Spatially-addressable immobilization of anti-ligands on surfaces

A method for attaching a plurality of anti-ligands having different ligand binding specificities to predefined regions on a surface, said method comprising the steps of:

• a) attaching to the surface caged biotin analogs having a low affinity for the anti-ligand or specific binding substances, said caged biotin analogs comprising a photolabile chemical protecting group;
• b) irradiating the caged biotin analogs on a first predefined region of the surface, thereby forming biotin analogs on the first predefined region having an affinity constant of 10.sup.7 or stronger for an anti-ligand or a specific binding substance;
• c) reacting the biotin analogs on the first predefined region with either (i)(a) the specific binding substance and a first anti-ligand or (b) a first anti-ligand attached to a specific binding substance, to form a biotin analog-specific binding substance-anti-ligand complex;
• or (ii) a first anti-ligand to form a biotin analog-anti-ligand complex;
• d) washing the surface to remove unbound specific binding substance or anti-ligand;
• e) repeating steps (b-d) on a different region of the surface with a different anti-ligand.

A method of screening a plurality of anti-ligands localized on predefined regions of a surface for affinity for ligands in solution comprising:

• a) attaching to the surface caged biotin analogs having a low affinity for the anti-ligand or specific binding substances, said caged biotin analogs comprising a photolabile chemical protecting group;
• b) irradiating the caged biotin analogs on a first predefined region of the surface, thereby forming biotin analogs on the first predefined region having an affinity constant of 10.sup.7 or stronger for an anti-ligand or a specific binding substance;
• c) reacting the biotin analogs on the first predefined region with either (i)(a) the specific binding substance and a first anti-ligand or (b) a first anti-ligand attached to a specific binding substance, to form a biotin analog-specific binding substance-anti-ligand complex;
• or (ii) a first anti-ligand to form a biotin analog-anti-ligand complex;
• d) washing the surface to remove unbound specific binding substance or anti-ligand;
• e) repeating steps (b-d) on a different region of the surface with a different anti-ligand to create a surface having a plurality of anti-ligands non-covalently bound on predefined regions of the surface;
• f) exposing the surface from step (e) to a solution containing at least one marked ligand;
• g) washing the surface free of unbound ligand;
• and
• h) identifying ligands binding to anti-ligands by determining regions on the surface where markers are located.

A method of performing a competitive assay of a ligand in solution by measuring the ligand's affinity for anti-ligands immobilized on predefined regions of a surface by the steps of:

• a) attaching to the surface caged biotin analogs having a low affinity for the anti-ligand or specific binding substances, said caged biotin analogs comprising a photolabile chemical protecting group;
• b) irradiating the caged biotin analogs on a first predefined region of the surface, thereby forming biotin analogs on the first predefined region having an affinity constant of 10.sup.7 or stronger for an anti-ligand or a specific binding substance;
• c) reacting the biotin analogs on the first predefined region with either (i)(a) the specific binding substance and a first anti-ligand or (b) a first anti-ligand attached to a specific binding substance, to form a biotin analog-specific binding substance-anti-ligand complex;
• or (ii) a first anti-ligand to form a biotin analog-anti-ligand complex;
• d) washing the surface to remove unbound specific binding substance or anti-ligand;
• e) repeating steps (b-d) on a different region of the surface with a different anti-ligand to create a surface having a plurality of anti-ligands non-covalently bound on predefined regions of the surface;
• f) incubating a solution containing at one or more marked ligands and unknown ligands with the surface of step (e);
• g) washing the surface free of unbound ligand;
• and
• h) measuring an amount of marked ligand remaining on the predefined regions of the surface.