SERS microscopic imaging as novel tool for assessing viability and enumerating yeast cells at various stages of cell cycle in lag,log, exponential and stationary phases of growth in culture

Surface enhanced Raman scattering (SERS) microscopic imaging was employed to enumerate the yeast cells in culture. We found this imaging method as an efficient tool for easily differentiating and quantitatively enumerating yeast cell at different stages of cell-division cycle (G1, S, G2 and M phase) at various stages of growth phases namely lag, log, exponential and stationary phases in culture. Apart from enumerating the cells at different stages of cell cycle under lag, log, exponential and stationary phases, it was possible using SERS microscopy to differentiate the live cells from dead ones. The dead cells were SERS inactive and gave enhanced autofluorescence compared with the live cells, which were SERS active. The results from the present investigation suggest that SERS microscopic imaging, using silver nanoparticles (AgNPs) as a sensitive tool to enumerate the yeast cells in culture.     

Highly sensitive detection of proteins and bacteria in aqueous solution using surface-enhanced Raman scattering and optical fibers

We report the detection of the proteins lysozyme and cytochrome c as well as the live bacterial cells of Shewanella oneidensis MR-1 in aqueous solutions with sensitivities order(s) of magnitude higher than those previously reported. Two highly sensitive surface-enhanced Raman scattering (SERS)-based biosensors using optical fibers have been employed for such label-free macromolecule detections. The first sensor is based on a tip-coated multimode fiber (TCMMF) with a double-substrate "sandwich" structure, and a detection limit of 0.2 μg/mL is achieved in protein detections. The second sensor is based on a liquid core photonic crystal fiber (LCPCF) with a better confinement of light inside the fiber core, and a detection limit of 10(6) cells/mL is achieved for the bacteria detection. Both SERS biosensors show great potential for highly sensitive and molecule-specific detection and identification of biomolecules.     

Molecular imaging with SERS-active nanoparticles

Raman spectroscopy has been explored for various biomedical applications (e.g., cancer diagnosis) because it can provide detailed information on the chemical composition of cells and tissues. For imaging applications, several variations of Raman spectroscopy have been developed to enhance its sensitivity. To date, a wide variety of molecular targets and biological events have been investigated using surface-enhanced Raman scattering (SERS)-active nanoparticles. The superb multiplexing capability of SERS-based Raman imaging, already successfully demonstrated in live animals, can be extremely powerful in future research where different agents can be attached to different Raman tags to enable the simultaneous interrogation of multiple biological events. Over the last several years, molecular imaging with SERS-active nanoparticles has advanced significantly and many pivotal proof-of-principle experiments have been successfully carried out. It is expected that SERS-based imaging will continue to be a dynamic research field over the next decade.     

高度结晶Ti3C2单层纳米片的表面增强拉曼散射

表面增强拉曼散射(SERS)作为一种优秀的免标记无损检测技术,已被广泛应用于环境污染物检测、生物组织传感及指纹级分子分析等领域.与以贵金属和半导体为代表的传统SERS基底不同,本文报道了一种新型的高灵敏SERS基底材料,即无核二维电子气(2DEG)结构的Ti3C2单层纳米片,其具有高稳定性、生物相容性和低成本等优点.我们通过一种化学剥离和微波加热相结合的方法合成了具有洁净表面的、高度结晶的单层Ti3C2纳米片.Ti3C2单层无核2DEG的独特电子结构提供了理想的无核散射传输通道,使单层Ti3C2纳米片的拉曼增强因子达到了3.82×108,其对典型污染物的检出限高达10-11mol L-1.初步的毒理学实验表明,该物质的细胞毒性非常低.考虑到碳化物纳米片合成简单、良好的生物相容性、低成本以及高的化学稳定性,Ti3C2单层对于设计和制造具有超高灵敏度的柔性SERS衬底具有很好的应用前景.     

