Au-MPY/DTNB@SiO₂ SERS nanoprobe for immunosorbent assay

Highlights

• Biocompatible SERS nanoprobe for protein detection.

• Silica-protected gold nanoaggregates for strong SERS enhancement.

• Sandwich (silver-proteins-Au-MPY/DTNB@SiO₂) model for ultrasensitive SERS immunoassay applications.

Abstract

In this paper, we developed a biocompatible surface-enhanced Raman scattering (SERS) nanoprobe that employs 4-mercaptopyridine (MPY)/5,5-dithiobis(2-nitrobenzoic acid) (DTNB)-decorated gold aggregates embedded in silicon dioxide. This highly sensitive SERS nanoprobe was applied in immunoassays. Furthermore, we constructed a sandwich (silver-proteins-Au-MPY/DTNB@SiO₂) model that contributes very strong electromagnetic (EM) fields. Compared with a single layer of the SERS-active substrate, the designed model exhibited highly sensitive detection capabilities. This method demonstrates considerable potential for ultrasensitive SERS immunoassay applications. In addition, the proposed silica-protected gold nanoaggregates possess good biocompatibility with proteins.

Introduction

Recently, surface-enhanced Raman scattering (SERS)-based high-sensitivity sensing technologies have become increasingly important because of the growing demands for biosystems, such as molecular biology analysis, disease diagnosis, and drug discovery [1], [2], [3], [4], [5], [6], [7]. The notable advantage of the SERS technique is that it is both surface selective and highly sensitive compared with other spectroscopic techniques. More importantly, SERS provides a large amount of fingerprint information for the identification of individual components in a mixture, which is a commonly exploited advantage of SERS [8], [9]. The most widely used method for a multiplex assay based on SERS involves using the encoding microbeads or nanoprobes with a unique code to identify the attached ligand molecules [10], [11], [12]. Although SERS-based encoding protocols have shown particular promise for multiplex detection, the amount of acquired molecular information is restricted by the number of distinguishable codes in the same array. In addition, the target always interferes with the SERS activity, which affects the accuracy and sensitivity of the detection. Moreover, with the development of the SERS technique, novel SERS-based nanoprobes have been developed and used for bioanalysis. These newly developed SERS-active nanoprobes produce strong and characteristic SERS signals that can be used for the indirect detection of target molecules through laser Raman spectrometry, demonstrating SERS labeling functions similar to the functions of external chromophores, such as organic dyes and fluorescent quantum dots. Furthermore, the proposed nanoprobes possess the ultrasensitive, multiplexing, and quantitative abilities of the SERS technique and demonstrate extraordinary features for bioanalysis.

In a SERS-based protein study, two independent approaches are most commonly employed to detect proteins directly and indirectly. For the direct determination of proteins, we can obtain the Raman information of the protein itself, which is direct, convenient, and more reliable than extrinsic SERS labeling. However, two major limitations include poor selectivity in some complex mixtures and poor sensitivity at relatively low sample concentrations [3], [13], [14], [15]. Therefore, most research focuses on the indirect determination of proteins based on the SERS method, which requires an appropriate dye label that also contributes to the Raman effect through resonance enhancement in addition to the surface enhancement effect, thus allowing multiplexed detection. The dye label-based method exhibits greater sensitivity and selectivity for the quantitative determination of proteins than direct protein detection. However, nonspecific adsorption in complex mixtures lowers the signal-to-background ratio and also generates false positive identifications. In addition, the reagent-based quantitative analysis of proteins using the SERS method can achieve much lower detection limits for the ultrasensitive detection of proteins and much wider linear concentration ranges for quantitative analysis, with excellent application to quantitative analysis of proteins.

In SERS-based studies of biomolecules, the stability and reproducibility of the SERS-active substrate are key points [16], [17], [18]. To date, many types of novel biocompatible SERS substrates have been developed. For example, silicon-dioxide-coated SERS-active substrates and tip-enhanced Raman scattering (TERS)-based methods for the analysis of protein structure, in which contact between the proteins and precious metals is avoided, have been developed for protein detection and analysis [19], [20], [21]. A shell-isolated nanoparticle-enhanced Raman spectroscopy (SHINERS)-based method was recently developed and has been widely used to study biomolecules [22], [23]. The SHINERS method employs ultrathin silica- or alumina shell-covered gold nanoparticles as the SERS-active substrate, which can avoid agglomeration and separate the substrate from direct contact with the target. Therefore, a high quantity of SERS results can be obtained based on the SHINERS method. To take advantage of the shell that is covered on the surface of the metal nanoparticles, herein, we report the development of a silica-covered gold nanoprobe for the quantitative determination of proteins. Based on the proposed method, picomolar detection can be achieved. In addition, the silica-covered gold nanoparticles show very good biocompatibility, which is useful in bimolecular studies [24], [25], [26], [27].