Surface-Enhanced Raman Spectroscopy of the Endothelial Cell Membrane

We applied surface-enhanced Raman spectroscopy (SERS) to cationic gold-labeled endothelial cells to derive SERS-enhanced spectra of the bimolecular makeup of the plasma membrane. A two-step protocol with cationic charged gold nanoparticles followed by silver-intensification to generate silver nanoparticles on the cell surface was employed. This protocol of post-labelling silver-intensification facilitates the collection of SERS-enhanced spectra from the cell membrane without contribution from conjugated antibodies or other molecules. This approach generated a 100-fold SERS-enhancement of the spectral signal. The SERS spectra exhibited many vibrational peaks that can be assigned to components of the cell membrane. We were able to carry out spectral mapping using some of the enhanced wavenumbers. Significantly, the spectral maps suggest the distribution of some membrane components are was not evenly distributed over the cells plasma membrane. These results provide some possible evidence for the existence of lipid rafts in the plasma membrane and show that SERS has great potential for the study and characterization of cell surfaces.     

Molecular conformation changes along the malignancy revealed by optical nanosensors

An interdisciplinary approach employing functionalized nanoparticles and ultrasensitive spectroscopic techniques is reported here to track the molecular changes in early stage of malignancy. Melanoma tissue tracking at molecular level using both labelled and unlabelled silver and gold nanoparticles has been achieved using surface enhanced Raman scattering (SERS) technique. We used skin tissue from ex vivo mice with induced melanoma. Raman and SERS molecular characterization of melanoma tissue is proposed here for the first time. Optical nanosensors based on Ag and Au nanoparticles with chemisorbed cresyl violet molecular species as labels revealed sensitive capability to tissues tagging and local molecular characterization. Sensitive information originating from surrounding native biological molecules is provided by the tissue SERS spectra obtained either with visible or NIR laser line. Labelled nanoparticles introduced systematic differences in tissue response compared with unlabelled ones, suggesting that the label functional groups tag specific tissue components revealed by proteins or nucleic acids bands. Vibrational data collected from tissue are presented in conjunction with the immunohistochemical analysis. The results obtained here open perspectives in applied plasmonic nanoparticles and SERS for the early cancer diagnostic based on the appropriate spectral databank.     

Method for Assessing the Reliability of Molecular Diagnostics Based on Multiplexed SERS-Coded Nanoparticles

Surface-enhanced Raman scattering (SERS) nanoparticles have been engineered to generate unique fingerprint spectra and are potentially useful as bright contrast agents for molecular diagnostics. One promising strategy for biomedical diagnostics and imaging is to functionalize various particle types (“flavors”), each emitting a unique spectral signature, to target a large multiplexed panel of molecular biomarkers. While SERS particles emit narrow spectral features that allow them to be easily separable under ideal conditions, the presence of competing noise sources and background signals such as detector noise, laser background, and autofluorescence confounds the reliability of demultiplexing algorithms. Results obtained during time-constrained in vivo imaging experiments may not be reproducible or accurate. Therefore, our goal is to provide experimentalists with a metric that may be monitored to enforce a desired bound on accuracy within a user-defined confidence level. We have defined a spectral reliability index (SRI), based on the output of a direct classical least-squares (DCLS) demultiplexing routine, which provides a measure of the reliability of the computed nanoparticle concentrations and ratios. We present simulations and experiments to demonstrate the feasibility of this strategy, which can potentially be utilized for a range of instruments and biomedical applications involving multiplexed SERS nanoparticles.     