Probing intrinsic and extrinsic components in single osteosarcoma cells by near-infrared surface-enhanced Raman scattering

We report on the capabilities of near-infrared surface-enhanced Raman scattering (SERS) using gold nanoparticles to obtain detailed chemical information with high spatial resolution from within single cancer cells, living or fixed. Colloidal gold particles, 60 nm in size, were introduced into live human osteosarcoma cells by endocytosis by adding them to the growth medium. Rapid SERS mapping of cells indicated that not only could rich vibrational spectra be obtained from intrinsic cellular constituents both in the cytoplasm and nucleus and but also the distribution of extrinsic molecules introduced into the cells, in this case, rhodamine 6G could be characterized, suggesting that the intracellular distribution of chemotherapeutic agents could potentially be measured by this technique. We show that the SERS signal intensity from the cellular components increases and more spectral detail is acquired from dried cells when compared with hydrated cells in buffer. The data also show spectral fluctuations, mainly in intensity but also in peak position, which are dependent upon the intensity of the excitation light and are probably due to diffusion of molecules on the surface of the gold nanoparticles. A detailed understanding of the origins of these effects is still not complete, but the ability to acquire very sensitive SERS inside living cancer cells indicates the potential of this technique as a useful tool in biomedicine.     

Highly Surface‐roughened “Flower‐like” Silver Nanoparticles for Extremely Sensitive Substrates of Surface‐enhanced Raman Scattering

Surface‐enhanced Raman scattering (SERS) is a new optical spectroscopic analysis technique with potential for highly sensitive detection of molecules. Recently, many efforts have been made to find SERS substrates with high sensitivity and reproducibility. In this Research News article, we provide a focused review on the synthesis of monodispersed silver particles with a novel, highly roughened, “flower‐like” morphology by reducing silver nitrate with ascorbic acid in aqueous solutions. The nanometer‐scale surface roughness of the particles can provide several hot spots on a single particle, which significantly increases SERS enhancement. The incident polarization‐dependent SERS of individual particles is also studied. Although the different “hot spots” on a single particle can have a strong polarization dependency, the total Raman signals from an individual particle usually have no obvious polarization dependency. Moreover, these flower‐like silver particles can be measured by SERS with high enhancement several times, which indicates the high stability of the hot spots. Hence, the flower‐like silver particles here can serve as highly sensitive and reproducible SERS substrates.     

Surface-enhanced Raman scattering-based nanoprobe for high-resolution, non-scanning chemical imaging

This work describes the development and demonstration of a non-scanning chemical imaging probe, capable of obtaining surface-enhanced Raman scattering (SERS) images of samples with which it is in direct contact. The SERS imaging arrays (i.e., nanoprobes) are used in a signal collection mode to obtain images by measuring as many as 30 000 individual sub-diffraction-limited locations on a sample's surface simultaneously. These SERS probes are fabricated from coherent fiber-optic imaging bundles, allowing for the formation of a highly ordered roughened metal surface, capable of providing uniform SERS enhancement (<2.0% relative standard deviation) across the entire imaging surface. These optimized SERS nanoprobes have potential application to a wide range of research fields from materials science to cellular biology.     

Gold nano-popcorn attached SWCNT hybrid nanomaterial for targeted diagnosis and photothermal therapy of human breast cancer cells

Breast cancer presents greatest challenge in health care in today's world. The key to ultimately successful treatment of breast cancer disease is an early and accurate diagnosis. Current breast cancer treatments are often associated with severe side effects. Driven by the need, we report the design of novel hybrid nanomaterial using gold nano popcorn-attached single wall carbon nanotube for targeted diagnosis and selective photothermal treatment. Targeted SK-BR-3 human breast cancer cell sensing have been performed in 10 cancer cells/mL level, using surface enhanced Raman scattering of single walls carbon nanotube's D and G bands. Our data show that S6 aptamer attached hybrid nanomaterial based SERS assay is highly sensitive to targeted human breast cancer SK-BR-3 cell line and it will be able to distinguish it from other non targeted MDA-MB breast cancer cell line and HaCaT normal skin cell line. Our results also show that 10 min of photothermal therapy treatment by 1.5 W/cm(2) power, 785 nm laser is enough to kill cancer cells very effectively using S6 aptamer attached hybrid nanomaterials. Possible mechanisms for targeted sensing and operating principle for highly efficient photothermal therapy have been discussed. Our experimental results reported here open up a new possibility for using aptamers modified hybrid nanomaterial for reliable diagnosis and targeted therapy of cancer cell lines quickly.     

