表面增强拉曼光谱技术在肿瘤病理中的应用

表面增强拉曼散射（Surface-enhanced Raman Scattering,SERS）技术作为鉴定生物分子种类最有力的分析工具之一,具有灵敏度高、特异性强、稳定性好及检测条件温和等优点。目前,SERS技术在肿瘤病理领域的应用尚处于起步阶段,但已显现出良好的应用前景和发展空间。该文简要介绍了SERS的机理、特性及活性基底,并对SERS技术在肿瘤病理的研究进展、局限性及潜在应用价值方面做较为全面的综述。     

Surface‐enhanced Raman spectroscopy for on‐site analysis: A review of recent developments

On‐site identification and quantification of chemicals is critical for promoting food safety, human health, homeland security risk assessment, and disease diagnosis. Surface‐enhanced Raman spectroscopy (SERS) has been widely considered as a promising method for on‐site analysis due to the advantages of nondestructive, abundant molecular information, and outstanding sensitivity. However, SERS for on‐site application has been restricted not only by the cost, performance, and portability of portable Raman instruments, but also by the sampling ability and signal enhancing performance of the SERS substrates. In recent years, the performance of SERS for on‐site analysis has been improved through portable Raman instruments, SERS substrates, and other combined technologies. In this review, popular commercial portable Raman spectrometers and the related technologies for on‐site analysis are compared. In addition, different types of SERS substrates for on‐site application are summarized. SERS combined with other technologies, such as electrochemical and microfluidics are also presented. The future perspective of SERS for on‐site analysis is also discussed.     

表面增强拉曼光谱技术在微生物鉴定中的应用进展

拉曼光谱自20世纪20年代被发现以来,经过近90年的发展,产生了许多分支。其中表面增强拉曼光谱是利用被测物质与粗糙金属表面的相互作用来提高拉曼光谱的信噪比,从而得到敏感度和精确度更高的图谱,可以将样品在不经过预处理的情况下对其进行快速检测。本文综述了表面增强拉曼光谱技术的原理、分类及鉴定特点,总结了该技术在食品、化学、医药、工业、病原等微生物学科的临床应用,进一步阐述了研究该技术的必要性和应用前景,旨在为从事该领域的科研人员提供参考依据。     

Characterization of multilayer-enhanced surface-enhanced raman scattering (SERS) substrates and their potential for SERS nanoimaging

Multilayer gold surface-enhanced Raman scattering (SERS) substrates, which consist of continuous gold films that are separated by self-assembled monolayers (SAMs) and cast over 430-nm diameter silica nanospheres on a glass slide, have been evaluated as a means of further enhancing the SERS signals produced from conventional metal film over nanostructure substrates. Evaluation of the effect of various SAMs, with different terminal functional groups, on the SERS enhancement factor were measured and compared to conventional single-layer gold film over nanostructure substrates, revealing relative enhancements as great as 22.4-fold in the case of 2-mercapto-ethanol spacer layers. In addition to evaluation of the effect of different terminal functionalities, the effect of spacer length was also investigated, revealing that the shorter chain length alcohols provided the greatest signals. Employing the optimal SERS multilayer geometry, SERS nanoimaging probes were fabricated and the SERS enhancement factor and variability in enhancement factor were measured over the SERS active imaging area, providing absolute enhancements similar to previous silver-based SERS nanoimaging probes (i.e., 1.2 × 10⁸). Varying the size of the multilayer gold islands that were deposited on the tip of the SERS active nanoimaging probe, it is possible to tune the optimal SERS excitation wavelength accurately and predictably over the range of approximately 450 to 600 nm, without coating the entire surface of the probe and significantly reducing the transmission and resulting signal-to-noise ratio of the images obtained.     