Therapeutic prognosis of prostate cancer using surface-enhanced Raman scattering of patient urine and multivariate statistical analysis

Surface-enhanced Raman scattering (SERS) is highly sensitive and label-free analytical technique based on Raman spectroscopy aided by field-multiplying plasmonic nanostructures. We report the use of SERS measurements of patient urine in conjunction with biostatistical algorithms to assess the treatment response of prostate cancer (PCa) in 12 recurrent (Re) and 63 nonrecurrent (NRe) patient cohorts. Multiple Raman spectra are collected from each urine sample using monodisperse silver nanoparticles (AgNPs) for Raman signal enhancement. Genetic algorithms-partial least squares-linear discriminant analysis (GA-PLS-LDA) was employed to analyze the Raman spectra. Comprehensive GA-PLS-LDA analyses of these Raman spectral features (p = 3.50 × 10⁻¹⁶ ) yield an accuracy of 86.6%, sensitivity of 86.0%, and specificity 87.1% in differentiating the Re and NRe cohorts. Our study suggests that SERS combined with multivariate GA-PLS-LDA algorithm can potentially be used to detect and monitor the risk of PCa relapse and to aid with decision-making for optimal intermediate secondary therapy to recurred patients.     

Electromagnetic Enhancement Effect Caused by Aggregation on SERS-Active Gold Nanoparticles

We report a morphology-correlated surface-enhanced Raman scattering (SERS) from molecules on the surface of individual clusters of gold nanoparticles of two types and compare the signal from clusters of two, three, four, and five nanoparticles with the signal from single particles. Cluster geometry and particle morphology are determined from transmission electron microscopy for both clusters of 78- to 133-nm nanospheres and clusters of ~250-nm-etched cylindrical particles with crevices and sharp edges, formed in templates. Scattering from molecules on etched cylinders, but not spheres, is sufficiently strong to allow spectra to be collected from single particles illuminated at 632.8 nm. SERS intensities from clusters of cylinders are found to scale linearly with particle number, whereas, for nanospheres, the scaling is non-linear. The linear scaling of SERS from cylinders is a reflection of the high enhancement provided by the sharp features of the individual particles; whereas, the non-linear scaling of SERS from clusters of spheres is found to be consistent with the near-field enhancement from inter-particle coupling simulated for clusters of spheres arranged in representative-observed geometries.     

A Real-Time Clinical Endoscopic System for Intraluminal,Multiplexed Imaging of Surface-Enhanced Raman Scattering Nanoparticles

The detection of biomarker-targeting surface-enhanced Raman scattering (SERS) nanoparticles (NPs) in the human gastrointestinal tract has the potential to improve early cancer detection; however, a clinically relevant device with rapid Raman-imaging capability has not been described. Here we report the design and in vivo demonstration of a miniature, non-contact, opto-electro-mechanical Raman device as an accessory to clinical endoscopes that can provide multiplexed molecular data via a panel of SERS NPs. This device enables rapid circumferential scanning of topologically complex luminal surfaces of hollow organs (e.g., colon and esophagus) and produces quantitative images of the relative concentrations of SERS NPs that are present. Human and swine studies have demonstrated the speed and simplicity of this technique. This approach also offers unparalleled multiplexing capabilities by simultaneously detecting the unique spectral fingerprints of multiple SERS NPs. Therefore, this new screening strategy has the potential to improve diagnosis and to guide therapy by enabling sensitive quantitative molecular detection of small and otherwise hard-to-detect lesions in the context of white-light endoscopy.     

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.     

Early diagnosis of oral cancer based on the surface plasmon resonance of gold nanoparticles

The high mortality rate in cancer such as oral squamous cell carcinoma is commonly attributed to the difficulties in detecting the disease at an early treatable stage. In this study, we exploited the ability of gold nanoparticles to undergo coupled surface plasmon resonance and set up strong electric fields when closely-spaced to improve the molecular contrast signal in reflectance-based imaging and also to enhance the Raman signal of bioanalytes in cancer. Colloidal gold nanoparticles were synthesized and conjugated to anti-epidermal growth factor receptor (EGFR) for imaging. A self-assembled surface enhanced Raman scattering (SERS)-active gold nanoparticle monolayer film was also developed as a biosensing surface using a simple drop-dry approach. We have shown that gold nanoparticles could elicit an optical contrast to discriminate between cancerous and normal cells and their conjugation with antibodies allowed them to map the expression of relevant biomarkers for molecular imaging under confocal reflectance microscopy. We have also shown that the SERS spectra of saliva from the closely-packed gold nanoparticles films was differentiable between those acquired from normal individuals and oral cancer patients, thus showing promise of a simple SERS-based saliva assay for early diagnosis of oral cancer.     

