On-line Continuous-flow Biochemical Interaction

Furthermore, when using 96 nM fluorescein-biotin and 32 nM streptavidin, an injection of 1 pmol of biotin results in an almost complete blocking of streptavidin and, consequently, the maximum peak height possible under the current conditions is about 95% of the highest point (3), indicating that apparent binding of biotin to streptavidin is in the order of 95%. 

Because the interaction between biotin and streptavidin is strong (fCa = 0.6 X 10 L mol ) with a relatively fast association rate (k+i = 2.4 x 10 L mol s ) and slow dissociation rate (k i = 0.4 x 10 s ), the reaction times are fast, i.e. 10-20 s. Furthermore, the addition of reporter ligand is performed only after the analyte protein reaction has taken place in coil I, avoiding a displacement reaction that would substantially increase the overall reaction time. 

Hence the reaction coil volumes were kept as small as possible to reduce bandbroadening, i.e. 17 pL and 33 pL, for coil I and coil II, respectively. 

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