Assay electrode having immobilized lipid/protein layers, methods of making the same and methods of using the same for luminescence test measurements
Improved systems, system components, and methods for performing assays involving biological membranes and/or components thereof. Preferred luminescence test measurements are conducted using an assay module with integrated electrodes having biological membranes and/or components thereof immobilized thereon with a reader apparatus adapted to receive assay modules, induce luminescence, preferably electrode induced luminescence, in the wells or assay regions of the assay modules and measure the induced luminescence.
1. A method of measuring an analyte in a lipid/protein layer, said method comprising:
(a) providing an electrode having a lipid/protein layer immobilized thereon, wherein said lipid/protein layer is comprised in or derived from whole cells, organelles, tissues, membrane fragments, liposomes, membrane ghosts, cell membranes, organelle membrane fragments, organelle membranes, virions, virion membrane fragments, virion membranes, liposomes, and combinations thereof;
(b) permeabilizing said lipid/protein layer;
(c) contacting said electrode with a solution comprising a binding partner of said analyte, said binding partner being labeled with an electrochemiluminescent label;
(d) forming a complex comprising said analyte and said binding partner; and
(e) measuring said complex using electrode induced luminescence.
2. The method of claim 1 , wherein said permeabilization step forms a stable tissue layer on said electrode.
3. The method of claim 1 , wherein said analyte comprises an internal cellular component.
4. The method of claim 1 , wherein said electrode induced luminescence is induced by applying electrical energy to said one or more electrodes.
5. The method of claim 1 , wherein said electrode induced luminescence is induced in the presence of a solution comprising an electrochemiluminescence coreactant.
6. The method of claim 5 , wherein said coreactant is a trialkylamine.
7. The method of claim 5 , wherein said solution does not comprise a surfactant.
8. The method of claim 5 , wherein said solution comprises a surfactant.
9. The method of claim 1 , wherein said analyte comprises a receptor.
10. The method of claim 9 , wherein said receptor is a membrane receptor.
11. The method of claim 10 , wherein said receptor is embedded into said lipid/protein layer.
12. The method of claim 9 , wherein said receptor remains active upon immobilization of said lipid/protein layer on said electrode.
13. The method of claim 9 , wherein said receptor is selected from a group consisting of transmembrane receptors with intrinsic tyrosine kinase activity, non-tyrosine kinase transmembrane receptors, G-protein coupled receptors, GPCR effector proteins, organelle-bound GTPases, guanine nucleotide exchange factors, GTPase activating proteins, cytokine/chemokine receptors, cell adhesion molecules, cytoplasmic peripheral membrane protein kinases, intracellular protein kinase adaptor/docking proteins, ion channels, passive transporter proteins, active ATP-driven transporters, ion-linked transporters, glycosyltranferases and glycoprotein modifying enzymes.
14. The method of claim 1 , wherein said lipid/protein layer is immobilized on said electrode via pokeweed mitogen.
15. The method of claim 1 , wherein a plurality of lipid/protein layers are immobilized on said electrode in the form of an array and at least two of the lipid/protein layers differ in composition and/or biological source.
16. The method of claim 15 , wherein said lipid/protein layer is comprised in or derived from a substance selected from virions, tissues, liposomes, membrane ghosts or combinations thereof.
17. The method of claim 16 , wherein said analyte is a receptor and said binding partner comprises a ligand of said receptor.
18. The method of claim 1 , wherein said lipid/protein layer is comprised in or derived from membrane fragments.
19. The method of claim 14 , wherein said pokeweed mitogen has a protein/carbohydrate ratio greater than or equal to 1.0.
20. The method of claim 19 , wherein said pokeweed mitogen has a protein/carbohydrate ratio greater than or equal to 2.5.
21. The method of claim 14 , wherein said pokeweed mitogen has a protein/carbohydrate ratio greater than or equal to 9.
22. The method of claim 1 , wherein said electrode is a screen-printed electrode.
23. The method of claim 1 , wherein said electrode is treated to enhance the adsorptive properties of said electrode.
24. The method of claim 23 , wherein said carbon ink electrode is plasma-treated.
25. The method of claim 1 wherein said electrode comprises a spacer layer between said electrode and said lipid/protein layer, wherein said spacer layer occupies a hydrophilic spacer volume.