Multiplex single‐cell quantification of rare RNA transcripts from protoplasts in a model plant system

Here we demonstrate multiplex and simultaneous detection of four different rare RNA species from plant, Arabidopsis thaliana, using surface‐enhanced Raman spectroscopy (SERS) and gold nanoprobes at single‐cell resolution. We show the applicability of nanoparticle‐based Raman spectroscopic sensor to study intracellular RNA copies. First, we demonstrate that gold‐nanoparticles decorated with Raman probes and carrying specific nucleic acid probe sequences can be uptaken by the protoplasts. We confirm the internalization of gold nanoprobes by transmission electron microscopy, inductively‐coupled plasma‐mass spectrometry and fluorescence imaging. Second, we show the utility of a SERS platform to monitor individual alternatively spliced (AS) variants and miRNA copies within single cells. Finally, the distinctive spectral features of Raman‐active dyes were exploited for multiplex analysis of AtPTB2, AtDCL2, miR156a and miR172a. Furthermore, single‐cell studies were validated by in vitro quantification and evaluation of nanotoxicity of gold probes. Raman tag functionalized gold nanosensors yielded an approach for the tracking of rare RNAs within the protoplasts. The SERS‐based approach for quantification of RNAs has the capability to be a highly sensitive, accurate and discerning method for single‐cell studies including AS variants quantification and rare miRNA detection in specific plant species.     

Characteristics of a novel deep red/infrared fluorescent cell-permeant DNA probe, DRAQ5, in intact human cells analyzed by flow cytometry, confocal and multiphoton microscopy

BACKGROUND: The multiparameter fluorometric analysis of intact and fixed cells often requires the use of a nuclear DNA discrimination signal with spectral separation from visible range fluorochromes. We have developed a novel deep red fluorescing bisalkylaminoanthraquinone, DRAQ5 (Ex(lambdamax) 646 nm; Em(lambdamax) 681 nm; Em(lambdarange) 665->800 nm), with high affinity for DNA and a high capacity to enter living cells. We describe here the spectral characteristics and applications of this synthetic compound, particularly in relation to cytometric analysis of the cell cycle. METHODS: Cultured human tumor cells were examined for the ability to nuclear locate DRAQ5 using single and multiphoton laser scanning microscopy (LSM) and multiparameter flow cytometry. RESULTS: Multiparameter flow cytometry shows that the dye can rapidly report the cellular DNA content of live and fixed cells at a resolution level adequate for cell cycle analysis and the cycle-specific expression of cellular proteins (e.g., cyclin B1). The preferential excitation of DRAQ5 by laser red lines (633/647 nm) was found to offer a means of fluorescence signal discrimination by selective excitation, with greatly reduced emission overlap with UV-excitable and visible range fluophors as compared with propidium iodide. LSM reveals nuclear architecture and clearly defines chromosomal elements in live cells. DRAQ5 was found to permit multiphoton imaging of nuclei using a 1,047-nm emitting mode-locked YLF laser. The unusual spectral properties of DRAQ5 also permit live cell DNA analysis using conventional 488 nm excitation and the single-photon imaging of nuclear fluorescence using laser excitation between 488 nm and low infrared (IR; 780 nm) wavelengths. Single and multiphoton microscopy studies revealed the ability of DRAQ5 to report three-dimensional nuclear structure and location in live cells expressing endoplasmic reticulum targeted-GFP, MitoTracker-stained mitochondria, or a vital cell probe for free zinc (Zinquin). CONCLUSION: The fluorescence excitation and emission characteristics of DRAQ5 in living and fixed cells permit the incorporation of the measurement of cellular DNA content into a variety of multiparameter cytometric analyses.     

