银/硅纳米孔柱阵列活性基底的SERS效应研究

以硅纳米孔柱阵列(Si-NPA)为衬底,采用浸渍法制备出一种具有规则表面形貌特征的银/硅纳米孔柱阵列(Ag/Si-NPA),并以R6G为探测目标材料,对其表面增强拉曼(SERS)效应进行了研究。结果表明,对于R6G浓度低至10-15M,Ag/Si-NPA均能表现出清晰的特征SERS峰。随着浓度的降低,R6G的荧光淬灭,所测拉曼光谱的基线降低,但特征峰峰位基本保持不变。在低浓度10-15M时得到的SERS光谱,理论上证明为单分子光谱。此外,Ag/Si-NPA活性基底具有较好的稳定性,在长达28天的自然老化过程中,Ag/Si-NPA能够保持对R6G较高的探测水平,光谱具有较好的信噪比和分辨率。Ag/Si-NPA是一种理想的SERS活性基底。     

银纳米有序阵列SERS活性基底的制备及在膀胱肿瘤细胞检测中的应用

以多孔阳极氧化铝(porous anodic alumina,PAA)膜为模板,采用真空电子束蒸镀技术,分别在PAA多孔层以及阻挡层表面形成了银纳米孔和银纳米帽有序阵列表面增强拉曼散射(surface-enhancedRaman scattering,SERS)活性基底,并以膀胱肿瘤细胞作为分子探针,测试和分析了这两种SERS活性基底的表面增强拉曼光谱的特性。结果表明,两种SERS活性基底对膀胱肿瘤细胞的拉曼散射信号均有很好的增强作用。银纳米帽有序阵列SERS活性基底不仅具有较高的SERS增强和荧光猝灭效应,而且不存在与PAA膜中草酸根杂质相关的干扰峰,可获得膀胱肿瘤细胞拉曼散射光谱的更多细节信息。     

银纳米粒子/碳纳米管阵列SERS基底增强因子分析和实验

为了直观、准确地定量分析表面拉曼增强散射基底结构的拉曼增强,利用磁控溅射和高温退火的方法制备了银纳米粒子修饰垂直排列的碳纳米管阵列三维复合结构样品;实验采用罗丹明6G(R6G)溶剂作为探针分子,结合共聚焦显微拉曼系统,开展了表面增强拉曼增强因子(EF)分析计算的相关实验。SEM结果表明：在有序碳纳米管阵列的表面和外壁均匀地负载了大量银纳米粒子。对退火温度为450 ℃,退火时间为30 min的样品进行了EF计算,得到其增强因子约为2.2×103,并分析了EF值低的原因主要是：在碳纳米管上溅射的银膜膜厚不均匀,导致退火后银颗粒分布不均,使得样品粗糙度值偏大,EF值较低;实验中所用的激励光源并非银纳米颗粒的优化光源。     

基于三明治SERS结构和酶剪切技术的肿瘤标志物miRNA-21的高灵敏检测

采用Langmiur-Bloggt膜技术和磁控溅射技术制备Ag覆盖聚苯乙烯小球六方密堆积阵列(Ag/PS HCA)基底,再将合成的海胆状Au纳米粒子与4-巯基苯甲酸(4-Mercaptobenzoic acid,4MBA)链接得到Au@4MBA探针,然后将单链寡核苷酸DNA21分别与基底和Au@4MBA探针链接,构建Au@4MBA-DNA21-Ag/PS HCA三明治结构,在DNA21与miRNA-21杂交后使用双链特异性剪切酶(DSN)剪切DNA磷酸二酯键,最后进行表面增强拉曼散射信号检测.实验结果表明,基于上述三明治表面增强拉曼散射结构和酶剪切技术进行肿瘤标志物miRNA-21的检测,在100pmol·L~(-1)到1fmol·L~(-1)的浓度范围内,检测极限达到0.853fmol·L~(-1),具有极高的灵敏度和优良的特异性.     

