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。     

SERS光谱技术在人体体液研究中的应用

SERS光谱技术具有高检测灵敏度和高特异性的优点,广泛应用于生物组织,细胞及生物分子的研究,表现出极大的应用潜力。本文首先介绍了拉曼及SERS光谱技术的原理,综述了SERS光谱技术在人体体液分析检测的研究概况,特别介绍了本课题组针对血液、精液、尿液开展的研究工作,最后简要讨论SERS光谱技术的发展前景。     

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 Raman 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。最后,对SERS这一技术作为实时、高效和可靠的病原菌检测工具的未来发展进行了展望。     

SERS标记方法在生化分析中的应用

表面增强拉曼散射(SERS)标记方法结合现代生物标记方法与SERS光谱技术,使吸附到金或银等贵金属表面的标记分子的拉曼信号显著增强,并将其作为标记示踪信号,具有生物兼容性好、灵敏度高、分子特征性强和快速简便等优点,已成为新颖的标记示踪技术的研究热点之一。本文综述近年来SERS标记技术应用于基因分析、蛋白质检测、微生物检测、肿瘤靶向和小分子物质的最新进展,着重介绍蛋白质和小分子物质的检测,并展望了今后的发展方向。     

非标记表面增强拉曼光谱在DNA检测中的应用

表面增强拉曼光谱(SERS)是一种超灵敏的生化分析技术,已经被广泛运用于细胞、核酸、蛋白质等生物分子的检测,在生物医学领域表现出了巨大的应用潜力。近年来,将表面增强拉曼光谱技术应用于遗传物质DNA的精准检测,引起了人们广泛的关注。本文简要叙述了表面增强拉曼光谱技术的基本原理及其在DNA检测中的优势,主要介绍了非标记的DNA-SERS检测应用进展,其中包括本项目组的相关工作。研究表明,非标记DNA-SERS技术有望成为一种快速、准确的临床诊断方式。     

有序金纳米柱SERS基底制备及苏丹Ⅱ的拉曼光谱

苏丹Ⅰ～Ⅳ是色泽鲜艳的化学物质,被非法用于食品添加剂,损害人们身体健康。表面增强拉曼光谱(SERS)借助金属纳米结构的表面等离激元共振(SPR)效应,具有很高的检测灵敏度,可用于苏丹分子的快速检测。本文中,笔者制备了有序金纳米柱二维阵列结构,作为SERS基底,用于苏丹Ⅱ的SERS检测。为了更好地理解金纳米柱阵列结构对苏丹Ⅱ的SERS增强效应,结合有限元方法计算,分析了金纳米阵列结构与SPR效应的关系,并结合密度泛函理论(DFT)计算分析了SERS光谱与分子结构的关系。这有利于进一步提高SERS检测灵敏度。结果表明,对于苏丹Ⅱ的SERS检测,最低浓度为0. 05 mmol/L,DFT计算确定苏丹Ⅱ的结构为trans-NH异构体。     

表面增强拉曼散射技术无标记检测乳腺癌细胞来源外泌体

外泌体含有释放细胞的特异性物质,反映了释放细胞的组成成分,成为液体活检的重要物质。为了鉴别正常乳腺上皮细胞和乳腺癌细胞来源的外泌体,本研究采用表面增强拉曼(SERS)技术检测乳腺癌细胞MCF-7和人正常乳腺上皮细胞MCF-10A来源的外泌体,并且对比两种细胞外泌体的SERS光谱,筛选出相应的特征拉曼光谱。结果发现在800~1800 cm-1区域内,相对MCF-10A细胞,MCF-7来源的外泌体中归属于核酸的拉曼峰相对强度明显增高,且部分脂类峰强有所降低或部分特征峰消失,同时发现MCF-7还表现出其自己独特的拉曼谱型。此结果表明SERS技术可高灵敏度检测不同细胞来源的外泌体细微分子变化,可区分肿瘤细胞来源的外泌体与正常细胞来源的外泌体。SERS技术可作为癌症的早期检测和诊断的一种快速、无标记和无损的方法。     

表面增强拉曼光谱技术在外泌体检测中的研究进展

外泌体是一种含有蛋白质、核酸和脂质等物质的小囊泡,与细胞间通信和肿瘤微环境的调节有关,因而成为一种新兴的无创早期癌症诊断标志物。目前用于外泌体分析的技术较复杂,并且耗时久、成本高。近年来,表面增强拉曼光谱(SERS)技术由于其灵敏度高、样品制备简单、快速无损等优点,在生物医学领域表现出了巨大的应用潜力。本文首先简要介绍了外泌体以及目前主要检测方法的优缺点,其次,阐述了SERS的基本原理及其在外泌体检测中的优势,重点介绍了非标记和标记SERS在外泌体检测中的应用进展。系列研究表明,基于SERS的外泌体检测技术有望成为一种无损、便捷、准确的临床诊断新方法。     