A new approach to solution-phase gold seeding for SERS substrates

Surface-enhanced Raman scattering (SERS) vastly improves signal-to-noise ratios as compared to traditional Raman scattering, making sensitive assays based upon Raman scattering a reality. However, preparation of highly stable SERS-active gold substrates requires complicated and expensive methodologies and instrumentation. Here, a general and completely solution-phase, seed-based approach is introduced, which is capable of producing gold films for SERS applications on a variety of substrates, not requiring surface modification or functionalization. SERS enhancement factors of ≈10(7) were observed. Moreover, solution-phase gold film deposition on highly complex surfaces, such as protein-coated bioassays, is demonstrated for the first time. Protein bioassays coated with such SERS-active gold films are combined with bioconjugated single-walled carbon nanotube Raman labels, affording highly sensitive detection of the cancer biomarker, carcinoembryonic antigen in serum, with a limit of detection of ≈5 fM (1 pg mL(-1) ).     

Multiplexed five-color molecular imaging of cancer cells and tumor tissues with carbon nanotube Raman tags in the near-infrared

Single-walled carbon nanotubes (SWNTs) with five different C13/C12 isotope compositions and well-separated Raman peaks have been synthesized and conjugated to five targeting ligands in order to impart molecular specificity. Multiplexed Raman imaging of live cells has been carried out by highly specific staining of cells with a five-color mixture of SWNTs. Ex vivo multiplexed Raman imaging of tumor samples uncovers a surprising up-regulation of epidermal growth factor receptor (EGFR) on LS174T colon cancer cells from cell culture to in vivo tumor growth. This is the first time five-color multiplexed molecular imaging has been performed in the near-infrared (NIR) region under a single laser excitation. Near zero interfering background of imaging is achieved due to the sharp Raman peaks unique to nanotubes over the low, smooth autofluorescence background of biological species.     

Highly uniform and reproducible surface-enhanced Raman scattering from DNA-tailorable nanoparticles with 1-nm interior gap

An ideal surface-enhanced Raman scattering (SERS) nanostructure for sensing and imaging applications should induce a high signal enhancement, generate a reproducible and uniform response, and should be easy to synthesize. Many SERS-active nanostructures have been investigated, but they suffer from poor reproducibility of the SERS-active sites, and the wide distribution of their enhancement factor values results in an unquantifiable SERS signal. Here, we show that DNA on gold nanoparticles facilitates the formation of well-defined gold nanobridged nanogap particles (Au-NNP) that generate a highly stable and reproducible SERS signal. The uniform and hollow gap (～1 nm) between the gold core and gold shell can be precisely loaded with a quantifiable amount of Raman dyes. SERS signals generated by Au-NNPs showed a linear dependence on probe concentration (R(2) > 0.98) and were sensitive down to 10 fM concentrations. Single-particle nano-Raman mapping analysis revealed that >90% of Au-NNPs had enhancement factors greater than 1.0 × 10(8), which is sufficient for single-molecule detection, and the values were narrowly distributed between 1.0 × 10(8) and 5.0 × 10(9).     

Chemical force microscopy of single live cells

Traditionally, cell surface properties have been difficult to study at the subcellular level, especially on hydrated, live cells. Here, we demonstrate the ability of chemical force microscopy to map the hydrophobicity of single live cells with nanoscale resolution. After validating the technique on reference surfaces with known chemistry, we probe the local hydrophobic character of two medically important microorganisms, Aspergillus fumigatus and Mycobacterium bovis, in relation with function. Applicable to a wide variety of cells, the chemically sensitive imaging method presented here provides new opportunities for studying the nanoscale surface properties of live cells and for understanding their roles in mediating cellular events.     

Tracking Drug‐Induced Epithelial–Mesenchymal Transition in Breast Cancer by a Microfluidic Surface‐Enhanced Raman Spectroscopy Immunoassay