银纳米粒子修饰三维碳纳米管阵列SERS实验

为了使表面增强拉曼散射(SERS)基底的三维聚焦体积内包含更多的“热点”,能吸附更多探针分子和金属纳米颗粒,以便获得更强的拉曼光谱信号,提出了银纳米粒子修饰垂直排列的碳纳米管阵列三维复合结构作为SERS基底,并对其进行了实验研究。利用化学气相沉积(CVD)方法制备了垂直排列的碳纳米管阵列;采用磁控溅射镀膜方法先在碳纳米管阵列上形成一层银膜,再通过设置不同的高温退火温度,使不同粒径的银纳米粒子沉积在垂直有序排列碳纳米管阵列的表面和外壁。SEM结果表明：在有序碳纳米管阵列的表面和外壁都均匀地负载了大量银纳米粒子,并且银纳米颗粒的粒径、形貌及颗粒间的间距随退火温度的不同而不同。采用罗丹明6G(R6G)分子作为探针分子,拉曼实验结果表明：R6G浓度越高,拉曼强度越强,但是R6G浓度的增加与拉曼强度增强并不呈线性变化;退火温度为450 ℃,银纳米颗粒平均粒径在100～120 nm左右,退火温度为400 ℃,银纳米颗粒平均粒径在70 nm左右,退火温度为450 ℃的拉曼信号强度优于退火温度400和350 ℃。     

以PAA为模板制备SERS基底及对三聚氰胺的检测

采用热蒸镀的方法直接在多孔氧化铝(porous anodic alumina,PAA)模板上蒸镀几微米的银膜,然后在HCl溶液中溶解掉模板,得到表面具有纳米尺度规则结构的银膜作为表面增强拉曼散射(surface-en-hanced Raman spectra,SERS)基底,并在该基底上测量了吡啶溶液(0.01 mol.L-1)的增强拉曼光谱,发现平均增强因子大于105。与直接在载玻片上蒸镀的银膜相比,具有纳米尺度规则结构银膜的增强效果提高了30倍。改变激发光功率测量吡啶的拉曼光谱,和普通拉曼散射一样,增强拉曼光谱的峰值强度随激发光强度线性变化,并在该基底上测量了三聚氰胺的拉曼光谱,发现在1 mW的激发功率下对于三聚氰胺的检出限为2.5 mg.L-1。     

银纳米阵列直径对SERS增强效果的影响

采用电化学沉积法分别在不同孔径的阳极氧化铝(AAO)模板上沉积一系列直径不同,排列规则的银纳米阵列。以对氨基苯甲酸(PABA)和三聚氰胺两种分子分别作为探针分子, 研究了银纳米阵列的直径大小对其表面增强拉曼散射(SERS)效果的影响。结果表明, 在波长为514.5 nm的激光激发下, 探针分子的SERS信号强度随银纳米阵列直径的改变而明显变化, 并在银纳米阵列直径约为53 nm时, SERS强度达到最大。利用电磁增强机制对此实验结果进行了分析和解释。     

基于SiC@Ag基底和银-生物素-链霉亲和素纳米聚集体双重SERS放大的miRNA-106a检测

基于SiC@Ag基底与Ag纳米颗粒的表面增强拉曼散射效应,提出了利用银-生物素-链霉亲和素纳米聚集体二次表面增强拉曼散射放大的超灵敏miRNA-106a检测方案.首先,将地高辛修饰的捕获DNA与固定在SiC@Ag基底上的抗地高辛链接,制备SiC@Ag@anti-digoxin/digoxin-DNA基底;将4-巯基苯甲酸(4MBA)标记的银纳米颗粒与修饰有氨基和生物素的探针DNA链接,制备Ag@4MBA@DNA-biotin探针.然后将制备的基底、探针与待测miRNA-106a组成"三明治"结构,获得表面增强拉曼散射信号放大.最后,依次加入链霉亲和素和制备的探针,形成银-生物素-链霉亲和素纳米聚集体,实现检测信号的二次放大.实验结果表明,利用SiC@Ag基底和银-生物素-链霉亲和素纳米聚集体双重表面增强拉曼散射放大,可以实现miRNA-106a的超灵敏检测,检测极限达到0.579fmol/L,对于肿瘤的早期诊断具有应用潜力.     

