Signatures of specificity of interactions of binary protein mixtures with citrate-stabilized gold nanoparticles{ Rumi Khandelia, a Jashmini Deka, a Anumita Paul* a and Arun Chattopadhyay* ab Received 17th January 2012, Accepted 26th March 2012 DOI: 10.1039/c2ra20096a In this article we report on the observation of specificity of interactions of binary mixtures of proteins with citrate-stabilized gold nanoparticles (Cit-Au NPs), by following the changes in the optical properties of the NPs. The protein mixtures consisted of a-amylase and bovine serum albumin (BSA) or a-amylase and amyloglucosidase (AMG) or glucose oxidase (GOD) and peroxidase (POD). The results observed herein indicated that interaction between a binary protein mixture and Cit-Au NPs depended on the nature and concentration of the component proteins. For example, addition of increasing concentrations of proteins containing a-amylase and BSA consistently broadened the extinction spectrum of Cit-Au NPs. The area under the curves when plotted against the concentration of either of the proteins increased linearly. FTIR, fluorescence, starch agar plate assay and gel electrophoresis results indicated that both a-amylase and BSA were present in the agglomerated structures of proteins and NPs, indicating that both of the proteins played a role in the association of NPs. On the other hand, when the mixture contained increasing concentration either of a-amylase or AMG, the broadening as well as the change in the area under the curve varied randomly rather than following any linearity. Interestingly, when the mixtures of GOD and POD were used—although broadening was observed—the change in the area was linear only for low concentrations of GOD in the medium and was very sensitive to its concentration. Transmission electron microscopy (TEM) results indicated agglomeration of the NPs in the presence of the protein mixtures as the primary reason behind the optical property change. Our observations indicated that the preferential attachment of one protein to Cit-Au NPs—in presence of the other—primarily depended on the overall charge of the protein in the medium. 1 Introduction Interaction of nanoparticles (NPs) with biomolecules (especially proteins) has been an important research area in the field of biomedicine with potential applications in diagnostics and therapeutics. When a biomolecule such as protein, DNA, or RNA interacts with NPs, the properties of the NP may undergo changes which can be probed using optical and magnetic techniques. This can form the basis for detection and assay of the biomolecule. For example, the optical properties of Au NPs have been used for the development of assays for important proteins and for studying the conformational changes of proteins consequent upon pH changes or heat. 1–8 Recently, based on their investigations of the interaction of Au NPs with common human blood proteins, Lacerda et al. concluded that the degree of co- operativity of particle-protein binding depends on particle size and the native protein structure. 9 Tang et al. have reported that in the presence of a mixture of Ag NPs and Au NPs the interaction between bovine serum albumin (BSA) and Ag NPs was reduced in comparison to that in absence of Au NPs. 10 In addition, the induced aggregation of a protein by Au NPs and the partial unfolding of a protein on the surface of a NP have recently been demonstrated. 4,11–12 Interestingly, while a recent report suggests affinity of a protein as the deciding factor in its attachment to Au NP in the presence of a mixture of proteins, 13 there is no other report on the behavior of a mixture of proteins with respect to their attachment to NPs. It would be important to know the consequence of attachment of a protein to a NP in relation to the other proteins present in the medium. Recent reports from our laboratory indicated that a protein at its lower concentration gets attached to Au NP. However, when the concentration of protein is increased these protein-NP compo- sites agglomerate forming larger structures. 4 The observations further indicated that the area under the extinction curve varied a Department of Chemistry, Indian Institute of Technology, Guwahati, 781039, India. E-mail: arun@iitg.ernet.in; anumita@iitg.ernet.in; Fax: + 91 361 2582349; Tel: +91 3612582304 b Centre for Nanotechnology, Indian Institute of Technology, Guwahati, 781039, India { Electronic Supplementary Information (ESI) available: Additional UV-vis, SDS-PAGE, enzymatic assay of a-amylase, fluorescence, FTIR and DLS experimental details, Bradford assay UV-vis and standard curve, additional UV-vis spectra and their analyses, TEM images, DLS- based particle size analysis results, time dependent fluorescence spectra, FTIR spectra, sensitivity graphs of individual GOD and POD, and various tables are available. See DOI: 10.1039/c2ra20096a/ RSC Advances Dynamic Article Links Cite this: RSC Advances, 2012, 2, 5617–5628 www.rsc.org/advances PAPER This journal is ß The Royal Society of Chemistry 2012 RSC Adv., 2012, 2, 5617–5628 | 5617 Downloaded on 28 June 2012 Published on 27 March 2012 on http://pubs.rsc.org | doi:10.1039/C2RA20096A View Online / Journal Homepage / Table of Contents for this issue