Non-fouling polymeric surface modification and signal amplification method for biomolecular detection

1. A method of detecting a second member of a specific binding pair nucleotide in a sample, comprising the steps of:

(a) providing a detector comprising:

(i) a substrate having a surface portion;

(ii) a linking layer on said surface portion; and

(iii) a polymer layer formed on said linking layer by the process of surface-initiated polymerization of monomeric units thereon, with each of said monomeric units comprising a monomer core group having at least one protein-resistant head group coupled thereto, to thereby form a brush molecule on said surface portion;

said brush molecule comprising a stem formed from the polymerization of said monomer core groups, and a plurality of branches formed from said head group projecting from said stem; and

(iv) a first member of a specific binding pair coupled to said brush molecule;

(b) contacting a sample suspected of containing said second member to said detector; and then

(c) determining the presence or absence of binding of said second member to said first member, the presence of binding indicating the presence of said second member in said sample.

2. The method of claim 1 , wherein said determining step comprises directly or indirectly conjugating an elongated nucleic acid to said second member, said elongated nucleic acid produced by the process of enzymatic extension with terminal transferase.

3. The method of claim 1 , wherein first member is a probe nucleotide and second member is a target nucleotide.

4. The method of claim 3 , wherein said determining step comprises (i) elongating said target nucleotide with terminal transferase to produce an elongated nucleic acid, and then (ii) detecting the presence or absence of said elongated nucleic acid.

5. The method of claim 1 ,

wherein said probe nucleotide comprises a beacon portion and a second portion, with said beacon portion folded by hybridization to said second portion, and wherein said binding of said target nucleotide to said probe nucleotide unfolds said beacon portion to produce a free end terminal for extension with terminal transferase;

and wherein said determining step comprises (i) elongating said beacon portion with terminal transferase to produce an elongated nucleic acid, and then (ii) detecting the presence or absence of said elongated nucleic acid.

6. The method of claim 2 , 4 or 5 , wherein said elongated nucleic acid is detected by: (a) atomic force microscopy, (b) detection of radiolabeled nucleotides incorporated in said elongated portion for radioactive detection, (c) detection of stable isotope labeled nucleotides in said elongated nucleic acid by mass spectrometry, (d) incorporation of fluorophores into said elongated nucleic acid and fluorescent detection, (e) incorporation of nucleotides containing amino acids to which fluorophores are attached into said elongated nucleic acid and fluorescent detection, or (e) indirectly by hybridization of the elongated nucleic acid with a labelled complementary nucleic acid, and subsequent detection of said complementary nucleic acid, wherein said complementary nucleic acid is labelled by coupling to a metal nanoparticle, quantum dot, fluorophore, or radionuclide.

7. The method of claim 1 , wherein said second member is a protein or peptide.

8. The method of claim 7 , wherein said determining step comprises: (i) specifically binding an antibody to said first member to form a sandwich, said

antibody having a first nucleic acid conjugated thereto, (ii) elongating said first nucleic acid with terminal transferase to produce an elongated nucleic acid, and then (iii) detecting the presence or absence of said elongated nucleic acid.

9. The method of claim 1 , wherein:

said surface portion comprises a material selected from the group consisting of metals, metal oxides, semiconductors, polymers, silicon, silicon oxide, and composites thereof;

said linking layer is a self-assembled monolayer;

said brush molecule is from 5 to 50 nanometers in length; and

said brush molecule is formed on said surface portion at a density from 40 to 100 milligrams per meter 2 .

10. The method of claim 1 , wherein said linking layer is formed from a compound comprising an anchor group coupled to an initiator.

11. The method of claim 10 , wherein said initiator is selected from the group consisting of bromoisobutyrate, polymethyl methacrylate-Cl, polystyrene-Cl, AIBN, 2-bromoisobutyrate, chlorobenzene, hexabromomethyl benzene, hexachloromethyl benzene, dibromoxylene, methyl bromoproprionate.

12. The method of claim 10 , wherein said initiator has the formula:

wherein:

R 1 and R 2 are each independently alkyl or aryl, or R 1 and R 2 together form an optionally alkyl-substituted cycloalkyl ring containing 4 to 7 carbon atoms; and

R is selected from the group consisting of alkyl, alkenyl, aryl, and aryl-substituted alkyl.

13. The method of claim 10 , wherein said initiator comprises at least one radically transferable atom or group.

14. The method of claim 13 , wherein said initiator is a small molecule initiator or a macromolecular initiator.