基于金纳米杆/PMMA基底SERS特性的高灵敏肿瘤标志物无标记检测

高品质贵金属纳米结构基底的制备是应用表面增强拉曼散射(SERS)技术进行高灵敏生物检测的关键。采用改进的Langmuir-Blodgett方法,通过在金纳米杆(Au NRs)溶胶注入乙醇,使得Au NRs迁移至溶胶与甲苯的交界面,并用聚甲基丙烯酸甲酯(PMMA)固定交界面处的Au NRs,形成大面积分布、均匀致密排列的二维畴状Au NRs/PMMA纳米结构薄膜基底。然后,采用等离子体清洗技术处理制备的基底,使得金纳米杆(Au NRs)的表面裸露,以增强基底的SERS特性。实验表明,Au NRs/PMMA基底具有优良的SERS特性,在785 nm波长的激光照射下,增强因子可以达到5.49×106。此外,利用制备的Au NRs/PMMA基底,开展前列腺癌症肿瘤标志物--前列腺特异性抗原(PSA)的高灵敏无标记定量检测研究。在PSA的无标记检测过程中,首先对PSA标准溶液和新生牛血清进行SERS光谱的直接检测,得到PSA分别位于823, 1 080, 1 385, 1 586和1 640 cm-1处的主要的拉曼特征峰;其次,通过对PSA标准溶液、临床男性血清样本及女性血清样本的SERS光谱进行测量和分析,筛选出在PSA的SERS光谱中与血清中PSA含量相关的拉曼特征峰,它们是分别位于649,680以及1 640 cm-1处的拉曼特征峰。进一步,通过对与PSA同属糖蛋白的肿瘤标志物甲胎蛋白(AFP)以及与PSA同源的人腺体激肽释放酶2(hK2)进行SERS光谱检测和分析,发现位于1 640 cm-1处的拉曼特征峰对于PSA具有高的特异性,将其作为临床血清样本中PSA无标记定量检测的具有特异性的拉曼特征峰,并以此为依据,对不同PSA浓度的标准溶液进行检测,得到位于1 640 cm-1处的拉曼特征峰强度与PSA样本溶液中PSA的浓度相关的剂量-响应曲线。最后,开展临床血清样本的应用检测。结果表明,基于Au NRs/PMMA基底的SERS检测结果与化学发光免疫分析(CLIA)方法的检测结果一致,且具有比CLIA更高的检测灵敏度,最低检测极限为0.06 ng·mL-1,且无标记检测范围为0.1 mg·mL-1～0.1 ng·mL-1。因此,基于Au NRs/PMMA SERS基底的高灵敏肿瘤标志物无标记检测具有重要应用前景。     

利用AAO模板制备SERS基底检测SudanⅠ

通过模板法制备大面积、可控的、可重复的、热点集中的金纳米结构阵列,并在纳米结构阵列上通过化学修饰分子,吸附更多苏丹红Ⅰ分子至金纳米的SERS增强区域,实现其高灵敏的表面增强拉曼分析检测。以多孔阳极氧化铝为模板,通过真空蒸镀金,约200 nm厚度,复制氧化铝的孔洞结构,用碱液将氧化铝模板腐蚀去除,可得到氧化铝模板的互补结构,即大面积的、均匀的金半球纳米结构阵列。在金纳米结构阵列上修饰十二硫醇,硫醇巯基端与纳米金相结合,碳链端自组装形成非极性的疏水环境,疏水环境可以捕获苏丹红Ⅰ分子,使其吸附至纳米金结构表面的SERS增强区域,实现苏丹红Ⅰ的SERS检测。由于SERS基底表面的金半球纳米结构均匀、规整,在激光光斑的区域内,苏丹红Ⅰ的SERS信号均匀、稳定,可以对苏丹红Ⅰ进行定量分析。苏丹红Ⅰ的拉曼峰强度对数与浓度对数之间呈线性关系,线性相关系数达0.99,线性范围为5×10-4～10-7mol·L-1,回收率范围77%～117%。此方法的检测限可达到4×10-8mol·L-1,与国标的高效液相色谱的检测限相当。     

