Electronic Supplementary Material
Simultaneous photometric microplate assay for free and total thiamine using gold nanoparticles and alkaline phosphatase
María Ángeles Molina-Delgado, María Paz Aguilar-Caballos, Agustina Gómez-Hens*
Department of Analytical Chemistry. Research Institute of Fine Chemistry and Nanochemistry (IUQFN). Campus of Rabanales. Annex to Marie Curie building. University of Cordoba. 14071-Cordoba. Spain
*Corresponding author: A. Gómez-Hens (e-mail: ). Phone number: +34-957218645. Fax number: +34-957218644
Optimization of the variables
The enzymatic hydrolysis of THPP was studied by assaying takadiastase and ALP. The results obtained showed that both enzymes are suitable for this purpose but takadiastase requires the incubation of the samples overnight at 37 ºC [1, 2]. However, the study of the incubation time required for the dephosphorylation of THPP in the presence of ALP, assaying different temperatures in the range of 20 – 45 ºC, showed that the hydrolysis was completed in only 15 min at 40 ºC, reaching the same absorbance as that obtained using the same TH concentration (Figure 1S). The influence of the enzyme activity was studied in the range of 0.6-203 U mL-1 ALP, obtaining the highest absorbance values in the range of 50.0-70.0 U mL-1 ALP.
The study of the influence of the pH on the system showed that the absorbance remains constant over the pH interval of 5.0-8.5 (Figure 2S.A). A pH of 8.0 was chosen because it is close to the optimum pH described for the maximum activity of ALP. Tris buffer was used to adjust this pH, choosing a 50 mM concentration as optimum value. This buffer was chosen instead of phosphate buffer, since phosphate ions may induce the aggregation of AuNPs and it can also inhibit ALP activity. Tris buffer contained 5 mM magnesium chloride and 0.2 mM zinc chloride according to manufacturer’s instructions. The influence of the AuNPs concentration on the system was assayed in the range of 0.24 – 8 nM (Figure 2S.B). For this purpose, AuNPs dispersions of higher concentration than that of stock (1 nM) were obtained by centrifugation and further re-dispersion of the AuNP pellet in a smaller volume. The best absorbance values were obtained using the 1 nM stock solution, which corresponds to 0.5 nM in the reaction mixture.
Figure 1S. Influence of the incubation time on the absorbance obtained for TH (1) and THPP (2) in the presence of AuNPs and ALP. [AuNPs] = 1.0 nM, [TH] = [THPP] = 1.5 µM, [ALP] = 63.2 U mL-1, pH = 8.0, 40 ºC.
Figure 2S. Influence of the pH (A) and the AuNP concentration (B) on the absorbance obtained for TH and THPP in the presence of AuNPs. [TH] = [THPP] = 1.5 µM, [ALP] = 63.2 U mL-1, 40 ºC. In (A) [AuNPs] = 1.0 nM, in (B) pH 8.0.
References
[1] Gratacós-Cubarsí M, Sárraga C, Clariana M, García Regueiro JA, Castellari M (2011) Analysis of vitamin B1 in dry-cured sausages by hydrophilic interaction liquid chromatography (HILIC) and diode array detection, Meat Sci 87:234-238.
[2] Tang X, Cronin DA, Brunton NP (2006) A simplified approach to the determination of thiamine and riboflavin in meats using reverse phase HPLC. J Food Compos Anal 19:831-837.