Large Uptake of Titania and Iron Oxide Nanoparticles in the Nucleus of Lung Epithelial Cells as Measured by Raman Imaging and Multivariate Classification

It is a challenging task to characterize the biodistribution of nanoparticles in cells and tissue on a subcellular level. Conventional methods to study the interaction of nanoparticles with living cells rely on labeling techniques that either selectively stain the particles or selectively tag them with tracer molecules. In this work, Raman imaging, a label-free technique that requires no extensive sample preparation, was combined with multivariate classification to quantify the spatial distribution of oxide nanoparticles inside living lung epithelial cells (A549). Cells were exposed to TiO₂ (titania) and/or α-FeO(OH) (goethite) nanoparticles at various incubation times (4 or 48 h). Using multivariate classification of hyperspectral Raman data with partial least-squares discriminant analysis, we show that a surprisingly large fraction of spectra, classified as belonging to the cell nucleus, show Raman bands associated with nanoparticles. Up to 40% of spectra from the cell nucleus show Raman bands associated with nanoparticles. Complementary transmission electron microscopy data for thin cell sections qualitatively support the conclusions.     

Fluorescent cell barcoding in flow cytometry allows high-throughput drug screening and signaling profiling

Flow cytometry allows high-content, multiparameter analysis of single cells, making it a promising tool for drug discovery and profiling of intracellular signaling. To add high-throughput capacity to flow cytometry, we developed a cell-based multiplexing technique called fluorescent cell barcoding (FCB). In FCB, each sample is labeled with a different signature, or barcode, of fluorescence intensity and emission wavelengths, and mixed with other samples before antibody staining and analysis by flow cytometry. Using three FCB fluorophores, we were able to barcode and combine entire 96-well plates, reducing antibody consumption 100-fold and acquisition time to 5-15 min per plate. Using FCB and phospho-specific flow cytometry, we screened a small-molecule library for inhibitors of T cell-receptor and cytokine signaling, simultaneously determining compound efficacy and selectivity. We also analyzed IFN-gamma signaling in multiple cell types from primary mouse splenocytes, revealing differences in sensitivity and kinetics between B cells, CD4+ and CD4- T cells and CD11b-hi cells.     

Silver salts of aromatic thiols applicable as core materials of molecular sensors operating via SERS and fluorescence

We have developed dual-tagging sensors, operating via both Raman and fluorescence spectroscopy, composed of silver aromatic thiolates (AgSRs) modified with fluorescent organic dye for multiplex immunoassays. Owing to the photo-induced production of SERS-active Ag nanoparticles, AgSRs exhibit the surface-enhanced Raman scattering (SERS) spectra of corresponding thiols. The fluorescence dye-modified AgSRs were accordingly fabricated using dye-grafted polyelectrolytes during layer-by-layer deposition of cationic and anionic polyelectrolytes onto AgSRs. In the final stage, the tagging sensors assembled with either specific biotin group or specific antibodies (anti-h-IgG or anti-r-IgG) were employed to detect either streptavidin molecules or target antigens (h-IgG or r-IgG), respectively. Since numerous AgSRs can be used as the core materials, multiple bioassays are expected to be accomplishable using the present methodology. The fluorescence signal may be used as an immediate indicator of molecular recognition, while the SERS signals can be used subsequently as the signature of specific molecular interactions.     

Bioanalytical application of surface‐ and tip‐enhanced Raman spectroscopy

Due to its fingerprint specificity and trace‐level sensitivity, surface‐enhanced Raman spectroscopy (SERS) is an attractive tool in bioanalytics. This review reflects the research in this highly interesting topic of the last 3–4 years. The detection of the SERS signature of biomolecules up to microorganisms and cells is introduced. Labeling using modified nanoparticles (SERS tags) is also introduced. In order to establish biomedical applications, SERS analysis is performed in complex matrices such as body fluids. Furthermore, the SERS technique is combined with other methods such as microfluidic devices for online monitoring and scanning probe microscopy (i.e. tip‐enhanced Raman spectroscopy, TERS) to investigate nanoscaled features. The present review illustrates the broad application fields of SERS and TERS in bioanalytics and shows the great potential of these methods for biomedical diagnostics.     

