Duncan Graham

The discovery of the enhancement of Raman scattering by molecules adsorbed on nanostructured metal surfaces is a landmark in the history of spectroscopic and analytical techniques. Significant experimental and theoretical effort has been directed toward understanding the surface-enhanced Raman scattering (SERS) effect and demonstrating its potential in various types of ultrasensitive sensing applications in a wide variety of fields. In the 45 years since its discovery, SERS has blossomed into a rich area of research and technology, but additional efforts are still needed before it can be routinely used analytically and in commercial products. In this Review, prominent authors from around the world joined together to summarize the state of the art in understanding and using SERS and to predict what can be expected in the near future in terms of research, applications, and technological development. This Review is dedicated to SERS pioneer and our coauthor, the late Prof. Richard Van Duyne, whom we lost during the preparation of this article.

The platinum-based anticancer drugs cisplatin, carboplatin, and oxaliplatin are an important component of chemotherapy but are limited by severe dose-limiting side effects and the ability of tumors to develop resistance rapidly. These drugs can be improved through the use of drug-delivery vehicles that are able to target cancers passively or actively. In this study, we have tethered the active component of the anticancer drug oxaliplatin to a gold nanoparticle for improved drug delivery. Naked gold nanoparticles were functionalized with a thiolated poly(ethylene glycol) (PEG) monolayer capped with a carboxylate group. [Pt(1R,2R-diaminocyclohexane)(H(2)O)(2)]2NO(3) was added to the PEG surface to yield a supramolecular complex with 280 (+/-20) drug molecules per nanoparticle. The platinum-tethered nanoparticles were examined for cytotoxicity, drug uptake, and localization in the A549 lung epithelial cancer cell line and the colon cancer cell lines HCT116, HCT15, HT29, and RKO. The platinum-tethered nanoparticles demonstrated as good as, or significantly better, cytotoxicity than oxaliplatin alone in all of the cell lines and an unusual ability to penetrate the nucleus in the lung cancer cells.

Surface-enhanced Raman scattering (SERS) and surface-enhanced resonance Raman scattering (SERRS) can provide positive identification of an analyte or an analyte mixture with high sensitivity and selectivity. Better understanding of the theory and advances in the understanding of the practice have led to the development of practical applications in which the unique advantages of SERS/SERRS have been used to provide effective solutions to difficult analytical problems. This review presents a basic theory and illustrates the way in which SERS/SERRS has been developed for practical use.

Taking the shortcut: Spectroscopic data (see picture) provide direct evidence that in non-aqueous Li+ electrolyte, O2 is reduced to O2−, which then forms LiO2 on the electrode surface which disproportionates to Li2O2. On charging, Li2O2 decomposes directly, in a one-step reaction to evolve O2 and does not pass through LiO2 as an intermediate. Detailed facts of importance to specialist readers are published as ”Supporting Information”. Such documents are peer-reviewed, but not copy-edited or typeset. They are made available as submitted by the authors. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.

In recent years, Surface-Enhanced Raman Spectroscopy (SERS) has experienced a tremendous increase of attention in the scientific community, expanding to a continuously wider range of diverse applications in nanoscience, which can mostly be attributed to significant improvements in nanofabrication techniques that paved the way for the controlled design of reliable and effective SERS nanostructures. In particular, the plasmon coupling properties of interacting nanoparticles are extremely intriguing due to the concentration of enormous electromagnetic enhancements at the interparticle gaps. Recently, great efforts have been devoted to develop new nanoparticle assembly strategies in suspension with improved control over hot-spot architecture and cluster structure, laying the foundation for the full exploitation of their exceptional potential as SERS materials in a wealth of chemical and biological sensing. In this review we summarize in an exhaustive and systematic way the state-of-art of plasmonic nanoparticle assembly in suspension specifically developed for SERS applications in the last 5 years, focusing in particular on those strategies which exploited molecular linkers to engineer interparticle gaps in a controlled manner. Importantly, the novel advances in this rather new field of nanoscience are organized into a coherent overview aimed to rationally describe the different strategies and improvements in the exploitation of colloidal nanoparticle assembly for SERS application to real problems.

Surface-enhanced Raman scattering (SERS) is of interest for biomedical analysis and imaging because of its sensitivity, specificity and multiplexing capabilities. The successful application of SERS for in vivo biosensing requires probes to be biocompatible and procedures to be minimally invasive, challenges that have respectively been met by developing new nanoprobes and instrumentation. This Review presents recent developments in these areas, describing case studies in which sensors have been implemented, as well as outlining shortcomings that must be addressed before SERS sees clinical use. Surface-enhanced Raman scattering (SERS) is a physical phenomenon first discovered in 1974. SERS has since been exploited for bioanalysis because of its high sensitivity and multiplexing capabilities. This Review describes the progress made and problems faced with respect to using in vivo SERS in humans.

Gold nanoparticles (AuNPs) have been extensively used as nanomaterials for theranostic applications due to their multifunctional characteristics in therapeutics, imaging, and surface modification. In this study, the unique functionalities of exosome-derived membranes were combined with synthetic AuNPs for targeted delivery to brain cells. Here, we report the surface modification of AuNPs with brain-targeted exosomes derived from genetically engineered mammalian cells by using the mechanical method or extrusion to create these novel nanomaterials. The unique targeting properties of the AuNPs after fabrication with the brain-targeted exosomes was demonstrated by their binding to brain cells under laminar flow conditions as well as their enhanced transport across the blood brain barrier. In a further demonstration of their ability to target brain cells, in vivo bioluminescence imaging revealed that targeted-exosome coated AuNPs accumulated in the mouse brain after intravenous injection. The surface modification of synthetic AuNPs with the brain-targeted exosome demonstrated in this work represents a highly novel and effective strategy to provide efficient brain targeting and shows promise for the future in using modified AuNPs to penetrate the brain.

Successful pathogen detection is crucial for public health as the threat of infectious disease is dramatically increasing globally due to bacteria developing resistance to many antimicrobial drugs. The increase in bacterial infections has led to urgent demands for simpler, faster, and more reliable detection methods to be developed allowing the most appropriate therapy to be provided. Surface enhanced Raman scattering (SERS) is an analytical technique which has gained a great deal of interest for biosensing due to its sensitivity, selectivity, and multiplexing capabilities. A new bionanosensor has been developed for the isolation and detection of multiple bacterial pathogens via magnetic separation and SERS. This novel assay format involves using lectin functionalized magnetic nanoparticles for capture and isolation of bacteria from the sample matrix followed by specifically detecting bacterial pathogens using SERS active nanoparticles functionalized with antibodies which are strain specific. Therefore, the sample is captured using a "magnetic plug" and interrogated with a laser allowing simple and fast optical detection. Three bacterial pathogens (Escherichia coli, Salmonella typhimurium, and methicillin-resistant Staphylococcus aureus) were successfully isolated and detected, with the lowest concentration for each of the strains detected at just 101 colony forming units per mL (CFU/mL). In addition to single pathogen detection, a mixture of all three bacterial strains was isolated and identified within the same sample matrix using SERS with the triplex detection also being confirmed using principal component analysis. Herein, we demonstrate that this multiplexed bionanosensor is capable of providing rapid and sensitive discrimination of bacterial pathogens both individually, and within a multiplex system, offering opportunities for future point of care devices and advancements in biomedical applications.

Surface-enhanced Raman scattering (SERS) generates molecularly specific fingerprints of analytes and when the experimental conditions are carefully controlled this is highly quantitative. This review critiques the development of quantitative SERS from simple univariate assessment of single vibrational modes to multivariate analysis of the whole spectrum for improved quantification. SERS has also been developed for direct multiplex detection and quantification of multiple analytes and this is also discussed, as is the need for LC-SERS for analyte separation should multivariate chemometric approaches fail to effect quantification. Finally, to effect absolute quantification with SERS, the concepts of isotopologues is introduced along with the standard addition method (SAM) and suitable examples that have been developed and exploit these techniques are presented. We believe that SERS will be routinely used for quantitative analysis and it is only a matter of time before this technique translates from the laboratory to the clinical environment.

Abstract Despite the clinical success of Androgen Receptor (AR)-targeted therapies, reactivation of AR signalling remains the main driver of castration-resistant prostate cancer (CRPC) progression. In this study, we perform a comprehensive unbiased characterisation of LNCaP cells chronically exposed to multiple AR inhibitors (ARI). Combined proteomics and metabolomics analyses implicate an acquired metabolic phenotype common in ARI-resistant cells and associated with perturbed glucose and lipid metabolism. To exploit this phenotype, we delineate a subset of proteins consistently associated with ARI resistance and highlight mitochondrial 2,4-dienoyl-CoA reductase (DECR1), an auxiliary enzyme of beta-oxidation, as a clinically relevant biomarker for CRPC. Mechanistically, DECR1 participates in redox homeostasis by controlling the balance between saturated and unsaturated phospholipids. DECR1 knockout induces ER stress and sensitises CRPC cells to ferroptosis. In vivo, DECR1 deletion impairs lipid metabolism and reduces CRPC tumour growth, emphasizing the importance of DECR1 in the development of treatment resistance.

Inspired by the role of intracellular liquid-liquid phase separation (LLPS) in formation of membraneless organelles, there is great interest in developing dynamic compartments formed by LLPS of intrinsically disordered proteins (IDPs) or short peptides. However, the molecular mechanisms underlying the formation of biomolecular condensates have not been fully elucidated, rendering on-demand design of synthetic condensates with tailored physico-chemical functionalities a significant challenge. To address this need, here we design a library of LLPS-promoting peptide building blocks composed of various assembly domains. We show that the LLPS propensity, dynamics, and encapsulation efficiency of compartments can be tuned by changes to the peptide composition. Specifically, with the aid of Raman and NMR spectroscopy, we show that interactions between arginine and aromatic amino acids underlie droplet formation, and that both intra- and intermolecular interactions dictate droplet dynamics. The resulting sequence-structure-function correlation could support the future development of compartments for a variety of applications.

Oligonucleotide-gold nanoparticle (OGN) conjugates are powerful tools for the detection of target DNA sequences due to the unique properties conferred upon the oligonucleotide by the nanoparticle. Practically all the research and applications of these conjugates have used gold nanoparticles to the exclusion of other noble metal nanoparticles. Here we report the synthesis of oligonucleotide-silver nanoparticle (OSN) conjugates and demonstrate their use in a sandwich assay format. The OSN conjugates have practically identical properties to their gold analogues and due to their vastly greater extinction coefficient both visual and absorption analyses can occur at much lower concentrations. This is the first report of OSN conjugates being successfully used for target DNA detection and offers improved sensitivity which is of interest to a range of scientists.

The labeling of biological species using dyes has become common practice to aid in their detection, and immediate positive identification of specific dyes in high dilution is a key requirement. Here the detection by surface-enhanced resonance Raman scattering (SERRS) of eight commercially available dye labels (ROX, rhodamine 6G, HEX, FAM, TET, Cy3, Cy5, TAMRA) attached to oligonucleotide strands is reported. Each of the eight labels was easily detected by using the SERRS from silver nanoparticles to produce a unique, molecularly specific spectrum. The conditions were optimized to obtain the best signal enhancement, and linear concentration graphs at low oligonucleotide concentrations were obtained. At higher concentrations (above approximately 10(-)(8) mol dm(-)(3)), curvature was introduced into the concentration graphs with the exception of rhodamine 6G, TET, and FAM, which gave linearity over the entire concentration range studied. Detection limits as low as 0.5 fmol were obtained, with lower possible if a smaller sample was analyzed. Investigation was also carried out into the effect of a Tris-HCl buffer containing the surfactant Tween 20 to aid in the prevention of surface adhesion of the oligonucleotides to the sample vessels at ultralow concentrations. The Tween 20 allowed lower detection limits to be obtained for each of the labels studied. This study shows that the different dyes commonly used with oligonucleotides can give quantitative SERRS at concentration levels not possible when the same dyes are used with fluorescence detection.

SESORS - Surface enhanced spatially offset Raman spectroscopy–imaging is explored for the first time in this study. Multiplexed surface enhanced Raman scattering (SERS) signals have been recovered non-invasively from a depth of 20 mm in tissues for the first time and reconstructed to produce a false colour image. Four unique 'flavours' of SERS nanoparticles (NPs) were injected into a 20 × 50 × 50 mm porcine tissue block at the corners of a 10 mm square. A transmission Raman data cube was acquired over an 11 × 11 pixel grid made up of 2 mm steps. The signals were reconstructed using the unique peak intensities of each of the nanoparticles. A false colour image of the relative signal levels was produced, demonstrating the capability of multiplexed imaging of SERS nanoparticles using deep Raman spectroscopy. A secondary but no less significant achievement was to demonstrate that Raman signals from SERS nanoparticles can be recovered non-invasively from samples of the order of 45–50 mm thick. This is a significant step forward in the ability to detect and identify vibrational fingerprints within tissue and offers the opportunity to adapt these particles and this approach into a clinical setting for disease diagnosis.

Abstract The exploration of the genetic information carried by DNA has become a major scientific challenge. Routine DNA analysis, such as PCR, still suffers from important intrinsic limitations. Surface‐enhanced Raman spectroscopy (SERS) has emerged as an outstanding opportunity for the development of DNA analysis, but its application to duplexes (dsDNA) has been largely hampered by reproducibility and/or sensitivity issues. A simple strategy is presented to perform ultrasensitive direct label‐free analysis of unmodified dsDNA with the means of SERS by using positively charged silver colloids. Electrostatic adhesion of DNA promotes nanoparticle aggregation into stable clusters yielding intense and reproducible SERS spectra at nanogram level. As potential applications, we report the quantitative recognition of hybridization events as well as the first examples of SERS recognition of single base mismatches and base methylations (5‐methylated cytosine and N6‐methylated Adenine) in duplexes.

