Chemistry: analytical and immunological testing – Involving kinetic measurement of antigen-antibody reaction
Reexamination Certificate
1999-10-28
2003-12-23
Le, Long V. (Department: 1641)
Chemistry: analytical and immunological testing
Involving kinetic measurement of antigen-antibody reaction
C435S006120, C435S007100, C436S523000, C436S527000, C436S537000, C436S546000, C436S164000, C436S165000, C436S172000, C436S805000
Reexamination Certificate
active
06667179
ABSTRACT:
FIELD OF THE INVENTION
The field of the invention is the use of semiconductor substrate quenchers of luminescence to detect proximal molecular binding events.
BACKGROUND OF THE INVENTION
Methods for detecting the binding of molecules to surfaces provide applications in numerous industries. For example, surface plasmon resonance has been used to detect molecular binding in a host of chemical and biotechnological fields, e.g. Deckert and Legay, 1999, Anal Biochem 1;274(1):81-9; Gomes et al., 1999, Vaccine, 18, 362-370; and Fierobe et al., 1999, Biochemistry 38, 12822-32.
Among certain physicists and material scientists, it is known that metals and semimetals can function as luminescence quenchers over short distances (see, e.g. Sluch et al., 1995
, Anomalous distance dependence of fluorescence lifetime quenched by a semiconductor
, Physical letters A200 (1995), 61-64). At least two distinct phenomenon are believed to contribute to proximal quenching: first, the excitation intensity is reduced for distances smaller than &lgr;/4 due to destructive interference of the incoming wave with the partially reflected wave from the surface (e.g. the silicon/silicon oxide boundary of silicon semiconductors); and second, the semiconductor functions as an acceptor by energy transfer. To date, the exploitation of this quenching property has been limited: Walczak et al. examined molecular distances from gold particles (Walczak et al., “
Golden ruler”: Very long
-
range resonance energy transfer to surface plasmon acceptors
, 1997, Biophysical Journal, Febuary 1997 Vol. 72, No. 2, Part 2, pp. TU367-TU367) and Nakache et al. measured mobility of a phospholipid in fluorescent recovery after photobleaching (FRAP) experiments (Nakache et al.,
Heterogeneity of membrane phospholipid mobility in endothelial cells depends on cell substrate
, 1985, Nature 317, 75-77). In fact, gold particles have been widely used in analytical applications, primarily for its heavy, electron-dense and inert properties, though its usefulness has been limited by the difficulty adhering to it biological molecules (see, e.g. Kramarcy and Sealock, 1991, J. Histochem Cytochem 39, 7-39).
Here we disclose that semiconductor luminescent quenching can be successfully applied to binding assays, and particularly to assaying binding and unbinding of receptor-ligand pairs. Our methods provide exceptionally large R
q
(distance at which quenching is 50%) values, e.g. approx. 50 nm, and more. This very large R
q
provides significant advantages over conventional fluorescence resonance energy transfer (FRET). For example, even with large receptors, the luminophore-ligand will be highly quenched—in contrast to conventional FRET which does not work well with large biomolecules. In addition, only the ligand need be labeled with luminophore in our technique, whereas in conventional FRET both ligand and receptor need be labeled.
The large R
o
also permits a number of hitherto impractical applications. For example, the semiconductor may be coated with a glass layer, a protein layer (e.g. biotinylated bovine serum albumin) can then attached to the glass layer, a second surface which acts as a crosslinker, such as streptavidin, can then be attached thereto, and finally, the biotinylated receptor then attached to the streptavidin. By forming a protein layer between the glass and receptor of interest, the receptor is much more likely to maintain its biological activity, since it is known that interaction with glass often denatures biomolecules.
Our technique also has advantages compared to fluorescence depolarization assays because the latter require that the ligand-bound fluorophore undergo a sizeable change in mobility upon ligand-binding to the receptor. This is often not the case and is difficult to optimize. In our technique, the luminophore's detailed interaction with the ligand and/or receptor is unimportant. The luminophore need only be attached to the ligand and the ligand upon binding to the receptor be brought in reasonably close proximity to the quenching surface.
SUMMARY OF THE INVENTION
The invention provides methods and compositions for detecting binding or unbinding of a molecule to a substrate. The molecule comprises a luminophore and the substrate comprises a semiconductor which acts as a luminescence quencher to provide distance-dependent quenching of the luminophore. Binding or unbinding of the molecule, which may be covalent or noncovalent, is detected as a decrease or increase, respectively, of the detectable luminescence of the luminophore.
The substrate may comprise a wide variety of semiconductors such as silicon, germanium, gallium-arsenide alloys, III/V alloys, etc., and take a wide variety of forms such as crystalline and amorphous materials, micro and nano particle suspensions, etc. The substrate may be coated, derivatized, etc. to provide improved and/or specific binding of the molecule. For example, the substrate may be chemically derivatized with thiol groups or coated with one or more layers of material such as glass, polyethylene glycol (PEG), protein, membrane, etc.
A wide variety of molecules and bindings/unbindings may be detected. In a particular embodiment, the detected binding or unbinding is specifically mediated, such as in a specific molecular binding pair, i.e. ligand and cognate receptor. In a particular aspect of this embodiment, the subject methods involve:
(a) forming a mixture comprising first and second molecules and a substrate, wherein:
the first molecule comprises a luminophore,
one of the first and second molecules is immobilized on the substrate, and
the substrate comprises a semiconductor which acts as a luminescence quencher to provide distance-dependent quenching of the luminophore, and between the semiconductor and the immobilized molecule, a molecular attachment layer,
whereby but for an incubation-induced change in binding of the first and second molecules, the luminophore and substrate are at a reference distance which provides a reference quenching, whereby the mixture provides a reference luminescence,
(b) incubating the mixture under conditions wherein the binding of the first and second molecules changes, whereby the luminophore and substrate are at a test distance which provides a test quenching, whereby the mixture provides a test luminescence, and
(c) detecting the test luminescence, wherein a difference between the test luminescence and the reference luminescence indicates binding or unbinding of the first and second molecules.
This assay may be constructed in a wide variety of ways with a wide variety of receptor ligand pairs. As examples, the assay encompasses methods wherein:
the forming step, the second molecule is immobilized on the substrate and the first molecule is bound to the second molecule, and
wherein the incubating step the first and second molecule unbind, releasing the luminophore from the substrate, e.g. wherein the first molecule is selected from an antigen, cytokine, hormone and a neurotransmitter and the second molecule is a corresponding receptor and optionally, wherein the forming step, the mixture further comprises a modulator which modulates binding of the first and second molecules;
the forming step, the first molecule is immobilized on the substrate and the second molecule is unbound to the first molecule, and wherein the incubating step the first and second molecule bind and release the luminophore from the substrate, e.g. wherein the second molecule is an enzyme selected from a protease, nuclease, helicase, kinase and phosphatase;
the molecular attachment layer comprises a material such as a glass; and
the substrate further comprises a phospholipid bilayer between the molecular attachment layer and the immobilized molecule, e.g. wherein the forming step, the immobilized molecule is bound to or in the membrane.
DETAILED DESCRIPTION OF SPECIFIC EMBODIMENTS OF THE INVENTION
The following descriptions of particular embodiments and examples are offered by way of illustration and not by way of limitation. As explained above, the subject binding assay
Gabel Gailene R.
Le Long V.
Osman Richard Aron
The Board of Trustees of the University of Illinois
LandOfFree
Semiconductor luminescence quenchers for detecting proximal... does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Semiconductor luminescence quenchers for detecting proximal..., we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Semiconductor luminescence quenchers for detecting proximal... will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-3120995