Epithelial–mesenchymal transition (EMT) is a primary mechanism for cancer metastasis. Detecting the activation of EMT can potentially convey signs of metastasis to guide treatment management and improve patient survival. One of the classic signatures of EMT is characterized by dynamic changes in cellular expression levels of E‐cadherin and N‐cadherin, whose soluble active fragments have recently been reported to be biomarkers for cancer diagnosis and prognosis. Herein, a microfluidic immunoassay (termed “SERS immunoassay”) based on sensitive and simultaneous detection of soluble E‐cadherin (sE‐cadherin) and soluble N‐cadherin (sN‐cadherin) for EMT monitoring in patients' plasma is presented. The SERS immunoassay integrates in situ nanomixing and surface‐enhanced Raman scattering readout to enable accurate detection of sE‐cadherin and sN‐cadherin from as low as 10 cells mL^?1. This assay enables tracking of a concurrent decrease in sE‐cadherin and increase in sN‐cadherin in breast cancer cells undergoing drug‐induced mesenchymal transformation. The clinical potential of the SERS immunoassay is further demonstrated by successful detection of sE‐cadherin and sN‐cadherin in metastatic stage IV breast cancer patient plasma samples. The SERS immunoassay can potentially sense the activation of EMT to provide early indications of cancer invasions or metastasis.     

Chitosan-coated anisotropic silver nanoparticles as a SERS substrate for single-molecule detection

Surface-enhanced Raman spectroscopy (SERS) is a technique that has become widely used for identifying and providing structural information about molecular species in low concentration. There is an ongoing interest in finding optimum particle size, shape and spatial distribution for optimizing the SERS substrates and pushing the sensitivity toward the single-molecule detection limit. This work reports the design of a novel, biocompatible SERS substrate based on small clusters of anisotropic silver nanoparticles embedded in a film of chitosan biopolymer. The SERS efficiency of the biocompatible film is assessed by employing Raman imaging and spectroscopy of adenine, a significant biological molecule. By combining atomic force microscopy with SERS imaging we find that the chitosan matrix enables the formation of small clusters of silver nanoparticles, with junctions and gaps that greatly enhance the Raman intensities of the adsorbed molecules. The study demonstrates that chitosan-coated anisotropic silver nanoparticle clusters are sensitive enough to be implemented as effective plasmonic substrates for SERS detection of nonresonant analytes at the single-molecule level.     

Silver nanoparticles deposited on anodic aluminum oxide template using magnetron sputtering for surface-enhanced Raman scattering substrate

Low-cost and highly sensitive surface-enhanced Raman scattering (SERS) substrates have been fabricated by a simple anodizing process and a magnetron sputtering deposition. The substrates, which consist of silver nanoparticles embedded on anodic aluminum oxide (AAO) templates, are investigated by a scanning electron microscope and a confocal Raman spectroscopy. The SERS activities are demonstrated by Raman scattering from adsorbed solutions of methylene blue and pyridine on the SERS substrate surface. The most optimized SERS substrate contains the silver nanoparticles, with a size distribution of 10-30 nm, deposited on the AAO template. From a calculation, the SERS enhancement factor is as high as 8.5 × 10⁷, which suggests strong potentials for direct applications in the chemical detection and analyses.     

Ultrasensitive Telomerase Activity Detection by Telomeric Elongation Controlled Surface Enhanced Raman Scattering

Telomerase is now considered to be a valuable biomarker and therapeutic target in the diagnosis and treatment of cancerous diseases, which brings an urgent need in the development of fast and efficient telomerase detection strategies. Here, a new surface enhanced Raman scattering (SERS) based protocol using telomeric elongation controlled SERS (TEC‐SERS) effect for the ultrasensitive telomerase detection is presented. The TEC‐SERS protocol not only provides an unprecedented high sensitivity but also avoids laborious PCR procedures. The detection limit is ≈2–3 orders of magnitude lower than those of previously reported methods. This highly sensitive and straightforward TEC‐SERS protocol can be developed as a routine telomerase detection method, which would greatly facilitate the telomerase based ultra‐early diagnosis of malignant tumors and the fast screening of anti‐cancer drugs.     

In vitro biomechanical properties,fluorescence imaging,surface-enhanced Raman spectroscopy,and photothermal therapy evaluation of luminescent functionalized CaMoO4:Eu@Au hybrid nanorods on human lung adenocarcinoma epithelial cells

Highly dispersible Eu³⁺-doped CaMoO₄@Au-nanorod hybrid nanoparticles (HNPs) exhibit optical properties, such as plasmon resonances in the near-infrared region at 790 nm and luminescence at 615 nm, offering multimodal capabilities: fluorescence imaging, surface-enhanced Raman spectroscopy (SERS) detection and photothermal therapy (PTT). HNPs were conjugated with a Raman reporter (4-mercaptobenzoic acid), showing a desired SERS signal (enhancement factor 5.0 × 10⁵). The HNPs have a heat conversion efficiency of 25.6%, and a hyperthermia temperature of 42°C could be achieved by adjusting either concentration of HNPs, or laser power, or irradiation time. HNPs were modified with antibody specific to cancer biomarker epidermal growth factor receptor, then applied to human lung cancer (A549) and mouse hepatocyte cells (AML12), and in vitro PTT effect was studied. In addition, the biomechanical properties of A549 cells were quantified using atomic force microscopy. This study shows the potential applications of these HNPs in fluorescence imaging, SERS detection, and PTT with good photostability and biocompatibility.     