SERRS is an extremely sensitive and selective technique which when applied to the detection of labelled DNA sequences allows detection limits to be obtained which rival, and in most cases are better than, fluorescence. In this tutorial review the conditions are explored which enable the successful detection of DNA using SERRS. The enhancing surface which is used is crucial and in this case suspensions of nanoparticles were the focus as they allow quantitative behaviour to be achieved in systems analogous to current fluorescence based approaches. The aggregation conditions required to obtain SERRS of DNA affect the sensitivity and the reproducibility and we describe the use of spermine as an effective aggregating agent to achieve excellent reproducibility and sensitivity. The nature of the label which is used, be it fluorescent or non-fluorescent, positively or negatively charged, also affects the SERRS response and these conditions are again discussed. Finally, we show how to detect a specific target DNA sequence in a meaningful diagnostic assay using SERRS and how the approaches described previously in the review are vital to the success of such approaches.

We report the use of a SERS based DNA detection assay for the multiplexed, quantification of three bacterial meningitis pathogens.

Dear SERRS: The quantitative detection of five labeled oligonucleotides by the title method (SERRS) without any separation is reported. The sensitivity of the multiplex analysis is the same as that for the individual dyes and indicates there is no compromise in the multiplexed format. Supporting information for this article is available on the WWW under http://www.wiley-vch.de/contents/jc_2002/2007/z604265_s.pdf or from the author. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.

Metallic nanoparticles of gold functionalized with oligonucleotides conventionally use a terminal thiol modification and have been used in a wide range of applications. Although readily available, the oligonucleotide–nanoparticle conjugates prepared in this way suffer from a lack of stability when exposed to a variety of small molecules or elevated temperatures. If silver is used in place of gold then this lack of stability is even more pronounced. In this study we report the synthesis of highly stabilized oligonucleotide–nanoparticle conjugates using a simple oligonucleotide modification. A modified solid support was used to generate 3′-thioctic acid modified oligonucleotides by treatment with an N -hydroxysuccimidyl ester of thioctic acid. Unusually, both gold and silver nanoparticles have been investigated in this study and show that these disulphide-modified oligonucleotide probes offer significant improvements in nanoparticle stability when treated with dithiothreitol (DTT) compared with monothiol analogues. This is a significant advance in oligonucleotide–nanoparticle conjugate stability and for the first time allows silver nanoparticles to be prepared that are more stable than standard gold-thiol functionalized nanoparticles. This opens up the possibility of using silver nanoparticles functionalized with oligonucleotides as an alternative to gold.

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Ohne Umweg: Spektroskopische Daten (siehe Bild) liefern direkte Belege, dass in nichtwässrigem Li+-Elektrolyt O2 zu O2− reduziert wird. Dieses bildet dann auf der Elektrodenoberfläche LiO2, das zu Li2O2 disproportioniert. Beim Ladevorgang zersetzt sich Li2O2 direkt in einer einstufigen Reaktion unter Bildung von O2, d. h., es wird keine LiO2-Zwischenstufe durchlaufen.

This letter discusses the potential of deep Raman spectroscopy, surface enhanced spatially offset Raman spectroscopy (SESORS and its variants), for noninvasively detecting small, deeply buried lesions using surface enhanced resonance Raman scattering (SERRS) active nanoparticles. An experimental demonstration of this concept is performed in transmission Raman geometry. This method opens prospects for in vivo, noninvasive, specific detection of molecular changes associated with disease up to depths of several centimeters representing significant improvement over traditionally detected Raman signals by 2 orders of magnitude. The disease specific signals can be achieved using uniquely tagged nanoparticles conjugated to target molecules, e.g., antibodies for production of the SERRS signal. This provides the molecular specific signal which is many orders of magnitude greater than normal biological Raman signals and can be easily multiplexed. To date, there have been no studies demonstrating the viability of deep Raman spectroscopy coupled to surface enhanced techniques for detecting low concentrations of molecules of interest at depths of greater than 5.5 mm in tissue. Such a breakthrough would open a host of new applications in medical diagnoses. Here we propose to facilitate such capability by combining SERRS (as a probe for disease specific changes) with deep Raman spectroscopy techniques. This permits noninvasive measurement of Raman signatures from conjugated SERRS nanoparticles at clinically relevant concentrations through tissues of between 15 and 25 mm thick.

Colorimetric detection of analytes using gold nanoparticles along with surface-enhanced Raman spectroscopy (SERS) are areas of intense research activity since they both offer sensing of very low concentrations of target species. Multimodal detection promotes the simultaneous detection of a sample by a combination of different techniques; consequently, surface chemistry design in the development of multimodal nanosensors is important for rapid and sensitive evaluation of the analytes by diverse analytical methods. Herein it is shown that nanoparticle size plays an important role in the design of functional nanoparticles for colorimetric and SERS-based sensing applications, allowing controlled nanoparticle assembly and tunable sensor response. The design and preparation of robust nanoparticle systems and their assembly is reported for trace detection of Ni(II) ions as a model system in an aqueous solution. The combination of covalently attached nitrilotriacetic acid moieties along with the L-carnosine dipeptide on the nanoparticle surface represents a highly sensitive platform for rapid and selective detection of Ni(II) ions. This systematic study demonstrates that significantly lower detection limits can be achieved by finely tuning the assembly of gold nanoparticles of different core sizes. The results clearly demonstrate the feasibility and usefulness of a multimodal approach.

Surface enhancement can provide improved detection sensitivity in a range of optical spectroscopies. When applied to bioanalysis these enhanced techniques allow for the detection of disease biomarkers at lower levels, which has a clear patient benefit. However, to achieve widespread clinical use of surface enhanced techniques there remain several “grand challenges”. In this review we consider the substrates employed to achieve enhancement before reviewing each enhanced optical technique in detail; surface plasmon resonance, localised surface plasmon resonance, surface enhanced fluorescence, surface enhanced infrared absorption spectroscopy and surface enhanced (resonance) Raman spectroscopy. Finally we set out the “grand challenges” currently facing the field.

The ability to detect chirality gives stereochemically attuned nanosensors the potential to revolutionize the study of biomolecular processes. Such devices may structurally characterize the mechanisms of protein-ligand binding, the intermediates of amyloidogenic diseases and the effects of phosphorylation and glycosylation. We demonstrate that single nanoparticle plasmonic reporters, or nanotags, can enable a stereochemical response to be transmitted from a chiral analyte to an achiral benzotriazole dye molecule in the vicinity of a plasmon resonance from an achiral metallic nanostructure. The transfer of chirality was verified by the measurement of mirror image surface enhanced resonance Raman optical activity spectra for the two enantiomers of both ribose and tryptophan. Computational modelling confirms these observations and reveals the novel chirality transfer mechanism responsible. This is the first report of colloidal metal nanoparticles in the form of single plasmonic substrates displaying an intrinsic chiral sensitivity once attached to a chiral molecule.

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Establishing a definitive diagnosis of pneumonia using conventional tests is difficult and expensive. Lateral flow immunoassays (LFIAs) are an advantageous point of care (POC) test option, but they have some limitations in terms of detection and quantification. In this work we have developed a lateral flow immunoassay for the ultrasensitive detection of penumolysin employing plasmonic Surface-Enhanced Resonance Raman Scattering (SERRS) tag as labelled probe. The combination of Au@Ag core–shell nanoparticles as plasmonic platform and Rhodamine B Isothiocyanate as Raman reporter has allowed us to fabricate a SERRS tag with high efficiency and reliability. The limit of detection of the SERRS-based LFIA was 1 pg mL−1. This could be a strong foundation for a pneumonia diagnosis test based on pneumolysin detection.

Since its discovery in 1974, surface-enhanced Raman scattering (SERS) has gained momentum as an important tool in analytical chemistry. SERS is used widely for analysis of biological samples, ranging from in vitro cell culture models, to ex vivo tissue and blood samples, and direct in vivo application. New insights have been gained into biochemistry, with an emphasis on biomolecule detection, from small molecules such as glucose and amino acids to larger biomolecules such as DNA, proteins, and lipids. These measurements have increased our understanding of biological systems, and significantly, they have improved diagnostic capabilities. SERS probes display unique advantages in their detection sensitivity and multiplexing capability. We highlight key considerations that are required when performing bioanalytical SERS measurements, including sample preparation, probe selection, instrumental configuration, and data analysis. Some of the key bioanalytical measurements enabled by SERS probes with application to in vitro, ex vivo, and in vivo biological environments are discussed.

Use of sensitive, non-destructive and straightforward 3D SERS for investigating the cellular uptake processes of functionalised nanotags in entire cell volume.

Carboxylesterases (CEs) are a class of enzymes that catalyze the hydrolysis of esters in a variety of endogenous and exogenous molecules. CEs play an important role in drug metabolism, in the onset and progression of disease, and can be harnessed for prodrug activation strategies. As such, the regulation of CEs is an important clinical and pharmaceutical consideration. Here, we report the first ratiometric sensor for CE activity using Raman spectroscopy based on a bisarylbutadiyne scaffold. The sensor was shown to be highly sensitive and specific for CE detection and had low cellular cytotoxicity. In hepatocyte cells, the ratiometric detection of esterase activity was possible, and the result was validated by multimodal imaging with standard viability stains used for fluorescence microscopy within the same cell population. In addition, we show that the detection of localized ultraviolet damage in a mixed cell population was possible using stimulated Raman scattering microscopy coupled with spectral phasor analysis. This sensor demonstrates the practical advantages of low molecular weight sensors that are detected using ratiometric Raman imaging and will have applications in drug discovery and biomedical research.

Metabolism of endothelial cells (ECs) depends on the availability of the energy substrates. Since the endothelium is the first line of defence against inflammation in the cardiovascular system and its dysfunction can lead to the development of cardiovascular diseases, it is important to understand how glucose metabolism changes during inflammation. In this work, glucose uptake was studied in human microvascular endothelial cells (HMEC-1) in high glucose (HG), and additionally in an inflammatory state, using Raman imaging. HG state was induced by incubation of ECs with a deuterated glucose analogue, while the EC inflammation was caused by TNF-α pre-treatment. Spontaneous and stimulated Raman scattering spectroscopy provided comprehensive information on biochemical changes, including lipids and the extent of unsaturation induced by excess glucose in ECs., induced by excess glucose in ECs. In this work, we indicated spectroscopic markers of metabolic changes in ECs as a strong increase in the ratio of the intensity of lipid / (protein + lipid) bands and an increase in the level of lipid unsaturation and mitochondrial changes. Inflamed ECs treated with HG, revealed enhanced glucose uptake, and intensified lipid production i.a. of unsaturated lipids. Additionally, increased cytochrome c signal in the mitochondrial region indicated higher mitochondrial activity and biogenesis. Raman spectroscopy is a powerful method for determining the metabolic markers of ED which will better inform understanding of disease onset, development, and treatment.

The effect of excitation frequency and state of aggregation on the sensitivity obtained in ultratrace analysis using colloidal suspensions of silver nanoparticles and surface-enhanced resonance Raman scattering (SERRS) detection is explored to define suitable conditions for quantitative analysis. Two structurally similar dyes, only one of which causes aggregation, were used as analytes without the use of external aggregating agents, thus simplifying the surface chemistry and removing a major source of error. Addition of the nonaggregating dye caused no change in particle charge or size and no time-dependent aggregation as measured by zeta potential and particle size analysis. The most intense single-particle scattering was obtained using excitation at the wavelength of the plasmon resonance. Molecular resonance added approximately 2 orders of magnitude in sensitivity. Addition of the aggregating dye caused a reduction in surface charge of the particles and initiated a time-dependent aggregation process. However, constant SERRS with time is obtained at some excitation wavelengths probably because a constant number of clusters active at these wavelengths is maintained in the dynamic aggregation process. The additional enhancement caused by aggregation and molecular resonance is spread over a range of excitation frequencies. However, electronic spectra suggested that plasmon resonance enhancement would be effective at the longest wavelength of excitation used (785 nm), but there was a significant drop in intensity this far away from the absorbance maximum of the dye (429 nm). Thus, sensitive analysis using suspensions of single nanoparticles is feasible provided the excitation frequency used is close to that of the plasmon resonance frequency. Aggregation adds only an enhancement of approximately 6 in the experiments performed since only some particles in aggregates will have an active plasmon at any one wavelength, but the range of excitation wavelengths at which good enhancement is obtained is wider giving more flexibility if more complexity.

Genetic research programs such as the human genome project have demonstrated the need for selective detection of nucleic acids at ultralow concentrations (McKusik, V. A. FASEB J. 1991, 5, 12−20. Cui, X.; et al. Proc. Natl. Acad. Sci. U.S.A. 1989, 89, 9389−9393). Current detection techniques require amplification of the genetic material by the polymerase chain reaction (PCR) and detection of the amplified product by fluorescence. A new reliable method for DNA detection has been developed based on adsorption of DNA on colloidal silver and subsequent signal detection using surface-enhanced resonance Raman scattering (SERRS). It uses specifically designed primers and can be applied directly to genetic analysis. Improved surface and aggregation chemistry, utilizing modified DNA and a novel aggregating agent, has resulted in acceptable reproducibility for biological detection. DNA was detected down to 8 × 10-13 M, which equates to less than one molecule in the interrogation volume at any one time, and an RSD of 10% was obtained. The increased sensitivity compared to fluorescence circumvents the need for an amplification step, and much greater selectivity is obtained due to the sharp vibrational spectra observed, thus reducing the need for separation procedures. The method is potentially sensitive enough to eliminate the need for time-consuming amplification steps and provide a new dimension to labeling chemistry and multiplex analysis of DNA.

