Hp. Jennissen et al., TIRF-rheometer for measuring protein adsorption under high shear rates: Constructional and fluid dynamic aspects, MATER WERKS, 30(12), 1999, pp. 850-861
In rural internal reflection fluorescence (TIRF) measurements an exponentia
lly decaying evanescent wave of light (285-290 nm) excites the Trp residues
in a protein to fluoresce at 350 nm when it is adsorbed to a transparent s
urface. A major problem in the measurement of protein adsorption kinetics i
n such systems is that the protein has to diffuse through a boundary layer
to reach the surface. The thickness of such a boundary layer can be reduced
by shearing the fluid phase. In the classical TIRF adsorption chamber only
a unidirectional flow of buffer through the chamber is possible. In such a
chamber the shear rates and shear stresses vary across the cross section a
nd only low shear rates are obtainable. Therefore based on a rheological sy
stem for studying fluid shear stress on cultured cells a TIRF-chamber was c
onstructed which allowed the installment of a rotating cone (max. rate: 120
0 rpm) and plate viscometer-type variable shear device. In this case a flow
field can be set up in which the shear rates and shear stresses are approx
imately constant. Cone angles (alpha) between 1.0-2.5 degrees allowed shear
rates (gamma) between 0 and 7200 s(-1). The TIRF-rheometer can be employed
in two different modes in the form of: (a) a closed system (no fluid flow
through the rheometer chamber), (b) an open system with continuous buffer f
low through the chamber. The flow conditions were checked by observing the
dissolution of a small spot of dried Coomassie blue as a function of the sh
ear rate and time. A significant secondary flow was found with all cone ang
les and was dependent on the square root of the shear rate. Ink injection s
tudies demonstrate that mixing times in the chamber below two seconds are o
btainable. The TIRF Rheometer thus provides a means for studying the shear
dependence of the adsorption of blood proteins and the generation of thin t
o ultra thin boundary layers for the measurement of protein adsorption kine
tics relevant to biomaterials.