Localization of PD-L1 on single cancer cells by iSERS microscopy with Au/Au core/satellite nanoparticles.
ABSTRACT: Programmed cell death-ligand 1 (PD-L1) is an important predictive biomarker. The detection of PD-L1 can be crucial for patients with advanced cancer where the use of immunotherapy is considered. Here, we demonstrate the use of immuno-SERS microscopy (iSERS) for localizing PD-L1 on single cancer SkBr-3 cells. A central advantage of iSERS is that the disturbing autofluorescence from cells and tissues can be efficiently minimized by red to near-infrared laser excitation. In this study we employed Au/Au core/satellite nanoparticles as SERS nanotags because of their remarkable signal brightness and colloidal stability upon red laser excitation. False-color iSERS images of the positive and negative controls clearly reveal the specific localization of PD-L1 with SERS nanotag-labeled antibodies.
Project description:The use of plasmonic nanotags based on the surface-enhanced Raman scattering (SERS) effect is highly promising for several applications in analytical chemistry, biotechnological assays and nanomedicine. To this end, a crucial parameter is the minimum number of SERS tags that allows for the collection of intense Raman signals under real operating conditions. Here, SERS Au nanotags (AuNTs) based on clustered gold nanoparticles are deposited on a substrate and analyzed in the same region using Raman spectroscopy and transmission electron microscopy. In this way, the Raman spectra and the surface density of the SERS tags are correlated directly, showing that 1 tag/µm2 is enough to generate an intense signal above the noise level at 633 nm with an excitation power of only 0.65 mW and an acquisition time of just 1 s with a 50× objective. The AuNT density can be even lower than 1 tag/µm2 when the acquisition time is extended to 10 s, but must be increased to 3 tags/µm2 when a 20× objective is employed under the same excitation conditions. In addition, in order to observe a linear response, it was found that 10 SERS AuNTs inside the probed area are required. These findings indicate that a better signal-to-noise ratio requires high-magnification optics, while linearity versus tag number can be improved by using low-magnification optics or a high tag density. In general the suitability of plasmonic SERS labels for ultrasensitive analytical and biomedical applications is evident.
Project description:We develop a facile and effective strategy to prepare monodispersed Au spherical nanoparticles by two steps. Large-scale monocrystalline Au nanooctahedra with uniform size were synthesized by a polyol-route and subsequently Au nanoparticles were transformed from octahedron to spherical shape in a liquid under ambient atmosphere by non-focused laser irradiation in very short time. High monodispersed, ultra-smooth gold nanospheres can be obtained by simply optimizing the laser fluence and irradiation time. Photothermal melting-evaporation model was employed to get a better understanding of the morphology transformation for the system of nanosecond pulsed-laser excitation. These Au nanoparticles were fabricated into periodic monolayer arrays by self-assembly utilizing their high monodispersity and perfect spherical shape. Importantly, such Au nanospheres arrays demonstrated very good SERS enhancement related to their periodic structure due to existence of many SERS hot spots between neighboring Au nanospheres caused by the electromagnetic coupling in an array. These gold nanospheres and their self-assembled arrays possess distinct physical and chemical properties. It will make them as an excellent and promising candidate for applying in sensing and spectroscopic enhancement, catalysis, energy, and biology.
Project description:Surfaced enhanced Raman scattering (SERS) nanotags operating with 1280 nm excitation were constructed from reporter molecules selected from a library of 14 chalcogenopyrylium dyes containing phenyl, 2-thienyl, and 2-selenophenyl substituents and a surface of hollow gold nanoshells (HGNs). These 1280 SERS nanotags are unique as they have multiple chalcogen atoms available which allow them to adsorb strongly onto the gold surface of the HGN thus producing exceptional SERS signals at this long excitation wavelength. Picomolar limits of detection (LOD) were observed and individual reporters of the library were identified by principal component analysis and classified according to their unique structure and SERS spectra.
