Volume 14 Issue 3 - June 4, 2010 PDF
Heme-protein Assisted Dispersion of Au-NP Multilayers on Chips: From Stabilization to High Density dsDNAs Fabricated In-situ for Protein/DNA Binding
Yu-Ting Li and Shu-Hui Chen*
Professor of Department of Chemistry, College of Sciences, National Cheng Kung University
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Immobilization of Au Nanoparticles (AuNPs) on a substrate is highly demanded in many analytical applications since they can be easily modified by a thiolated molecule.  Stable and high density of AuNPs on a substrate can be used to fabricate high density ligands with high capacity and high sensitivity for sensing.  Whereas, by current methods using polymer linkers such as aminopropyltriethoxysilane (APTES) and (3-mercaptopropyl)trimethoxysilane (MPTS) the coating density of AuNPs on a substrate is low and the stability is also poor; requiring surface activation to create hydroxyl groups.  Recent studies indicate that porphyrin can form a self-organized monolayer on the Au(111) surface due to the coordination of the porphyrin macrocycle to Au(111).  Macrocyclic porphyrin ligands have also been synthesized to form quite stable Au(0) porphyrin nanocomplexes, with porphyrin rings parallel to the AuNP surface.  The existence of the Soret band, tunable by the distance between the ring and the AuNP surface, implies an electronic interaction between AuNP and porphyrin.  In this study, we demonstrated that heme proteins, which have porphyrin moieties, are effective linking agents in stabilizing AuNPs.  Unlike porphyrins, however, multiple interaction forces could happen for a protein due to its multi-functionalties.
Figure 1 Protein-anchored AuNP multilayers (n=4) and APTES-anchored monolayer on glass.

In order to investigate whether heme proteins in general are unique anchoring agents for forming stable AuNP multilayers on chips, we first examined several heme proteins (Mb, hemoglobin, cytochrome C) and non-heme acidic proteins (β-casein (Cas), bovine serum albumin, α-lactalbumin) as well as a non-heme basic protein (lysozyme).  As a comparison, we also investigated APTES-functionalized surfaces.  As shown in Figure 1, for all heme-protein anchored surfaces, AuNPs could be uniformly coated and exhibit pink or deep pink color.  No significant aggregation was noted throughout the multilayer (n≥4) formation, indicating that all heme proteins investigated could act as an effect linking agent for forming stable AuNP multilayers.  However, on non-heme protein anchored surfaces, AuNPs were either coated with very low density such as Cas and α-lactalbumin or appeared to show dark blue color such as lysozyme and bovine serum albumin, an indication of aggregations.  Lysozyme is a strong basic protein with an isoelectric point around 11 and was suspected to be a good linking agent for AuNPs due to electrostatic interactions with the negatively charged citrate-protected AuNP surface.  However, instead of stabilization, our observations suggest that such strong electrostatic interactions arising from charged protein functional groups could lead to aggregation.  On the APTES-functionalized substrate, AuNPs were coated with a low density as observed for non-heme proteins.  In order to examine whether the heme group could assist in AuNP dispersion, we cross linked Cas with hemin, an iron-chelated porphyrin, and used the cross-linked hemin-β-casein (H-Cas) as the linking agent for AuNPs.  On the H-Cas surface, AuNPs were uniformly coated on both the monolayer and the multilayers as on the Mb surface (Figure 1), indicating the contribution of heme groups in AuNPs stabilization.  SEM was also used to examine the monolayer and multilayer surfaces.  As shown in Figures 2a and 2b, the coating density of AuNPs on Mb-anchored surface greatly increased from monolayer to multilayers with good dispersion.  Moreover, the heme protein-anchored multilayers were all stable when exposed to air for more than one week.  As shown in Figure 2c, AuNPs on the Cas-anchored monolayer are much lower in density than those on the Mb-anchored surface (Figure 2a), and some local aggregation was noted.  Whereas, on the H-Cas surface, AuNPs were densely coated and well dispersed on the monolayer (Figure 1d) as well as on the multilayers without notable aggregation.  These results strongly suggest that the heme group of anchored proteins play an important role in stabilizing AuNP composites on a surface.

