Reflective FT-NIR and SERS studies of HER-II breast cancer biomarker using plasmonic-active nanostructured thin film immobilized oriented antibody

We describe the fabrication of plasmonic-active nanostructured thin film substrate as a label-free surface-enhanced Raman scattering (SERS)-based biosensor immobilized covalently with monoclonal HER-II antibody (mAb) to detect overexpressed HER-II as a biomarker in breast cancer serum (BCS). Oriented conjugation of mAb via hydrazone linkage to provide higher mAb accessibility was characterized by UV-vis and reflective Fourier transform near-infrared (FT-NIR) spectroscopic techniques. The interaction of BCS with mAb was studied by FT-NIR and nonresonant SERS at 637 nm. The results showed detection of glycoprotein content at different laser powers including a rise in amino acid and glycan content with varying results at higher power. With nonresonant SERS we observed nonlinear behavior of peak intensity. Analysis of variance was implemented to determine the effect of laser power which was found not to be a contributing factor. However, at the nanoscale, factors including the heating effect and aggregation of molecules can contribute to the nonlinearity of peak intensity.     

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。     

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.     

在银溶胶中甘氨酸和甘氨酸二肽的表面增强拉曼散射

甘氨酸及Gly-Gly和Gly-L-Pro二肽的表面增强拉曼散射谱与溶液内的普通拉曼谱的对比研究表明,银胶对COO、NH_2、C-N和C-C基团的振动有明显的增强.对CH_2基团无增强.Gly-Gly的SERS与pH有关,接近中性pH值时增强效果好,在酸性和碱性溶液内效果差.在 Gly-L-Pro的SERS谱中有强的酰胺Ⅰ带,而 Gly-Gly则几乎观察不到.     

疏水性SERS基底检测应用研究进展

本文简要介绍了表面增强拉曼散射(SERS)的拉曼信号增强原理,常规SERS增强基底的研究应用进展;同时介绍了疏水性基底的定义及优势,并重点综述了疏水性SERS基底的分类及研究应用的进展;最后展望了疏水性SERS基底的研究方向和发展趋势。疏水性SERS基底的发展将有望为今后开展面向超低浓度的生化物质检测以及基于人体体液的疾病的灵敏、客观检测诊断提供新方法和新思路。     

The Use of Polarization-dependent SERS from Scratched Gold Films to Selectively Eliminate Solution-phase Interference

Polarization-dependent surface-enhanced Raman scattering (SERS) was studied for oxazine 720 molecules adsorbed on a scratched gold surface placed in situ and under electrochemical control. A quantitative method for evaluating the observed polarization dependence will be introduced. This method takes into account the polarization artifacts caused by optical elements in the light microscope used for Raman microscopy. Intensity of the SERS obtained from oxazine 720 adsorbed on scratches in gold showed a polarization dependence after correction was made for these artifacts. In contrast, intensity of the ordinary Raman signal obtained from perchlorate ions in the solution above a scratched gold surface was found to be polarization-independent. Therefore, polarization effects can be used to selectively remove solution-phase interference signals from the SERS spectrum of an adsorbed analyte. These polarization effects were found to be independent of the applied potential, meaning the methodology is applicable to electrochemical SERS studies.     

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。     

Raman microprobe spectroscopy: A new tool for biofouling diagnostics

Raman vibrational spectra were obtained from crystalline amino acids and acid molecules adsorbed at the surfaces of silver electrodes in aqueous solutions. Results revealed that aromatic acids such as phenylalanine adsorbed via their aromatic ring while bases such as alanine were coordinated by their amine functional groups. Sulphur containing acids (cysteine and cystine) were found to bond through their sulphur groups. In all cases, adsorption increased towards the point of zero charge of silver, as would be expected for uncharged species. Similar experiments carried out on α‐amylase solutions showed that the enzyme molecule changes its coordination to the silver surface as a function of electrode potential, indicating that C‐S, C‐N, and aromatic ring functional groups are all present on the outer surface of the enzyme structure.     

对硝基苯硫酚分子内嵌的星形表面增强拉曼散射金 "套娃 "纳米颗粒用于肿瘤拉曼影像的初步研究

目的:制备对硝基苯硫酚(4-Nitrobenzenethiol,4-NBT)分子内嵌的星形表面增强拉曼散射(Surface enhanced Raman Scattering,SERS)金"套娃"纳米颗粒,测定其拉曼增强效果和应用于细胞以及活体肿瘤拉曼影像的可行性。方法:以种子介导法先后制备金纳米星及星形SERS金"套娃"纳米颗粒,采用透射电镜观察其形貌,激光粒度分析仪测定其粒径及Zeta电位,拉曼光谱仪测定其拉曼光谱,考察其对A549细胞的拉曼成像效果,建立A549皮下瘤模型,考察其对活体皮下瘤的成像效果。结果:制备并优化的金纳米星粒径较小,为60.5 nm,其针尖密度较高,以此为核心制备的星形SERS金"套娃"纳米颗粒形态规整,粒径约为66.7nm,Zeta电位约为-16.6 m V,拉曼增强效果提升至其前驱体金纳米星的5.3倍,能够实现对A549细胞及A549皮下瘤的拉曼成像。结论:所制备的星形SERS金"套娃"纳米颗粒形态规整均一,拉曼增强效果较好,能实现对细胞及活体肿瘤的拉曼影像。     