Journal Article Synthesis and physical properties of anti -HIV antisense oligonucleotides bearing terminal lipophilic groups Get access Calum MacKellar, Calum MacKellar Search for other works by this author on: Oxford Academic PubMed Google Scholar Duncan Graham, Duncan Graham Search for other works by this author on: Oxford Academic PubMed Google Scholar David W. Will, David W. Will Search for other works by this author on: Oxford Academic PubMed Google Scholar Stephen Burgess, Stephen Burgess 1Institute of Cell, Animal and Population Biology, Division of Biological Sciences, University of EdinburghWest Mains Road, Edinburgh EH9 3JJ, UK Search for other works by this author on: Oxford Academic PubMed Google Scholar Tom Brown Tom Brown * * To whom correspondence should be addressed Search for other works by this author on: Oxford Academic PubMed Google Scholar Nucleic Acids Research, Volume 20, Issue 13, 11 July 1992, Pages 3411–3417, https://doi.org/10.1093/nar/20.13.3411 Published: 11 July 1992 Article history Received: 19 March 1992 Revision received: 05 June 1992 Accepted: 05 June 1992 Published: 11 July 1992

This communication contains data from a comparison between the detection limits obtained using surface enhanced resonance Raman scattering (SERRS) and fluorescence detection of dye labelled oligonucleotides. The results show that the detection limits for SERRS are generally at least three orders of magnitude lower than those obtained for fluorescence.

The accurate detection of DNA sequences is essential for a variety of post human genome projects including detection of specific gene variants for medical diagnostics and pharmacogenomics. A specific DNA sequence detection assay based on surface-enhanced resonance Raman scattering (SERRS) and an amplification refractory mutation system (ARMS) is reported. Initially, generation of PCR products was achieved by using specifically designed allele-specific SERRS active primers. Detection by SERRS of the PCR products confirmed the presence of the sequence tested for by the allele-specific oligonucleotides. This lead directly to the multiplex genotyping of human DNA samples for the deltaF508 mutational status of the cystic fibrosis transmembrane conductance regulator gene using SERRS active primers in an ARMS assay. Removal of the unincorporated primers allowed fast and accurate analysis of the three genotypes possible in this system in a multiplex format without any separation of amplicons. The results indicate that SERRS can be used in modern genetic analysis and offers an opportunity for the development of novel assays. This is the first demonstration of the use of SERRS in multiplex genotyping and shows potential advantages over fluorescence as a detection technique with considerable promise for future development.

Methods of detection of amphetamine sulfate using surface enhanced Raman scattering (SERS) from colloidal suspensions and vapour deposited films of both silver and gold are compared. Different aggregating agents are required to produce effective SERS from silver and gold colloidal suspensions. Gold colloid and vapour deposited gold films give weaker scattering than the equivalent silver substrates when high concentrations of drug are analysed but they also give lower detection limits, suggesting a smaller surface enhancement but stronger surface adsorption. A 10−5 mol dm−3 solution (the final concentration after addition of colloid was 10−6 mol dm−3) of amphetamine sulfate was detected from gold colloid with an RSD of 5.4%. 25 μl of the same solution could be detected on a roughened gold film. The intensities of the spectra varied across the film surface resulting in relatively high RSDs. The precision was improved by averaging the scattering from several points on the surface. An attempt to improve the detection limit and precision by concentrating a suspension of gold colloid and amphetamine sulfate in aluminium wells did not give effective quantitation. Thus, positive identification and semi-quantitative estimation of amphetamine sulfate can be made quickly and easily using SERS from suspended gold colloid with the appropriate aggregating agents.

Abstract Surface‐enhanced resonance Raman scattering (SERRS) from silver nanoparticles using 514.5‐nm excitation has been shown to offer huge potential for applications in highly sensitive multiplexed DNA assays. If the technique is to be applied to real biological samples and integrated with other methods, then the use of gold nanoparticles and longer wavelengths of excitation are desirable. The data presented here demonstrate that dye‐labeled oligonucleotide sequences can be directly detected by SERRS using gold nanoparticles in a quantitative manner for the first time. The performance of gold and silver nanoparticles as SERRS substrates was assessed using 514.5‐, 632.8‐, and 785‐nm excitation and a range of 13 commercially available dye‐labeled oligonucleotides. The quantitative response allowed the limit of detection to be determined for each case and demonstrates that the technique is highly effective, sensitive, and versatile. The possibility of excitation at multiple wavelengths further enhances the multiplexing potential of the technique. The importance of effectively combining the optical properties of the nanoparticle and the dye label is demonstrated. For example, at 632.8‐nm excitation, the dye BODIPY TR‐X and gold nanoparticles make a strong SERRS combination with very little background fluorescence. This study allows the choice of nanoparticle and dye label for particular experimental setups, and significantly expands the applicability of enhanced Raman scattering for use in many disciplines.

The labelling of target biomolecules followed by detection using some form of optical spectroscopy has become common practice to aid in their detection. This approach has allowed the field of bioanalysis to dramatically expand; however, most methods suffer from the lack of the ability to discriminate between the components of a complex mixture. Currently, fluorescence spectroscopy is the method of choice but its ability to multiplex is greatly hampered by the broad overlapping spectra which are obtained. Surface enhanced resonance Raman scattering (SERRS) holds many advantages over fluorescence both in sensitivity and, more importantly here, in its ability to identify components in a mixture without separation due to the sharp fingerprint spectra obtained. Here the first multiplexed simultaneous detection of six different DNA sequences, corresponding to different strains of the Escherichia coli bacterium, each labelled with a different commercially available dye label (ROX, HEX, FAM, TET, Cy3, or TAMRA) is reported. This was achieved with the aid of multivariate analysis, also known as chemometrics, which can involve the application of a wide range of statistical and data analysis methods. In this study, both exploratory discriminant analysis and supervised learning, by partial least squares (PLS) regression, were used and the ability to discriminate whether a particular labelled oligonucleotide was present or absent in a mixture was achieved using PLS with very high sensitivity (0.98–1), specificity (0.98–1), accuracy (range 0.99–1), and precision (0.98–1).

The solid-state structures of a series of azine or azo chromophore containing bipyridyl ligand complexes of Ag(I) salts have been determined by single-crystal X-ray diffraction. The supramolecular structures are dominated by one-dimensional chains formed through pyridyl–Ag–pyridyl bonding, but the packing of these chains through non-covalent intermolecular interactions is unpredictable. Ag⋯anion interactions are shown to be important, especially for nitrate and perchlorate species, but these may be supported or replaced by Ag⋯Ag, Ag⋯solvent, Ag⋯azine or Ag⋯ π contacts. The molecular structures of the ligands show little alteration on complex formation, except for the AgNO3 complex of N,N′-bis-pyridin-4-ylmethylene-hydrazine where the normally planar azine ligand adopts a twisted geometry.

Silver nanoparticles can be used to provide excellent surface enhanced resonance Raman scattering. Control of the surface chemistry and the use of appropriate protocols enables effective sensing of biomolecules.

Tumor necrosis factor α is an inflammatory cytokine which has been linked with many infectious and inflammatory diseases. Detection and quantification of this key biomarker is commonly achieved by use of an enzyme-linked immunosorbent assay (ELISA). This fundamental technique uses the spectroscopic detection of a chromogen such as 3,3′,5,5′-tetramethylbenzidine (TMB). Horseradish peroxidase (HRP), bound to the detection antibody, catalyzes the oxidation of TMB by hydrogen peroxide to generate colored products which may be measured spectrophotometrically. In this study we have used a conventional ELISA kit and shown that, by replacing the traditional colorimetric detection with resonance Raman spectroscopy, we can achieve 50 times lower detection limits and the potential for multiplexed analysis is increased. In this approach, the laser wavelength was tuned to be in resonance with an electronic transition of the oxidized TMB. The relative intensity of the enhanced Raman bands is proportional to the amount of TMB, thus providing a means of improved quantification. Furthermore, TMB is one of the most widely used chromogenic substrates for HRP-based detection and commercial ELISA test kits, indicating that this detection technique is applicable to a large number of target analytes.

Reproducible control of stem cell populations, regardless of their original source, is required for the true potential of these cells to be realised as medical therapies, cell biology research tools and in vitro assays. To date there is a lack of consistency in successful output when these cells are used in clinical trials and even simple in vitro experiments, due to cell and material variability. The successful combination of single chemistries in nanoarray format to control stem cell, or any cellular behaviour has not been previously reported. Here we report how homogenously nanopatterned chemically modified surfaces can be used to initiate a directed cellular response, particularly mesenchymal stem cell (MSC) differentiation, in a highly reproducible manner without the need for exogenous biological factors and heavily supplemented cell media. Successful acquisition of these data should lead to the optimisation of cell selective properties of materials, further enhancing the role of nanopatterned substrates in cell biology and regenerative medicine. The successful design and comparison of homogenously molecularly nanopatterned surfaces and their direct effect on human MSC adhesion and differentiation are reported in this paper. Planar gold surfaces were patterned by dip pen nanolithography (DPN) to produce arrays of nanodots with optimised fixed diameter of 70 nanometres separated by defined spacings, ranging from 140 to 1000 nm with terminal functionalities of simple chemistries including carboxyl, amino, methyl and hydroxyl. These nanopatterned surfaces exhibited unprecedented control of initial cell interactions and subsequent control of cell phenotype and offer significant potential for the future.

The implementation of Raman and surface enhanced Raman spectroscopy (SERS) for the detection of disease has increased in recent years. The reasons for their increased implementation have often been attributed to their well-known advantages, including the production of narrow spectral bands, which are characteristic of the molecular components present, their non-destructive method of analysis and the sensitivity and specificity which they can confer. This review analyses a range of diseases which can be detected by Raman or SERS, particularly those in vitro, ex vivo and in vivo. The sophistication of the investigated systems varied widely but the suitability of Raman and SERS for medical diagnostics and future implementation in a clinical environment is clearly demonstrated.

Improved positively charged nanoparticles are described to provide a simplified SERS substrate for DNA detection. Complete flocculation of the nanoparticles is prevented due to the controlled analyte induced aggregation. This provides a stable aggregation state which significantly extends the analysis window simplifying DNA detection by SERS.

This paper describes the first report of the combination of functionalised silver nanoparticles and silver-coated magnetic nanoparticles in a stable sandwich assay for DNA detection using SERS, providing robust multi-target recognition.

Guest editors Duncan Graham, Martin Moskovits and Zhong-Qun Tian introduce the surface and tip enhanced spectroscopies issue of <italic>Chemical Society Reviews</italic>.

Vascular immune-inflammatory responses play a crucial role in the progression and outcome of atherosclerosis. The ability to assess localized inflammation through detection of specific vascular inflammatory biomarkers would significantly improve cardiovascular risk assessment and management; however, no multi-parameter molecular imaging technologies have been established to date. Here, we report the targeted in vivo imaging of multiple vascular biomarkers using antibody-functionalized nanoparticles and surface-enhanced Raman scattering (SERS). Methods: A series of antibody-functionalized gold nanoprobes (BFNP) were designed containing unique Raman signals in order to detect intercellular adhesion molecule 1 (ICAM-1), vascular cell adhesion molecule 1 (VCAM-1) and P-selectin using SERS. Results: SERS and BFNP were utilized to detect, discriminate and quantify ICAM-1, VCAM-1 and P-selectin in vitro on human endothelial cells and ex vivo in human coronary arteries. Ultimately, non-invasive multiplex imaging of adhesion molecules in a humanized mouse model was demonstrated in vivo following intravenous injection of the nanoprobes. Conclusion: This study demonstrates that multiplexed SERS-based molecular imaging can indicate the status of vascular inflammation in vivo and gives promise for SERS as a clinical imaging technique for cardiovascular disease in the future.

Abstract The potential to bioprint and study 3D bacterial biofilm constructs could have great clinical significance at a time when antimicrobial resistance is rising to dangerously high levels worldwide. In this study, clinically relevant bacterial species including Escherichia coli, Staphylococcus aureus (MSSA), Methicillin-resistant Staphylococcus aureus (MRSA) and Pseudomonas aeruginosa were 3D bioprinted using a double-crosslinked alginate bioink to form mature bacteria biofilms, characterized by confocal laser scanning microscopy (CLSM) and fluorescent staining. Solid and porous bacteria-laden constructs were reproducibly bioprinted with thicknesses ranging from 0.25 to 4 mm. We demonstrated 3D bioprinting of thicker biofilms (&gt;4 mm) than found in currently available in vitro models. Bacterial viability was excellent in the bioprinted constructs, with CLSM observation of bacterial biofilm production and maturation possible for at least 28 d in culture. Importantly, we observed the complete five-step biofilm life cycle in vitro following 3D bioprinting for the first time, suggesting the formation of mature 3D bioprinted biofilms. Bacterial growth was faster in thinner, more porous constructs whilst constructs crosslinked with BaCl 2 concentrations of above 10 mM had denser biofilm formation. 3D MRSA and MSSA biofilm constructs were found to show greater resistance to antimicrobials than corresponding two-dimensional (2D) cultures. Thicker 3D E. coli biofilms had greater resistance to tetracycline than thinner constructs over 7 d of treatment. Our methodology allowed for the precise 3D bioprinting of self-supporting 3D bacterial biofilm structures that developed biofilms during extended culture. 3D biofilm constructs containing bacterial biofilms produce a model with much greater clinical relevance compared to 2D culture models and we have demonstrated their use in antimicrobial testing.

