Bioconjugate-nanoparticle probes
View Patent ↗The invention provides nanoparticle-bioconjugate probes that are useful for detecting target analytes such as nucleic acids. The probes of the invention are stable towards heat and resistant to displacement by thiol containing compounds such as DTT (dithiothreitol).
1. A method for preparing a nanoparticle-probe, the nanoparticle-probe comprising a bioconjugate coupled to a nanoparticle, the method comprising:
(i) reacting a thiol modifier reagent having a structure of formula:
wherein n is 1–10, with a structure of formula:
to produce a disulfide having the formula
(ii) reducing the disulfides a″, b″, c″, d″ or e″ of (i) to produce a bioconjugate having formula:
(iii) contacting one or more of the bioconjugates of (ii) with a nanoparticle to form the nanoparticle-probe, wherein the bioconjugate is coupled to the nanoparticle through the sulfur groups: wherein
n is 2–100;
m is 0–100;
X is a nucleotide or modified nucleotide;
Z is a nucleotide, modified oligonucleotide, or polyanion;
Q is a recognition group;
R is linear or branched C 1 –C 8 alkyl;
W is a steroid; and
each L is a linker formed by the coupling of two moieties selected from the group consisting of COOH, NH 2, CHO, F, Cl, Br, I, NCO, NCS, allyl, vinyl, and CH 3 CO 2 − , or L is —C(═NH 2 Cl)(CH 2 ) 3 —.
2. The method of claim 1 , wherein the bioconjugate is bioconjugate (B) and step (ii) comprises:
contacting the bioconjugate (B) with the nanoparticle in water;
adding a salt to the water to form a salt solution;
incubating the bioconjugate and nanoparticle in the salt solution to form a bioconjugate-nanoparticle probe; and
isolating the bioconjugate-nanoparticle probe from the salt solution.
3. The method of claim 2 , wherein all of the salt is added to the water in a single addition.
4. The method of claim 2 , wherein the salt is added gradually over time.
5. The method of claim 2 , wherein the salt is selected from the group consisting of sodium chloride, magnesium chloride, potassium chloride, ammonium chloride, sodium acetate, ammonium acetate, a combination of two or more of these salts, one of these salts in a phosphate buffer, and a combination of two or more these salts in a phosphate buffer.
6. The method of claim 5 , wherein the salt is sodium chloride in a phosphate buffer.
7. The method of claim 2 , wherein the nanoparticle is selected from the group consisting of a metal, a semiconductor, a magnetic material, a colloidal material, ZnS, ZnO, TiO 2, AgI , AgBr, HgI 2, PbS, PbSe, ZnTe, CdTe, In 2 S 3, In 2 Se 3, Cd 3 P 2 , Cd 3 As 2, InAs, GaAs, GaP, BaTiO 3, Co, Fe 2 O 3, core shell particles and alloyed particles.
8. The method of claim 7 , wherein the nanoparticle is gold.
9. The method of claim 1 , wherein the bioconjugate is bioconjugate (A).
10. The method of claim 1 , wherein Q in the bioconjugate comprises an organic molecule, an organic or inorganic polymer, a receptor, a nucleotide, a nucleoside, a polynucleotide, an oligonucleotide, a protein, an antibody, a peptide, a carbohydrate, a sugar, a hapten, a nucleic acid, an amino acid, a peptide nucleic acid, a linked nucleic acid, a nucleoside triphosphate, a lipid, a lipid bound protein, an aptamer, a virus, a cell fragment, or a whole cell.
11. The method of claim 1 , wherein the recognition group comprises a first and a second recognition group, wherein the first recognition group is bound to the second recognition group and wherein the second recognition group is a member of a recognition couple that specifically binds to a target analyte.
12. The method of claim 11 , wherein the first and second recognition groups comprise a specific binding pair.
13. The method of claim 12 , wherein the binding pair is an antibody-antigen or a receptor-ligand.
14. The method of claim 11 , wherein the first and second recognition groups are oligonucleotides having at least a portion of its sequence complementary to each other.
15. The method of claim 1 , wherein X and Z are independently adenine, guanine, cystosine, thymine, uracil, a nucleotide derivative, a modified nucleotide, or other molecules that may be incorporated into the oligonucleotide synthesis process.
16. The method of claim 15 , wherein X is thymine.
17. The method of claim 1 , wherein L is —NH—C(═O)—.
18. The method of claim 1 , wherein n from bioconjugate D is 2.
19. The method of claim 10 , wherein Q is an oligonucleotide.
20. The method of claim 10 , wherein Q is a cDNA.
21. The method of claim 10 , wherein Q is a polynucleotide.
22. The method of claim 1 , wherein the nanoparticle is selected from nanoparticles that have an affinity for thiol groups.
23. The method of claim 22 , wherein the nanoparticle is selected from the group consisting of a metal, a semiconductor, a magnetic material, including Au, Ag, Pt, Co, CdSe, CdS, or Si.
24. The method of claim 23 , wherein the nanoparticle is gold.
25. The method of claim 1 , wherein prior to step (iii), further comprising reacting bioconjugates A or D of step (ii) with
to produce a bioconjugate having the formula