Chemistry: electrical and wave energy – Apparatus – Electrolytic
Reexamination Certificate
1999-03-02
2004-08-03
Warden, Jill (Department: 1743)
Chemistry: electrical and wave energy
Apparatus
Electrolytic
C435S817000
Reexamination Certificate
active
06770179
ABSTRACT:
BACKGROUND OF THE INVENTION
(a) Field of the Invention
The invention relates to biosensors in which a monomolecular biological conjugate layer is attached to a transducing device.
(b) Description of Prior Art
Biosensors are a rapidly emerging technology for detecting and/or measuring the occurrence of biological phenomena or the presence of biological molecules or organisms. A biosensor is any analytical device incorporating a biological material, a biologically derived material or biomimetic intimately associated with or integrated within a physiochemical transducer or transducing microsystem, which may be optical, electrochemical, thermoelectric, piezoelectric or magnetic (Fishman et al.,
Annu. Rev. Biophys. Biomol. Struct
., 27:165-198, 1998). Typical biosensors are formed by attaching a biological molecule, such as an enzyme or an antibody, to a transducer. The biosensor is exposed to an environment in which it is desired to detect bioactivity or a specific biological entity, and signals emitted by the transducer reflect the involvement of the biological molecule in a bioreaction or biointeraction.
Examples of biosensor technology are disclosed in Ahluwalia et al., “A comparative study of protein immobilization techniques for optical immunosensors,”
Biosensors & Bioelectronics
, 7:207-214(1991) and Geddes et al., “Immobilisation of IgG onto gold surfaces and its interaction with anti-IgG studied by surface plasmon resonance,”
Journal of Immunological Methods
, 175:149-60 (1994).
An important issue in the construction of biosensors is the attachment and immobilization of a biological material in relation to the transducer. If the biological molecule is not attached in proper relation to the transducer or in sufficient amount, then the sensor may not operate satisfactorily. Problems encountered with prior biosensor constructions include maintaining sufficient accessibility, density and/or orientation of the biologically active molecule or organism used in the biosensor. Some sensor designs enclose the bioactive molecule in a matrix or membrane. Such designs tend to restrict the accessibility of the bioactive molecule to the moiety which it is intended to interact with and sense and thereby limit the sensitivity of the resulting sensor. Attempts have also been made to directly attach bioactive molecules to sensors and/or substrates. However, such techniques do not necessarily result in optimum density of the bioactive sensor molecules or in a uniform orientation of the bioactive sensor molecule with the active site or epitope in a properly exposed position for effective interaction with the intended moiety. None of the prior art biosensors is provided with a chemical coating which would promote optimum biochemical interactions reactions at the biosensor/test medium interface, thereby enhancing the sensitivity and usefulness of the biosensor. Consequently, despite the efforts of the prior art, there remains a substantial need for improved biosensor designs.
In various fields, attempts have been made to affix molecules on the surfaces of articles or materials to modify their surface properties. For example, Sukenik, C. N. et al. (J. Biomed. Materials Res., 24:1307-1323, 1990) describes the modulation of cell adhesion by modification of titanium surfaces with covalently attached self-assembled monolayers. Wieserman et al., U.S. Pat. No. 4,788,176 teaches a process for chemically bonding a monomolecular layer of phosphorous- containing material to metal oxide/hydroxide particles to form an active material suitable for use as an absorbent. Rhee et al., U.S. Pat. No. 5,328,955 discloses covalent attachment of collagen to organic polymers and their use with implants. However, none of these documents discloses or suggests the use of bioactive conjugates which include covalently attached biologically active molecules to a biosensor surface in order to enhance the sensitivity and/or usefulness of a biosensor.
SUMMARY OF THE INVENTION
Advances in the immobilization of affinity ligands and innovation in the merging field of bioelectronics have combined to produce revolutionary new detection devices. Affinity electrodes and biosensors are based on the specific interaction between receptors, enzymes, or antibody molecules and their specific target analytes. The application of this technology to measurement systems has created novel analytical detection devices for such diverse fields as diagnostics, therapeutics, process control, waste and environmental monitoring, computer technology, and the kinetic analysis of the interaction of various biological substances.
Common to every device is a support material to which an immobilized affinity ligand is attached. This ligand may be an enzyme that is designed to monitor the presence of its specific substrate in solution. It may be an antibody that can measure its complementary antigen, or an antigen to detect specific antibodies. The affinity ligand may also be any biospecific molecule that interacts with a particular receptor protein (or vice versa). It can even be an immobilized intact living organism (cellular) that can act on specific substances in the solutions with which it comes in contact.
To detect the interaction of these affinity pairs, a functional biosensor needs an electronic transducer that senses the subtle chemical changes that take place between the immobilized ligand and the specific analyte. The detection process may involve the monitoring of electrical effects such as potentiometric changes, amperometric fluctuations, or capacitance differences; optical effects such as light absorption, scattering, or refractive index; changes in density or mass; acoustical effects such as changes amplitude, frequency, or phase of a sound wave; or thermal differences using sensitive calorimeters. The electronic detector then sends its signals an amplification device that also may process and compute the concentration of the analyte in solution. The output and control of these instruments may be as simple as reading a needle gauge on a device such as a pH meter as complex as sophisticated computerized instruments with programmable interfaces.
The principles of biosensor operation have been employed for decades with oscilloscopes designed to monitor slight changes in electrical phenomena. For instance, an olfactory organ such as the antenna of a butterfly can be placed between the input leads of an oscilloscope and used to detect the interaction of olfactory receptors with various volatile substances. In this case, the initial amplifiers of the receptor-ligand interaction are the olfactory nerves that generate action potentials along their length in response to the binding of specific substances. Important qualitative information can be obtained in such a system, but is of little quantitative use.
In modern biosensor design, a synthetic receptor-ligand surface is constructed that has specificity for a single substance. Since the surface is monospecific and the response varies in proportion to the quantity of ligand in the sample solution, quantitative analytical measurements are possible.
The goal is to form a local concentration of the affinity ligand across the biospecific surface. Correct orientation and retention of activity are important in this process, especially for ligands containing active sites that must interact with specific analytes after immobilization.
In general, entrapment or absorption procedures do not yield stable affinity systems for biosensor design. Such sensors may work for brief periods in the laboratory, but the weak bonds created by noncovalent attachment usually cause severe leakage of the biomolecule off the surface and degradation of performance with use. Entrapment, however does provide a viable immobilization means when attaching cellular ligands to a surface, since the cells are typically surrounded by a polymerized or gelatinous membrane and are unable to break free.
The aim of the present invention is to provide biosensors in which a monomolecular biological conjugate layer is covalently attached either
Crowell & Moring LLP
Noguerola Alex
Universite de Montreal
Warden Jill
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