Surface-enhanced resonance Raman spectroscopy of porphyrin and metalloporphyrin species in systems with Ag nanoparticles and their assemblies

The advantages of systems with Ag nanoparticles and their assemblies for surface-enhanced resonance Raman scattering (SERRS) spectral investigation, detection and determination of porphyrin species are demonstrated. SERRS spectral detection limits of the testing porphyrin species (including porphyrin aggregates) in these systems are shown to be, on average, 10(2)-10(3) lower than detection limits by resonance Raman scattering (RRS). Systems with Ag nanoparticles modified by anionic organosulfur spacers enable us to obtain SERRS spectra of unperturbed cationic porphyrin species. In the case of thiopheneacetate-modified Ag particles prepared by laser ablation, no negative effect of the spacer on the spectral detection limit of the porphyrin was observed. Systems with isolated Ag nanoparticles allow for obtaining SERRS spectra of porphyrin species upon excitation into the Soret electronic absorption band which leads to at least a 10-fold decrease in the detection limit.     

Ultrasensitive and selective detection of copper (II) and mercury (II) ions by dye-coded silver nanoparticle-based SERS probes

A simple and distinctive method for the ultrasensitive detection of Cu(2+) and Hg(2+) based on surface-enhanced Raman scattering (SERS) using cysteine-functionalized silver nanoparticles (AgNPs) attached with Raman-labeling molecules was developed. The glycine residue in a silver nanoparticle-bound cysteine can selectively bind with Cu(2+) and Hg(2+) and form a stable inner complex. Silver nanoparticles co-functionalized with cysteine and 3,5-Dimethoxy-4-(6'-azobenzotriazolyl)phenol (AgNP conjugates) can be used to detect Cu(2+) and Hg(2+) based on aggregation-induced SERS of the Raman tags. The addition of SCN(-) to the analyte can successfully mask Hg(2+) and allow for the selective detection of Cu(2+). This SERS-based assay showed an unprecedented limit of detection (LOD) of 10pM for Cu(2+) and 1pM for Hg(2+); these LODs are a few orders of magnitude more sensitive than the typical colorimetric approach based on the aggregation of noble nanoparticles. The analysis of real water samples diluted with pure water was performed and verified this conclusion. We envisage that this SERS-based assay may provide a general and simple approach for the detection of other metal ions of interest, which can be adopted from their corresponding colorimetric assays that have already been developed with significantly improved sensitivity and thus have wide-range applications in many areas.     

FLOW CYTOMETRY AND THE SINGLE CELL IN PHYCOLOGY

Flow cytometers measure light scattering and fluorescence characteristics from individual particles in a fluid stream as they cross one or more light beams at rates of up to thousands of events per second. Flow cytometrically detectable optical signals may arise naturally from algae, reflecting cell size, structure, and endogenous pigmentation, or may be generated by fluorescent stains that report the presence of otherwise undetected cellular constituents. Some flow cytometers can physically sort particles with desired optical characteristics out of the flow stream and collect them for subsequent culture or other analyses. The statistically rigorous, cell‐level perspective provided by flow cytometry has been advantageous in experimental investigations of phycological problems, such as the regulation of cell cycle progression. The capacity of flow cytometry to measure large numbers of cells in large numbers of samples rapidly and quantitatively has been used extensively by biological oceanographers to define the distributions and dynamics of marine picophytoplankton. Recent work has shown that flow cytometry can be used to elucidate relationships between the optical properties of individual cells and the bulk optical properties of the water they live in, and thereby may provide an explicit link between algal physiology and global biogeochemistry. Unfortunately, commercially available flow cytometers that are optimized for biomedical applications have a limited capacity to analyze larger phytoplankton. To circumvent these limitations, many investigators are developing flow cytometers specifically designed for analyzing the broad range of sizes, shapes, and pigments found among algae. These new instruments can perform some novel measurements, including simple fluorescence excitation spectra, detailed angular scattering measurements, and in‐flow digital imaging. The growing accessibility and power of flow cytometers may allow the technology to be applied to a wider array of problems in phycology, including investigations of nonplanktonic and multicellular algae, but also presents new challenges for effectively analyzing the large quantity of multiparameter data produced. Ultimately, the detection of molecular probes by flow cytometry may allow single‐cell taxonomic and physiological information to be garnered for a variety of algae, both in culture and in nature.     