表面增强拉曼散射机理研究进展

简要回顾表面增强拉曼散射（Surface—enhanced Raman Scattering,SERS）的发展历史,主要综述近年来SERS物理增强机理及理论计算方面的研究进展,并对SERS机理理论研究发展作了展望。     

Surface-Enhanced Raman Scattering from Individual Au Nanoparticles on Au Films

We investigated the effect of optical thick metal films on the surface-enhanced Raman scattering (SERS) activity of individual Au nanoparticle (NP) monomers and dimers. The film presence is revealed to be positive for the SERS activity of individual NP monomers, while it is not always positive for the electromagnetic enhancement at hot spots for SERS of the dimer, which is explained well by our numerical simulations. The polarized SERS signals from the NP dimer are elucidated well in terms of the plasmon hybridization of the dimer. SERS contributions both from individual NP surfaces and the junction between the NP and its supporting substrate were discussed as well.     

Bioanalytical application of surface‐ and tip‐enhanced Raman spectroscopy

Due to its fingerprint specificity and trace‐level sensitivity, surface‐enhanced Raman spectroscopy (SERS) is an attractive tool in bioanalytics. This review reflects the research in this highly interesting topic of the last 3–4 years. The detection of the SERS signature of biomolecules up to microorganisms and cells is introduced. Labeling using modified nanoparticles (SERS tags) is also introduced. In order to establish biomedical applications, SERS analysis is performed in complex matrices such as body fluids. Furthermore, the SERS technique is combined with other methods such as microfluidic devices for online monitoring and scanning probe microscopy (i.e. tip‐enhanced Raman spectroscopy, TERS) to investigate nanoscaled features. The present review illustrates the broad application fields of SERS and TERS in bioanalytics and shows the great potential of these methods for biomedical diagnostics.     

Rapid detection of food- and waterborne bacteria using surface-enhanced Raman spectroscopy coupled with silver nanosubstrates

Development of rapid and sensitive methods to detect pathogens is important to food and water safety. This study aimed to detect and discriminate important food- and waterborne bacteria (i.e., Escherichia coli O157:H7, Staphylococcus epidermidis, Listeria monocytogenes, and Enterococcus faecelis) by surface-enhanced Raman spectroscopy (SERS) coupled with intracellular nanosilver as SERS substrates. An in vivo molecular probing using intracellular nanosilver for the preparation of bacterial samples was established and assessed. Satisfactory SERS performance and characteristic SERS spectra were obtained from different bacterial samples. Distinctive differences were observed in SERS spectral data, specifically in the Raman shift region of 500–1,800 cm⁻¹, and between bacterial samples at the species and strain levels. The detection limit of SERS coupled with in vivo molecular probing using silver nanosubstrates could reach the level of single cells. Experiments with a mixture of E. coli O157:H7 and S. epidermidis for SERS measurement demonstrate that SERS could be used for classification of mixed bacterial samples. Transmission electron microscopy was used to characterize changes of morphology and cellular composition of bacterial cells after treatment of intracellular nanosilver. The results indicate that SERS coupled with intracellular silver nanosubstrates is a promising method for detection and characterization of food- and waterborne pathogenic and non-pathogenic bacterial samples.     

基于纳米信号标签的表面增强拉曼散射在病原菌检测中的应用

表面增强拉曼散射(surface-enhanced Ranan scattering,SERS)技术由于其灵敏度高、检测速度快、高特异性和无损等优点,在病原菌检测领域受到了广泛的关注.主要总结了近年来基于纳米信号标签的SERS方法在检测病原菌领域中的研究进展,并介绍了多功能SERS检测平台的构建及在病原菌检测中的应用....     

