Development of au nanostructure complex based surface enhanced raman scattering (SERS) sensor for disease biomarker detection and study of nanomaterial biotoxicity

Abstract

Gold is very stable in biochemical environments and can immobilize easily bioactive molecules including DNAs, aptamers, antibodies, and peptides through Au-S bonding. In addition, gold is an excellent plasmonic material and has been widely used for fabrication of sensitive sensors. However, intrinsic surface defects of gold, such as step, terrace, vacancy, and grain boundary, are major problems for uniform immobilization of the biochemical molecules. Top-down polishing including thermal annealing, UV-ozone cleaning, and hydroxyl radical etching has been employed to reduce the surface defects, but being hardly possible to eliminate them completely. On the other hand, Au nanoplates synthesized in vapor phase have atomically smooth surfaces without any surface defects. Atomically flat surfaces of single-crystalline Au nanoplates can maximize the functionality of biomolecules, thus realizing extremely high-performance biosensors. Here, this paper reports both highly specific and supersensitive detection of C-reactive protein (CRP) and anti-cyclic citrullinated peptides (anti-CCP) by employing atomically flat Au nanoplates. Therefore, this thesis is organized as follows. Chapter 1 demonstrates Synthesis of two dimensional single crystalline Au nanostructures for applications of biosensors. Chapter 2 reports ultrasensitive CRP detection method employing Au nanoplate and protein G. Chapter 3 reports ultrasensitive anti-CCP detection method employing Au nanoplate and cyclic citrullinated peptides (CCP).

In chapter 1, we demonstrate synthesis of single crystalline Au nanoplates using CVD method, and report surface roughness measurements with atomic force microscopy (AFM).

In chapter 2, we report super-sensitive detection for CRP employing an ultraclean and attomically flat Au nanoplate. CRP is a protein biomarker for inflammation and infection and can be used as a predictive or prognostic marker for various cardiovascular diseases. To maximize the binding capacity for CRP, we optimized the Au nanoplate-Cys3-protein G-anti-CRP structure by observing atomic force microscopy (AFM) images. The optimally anti-CRP-immobilized Au nanoplates allowed extremely specific detection of CRP at the attomolar level. To confirm the binding of CRP onto the Au nanoplate, we assembled Au nanoparticles (NPs) onto the CRP-captured Au nanoplate by a sandwich immunoreaction and obtained surface-enhanced Raman scattering (SERS) spectra and scanning electron microscopy (SEM) images. Both the SERS and SEM results showed that we completely eliminated the nonspecific binding of Au NPs onto the optimally anti-CRP-immobilized Au nanoplate. Compared with the anti-CRP-immobilized rough Au film and the randomly anti-CRP-attached Au nanoplate, the optimally anti-CRP-immobilized Au nanoplate provided a highly improved detection limit of 10-17 M.

In chapter 3, we report super-sensitive detection for anti-CCP employing an ultraclean and atomically flat Au nanoplate. Recently, it has been reported that a small amount of anti-CCPs exists at early-stage of the rheumatoid arthritis (RA). So, ultrasensitive sensor to detect them has been quite desirable for early-diagnosis of RA. Here, we produced a highly sensitive SERS sensor against anti-CCPs employing ultraflat, ultraclean single-crystalline Au nanoplates. Interestingly, the atomically flat Au nanoplates without surface defects can immobilize thiolated CCPs and blocking molecules uniformly and reduce the non-specific bindings as 50 times compared with a commercial Au film. Therefore, the Au Particle-on-Plate SERS platform employing the Au nanoplates can analyze anti-CCPs quantitatively with 4×10-17 M (40aM) of detection limit.

In chapter 4, we report the bioaccumulation of nanoplastics and their effect on the toxicity of a metal ion individually and in combination, using polystyrene (PS) nanoplastics and Au ions as representative model materials, respectively, in zebrafish embryos (ZFEs). We demonstrated that PS nanoplastics induced only marginal alterations in the survival, hatching rate, developmental abnormality, and cell death of ZFEs but that these effects became synergistically exacerbated in a size- and dose-dependent manner when the PS nanoplastics were concomitant with the Au ion. Such exacerbation of toxicity was correlated with the oxidative stress and pro-inflammatory responses synergized by the presence of PS, which induced mitochondrial damages at the subcellular level. Taken together, we propose a role of PS mainly as a potential risk factor for environments and organisms.