Chemistry: molecular biology and microbiology – Enzyme – proenzyme; compositions thereof; process for...
Reexamination Certificate
2000-12-04
2002-05-14
Jones, W. Gary (Department: 1655)
Chemistry: molecular biology and microbiology
Enzyme , proenzyme; compositions thereof; process for...
C436S172000, C436S501000, C436S546000, C252S301160, C252S301220
Reexamination Certificate
active
06387672
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Area of the Art
The invention relates generally to fluorescence sensor molecules and specifically to a new group of modular fluorescence sensor molecules.
2. Description of the Prior Art
Numerous assay methods have been developed for the detection and quantitative determination of analytes contained in biochemical samples. A substantial portion of the currently used assay methods relies on specific binding reactions between analytes and assay reagents. The analytes may be large complex molecules, such as proteins, viruses, viral antigens, bacterial cells, cell surface receptors, enzymes, hormones, polysaccharides, glycoproteins, lipoproteins, or small haptenic molecules, such as peptides, certain hormones, therapeutic drugs, and drugs of abuse, to name a few.
The binding assays can be divided into two major groups based on their format: homogeneous and heterogeneous assays. Homogeneous assays are based on a single-phase reaction between analyte and assay reagents. Heterogeneous assays, typically, involve binding of an analyte contained in the liquid sample to assay reagents, which are attached to a solid support. Various materials have been used as support surfaces, including glass rods, glass beads, silica impregnated strips, glass fiber, and microparticles.
Dyes in general, and fluorescent dyes in particular, are commonly utilized in both homogeneous and heterogeneous binding assays to provide a detectable signal. However, an accurate detection of fluorescent signals produced by analytes bound to the labels is often hindered by a high and variable background due to the fluorescence of the biological sample itself.
Liquid flow cytometry helps to overcome this problem. In flow cytometry, labeled particles with bound analyte are passed through a laser beam. The emitted fluorescent signals of the particles are measured and correlated to the presence and quantity of the analyte. The main advantage of this method is its capability of accurate detection and measurement of the fluorescent signals associated with the bound analyte in the presence of other unbound constituents of the sample.
Saccharides represent an important group of biochemical analytes. Current methods for determining their concentrations in a sample typically rely on enzymatic assays. Although enzymatic assays have proven to be reliable, they must utilize rather unstable enzymes, which become exhausted in the presence of their substrates. Additionally, conventional enzymatic assay methods cannot be utilized in a convenient flow cytometry format. Particle-based assays, such as the ones used in flow cytometry, require a signal change confined to the particle. Normal enzymatic analysis methods use freely diffusable intermediates that violate this requirement.
Determination of saccharides is particularly important in clinical settings. Treatment of diabetes and hypoglycemia requires frequent measurement of tissue glucose concentration. This is commonly accomplished by drawing a small blood sample (as by a fingerstick) several times daily. A patient typically uses a lancet to draw a droplet of blood and applies the droplet to a reagent strip which is read in a small meter. This process is painful, invasive, and time-consuming.
Recently, a minimally invasive method for measuring glucose in vivo has been disclosed in U.S. patent application Ser. No. 09/393,738 filed on Sep. 10, 1999, which has been commonly assigned to the assignee of the present invention and is incorporated by reference herein. The method is based on the use of implanted sensor particles capable of generating a detectable analyte signal in response to the analyte concentration in the body. The proposed method is less intrusive than the conventional fingerstick technique for measuring blood D-glucose. It requires only periodical replacement of the sensor particles in the skin.
The sensor particles typically comprise fluorescence sensors either attached to the surface or incorporated into the body of the particles. The sensors are specific to the target analyte. The binding of the sensor to the analyte generates a detectable signal that is responsive to the concentration of the analyte. When the analyte is glucose, diboronic acids conjugated to fluors are used.
Similar fluorescent sensors, which are specific to glucose, are also described by James et al., in the Journal of the American Chemical Society, 1995, vol. 117 pp. 8982-8987, by James et al. in U.S. Pat. No. 5,503, 770, and by Takeuchi et al. in Tetrahedron, vol. 52, No. 4, pp. 1195-1204. Briefly, the fluorescent sensors of James and Takeuchi have the following general formula:
In the formula, F designates a fluorophore, R is a lower aliphatic or aromatic group, and n+m is 2 or 3. The fluorescent intensity of the sensor changes in response to photo-induced electron transfer (PET) between the amine group and the fluorophore as modulated by binding of glucose hydroxyls to a pair of boronic acids. In the absence of glucose binding, the fluorescence by the fluorescent group is quenched by the unshared electron pair of the nitrogen atom. When glucose is bound, the unshared electron pair is utilized in the bond formation and does not participate in fluorescence-quenching. Consequently, intrinsic fluorescence of the sensor is expressed.
SUMMARY OF THE INVENTION
While an application of above-described fluorescent sensors in biochemical assays and clinical tests provide certain advantages over earlier enzyme-based in vitro methods, it, nevertheless, suffers from serious shortcomings. Because the fluorophore is used as a spacer to separate the saccharide binding groups and to provide desired analyte selectivity, strict limitations on the type of the fluorophore, its size and conformation are imposed. For the same reason, there are also significant limitations on the type, size, and conformation of analyte that can specifically bind to the disclosed fluorescent compounds to produce a detectable signal. Furthermore, although the referenced art suggests using the PET-type fluorescent compounds in a heterogeneous assay format, no means for attaching the compound to a solid support are provided. Again, such attachment would be particularly difficult in view of the strict limitations imposed on the conformation and structure of the fluorescent molecule.
Accordingly, it is an object of the present invention to provide a fluorescent sensor, particularly PET-type sensor, with a modular structure, which allows independent selection of fluorescent and binding groups. It is also an object of the present invention to provide a fluorescent sensor that can be easily adapted for specific binding to a broad range of analytes. It is a further object of the present invention to provide a fluorescent sensor that can be used in both homogeneous and heterogeneous binding assay formats and can be easily attached to solid surfaces. Finally, it is an object of the present invention to provide convenient methods of making and using the fluorescent sensors.
These and other objects and advantages are achieved in a modular fluorescence sensor of the present invention having the following general formula:
In the above formula, Fl is a fluorohore, N is a nitrogen atom, Bd1 and Bd2 are independently selected binding groups, Sp is an aliphatic spacer, and An is an anchor group for attaching the sensor to solid substrates. n=1 or 2, m=1 or 2, and x is an integer. The binding groups are capable of binding an analyte molecule to form a stable 1:1 complex. Examples of binding groups include, but are not limited to, boronic acid, crown ether, and aza-crown ether, such as 1,4,7,10,13-Pentaoxa-16-aza-cyclooctadecane (aza 18-crown-6) and 1,4,7,13-tetraoxa-10-aza-cyclohexadecane (aza 15-crown-5). In a preferred embodiment, the Bd1 is R1—B(OH)2 and Bd2 is R2—B(OH)2. R1 and R2 are aliphatic or aromatic functional groups selected independently from each other and B is a boron atom.
In another aspect, the present invention provides a method of synthesizing a modular fluorescence sensor. The method co
Arimori Susumu
James Tony D.
Beckman Coulter Inc.
Forman B J
Grant Arnold
Hogan & Hartson
Jones W. Gary
LandOfFree
Photo-induced electron transfer fluorescent sensor molecules does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Photo-induced electron transfer fluorescent sensor molecules, we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Photo-induced electron transfer fluorescent sensor molecules will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-2839507