Military-grade explosives such as 2,4,6-trinitroluene (TNT) are still a major worldwide concern in terms of terror threat and environmental impact. The most common methods currently employed for the detection of explosives involve colorimetric tests, which are known to be rapid and portable; however, they often display false positives and lack sensitivity. Other methods used include ion mobility mass spectrometry, gas chromatography-mass spectrometry (GC-MS), and liquid chromatography-mass spectrometry (LC-MS), which despite producing more reliable results often require large, expensive instrumentation and specially trained staff. Here we demonstrate an alternative approach that utilizes the formation of a colored Janowsky complex with nitroaromatic explosives through reaction of the enolate ion of 3-mercapto-2-butanone. The colored complex is formed rapidly and can then be detected sensitively using surface-enhanced Raman scattering (SERS). We demonstrate that SERS can be used as a quick, sensitive, and selective technique for the detection of 2,4,6-trinitrotoluene (TNT), hexanitrostillbene (HNS), and 2,4,6-trinitrophenylmethylnitramine (tetryl) with a detection limit of 6.81 ng mL–1 achieved for TNT, 17.2 ng mL–1 for tetryl, and 135.1 ng mL–1 for HNS. This method of detection also requires minimal sample preparation, can be done in a solution-based format, and utilizes the same precursor reagents for complex formation with each of the explosives which can then be identified due to the specificity of the unique SERS response obtained. We demonstrate the ability to simultaneously identify three explosive compounds within a total analysis time of 10 min. This method of detection shows promise for the development of rapid and portable SERS-based assays which can be utilized in the field in order to achieve reliable and quantitative detection.

Surface-enhanced Raman scattering (SERS) is a powerful and sensitive technique for the detection of fingerprint signals of molecules and for the investigation of a series of surface chemical reactions. Many studies introduced quantitative applications of SERS in various fields, and several SERS methods have been implemented for each specific application, ranging in performance characteristics, analytes used, instruments, and analytical matrices. In general, very few methods have been validated according to international guidelines. As a consequence, the application of SERS in highly regulated environments is still considered risky, and the perception of a poorly reproducible and insufficiently robust analytical technique has persistently retarded its routine implementation. Collaborative trials are a type of interlaboratory study (ILS) frequently performed to ascertain the quality of a single analytical method. The idea of an ILS of quantification with SERS arose within the framework of Working Group 1 (WG1) of the EU COST Action BM1401 Raman4Clinics in an effort to overcome the problematic perception of quantitative SERS methods. Here, we report the first interlaboratory SERS study ever conducted, involving 15 laboratories and 44 researchers. In this study, we tried to define a methodology to assess the reproducibility and trueness of a quantitative SERS method and to compare different methods. In our opinion, this is a first important step toward a “standardization” process of SERS protocols, not proposed by a single laboratory but by a larger community.

Abstract Melanin pigments have various properties that are of technological interest including photo‐ and radiation protection, rich coloration, and electronic functions. Nevertheless, laboratory‐based synthesis of melanin and melanin‐like materials with morphologies and chemical structures that are specifically optimized for these applications, is currently not possible. Here, melanin‐like materials that are produced by enzymatic oxidation of a supramolecular tripeptide structures that are rich in tyrosine and have a 1D morphology are demonstrated, that are retained during the oxidation process while conducting tracks form through oxidative tyrosine crosslinking. Specifically, a minimalistic self‐assembling peptide, Lys–Tyr–Tyr (KYY) with strong propensity to form supramolecular fibers, is utilized. Analysis by Raman spectroscopy shows that the tyrosines are pre‐organized inside these fibers and, upon enzymatic oxidation, result in connected catechols. These form 1D conducting tracks along the length of the fiber, which gives rise to a level of internal disorder, but retention of the fiber morphology. This results in highly conductive structures demonstrated to be dominated by proton conduction. This work demonstrates the ability to control oxidation but retain a well‐defined fibrous morphology that does not have a known equivalent in biology, and demonstrate exceptional conductivity that is enhanced by enzymatic oxidation.

A paper-based microfluidics self-testing device capable of colorimetric and SERS-based sensing of cardiovascular disease associated miR-29a has been developed for improving patient care and triage.

Statins have displayed significant, although heterogeneous, anti-tumour activity in breast cancer disease progression and recurrence. They offer promise as a class of drugs, normally used for cardiovascular disease control, that could have a significant impact on the treatment of cancer. Understanding their mode of action and accurately assessing their efficacy on live cancer cells is an important and significant challenge. Stimulated Raman scattering (SRS) microscopy is a powerful, label-free imaging technique that can rapidly characterise the biochemical responses of live cell populations following drug treatment. Here, we demonstrate multi-wavelength SRS imaging together with spectral phasor analysis to characterise a panel of breast cancer cell lines (MCF-7, SK-BR-3 and MDA-MB-231 cells) treated with two clinically relevant statins, atorvastatin and rosuvastatin. Label-free SRS imaging within the high wavenumber region of the Raman spectrum (2800-3050 cm-1) revealed the lipid droplet distribution throughout populations of live breast cancer cells using biocompatible imaging conditions. A spectral phasor analysis of the hyperspectral dataset enables rapid differentiation of discrete cellular compartments based on their intrinsic SRS characteristics. Applying the spectral phasor method to studying statin treated cells identified a lipid accumulating phenotype in cell populations which displayed the lowest sensitivity to statin treatment, whilst a weaker lipid accumulating phenotype was associated with a potent reduction in cell viability. This study provides an insight into potential resistance mechanisms of specific cancer cells towards treatment with statins. Label-free SRS imaging provides a novel and innovative technique for phenotypic assessment of drug-induced effects across different cellular populations and enables effective analysis of drug-cell interactions at the subcellular scale.

Optical techniques are widely used tools in the visualisation of biological species within complex matrices, including biopsies, tissue resections and biofluids. Raman spectroscopy is an emerging analytical approach that probes the molecular signature of endogenous cellular biomolecules under biocompatible conditions with high spatial resolution. Applications of Raman spectroscopy in prostate cancer include biopsy analysis, assessment of surgical margins and monitoring of treatment efficacy. The advent of advanced Raman imaging techniques, such as stimulated Raman scattering, is creating opportunities for real-time in situ evaluation of prostate cancer. This review provides a focus on the recent preclinical and clinical achievements in implementing Raman-based techniques, highlighting remaining challenges for clinical applications. The research and clinical results achieved through in vivo and ex vivo Raman spectroscopy illustrate areas where these evolving technologies can be best translated into clinical practice.

Hyperspectral stimulated Raman scattering (SRS) microscopy is a powerful imaging modality for the analysis of biological systems. Here, we report the application of k-means cluster analysis (KMCA) of multi-wavelength SRS images in the high-wavenumber region of the Raman spectrum as a robust and reliable method for the segmentation of cellular organelles based on the intrinsic SRS spectrum. KMCA has been applied to the study of the endogenous lipid biochemistry of prostate cancer and prostate healthy cell models, while the corresponding SRS spectrum of the lipid droplet (LD) cluster enabled direct comparison of their composition. The application of KMCA in visualizing the LD content of prostate cell models following the inhibition of de novo lipid synthesis (DNL) using the acetyl-coA carboxylase inhibitor, 5-(tetradecyloxy)-2-furoic acid (TOFA), is demonstrated. This method identified a reliance of prostate cancer cell models upon DNL for metabolic requirements, with a significant reduction in the cellular LD content after treatment with TOFA, which was not observed in normal prostate cell models. SRS imaging combined with KMCA is a robust method for investigating drug-cell interactions in a label-free manner.

Surface-enhanced Raman scattering (SERS) is widely explored for the elucidation of underlying mechanisms behind biological processes. However, the capability of absolute quantitation of the number of nanoparticles from the SERS response remains a challenge. Here, we show for the first time the development of a new 2D quantitation model to allow calibration of the SERS response against the absolute concentration of SERS nanotags, as characterized by single particle inductively coupled plasma mass spectrometry (spICP-MS). A novel printing approach was adopted to prepare gelatin-based calibration standards containing the SERS nanotags, which consisted of gold nanoparticles and the Raman reporter 1,2-bis(4-pyridyl)ethylene. spICP-MS was used to characterize the Au mass concentration and particle number concentration of the SERS nanotags. Results from laser ablation inductively coupled plasma time-of-flight mass spectrometry imaging at a spatial resolution of 5 μm demonstrated a homogeneous distribution of the nanotags (between-line relative standard deviation < 14%) and a linear response of 197Au with increasing nanotag concentration (R2 = 0.99634) in the printed gelatin standards. The calibration standards were analyzed by SERS mapping, and different data processing approaches were evaluated. The reported calibration model was based on an "active-area" approach, classifying the pixels mapped as "active" or "inactive" and calibrating the SERS response against the total Au concentration and the particle number concentration, as characterized by spICP-MS. This novel calibration model demonstrates the potential for quantitative SERS imaging, with the capability of correlating the nanoparticle concentration to biological responses to further understand the underlying mechanisms of disease models.

Hyperspectral stimulated Raman scattering (SRS) microscopy coupled to spectral phasor analysis is a powerful method for the detection of fatty acids in solution and in cellular lipid droplets.

DNA analysis often requires a separation step; this step is avoided if surface-enhanced resonance Raman scattering (SERRS) is used. SERRS is shown to differentiate between two labeled oligonucleotide strands, in varying proportions, in a mixture, and at low concentration levels. The method is discriminatory, sensitive, and requires only commonly available equipment.

Melanoma-reactive HLA-A x 0201-restricted cytotoxic T lymphocyte (CTL) lines generated in vitro lyse autologous and HLA-matched allogeneic melanoma cells and recognize multiple shared peptide antigens from tyrosinase, MART-1, and Pme117/gp100. However, a subset of melanomas fail to be lysed by these T cells. In the present report, four different HLA-A x 0201+ melanoma cell lines not lysed by melanoma-reactive allogeneic CTL have been evaluated in detail. All four are deficient in expression of the melanocytic differentiation proteins (MDP) tyrosinase, Pme117/gp100, gp75/ trp-1, and MART-1/Melan-A. This concordant loss of multiple MDP explains their resistance to lysis by melanoma-reactive allogeneic CTL and confirms that a subset of melanomas may be resistant to tumor vaccines directed against multiple MDP-derived epitopes. All four melanoma lines expressed normal levels of HLAA x 0201, and all were susceptible to lysis by xenoreactive-peptide-dependent HLA-A x 0201-specific CTL clones, indicating that none had identifiable defects in antigen-processing pathways. Despite the lack of shared MDP-derived antigens, one of these MDP-negative melanomas, DM331, stimulated an effective autologous CTL response in vitro, which was restricted to autologous tumor reactivity. MHC-associated peptides isolated by immunoaffinity chromatography from HLA-A1 and HLA-A2 molecules of DM331 tumor cells included at least three peptide epitopes recognized by DM331 CTL and restricted by HLA-A1 or by HLA-A x 0201. Recognition of these CTL epitopes cannot be explained by defined, shared melanoma antigens; instead, unique or undefined antigens must be responsible for the autologous-cell-specific anti-melanoma response. These findings suggest that immunotherapy directed against shared melanoma antigens should be supplemented with immunotherapy directed against unique antigens or other undefined antigens, especially in patients whose tumors do not express MDP.

Surface enhanced resonance Raman scattering (SERRS) is an alternative to fluorescence for use in bioanalysis however due to the different optical mechanism it requires specifically designed reporters. Recently we have reported the use of 8-hydroxyquinolinyl azo dyes and their ester derivatives as reporters of lipase activity using SERRS. Acylation of the 8-hydroxy moiety significantly reduces surface enhancement of the Raman response and subsequent lipase catalysed ester hydrolysis enables the analyte to bind to silver nanoparticles, thus providing surface enhancement and the SERRS signal is ‘switched on’. By following this principle, phosphorylated and galactosylated analogues of 8-hydroxyquinolinylazo dyes were prepared and shown to act as reporters of enzymatic activity for alkaline phosphatase and β-galactosidase respectively when using SERRS.

Surface-enhanced Raman scattering (SERS) is shown to give linear and sensitive concentration-dependent detection of folic acid using silver nanoparticles created via ethylene-diaminetetraacetic acid (EDTA) reduction. Optical detection by SERS overcomes the primary limitation of photodissociation encountered during the application of other shorter wavelength ultraviolet (UV)/near-UV techniques such as fluorescence based microscopy. The SERS approach in water-based samples was demonstrated and optimized using several longer wavelengths of excitation (514.5, 632.8, and 785 nm). Excitation in the green (514.5 nm) was found to achieve the best balance between photodissociation and SERS efficiency. Linear concentration dependence was observed in the range of 0.018 to 1 microM. The importance of folic acid in a clinical setting and the potential applications of this technique in a biological environment are highlighted. We demonstrate the potential to transfer this technique to real biological samples by the detection of folic acid in human serum samples by SERS.

Shining light: Surface enhanced Raman scattering (SERS) offers much in terms of molecularly specific information at ultrasensitive levels. Two recent breakthroughs have occured in this field. First, a new surface was developed that provides SERS information previously inaccessible, and second, a nanoparticle SERS label was introduced to image antigens relating to cancer in tissue.

Detection of specific DNA sequences is central to modern molecular biology and also to molecular diagnostics where identification of a particular disease is based on nucleic acid identification. Many methods exist, and fluorescence spectroscopy dominates the detection technologies employed with different assay formats. This study demonstrates the use of surface-enhanced resonance Raman scattering (SERRS) to detect specific DNA sequences when coupled with modified SERRS-active probes that have been designed to modify the affinity of double- and single-stranded DNA for the surface of silver nanoparticles resulting in discernible differences in the SERRS which can be correlated to the specific DNA hybridization event. The principle of the assay lies on the lack of affinity of double-stranded DNA for silver nanoparticle surfaces; therefore, hybridization of the probe to the target results in a reduction in the SERRS signal. Use of locked nucleic acid (LNA) residues in the DNA probes resulted in greater discrimination between exact match and mismatches when used in comparison to unmodified labeled DNA probes. Polymerase chain reaction (PCR) products were detected using this methodology, and ultimately a multiplex detection of sequences relating to a hospital-acquired infection, namely, methicillin-resistant Staphylococcus aureus (MRSA), demonstrated the versatility and applicability of this approach to real-life situations.

