Photoactivatable o-nitrobenzyl polyethylene glycol-silane for the production of patterned biomolecular arrays

• US 5,773,308 A
• Filed: 02/10/1997
• Issued: 06/30/1998
• Est. Priority Date: 02/10/1997
• Status: Expired due to Fees

• Current Assignee: United States Navy (U.S. Department Of Defense)
• Original Assignee: the united states of america as represented by the secretary of the navy
• Inventors: Bart, John C., Golightley, Sara K., Conrad, David W.
• Primary Examiner(s): Housel, James C.
• Assistant Examiner(s): PORTNER, VIRGINIA ALLEN
• Application Number:
  US08/797,325
• Time in Patent Office: 505 Days
• Field of Search: 436/527, 436/518, 435/6
• US Class Current: 436/527
• CPC Class Codes: C07C 271/16 to carbon atoms of hydrocarbon radicals substituted by singly-bound oxygen atoms
• CPC Class Codes: C07F 7/0836 Compounds with one or more Si-OH or Si-O-metal linkage
• CPC Class Codes: C07F 7/12 Organo silicon halides
• CPC Class Codes: C07F 7/1804 Compounds having Si-O-C linkages Si-O-acyl linkages C07F7/1896
• CPC Class Codes: G01N 33/54353 with ligand attached to the carrier via a chemical coupling agent coatings G01N33/54393
• CPC Class Codes: Y02P 20/55 Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups

Abstract

O-nitrobenzyl analogs that include a photoremovable protecting group that resists the nonspecific adsorption of biomolecules and a linking group for attaching the o-nitrobenzyl analog to a substrate are disclosed. Also disclosed are methods for using o-nitrobenzyl analogs for creating patterned arrays of anti-ligands on a substrate so that a plurality of bioassays can be conducted simultaneously. In particular, the compounds disclosed are o-nitrobenzyl-polyethylene glycol-silanes, wherein the silane group serves to attach the compound to a substrate, the polyethylene glycol group resists the nonspecific adsorption of biomolecules. The o-nitrobenzyl group provides a photoactivatable functionality that allows the polyethylene glycol group to be selectively removed upon exposure to UV radiation and be replaced by an anti-ligand.

Claims

1. A compound having the formula:

##STR9## wherein R¹ is --(CH₂ CH₂ O)_n CH₃, where n is an integer from about 6 to about 300, R² is a linear linking group having a distal end that is capable of forming a covalent bond with a substrate, and R³ is NO₂ or H.

2. The compound of claim 1 wherein R² is --O(CH₂)_m SiCl₃, --O(CH₂)_m Si(OCH₃)₃ or --O(CH₂)_m Si(OCH₂ CH₃)₃ where m is an integer from about 9 to about 25.

7. A patterned substrate for performing a plurality of biological assays simultaneously utilizing the compound of claim 2, wherein, the patterned substrate comprising a plurality of predefined regions, each predefined region having bound thereto a compound of the formula:

##STR12## wherein Si forms at least one covalent bond with the predefined region of the substrate and x is an integer between 1 and 3 representing the number of covalent bonds between Si and the predefined region of the substrate, m is an integer from about 9 to about 25, R³ is NO₂ or H and wherein the anti-ligand is different in each predefined region of the substrate.

8. The patterned substrate of claim 7 wherein the patterned substrate is glass, fused silica, silicon, ceramic, metal oxide or a polymer having surface hydroxyl groups.

9. The patterned substrate of claim 7 wherein the patterned surface is an ELISA plate well.

10. The patterned substrate of claim 7 wherein the patterned substrate is the interior of a capillary tube.

11. The patterned substrate of claim 7 wherein each anti-ligand is an antibody.

12. A method for attaching a plurality of anti-ligands having different binding specificities to predefined regions on a substrate, the method comprising the steps of

(a) covalently binding to the substrate a protected o-nitrobenzyl analog wherein the anolog is the compound of claim 1, having a photoremovable protecting group that has a resistance to nonspecific biomolecule adsorption, to create a modified substrate having a resistance to nonspecific biomolecule adsorption

(b) irradiating the protected o-nitrobenzyl analog on a first predefined region of the substrate to remove the photoremovable protecting group and to form unprotected o-nitrosobenzyl analog having a reactive group capable of covalently binding an anti-ligand,

(c) reacting the unprotected o-nitrosobenzyl analog with an anti-ligand to covalently bind the anti-ligand to the unprotected o-nitrosobenzyl analog to form a bridging o-nitrosobenzyl analog that is covalently bound to the first predefined region of the substrate and that is covalently bound to the anti-ligand,

(d) washing the substrate to remove unbound anti-ligand, whereby steps (a) to (d) result in a first predefined region of the modified substrate having a bridging o-nitrosobenzyl analog covalently bound thereto and having an anti-ligand bound to the bridging o-nitrosobenzyl analog, and a remainder region of the modified that has not been irridiated and has the protected o-nitrobenzyl analog bound thereto, the remainder region having a resistance to nonspecific biomolecule adsorption,

(e) repeating steps (b) to (d) at least one additional time on a different region of the substrate with a different anti-ligand.

