TIRF-rheometer for measuring protein adsorption under high shear rates: Constructional and fluid dynamic aspects

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.