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Letter
A Simple Method to Determine Protein Concentration in the Protein-Nanoparticle Conjugates Aqueous Solution Using Circular Dichroism Spectroscopy Shanghao Li, Zhili Peng, and Roger M. Leblanc Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.5b01451 • Publication Date (Web): 12 Jun 2015 Downloaded from http://pubs.acs.org on June 14, 2015
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Analytical Chemistry
A Simple Method to Determine Protein Concentration in the Protein-Nanoparticle Conjugates Aqueous Solution Using Circular Dichroism Spectroscopy Shanghao Li1, Zhili Peng1, and Roger M. Leblanc1,* 1
Department of Chemistry, 1301 Memorial Drive, University of Miami, Coral Gables, Florida,
33146, United States Keywords: Protein Concentration Determination; Protein-Nanoparticle Conjugates; Circular Dichroism Spectroscopy; Carbon Dots; Gold Nanoparticles; Polyethylene Glycol *Corresponding author (R.M.L). Tel.: +1–305–284–2194; Fax: + 1–305–284–6367. E–mail:
[email protected].
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Analytical Chemistry
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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 non-destructive approach to quantify the protein concentration in the proteinNPs 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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Analytical Chemistry
One of the most promising interfaces of nanotechnology is to conjugate proteins with nanoparticles (protein-NPs) since the conjugated system brings together at the same time the unique properties of nanoparticles and the biological functions of proteins.1 These conjugates hold exciting potentials in the fields of biotechnology and medicine, such as sensing, imaging, diagnostics, catalysis, drug delivery and the control of protein activity.2-6 The conjugates usually require the retention of the protein structure and function.7 To this end, tremendous efforts have been made to synthesize and characterize protein-NPs without compromising functions of the proteins. Two main strategies have been widely applied to conjugate proteins to nanoparticles: (1) passive adsorption via non-covalent interactions; and (2) covalent binding by exploiting functional groups between the nanoparticles and the target proteins.2, 7-10 For the classic passive adsorption strategy, the binding is usually governed by ionic, hydrogen bonding, hydrophobic, or van der Waals interactions, and therefore the conjugation is not specific. Furthermore, desorption from the surface and partial denaturation of the conjugated proteins might occur over time. On the contrary, covalent conjugation of proteins to nanoparticles can minimize the drawbacks encountered with the passive conjugation strategy.11 Therefore, the formation of covalent protein-NPs conjugates provides 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 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
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Analytical Chemistry
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very strong UV-vis absorbance and often overlap with the absorbance of proteins or the dye, making extremely difficult the UV-vis quantification of the conjugated protein. Furthermore, nanoparticles with quantum size (
