A novel nanolayer biosensor principle.

A method for eliminating the mass transport limitation on biosensor surfaces is introduced. The measurement of macromolecular binding kinetics on plane surfaces is the key objective of many evanescent wave (e.g. total internal reflection fluorescence (TIRF)), and surface plasmon resonance (SPR) based biosensor systems, allowing the determination of binding constants within minutes or hours. However, these methods are limited in not being rigorously applicable to large macromolecules like proteins or DNA, since the on-rates are transport limited due to a Nernst diffusion layer of 5-10 microm thickness. Thus, for the binding of fibrinogen (340 kDa) to a surface current SPR biosensors will show a mass transport coefficient of ca. 2 x 10(-6) m/s. In a novel approach with an immiscible fluid vesicle (e.g. air bubble), it has been possible to generate nanoscopic fluid films of ca. 200 nm thickness on the sensor surface of an interfacial TIRF rheometer system. The thickness of the liquid film can be can be easily probed and measured by evanescent wave technology. This nanofilm technique increases the mass transport coefficient for fibrinogen to ca. 1 x 10(-4) m/s eliminating the mass transport limitation, making the binding rates reaction-rate limited. From the resulting exponential kinetic functions, lasting only 20-30s, the kinetic constants for the binding reaction can easily be extracted and the binding constants calculated. As a possible mechanism for the air bubble effect it is suggested that the aqueous fluid flow in the rheometer cell is separated by the air bubble below the level of the Nernst boundary layer into two independent laminar fluid flows of differing velocity: (i) a slow to stationary nanostream ca. 200 nm thick strongly adhering to the surface; and (ii) the bulk fluid streaming over it at a much higher rate in the wake of the air bubble. Surprising properties of the nanofluidic film are: (i) its long persistence for at least 30-60s after the air bubble has passed (2.5s); and (ii) the absence of solute depletion. It is suggested that a new liquid-liquid interface (i.e. a "vortex sheet") between the two fluid flows plays a decisive role, lending metastability to the nanofluidic film and replenishing its protein concentration via the vortices-thus upholding exponential binding kinetics. Finally, the system relaxes via turbulent reattachment of the two fluid flows to the original velocity profile. It is concluded that this technique opens a fundamentally novel approach to the construction of macromolecular biosensors.

[1]  H. Jennissen,et al.  Base-atom recognition in protein adsorption to alkyl agaroses. , 1992, Journal of chromatography.

[2]  M. Doyle,et al.  Kinetic analysis of a protein antigen-antibody interaction limited by mass transport on an optical biosensor. , 1997, Biophysical chemistry.

[3]  V. Hlady,et al.  Methods for studying protein adsorption. , 1999, Methods in enzymology.

[4]  D. Myszka,et al.  Kinetic analysis of macromolecular interactions using surface plasmon resonance biosensors. , 1997, Current opinion in biotechnology.

[5]  J. Lumley,et al.  Fluid Dynamics for Physicists , 1996 .

[6]  J. Israelachvili Intermolecular and surface forces , 1985 .

[7]  L. J. Gosting Measurement and interpretation of diffusion coefficients of proteins. , 1956, Advances in protein chemistry.

[8]  H. Jennissen,et al.  Monitoring fibrinogen adsorption kinetics by interfacial TIRF rheometry , 1996, Journal of molecular recognition : JMR.

[9]  W. Nernst,et al.  Theorie der Reaktionsgeschwindigkeit in heterogenen Systemen , 1904 .

[10]  L. Prandtl,et al.  Essentials of fluid dynamics , 1952 .

[11]  H. Jennissen Protein binding to two-dimensional hydrophobic binding-site lattices: Sorption kinetics of phosphorylase b on immobilized butyl residues , 1986 .

[12]  K. Laki,et al.  The polymerization of proteins; the action of thrombin on fibrinogen. , 1951, Archives of biochemistry and biophysics.

[13]  J. Andrade Surface and Interfacial Aspects of Biomedical Polymers , 1985 .

[14]  H. Jennissen General aspects of protein adsorption , 1988 .

[15]  K. Jacobsson I. Studies on the determination of fibrinogen in human blood plasma. II. Studies on the trypsin and plasmin inhibitors in human blood serum. , 1955, Scandinavian journal of clinical and laboratory investigation.

[16]  D G Myszka,et al.  Extending the range of rate constants available from BIACORE: interpreting mass transport-influenced binding data. , 1998, Biophysical journal.

[17]  J. Voegel,et al.  Adsorption kinetics of plasma proteins onto silica , 1984 .

[18]  F. Bretherton The motion of long bubbles in tubes , 1961, Journal of Fluid Mechanics.

[19]  R. Doolittle THE STRUCTURE AND EVOLUTION OF VERTEBRATE FIBRINOGEN , 1983, Annals of the New York Academy of Sciences.

[20]  Joseph D. Andrade,et al.  The Contact Angle and Interface Energetics , 1985 .

[21]  R. Karlsson,et al.  Kinetic and Concentration Analysis Using BIA Technology , 1994 .

[22]  P. Dean Affinity chromatography and related techniques : Analytical chemistry symposia series, volume 9 Edited by T. C. J. Gribnau, J. Visser and R. J. F. Nivard Elsevier Scientific; Amsterdam, New York, 1982 xviii + 584 pages. $83.00, Dfl 170.15 , 1982 .

[23]  H. Jennissen,et al.  The binding of phosphorylase kinase to immobilized calmodulin , 1993, Journal of molecular recognition : JMR.

[24]  E. Pefferkorn,et al.  Adsorption and Desorption of Synthetic and Biological Macromolecules at Solid-Liquid Interfaces: Equilibrium and Kinetic Properties , 1987 .

[25]  H. Jennissen,et al.  Protein binding to two-dimensional hydrophobic binding-site lattices: adsorption hysteresis on immobilized butyl-residues , 1979 .

[26]  J. Andrade,et al.  FLUORESCENCE OF ADSORBED PROTEIN LAYERS: I. QUANTITATION OF TOTAL INTERNAL REFLECTION FLUORESCENCE. , 1986 .

[27]  J. Kop,et al.  The role of intrinsic binding rate and transport rate in the adsorption of prothrombin, albumin, and fibrinogen to phospholipid bilayers , 1986 .

[28]  E. Bänsch,et al.  Quasi-stability of the primary flow in a cone and plate viscometer , 2004 .

[29]  D G Myszka,et al.  Kinetic analysis of macromolecular interactions using surface plasmon resonance biosensors. , 1997, Methods in enzymology.

[30]  V. Hlady,et al.  Human serum albumin adsorption onto octadecyldimethylsilyl-silica gradient surface. , 1994, Colloids and surfaces. B, Biointerfaces.

[31]  H. Schlichting Boundary Layer Theory , 1955 .