26. The method of claim 1 wherein said lipid/protein layer retain at least 20% of its biological activity after immobilization to said electrode.
27. The method of claim 1 wherein said lipid/protein layer retain at least 30% of its biological activity after immobilization to said electrode.
28. The method of claim 1 wherein said lipid/protein layer retain at least 40% of its biological activity after immobilization to said electrode.
29. The method of claim 1 wherein said lipid/protein layer retain at least 50% of its biological activity after immobilization to said electrode.
30. The method of claim 1 wherein lipid/protein layer is immobilized on said electrode via lectin.
31. A method of measuring a plurality of analytes in a lipid/protein layer, wherein said plurality of analytes comprises internal cellular components, external cellular components, or combinations thereof, said method comprising:
(f) providing an electrode having a lipid/protein layer immobilized thereon, wherein said lipid/protein layer is comprised in or derived from whole cells, organelles, tissues, membrane fragments, liposomes, membrane ghosts, cell membranes, organelle membrane fragments, organelle membranes, virions, virion membrane fragments, virion membranes, liposomes, and combinations thereof;
(g) permeabilizing said lipid/protein layer;
(h) contacting said electrode with a solution comprising a plurality of binding partners of said plurality of analytes, wherein said plurality of binding partners are labeled with an electrochemiluminescent label;
(i) forming a plurality of complexes each comprising an analyte bound to a binding partner of said cellular component; and
(j) measuring said plurality of complexes using electrode induced luminescence.
32. The method of claim 31 wherein said plurality of analytes comprises a combination of internal and external cellular components.
33. The method of claim 31 , wherein said electrode induced luminescence is induced by applying electrical energy to said one or more electrodes.
34. The method of claim 31 , wherein said electrode induced luminescence is induced in the presence of a solution comprising an electrochemiluminescence coreactant.
35. The method of claim 34 , wherein said coreactant is a trialkylamine.
36. The method of claim 34 , wherein said solution does not comprise a surfactant.
37. The method of claim 34 , wherein said solution comprises a surfactant.
38. The method of claim 31 , wherein said plurality of analytes comprises a receptor.
39. The method of claim 38 , wherein said receptor is a membrane receptor.
40. The method of claim 39 , wherein said receptor is embedded into said lipid/protein layer.
41. The method of claim 39 , wherein said receptor remains active upon immobilization of said lipid/protein layer on said electrode.
42. The method of claim 38 , wherein said receptor is selected from a group consisting of transmembrane receptors with intrinsic tyrosine kinase activity, non-tyrosine kinase transmembrane receptors, G-protein coupled receptors, GPCR effector proteins, organelle-bound GTPases, guanine nucleotide exchange factors, GTPase activating proteins, cytokine/chemokine receptors, cell adhesion molecules, cytoplasmic peripheral membrane protein kinases, intracellular protein kinase adaptor/docking proteins, ion channels, passive transporter proteins, active ATP-driven transporters, ion-linked transporters, glycosyltranferases and glycoprotein modifying enzymes.
43. The method of claim 31 , wherein a plurality of lipid/protein layers are immobilized on said electrode in the form of an array and at least two of the lipid/protein layers differ in composition and/or biological source.
44. The method of claim 31 , wherein said permeabilization step forms a stable tissue layer on said electrode.
45. The method of claim 31 , wherein said electrode is a screen-printed electrode.
46. The method of claim 31 , wherein said electrode is treated to enhance the adsorptive properties of said electrode.
47. The method of claim 46 , wherein said electrode is plasma-treated.
48. The method of claim 31 , wherein said electrode comprises a spacer layer between said electrode and said lipid/protein layer, wherein said spacer layer occupies a hydrophilic spacer volume.
49. The method of claim 31 , wherein said lipid/protein layer retains at least 20% of its biological activity after immobilization to said electrode.
50. The method of claim 31 , wherein said lipid/protein layer retains at least 30% of its biological activity after immobilization to said electrode.
51. The method of claim 31 , wherein said lipid/protein layer retains at least 40% of its biological activity after immobilization to said electrode.
52. The method of claim 31 , wherein said lipid/protein layer retains at least 50% of its biological activity after immobilization to said electrode.
53. The method of claim 31 wherein said lipid/protein layer is immobilized on said electrode via lectin or pokeweed mitogen.