鼻咽癌细胞组织与血液的SERS光谱研究进展

基于表面增强拉曼光谱(SERS)技术对于人体细胞组织与血液的检测和研究,SERS光谱技术能够发现正常组织与病变组织的差异性,为医学临床上实现癌症的早期诊断提供了科学依据。由于鼻咽癌不具有明显的病变特征、病灶的位置难以通过常规医学手段检测。因此利用SERS光谱技术,应用于鼻咽癌细胞组织与血液的研究,可提高鼻咽癌患者的生存率。对鼻咽癌细胞组织与血液SERS光谱分析和诊断的探索研究,有助于SERS光谱技术发展成为一种在生物医学领域中的分析检测手段,在医学临床诊断上具有潜在的应用前景。     

基于固态基底的SERS免疫检测新方法研究

目的:通过引入新型表面增强拉曼散射(SERS)检测探针(Au-DTNB-Tyr NPs)和金标银染技术,建立基于固态硅片基底的SERS免疫检测新技术。方法:羊抗人IgM-HRP作为检测抗体,在硅片基底上检测不同浓度的人IgM,HRP催化SERS检测探针沉积,利用金标银染技术增强SERS信号。结果:所建立的SERS免疫检测新方法检测人IgM的检测限为10 pg/mL,且SERS信号强度与人IgM浓度具有良好的线性关系(R2=0.993)。结论:基于硅片基底的SERS免疫检测新技术可高灵敏地定量检测人IgM,为实现固态硅片基底对多种抗原的高通量集成化检测奠定了基础。     

New biochip technology for label-free detection of pathogens and their toxins

microSERS is a new biochip technology that uses surface-enhanced Raman scattering (SERS) microscopy for label-free transduction. The biochip itself comprises pixels of capture biomolecules immobilized on a SERS-active metal surface. Once the biochip has been exposed to the sample and the capture biomolecules have selectively bound their ligands, a Raman microscope is used to collect SERS fingerprints from the pixels on the chip. SERS, like other whole-organism fingerprinting techniques, is very specific. Our initial studies have shown that the Gram-positive Listeria and Gram-negative Legionella bacteria, Bacillus spores and Cryptosporidium oocysts can often be identified at the subspecies/strain level on the basis of SERS fingerprints collected from single organisms. Therefore, pathogens can be individually identified by microSERS, even when organisms that cross-react with the capture biomolecules are present in a sample. Moreover, the SERS fingerprint reflects the physiological state of a bacterial cell, e.g., when pathogenic Listeria and Legionella were cultured under conditions known to affect virulence, their SERS fingerprints changed significantly. Similarly, nonviable (e.g., heat- or UV-killed) microorganisms could be differentiated from their viable counterparts by SERS fingerprinting. Finally, microSERS is also capable of the sensitive and highly specific detection of toxins. Toxins that comprised as little as 0.02% by weight of the biomolecule-toxin complex produced strong, unique fingerprints when spectra collected from the complexes were subtracted from the spectra of the uncomplexed biomolecules. For example, aflatoxins B(1) and G(1) could be detected and individually identified when biochips bearing pixels of antibody or enzyme capture biomolecules were incubated in samples containing one or both aflatoxins, and the spectra were then collected for 20 s from an area of the biomolecule pixel approximately 1 microm in diameter. In the future, we plan to investigate the use of hyperspectral imaging Raman microscopy for collecting fingerprints from all the pixels on the biochip, individually yet simultaneously, to enable the rapid detection of diverse pathogens and their toxins in a sample, using a single biochip.     

Rapid and Sensitive Detection of Rotavirus Molecular Signatures Using Surface Enhanced Raman Spectroscopy

Human enteric virus infections range from gastroenteritis to life threatening diseases such as myocarditis and aseptic meningitis. Rotavirus is one of the most common enteric agents and mortality associated with infection can be very significant in developing countries. Most enteric viruses produce diseases that are not distinct from other pathogens, and current diagnostics is limited in breadth and sensitivity required to advance virus detection schemes for disease intervention strategies. A spectroscopic assay based on surface enhanced Raman scattering (SERS) has been developed for rapid and sensitive detection of rotavirus. The SERS method relies on the fabrication of silver nanorod array substrates that are extremely SERS-active allowing for direct structural characterization of viruses. SERS spectra for eight rotavirus strains were analyzed to qualitatively identify rotaviruses and to classify each according to G and P genotype and strain with >96% accuracy, and a quantitative model based on partial least squares regression analysis was evaluated. This novel SERS-based virus detection method shows that SERS can be used to identify spectral fingerprints of human rotaviruses, and suggests that this detection method can be used for pathogen detection central to human health care.     

Single-molecule fluorescence characterization in native environment

Single-molecule detection (SMD) with fluorescence is a widely used microscopic technique for biomolecule structure and function characterization. The modern light microscope with high numerical aperture objective and sensitive CCD camera can image the brightly emitting organic and fluorescent protein tags with reasonable time resolution. Single-molecule imaging gives an unambiguous bottom-up biomolecule characterization that avoids the "missing information" problem characteristic of ensemble measurements. It has circumvented the diffraction limit by facilitating single-particle localization to ~1 nm. Probes developed specifically for SMD applications extend the advantages of single-molecule imaging to high probe density regions of cells and tissues. These applications perform under conditions resembling the native biomolecule environment and have been used to detect both probe position and orientation. Native, high density SMD may have added significance if molecular crowding impacts native biomolecule behavior as expected inside the cell.     