Protein separation and identification using magnetic beads encoded with surface-enhanced Raman spectroscopy

This article presents a prototype of a surface-enhanced Raman spectroscopy (SERS)-encoded magnetic bead of 8 μm diameter. The core part of the bead is composed of a magnetic nanoparticle (NP)-embedded sulfonated polystyrene bead. The outer part of the bead is embedded with Ag NPs on which labeling molecules generating specific SERS bands are adsorbed. A silica shell is fabricated for further bioconjugation and protection of SERS signaling. Benzenethiol, 4-mercaptotoluene, 2-naphthalenethiol, and 4-aminothiophenol are used as labeling molecules. The magnetic SERS beads are used as substrates for protein sensing and screening with easy handling. As a model application, streptavidin-bound magnetic SERS beads are used to illustrate selective separation in a flow cytometry system, and the screened beads are spectrally recognized by Raman spectroscopy. The proposed magnetic SERS beads are likely to be used as a versatile solid support for protein sensing and screening in multiple assay technology.     

Interface-Induced Ag Monolayer Film for Surface-Enhanced Raman Scattering Detection of Water-Insoluble Enrofloxacin

Water-insoluble molecules usually show poor surface-enhanced Raman scattering (SERS) signals, because they are hardly adsorbed on the surface of most commonly used SERS substrates, such as aqueous Ag or Au colloids. In this work, a highly sensitive and reproducible Ag monolayer film (Ag MLF) SERS substrate prepared by self-assembly of Ag nanoparticles (Ag NPs) on water/oil interface can realize the trace SERS detection of water-insoluble enrofloxacin. The positively charged phase transfer catalyst can transfer the negatively charged Ag nanoparticles in aqueous solution to the water/oil interface. At the same time, the water-insoluble enrofloxacin can also be attracted to the interface because of its lipophilic group. The type/volume of the oil phase and phase transfer catalyst and the vortex mixing time were all optimized to maximize the SERS effect of Ag MLF. Results showed that trace water-insoluble enrofloxacin can be identified by Ag MLF and its detection sensitivity was significantly improved. The proposed novel Ag MLF can be further applied to detect other water-insoluble molecules in SERS.

     

Single cell analysis using surface enhanced Raman scattering (SERS) tags

Fluorescence is a mainstay of bioanalytical methods, offering sensitive and quantitative reporting, often in multiplexed or multiparameter assays. Perhaps the best example of the latter is flow cytometry, where instruments equipped with multiple lasers and detectors allow measurement of 15 or more different fluorophores simultaneously, but increases beyond this number are limited by the relatively broad emission spectra. Surface enhanced Raman scattering (SERS) from metal nanoparticles can produce signal intensities that rival fluorescence, but with narrower spectral features that allow a greater degree of multiplexing. We are developing nanoparticle SERS tags as well as Raman flow cytometers for multiparameter single cell analysis of suspension or adherent cells. SERS tags are based on plasmonically active nanoparticles (gold nanorods) whose plasmon resonance can be tuned to give optimal SERS signals at a desired excitation wavelength. Raman resonant compounds are adsorbed on the nanoparticles to confer a unique spectral fingerprint on each SERS tag, which are then encapsulated in a polymer coating for conjugation to antibodies or other targeting molecules. Raman flow cytometry employs a high resolution spectral flow cytometer capable of measuring the complete SERS spectra, as well as conventional flow cytometry measurements, from thousands of individual cells per minute. Automated spectral unmixing algorithms extract the contributions of each SERS tag from each cell to generate high content, multiparameter single cell population data. SERS-based cytometry is a powerful complement to conventional fluorescence-based cytometry. The narrow spectral features of the SERS signal enables more distinct probes to be measured in a smaller region of the optical spectrum with a single laser and detector, allowing for higher levels of multiplexing and multiparameter analysis.     

Highly reproducible immunoassay of cancer markers on a gold-patterned microarray chip using surface-enhanced Raman scattering imaging

