Rapid detection of food- and waterborne bacteria using surface-enhanced Raman spectroscopy coupled with silver nanosubstrates

Development of rapid and sensitive methods to detect pathogens is important to food and water safety. This study aimed to detect and discriminate important food- and waterborne bacteria (i.e., Escherichia coli O157:H7, Staphylococcus epidermidis, Listeria monocytogenes, and Enterococcus faecelis) by surface-enhanced Raman spectroscopy (SERS) coupled with intracellular nanosilver as SERS substrates. An in vivo molecular probing using intracellular nanosilver for the preparation of bacterial samples was established and assessed. Satisfactory SERS performance and characteristic SERS spectra were obtained from different bacterial samples. Distinctive differences were observed in SERS spectral data, specifically in the Raman shift region of 500–1,800 cm⁻¹, and between bacterial samples at the species and strain levels. The detection limit of SERS coupled with in vivo molecular probing using silver nanosubstrates could reach the level of single cells. Experiments with a mixture of E. coli O157:H7 and S. epidermidis for SERS measurement demonstrate that SERS could be used for classification of mixed bacterial samples. Transmission electron microscopy was used to characterize changes of morphology and cellular composition of bacterial cells after treatment of intracellular nanosilver. The results indicate that SERS coupled with intracellular silver nanosubstrates is a promising method for detection and characterization of food- and waterborne pathogenic and non-pathogenic bacterial samples.     

Rational design of thiolated polyenes as trifunctional Raman reporter molecules in surface‐enhanced Raman scattering nanotags for cytokine detection in a lateral flow assay

The characteristic vibrational spectroscopic fingerprint of Raman reporter molecules adsorbed on noble metal nanoparticles is employed for the identification of target proteins by the corresponding surface‐enhanced Raman scattering (SERS) nanotag‐labeled antibodies. Here, we present the modular synthesis of thiolated polyenes with two to five C═C double bonds introduced via stepwise Wittig reactions. The experimental characterization of their electronic and vibrational properties is complemented by density functional theory calculations. Highly SERS‐active nanotags are generated by using the thiolated polyenes as Raman reporter molecules in Au/Au core/satellite supraparticles with multiple hot spots. The cytokines IL‐1β and IFN‐γ are detected in a duplex SERS‐based lateral flow assay on a nitrocellulose test strip by Raman microscopy. The thiolated polyenes are suitable for use in immuno‐SERS applications such as point‐of‐care testing as well as cellular and tissue imaging.     

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.     

Single cell analysis using surface enhanced Raman scattering (SERS) tags

Fluorescence is a mainstay of bioanalytical methods, offering sensitive and quantitative reporting, often in multiplexed or multiparameter assays. Perhaps the best example of the latter is flow cytometry, where instruments equipped with multiple lasers and detectors allow measurement of 15 or more different fluorophores simultaneously, but increases beyond this number are limited by the relatively broad emission spectra. Surface enhanced Raman scattering (SERS) from metal nanoparticles can produce signal intensities that rival fluorescence, but with narrower spectral features that allow a greater degree of multiplexing. We are developing nanoparticle SERS tags as well as Raman flow cytometers for multiparameter single cell analysis of suspension or adherent cells. SERS tags are based on plasmonically active nanoparticles (gold nanorods) whose plasmon resonance can be tuned to give optimal SERS signals at a desired excitation wavelength. Raman resonant compounds are adsorbed on the nanoparticles to confer a unique spectral fingerprint on each SERS tag, which are then encapsulated in a polymer coating for conjugation to antibodies or other targeting molecules. Raman flow cytometry employs a high resolution spectral flow cytometer capable of measuring the complete SERS spectra, as well as conventional flow cytometry measurements, from thousands of individual cells per minute. Automated spectral unmixing algorithms extract the contributions of each SERS tag from each cell to generate high content, multiparameter single cell population data. SERS-based cytometry is a powerful complement to conventional fluorescence-based cytometry. The narrow spectral features of the SERS signal enables more distinct probes to be measured in a smaller region of the optical spectrum with a single laser and detector, allowing for higher levels of multiplexing and multiparameter analysis.     

SERS Properties of Different Sized and Shaped Gold Nanoparticles Biosynthesized under Different Environmental Conditions by Neurospora crassa Extract

Surface-enhanced Raman scattering (SERS) is a surface-sensitive technique that enhances Raman scattering by molecules adsorbed on rough metal surfaces. It is known that metal nanoparticles, especially gold and silver nanoparticles, exhibit great SERS properties, which make them very attractive for the development of biosensors and biocatalysts. On the other hand, the development of ecofriendly methods for the synthesis of metallic nanostructures has become the focus of research in several countries, and many microorganisms and plants have already been used to biosynthesize metallic nanostructures. However, the majority of these are pathogenic to plants or humans. Here, we report gold nanoparticles with good SERS properties, biosynthesized by Neurospora crassa extract under different environmental conditions, increasing Raman signals up to 40 times using methylene blue as a target molecule. Incubation of tetrachloroauric acid solution with the fungal extract at 60°C and a pH value of a) 3, b) 5.5, and c) 10 resulted in the formation of gold nanoparticles of a) different shapes like triangles, hexagons, pentagons etc. in a broad size range of about 10-200 nm, b) mostly quasi-spheres with some different shapes in a main size range of 6-23 nm, and c) only quasi-spheres of 3-12 nm. Analyses included TEM, HRTEM, and EDS in order to corroborate the shape and the elemental character of the gold nanoparticles, respectively. The results presented here show that these ‘green’ synthesized gold nanoparticles might have potential applicability in the field of biological sensing.     

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.     

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.     

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.     

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.     