Project description:We report a quantitative evaluation of the choice of reporters for multiplexed surface-enhanced Raman spectroscopy (SERS). An initial library consisted of 15 reporter molecules that included commonly used Raman dyes, thiolated reporters, and other small molecules. We used a correlation matrix to downselect Raman reporters from the library to choose five candidates: 1,2-bis(4-pyridyl)ethylene, 4-mercaptobenzoic acid, 3,5-dichlorobenzenthiol, pentachlorothiophenol, and 5,5'-dithiobis(2-nitrobenzoic acid). We evaluated the ability to distinguish the five SERS reporters in a dipstick immunoassay for the biomarker human IgG. Raman nanotags, or gold nanostars conjugated to the five reporters and anti-human IgG polyclonal antibodies were constructed. A linear discriminant analysis approach was used to evaluate the separation of the nanotag spectra in mixtures of fixed ratios.
Project description:Single-molecule detection by surface-enhanced Raman scattering (SERS) is a powerful spectroscopic technique that is of interest for the sensor development field. An important aspect of optimizing the materials used in SERS-based sensors is the ability to have a high density of "hot spots" that enhance the SERS sensitivity to the single-molecule level. Photodeposition of gold (Au) nanoparticles through electric-field-directed self-assembly on a periodically proton-exchanged lithium niobate (PPELN) substrate provides conditions to form well-ordered microscale features consisting of closely packed Au nanoparticles. The resulting Au nanoparticle microstructure arrays (microarrays) are plasmon-active and support nonresonant single-molecule SERS at ultralow concentrations (<10-9-10-13 M) with excitation power densities <1 × 10-3 W cm-2 using wide-field imaging. The microarrays offer excellent SERS reproducibility, with an intensity variation of <7.5% across the substrate. As most biomarkers and molecules do not support resonance enhancement, this work demonstrates that PPELN is a suitable template for high-sensitivity, nonresonant sensing applications.
Project description:Noble metal nanoparticles (NPs) such as silver (Ag) and gold (Au) have unique plasmonic properties that give rise to surface enhanced Raman scattering (SERS). Generally, Ag NPs have much stronger plasmonic properties and, hence, provide stronger SERS signals than Au NPs. However, Ag NPs lack the chemical stability and biocompatibility of comparable Au NPs and typically exhibit the most intense plasmonic resonance at wavelengths much shorter than the optimal spectral region for many biomedical applications. To overcome these issues, various experimental efforts have been devoted to the synthesis of Ag/Au hybrid NPs for the purpose of SERS detections. However, a complete understanding on how the SERS enhancement depends on the chemical composition and structure of these nanoparticles has not been achieved. In this study, Mie theory and the discrete dipole approximation have been used to calculate the plasmonic spectra and near-field electromagnetic enhancements of Ag/Au hybrid NPs. In particular, we discuss how the electromagnetic enhancement depends on the mole fraction of Au in Ag/Au alloy NPs and how one may use extinction spectra to distinguish between Ag/Au alloyed NPs and Ag-Au core-shell NPs. We also show that for incident laser wavelengths between ∼410 nm and 520 nm, Ag/Au alloyed NPs provide better electromagnetic enhancement than pure Ag, pure Au, or Ag-Au core-shell structured NPs. Finally, we show that silica-core Ag/Au alloy shelled NPs provide even better performance than pure Ag/Au alloy or pure solid Ag and pure solid Au NPs. The theoretical results presented will be beneficial to the experimental efforts in optimizing the design of Ag/Au hybrid NPs for SERS-based detection methods.