Fabrication and characterization of a multiwell array SERS chip with biological applications

Uniform, large surface area substrates for surface-enhanced Raman spectroscopy (SERS) are fabricated by oblique angle deposition. The SERS-active substrates are patterned by a polymer-molding technique to provide a uniform array for high throughput biosensing and multiplexing. Using a conventional SERS-active molecule, 1,2-di(4-pyridyl)ethylene (BPE) ≥98%, we show that this device provides a uniform Raman signal enhancement from well to well with a detection limit of at least 10⁻⁸ M of the BPE solution or 10⁻¹⁸ mol of BPE. The SERS intensity is also demonstrated to vary logarithmically with the log of BPE concentration and the apparent sensitivity of the patterned substrate is compared to previous reports from our group on non-patterned substrates. Avian influenza is analyzed to demonstrate the utility of SERS multiwell patterned substrates for biosensing. The spectra acquired from patterned substrates show better reproducibility and less variation compared to the unpatterned substrates according to multivariate analysis. Our results highlight potential advantages of the patterned substrate.     

金溶胶基底的 SERS 增强效果的实验研究

本文以结晶紫作为探针分子,研究了以金溶胶膜、pH ＝6以及 pH ＝13的金溶胶溶液为活性基底的表面增强拉曼光谱的增强效果。采用化学还原法制备金溶胶,加入氢氧化钠改变其 pH 值,并以自组装法制备金溶胶膜。通过比较金溶胶膜、pH ＝6及 pH ＝13时金溶胶溶液的增强因子以及在这三种金溶胶基底上结晶紫的检测限,分析不同活性基底增强效果的差异。三种活性基底的增强因子分别可达到5．9×103、1．5×105、2．3×107,pH ＝13的金溶胶溶液有最佳的增强效果。以这三种金溶胶为基底对结晶紫进行表面增强拉曼光谱探测,可得到检测限为70．7 nmol／L、9．6 nmol／L、1．8 nmol／L。结果表明,金溶胶溶液的增强效果明显优于金溶胶膜,而通过改变金溶胶体系的 pH 值可以改变金纳米颗粒的聚合程度及对探测物的吸附特性从而获得更高灵敏度的活性基底。     

SERS and integrative imaging upon internalization of quantum dots into human oral epithelial cells

CdTe quantum dots (QDs) are widely used in bio‐applications due to their size and highly efficient optical properties. However internalization mechanisms thereof for the variety of freshly extracted, not cultivated human cells and their specific molecular interactions remains an open topic for discussion. In this study, we assess the internalization mechanism of CdTe quantum dots (3.3 nm) capped with thioglycolic acid using non cultivated oral epithelial cells obtained from healthy donors. Naked gold nanoparticles (40 nm) were successfully used as nanosensors for surface‐enhanced Raman spectroscopy to efficiently identify characteristic Raman peaks, providing new evidence indicating that the first interactions of these QDs with epithelial cells occurred preferentially with aromatic rings and amine groups of amino acid residues and glycans from trans‐membrane proteins and cytoskeleton. Using an integrative combination of advanced imaging techniques, including ultra‐high resolution SEM, high resolution STEM coupled with EDX spectroscopy together with the results obtained by Raman spectroscopy, it was determined that thioglycolic acid capped CdTe QDs are efficiently internalized into freshly extracted oral epithelial cells only by facilitated diffusion, distributed into cytoplasm and even within the cell nucleus in three minutes.

     

A new silver nanochain SERS analytical platform to detect trace hexametaphosphate with a rhodamine S molecular probe

Using AgNO₃ as the precursor, stable silver nanochain (AgNC) sols, orange‐red in color, were prepared using hydrazine hydrate. A strong surface plasmon resonance Rayleigh scattering (RRS) peak occurred at 420 nm plus two surface plasmon resonance (SPR) absorption peaks at 410 nm and 510 nm. Rhodamine S (RhS) cationic dye was absorbed on the as‐prepared AgNC substrate to obtain a RhS–AgNC surface‐enhanced Raman scattering (SERS) nanoprobe that exhibited a strong SERS peak at 1506 cm^–1 and a strong RRS peak at 375 nm. Upon addition of the analyte sodium hexametaphosphate (HP), it reacted with RhS, which resulted in a decrease in the SERS and RRS peaks that was studied in detail. The decreased SERS and RRS intensities correlated linearly with HP concentration in the range of 0.0125–0.3 µmol/L and 0.05–1.0 µmol/L, with a detection limit of 6 nmol/L and 20 nmol/L HP respectively. Due to advantages of high sensitivity, good selectivity and simple operation, the RhS molecular probes were used to determine HP concentration in real samples. Copyright © 2015 John Wiley & Sons, Ltd.     