13. The method of claim 12 wherein the unprotected o-nitrosobenzyl analog is a compound of the formula:

##STR13## wherein R³ is NO₂ or H and R² is a linear linking group having a distal end that is covalently bonded to the first predefined region of the substrate, and

wherein the bridging o-nitrosobenzyl analog is a compound of the formula;

##STR14## wherein R³ is NO₂ or H and R² is a linear linking group having a distal end that is covalently bonded to the first predefined region of the substrate.

3. A compound having the formula:

##STR10## wherein R is a leaving group, m is an integer from about 9 to about 25, n is an integer from about 6 to about 300 and R³ is NO₂ or H.

4. The compound of claim 3 wherein R is Cl, OCH₃, or OCH₂ CH₃.

5. A modified substrate having a compound covalently bonded thereto, the compound having the formula:

##STR11## wherein R¹ is --(CH₂ CHO)_n CH₃, where n is an integer from about 6 to about 300, R² is a linear linking group having a distal end that is covalently bonded to the solid substrate and R³ is NO₂ or H.

6. The modified substrate of claim 5 wherein R² is --O(CH₂)_m Si(OH)_3-x where Si forms at least one covalent bond with the substrate and x is an integer between 1 and 3 representing the number of covalent bonds between Si and the substrate.

14. A method for attaching a plurality of anti-ligands having different binding specificities to predefined regions on a substrate, the method comprising the steps of

(a) covalently binding to the substrate a protected o-nitrobenzyl analog of the formula:

##STR15## wherein R is a leaving group, m is an integer from about 9 to about 25, and n is an integer from about 6 to about 300, and R³ is NO₂ or H to form a modified substrate having bound thereto a compound of the formula;

##STR16## wherein Si forms at least one covalent bond with the substrate and x is an integer between 1 and 3 representing the number of covalent bonds between Si and the substrate, m is an integer from about 9 to about 25, and n is an integer from about 6 to about 300, and R³ is NO₂ or H,

(b) irradiating the protected o-nitrobenzyl analog on a first prefined region of the modified substrate to form unprotected o-nitrosobenzyl analog of the formula;

##STR17## wherein Si forms at least one covalent bond with the first predefined region of the modified substrate and x is an integer between 1 and 3 representing the number of covalent bonds between Si and the first predefined region of the modified substrate and m is an integer from about 9 to about 25 and R³ is NO₂ or H,

(c) reacting the unprotected o-nitrosobenzyl analog with an anti-ligand to covalently bind the anti-ligand to the unprotected o-nitrosobenzyl analog to form a bridging o-nitrosobenzyl analog of the formula;

##STR18## where Si forms at least one covalent bond with the first predefined region of the modified substrate and x is an integer between 1 and 3 representing the number of covalent bonds between Si and the first predefined region of the modified substrate, m is an integer from about 9 to about 25 and R³ is NO₂ or H

(d) washing the substrate to remove unbound anti-ligand,

(e) repeating steps (b) to (d) at least one additional time on a different region of the modified substrate with a different anti-ligand.

15. The method of claim 14 wherein the substrate is glass, fused silica, silicon, ceramic, metal oxide or a polymer having surface hydroxyl groups.

16. The method of claim 14 wherein the substrate is an ELISA plate.

17. The method of claim 14 wherein the substrate is the interior of a capillary tube.

18. The method of claim 14 wherein each anti-ligand is an antibody.

19. The method of claim 14 wherein R is Cl, OCH₃ or OCH₂ CH₃.

20. A patterned substrate for performing a plurality of biological assays simultaneously, made by a process comprising the steps of:

(a) covalently binding to the substrate a protected o-nitrobenzyl analog of the formula;

##STR19## wherein R is a leaving group, m is an integer from about 9 to about 25, and n is an integer from about 6 to about 300, and R³ is NO₂ or H, to form a modified substrate having bound thereto a compound of the formula;

##STR20## wherein Si forms at least one covalent bond with the substrate and x is an integer between 1 and 3 representing the number of covalent bonds between Si and the substrate, m is an integer from about 9 to about 25, and n is an integer from about 6 to about 300, and R³ is NO₂ or H,

(b) irradiating the protected o-nitrobenzyl analog on a first predefined region of the substrate to form unprotected o-nitrosobenzyl analog of the formula;

##STR21## where Si forms at least one covalent bond with the first predefined region of the modified substrate and x is an integer between 1 and 3 representing the number of covalent bonds between Si and the first predefined region of the modified substrate and m is an integer from about 9 to about 25 and R³ is NO₂ or H,

(c) reacting the unprotected o-nitrosobenzyl analog with an anti-ligand to covalently bind the anti-ligand to the unprotected o-nitrosobenzyl analog to form a bridging o-nitrosobenzyl analog of the formula;

##STR22## where Si forms at least one covalent bond with the first predefined region of the modified substrate and x is an integer between 1 and 3 representing the number of covalent bonds between Si and the first predefined region of the modified substrate and m is an integer from about 9 to about 25 and R³ is NO₂ or H,

(d) washing the substrate to remove unbound anti-ligand,

(e) repeating steps (b) to (d) at least one additional time on a different region of the modified substrate with a different anti-ligand.

21. The patterned substrate of claim 20 wherein R is Cl, OCH₃ or OCH₂ CH₃.