This paper reports a highly reproducible immunoassay of cancer markers using surface-enhanced Raman scattering (SERS) imaging. SERS is a highly sensitive detection method but it is limited in its ability to achieve reproducible signal enhancement because of the difficulty with precisely controlling the uniform distribution of hot junctions. Consequently, inconsistent enhancement prevents the wide exploitation of SERS detection as a bio-detection tool for quantitative analysis. To resolve this problem, we explored the use of a SERS imaging-based immunoassay. For this purpose, Raman reporter-labeled hollow gold nanospheres (HGNs), were manufactured and antibodies were immobilized onto their surfaces for targeting specific antigens. After the formation of sandwich immunocomplexes using these functional HGNs on the surfaces of gold patterned wells, the SERS mapping images were measured. For target protein markers, 12×9 pixels were imaged using a Raman mapping technique in the 0-10(-4) g/mL concentration range, and the SERS signals for 66 pixels were averaged. Here, the SERS imaging-based assay shows much better correlations between concentration and intensity than does the conventional point-based assay. The limits of detection were determined to be 0.1 pg/mL and 1.0 pg/mL for angiogenin (ANG) and alpha-fetoprotein (AFP), respectively. This detection sensitivity is increased by three or four orders of magnitude over that of conventional ELISA method. The detectable dynamic range for SERS imaging (10(-4)-10(-12) g/mL) is also much wider than that for ELISA (10(-6)-10(-9) g/mL).     

Quantitative Surface-Enhanced Raman for Gene Expression Estimation

We demonstrate for the first time, to our knowledge, a unique gene expression assay by surface-enhanced Raman scattering (SERS) using nonfluorescent Raman labels to quantify gene expression at the resolution of alternative splicing using RNA extracted from cancer cells without any amplification steps. Our approach capitalizes on the inherent plasmon-phonon mode of SERS substrates as a self-referencing standard for the detection and quantification of genetic materials. A strategy integrating S1 nuclease digestion with SERS detection was developed to quantify the expression levels of splice junction Δ(9,10), a segment of the breast cancer susceptibility gene 1 (BRCA1) from MCF-7 and MDA-MB-231 cells. Quantification results were cross-validated using two Raman tags and qualitatively confirmed by RT-PCR. Our methodology based on SERS technology provides reliable gene expression data with high sensitivity, bypassing the intricacies involved in fabricating a consistent SERS substrate.     

Study of Langmuir-Blodgett phospholipidic films deposited on surface enhanced Raman scattering active gold nanoparticle monolayers

Surface enhanced Raman scattering (SERS) was used to study phospholipid monolayers transferred by the Langmuir-Blodgett (LB) technique to SERS active substrates. These substrates, which were constituted of gold colloidal nanoparticles bound to polysilane films grafted onto glass plates, showed a uniform and homogeneous layer with strong interacting particles as revealed from UV-visible extinction spectra and atomic force microscopy images. Laser excitation at 632.8 nm within the red part of the localized surface plasmon resonance leads to intense and reproducible SERS spectra of trans-1,2-bis(4-pyridyl)ethylene (BPE). From SERS measurements at different pHs it was possible to determine the apparent pK(a) of BPE adsorbed on gold-coated silanized substrates in the absence and presence of one LB monomolecular layer of phospholipids. These SERS titrations allowed the estimation of the pH at the metal-LB film interface.     

Tapered Fiber Probe Modified by Ag Nanoparticles for SERS Detection

A tapered optical fiber fabricated by a simple chemical etching method and modified with Ag nanoparticles (AgNPs) by chemical deposition was evaluated for surface-enhanced Raman scattering (SERS). The fiber probe was used for SERS measurements in both direct and remote scattering modes, yielding desired performance in both scattering configurations. The state of the obtained AgNPs made a significant contribution to the high sensitivity of SERS to Rhodamine 6G (R6G) molecules (down to a concentration of 10^?7 M), and the substrate had an analyst enhancement factor (AEF) on the order of ～10⁸. Meanwhile, the SERS intensity during the evaporation process was investigated, showing a good stability at the later stage of the evaporation process. The fiber SERS probes demonstrated good reproducibility with the average relative standard deviation (RSD) values being less than 0.2 for the major Raman peaks.     