Surface enhanced raman scattering (SERS) based molecular diagnostic assays for the detection of specific DNA sequences have been developed in recent years to compete with the more common fluorescence based approaches. Current SERS assays either require time-consuming separation steps that increase assay cost and can also increase the risk of contamination or they are negative assays, where the signal intensity decreases in the presence of target DNA. Herein, we report a new separation free SERS assay with an increase in signal intensity when target DNA is present using a specifically designed SERS primer. The presence of specific bacterial DNA from Staphylococcus epidermidis was detected using polymerase chain reaction (PCR) and SERS and indicates a new opportunity for exploration of SERS assays requiring minimal handling steps.

The detection of inflammatory changes is a key aim for the early diagnosis and treatment of several autoimmune, infectious, and metastatic diseases. While surface-enhanced Raman scattering (SERS) has the capability to provide noninvasive, in vivo imaging at sufficient depth to achieve this goal, this approach has not been exploited in the study of inflammation. SERS-active nanoparticles were coded with a unique Raman signal that was protected under a wide range of conditions and stimuli. To detect early-stage inflammation, gold nanoparticle clusters containing Raman-active molecules were conjugated to intercellular adhesion molecule 1- (ICAM-1-) specific monoclonal antibodies. SERS allowed noninvasive measurement of ICAM-1 expression in vivo with twice the sensitivity of two-photon fluorescence. This is the first time SERS has been used for in vivo detection of inflammation and is a major advance in the ever-growing toolkit of approaches for use in noninvasive, next-generation in vivo imaging.

Recent studies have clearly demonstrated that Raman and surface enhanced Raman scattering (SERS) spectroscopies are information rich, non-destructive techniques for the monitoring of subtle intracellular changes. However, despite the demonstrated and sophisticated applications of these techniques in cell studies there still remains a lack of accompanying 3D images. Herein we demonstrate for the first time combined 3D Raman and SERS imaging for the simultaneous confirmation of the cellular inclusion and multiple component detection of SERS nanotags. We also report on the 3D elucidation of the cell nuclei by multivariate analysis methods. Imaging in 3D will be critical to understanding architectural changes between diseased and healthy cells, and tissues. It will also provide non-destructive definitive proof of cellular uptake whilst simultaneously confirming targeting of SERS nanotags to their intended destinations.

The increasing popularity of surface plasmon resonance (SPR) and surface enhanced Raman scattering (SERS) sensor design based on nanotriangle or nanohole arrays, and the possibility to manufacture substrates at the transition between these plasmonic substrates, makes them ideal candidates for the establishment of structure-property relationships. This work features near diffraction-limited Raman images and FDTD simulations of nanotriangle and nanohole arrays substrates, which clearly demonstrate that the localization of the hot spot on these SERS substrates is significantly influenced by the ratio of diameter/periodicity (D/P). The experimental and simulation data reveal that the hot spots are located around nanotriangles (D/P = 1), characteristic of localized SPR. Decreasing the D/P ratio to 0.75-0.7 led to the creation of nanohole arrays, which promoted the excitation of a propagating surface plasmon (SP) delocalized over the metal network. The optimal SERS intensity was consistently achieved at this transition from nanotriangles to nanoholes, for every periodicity (650 nm to 1.5 μm) and excitation wavelength (633 and 785 nm) investigated, despite the presence or absence of a plasmonic band near the laser excitation. Further decreasing the D/P ratio led to excitation of a localized SP located around the rim of nanohole arrays for D/P of 0.5-0.6, in agreement with previous reports. In addition, this manuscript provides the first evidence that the hot spots are positioned inside the hole for D/P of 0.4, with the center being the region of highest electric field and Raman intensity. The compelling experimental evidence and FDTD simulations offer an overall understanding of the plasmonic properties of nanohole arrays as SERS and SPR sensors, which is of significant value in advancing the diversity of applications from such surfaces.

The understanding of the relationship between plasmonic and surface-enhanced Raman scattering (SERS) properties of dynamic nanoparticle assemblies is of paramount importance for the optimal design of related plasmonic nanostructures, especially for SERS applications. In this regard, recent studies have provided new important insights for well-ordered nanoparticle assemblies but little is known about the relationship between the physical and optical properties for large ensembles of randomly aggregated metal nanoparticles in suspension, which still represents the simplest and most common route to obtain highly effective SERS substrates. Here we exploit the triplex-assembling ability of DNA-conjugated silver nanoparticles to engineer interparticle junctions with controlled interparticle distance and tune the aggregation rate to allow accurate investigation into the correlation between the averaged time-dependent plasmonic and SERS responses within a complex ensemble of nanoparticles in suspension. Solution-based single particle tracking was used to characterize the heterogeneity of the nanoparticle assembly with statistical reliability, acting as a key tool to unravel the connection between these two bulk responses. To achieve this, we report the first example of the parallel hybridization of dye-labeled locked nucleic acid (LNA) silver nanoparticle probes to double stranded DNA bridges of different lengths to form a triplex assembly, that provides SERS enhancements directly related to the interparticle distance imposed by the high structural rigidity of the double stranded linker. This is also a crucial step towards utilising SERS for the study of DNA in its natural double stranded state and, ultimately, to obtain nanoscale distance-dependent information in challenging biological environments using specially designed nanoparticles.

Abstract The exploration of the genetic information carried by DNA has become a major scientific challenge. Routine DNA analysis, such as PCR, still suffers from important intrinsic limitations. Surface‐enhanced Raman spectroscopy (SERS) has emerged as an outstanding opportunity for the development of DNA analysis, but its application to duplexes (dsDNA) has been largely hampered by reproducibility and/or sensitivity issues. A simple strategy is presented to perform ultrasensitive direct label‐free analysis of unmodified dsDNA with the means of SERS by using positively charged silver colloids. Electrostatic adhesion of DNA promotes nanoparticle aggregation into stable clusters yielding intense and reproducible SERS spectra at nanogram level. As potential applications, we report the quantitative recognition of hybridization events as well as the first examples of SERS recognition of single base mismatches and base methylations (5‐methylated cytosine and N6‐methylated Adenine) in duplexes.

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.

Artificial enzymes have become an increasingly interesting area of research due to their many advantages over natural protein enzymes which are expensive, difficult to isolate and unable to stand harsh environments.

In order to improve patient survival and reduce the amount of unnecessary and traumatic biopsies, non-invasive detection of cancerous tumours is of imperative and urgent need. Multicellular tumour spheroids (MTS) can be used as an ex vivo cancer tumour model, to model in vivo nanoparticle (NP) uptake by the enhanced permeability and retention (EPR) effect. Surface enhanced spatially offset Raman spectroscopy (SESORS) combines both surface enhanced Raman spectroscopy (SERS) and spatially offset Raman spectroscopy (SORS) to yield enhanced Raman signals at much greater sub-surface levels. By utilizing a reporter that has an electronic transition in resonance with the laser frequency, surface enhanced resonance Raman scattering (SERRS) yields even greater enhancement in Raman signal. Using a handheld SORS spectrometer with back scattering optics, we demonstrate the detection of live breast cancer 3D MTS containing SERRS active NPs through 15 mm of porcine tissue. False color 2D heat intensity maps were used to determine tumour model location. In addition, we demonstrate the tracking of SERRS-active NPs through porcine tissue to depths of up to 25 mm. This unprecedented performance is due to the use of red-shifted chalcogenpyrylium-based Raman reporters to demonstrate the novel technique of surface enhanced spatially offset resonance Raman spectroscopy (SESORRS) for the first time. Our results demonstrate a significant step forward in the ability to detect vibrational fingerprints from a tumour model at depth through tissue. Such an approach offers significant promise for the translation of NPs into clinical applications for non-invasive disease diagnostics based on this new chemical principle of measurement.

The integration of surface-enhanced Raman scattering (SERS) with droplet microfluidics has the potential to improve our understanding of cellular systems. Herein, we present the first application of SERS droplet microfluidics for single-cell analysis. A microfluidic device was used to encapsulate single prostate cancer cells and wheat germ agglutin (WGA)-functionalized SERS nanoprobes in water-in-oil droplets that were subsequently locked into a storage droplet array for spectroscopic investigation. The stationary droplets enabled the rapid identification of SERS regions of interest in live cancer cells by allowing collection of “fast” coarse maps over an area of several square millimeters followed by “slower” detailed interrogation of the identified hotspots. We demonstrate SERS at cellular resolution via a proof-of-concept assay that detects glycan expression on the surface of prostate cancer cells using WGA-modified metallic nanoparticles. The data illustrates the potential of SERS optofluidic systems for high-throughput cell screening and illustrates a previously unobserved high degree of cell-to-cell variability in the size and number of glycan islands.

Raman spectroscopy has been used extensively for the analysis of biological samples in vitro , ex vivo and in vivo . While important progress has been made towards using this analytical technique in clinical applications, there is a limit to how much chemically specific information can be extracted from a spectrum of a biological sample, which consists of multiple overlapping peaks from a large number of species in any particular sample. In an attempt to elucidate more specific information regarding individual biochemical species, as opposed to very broad assignments by species class, we propose a bottom-up approach beginning with a detailed analysis of pure biochemical components. Here, we demonstrate a simple ratiometric approach applied to fatty acids, a subsection of the lipid class, to allow the key structural features, in particular degree of saturation and chain length, to be predicted. This is proposed as a starting point for allowing more chemically and species-specific information to be elucidated from the highly multiplexed spectrum of multiple overlapping signals found in a real biological sample. The power of simple ratiometric analysis is also demonstrated by comparing the prediction of degree of unsaturation in food oil samples using ratiometric and multivariate analysis techniques which could be used for food oil authentication.

Unraveling the role played by the surface chemistry of silver colloids in the direct SERS analysis of DNA.

Lectin-functionalized silver nanoparticles have been successfully designed for use as molecular imaging agents to investigate carbohydrate–lectin interactions at the surface of mammalian cells, using surface-enhanced Raman scattering (SERS). Carbohydrate-lectin interactions are key to many cellular processes and are responsible for controlling an array of cellular interactions. In this study, lectin-functionalized silver nanoparticles were used to detect the expression of carbohydrate species at the cellular interface. The carbohydrate–lectin interactions were demonstrated using three different lectin species for three distinct cell types. Due to the known difference between the expressions of glycans in cancerous versus noncancerous cells of the same origin, this approach has been expanded to study both cancerous and noncancerous prostate cells. This has been achieved via confocal SERS mapping of the expression of the key glycan, sialic acid, on the surface of each of these cell types. In achieving such discrimination, a novel method has been created by which glycan expression can be reproducibly monitored. Comparative studies were performed using both fluorescence and SERS. SERS provided an increased discrimination over fluorescence when analyzing cell subsets to discriminate between cancerous and noncancerous cells. The success of this method means that it could be used to complement the current gold standard histopathological techniques.

Surface-enhanced, spatially offset Raman spectroscopy (SESORS) combines the remarkable enhancements in sensitivity afforded by surface-enhanced Raman spectroscopy (SERS) with the non-invasive, subsurface sampling capabilities of spatially offset Raman spectroscopy. Taken together, these techniques show great promise for in vivo Raman measurements. Herein, we present a step forward for this technique, demonstrating SESORS through tissue analogues of six known and varied thicknesses, with a large number of distinct spatial offsets, in a backscattering optical geometry. This is accomplished by spin-coating SERS-active nanoparticles (NPs) on glass slides and monitoring the relative spectral contribution from the NPs and tissue sections, respectively, as a function of both the tissue thickness and the spatial offset of the collection probe. The results show that SESORS outperforms SERS alone for this purpose, the NP signal can be attained at tissue thicknesses of >6.75 mm, and greater tissue thicknesses require greater spatial offsets to maximize the NP signal, all with an optical geometry optimized for utility. This demonstration represents a step forward toward the implementation of SESORS for non-invasive, in vivo analysis.

Breast cancer is one of the leading causes of cancer death in women. Novel in vitro tools that integrate three-dimensional (3D) tumor models with highly sensitive chemical reporters can provide useful information to aid biological characterization of cancer phenotype and understanding of drug activity. The combination of surface-enhanced Raman scattering (SERS) techniques with microfluidic technologies offers new opportunities for highly selective, specific, and multiplexed nanoparticle-based assays. Here, we explored the use of functionalized nanoparticles for the detection of estrogen receptor alpha (ERα) expression in a 3D tumor model, using the ERα-positive human breast cancer cell line MCF-7. This approach was used to compare targeted versus nontargeted nanoparticle interactions with the tumor model to better understand whether targeted nanotags are required to efficiently target ERα. Mixtures of targeted anti-ERα antibody-functionalized nanotags (ERα-AuNPs) and nontargeted (against ERα) anti-human epidermal growth factor receptor 2 (HER2) antibody-functionalized nanotags (HER2-AuNPs), with different Raman reporters with a similar SERS signal intensity, were incubated with MCF-7 spheroids in microfluidic devices and spectroscopically analyzed using SERS. MCF-7 cells express high levels of ERα and no detectable levels of HER2. 2D and 3D SERS measurements confirmed the strong targeting effect of ERα-AuNP nanotags to the MCF-7 spheroids in contrast to HER2-AuNPs (63% signal reduction). Moreover, 3D SERS measurements confirmed the differentiation between the targeted and the nontargeted nanotags. Finally, we demonstrated how nanotag uptake by MCF-7 spheroids was affected by the drug fulvestrant, the first-in-class approved selective estrogen receptor degrader (SERD). These results illustrate the potential of using SERS and microfluidics as a powerful in vitro platform for the characterization of 3D tumor models and the investigation of SERD activity.

Duplex SERS-based lateral flow testing for <italic>C. diff</italic> bacterial infection using new biomarker, SlpA, and ToxB within 20 minutes.

