Optics: measuring and testing – For light transmission or absorption
Reexamination Certificate
2001-07-23
2004-03-23
Lee, Diane I. (Department: 2876)
Optics: measuring and testing
For light transmission or absorption
C356S336000, C356S445000
Reexamination Certificate
active
06710877
ABSTRACT:
FIELD OF THE INVENTION
This invention relates to sensing of diffusion of biomolecules. More particularly, the present invention relates to substrates, apparatus and methods for monitoring and detecting interactions between a ligand and a receptor.
BACKGROUND OF THE INVENTION
The drug discovery process is a highly risky venture. The Pharmaceutical Research and Manufacturers of America estimate the average time to bring a drug to market is approximately 12 to 15 years at an average cost of approximately 500 million dollars. Among US pharmaceutical companies, more than 100 new therapeutic treatments have been added to the available medicines in the last two years. Among the many sequence of events applied in the pharmaceutical industry to realize commercially successful products, high-throughput screening (HTS) is believed to be an essential cornerstone of an effective drug discovery strategy. The HTS market in 1998 was estimated at 1.6 billion dollars.
High-throughput screening (HTS) refers to the initial activity in the pharmaceutical development process that systematically compares the binding of a target molecule or compound with each compound archived in a pharmacophore compound library. These libraries may contain millions of potential drugs acquired from a variety of sources, natural or otherwise, that are systematically screened for bioactivity against target molecules or compounds. The remarkable biological and biochemical advances of the last decade at the cellular and molecular levels have created numerous opportunities for discovery by uncovering an abundance of new receptors and enzymes that are mechanistically associated with disease pathologies. As these new targets emerge, a demand on analytic capacity to screen the targets against the large compound libraries for “hits” becomes a tremendous logistical effort.
The development of a technology applying direct binding assays (DBA) to high-throughput screening (HTS) could capture a significant share of the market for HTS. Surface-plasmon resonance (SPR) is a popular DBA technique in the pharmaceutical industry. SPR is but one of a large class of optical biosensors collectively referred to as evanescent wave detectors. This class includes film waveguide grating couplers, film prism waveguide couplers and long-period fiber waveguide couplers. The essential feature of all these techniques is that a standing “evanescent” wave is generated above the sensing surface by a wavelength's distance from the surface (approximately 100-200 nm) that is sensitive to the local dielectric environment. By changing the local refractive index, the standing wave is altered, requiring either a new angle of incident light to set up the “resonance condition” or inducing a phase shift of the reflected light. Since all proteins, independent of sequence, contribute the same refractive index per unit mass, this technique can serve as a mass detector. A linear correlation between resonance angle shift and surface protein concentration has been demonstrated, allowing real time detection of mass change without the need for labeling. All evanescent wave techniques are variations on this essential theme.
To distinguish between molecules floating in the bulk solution from those molecules attached to surface-bound target molecules, evanescent-based biosensors require that one flows buffer solution over the sensing surface after having flowed the buffer/analyte solution to infer the presence of surface bound analyte. Measurement of the adsorption in real time requires complex fluidic control of different flow conditions and solutions to infer the association rate constants from the observed binding rates. This is because evanescent-based methods do not distinguish between molecules floating in the evanescent standing wave and molecules anchored to the substrate also present in the evanescent field.
Another limitation of evanescent wave methods is that they do not readily lend themselves to miniaturization. In addition, light is invariably coupled into substrates with non-zero angles-of-incidence, often requiring complex schemes to measure extremely small shifts in reflected light or inducing small angular shifts in the incident light. Such limitations make massive deployment of similar sensing elements on small chips extremely problematic.
It would be desirable to provide substrates, apparatus and methods that do not encounter the drawbacks of evanescent-wave based methods. Many drugs are small molecules in the range between 200-1000 Dalton with binding affinities in the range between 10
−6
and 10
−12
M. Therefore, it would be advantageous to provide methods and apparatus capable of characterizing small-drug and target-protein binding interactions. It would also be advantageous to provide HTS methods and apparatus that are capable of being “homogeneous and label-free” and provide the same information content as the more labor-intensive methods discussed above.
SUMMARY OF INVENTION
The invention relates to assay methods and apparatus for monitoring biological or chemical interactions by providing means to monitor and/or detect diffusion proximate a sensing area. According to the present invention, the interactions between and among chemicals, cells and biomolecules can be detected by monitoring the diffusion of a molecule or chemical proximate a sensing area. Such diffusion monitoring provides the ability to detect and measure interactions between ligands and receptors, such as a protein molecule or a cell.
According to one aspect of the invention, an apparatus is provided that measures rate of diffusion proximate a sensing area. According to another aspect of the invention, the rate of diffusion may be measured by several means. For example, the rate of diffusion proximate the sensing area may be measured by monitoring the concentration of a molecule proximate the sensing area. One example of such an apparatus may include a first area that may optionally contain a matrix material and receptor molecule or cell contained in the first area. The matrix material can be any suitable matrix for the receptor, such as a polymeric matrix material. According to this aspect of the invention, the apparatus preferably includes a second area adjacent the first area and a boundary area is disposed between the first and second areas. According to this aspect of the invention, preferably a ligand molecule is contained in the first area with the receptor and the matrix material. The second area preferably contains a solution such as a buffer solution.
According to another aspect of the invention, the ligand is smaller than the receptor. For example, the ligand molecule may be a drug molecule having a molecular weight less than 1000 Daltons, and the receptor has a molecular weight greater than 5 kiloDaltons. According to another aspect, the boundary area includes a membrane operative to allow ligand molecules to pass therethrough and to prevent passage of receptor molecules In another aspect of the invention, the receptor includes a protein molecule. It is understood that the invention is not limited to any particular ligands and receptors. As such, the receptors could include a wide variety of biomolecules including, but not limited to proteins, nucleic acids, and cells.
According to still another aspect of the invention, the means for detecting the diffusion of the molecules or cells includes an optical detector. A wide variety of suitable optical detection systems can be used in accordance with the present invention. For example, a light source and a light detector can be used. The light source may be, for example, an ultraviolet (UV) light source, and the light detector can be a charge-coupled light detector. According to this aspect, the light may be directed towards the second area, and the optical detector is positioned and operative to measure the change in light absorbed by the second area.
According to another embodiment of the invention, the means for detecting the rate of diffusion of the molecules or cells includes a diffraction device that
Chase Christopher J.
Kalal Peter J.
Quesada Mark A.
Shi Youchun
Corning Incorporated
Kim Ahshik
Kung Vincent T.
Lee Diane I.
Servilla Scott S.
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