A Shape-Engineered Surface-Enhanced Raman Scattering Optical Fiber Sensor Working from the Visible to the Near-Infrared

Surface-enhanced Raman scattering (SERS) takes advantage of the giant electromagnetic field enhancement provided by localized surface plasmons in metal nanoparticles to amplify the weak Raman scattering of the molecules. Optical fibers coated with noble metal nanoparticles can therefore be used as SERS-based sensors for remote detection of molecular species. In this article, we report on the development of an optical fiber SERS sensor capable to operate on a range of excitation wavelengths from the visible to the near-infrared. We introduce a quasistatic chemical etching protocol to engineer the tip shape and investigate the effects of the tip shape on the sensor performances.     

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.     

Selective analysis of antitumor drug interaction with living cancer cells as probed by surface-enhanced Raman spectroscopy

A new technique for the selective measurement of small amounts of antitumor drugs in the nucleus and cytoplasm of a living cancer cell, based on surface-enhanced Raman spectroscopy (SERS), is proposed. The ability to detect SERS signals from very dilute (up to 10^–10 M) solutions of doxorubicin or adriamycin (DOX), and 4O-tetrahydropyranyl-adriamycin (THP-ADM), as well as from their complexes with targets in vitro and in vivo, has been demonstrated. SERS spectra were obtained from a population as well as from single living erythroleukaemic K562 cells treated with DOX. The results of the measurements on the population of cells containing DOX in nuclei or in the cytoplasm are well correlated with the microscopic SERS measurements on the single cells treated with DOX, obtained by selectively recording signals from the living cell nucleus or from the cytoplasm. Possibilities for the application of this new technique in different aspects of cancer research are discussed.Abbreviations DNA deoxyribonucleic acid - DOX doxorubicin - SERS surface-enhanced Raman spectroscopy - THP-ADM 4O-tetrahydropyranyl adriamycin - PBS phosphate buffered saline Offprint requests to: M. Manfait     

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.     

Supervised learning methods for the recognition of melanoma cell lines through the analysis of their Raman spectra

Malignant melanoma is an aggressive form of skin cancer, which develops from the genetic mutations of melanocytes – the most frequent involving BRAF and NRAS genes. The choice and the effectiveness of the therapeutic approach depend on tumour mutation; therefore, its assessment is of paramount importance. Current methods for mutation analysis are destructive and take a long time; instead, Raman spectroscopy could provide a fast, label-free and non-destructive alternative. In this study, confocal Raman microscopy has been used for examining three in vitro melanoma cell lines, harbouring different molecular profiles and, in particular, specific BRAF and NRAS driver mutations. The molecular information obtained from Raman spectra has served for developing two alternative classification algorithms based on linear discriminant analysis and artificial neural network. Both methods provide high accuracy (≥90%) in discriminating all cell types, suggesting that Raman spectroscopy may be an effective tool for detecting molecular differences between melanoma mutations.     

Gold nanoparticle-based surface-enhanced Raman scattering for noninvasive molecular probing of embryonic stem cell differentiation

This study reports the use of gold nanoparticle-based surface-enhanced Raman scattering (SERS) for probing the differentiation of mouse embryonic stem (mES) cells, including undifferentiated single cells, embryoid bodies (EBs), and terminally differentiated cardiomyocytes. Gold nanoparticles (GNPs) were successfully delivered into all 3 mES cell differentiation stages without affecting cell viability or proliferation. Transmission electron microscopy (TEM) confirmed the localization of GNPs inside the following cell organelles: mitochondria, secondary lysosome, and endoplasmic reticulum. Using bright- and dark-field imaging, the bright scattering of GNPs and nanoaggregates in all 3 ES cell differentiation stages could be visualized. EB (an early differentiation stage) and terminally differentiated cardiomyocytes both showed SERS peaks specific to metabolic activity in the mitochondria and to protein translation (amide I, amide II, and amide III peaks). These peaks have been rarely identified in undifferentiated single ES cells. Spatiotemporal changes observed in the SERS spectra from terminally differentiated cardiomyocyte tissues revealed local and dynamic molecular interactions as well as transformations during ES cell differentiation.     

Highly sensitive SERS detection of cancer proteins in low sample volume using hollow core photonic crystal fiber

Enzyme-linked immunosorbent assays (ELISA) are commonly used for detecting cancer proteins at concentration in the range of about ng-μg/mL. Hence it often fails to detect tumor markers at the early stages of cancer and other diseases where the amount of protein is extremely low. Herein, we report a novel photonic crystal fiber (PCF) based surface enhanced Raman scattering (SERS) sensing platform for the ultrasensitive detection of cancer proteins in an extremely low sample volume. As a proof of concept, epidermal growth factor receptors (EGFRs) in a lysate solution from human epithelial carcinoma cells were immobilized into the hollow core PCF. Highly sensitive detection of protein was achieved using anti-EGFR antibody conjugated SERS nanotag. This SERS nanotag probe was realized by anchoring highly active Raman molecules onto the gold nanoparticles followed by bioconjugation. The proposed sensing method can detect low amount of proteins at ～100 pg in a sample volume of ～10 nL. Our approach may lead to the highly sensitive protein sensing methodology for the early detection of diseases.     

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.     

Porphyrin-lipid stabilized gold nanoparticles for surface enhanced Raman scattering based imaging

A porphyrin-phospholipid conjugate with quenched fluorescence was utilized to serve as both the Raman dye and a stabilizing, biocompatible surface coating agent on gold nanoparticles. Through simple synthesis and validation with spectroscopy and confocal microscopy, we show that this porphyrin-lipid stabilized AuNP is a novel SERS probe capable of cellular imaging. To date, this is the first use of porphyrin as a Raman reporter molecule for SERS based imaging.