Figure 3.        5/2Au and 7/2Au ESCA spectra for (a) Mb-AuNPs, (b) H-Cas-AuNPs, (c) Cas-AuNPs, and (d) aminosilane-functionalized AuNPs.
Figure 2. SEM and photo images of (a) Mb anchored AuNP monolayer, (b) Mb anchored AuNP multilayer (n=4), (c) Cas anchored AuNP monolayer, and (d) H-Cas anchored AuNP monolayer on glass.
ESCA were measured to investigate the molecular interactions between AuNPs and heme-proteins.  As shown in Figure 3, both 5/2Au and 7/2Au bands were detected from various AuNP surfaces which were anchored by molecules with or without heme groups.  Notably, for heme-proteins, Mb and H-Cas anchored AuNPs, the fitted spectra show two chemical states for each Au band; however, for non heme molecules, APTES and Cas anchored AuNPs, the fitted spectra exhibit only the elemental Au bands (5/2Au and 7/2Au).  Apparently, cross linking Cas with hemin resulted in an additional chemical state for AuNPs.  These results indicate that the additional chemical state of Au is associated with the heme groups.  Since the binding energy of the additional state is lower than that of the elemental states, Au with lower oxidation states, such as π-orbital interactions, is likely to occur in order to reduce the tunneling resistance of the surrounding ligands.  Another way to exploit possible π-coordination between AuNPs and ligands is to examine the ESCA spectra of atoms that could serve as Lewis bases, such as N and O on proteins.  By comparison with the N1s spectra of amino acids, we found multiple chemical states of N1s for Cas including those correspond to the N atoms on the ε-amino group and the N-terminus of the amide bond, 400 and 399 eV, respectively.  On the other hand, we observed a 401 eV peak which was concluded to be related to the interaction between AuNPs and the exposed heme groups.  Taken together, we propose that the stabilization power arises from π conjugation between AuNPs and the heme group.  We also propose that such conjugation must be facilitated by the exposure of the heme group through a conformational change of the protein as well as interactions of other functional groups with AuNPs to bring the heme moiety to a close face-to-face distance with AuNPs. (left of Scheme 1).
Scheme 1. Heme-protein stabilized AuNP multilayers formed through π conjugation between AuNPs and the heme groups and high density dsDNAs fabricated by in-situ hybridization induced fluorescence restoration for sensing transcription factor binding.

We then used the stable AuNPs multilayers anchored by Mb for fabricating double stranded DNA (dsDNA) containing the sequence of estrogen response element (ERE) by an in-situ method that can be tracked via hybridization-induced fluorescence restoration (Right of Scheme 1).  ERE is the cis regulatory sequence of estrogen receptors (ERs) located in the promoter region of the target genes and the ERE-ER binding is the key for transcriptional activation.  DNA probes were bonded to the monolayer via thiol groups at one end, and a fluorophore dye (Cy3) was attached to the other end of the probe.  The fluorescence was restored when the complementary DNA was hybridized on chip, and no fluorescence could be detected when buffers or non-complementary DNA was added (Right of Scheme 1). Two cell lines, breast cancer cell MCF7 and lung cancer cell A549, which are known to express two different isoforms of estrogen receptors (ERα and β), were tested.  MCF-7 cells are known to express more ERα than ERβ; A549 cells are known to express ERβ but very little ERα.  Both cells were treated with 17β-estradiol for 24 hours, and the binding of activated ERα and β to ERE on chips was detected using the Enzyme-Linked ImmunoSorbent Assay (ELISA).  Based on the comparison of signals detected from the control and the standard made of recombinant ERα, as expected, MCF-7 cells were detected with more ERα than ERβ, and A549 cells were detected with ERβ but very little ERα.  These results suggest that the ERE chips fabricated on heme protein anchored Au-NP multilayers are suitable for quantitative analysis with high sensitivity and specificity.
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