Patent
This invention relates to enzyme-sensitive biosensors and methods and kits using the same. Specifically, the invention relates to methods, systems and kits for the detection of enzymes using the chemical shift observed in an isotope complexed to the biosensor resulting from a change in the biosensor as the result of the enzyme's activity.
1. A chemical reaction-detection system comprising a biosensor, said biosensor comprises: a hyperpolarized noble element complexed with a monopropargyl-cryptophane-A coupled peptide, wherein the peptide is a reactant, specific to said detected chemical reaction and whereby modulation of the reactant induces change in the chemical environment of the noble element resulting in a chemical shift detectable by 29 Xe NMR spectroscopy.
2. The biosensor of claim 1 , wherein said hyperpolarized noble element is Xe.
3. The biosensor of claim 2 , wherein the hyperpolarized noble element is 129 Xe.
4. The biosensor of claim 1 , wherein the reactant is an enzyme and wherein the detected enzyme and its specific substrates is any one of the enzymes and their substrates in Table III.
5. A method of detecting and/or analyzing the activity of a matrix metalloproteinase, a caspase, an enzyme, or an integrin in a biological sample of a subject, comprising the steps of: contacting the biological sample with a biosensor responsive to said matrix metalloproteinase, caspase, enzyme, or integrin, said biosensor comprising a hyperpolarized noble element complexed with a monopropargyl-cryptophane-A coupled peptide, wherein the peptide is a substrate; specific to said detected matrix metalloproteinase, caspase, enzyme, or capable of interacting with the integrin and whereby cleavage of the substrate by the enzyme or internalization of the biosensor coupled to the integrin induces change in the chemical environment of the noble element resulting in a chemical shift detectable by NMR; and analyzing the chemical shift in said element, whereby a chemical shift indicates activity of said metalloproteinase or caspase.
6. The method of claim 5 , wherein said noble element is Xe.
7. The method of claim 6 , wherein the hyperpolarized noble element is 129 Xe.
8. The method of claim 5 , wherein said biological sample is blood, sputum, sera, urine, mucosa, feces, epidermal sample, skin sample, cheek swab, sperm, semen, amniotic fluid, cultured cells, bone marrow sample, chorionic villi, primary tumor biopsies, metastases biopsies, diffuse tumor biopsies, or a combination thereof.
9. A device for screening for the activity of a matrix metalloproteinase, a caspase, an enzyme, or an integrin in a biological sample, comprising a biosensor responsive to said matrix metalloproteinase, caspase, enzyme, or integrin, said biosensor comprising a hyperpolarized noble element complexed with a monopropargyl-cryptophane-A coupled peptide, wherein the peptide is a substrate, specific to said detected matrix metalloproteinase, caspase, enzyme, or capable of interacting with the integrin and whereby cleavage of the substrate by the enzyme or internalization of the biosensor coupled to the integrin induces change in the chemical environment of the noble element resulting in a chemical shift detectable by NMR; and instructions for use.
10. The device of claim 9 , wherein the matrix metalloproteinase (MMP) or enzyme is any MMP or enzyme described in Table III with its corresponding substrate as the peptide in said biosensor.
11. The device of claim 9 , further comprising an additional MMP, enzyme or integrin-responsive biosensor.
12. The device of claim 11 , wherein the additional matrix metalloproteinase (MMP) or enzyme is any MMP or enzyme described in Table III with its corresponding substrate as a peptide in said additional MMP-responsive biosensor.
13. The device of claim 9 , wherein said instructions, further comprise a library of chemical shifts identifying each matrix metalloproteinase or enzyme described in Table III.
14. The device of claim 9 , wherein said biological sample is blood, sputum, sera, urine, mucosa, feces, epidermal sample, skin sample, cheek swab, sperm, semen, amniotic fluid, cultured cells, bone marrow sample, chorionic villi, primary tumor biopsies, metastases biopsies, diffuse tumor biopsies, or a combination thereof.
15. A method of diagnosing a suspected cancer cell in vivo in a subject as malignant or benign, comprising the step of contacting a suspected cell with a composition comprising a fluorescently labeled biosensor wherein said biosensor comprises: a hyperpolarized noble element; complexed with a monopropargyl-cryptophane-A coupled peptide, wherein the peptide is capable of binding an integrin expressed on the surface of the suspected cancer cell and analyzing the change in fluorescent intensity of the cell before and after administration of the composition, whereby increase in fluorescent intensity, indicates the cell is malignant.
16. The method of claim 15 , wherein the peptide is tetra-RGD (SEQ ID. No. 30).
17. The method of claim 15 , wherein the detectable label is Cy3 near-infrared (NIRS) dye.
18. The method of claim 15 , wherein the cancer cell is melanoma, glioma, ovarian, prostate, breast, pancreatic or lung cancer cell.
19. The method of claim 15 , wherein the integrin is α v β33.