Abstract Supramolecular self‐assembly in biological systems holds promise to convert and amplify disease‐specific signals to physical or mechanical signals that can direct cell fate. However, it remains challenging to design physiologically stable self‐assembling systems that demonstrate tunable and predictable behavior. Here, the use of zwitterionic tetrapeptide modalities to direct nanoparticle assembly under physiological conditions is reported. The self‐assembly of gold nanoparticles can be activated by enzymatic unveiling of surface‐bound zwitterionic tetrapeptides through matrix metalloprotease‐9 (MMP‐9), which is overexpressed by cancer cells. This robust nanoparticle assembly is achieved by multivalent, self‐complementary interactions of the zwitterionic tetrapeptides. In cancer cells that overexpress MMP‐9, the nanoparticle assembly process occurs near the cell membrane and causes size‐induced selection of cellular uptake mechanism, resulting in diminished cell growth. The enzyme responsiveness, and therefore, indirectly, the uptake route of the system can be programmed by customizing the peptide sequence: a simple inversion of the two amino acids at the cleavage site completely inactivates the enzyme responsiveness, self‐assembly, and consequently changes the endocytic pathway. This robust self‐complementary, zwitterionic peptide design demonstrates the use of enzyme‐activated electrostatic side‐chain patterns as powerful and customizable peptide modalities to program nanoparticle self‐assembly and alter cellular response in biological context.

The rapid detection of biomolecules in a point of care (POC) setting is very important for diagnostic purposes. A platform which can provide this, whilst still being low cost and simple to use, is paper-based lateral flow immunoassays (LFIA). LFIA combine immunology and chromatography to detect a target by forming an immunocomplex with a label which traps them in a test zone. Qualitative analysis can be performed using the naked eye whilst quantitative analysis takes place by measuring the optical signal provided by the label at the test zone. There are numerous detection methods available; however, many suffer from low sensitivity and lack of multiplexing capabilities or are poor at providing POC quantitative analysis. An attractive method to overcome this is to use nanoparticles coated in Raman reporters as the labelled species and to analyse test zones using surface-enhanced Raman scattering (SERS). Due to the wide variety of metal nanoparticles, Raman reporter and laser excitations that are available, SERS-based LFIA have been adapted to identify and quantify multiple targets at once. Large Raman microscopes combined with long mapping times have limited the platform to the lab; however, by transferring the analysis to portable Raman instruments, rapid and quantitative measurements can be taken at the POC without any loss in sensitivity. Portable or handheld SERS-LFIA platforms can therefore be used anywhere, from modern clinics to remote and resource-poor settings. This review will present an overview of SERS-based LFIA platforms and the major recent advancements in multiplexing and portable and handheld detection with an outlook on the future of the platform.

Antibiotic resistant bacteria constitute a global health threat. It is essential for healthcare professionals to prescribe the correct dose of an effective antibiotic to mitigate the bacterial infection in a timely manner to improve the therapeutic outcomes to the patient and prevent the dissemination of antibiotic resistance. To achieve this, there is a need to implement a rapid and ultra-sensitive clinical diagnosis to identify resistant bacterial strains and monitor the effect of antibiotics. In this review, we highlight the use of surface enhanced Raman scattering (SERS) as a powerful diagnostic technique for bacterial detection and evaluation. Initially, this is viewed through a lens covering why SERS can surpass other traditional techniques for bacterial diagnosis. This is followed by different SERS substrates design, detection strategies that have been used for various bacterial biomarkers, how SERS can be combined with other diagnostic platforms to improve its performance towards the bacterial detection and the application of SERS for antibiotic resistance diagnosis. Finally, the recent progress in SERS detection methods in the last decade for the "Big 5" antibiotic resistant challenges as demonstrators of public health major threats is reviewed, namely: Methicillin-resistant Staphylococcus aureus (MRSA), Carbapenem-resistant Enterobacteriaceae (CRE)/Extended-spectrum beta-lactamases (ESBLs), Mycobacterium tuberculosis (TB), Vancomycin-resistant Enterococcus (VRE) and Neisseria Gonorrhoea (NG). This review provides a comprehensive view of the current state of the art with regard to using SERS for assessing antibiotic resistance with a future outlook on where the field go head in the coming years.