Designable nanoplasmonic biomarkers for direct microscopy cytopathology diagnostics

Direct microscopy interpretation of fine‐needle biopsy cytological samples is routinely used by practicing cytopathologists. Adding possibility to identify selective and multiplexed biomarkers on the same samples and with the same microscopy technique can greatly improve diagnostic accuracy. In this article, we propose to use biomarkers based on designable plasmonic nanoparticles (NPs) with unique optical properties and excellent chemical stability that can satisfy the above‐mentioned requirements. By finely controlling the size and composition of gold‐silver alloy NPs and gold nanorods, the NPs plasmonic resonance properties, such as scattering efficiency and resonance peak spectral position, are adjusted in order to provide reliable identification and chromatic differentiation by conventional direct microscopy. Efficient darkfield NPs imaging is performed by using a novel circular side illumination adaptor that can be easily integrated into any microscopy setup while preserving standard cytopathology visualization method. The efficiency of the proposed technology for fast visual detection and differentiation of three spectrally distinct NP‐markers is demonstrated in different working media, thus confirming the potential application in conventional cytology preparations. It is worth emphasizing that the presented technology does not interfere with standard visualization with immunohistochemical staining, but should rather be considered as a second imaging modality to confirm the diagnostics.      

A structural overview of mycobacterial adhesins: Key biomarkers for diagnostics and therapeutics

Adherence, colonization, and survival of mycobacteria in host cells require surface adhesins, which are attractive pharmacotherapeutic targets. A large arsenal of pilus and non‐pilus adhesins have been identified in mycobacteria. These adhesins are capable of interacting with host cells, including macrophages and epithelial cells and are essential to microbial pathogenesis. In the last decade, several structures of mycobacterial adhesins responsible for adhesion to either macrophages or extra cellular matrix proteins have been elucidated. In addition, key structural and functional information have emerged for the process of mycobacterial adhesion to epithelial cells, mediated by the Heparin‐binding hemagglutinin (HBHA). In this review, we provide an overview of the structural and functional features of mycobacterial adhesins and discuss their role as important biomarkers for diagnostics and therapeutics. Based on the reported data, it appears clear that adhesins are endowed with a variety of different structures and functions. Most adhesins play important roles in the cell life of mycobacteria and are key virulence factors. However, they have adapted to an extracellular life to exert a role in host‐pathogen interaction. The type of interactions they form with the host and the adhesin regions involved in binding is partly known and is described in this review.     

蛋白质芯片在肿瘤血清标识物发现中的应用

蛋白质芯片是一种新型的高通量蛋白质组学技术,由于其具有高通量、微型化、可平行快速分析等优点,因此在肿瘤血清标识物发现研究方面具有广泛的应用前景。本文综述了蛋白质芯片的基本原理、类型及其在肿瘤血清标记物发现研究中的应用,将蛋白质芯片技术与传统的肿瘤标志物发现技术进行了比较,并对蛋白质芯片技术在肿瘤标识物发现研究上的进一步应用进行了展望。     

Highly sensitive fluorescence and SERS detection of azide through a simple click reaction of 8‐chloroquinoline and phenylacetylene

In 0.19 mol/L acetic acid (HAc), a click reaction of 8‐chloroquinoline/azide/phenylacetylene take places in aqueous solution without Cu(I) as a catalyst. 8‐Chloroquinoline (CQN) exhibited a strong fluorescence peak at 430 nm that was quenched linearly as the concentration of azide increased from 20 to 1000 ng/mL. This quenching was due to consumption of CQN in the click reaction and a decrease in the number of efficiently excited photons due to the presence of triazole–quinoline ramification molecules with strong hydrophobicity. Using blue nanosilver sol as the substrate, CQN absorbed onto the surface of nanosilver particles, showing a strong surface‐enhanced Raman scattering (SERS) peak at 1585 cm^‐1 that decreased linearly as the azide concentration increased from 8 to 500 ng/mL; the detection limit was 4 ng/mL. Thus, two new, simple and sensitive fluorescence and SERS methods have been developed for the determination of azide via the click reaction. Copyright © 2014 John Wiley & Sons, Ltd.     

SERS Study of Tetrodotoxin (TTX) by Using Silver Nanoparticle Arrays

The optical properties of tetrodotoxin (TTX), a scarce toxin with anesthetic properties, were studied using nanoparticle arrays-assisted surface-enhanced Raman scattering (SERS). The nanoparticles arrays were fabricated using nanosphere lithography and a metallic lift-off process to control the particle size, shape, and spacing in the arrays. Using density functional methods, the Raman spectrum of TTX was also calculated with Gaussian03 software. The main peaks of the spectrum are originated from the vibration of the NH₂ molecule group. In the SERS experiment, we were able to measure the Raman spectrum with a TTX concentration as less as 0.9 ng/mL. This sensitivity is comparable to that from high performance liquid chromatography.