金纳米颗粒阵列基底的化学置换制备及其表面增强拉曼散射特性研究

表面增强拉曼(SERS)作为一种分析手段,具有高灵敏度、高选择性、高重复性、非破坏性等优点,在过去的几十年中,被广泛应用在成分检测、环境科学、生物医药及传感器等领域。其中以金、银等贵金属纳米颗粒薄膜在表面增强拉曼(SERS)活性基底方面得到了更为广泛的应用。SERS技术一个关键的因素是如何制设计并备具有大面积、高增强能力及高重复性、可循环使用的SERS基底。通常,贵金属纳米颗粒规则阵列结构的单元颗粒电磁增强特性及其颗粒间的电磁耦合增强特性的综合作用可大力提升SERS基底的探测性能。然而,利用传统微纳米加工方法如光刻、电子束光刻等方法制备得到的贵金属纳米阵列结构的表面粗糙度不够理想。结合光刻与化学置换方法制备金纳米颗粒四方点阵列孔洞结构,并研究其作为SERS基底的电磁增强特性。具体研究利用光刻法在硅衬底上制备了规则排列的四方点阵列孔洞结构,用磁控溅射在其表面镀上金属铁膜;接着在衬底上旋涂浓度为1.893 8 mol·L-1的氯金酸液膜,在孔洞内铁和氯金酸发生置换反应,进而孔洞生成金纳米颗粒,最终得到金纳米颗粒四方点阵SERS活性基底。采用罗丹明6G(R6G)分子作为探测分子测试不同金纳米颗粒阵列结构基底的SERS谱。实验结果表明,随着化学置换反应时间的延长,金纳米颗粒排列更加紧凑有序,SERS谱增强性能更好。     

滤纸SERS基底的制备及其在精浆分析中的应用

本文介绍浸泡法制备基于滤纸的SERS基底,并分析滤纸SERS基底表面银纳米粒子(AgNP)的分布与浸泡时间的关系。以精浆为检测对象,相比于514nm波长激发,785nm激发可获得更好的光谱数据,同时还比较了该波长激发下精浆的常规拉曼光谱与SERS光谱。更为重要的是,通过采用精浆中654cm-1谱峰强度评估不同浸泡时间下(6h,12h,24h)滤纸SERS基底的增强性能和测量结果的重复性。实验结果表明,12h浸泡获得的滤纸SERS基底表面具有均匀的AgNP分布,在785nm波长激发下,纸基SERS基底可提供增强效果及光谱重复性俱佳的精浆SERS光谱。     

二维银球腔阵列的制备及其在SERS检测中的应用

以密堆积的700 nm单分散聚苯乙烯微球为模板,采用多电流阶跃方法制备了不同深度的二维有序微/纳尺度银球腔阵列。通过扫描电子显微镜,反射紫外对球腔形貌及表面等离子体共振进行了表征,以对氨基苯硫酚及罗丹明6G为探针分子进行了表面增强拉曼光谱(SERS)的研究。结果表明,通过控制电化学沉积的条件可以实现对球腔形貌的控制。以该种球腔阵列作为SERS基底,其增强因子可达107,并具有良好的信号重现性,信号间相对标准偏差小于8%。该基底用于对罗丹明6G的定量检测,检测限可达0.1 ng·mL-1。     

Fabrication and surface-enhanced Raman scattering (SERS) of Ag/Au bimetallic films on Si substrates

Ag films on Si substrates were fabricated by immersion plating and served as sacrificial materials for preparation of Ag/Au bimetallic films by galvanic replacement reaction. The formation procedure of films on the surface of Si was studied by scanning electron microscopy (SEM), which revealed Ag films with island and dendritic morphologies experienced novel structural evolution process during galvanic replacement reaction, and nanostructures with holes were produced within the resultant Ag/Au bimetallic films. SERS activity both of sacrificial Ag films and resultant Ag/Au bimetallic films was investigated by using crystal violet as an analyte. It has been shown that SERS signals increased with the process of galvanic substitution and reached intensity significantly stronger than that obtained from pure Ag films.     

高性能三维纳米结构SERS芯片设计、批量制备与痕量汞离子检测

表面增强拉曼散射（SERS）是一种无损、高灵敏、快速检测痕量重金属离子的光谱技术。通过调控和优化纳米结构图案和尺寸可显著增强局域表面等离子体共振（LSPR）与表面等离子体激元（SPP）的耦合以提升电磁场强度,是获得高性能SERS芯片的重要新途径。提出一种用于检测痕量汞离子的新型金属/介质三维周期纳米结构高性能SERS芯片。利用新型应力分化式双层模板纳米压印方法实现了大面积高均一纳米结构SERS芯片的低成本、批量制备。该芯片成功用于痕量汞离子的高灵敏快速检测。采用有限元方法对压印过程界面微区应力进行模拟,通过调控压印模板纵向结构和横向尺寸对模板进行设计。模拟结果表明,纵向有台阶结构的双层模板图案区域呈现高、低两个应力分区,其中,高应力区占图案~72%的面积,其应力均匀性比单层模板提升17%;低应力区分布在图案边缘~28%的区域,可有效减小脱模切应力。当模板横向尺寸从15 mm缩减至7 mm,界面应力整体提升1～2个数量级,将显著提高压印成功率。使用不同横向尺寸的单、双层模板进行压印实验结果表明,尺寸为7 mm的压力分化式双层模板实现了大面积高均一纳米结构的高质量制备,这与模拟结果高度一致。在压印胶纳米结构上构筑金纳米颗粒得到金属/介质三维周期纳米结构SERS芯片。此芯片对罗丹明6G分子的检测极限为2.08×10-12 mol·L-1,增强因子达3×108,检测均一性RSD为8.07%。该芯片对汞离子的探测限浓度仅为10 ppt(5.0×10-11 mol·L-1),浓度线性工作范围为5.0×10-11～5.0×10-5 mol·L-1,跨度达6个数量级,呈现良好的线性关系(R2=0.966),在目前汞离子检测技术中具有显著优势。提出一种通用的高灵敏快速检测痕量物质的SERS芯片设计和制备方法。这种基于光学原理芯片“结构设计-批量制备-实际应用”的研究范式将连接芯片设计和批量制备两个关键点,推动其实际应用。     