Article11 January 2022Open Access Source DataTransparent process THEM6-mediated reprogramming of lipid metabolism supports treatment resistance in prostate cancer Arnaud Blomme Corresponding Author Arnaud Blomme [email protected] orcid.org/0000-0003-4183-8726 CRUK Beatson Institute, Garscube Estate, Glasgow, UK Contribution: Conceptualization, Data curation, Formal analysis, ​Investigation, Methodology, Writing - original draft, Project administration, Writing - review & editing Search for more papers by this author Coralie Peter Coralie Peter CRUK Beatson Institute, Garscube Estate, Glasgow, UK Contribution: ​Investigation Search for more papers by this author Ernest Mui Ernest Mui Institute of Cancer Sciences, University of Glasgow, Garscube Estate, Glasgow, UK Contribution: ​Investigation Search for more papers by this author Giovanny Rodriguez Blanco Giovanny Rodriguez Blanco CRUK Beatson Institute, Garscube Estate, Glasgow, UK Contribution: Formal analysis, Methodology Search for more papers by this author Ning An Ning An Laboratory of Cancer Signaling, GIGA-Institute, University of Liège, Liège, Belgium Contribution: ​Investigation Search for more papers by this author Louise M Mason Louise M Mason School of Engineering, University of Glasgow, Glasgow, UK Contribution: ​Investigation, Methodology Search for more papers by this author Lauren E Jamieson Lauren E Jamieson Centre for Molecular Nanometrology, Department of Pure and Applied Chemistry, Technology and Innovation Centre, University of Strathclyde, Glasgow, UK Contribution: Formal analysis, Methodology Search for more papers by this author Grace H McGregor Grace H McGregor CRUK Beatson Institute, Garscube Estate, Glasgow, UK Institute of Cancer Sciences, University of Glasgow, Garscube Estate, Glasgow, UK Contribution: Formal analysis, Methodology Search for more papers by this author Sergio Lilla Sergio Lilla CRUK Beatson Institute, Garscube Estate, Glasgow, UK Contribution: Formal analysis, ​Investigation, Methodology Search for more papers by this author Chara Ntala Chara Ntala CRUK Beatson Institute, Garscube Estate, Glasgow, UK Institute of Cancer Sciences, University of Glasgow, Garscube Estate, Glasgow, UK Contribution: Formal analysis Search for more papers by this author Rachana Patel Rachana Patel CRUK Beatson Institute, Garscube Estate, Glasgow, UK Contribution: Resources, ​Investigation, Methodology Search for more papers by this author Marc Thiry Marc Thiry orcid.org/0000-0002-4944-584X GIGA-Neurosciences, Unit of Cell and Tissue Biology, University of Liège, Liège, Belgium Contribution: Formal analysis, ​Investigation, Methodology Search for more papers by this author Sonia H Y Kung Sonia H Y Kung Department of Urologic Sciences, Faculty of Medicine, University of British Columbia, Vancouver, BC, Canada Vancouver Prostate Centre, Vancouver, BC, Canada Search for more papers by this author Marine Leclercq Marine Leclercq orcid.org/0000-0002-3974-0060 Laboratory of Cancer Signaling, GIGA-Institute, University of Liège, Liège, Belgium Contribution: Data curation, Formal analysis Search for more papers by this author Catriona A Ford Catriona A Ford CRUK Beatson Institute, Garscube Estate, Glasgow, UK Contribution: ​Investigation Search for more papers by this author Linda K Rushworth Linda K Rushworth CRUK Beatson Institute, Garscube Estate, Glasgow, UK Institute of Cancer Sciences, University of Glasgow, Garscube Estate, Glasgow, UK Contribution: ​Investigation Search for more papers by this author David J McGarry David J McGarry CRUK Beatson Institute, Garscube Estate, Glasgow, UK Contribution: ​Investigation Search for more papers by this author Susan Mason Susan Mason CRUK Beatson Institute, Garscube Estate, Glasgow, UK Contribution: Methodology Search for more papers by this author Peter Repiscak Peter Repiscak CRUK Beatson Institute, Garscube Estate, Glasgow, UK Contribution: Data curation, Formal analysis Search for more papers by this author Colin Nixon Colin Nixon orcid.org/0000-0002-8085-2160 CRUK Beatson Institute, Garscube Estate, Glasgow, UK Contribution: Methodology Search for more papers by this author Mark J Salji Mark J Salji orcid.org/0000-0003-4261-3086 Institute of Cancer Sciences, University of Glasgow, Garscube Estate, Glasgow, UK Contribution: Resources Search for more papers by this author Elke Markert Elke Markert Institute of Cancer Sciences, University of Glasgow, Garscube Estate, Glasgow, UK Contribution: Data curation, Formal analysis Search for more papers by this author Gillian M MacKay Gillian M MacKay CRUK Beatson Institute, Garscube Estate, Glasgow, UK Contribution: Methodology Search for more papers by this author Jurre J Kamphorst Jurre J Kamphorst CRUK Beatson Institute, Garscube Estate, Glasgow, UK Institute of Cancer Sciences, University of Glasgow, Garscube Estate, Glasgow, UK Contribution: Supervision Search for more papers by this author Duncan Graham Duncan Graham orcid.org/0000-0002-6079-2105 Centre for Molecular Nanometrology, Department of Pure and Applied Chemistry, Technology and Innovation Centre, University of Strathclyde, Glasgow, UK Contribution: Supervision, Methodology Search for more papers by this author Karen Faulds Karen Faulds Centre for Molecular Nanometrology, Department of Pure and Applied Chemistry, Technology and Innovation Centre, University of Strathclyde, Glasgow, UK Contribution: Supervision, Methodology Search for more papers by this author Ladan Fazli Ladan Fazli Department of Urologic Sciences, Faculty of Medicine, University of British Columbia, Vancouver, BC, Canada Vancouver Prostate Centre, Vancouver, BC, Canada Contribution: Resources, Formal analysis Search for more papers by this author Martin E Gleave Martin E Gleave orcid.org/0000-0003-4235-0167 Department of Urologic Sciences, Faculty of Medicine, University of British Columbia, Vancouver, BC, Canada Vancouver Prostate Centre, Vancouver, BC, Canada Contribution: Resources Search for more papers by this author Edward Avezov Edward Avezov orcid.org/0000-0002-2894-0585 UK Dementia Research Institute at University of Cambridge, Department of Clinical Neurosciences, University of Cambridge, Cambridge, UK Contribution: Resources Search for more papers by this author Joanne Edwards Joanne Edwards orcid.org/0000-0002-7192-6906 Institute of Cancer Sciences, University of Glasgow, Garscube Estate, Glasgow, UK Contribution: Resources, Formal analysis Search for more papers by this author Huabing Yin Huabing Yin School of Engineering, University of Glasgow, Glasgow, UK Contribution: Supervision, Methodology Search for more papers by this author David Sumpton David Sumpton orcid.org/0000-0002-9004-4079 CRUK Beatson Institute, Garscube Estate, Glasgow, UK Contribution: Formal analysis, ​Investigation, Methodology Search for more papers by this author Karen Blyth Karen Blyth CRUK Beatson Institute, Garscube Estate, Glasgow, UK Institute of Cancer Sciences, University of Glasgow, Garscube Estate, Glasgow, UK Contribution: Resources, Supervision Search for more papers by this author Pierre Close Pierre Close Laboratory of Cancer Signaling, GIGA-Institute, University of Liège, Liège, Belgium Contribution: Supervision Search for more papers by this author Daniel J Murphy Daniel J Murphy orcid.org/0000-0002-5538-5468 CRUK Beatson Institute, Garscube Estate, Glasgow, UK Institute of Cancer Sciences, University of Glasgow, Garscube Estate, Glasgow, UK Contribution: Resources, Supervision Search for more papers by this author Sara Zanivan Sara Zanivan orcid.org/0000-0002-9880-9099 CRUK Beatson Institute, Garscube Estate, Glasgow, UK Institute of Cancer Sciences, University of Glasgow, Garscube Estate, Glasgow, UK Contribution: Supervision, Methodology Search for more papers by this author Hing Y Leung Corresponding Author Hing Y Leung [email protected] orcid.org/0000-0002-3933-3975 CRUK Beatson Institute, Garscube Estate, Glasgow, UK Institute of Cancer Sciences, University of Glasgow, Garscube Estate, Glasgow, UK Contribution: Conceptualization, Funding acquisition, Project administration, Writing - review & editing Search for more papers by this author Arnaud Blomme Corresponding Author Arnaud Blomme [email protected] orcid.org/0000-0003-4183-8726 CRUK Beatson Institute, Garscube Estate, Glasgow, UK Contribution: Conceptualization, Data curation, Formal analysis, ​Investigation, Methodology, Writing - original draft, Project administration, Writing - review & editing Search for more papers by this author Coralie Peter Coralie Peter CRUK Beatson Institute, Garscube Estate, Glasgow, UK Contribution: ​Investigation Search for more papers by this author Ernest Mui Ernest Mui Institute of Cancer Sciences, University of Glasgow, Garscube Estate, Glasgow, UK Contribution: ​Investigation Search for more papers by this author Giovanny Rodriguez Blanco Giovanny Rodriguez Blanco CRUK Beatson Institute, Garscube Estate, Glasgow, UK Contribution: Formal analysis, Methodology Search for more papers by this author Ning An Ning An Laboratory of Cancer Signaling, GIGA-Institute, University of Liège, Liège, Belgium Contribution: ​Investigation Search for more papers by this author Louise M Mason Louise M Mason School of Engineering, University of Glasgow, Glasgow, UK Contribution: ​Investigation, Methodology Search for more papers by this author Lauren E Jamieson Lauren E Jamieson Centre for Molecular Nanometrology, Department of Pure and Applied Chemistry, Technology and Innovation Centre, University of Strathclyde, Glasgow, UK Contribution: Formal analysis, Methodology Search for more papers by this author Grace H McGregor Grace H McGregor CRUK Beatson Institute, Garscube Estate, Glasgow, UK Institute of Cancer Sciences, University of Glasgow, Garscube Estate, Glasgow, UK Contribution: Formal analysis, Methodology Search for more papers by this author Sergio Lilla Sergio Lilla CRUK Beatson Institute, Garscube Estate, Glasgow, UK Contribution: Formal analysis, ​Investigation, Methodology Search for more papers by this author Chara Ntala Chara Ntala CRUK Beatson Institute, Garscube Estate, Glasgow, UK Institute of Cancer Sciences, University of Glasgow, Garscube Estate, Glasgow, UK Contribution: Formal analysis Search for more papers by this author Rachana Patel Rachana Patel CRUK Beatson Institute, Garscube Estate, Glasgow, UK Contribution: Resources, ​Investigation, Methodology Search for more papers by this author Marc Thiry Marc Thiry orcid.org/0000-0002-4944-584X GIGA-Neurosciences, Unit of Cell and Tissue Biology, University of Liège, Liège, Belgium Contribution: Formal analysis, ​Investigation, Methodology Search for more papers by this author Sonia H Y Kung Sonia H Y Kung Department of Urologic Sciences, Faculty of Medicine, University of British Columbia, Vancouver, BC, Canada Vancouver Prostate Centre, Vancouver, BC, Canada Search for more papers by this author Marine Leclercq Marine Leclercq orcid.org/0000-0002-3974-0060 Laboratory of Cancer Signaling, GIGA-Institute, University of Liège, Liège, Belgium Contribution: Data curation, Formal analysis Search for more papers by this author Catriona A Ford Catriona A Ford CRUK Beatson Institute, Garscube Estate, Glasgow, UK Contribution: ​Investigation Search for more papers by this author Linda K Rushworth Linda K Rushworth CRUK Beatson Institute, Garscube Estate, Glasgow, UK Institute of Cancer Sciences, University of Glasgow, Garscube Estate, Glasgow, UK Contribution: ​Investigation Search for more papers by this author David J McGarry David J McGarry CRUK Beatson Institute, Garscube Estate, Glasgow, UK Contribution: ​Investigation Search for more papers by this author Susan Mason Susan Mason CRUK Beatson Institute, Garscube Estate, Glasgow, UK Contribution: Methodology Search for more papers by this author Peter Repiscak Peter Repiscak CRUK Beatson Institute, Garscube Estate, Glasgow, UK Contribution: Data curation, Formal analysis Search for more papers by this author Colin Nixon Colin Nixon orcid.org/0000-0002-8085-2160 CRUK Beatson Institute, Garscube Estate, Glasgow, UK Contribution: Methodology Search for more papers by this author Mark J Salji Mark J Salji orcid.org/0000-0003-4261-3086 Institute of Cancer Sciences, University of Glasgow, Garscube Estate, Glasgow, UK Contribution: Resources Search for more papers by this author Elke Markert Elke Markert Institute of Cancer Sciences, University of Glasgow, Garscube Estate, Glasgow, UK Contribution: Data curation, Formal analysis Search for more papers by this author Gillian M MacKay Gillian M MacKay CRUK Beatson Institute, Garscube Estate, Glasgow, UK Contribution: Methodology Search for more papers by this author Jurre J Kamphorst Jurre J Kamphorst CRUK Beatson Institute, Garscube Estate, Glasgow, UK Institute of Cancer Sciences, University of Glasgow, Garscube Estate, Glasgow, UK Contribution: Supervision Search for more papers by this author Duncan Graham Duncan Graham orcid.org/0000-0002-6079-2105 Centre for Molecular Nanometrology, Department of Pure and Applied Chemistry, Technology and Innovation Centre, University of Strathclyde, Glasgow, UK Contribution: Supervision, Methodology Search for more papers by this author Karen Faulds Karen Faulds Centre for Molecular Nanometrology, Department of Pure and Applied Chemistry, Technology and Innovation Centre, University of Strathclyde, Glasgow, UK Contribution: Supervision, Methodology Search for more papers by this author Ladan Fazli Ladan Fazli Department of Urologic Sciences, Faculty of Medicine, University of British Columbia, Vancouver, BC, Canada Vancouver Prostate Centre, Vancouver, BC, Canada Contribution: Resources, Formal analysis Search for more papers by this author Martin E Gleave Martin E Gleave orcid.org/0000-0003-4235-0167 Department of Urologic Sciences, Faculty of Medicine, University of British Columbia, Vancouver, BC, Canada Vancouver Prostate Centre, Vancouver, BC, Canada Contribution: Resources Search for more papers by this author Edward Avezov Edward Avezov orcid.org/0000-0002-2894-0585 UK Dementia Research Institute at University of Cambridge, Department of Clinical Neurosciences, University of Cambridge, Cambridge, UK Contribution: Resources Search for more papers by this author Joanne Edwards Joanne Edwards orcid.org/0000-0002-7192-6906 Institute of Cancer Sciences, University of Glasgow, Garscube Estate, Glasgow, UK Contribution: Resources, Formal analysis Search for more papers by this author Huabing Yin Huabing Yin School of Engineering, University of Glasgow, Glasgow, UK Contribution: Supervision, Methodology Search for more papers by this author David Sumpton David Sumpton orcid.org/0000-0002-9004-4079 CRUK Beatson Institute, Garscube Estate, Glasgow, UK Contribution: Formal analysis, ​Investigation, Methodology Search for more papers by this author Karen Blyth Karen Blyth CRUK Beatson Institute, Garscube Estate, Glasgow, UK Institute of Cancer Sciences, University of Glasgow, Garscube Estate, Glasgow, UK Contribution: Resources, Supervision Search for more papers by this author Pierre Close Pierre Close Laboratory of Cancer Signaling, GIGA-Institute, University of Liège, Liège, Belgium Contribution: Supervision Search for more papers by this author Daniel J Murphy Daniel J Murphy orcid.org/0000-0002-5538-5468 CRUK Beatson Institute, Garscube Estate, Glasgow, UK Institute of Cancer Sciences, University of Glasgow, Garscube Estate, Glasgow, UK Contribution: Resources, Supervision Search for more papers by this author Sara Zanivan Sara Zanivan orcid.org/0000-0002-9880-9099 CRUK Beatson Institute, Garscube Estate, Glasgow, UK Institute of Cancer Sciences, University of Glasgow, Garscube Estate, Glasgow, UK Contribution: Supervision, Methodology Search for more papers by this author Hing Y Leung Corresponding Author Hing Y Leung [email protected] orcid.org/0000-0002-3933-3975 CRUK Beatson Institute, Garscube Estate, Glasgow, UK Institute of Cancer Sciences, University of Glasgow, Garscube Estate, Glasgow, UK Contribution: Conceptualization, Funding acquisition, Project administration, Writing - review & editing Search for more papers by this author Author Information Arnaud Blomme *,1, Coralie Peter1, Ernest Mui2, Giovanny Rodriguez Blanco1, Ning An3, Louise M Mason4, Lauren E Jamieson5, Grace H McGregor1,2, Sergio Lilla1, Chara Ntala1,2, Rachana Patel1, Marc Thiry6, Sonia H Y Kung7,8, Marine Leclercq3, Catriona A Ford1, Linda K Rushworth1,2, David J McGarry1, Susan Mason1, Peter Repiscak1, Colin Nixon1, Mark J Salji2, Elke Markert2, Gillian M MacKay1, Jurre J Kamphorst1,2, Duncan Graham5, Karen Faulds5, Ladan Fazli7,8, Martin E Gleave7,8, Edward Avezov9, Joanne Edwards2, Huabing Yin4, David Sumpton1, Karen Blyth1,2, Pierre Close3, Daniel J Murphy1,2, Sara Zanivan1,2 and Hing Y Leung *,1,2 1CRUK Beatson Institute, Garscube Estate, Glasgow, UK 2Institute of Cancer Sciences, University of Glasgow, Garscube Estate, Glasgow, UK 3Laboratory of Cancer Signaling, GIGA-Institute, University of Liège, Liège, Belgium 4School of Engineering, University of Glasgow, Glasgow, UK 5Centre for Molecular Nanometrology, Department of Pure and Applied Chemistry, Technology and Innovation Centre, University of Strathclyde, Glasgow, UK 6GIGA-Neurosciences, Unit of Cell and Tissue Biology, University of Liège, Liège, Belgium 7Department of Urologic Sciences, Faculty of Medicine, University of British Columbia, Vancouver, BC, Canada 8Vancouver Prostate Centre, Vancouver, BC, Canada 9UK Dementia Research Institute at University of Cambridge, Department of Clinical Neurosciences, University of Cambridge, Cambridge, UK *Corresponding author. Tel: +32 4 366 2548; E-mail: [email protected] *Corresponding author. Tel: +44 141 330 3658; E-mail: [email protected] EMBO Mol Med (2022)14:e14764https://doi.org/10.15252/emmm.202114764 See also: M Chattopadhyay & D Germain (March 2022) PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Despite the clinical benefit of androgen-deprivation therapy (ADT), the majority of patients with advanced prostate cancer (PCa) ultimately develop lethal castration-resistant prostate cancer (CRPC). In this study, we identified thioesterase superfamily member 6 (THEM6) as a marker of ADT resistance in PCa. THEM6 deletion reduces in vivo tumour growth and restores castration sensitivity in orthograft models of CRPC. Mechanistically, we show that the ER membrane-associated protein THEM6 regulates intracellular levels of ether lipids and is essential to trigger the induction of the ER stress response (UPR). Consequently, THEM6 loss in CRPC cells significantly alters ER function, reducing de novo sterol biosynthesis and preventing lipid-mediated activation of ATF4. Finally, we demonstrate that high THEM6 expression is associated with poor survival and correlates with high levels of UPR activation in PCa patients. Altogether, our results highlight THEM6 as a novel driver of therapy resistance in PCa as well as a promising target for the treatment of CRPC. Synopsis Resistance to androgen-deprivation therapy (ADT) leads to the development of lethal castration-resistant prostate cancer. This study highlights THEM6 as a clinically relevant protein that supports ADT-induced rewiring of lipid metabolism and sustains UPR activation, thus promoting ADT resistance. THEM6 is overexpressed in ADT-resistant tumours and supports PCa tumour growth in androgen-deprived conditions. Loss of THEM6 significantly alters the cellular lipidome of CRPC cells, resulting in decreased levels of ether lipids. THEM6 is an ER-membrane protein that physically interacts with several components of the protein trafficking machinery. Loss of THEM6 significantly affects ER function, reducing de novo sterol synthesis and impairing lipid-mediated activation of the UPR, two processes that promote ADT resistance. High THEM6 expression in tumour biopsies is associated with shortened patient survival and correlates with sustained UPR activation, thus highlighting the potential of THEM6 as a therapeutic target in CRPC. The paper explained Problem Androgen-deprivation therapy (ADT) is the standard of care for patients with non-resectable prostate cancer (PCa). Despite initial clinical benefit, patients ultimately relapse and develop lethal castration-resistant prostate cancer (CRPC). Results We identified the uncharacterised protein THEM6 as a protein associated with ADT resistance in several in vitro and in vivo models of CRPC. Functionally, THEM6 loss impaired tumour growth and increased tumour response to ADT in PCa orthografts. Mechanistically, THEM6 was located at the membrane of the endoplasmic reticulum (ER) and primarily regulated ether lipid metabolism. Consequently, THEM6 expression was required to maintain sterol biosynthesis and to sustain activation of the ER stress response (UPR), two ER-related processes that promote ADT resistance. Finally, we demonstrated that THEM6 levels were elevated in treatment-resistant patient biopsies, associated with unfavourable patient outcome and correlated with sustained UPR activation in PCa patients. Impact The results highlight THEM6 as a clinically relevant marker of ADT resistance in prostate cancer. Because of its role in the regulation of lipid metabolism and ER biology, THEM6 represents a potential novel therapeutic target for the treatment of CRPC. Introduction Androgen-deprivation therapy (ADT) and androgen receptor (AR)-targeted therapies have significantly improved outcomes for patients suffering from advanced prostate cancer (PCa). However, treatment resistance ultimately leads to the development of lethal castration-resistant prostate cancer (CRPC), which remains a major therapeutic challenge. Resistance to treatment is accompanied by a plethora of cellular and metabolic adaptations that allow cancer cells to cope with stress-inducing factors (Marine et al, 2020). Together with mitochondria, the endoplasmic reticulum (ER) plays a central role in the regulation of stress-signalling pathways. Indeed, the ER is critical for the establishment of a complex stress response, termed the unfolded protein response (UPR), that orchestrates the cellular adaptation to various perturbations such as impairment of protein or lipid homeostasis. Activation of the UPR relies on the coordinated action of three major branches, each of them characterised by the activity of a specific ER stress sensor: Inositol Requiring Enzyme 1 (IRE1α), PRKR-like Endoplasmic Reticulum Kinase (PERK) and Activating Transcription Factor 6 (ATF6). In cancer, correct induction of the UPR is required to support oncogenic transformation (Hart et al, 2012). Persistent activation of ER stress responses can further promote tumour progression, metastasis dissemination and resistance to therapy (Chen & Cubillos-Ruiz, 2021). Therefore, clinical targeting of the UPR, alone or in combination with other treatment modalities, is regarded as a promising strategy for the treatment of aggressive cancer (Logue et al, 2018; Xie et al, 2018; Zhao et al, 2018; Jin & Saatcioglu, 2020). This strategy is particularly effective in the context of PCa, where inhibition of the IRE1a branch of the UPR significantly impairs tumour growth and activation of c-MYC signalling (Sheng et al, 2019), an important feature of ADT resistance (Bernard et al, 2003). Similarly, targeting of the PERK-eIF2a-ATF4 branch of the UPR effectively reduces tumour progression and metastasis dissemination in preclinical models of CRPC (Nguyen et al, 2018), while ATF4 signalling is essential for PCa growth and survival (Pallmann et al, 2019). The ER is also the primary site of lipid and cholesterol biosynthesis. Changes in lipid homeostasis, such as impaired membrane lipid saturation (Pineau et al, 2009) or imbalance in the levels of phospho- and sphingolipid species (Han et al, 2010; Thibault et al, 2012), can also contribute to ER stress. Recently, we and others demonstrated the importance of lipid remodelling in tumour resistance to antiandrogen therapy (Blomme et al, 2020; Tousignant et al, 2020). Thus, targeting lipid-mediated ER stress could be considered as a potential therapeutic option for the treatment of CRPC. Acyl-CoA thioesterases (ACOTs) are a class of enzymes that hydrolyse acyl-CoA molecules. In contrast with type I enzymes, type II ACOTs are related by structure rather than sequence, as evidenced by their low levels of sequence similarity (Cohen, 2013). By contrast, type II ACOTs are characterised by the presence of an evolutionarily conserved domain, the “Hotdog” domain, which confers the thioesterase enzymatic activity (Pidugu et al, 2009). Type II ACOTs also include members of the Thioesterase Superfamily (THEM), which primarily function to deactivate fatty acyl-CoA thioesters and generate free fatty acids (FA) and CoA. THEM proteins are involved in the regulation of intracellular FA trafficking and have been shown to influence both lipogenesis and beta-oxidation depending on the physiological context (Tillander et al, 2017). Interestingly, overexpression of thioesterase superfamily member 6 (THEM6/c8orf55) has been reported in several cancer types in a proteomic study focusing on the identification of colon cancer biomarkers (Kume et al, 2014). However, the biological role of THEM6 in normal and pathological physiology remains unexplored. In this study, we identified THEM6 as a clinically relevant protein associated with ADT resistance in PCa. Mechanistically, we show that THEM6 maintains lipid homeostasis by controlling intracellular levels of ether lipids. Consequently, THEM6 expression is critical for ER membrane integrity, sterol biosynthesis and ATF4 activation under ADT conditions. Importantly, high THEM6 expression is frequently observed in CRPC patient biopsies, correlates with high levels of UPR activation and is associated with shortened patient survival. Taken together, our results highlight the potential of THEM6 as a future therapeutic option for the treatment of CRPC. Results THEM6 expression is increased upon ADT resistance We previously performed an in-depth proteomic analysis comparing in vivo hormone-naïve (HN) and castration-resistant (CRPC) orthograft models of PCa (Martinez et al, 2021). For this purpose, we injected matched pairs of isogenic human PCa cell lines (namely 22rv1/CWR22res and LNCaP AI/LNCaP) into the prostate of immuno-deficient mice to generate tumour orthografts. Importantly, CRPC cells (22rv1 and LNCaP AI) were routinely cultured in absence of androgens and subsequently injected into castrated mice, while the matched HN cells (CWR22res and LNCaP) were cultured in androgen-containing medium and further injected into intact mice. We then sought to identify the proteins that were commonly regulated following ADT in the different models. From this comparison, we identified THEM6/c8orf55 as a protein significantly upregulated in CRPC tumours (22rv1 and LNCaP AI) when compared to HN counterparts (CWR22res and LNCaP, respectively; Figs 1A and EV1A). Increased THEM6 levels in CRPC conditions were validated in vivo (Fig 1B and C) and in vitro (Fig EV1B). Of note, THEM6 expression was the lowest in normal prostate epithelial cells (RWPE-1) when compared with multiple PCa cell lines (Fig EV1B). Immunohistochemistry (IHC) staining of prostate orthografts confirmed THEM6 over-expression in CRPC tumours and highlighted strong cytoplasmic and perinuclear staining in tumour epithelial cells (Fig 1C). Figure 1. Loss of THEM6 impairs PCa tumour growth following ADT Volcano plot of the differentially modulated proteins in 22rv1 (CRPC) versus CWR22res (HN) tumours. Red and blue dots represent the proteins that are significantly up- and down-regulated, respectively (P-value < 0.05, FC = 1.5). Western blot analysis of THEM6 expression in CRPC (22rv1 and LNCaP AI) and HN (CWR22res and LNCaP) prostate orthografts. VCL was used as a sample loading control. IHC staining of THEM6 expression in CWR22res and 22rv1 orthografts. Scale bar represents 100 µm. Western blot analysis of THEM6 expression in CTL and THEM6 KO CRPC cells. HSC70 was used as a sample loading control. Proliferation of CTL and THEM6 KO CRPC cells after 72 h. Data are expressed as a percentage of CTL cells. Cell stiffness (Young's modulus) of CTL and THEM6 KO CRPC cells measured by atomic force microscopy. Tumour volume (measured by ultrasound) of CTL and THEM6 KO 22rv1-derived orthografts developed in surgically castrated mice. Tumour volume of CTL and THEM6 KO LNCaP AI-derived xenografts developed in surgically castrated mice. Tumour volume (measured by ultrasound) of CTL and THEM6 KO CWR22res-derived orthografts. Orchidectomy was performed 3 weeks after cell implantation. Data information: Panels (E, G, H, I) Data are presented as mean values ± SD. Panel (F) Centre line corresponds to median of data, top and bottom lines correspond to maximal and minimal values. Statistical analysis: (E, F) One-way ANOVA with a Dunnett's multiple comparisons test. (G, H) two-tailed Mann–Whitney U-test. (I) Kruskal–Wallis test. Data reproducibility: Panel (A) n = 3 tumours per group. Panel (B) n = 1 gel loaded with three prostate orthografts per condition. Panels (C, D) representative image from 3 independent biological experiments. Panel (E) n = 3 independent biological experiments. Panel (F) (up): n = 46 (CTL); 47 (KO1); 49 (KO2) cells measured. Panel (F) (down): n = 31 (CTL); 4

