This is one important reason why statistical experimental design is needed. Design of experiments (DOE) originated as a method to maximize the knowledge gained from experimental data. Compared with conventional methods, multivariate approaches based on DOE allow studying all possible interactions between experimental variables and can significantly reduce the experimental effort needed

to investigate the experimental factors and their interactions. These methods are especially valuable for optimization of chemical processes. The examples of application of multivariate DOE include using MODDE 6 software for optimization of supercritical fluid extraction, conditions for the SYN-117 in vitro extraction of indole alkaloids from the dried leaves of Catharanthus roseus, and GC/MS-based analysis of amino acids and organic

acids in rat brain tissue samples [9, 10]. Only a few reports discussing the chemometrics JPH203 approach in rational design of MIPs have appeared. Thus, Kempe and Kempe [11] ABT-888 employed multivariate data analysis (MODDE 6.0 software, Umetrics, Umea, Sweden) for the optimization of monomer and cross-linker ratios in the design of a polymer specific for propranolol. Mijangos et al. [12] used chemometrics (MODDE 6.0 software, Umetrics, Sweden) to optimize several parameters such as concentration of initiator (1,1′-azobis(cyclohexane-1-carbonitrile) and 2,2-dimethoxy-2-phenylacetophenone) and polymerization time required for

the design of high-performance MIP for ephedrine. Phospholipase D1 In the present work, we demonstrate the use of the multivariate DOE approach and MODDE 9.0 software (Umetrics, Sweden) for increasing the yield of MIP nanoparticles synthesized in the automatic photoreactor developed by our team. Methods Reagents and materials N,N′-methylene-bis-acrylamide, ethylene glycol methacrylate phosphate, 3-aminopropyltrimethyloxysilane (APTMS), fluorescein O-methacrylate, and acetone were purchased from Sigma-Aldrich, Gillingham, UK. Acetonitrile was obtained from Fisher Scientific (Bromborough, UK). N,N-diethyldithiocarbamic acid benzyl ester was obtained from TCI Europe (Boerenveldseweg 6, 2070 Zwijndrecht, Belgium). Vancomycin was chosen as the model template in solid-phase synthesis of MIP nanoparticles. All chemicals and solvents were of analytical or HPLC grade and were used without further purification. Phosphate buffered saline (PBS) was prepared from PBS buffer tablets (Sigma-Aldrich, Gillingham, UK) and comprised 0.01 M phosphate buffer, 0.0027 M potassium chloride, and 0.137 M sodium chloride, with pH 7.4, at 25°C. Where necessary, the pH of the buffer was adjusted to pH 7.2 by the addition of HCl. Preparation of template-derivatized glass beads Glass beads (75-μm diameter from Sigma-Aldrich) were activated by boiling in 4 M NaOH for 10 min, then washed with double-distilled water followed by acetone, and dried at 80°C.