Measuring and testing – Gas analysis
Reexamination Certificate
1999-03-30
2001-09-18
Williams, Hezron (Department: 2856)
Measuring and testing
Gas analysis
C073S024060, C422S082010, C422S068100, C436S163000
Reexamination Certificate
active
06289717
ABSTRACT:
BRIEF SUMMARY OF THE INVENTION
This invention relates to novel methods for the detection of biological and biologically derived materials using micromechanical devices as the detection mechanism. The measurement of small changes in the deflection of a micromechanical device such as a cantilever spring element which has been coated with one member of a specific binding partner provides qualitative and quantitative determination of the presence of the other specific binding partner. A specific binding partner is a member of a pair of biochemical entities which exhibit specific binding, i.e., a high intrinsic affinity or high total avidity (q.v. Kabat, E. A., Structural Concepts in Immunology and Immuno Chemistry, 2
nd
ed. New York; Holt Rinehart & Winston, 1976). Non-limiting examples are Ag-Ab (Antigen-Antibody) complexes, nucleic acid probes and targets, steroid hormone-peptide binding pairs, etc.
BACKGROUND OF THE INVENTION
Microcantilevers can be utilized in biophysical and biochemical studies to determine energy changes as indications of biochemical reactions in a medium. There is a great interest in developing ultra-miniature probes and assays that require very small volumes of sampled media for accurate qualitative and quantitative analysis of biochemical reactions and these assays can be performed using a microcantilever bound substrate.
In Thundat et al., U.S. Pat. No. 5,719,324, a piezoelectric transducer is disclosed that is fabricated with a cantilever having a spring element treated with a chemical which reacts with a specific vapor phase chemical. An oscillator means maintains a resonant vibrational frequency during detection of a chemical, with changes in resonant frequency indicating amounts of targeted chemical detected in the monitored atmosphere. Alternatively, the rate of cantilever bending is monitored to indicate the target chemical concentration.
In Wachter et al., U.S. Pat. No. 5,445,008, a mass microsensor is disclosed that is fabricated with a microcantilever that oscillates due to a piezoelectric transducer, with a chemical coating on the microcantilever that absorbs a targeted chemical from the monitored atmosphere. The resonant frequency of the microcantilever is analyzed to determine changes that indicate amounts of targeted chemical detected in the monitored atmosphere.
In Marcus et al., U.S. Pat. No. 5,475,318, a microprobe is disclosed that includes a microcantilever, a base, a probe tip projecting from the base, and a heating element that heats the probe tip, which comes into contact with a material to be investigated.
In Hafeman, U.S. Pat. No. 4,963,815, a device and method is provided for determining an analyte by measuring a redox potential-modulated photoinducing electrical signal from an electronically conducting layer on a semiconductor device.
In Kolesar, U.S. Pat. No. 4,549,427, a chemical nerve agent detector is disclosed that includes a transducer having two microcantilever oscillators. The active microcantilever of the two microcantilevers has a chemically selective substance that absorbs chemical nerve agents from the atmosphere, with modifications in the oscillation of the active microcantilever, and comparisons are made between the frequency of the active cantilever and the reference cantilever.
The above described methods and devices of measuring chemical and micromechanical parameters are not directed towards immunoassays which can detect the binding of a few molecules upon a substrate. The present invention describes a novel and sensitive technique which measures adsorption-induced forces for detecting proteins, other biopolymers, nucleic acid sequences, and micro-organisms in a gas or liquid medium using microcantilever.
SUMMARY OF THE INVENTION
It is an object of this invention to provide a detection and quantitation method for antibody-antigen binding reactions which are capable of detecting low levels of chemical binding.
It is a further object of this invention to provide a microcantilever assay for the detection of reactions involving biomolecules coupled to the microcantilever which results in a stress-induced bending of the cantilever.
It is an additional object of this invention to provide an ultra-miniature assay template which requires reduced sample volumes for operation.
It is a further and more particular object of this invention to provide an ultra-miniature microcantilever that provides a sensitivity sufficient to detect a single microorganism from a sample.
These and other objects of the invention are accomplished by an apparatus and a method for detecting and measuring highly specific binding reactions in a sampled media. The present invention provides a cantilever with one of its surfaces coated with specific binding partners such as antibody or antigen molecules, or with specific binding peptides identified from display libraries, while the other surface is covered with a different, possibly inert, material. As long as the amount of adsorption is different on the opposing surfaces, or there are different interactions of monitored molecules on opposing surfaces, there will be a differential stress. Since the cantilever thickness is very small, an antibody-antigen (Ab-Ag) type of interaction is manifested as changes in the differential surface stress of the microcantilever surface. This surface stress, for example, can originate from changes in volume, or charge,or polarization, or induced polarization due to the formation of chemical interactions. If a specific interaction does not take place, there will not be any change in surface stress when compared to a reference microcantilever. These changes in differential surface stress manifest themselves as changes in cantilever deflection which can be measured with a sub-angstrom sensitivity. The cantilever technique offers more simplicity and higher sensitivity than any currently used techniques.
Upon interaction with specific agents a stress is induced which deflects the spring element. The apparatus and the method provides a means for detection of the changes in deflection of the cantilevered spring element created by the physical binding or biochemical interaction stress. The deflection is measured and provides a basis for quantitative and qualitative analysis. The microcantilever assays provide sensitivities in the sub-nanometer range for deflections in response to stresses on the spring element.
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Photothermal Spectroscopy with
Doktycz Mitchel J.
Jacobson K. Bruce
Kennel Stephen J.
Thundat Thomas G.
Warmack Robert J.
Hardaway/Mann IP Group
O'Toole J. Herbert
Politzer Jay L.
U. T. Battelle, LLC
Williams Hezron
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