Project description:Cis-regulatory DNA sequences causally mediate patterns of gene expression, but efficient experimental analysis of these control systems has remained challenging. Here we develop a new version of "barcoded" DNA-tag reporters, "Nanotags" that permit simultaneous quantitative analysis of up to 130 distinct cis-regulatory modules (CRMs). The activities of these reporters are measured in single experiments by the NanoString RNA counting method and other quantitative procedures. We demonstrate the efficiency of the Nanotag method by simultaneously measuring hourly temporal activities of 126 CRMs from 46 genes in the developing sea urchin embryo, otherwise a virtually impossible task. Nanotags are also used in gene perturbation experiments to reveal cis-regulatory responses of many CRMs at once. Nanotag methodology can be applied to many research areas, ranging from gene regulatory networks to functional and evolutionary genomics.
Project description:Insight into the nature of transient reaction intermediates and mechanistic pathways involved in heterogeneously catalyzed chemical reactions is obtainable from a number of surface spectroscopic techniques. Carrying out these investigations under actual reaction conditions is preferred but remains challenging, especially for catalytic reactions that occur in water. Here, we report the direct spectroscopic study of the catalytic hydrodechlorination of 1,1-dichloroethene in H2O using surface-enhanced Raman spectroscopy (SERS). With Pd islands grown on Au nanoshell films, this reaction can be followed in situ using SERS, exploiting the high enhancements and large active area of Au nanoshell SERS substrates, the transparency of Raman spectroscopy to aqueous solvents, and the catalytic activity enhancement of Pd by the underlying Au metal. The formation and subsequent transformation of several adsorbate species was observed. These results provide the first direct evidence of the room-temperature catalytic hydrodechlorination of a chlorinated solvent, a potentially important pathway for groundwater cleanup, as a sequence of dechlorination and hydrogenation steps. More broadly, the results highlight the exciting prospects of studying catalytic processes in water in situ, like those involved in biomass conversion and proton-exchange membrane fuel cells.
Project description:We present a systematic study on the fabrication, characterization of versatile, and low-cost filter paper-based surface-enhanced Raman spectroscopy (SERS) substrates loaded with salt-induced aggregated Ag/Au nanoparticles (NPs). These were demonstrated as efficient SERS substrates for the detection of multiple explosive molecules such as picric acid (5 ?M), 2,4-dinitrotoluene (1 ?M), and 3-nitro-1,2,4-triazol-5-one (10 ?M) along with a common dye molecule (methylene blue, 5 nM). The concentrations of the dye and explosive molecules in terms of mass represent 31.98 pg, 11.45 ng, 1.82 ng, and 13.06 ng, respectively. Silver (Ag) and gold (Au) colloidal NPs were prepared by femtosecond laser (?50 fs, 800 nm, 1 kHz) ablation of Ag/Au-target immersed in distilled water. Subsequently, the aggregated nanoparticles were achieved by mixing the pure Ag and Au NPs with different concentrations of NaCl. These aggregated NPs were characterized by UV-visible absorption and high-resolution transmission electron microscopy techniques. The SERS substrates were prepared by soaking the filter paper in aggregated NPs. The morphologies of the paper substrates were investigated using field-emission scanning electron microscopy technique. We have achieved superior enhancements with high reproducibility and sensitivity for filter paper substrates loaded with Ag/Au NPs mixed for an optimum concentration of 50 mM NaCl.
Project description:There is a strong correlation between the surface enhanced Raman scattering (SERS) enhancement factor (EF), the excitation wavelength, and the feature properties (composition, size, geometry, and analytes). The prediction of the EF of specific substrates, crucial to the quantitative SERS detection, is however still very difficult. The present work presents smart liquid SERS substrates consisting of suspensions of Fe3O4/Au nanoparticles, which provide high spot-to-spot uniformity, reproducibility and good reversibility. The EF of these substrates can be reversibly tuned by applying an external magnetic field. The EF magnetic tuning is within 2 orders of magnitude per substrate in the range of 10(4)-10(7). The ability to reversibly adjust the SERS EF enables to reduce EF variations caused by external effects such as substrate-to-substrate differences and long-term-storage degradation. This improves the quantitative detection of analytes and might be a significant step forward in employing SERS for practical applications.