<Emphasis Type="Italic">In situ</Emphasis> probing of cell–cell communications with surface-enhanced Raman scattering (SERS) nanoprobes and microfluidic networks for screening of immunotherapeutic drugs

Discovering novel drugs for cancer immunotherapy requires a robust in vitro drug screening platform that allows for straightforward probing of cell-cell communications.Here,we combined surface-enhanced Raman scattering (SERS) nanoprobes with microfluidic networks to monitor in situ the cancer-immune system intercellular communications.The microfluidic platform links up immune cells with cancer cells,where the cancer-cell secretions act as signaling mediators.First,gold@silver core-shell nanorods were employed to fabricate SERS immunoprobes for analysis of the signaling molecules.Multiple cancer secretions in a tumor microenvironment were quantitatively analyzed by a SERS-assisted three-dimensional (3D) barcode immunoassay with high sensitivity (1 ng/mL).Second,in an on-chip cell proliferation assay,multiple immunosuppressive proteins secreted by cancer cells were found to inhibit activation of immune cells,indicating that the platform simulates the physiological process of cancer-immune system communications.Furthermore,potential drug candidates were tested on this platform.A quantitative SERS immunoassay was performed to evaluate drug efficacy at regulating the secretion behavior of cancer cells and the activity of immune cells.This assay showed the suitability of this platform for in vitro drug screening.It is expected that the fully integrated and highly automated SERS-microfluidic platform will become a powerful analytical tool for probing intercellular communications and should accelerate the discovery and clinical validation of novel drugs.     

Multifunctional Silver‐Embedded Magnetic Nanoparticles as SERS Nanoprobes and Their Applications

In this study, surface‐enhanced Raman spectroscopy (SERS)‐encoded magnetic nanoparticles (NPs) are prepared and utilized as a multifunctional tagging material for cancer‐cell targeting and separation. First, silver‐embedded magnetic NPs are prepared, composed of an 18‐nm magnetic core and a 16‐nm‐thick silica shell with silver NPs formed on the surface. After simple aromatic compounds are adsorbed on the silver‐embedded magnetic NPs, they are coated with silica to provide them with chemical and physical stability. The resulting silica‐encapsulated magnetic NPs (M‐SERS dots) produce strong SERS signals and have magnetic properties. In a model application as a tagging material, the M‐SERS dots are successfully utilized for targeting breast‐cancer cells (SKBR3) and floating leukemia cells (SP2/O). The targeted cancer cells can be easily separated from the untargeted cells using an external magnetic field. The separated targeted cancer cells exhibit a Raman signal originating from the M‐SERS dots. This system proves to be an efficient tool for separating targeted cells. Additionally, the magnetic‐field‐induced hot spots, which can provide a 1000‐times‐stronger SERS intensity due to aggregation of the NPs, are studied.     

High Surface‐Enhanced Raman Scattering Performance of Individual Gold Nanoflowers and Their Application in Live Cell Imaging

Molecular imaging techniques based on surface‐enhanced Raman scattering (SERS) face a lack of reproducibility and reliability, thus hampering its practical application. Flower‐like gold nanoparticles have strong SERS enhancement performance due to having plenty of hot‐spots on their surfaces, and this enhancement is not dependent on the aggregation of the particles. These features make this kind of particle an ideal SERS substrate to improve the reproducibility in SERS imaging. Here, the SERS properties of individual flower‐like gold nanoparticles are systematically investigated. The measurements reveal that the enhancement of a single gold nanoparticle is independent of the polarization of the excitation laser with an enhancement factor as high as 10⁸. After capping with Raman signal molecules and folic acid, the gold nanoflowers show strong Raman signal in the living cells, excellent targeting properties, and a high signal‐to‐noise ratio for SERS imaging.