An analysis of avidin, biotin and their interaction at attomole levels by voltammetric and chromatographic techniques.
The electroanalytical determination of avidin in solution, in a carbon paste, and in a transgenic maize extract was performed in acidic medium at a carbon paste electrode (CPE). The oxidative voltammetric signal resulting from the presence of tyrosine and tryptophan in avidin was observed using square-wave voltammetry. The process could be used to determine avidin concentrations up to 3 fM (100 amol in 3 microl drop) in solution, 700 fM (174 fmol in 250 microl solution) in an avidin-modified electrode, and 174 nM in a maize seed extract. In the case of the avidin-modified CPE, several parameters were studied in order to optimize the measurements, such as electrode accumulation time, composition of the avidin-modified CPE, and the elution time of avidin. In addition, the avidin-modified electrode was used to detect biotin in solution (the detection limit was 7.6 pmol in a 6 mul drop) and to detect biotin in a pharmaceutical drug after various solvent extraction procedures. Comparable studies for the detection of biotin were developed using HPLC with diode array detection (HPLC-DAD) and flow injection analysis with electrochemical detection, which allowed biotin to be detected at levels as low as 614 pM and 6.6 nM, respectively. The effects of applied potential, acetonitrile content, and flow rate of the mobile phase on the FIA-ED signal were also studied.
Kizek
Rene
R
Department of Chemistry and Biochemistry, Mendel University of Agriculture and Forestry, Zemedelska 1, 613 00, Brno, Czech Republic. kizek@sci.muni.cz
Masarik
Michal
M
Kramer
Karl J
KJ
Potesil
David
D
Bailey
Michele
M
Howard
John A
JA
Klejdus
Borivoj
B
Mikelova
Radka
R
Adam
Vojtech
V
Trnkova
Libuse
L
Jelen
Frantisek
F
D001360:Avidin / Q000032:analysis* / Q000737:chemistry; D001710:Biotin / Q000032:analysis* / Q000737:chemistry; D002244:Carbon / Q000737:chemistry; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D004347:Drug Interactions; D004563:Electrochemistry / Q000379:methods*; D004566:Electrodes; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
D001360:Avidin; D001710:Biotin; D002244:Carbon