Experimental parameters for the SERS of nitrate ion for label‐free semi‐quantitative detection of proteins and mechanism for proteins to form SERS hot sites: a SERS study

We have explored the effects of the experimental parameters on the surface‐enhanced Raman scattering (SERS) intensities of NO₃⁻ and proteins observed by a heat‐induced SERS method developed by our group. The results have shown that a strong SERS signal can be obtained at pH 4.0, using an Ag colloid prepared with the reduction time of 15 min (the average size of Ag nanoparticle is 56.5 nm) dilution prepared Ag colloid by a factor of 2 by use of a 5 mM citrate buffer, using 6 mM NaNO₃ and drying the sample at 100 °C, respectively. Based on the results, two possible mechanisms for proteins to form SERS hot sites during the sample preparations are proposed. A semi‐quantitative SERS detection of ribonuclease B has been investigated. Also, NaNO₂, Mg (NO₃)₂, MgSO₄ and Na₂SO₄ have been found to be suitable for the heat‐induced SERS method. Importantly, samples prepared by the heat‐induced SERS method are so stable that these samples can be used as a standard and transferred to different laboratories for direct comparison. Namely, it can overcome uncontrollable aggregation of Ag colloids in a solution sample. All these advantages and the simplicity of experimental setup have demonstrated that the heat‐induced SERS method using NaNO₃ as an electrolyte is very promising for label‐free routine and quantitative detection of proteins. Copyright © 2011 John Wiley & Sons, Ltd.     

Large‐area Ag nanorod array substrates for SERS: AAO template‐assisted fabrication,functionalization, and application in detection PCBs

Highly ordered arrays of thiolated β‐cyclodextrin (HS‐β‐CD) functionalized Ag‐nanorods (Ag‐NRs) with plasmonic antennae enhancement of electrical field have been achieved for encapsulation and rapid detection of polychlorinated biphenyls (PCBs). The large‐area ordered arrays of rigid Ag‐NRs supported on copper base were fabricated via porous anodic aluminum oxide (AAO) template‐assisted electrochemical deposition. The inter‐nanorod gaps between the neighboring Ag‐NRs were tuned to sub‐10 nm by thinning the pore‐wall thickness of the AAO template using diluted H₃PO₄. The nearly perfect large‐area ordered arrays of Ag‐NRs supported on copper base render these systems excellent in surface‐enhanced Raman scattering (SERS) performance with uniform electric field enhancement, as testified by the SERS spectra and Raman mappings of rhodamine 6 G. Furthermore, the Ag‐NRs were functionalized with HS‐β‐CD molecules so as to capture the apolar PCB molecules in the hydrophobic cavity of the CD. Compared to the ordinary undecorated SERS substrates, the HS‐β‐CD modified Ag‐NR arrays exhibit better capture ability and higher sensitivity in rapid detection of PCBs. Copyright © 2012 John Wiley & Sons, Ltd.     

