Antibody-nanoparticle conjugates and methods for making and using such conjugates
Disclosed herein are antibody-nanoparticle conjugates that include two or more nanoparticles (such as gold, palladium, platinum, silver, copper, nickel, cobalt, iridium, or an alloy of two or more thereof) directly linked to an antibody or fragment thereof through a metal-thiol bond. Methods of making the antibody-nanoparticle conjugates disclosed herein include reacting an arylphosphine-nanoparticle composite with a reduced antibody to produce an antibody-nanoparticle conjugate. Also disclosed herein are methods for detecting a target molecule in a sample that include using an antibody-nanoparticle conjugate (such as the antibody-nanoparticle conjugates described herein) and kits for detecting target molecules utilizing the methods disclosed herein.
1. A method for detecting a target molecule in a sample, comprising:
(a) contacting the sample with a first antibody that specifically binds to the target molecule;
(b) contacting the sample after step (a) with a second antibody conjugated to one or more enzyme molecules, wherein the second antibody specifically binds to the first antibody thereby forming a complex;
(c) contacting the complex formed in step (b) above with a third antibody-nanoparticle conjugate wherein the third antibody specifically binds to the second antibody and wherein said third antibody-nanoparticle conjugate comprises three or more metal nanoparticles directly linked to a single third antibody through three or more metal-thiol bonds, wherein the metal-thiol bonds are between thiol of cysteine residues of the antibody and the three or more metal nanoparticles without an intervening linker;
(d) contacting the sample after step (c) with a substrate of the enzyme and a metal ion, such that a metal precipitate forms and colocalizes with the target molecule; and
(e) detecting the metal precipitate, thereby detecting the target molecule.
2. The method of claim 1 , wherein the target molecule comprises a hapten-labeled nucleic acid probe that specifically binds to the target molecule and wherein the first antibody specifically binds the hapten-labeled nucleic acid probe.
3. The method of claim 1 , wherein the nanoparticles comprise gold, palladium, platinum, silver, copper, nickel, cobalt, iridium, or an alloy of two or more thereof.
4. The method of claim 1 , wherein the metal ion comprises silver ion, gold ion, copper ion, nickel ion, platinum ion, palladium ion, cobalt ion, or iridium ion.
5. The method of claim 1 , further comprising a gold toning step comprising contacting the sample after step (d) with a gold halide salt.
6. The method of claim 5 , wherein the method further comprises amplifying the signal after the gold toning step.
7. The method of claim 6 , wherein the amplifying includes contacting with a silver salt selected from the group consisting of silver nitrate, silver oxide, silver acetate, and silver perchlorate.
8. The method according to claim 1 , further comprising contacting the sample with a reducing agent.
9. The method of claim 8 , wherein the reducing agent comprises sodium thiosulfate.
10. The method according to claim 1 , wherein the metal nanoparticles are about 5 nm or less in diameter.
11. The method according to claim 1 , wherein the single third antibody is directly linked to three to seven nanoparticles through said metal thiol bond without an intervening linker.
12. The method according to claim 1 , wherein the second antibody comprises a goat anti-rabbit immunoglobulin G antibody.
13. The method according to claim 1 , wherein the enzyme comprises an alkaline phosphatase, an acid phosphatase, a β-galactosidase, a β-glucosidase, a β-lactamase, or an esterase.
14. The method of claim 13 , wherein the enzyme is alkaline phosphatase and the substrate of the enzyme comprises 5-bromo-4-chloro-3-indolyl phosphate, ascorbic acid phosphate, or a hydroquinone phosphate derivative.
15. The method of claim 14 , wherein the second antibody is conjugated to three alkaline phosphate molecules.
16. The method according to claim 1 , wherein the target molecule comprises HER2, c-Myc, n-Myc, Abl, EGFR, IGF1R, c-Met, TOP2A, Bcl2, Bcl6, Rb1, p53, or MET.