Multiplex analysis using membrane-bound sensitizers
View Patent ↗The present invention is directed to methods and compositions for determining the presence, absence, and/or amounts of one or more membrane-associated analytes in a sample. In accordance with the invention, binding compounds derivatized with releasable molecular tags specifically bind to selected membrane-associated analytes, after which the molecular tags are released upon activation of cleavage moieties, or sensitizers, anchored in the same membrane as the membrane-associated analytes. The released molecular tags are then identified by their distinct separation and detection characteristics.
1. A method for determining the presence or absence of one or more membrane-associated analytes, the method comprising the steps of:
providing a lipid membrane comprising one or more membrane-associated analytes and a lipophilic sensitizer anchored therein;
providing one or more binding compounds, such that there is at least one binding compound specific for each of the one or more membrane-associated analytes, each binding compound having one or more molecular tags, each molecular tag being attached to the binding compound by a cleavable linkage, and the molecular tags of each binding compound being distinguishable from those of every other binding compound by one or more physical and/or optical characteristcs;
combining the lipid membrane and the plurality of binding compounds specific for the one or more membrane-associated analytes such that in the presence of a membrane-associated analyte a complex is formed between such membrane-associated analyte and a binding compound specific therefor;
activating the lipophilic sensitizer in the lipid membrane so that an active species is produced that cleaves cleavable linkages between molecular tags and binding compounds that form said complexes so that molecular tags are released; and
separating and identifying the released molecular tags by the one or more physical characteristics to determine the one or more membrane-associated analytes in the sample.
2. The method of claim 1 wherein said step of separating includes electrophoretically separating said released molecular tags, and wherein each of said molecular tags has a molecular weight in the range of from 50 to 1500 daltons.
3. The method of claim 2 , wherein said active species is singlet oxygen, and wherein each of said molecular tags attached to said binding compounds are selected from a group defined by the formula:
-L-(M,D)
wherein:
L is a cleavable linkage;
D is a detection moiety; and
M is a bond or a water soluble organic compound consisting of from 1 to 100 atoms, not including hydrogen, that are selected from the group consisting of carbon, oxygen, nitrogen, phosphorus, boron, and sulfur.
4. The method of claim 3 wherein D is a fluorescent label, a chromogenic label, or an eleotrochemical label.
5. The method of claim 4 wherein M is a polymer selected from anyone of polyethers, polyesters, polypeptides, oligosacoharides, polyurethanes, polyamides, polysulfonamides, polysulfoxides, polyphosphonates, and block copolymers thereof.
6. The method of claim 5 wherein D is a fluorescein.
7. The method of claim 6 wherein said fluorescein is selected from the group consisting of 5- and 6-carboxyfluorescein, 5- and 6-carboxy-4,7-dichlorofluorescein, 2′,7′-dimethoxy-5- and 6-carboxy-4,7-dichlorofluorescein, 2′,7′-dimethoxy-4′,5′-dichloro-5- and 6-carboxyflurescein, 2′,7′-dimethoxy-4′,5′-dichloro-5- and 6-carboxy-4,7-dichlorofluorescein, 1′,2′,7′,8′-dibenzo-5- and 6-carboxy-4,7-dichlorofluorescein, 1′,2′,7′,8′-dibenzo-4′,5′,-dichloro-5- and 6-carboxy-4,7-dichloroflurescein, 2′,7′-dichloro-5- and 6-carboxy-4,7-dichlorofluorescein, and 2′,4′,5′,7′-tetrachloro-5- and 6-carboxy-4,7-dichlorofluorescein.
8. The method of claim 3 wherein L is selected from the group consisting of olefins, thioethers, selenoethers, thaizoles, oxazoles, and imidazoles.
9. The method in accordance with claims 1 , 2 , 3 , 4 , 5 , 6 , 7 , or 8 wherein said one or more binding compounds are a plurality of binding compounds in the range of from 2 to 50.
10. The method of claim 9 wherein each of said binding compounds is an antibody binding composition.
11. The method of claim 9 wherein said one or more physical characteristics is electrophoretic mobility.
12. The method of claim 11 wherein said step of separating and identifying include forming an electropherogram in which each of said released molecular tags forms a distinct peak.
13. The method in accordance with claim 9 wherein said lipid membrane is a lipid bilayer membrane.
14. The method of claim 13 wherein said lipid bilayer membrane is a biological membrane, a micelle, or a liposome.
15. The method of claim 13 wherein said lipophilic sensitizer is a lipophilic photosensitizer.