Influence of backbone length and synthetic mutations on orientation of neurotensin fragments adsorbed onto a colloidal silver surface: SERS studies

Neurotensin (NT) is a naturally occurring neurotransmitter that mediates the metabotropic seven‐transmembrane G protein‐coupled receptors, namely NTR1s, richly expressed on tumor surface. Therefore, mutated active molecular fragments of NT that possess selective antagonist or weak agonist properties and the high affinity to NTR1 have attracted considerable interest for use in thrombus, inflammation, and imaging/treatment of tumors. In this work, SERS spectra of three N‐terminal fragments of human NT (NT^1‐6, NT^1‐8, and NT^1‐11) and six specifically mutated C‐terminal fragments of human NT, including NT^8‐13, [Dab⁹]NT^8‐13, [Lys⁸,Lys⁹]NT^8‐13, [Lys⁸‐(®)‐Lys⁹]NT^8‐13, [Lys⁹,Trp¹¹,Glu¹²]NT^8‐13, and NT^9‐13, adsorbed onto nanometer‐sized colloidal silver particles in an aqueous solution at pH level of the solution 2 are presented. A comparison was made between the structures of the native and mutated fragments to determine how changes in peptide length and mutations of the structure influenced the NT adsorption properties. Based on the interpretation of the obtained data, we showed that all of the investigated NT fragments, excluding [Lys⁹,Trp¹¹,Glu¹²]NT^8‐13, tended to adsorb on the silver surface mainly through the L‐tyrosine residue and the carboxylate group. The Tyr ring lied more‐or‐less flat on the silver surface. The hydrogen atom from the phenol group dissociated upon binding. On the other hand, [Lys⁹,Trp¹¹,Glu¹²]NT^8‐13 bound to this substrate through the close to vertical co‐pyrrole ring of the indole ring (Trp¹¹) and –COO^‐ . Comparison of the presented data with those obtained earlier for NT allows to suggest that in the case of naturally occurring neurotensin, both Tyr residues together with the carboxylate group play crucial role in the binding to the nanometer‐sized colloidal silver particles. This geometry of binding forces the NT molecule to lay flat on the surface. Copyright © 2012 John Wiley & Sons, Ltd.     

Coadsorption of trimethyl phosphine and thiocyanate on colloidal silver: a SERS study combined with theoretical calculations

The adsorption of trimethyl phosphine (TMP) on colloidal silver has been investigated by means of surface‐enhanced Raman scattering spectroscopy (SERS). On the basis of surface selection rules, it is deduced from the SERS results that TMP adsorbs on silver surface via its P atom. The electron donor effect of TMP can be sensitively probed by the coadsorbed SCN⁻. The Raman wavenumber of νCN of the adsorbed SCN⁻ shifts to lower wavenumbers when TMP is coadsorbed with SCN⁻ and the red shift of C≡N stretching wavenumber is found to increase with increasing surface coverage of TMP. This could be explained in terms of the electron donor effect of TMP. Density functional theory (DFT) calculations further confirm the experimental results that the charge transfer is from TMP to silver surface rather than reversely. Natural bond orbital (NBO) analysis indicates that the red shift of C≡N stretching mode is due the increase of electronic populations of π* orbital of C≡N bond induced by coadsorbed TMP, consequently the C≡N bond is weakened, and the νCN shifts to lower wavenumbers. An NBO analysis also indicates that the conjugated effect between S atom and C≡N bond could easily make the charge transfer from silver surface to C≡N bond. Copyright © 2008 John Wiley & Sons, Ltd.     

Understanding tip‐enhanced Raman spectra of biological molecules: a combined Raman,SERS and TERS study

Although conventional Raman, surface‐enhanced Raman (SERS) and tip‐enhanced Raman spectroscopy (TERS) have been known for a long time, a direct, thorough comparison of these three methods has never been carried out. In this paper, spectra that were obtained by conventional Raman, SERS (on gold and silver substrates) and TERS (in ‘gap mode’ with silver tips and gold substrates) are compared to learn from their differences and similarities. Because the investigation of biological samples by TERS has recently become a hot topic, this work focuses on biologically relevant substances. Starting from the TER spectra of bovine serum albumin as an example for a protein, the dipeptides Phe–Phe and Tyr–Tyr and the tripeptide Tyr–Tyr–Tyr were investigated. The major findings were as follows. (1) We show that the widely used assumption that spectral bands do not shift when comparing SER, TER and conventional Raman spectra (except due to binding to the metal surface in SERS or TERS) is valid. However, band intensity ratios can differ significantly between these three methods. (2) Marker bands can be assigned, which should allow one to identify and localize proteins in complex biological environments in future investigations. From our results, general guidelines for the interpretation of TER spectra are proposed. Copyright © 2012 John Wiley & Sons, Ltd.