Direct Fabrication Route to Plastic-Supported Gold Nanoparticles for Flexible NIR-SERS

Stable gold nanoparticles with surface plasmon resonance tunable from visible (Vis) to near-infrared (NIR) are deposited via a direct sputtering methodology on large area polyethylene terephthalate (PET) to be used as effective, flexible NIR surface-enhanced Raman scattering (SERS) substrates. An O₂ plasma treatment of PET is used to tailor growth dynamics, geometry, and plasmonic properties of nanoparticles. The O₂ plasma treatment of PET results also in effective improvement of nanoparticle anchoring on the plastic substrate, providing more stable, flexible SERS systems. The functionality of fabricated SERS substrates has been tested using benzylthiol, and SERS enhancement factors in the range 10⁴ have been achieved, which are comparable with those reported in literature for gold nanostructures fabricated on silicon substrate. These results attest the great potentiality of this methodology for the production of cost-effective flexible and reusable large-scale SERS substrates.     

Structural analysis of biological aliphatic compounds using surface-enhanced Fourier transform Raman spectroscopy

The surface-enhanced Raman scattering (SERS) technique for Fourier transform Raman spectrometry is employed to reveal the chemical structure of biological aliphatic compounds consisting of folded, long aliphatic chains. The structural analysis is performed via the measurements of the accordion-vibration modes generated in the ordered, long aliphatic chain. The SERS spectra after subtraction of a background spectrum give segment lengths that are almost perfectly consistent with the chemical structures studied by mass spectrometry. The agreement of the SERS results with those of mass spectrometry suggests the positions of kinks in the long hydrocarbon chain. The combination technique of SERS and mass spectrometry is useful to discuss the structure of folded, long biological lipids.     

SERS观察在Pd OECC薄膜中β-胡萝卜素的基频和高阶拉曼模

利用有极高检测灵敏度的表面增强拉曼散射(SERS)技术,对吸附在银镜表面上的浓度较低的纯化的放氧核心复合物(Pd OECC)薄层进行了频移在250～3 100 cm-1范围内的拉曼光谱测量,除得到β-胡萝卜素分子的基频拉曼振动模外,在高频端还得到了许多弱峰.根据泛音和组合谱带选择定则分析,这些振动模式来自β-胡萝卜素分子的高阶拉曼光谱.还进行了Pd OECC在强光破坏前后的SERS光谱研究.在强光照射下,β-胡萝卜素分子的SERS光谱的散射强度明显降低,且线宽增加,说明强光照射不但改变了β-胡萝卜素的构象,而且也改变了β-胡萝卜素分子所处的微环境.其结果与强光照射前后吸收光谱的变化一致.另外,没有观察到Pd OECC薄层与银镜相互作用的其他新振动峰或Pd OECC中其他振动峰峰型的变化,可见Pd OECC在银镜表面保持原来的状态.这证明SERS技术在光合作用光破坏机理研究中的可行性.     

SERS quantitative detection of trace human chorionic gonadotropin using a label‐free Victoria blue B as probe in the aggregated immunonanogold sol substrate

Nanogold particles (NG) were modified by anti‐rabbit antibody (RAb) against human chorionic gonadotropin to obtain an immunonanogold probe (ING). In pH 7.0 Na₂HPO₄‐citrate buffer solution containing KCl, ING probes formed large aggregates in which Victoria blue B (VBB) molecules were adsorbed on the surface and which exhibited strong surface‐enhanced Raman scattering (SERS) at a peak of 1612 cm^–1. After addition of human chorionic gonadotropin (hCG) an immune reaction with the ING probe occurred to form dispersive ING–hCG complexes with non‐SERS activity that led to a decreased SERS peak at 1612 cm^–1. The decreased SERS intensity was linear to the concentration of hCG over 2.4–73.2 ng/mL. The ING reaction was studied in detail by SERS, scanning electron microscope (SEM), resonance Rayleigh scattering (RRS), surface plasmon resonance (SPR) absorption and laser scattering techniques. SERS quenching was observed and discussed. Copyright © 2014 John Wiley & Sons, Ltd.