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Title: AN ANALYSIS OF AVIDIN, BIOTIN AND THEIR INTERACTION AT ATTOMOLE LEVELS BY VOLTAMMETRIC AND CHROMATOGRAPHIC TECHNIQUES

Author
item KIZEK, RENE - MENDEL UNIV, CZECH REP
item MASARIK, MICHAL - INST BIOPHYSICS, CZECH RE
item KRAMER, KARL - 5430-05-30 RETIRED
item POTESIL, DAVID - MENDEL UNIV, CZECH REP
item BAILEY, MICHELE - PRODIGENE INC
item HOWARD, JOHN - PRODIGENE INC
item KLEJDUS, BORIVOJ - MENDEL UNIV, CZECH REP
item MIKELOVA, RADKA - MENDEL UNIV, CZECH REP
item ADAM, VOJTECH - MENDEL UNIV, CZECH REP
item TRNKOVA, LIBUSE - DEPT THEORET CHEM, CZECH
item JELEN, FRANTISEK - INST BIOPHYSICS, CZECH RE
item Beeman, Richard

Submitted to: Analytical and Bioanalytical Chemistry
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 12/10/2004
Publication Date: 3/4/2005
Citation: Kizek, R., Masarik, M., Kramer, K.J., Potesil, D., Bailey, M., Howard, J.A., Klejdus, B., Mikelova, R., Adam, V., Trnkova, L., Jelen, F., 2005. An analysis of avidin, biotin and their interaction at attomole levels by voltammetric and chromatographic techniques. Analytical and Bioanalytical Chemistry 381: 1167-1178.

Interpretive Summary: We describe a potentially very useful ultra-sensitive biotechnology for detection of the vitamin biotin, the biopesticide avidin, and the complex that forms between the two. Results of collaborative research with scientists from the Czech Republic and ProdiGene, Inc. (College Station, TX) demonstrate that electrochemical procedures can detect these materials at attomolar levels (10-15 M). An electrochemical method was developed to analyze for the vitamin in drugs and vitamin supplements. Another electrochemical procedure was developed for avidin detection in transgenic corn seeds, which is relatively easy, rapid, and easily applicable. Avidin has been previously developed as a biopesticide in transgenic plants for insect pest management. This analytical technology offers useful and promising possibilities in the field of biosensor development.

Technical Abstract: 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 microliter drop) in solution, 700 fM (174 fmol in 250 microliter 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 for the optimization of the measurements such as electrode accumulation time, composition of the avidin-modified CPE, and elution time of avidin. In addition, the avidin-modified electrode was used for the detection of biotin in solution (detection limit was 7.6 pmol in 6 microliter drop) and for the detection of biotin in a pharmaceutical drug after different solvent extraction procedures. Comparable studies for the detection of biotin were developed using HPLC with diode array detection (HPLC-DAD) and HPLC with electrochemical detection (HPLC-ED), which allowed biotin to be detected to a level 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 HPLC-ED signal was also studied.