| Sensor constructs and detection methods -> Monitor Keywords |
|
|
  Sensor constructs and detection methodsUSPTO Application #: 20070248994Title: Sensor constructs and detection methods Abstract: Sensor constructs for detecting the presence of an analyte in a sample comprising a molecular scaffold, a pair of labels which interact via Förster resonance energy transfer, and at least one molecular recognition domain for binding the analyte. A changed optical signal is produced when the analyte is bound by the molecular recognition domain. (end of abstract) Agent: Sheldon Mak Rose & Anderson PC - Pasadena, CA, US Inventor: Wei-Wen SU USPTO Applicaton #: 20070248994 - Class: 435007100 (USPTO) Related Patent Categories: Chemistry: Molecular Biology And Microbiology, Measuring Or Testing Process Involving Enzymes Or Micro-organisms; Composition Or Test Strip Therefore; Processes Of Forming Such Composition Or Test Strip, Involving Antigen-antibody Binding, Specific Binding Protein Assay Or Specific Ligand-receptor Binding Assay The Patent Description & Claims data below is from USPTO Patent Application 20070248994. Brief Patent Description - Full Patent Description - Patent Application Claims
[0001] This application claims the benefit of priority from co-pending U.S. patent application Ser. No. 11/083,461, filed Mar. 17, 2005 and titled "Sensor Constructs and Detection Methods," which claims the benefit of priority from U.S. Provisional Patent Application No. 60/554,313, filed Mar. 17, 2004 and titled "Novel Fluorescent Nanosensor Proteins." The disclosures of each of the foregoing patent applications are incorporated herein by reference in their entirety. BACKGROUND [0002] Immunoassays and assays based on the polymerase chain reaction (PCR) are among the most widely used techniques for detecting analytes. While PCR-based techniques generally give good detection sensitivity, they have not been widely adopted for routine screening in many laboratories. One reason is that when PCR is used for detecting analytes in biological materials, its accuracy is strongly influenced not only by the performance of the PCR assay itself, but also by the quality of the nucleic acids extracted from the biological materials being tested. In addition, the detection sensitivity of PCR-based methods can be reduced by inhibitors in the extract that may interfere with the amplification process. Trained lab personnel are also required to conduct PCR assays in order to assure their accuracy. [0003] Immunoassays are generally less expensive and require less training to perform. They have been adopted in a variety of formats, with enzyme linked immunosorbent assays (ELISAs), lateral-flow immunoassays, and Western-blot assays being the most common. Current ELISA and Western-blot techniques, however, require multiple incubation steps and are prone to operator error. Lateral-flow immunoassays are generally faster but are not as accurate as conventional ELISAs. [0004] An alternative assay format has been proposed by Tsien, et al. for detecting analytes. This technique, described in U.S. Pat. No. 5,998,204, makes use of a protein having an analyte-binding region and two fluorescent labels. When the analyte-binding region binds an analyte, a conformational change occurs which causes the two fluorescent labels to change position relative to each other. This alters a fluorescence resonance energy interaction between the labels, which is detected in order to determine analyte binding. [0005] Frommer, et al. have proposed a similar assay format in PCT International Application No. 03/025220. This assay makes use of a fusion protein that includes a periplasmic binding protein portion and two fluorescent protein portions. The fusion protein changes conformation upon binding an analyte, changing the relative positions of the two fluorescent protein portions. An altered fluorescence resonance energy interaction is thereby induced between the fluorescent protein moieties. [0006] The assay systems disclosed by Tsien and Frommer both require the use of sensor constructs which change conformation upon binding an analyte of interest. Both, moreover, detect only small analytes such as simple sugars and amino acids. There remains a need, therefore, for a more general approach to analyte detection which takes advantage of resonance energy transfer interactions but which is not limited to constructs that must change conformation in order to detect an analyte or that detect only small analytes. SUMMARY [0007] The present method of detecting an analyte in a sample involves the use of a sensor construct that comprises: (i) a molecular recognition domain which specifically binds the analyte; (ii) a first label comprising an RET donor; and (iii) a second label comprising an RET acceptor for the RET donor. The RET acceptor is separated from the RET donor by a distance that allows Forster resonance energy transfer from the donor to the acceptor to occur, preferably by a distance of from about 1 to about 25 nanometers. When the molecular recognition domain of the sensor construct binds the analyte, proximity of the analyte to the RET donor and/or the RET acceptor interferes with a FRET interaction between the RET donor and RET