Chemical apparatus and process disinfecting – deodorizing – preser – Analyzer – structured indicator – or manipulative laboratory... – Means for analyzing liquid or solid sample
Reexamination Certificate
1999-05-21
2003-03-25
Warden, Jill (Department: 1743)
Chemical apparatus and process disinfecting, deodorizing, preser
Analyzer, structured indicator, or manipulative laboratory...
Means for analyzing liquid or solid sample
C422S050000, C422S063000, C422S068100, C436S501000, C436S513000, C436S518000, C436S043000
Reexamination Certificate
active
06537499
ABSTRACT:
TECHNICAL FIELD
The present invention relates to a method as well as a device for identification of a substance, preferably comprising biochemical molecules. In particular, it is the aim of the present invention to improve the technique of analyzing unknown biomolecules for achieving results in an easier and quicker way so that the method can be used without investing great effort and high costs. A preferred embodiment of the invention is a biosensor for easy detection of unknown biomolecules.
BACKGROUND OF THE INVENTION
Many approaches have been undertaken for determining the type of substance such as a chemical substance, in particular a biomolecule, which provides a very high degree of selective binding properties.
There are methods of analyzing the optical properties of individual substances such as absorption or transmission behavior, all these methods require very accurate measurement equipment comprising light sources for emitting defined wavelengths as well as very accurate detection devices, rendering these methods very expensive:
Methods are known for identification of substances by measuring their surface properties such as surface forces or work of adhesion. The work of adhesion of a substance coated onto a solid surface can be determined by measuring the contact angle between the surface and a fluid droplet applied onto it. This technique is described in an article by Israelachvili, ,“Interfacial Forces”, J.Vac.Sci. Technol. A 10(5), September/October 1992, pp. 2961-2971.
Another approach for estimating the surface free energies of solid surfaces requires direct examination of solid-solid interfaces. In an article by Chaudhury and Whitesides, Langmuir, vol. 7,. No. 5, 1991, pp. 1013-1025, measurement of the surface free energy of elastomeric polymers is deduced by measurement of the deformation of semispherical lenses of poly-dimethylsiloxane elastomers and their chemical derivatives. Analyses of the deformation of the lens were conducted using Young's equation and the Johnson, Kendall Roberts (YRK) model. The analyses included balancing the adhesion forces acting across the interface with the restoring forces that oppose the deformation of the lens. Accurate results require the Young's modulii and the Poisson ratios of each material. In addition, spherical lenses with a well-defined radius and smooth surface have to be produced for one or both materials involved in the measurement.
By determining the work of adhesion of unknown substances which are derivatized onto a sample surface, an estimation can be made about the nature and the type of the derivatized substance. However, the aforementioned methods are complicated and drawn out.
Another aspect in determining binding constants of substances by measuring their surface properties is the direct interaction between at least two molecules. Such interactions, derived from multiple weak bonds between geometrically complementary surfaces which are coated with different chemical groups, can be very strong. In the field of biomolecules, the sensitivity of binding properties is very distinct so that molecular recognition between a receptor and a ligand, an antibody and an antigen as well as complementary strands of DNA provides a basis for identification of biochemical substances such as biomolecules.
With the development of scanning probe microscopes, in particular the atomic force microscope (AFM), surfaces can be probed in physiological environments with molecular resolution and forces down to the piconewton range as is disclosed in an article by G. Binnig, C. F. Quate and Ch. Gerber, Phys. Ref. Lett. 56, 930 (1986).
Using the atomic force microscope, Florin et al. measured the adhesion force between the tip of an atomic force microscope cantilever derivatized with avidin and agarose beads functionalized with biotin. They found the force required to separate the tip and the bead to be quantized in integer multiples of 160 pN (E.-L. Florin, V. T. Moy and H. E. Gaub, Science 264, 415 (1994)).
It is an object of the present invention to provide a method for identification of unknown substances which requires less effort with respect to necessary equipment and fewer procedure steps. The method should be easy to control and should produce reliable results.
It is a further object of the present invention to provide a device for identification of substances based on it's binding characteristics to a known probe, in particular comprising a biochemical molecule, without the need of highly sophisticated analyzing devices, which usually are very expensive. Furthermore such a device should be easy to handle and should be used as a biosensor enabling the identification of a plurality of unknown substances.
SUMMARY OF THE INVENTION
The present invention relates to a method for identification of a substance, preferably comprising biochemical molecules, which is usually derivatized onto a sample surface. Furthermore, a well-known probe is used which is favorably derivatized onto a contact surface for the purpose of better handling. Both surfaces coated with the aforementioned layers are brought into contact and then separated from each other while measuring physical parameters characterizing the interaction between said substance and said probe. The obtained results are compared with reference values for identification. These reference values can be listed or measured with reference techniques, for example, as is described above in connection with the cited state of the art.
For characterization and identification of an unknown substance, the binding related work of adhesion is a suitable parameter which can be measured with the aforementioned invented method. In a preferred implementation of the invented method a molded elastomeric pillar is used with a well-defined contact surface onto which a known probe is derivatized. This probe is pushed towards the sample surface onto which the unknown substance, preferably biochemical molecules, is derivatized, until both surfaces have a well-defined conformal contact.
The term “conformal contact” implies that the surface shapes of the two media put on top of each other are similar to such an extent that fluids can essentially not penetrate into the plane where the surfaces meet each other except for immobilized water strongly associated with the molecules. The term “fluid” refers to both liquids and gases.
While both surfaces are brought together until conformal contact is reached the force during this loading phase is determined as a function of the travelling distance from the point of which the two surfaces make the first contact until the state of conformal contact which is indicated by a given force. Subsequently the two surfaces are withdrawn from each other until both surfaces are completely separated. Also the force during the separation phase is determined as a function of the travelling distance from the state of conformal contact until the point of becoming totally separated.
The force applied during the loading phase is summed up from the point of contact until the maximum load. This integral represents the energy stored in the elastic deformation of the pillar. The applied force during the unloading phase is similarly summed up from maximal load to zero load after backlesh compensation.
Surfaces having substances with great work of adhesion have to be pulled away from each other until these snap apart. The difference between the two energies (loading integral, unloading integral) is the work of adhesion and corresponds individually to the substance coated on the sample surface. The obtained value of the work of adhesion can now be compared with a reference value derived from stored data or a reference measurement.
Another alternative method for identification of a substance by obtaining results of high quality and expressiveness about the nature and the type of substance can be reached in the following manner:
As is the case in the aforementioned method the unknown substance is derivatized onto a sample surface which comes into contact with a contact surface onto
Bernard Andre
Biebuyck Hans
Delamarche Emmanuel
Michel Bruno
Schmid Heinz
Kaufman Stephen C.
Sines Brian
Warden Jill
Whitham Curtis & Christofferson, P.C.
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