Ultraclean, ultraflat, perfect-crystal gold nanoplate for surface modification and biosensor applications

Abstract

Gold nanostructures are considered to be the best material for a very stable and superior bio-active surface in a variety of organic, inorganic, and biochemical environments, and Au-S bonds have been widely employed as substrates since various functional molecules can be easily and densely fixed on a substrate. We synthesized single-crystalline Au nanoplates that were smooth at the atomic level without surface contamination and defects using only gold slug. Unlike conventional gold films, this Au nanoplate has an atomically smooth surface over a range of hundreds of microns in size and can be constructed that are very well aligned from coupled metal-molecule interfaces. In addition, we have developed a high sensitive and selectivity SERS sensor for the diagnosis of disease using the combination of [Au nanoplate + Au nanoparticle] based on the ultraclean and ultraflat geometry of Au nanoplate. It can immobilize the bioreceptors very uniformly on the surface, maximizing their ability to regulate antibody orientation.

In chapter 2, we demonstrate the charge transport properties of a self-assembled organic monolayer on Au nanoplates with conductive probe atomic force microscopy (CP-AFM). Atomically flat Au nanoplates, a few hundred micrometers on each side, that have only (111) surfaces, were synthesized using the chemical vapor transport method

these nanoplates were employed as the substrates for hexadecanethiol (HDT) self-assembled monolayers (SAMs). Atomic-scale high resolution images show ($\surd$3×$\surd$3)R30$^\circ$ molecular periodicity, indicating a well-ordered structure of the HDT on the Au nanoplates. We observed reduced friction and adhesion forces on the HDT SAMs on Au nanoplates, compared with Si substrates, which is consistent with the lubricating nature of HDT SAMs. The electrical properties, such as I–V characteristics and current as a function of load, were measured using CP-AFM. We obtained a tunneling decay constant ($\beta$) of 0.57 $\AA^{−1}$, including through-bond ($\beta$tb = 0.99 $\AA^{−1}$) and through-space ($\beta$ts = 1.36 $\AA^{−1}$) decay constants for the two-pathway model. This indicates that the charge transport properties of HDT SAMs on Au nanoplates are consistent with those on a Au (111) film, suggesting that SAMs on nanoplates can provide a new building block for molecular electronics.

In chapter 3, We report the characterization and formation of catechol-terminated molecules immobilized on gold nanoplates (Au NPLs) using N-(3,4-dihydroxyphenethyl)-2-mercaptoacetamide (Cat-EAA-SH). Single-crystalline Au NPLs, synthesized using a one-step chemical vapor transport method, have ultraclean and ultraflat surfaces that make Cat-EAA-SH molecules aligned into a well-ordered network of a large-scale. Topographic study of the catechol-terminated molecules on Au NPLs using atomic force microscopy (AFM) showed more orderly orientation and higher density, leading to significantly higher adhesion as observed from local force-distance curves than those on other Au surfaces. These coherently aligned catechol-terminated molecules on the atomically smooth gold surface led to significantly more reproducible and thus more physico-chemically meaningful measurements than was possible before by employing rough gold surfaces.

In chapter 4, we report a ultrasensitive SERS platform using a ultraclean and ultraflat single-crystalline Au nanoplate to detect GPC1, which is a new biomarker of pancreatic cancer, and expressed on the surface of tumor-derived exosomes. Unlike conventional Au films, Cys3-protein G was uniformly immobilized by Au-S bonds on Au nanoplate with atomically smooth surfaces without defects over the entire nanoplate that has a size of several hundred microns, and the antigen binding site of the antibody can be immobilized on the substrate without denaturation as protein G binds specifically to the Fc region of the antibody. We fabricated a SERS sensor using a substrate composed of [Au nanoplate + protein G + anti-GPC1] and modified Au nanoparticles. The SERS signals intensified as the GPC1 peptide concentration increased. This intensity increased linearly within a concentration range of 10 aM to 10 fM, enabling the quantitative detection of GPC1 peptide, and estimated the detection limit of this method to be 10 aM. SERS analysis can distinguish between positive and negative GPC1 in a variety of samples from peptides, cell lines, and clinical samples with high sensitivity and specificity. In addition, we was performed to dilute the serum to detect exosomes at a concentration as low as about 2,000 times and to improve sensitivity and specificity. This indicates that the usefulness of pancreatic cancer as a biomarker and the possibility of early diagnosis of disease.

In the last chapter, Multivalency can improve the sensitivity of biosensor because the increased valency can strengthen the binding affinity between the receptor and target biomolecule. Here, we report surface-enhanced Raman scattering (SERS)-based immunoassay by using multivalent antibody-conjugated nanoparticles (NPs) for the first time. Multivalent antibody was generated through the ligation of Fab fragment-fused Fc binding peptide to Immunoglobulin G. This synthetic method is easy and fast due to the elimination of heterologous expression, fusion of antibody, or chemical modification steps. We constructed multivalent antibody-nanoparticle conjugate (MANC) and employed them as a SERS immunoprobe. MANCs improved the sensitivity of SERS-based immunoassay 100 times than the standard antibody-NP conjugates. We anticipate that the MANCs make us one-step closer to the practical SERS-based immunoassay.