acceptor, resulting in a detectable optical signal. The presence of the analyte in the sample is indicated by the detection of this optical signal. [0008] The present methods can further include the more specific steps of measuring a control optical signal generated by a Forster resonance energy transfer interaction between the RET donor and the RET acceptor of the sensor construct prior to contacting the sensor construct with the sample, and then determining the difference between the control optical signal and the optical signal detected after contacting the sensor construct with the sample. The difference between this signal and the control optical signal is indicative of the amount of the analyte in the sample, thereby allowing quantitative detection of the analyte. Preferably, a change in the intensity or decay kinetics of the fluorescence or luminescence of the sensor construct is measured as the optical signal. [0009] In the present methods, the RET donor can be any of a number of fluorescent or luminescent moieties, such as a fluorescent protein, a luminescent protein, a non-protein fluorophore, a non-protein chemiluminescent compound, or a fluorescent nanocrystal. The RET acceptor can be a molecule which quenches a signal from the RET donor, and in some embodiments displays an increased, sensitized acceptor signal. In order to increase the intensity of the optical signal, multiple RET donors and acceptors can also be included on each sensor construct. The sensor construct itself can be a polypeptide, and can comprise an antibody or antibody fragment such as a Fv portion of an antibody. In such embodiments, the molecular recognition domain is a CDR region of the Fv portion of the sensor construct. The molecular recognition domain can alternatively comprise a metal, such as a chelated metal, or an oligonucleotide. [0010] In some embodiments of the sensor construct, the molecular scaffold, the molecular recognition domain, and/or the labels can be provided as separate molecules and then combined to form the sensor construct. For example, the molecular scaffold can comprise a heterodimeric coiled coil polypeptide pair and the molecular recognition domain can comprise the binding domain of a Fv fragment of an antibody (comprising a V.sub.H and a V.sub.L fragment). The molecular recognition domain in this embodiment can also be a chelated metal, an oligonucleotide, a peptide, a biotin molecule, or a non-peptide enzyme substrate/inhibitor molecule. The different components of the sensor construct in such embodiments are self-assembling when brought into contact with one another and allow Forster resonance energy transfer from the RET donor to the RET acceptor to occur. DRAWINGS [0011] These and other features, aspects and advantages of the present invention will become better understood with regard to the following description, appended claims, and accompanying figures where: [0012] FIG. 1 illustrates the binding of an analyte to a sensor construct according to the present methods. [0013] FIG. 2 illustrates an alternative embodiment of the present sensor construct in which the MRD is attached to a label. [0014] FIG. 2A illustrates another embodiment of the present sensor construct in which the MRD is attached to a label. [0015] FIG. 3 illustrates the binding of an analyte to a further alternative embodiment of the present sensor construct. [0016] FIG. 3A illustrates the binding of an analyte to a yet another embodiment of the present sensor construct. [0017] FIG. 4 illustrates a further alternative embodiment of the present sensor construct in which the MRD is self-assembling. [0018] FIG. 4A illustrates an embodiment of the sensor construct depicted in FIG. 4 in which the labels are also self-assembling. [0019] FIG. 5A illustrates the assembly of another self-assembling embodiment of the present sensor construct. [0020] FIG. 5B illustrates the binding of an analyte to the sensor construct depicted in FIG. 5A. [0021] FIG. 6 illustrates an embodiment of the present sensor construct in which the molecular scaffold is self-assembling. Continue reading... Full patent description for Sensor constructs and detection methods Brief Patent Description - Full Patent Description - Patent Application Claims Click on the above for other options relating to this Sensor constructs and detection methods patent application. ###  How KEYWORD MONITOR works... a FREE service from FreshPatents1. Sign up (takes 30 seconds). 2. Fill in the keywords to be monitored. 3. Each week you receive an email with patent applications related to your keywords. Start now! - Receive info on patent apps like Sensor constructs and detection methods or other areas of interest. ### Previous Patent Application: Methods and compositions for separating cells Next Patent Application: Systems and methods for predicting an individual's risk of developing rheumatoid arthritis Industry Class: Chemistry: molecular biology and microbiology ### FreshPatents.com Support Thank you for viewing the Sensor constructs and detection methods patent info. IP-related news and info Results in 0.85854 seconds Other interesting Feshpatents.com categories: Software: Finance , AI , Databases , Development , Document , Navigation , Error |
||