Design of Polarization Independent SERS Substrate with Raman Gain Evaluated Using Purcell Factor

Surface-enhanced Raman scattering (SERS) is a very promising detection/diagnostic technique at trace levels as the molecules exhibit a significant increase in their Raman signals when they are attached or are in proximity to plasmonic structures. In this study, a numerical design of SERS substrate as a probe has been demonstrated for detection and diagnosis of blood, water and urea samples. The proposed nanospiral design is polarization independent, and it offers the enhancement of the electric field strength ~ 10⁹. The substrate design is based on 3D finite difference time domain simulations and is robust, versatile and sensitive even at low concentrations of the analyte. It works equally well when used in the reflection mode. In this study, the cavity quantum electrodynamics (CQED) Purcell factor has also been transposed to plasmonics. The Purcell factor in corroboration with CQED has been used to achieve efficient light–matter interaction at nanoscale by providing a more realistic result. It takes into account the randomness of incident wave polarizations and arbitrary orientations of interacting molecules. This gives a deeper insight into electromagnetic Raman gain in SERS and can be used to design novel SERS substrates.

     

Antibody-based SERS diagnostics of fhit protein without label

Surface-enhanced Raman scattering (SERS) is a particularly promising technique that has the potential to perform highly selective and sensitive in situ measurements of antibody-antigen reactions. This work describes the use of silver (Ag) colloids for immunoassay-based SERS detection of the fragile histidine triad (Fhit) protein. Alterations in Fhit protein expression have been associated with several human cancers, and, thus, the detection of Fhit protein is important because it can potentially be used as a cancer diagnostic biomarker, for both cancer detection and therapy.     

Study on gastric cancer blood plasma based on surface-enhanced Raman spectroscopy combined with multivariate analysis

A surface-enhanced Raman spectroscopy (SERS) method combined with multivariate analysis was developed for non-invasive gastric cancer detection. SERS measurements were performed on two groups of blood plasma samples: one group from 32 gastric patients and the other group from 33 healthy volunteers. Tentative assignments of the Raman bands in the measured SERS spectra suggest interesting cancer-specific biomolecular changes, including an increase in the relative amounts of nucleic acid, collagen, phospholipids and phenylalanine and a decrease in the percentage of amino acids and saccharide in the blood plasma of gastric cancer patients as compared with those of healthy subjects. Principal components analysis (PCA) and linear discriminant analysis (LDA) were employed to develop effective diagnostic algorithms for classification of SERS spectra between normal and cancer plasma with high sensitivity (79.5%) and specificity (91%). A receiver operating characteristic (ROC) curve was employed to assess the accuracy of diagnostic algorithms based on PCA-LDA. The results from this exploratory study demonstrate that SERS plasma analysis combined with PCA-LDA has tremendous potential for the non-invasive detection of gastric cancers.     

鱼肉中磺胺类抗生素的表面增强拉曼光谱探测与分析

本文以银溶胶为表面增强拉曼活性基底,实现了鱼肉中磺胺类抗生素的痕量检测。采用微波加热法制备银溶胶,比较了两种提取剂（氨水、乙酸乙酯）对鱼肉中抗生素的提取及探测效果。实验发现,鱼肉中的物质对抗生素检测有较大干扰,乙酸乙酯作为提取剂的效果要明显好于氨水。以银溶胶为基底,乙酸乙酯作为提取剂对两种限制使用的磺胺类抗生素（磺胺甲基嘧啶、磺胺二甲基嘧啶）检测的最低浓度皆为1 ppm,检测限分别为0．16 ppm、0．59 ppm。结果表明,利用此方法,可以实现鱼肉中一定浓度抗生素的检测,为实现水产品中抗生素的检测提供了实验基础。     

Highly sensitive SERS detection of cancer proteins in low sample volume using hollow core photonic crystal fiber

Enzyme-linked immunosorbent assays (ELISA) are commonly used for detecting cancer proteins at concentration in the range of about ng-μg/mL. Hence it often fails to detect tumor markers at the early stages of cancer and other diseases where the amount of protein is extremely low. Herein, we report a novel photonic crystal fiber (PCF) based surface enhanced Raman scattering (SERS) sensing platform for the ultrasensitive detection of cancer proteins in an extremely low sample volume. As a proof of concept, epidermal growth factor receptors (EGFRs) in a lysate solution from human epithelial carcinoma cells were immobilized into the hollow core PCF. Highly sensitive detection of protein was achieved using anti-EGFR antibody conjugated SERS nanotag. This SERS nanotag probe was realized by anchoring highly active Raman molecules onto the gold nanoparticles followed by bioconjugation. The proposed sensing method can detect low amount of proteins at ～100 pg in a sample volume of ～10 nL. Our approach may lead to the highly sensitive protein sensing methodology for the early detection of diseases.