koff - LEPA

! = ! eq
t
t d
Exercise : Calculate t a , t d , and τ. Please comment. Plot Γ(t)/Γ eq .
To give some orders of magnitude, Table 1 gives some kinetic values for some classical systems.
k on / M –1 ·s –1 k off / s –1
IgE 10 6 10 –3
IgG4 10 6 10 –2
Avidin-Biotin 10 8 10 –7
ss-DNA 6·10 4 5·10 –5
Exercise : Please comment on the different values
1.3 Adsorption measurement
To monitor the adsorption of target species onto a substrate, one can distinguish label-free
techniques and techniques relying on a chemical labelling step.
Label free techniques include wire conductivity measurements, capacitance measurements,
quartz microbalance measurements, resonators and surface plasmon resonance measurements.
Chemical labelling is used for immunoassays and protein arrays using often a secondary
antibody labelled with an enzyme. Enzyme activity is then monitored by addition of a substrate.
In the case of DNA, the complementary oligo sequence is itself labelled often with a
fluorophore.
Label-free techniques are very useful to measure the kinetics of binding. For example, a flow
system combined to SPR can be used to measure a sensorgram. The measurement is done in two
steps. First, a buffer solution is passed on the detector, and at time t=0, a solution of target
species is injected, the adsorption is then monitored. At a given time, the flowing solution is
switched to the eluent and the desorption is monitored as shown below.
Adsorption
Desorption
Exercise : Please explain the physical principles of a label-free technique (min 1 A4 page).
Derive the equation and draw a sensorgram for the system used above, namely K=10 9 M –1 ,
k on =10 6 M –1 ·s –1 , a bulk analyte concentration of 0.1 pM.
Time
(11)
4