Method for quantifying biomolecules conjugated to a nanoparticle

Abstract

Disclosed embodiments concern quantifying a biomolecule conjugated to a nanoparticle. Quantifying typically comprises determining the number of biomolecules per nanoparticle. Any suitable biomolecule can be used, including but not limited to, amino acids, peptides, proteins, haptens, nucleic acids, oligonucleotides, DNA, RNA, and combinations thereof. A single type of biomolecule may be conjugated to the nanoparticle, more than one biomolecule of a particular class may be conjugated to the nanoparticle, or two or more classes of biomolecules may be conjugated to the nanoparticle. Certain disclosed embodiments comprise enzymatically or chemically digesting a biomolecule conjugated to the nanoparticle, or displacing a biomolecule using ligand-exchange chemistry. Where biomolecule concentrations are determined, any technique suitable for determining biomolecule concentration can be used, such as spectrophotometric techniques, including measuring tryptophan fluorescence and using a standard fluorescence intensity versus biomolecule concentration curve.

1 . A method for using a protein-quantum dot conjugate for an assay, comprising:
 displacing, and optionally digesting, protein from a first aliquot of a protein-quantum dot conjugate sample to produce displaced protein;   substantially separating the quantum dot from the displaced protein;   determining a concentration of displaced protein spectrophotometrically; and   using a second aliquot of the protein-quantum dot sample for the assay.   
     
     
         2 . The method according to  claim 1  where the protein is an antibody, an antibody fragment, a genetically engineered antibody, a chimeric antibody, a heteroconjugate antibody, or a combination thereof. 
     
     
         3 . The method according to  claim 1  where the protein is displaced either simultaneously with or followed by a digestion process to produce smaller constituent units. 
     
     
         4 . The method according to  claim 1  further comprising enzymatically digesting protein conjugated to the quantum dot using an enzyme selected from proteinase K, trypsin, clostripain, staphylococcal protease, thrombin, chymotrypsin, carboxypeptidase a, pepsin, papain, and combinations thereof. 
     
     
         5 . The method according to  claim 1  further comprising chemically digesting protein conjugated to the quantum dot using a mineral acid, organic acid, or cyanogen bromide. 
     
     
         6 . The method according to  claim 1  further comprising separating the quantum dot from the displaced protein by centrifugation. 
     
     
         7 . The method according to  claim 1  further comprising displacing protein from the quantum dot by ligand exchange using an amine, a polyamine, a phosphine, phosphine oxide, an alkyl phosphine, a derivatized alkyl phosphine, an alkyl phosphine oxide, a derivatized alkyl phosphine oxide, a thiol, or a combination thereof. 
     
     
         8 . The method according to  claim 7  comprising using a thiol ligand exchange compound selected from dithiothreitol, erythritol, dierythritol, trierythritol, pentaerythritol, dipentaerythritol, tripentaerythritol, dihydrolipoic acid, or a combination thereof. 
     
     
         9 . The method according to  claim 1  comprising quantifying protein concentration using tryptophan fluorescence. 
     
     
         10 . The method according to  claim 1 , further comprising:
 reacting displaced protein with a compound to form a detectable moiety; and   determining detectable moiety concentrations.   
     
     
         11 . The method according to  claim 10  where the detectable moiety is a fluorophore or a chromophore. 
     
     
         12 . The method according to  claim 11  where the detectable moiety is a fluorophore and further comprising quenching quantum dot fluorescence using a mineral acid or a transition metal. 
     
     
         13 . The method according to  claim 10 , further comprising:
 separating excess compound from products produced by reacting displaced protein with the compound; and   subsequently determining detectable moiety concentrations.   
     
     
         14 . The method according to  claim 13  where the compound is fluorescamine, (3-(4-carboxybenzoyl)quinoline-2-carboxaldehyde, o-phthaldialdehyde, or a combination thereof. 
     
     
         15 . The method according to  claim 13  where the compound is a reactive derivative of Texas Red, fluorescein isothiocyanate, 2′,7′-difluorofluorescein, coumarin, or a combination thereof. 
     
     
         16 . The method according to  claim 1  where the quantum dot is an alloyed quantum dot. 
     
     
         17 . The method according to  claim 16  where the quantum dot comprises CdSe, ZnSSe, ZnSeTe, ZnSTe, CdSSe, CdSeTe, ScSTe, HgSSe, HgSeTe, HgSTe, ZnCdS, ZnCdSe, ZnCdTe, ZnHgS, ZnHgSe, ZnHgTe, CdHgS, CdHgSe, CdHgTe, ZnCdSSe, ZnHgSSe, ZnCdSeTe, ZnHgSeTe, CdHgSSe, CdHgSeTe, InGaAs, GaAlAs, or InGaN. 
     
     
         18 . The method according to  claim 1  where the protein is an immunoglobulin conjugated to a quantum dot, the protein-quantum dot conjugate is digested using proteinase K, and an immunoglobulin concentration is determined by measuring tryptophan fluorescence. 
     
     
         19 . The method according to  claim 1  where the protein is an immunoglobulin conjugated to a quantum dot, a reaction mixture comprising the protein-quantum dot conjugate is heated in a solution comprising a thiol ligand exchange compound, and an immunoglobulin concentration is determined by measuring tryptophan fluorescence. 
     
     
         20 . The method according to  claim 1  where the protein is streptavidin or an immunoglobulin conjugated to a quantum dot, a reaction mixture comprising the protein-quantum dot conjugate is digested with an acid, released amino acids are reacted with fluorescamine, and a streptavidin or immunoglobulin concentration is determined using fluorescence spectroscopy.