Gastric cancer detection based on blood plasma surface-enhanced Raman spectroscopy excited by polarized laser light

We have recently applied surface-enhanced Raman spectroscopy (SERS) for blood plasma analysis for non-invasive nasopharyngeal cancer detection and obtained good preliminary results. The aim of this study was to develop a more robust SERS spectroscopy based blood plasma analysis method for non-invasive gastric cancer detection. The effect of different laser polarizations (non-polarized, linear-polarized, right-handed circularly polarized, and left-handed circularly polarized) on blood plasma SERS spectroscopy was explored for the first time. Silver nanoparticles as the SERS-substrate were directly mixed with blood plasma to enhance the Raman scattering of various biomolecular constituents. High quality SERS spectra were obtained using a fiber optic probe and a dispersive type near infrared Raman system. Blood plasma samples from gastric cancer patients (n=32) and healthy subjects (n=33) were analyzed. The diagnostic performance for differentiating gastric cancer plasma from normal plasma was evaluated. Principal component analysis combined with linear discriminant analysis (LDA) of the obtained spectral data was used to develop diagnostic algorithms. Classification results obtained from cross-validation of the LDA model based on the four spectral data sets of different laser polarizations demonstrated different diagnostic sensitivities and specificities: 71.9% and 72.7% for non-polarized laser excitation, 75% and 87.9% for linear-polarized laser excitation, 81.3% and 78.8% for right-handed circularly polarized laser excitation, 100% and 97% for left-handed circularly polarized laser excitation. The results from this exploratory study demonstrated that plasma SERS spectroscopy with left-handed circularly polarized laser excitation has great promise of becoming a clinically useful diagnostic tool for non-invasive gastric cancer detection.     

Theoretical and pH dependent surface enhanced Raman spectroscopy study on caffeine

The surface enhanced Raman spectroscopy (SERS) spectrum of caffeine is recorded on a silver colloid at different pH values. It is discussed on the basis of the SERS "surface selection rules" in order to characterize its vibrational behavior on such a biological artificial model. To improve the previous assignments in the Raman spectrum and for a reliable, detailed analysis of SERS spectra, density functional theory calculations (structural parameters, harmonic vibrational wavenumbers, total electron density, and natural population analysis of the molecule) are performed for the anhydrous form of caffeine and the results are discussed. The predicted geometry and vibrational Raman spectra are in good agreement with the experimental data. The flat orientation of the mainly chemisorbed caffeine attached through the pi electrons and the lone pair of nonmethylated N atoms of the imidazole ring are proposed to occur at neutral and basic pH values. At acid pH values caffeine is probably adsorbed on the Ag surface through one or both oxygen atoms, more probably through the O atom of the conjugated carbonyl group with an end-on orientation. However, the changes in the overall SERS spectral pattern seem to indicate the electromagnetic mechanism as being the dominant one.     

Primer-design for multiplexed genotyping

Single-nucleotide polymorphism (SNP) analysis is a powerful tool for mapping and diagnosing disease-related alleles. Mutation analysis by polymerase-mediated single-base primer extension (minisequencing) can be massively parallelized using DNA microchips or flow cytometry with microspheres as solid support. By adding a unique oligonucleotide tag to the 5′ end of the minisequencing primer and attaching the complementary antitag to the array or bead surface, the assay can be ‘demultiplexed’. Such high-throughput scoring of SNPs requires a high level of primer multiplexing in order to analyze multiple loci in one assay, thus enabling inexpensive and fast polymorphism scoring. We present a computer program to automate the design process for the assay. Oligonucleotide primers for the reaction are automatically selected by the software, a unique DNA tag/antitag system is generated, and the pairing of primers and DNA tags is automatically done in a way to avoid any crossreactivity. We report results on a 45-plex genotyping assay, indicating that minisequencing can be adapted to be a powerful tool for high-throughput, massively parallel genotyping. The software is available to academic users on request.