Fusion protein arrays on metal substrates for surface plasmon resonance imaging
Disclosed are methods for making surface plasmon resonance-capable arrays wherein molecules, such as proteins or nucleic acids, or cells, are adhered to a metal substrate. The metal substrates are modified by depositing an ω-modified alkanethiol monolayer to the substrate and then contacting the ω-modified monolayer with a heterobifunctional linking compound. Biomolecules or cells can then be attached to the heterobifunctional linking compound. Also disclosed are arrays wherein glutathione-containing molecules are immobilized on the substrate and GST-containing molecules are then specifically immobilized onto the substrate, taking advantage of the affinity between glutathione and GST.
1. A method of immobilizing biomolecules or cells to a metal substrate comprising:
(a) depositing an amino-C 8 –C 24 -alkanethiol monolayer on a metal substrate;
(b) contacting the ω-modified monolayer of step (a) with a heterobifunctional compound comprising a first moiety reactive with the ω-modified alkanethiol monolayer and a second moiety comprising a protected thiol moiety, under conditions wherein the heterobifunctional compound binds to the ω-modified alkanethiol monolayer via the first moiety;
(c) deprotecting the second moiety of the heterobifunctional compound to yield an unprotected thiol moiety; and then
(d) attaching a biomolecule or cell to the unprotected thiol moiety of step (c).
2. The method of claim 1 , wherein in step (a), 11-mercaptoundecylamine (MUAM) is deposited on the metal substrate.
3. The method of claim 1 , wherein in step (b), the ω-modified monolayer is contacted with a heterobifunctional compound wherein the first moiety comprises a succinimidyl group.
4. The method of claim 1 , wherein in step (b), the ω-modified monolayer is contacted with N-succinimidyl-S-acetylthiopropionate.
5. The method of claim 1 , wherein in step (d), a deoxyribonucleic acid (DNA) molecule is attached to the unprotected thiol moiety.
6. The method of claim 1 , wherein in step (d), a ribonucleic acid (RNA) molecule is attached to the unprotected thiol moiety.
7. The method of claim 1 , wherein in step (d), a polypeptide molecule is attached to the unprotected thiol moiety.
8. The method of claim 1 , wherein in step (d), a protein molecule is attached to the unprotected thiol moiety.
9. The method of claim 1 , wherein in step (d), a glutathione-containing molecule is attached to the unprotected thiol moiety.
10. A method of immobilizing biomolecules or cells to a metal substrate comprising:
(a) depositing an ω-modified alkanethiol monolayer on a metal substrate;
(b) contacting the ω-modified monolayer of step (a) with a heterobifunctional compound comprising a first moiety reactive with the ω-modified alkanethiol monolayer and a second moiety comprising a protected thiol moiety, under conditions wherein the heterobifunctional compound binds to the ω-modified alkanethiol monolayer via the first moiety;
(c) deprotecting the second moiety of the heterobifunctional compound to yield an unprotected thiol moiety;
(d) attaching a glutathione-containing molecule to the unprotected thiol moiety of step (c); and then
(e) contacting a glutathione-S-transferase-containing (GST-containing) molecule to the glutathione-containing molecule, whereby the GST-containing molecule is reversibly adhered to the glutathione-containing molecule.
11. The method of claim 10 , wherein in step (e), a glutathione-S-transferase (GST) fusion protein is contacted to the glutathione-containing molecule.
12. A method of immobilizing biomolecules or cells to a metal substrate comprising:
(a) depositing an ω-modified alkanethiol monolayer on a metal substrate;
(b) contacting the ω-modified monolayer of step (a) with a heterobifunctional compound comprising a first moiety reactive with the ω-modified alkanethiol monolayer and a second moiety comprising a protected thiol moiety, under conditions wherein the heterobifunctional compound hinds to the ω-modified alkanethiol monolayer via the first moiety;
(c) deprotecting the second moiety of the heterobifunctional compound to yield an unprotected thiol moiety;
(d) attaching a biomolecule or cell to the unprotected thiol moiety of step (c), wherein the biomolecule or cell is attached to the unprotected thiol moiety via a linker molecule.
13. The method of claim 12 , wherein in step (d), the biomolecule or cell is attached to the unprotected thiol moiety via a maleimide-containing linker molecule.