at Association of the Chemical Engineers of Serbia AChE www.ache.org.rs/CICEQ Chemical Industry & Chemical Engineering Quarterly 19 (4) 553-561 (2013) CI&CEQ 553 DRAGAN R. ŽIVANČEV 1 BRANISLAVA G. NIKOLOVSKI 2 ALEKSANDRA M. TORBICA 1 JASNA S. MASTILOVIĆ 1 NEVENA H. UKIĆ 3 1 University of Novi Sad, Institute of Food Technology, Novi Sad, Serbia 2 University of Novi Sad, Faculty of Technology, Novi Sad, Novi Sad, Serbia 3 University of Kragujevac, Faculty of Science, Kragujevac, Serbia SCIENTIFIC PAPER UDC 633.11:631.526.2(497.11):664.6/.7 DOI 10.2298/CICEQ120517090Z LAB-ON-A-CHIP METHOD UNCERTAINTIES IN DETERMINATION OF HIGH-MOLECULAR- -WEIGHT GLUTENIN SUBUNITS Polymeric wheat endosperm proteins, especially the high-molecular-weight glutenin subunits (HMW-GS), are probably the most interesting protein fraction giving the essential information about the bread-making quality of wheat flour. A relatively new method that shows great potential for a fast, reliable and auto- matable analysis of protein purity, sizing and quantification is microfluidic or Lab-on-a-Chip (LoaC) capillary electrophoresis. This aim of this work was to explore the possibilities of implementation of LoaC method to analysis of pro- tein samples isolated from a Serbian common wheat variety, emphasizing the steps that might bring uncertainties and affect reproducibility of obtained glu- tenin subunits quantitation results. A good resolution of protein bands in a molecular weight range of 14.0 to 220.0 kDa was achieved. The reproducibility of HMW-GS sizing and quantitation were good, with the average coefficient of variation values of 1.2 and 12.2%. The ratio of HMW-GS to low-molecular-weight glutenin subunits (LMW-GS) was about 20%. The investigation ruled out influ- ences of the extract solution addition and the buffer addition steps of the applied method, as well as the individual chip influence on GS quantitation results. However, there was statistically significant difference between HMW-GS quan- titation results of multi-step and one-step extraction procedures applied prior to glutenin subunits extraction step. Keywords: wheat, glutenin subunits, Lab-on-a-Chip, Arija wheat variety, LoaC Lab-on-a-Chip. Wheat gluten has a major effect on the end-use quality of baking industry products, since it is respon- sible for the visco-elastic properties of the dough. This protein macromolecule is composed of two compo- nents, gliadins and glutenins. Gliadins are viscosous and affect the extensibility of dough, whereas glute- nins are responsible for the dough elasticity [1]. Also, gliadins are single-chain polypeptides with molecular weights (MW) between 30,000 and 80,000 Da, whereas glutenins are multichain polypeptides with MW ranging from 80,000 to several millions Da [2,3]. Glutenins could further be divided into two groups, high molecular weight glutenin subunits (HMW-GS) and low molecular weight glutenin subunits (LMW- -GS). It is a well-known fact, that diverse HMW-GS Correspondence: D.R. Živančev, University of Novi Sad, Insti- tute of Food Technology, Bul. cara Lazara 1, 21000 Novi Sad, Serbia. E-mail: dragan.zivancev@fins.uns.ac.rs Paper received: 17 May, 2012 Paper revised: 5 September, 2012 Paper accepted: 6 October, 2012 are correlated with bread-making quality [4], for ins- tance, 5+10 depict good quality and 2+12, 4+12 sub- units are connected with lack of dough strength. Hou et al. [5] confirmed that a positive correlation exists between HMW-GS content and rheological properties of wheat dough, whereas different scientists empha- size significant influence of HMW-GS quantity in pre- diction of dough or gluten strength [6,7]. MacRitchie [8] also showed that the glutenin:gliadin (Glu:Gli) ratio shows considerable influence on dough and pan bread loaf quality. Electrophoresis and liquid chromatography are techniques that have been commonly applied for cereal proteins separation [9]. The most common electrophoretic methods for examination of cereal proteins is sodium-dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE). However, this form has several disadvantages. For instance, SDS-PAGE is time-consuming and includes a number of necessary manual steps, such as staining, destaining, imaging, analyzing [10]. Quantification can also be difficult [11]