Letter pubs.acs.org/ac
Method To Determine Protein Concentration in the Protein− Nanoparticle Conjugates Aqueous Solution Using Circular Dichroism Spectroscopy Shanghao Li, Zhili Peng, and Roger M. Leblanc* Department of Chemistry, University of Miami, 1301 Memorial Drive, Coral Gables, Florida 33146, United States S Supporting Information *
ABSTRACT: Considerable efforts have been made to synthesize and characterize protein−nanoparticle conjugates (protein− NPs) for their promising applications in bionanotechnology. However, protein concentration determination in the protein−NPs has so far not been reported. In this Letter, we present a simple and nondestructive approach to quantify the protein concentration in the protein−NPs aqueous solution using circular dichroism (CD) spectroscopy. Carbon dots (∼4 nm), gold nanoparticles (∼10 nm), and polyethylene glycol (PEG, molecular weight ∼3000) were either physically mixed or covalently conjugated (not in the case of gold nanoparticles) with proteins (human transferrin, human serum albumin, and ovalbumin). We were able to quantify the protein concentration in the protein−nanoparticle conjugates using a calibration curve from the CD spectra.
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more control over the properties of both nanoparticles and proteins, gaining more attention in the research.7 In principle, any protein has one carboxylic and one primary amino functional group at its terminals, while the side chains may introduce additional functional groups for conjugation. These functional groups can be used to covalently conjugate proteins to the nanoparticles. Significant efforts have recently been made to characterize protein−NPs (i.e., size, composition, and morphology).12,13 It is worth noting the great importance of accurate quantification of protein concentrations in the protein−NPs conjugates for applications, ranging from enzymatic studies to pharmaceutical testing.14,15 UV−vis absorbance and the dye-binding assay are usually applied to determine the concentration of free (i.e., not conjugated) proteins in an assay based on a known extinction coefficient or a standard calibration curve.16 Unfortunately, most of nanoparticles have very strong UV−vis absorbance and often overlap with the absorbance of proteins or the dye, making the UV−vis quantification of the conjugated protein extremely difficult. Furthermore, nanoparticles with a quantum size (