A relatively new approach to subcellular research is Raman microscopy with the application of sensors called Raman probes. This paper describes the use of the sensitive and specific Raman probe, 3-O-propargyl-d-glucose (3-OPG), to track metabolic changes in endothelial cells (ECs). ECs play a significant role in a healthy and dysfunctional state, the latter is correlated with a range of lifestyle diseases, particularly with cardiovascular disorders. The metabolism and glucose uptake may reflect the physiopathological conditions and cell activity correlated with energy utilization. To study metabolic changes at the subcellular level the glucose analogue, 3-OPG was used, which shows a characteristic and intense Raman band at 2124 cm-1.3-OPG was applied as a sensor to track both, its accumulation in live and fixed ECs and then metabolism in normal and inflamed ECs, by employing two spectroscopic techniques, i.e. spontaneous and stimulated Raman scattering microscopies. The results indicate that 3-OPG is a sensitive sensor to follow glucose metabolism, manifested by the Raman band of 1602 cm-1. The 1602 cm-1 band has been called the "Raman spectroscopic signature of life" in the cell literature, and here we demonstrate that it is attributed to glucose metabolites. Additionally, we have shown that glucose metabolism and its uptake are slowed down in the cellular inflammation. We showed that Raman spectroscopy can be classified as metabolomics, and its uniqueness lies in the fact that it allows the analysis of the processes of a single living cell. Gaining further knowledge on metabolic changes in the endothelium, especially in pathological conditions, may help in identifying markers of cellular dysfunction, and more broadly in cell phenotyping, better understanding of the mechanism of disease development and searching for new treatments.

The demonstration that quantitative and sensitive analysis can be carried out using surface enhanced resonance Raman scattering (SERRS) prompted a discussion and investigation of the main variables which are within the control of the analyst using colloidal silver as the substrate. Previous papers have dealt with the crucial need to obtain good chemisorption of the analyte to the surface and have reported the use of specially designed dyes for SERRS. One of the most variable processes is the aggregation of the colloid. Here, we investigate the addition of controlled amounts of an organic aggregating agent, poly-L-lysine, at concentrations which reduce the zeta potential in a controlled manner, thus aiding aggregation control. The relationship between the excitation frequency, the surface plasmon resonance frequency of the silver colloid and the frequency of the maximum absorbance of the molecular chromophore is studied using low concentrations of dye and no aggregating agent. Under these conditions, little to no aggregation is expected. The magnitude of the enhancement is strongly dependent on the frequency of the molecular chromophore as well as the plasmon resonance frequency. However, when sodium chloride is used to aggregate the colloid, a larger enhancement is obtained and the strong dependence on the molecular chromophore largely disappears. A much broader enhancement profile is obtained which appears to be related more to the specific enhancement processes caused by aggregation than the frequency of the chromophore. However, the total enhancement for SERRS is higher than for SERS thus indicating that the chromophore is still important to the process.

Beads labelled using surface enhanced resonance Raman scattering (SERRS) are highly sensitive and specific tags, with potential applications in biological assays, including molecular diagnostics. The beads consist of a nucleus containing dye labelled silver-nanoparticle aggregates surrounded by a polymer core. The nuclei generate strong SERRS signals. To illustrate the coding advantage created by the sharp, molecularly specific SERRS signals, four specially designed SERRS dyes have been used as labels and three of these have been combined in a multiplex analysis. These dyes use specific groups such as benzotriazole and 8-hydroxyquinoline to improve binding to the surface of the silver particles. The aggregation state of the particles is held constant by the polymer core, this nucleus also contains many dye labels, yielding a very high Raman scattering intensity for each bead. To functionalise these beads for use in biological assays an outer polymer shell can be added, which allows the attachment of oligonucleotide probes. Oligonucleotide modified beads can then be used for detection of specific oligonucleotide targets. The specificity of SERRS will allow for the detection of multiple targets within a single assay.

There is a continued interest in the development of new on-chip protocols for the determination of the causes of infectious disease. In this paper, we demonstrate the use of surface-enhanced resonance Raman scattering (SERRS) for detecting the clinically relevant nucleic acid sequences of Chlamydia trachomatis in a bead-based lab-on-a-chip format, incorporating a solid-phase sample clean-up on-chip. The assay uses streptavidinated polymer microspheres to capture a biotinylated PCR product of the oligonucleotide sequence, which was subsequently hybridized against a complementary rhodamine-labeled, Raman active probe. Central to the assay is an in-channel integrated microfilter, which was used to retain the microspheres, enabling the bound target to be separated from the rest of the sample as part of a solid-phase clean-up (thereby removing any contributions from the background). After washing, the bound Rhodamine labeled detection probe was released thermally from the microspheres by heating and was subsequently mixed on-chip with a stream of silver nanoparticles. The signal was detected downstream using a Raman spectrometer to collect the SERRS response. The assay offers several advantages over traditional laboratory methods, including: the speed of the assay on-chip, the potential for sample clean-up; and the low volume of sample required.

A comparison is made of the quantitative detection of a labeled antibody by surface-enhanced resonance Raman scattering (SERRS) and by fluorescence using the same instrument with the same laser excitation source. The area under the curve for the fluorescence band is greater than for any single peak in the SERRS spectrum, but the broad fluorescence band is more difficult to discriminate from the background at low concentrations. Using the peak height of one SERRS band and the peak height at the fluorescence maximum, the detection limit for SERRS was lower (1.19 × 10-11 mol·dm-3) than that obtained using fluorescence (3.46 × 10-10 mol·dm-3). The SERRS detection limit is calculated for the concentration of the sample added, but compared to fluorescence, there is an additional dilution step due to the addition of the colloid and the extent of this dilution is dependent on assay format. For comparison with the detection limits found earlier with labeled oligonucleotides, SERRS was remeasured with a 10 s accumulation time, and the final concentration in the cuvette after colloid addition and before any adsorption to the silver was used to calculate a detection limit of 2.79 × 10-13 mol·dm-3. This is comparable to the detection limit found using a similar SERRS procedure for an oligonucleotide labeled with the same dye. This experiment is dependent on many parameters that could affect this result, including the nature of the SERRS substrate, the excitation wavelength, and the dye chosen. However, the result indicates that SERRS can give assay sensitivities comparable or better than fluorescence for quantitative direct assay determination, suggesting that the much greater potential for multiple analyte detection could be exploited.

The ability to accurately and sensitively measure the activity of specific enzymes is central to many aspects of modern chemistry and when combined with new nanoscience based approaches, offers significant opportunities for advancing other scientific disciplines. We review the development of surface enhanced resonance Raman scattering (SERRS) for the detection of enzymes, from the initial direct spectroscopy of enzymes, substrate/product and inhibitors adsorbed onto metallic colloids, to the current approach of measuring enzymatic activity by recording the SERRS spectra of a product which is only 'switched on' after enzyme activity. Developments focused on improvements to modular masked SERRS substrates, which are unmasked by specific enzymes, are also reviewed. Finally, we set out the remaining grand challenges within the area of enzymatic analysis by SERRS which include single molecule detection, in vivo studies and increased multiplexing for screening of evolved enzyme libraries.

We report the first use of a commonly used ELISA colorimetric substrate as a SERRS marker and show how it can be used for the detection of pg ml−1 levels of human prostate specific antigen (PSA) in clinical samples. The technique is amenable over a wide range of concentrations and lends itself to future multiplexing analysis.

Optical analysis in the near infrared region is of significant biological importance due to better tissue penetration and reduced autofluorescence. In this work, an improved synthesis of hollow gold nanospheres (HGNs), which provides a tunable localized surface plasmon resonance (LSPR) from 610 nm up to 1320 nm, is demonstrated. The scattering properties of these nanoparticles are shown using surface enhanced Raman scattering (SERS) at 1064 nm excitation wavelength and are compared to citrate reduced gold and silver nanoparticles of similar physical sizes and surface properties. After the addition of salts, a strong signal was observed from hollow gold with a LSPR of 650 nm and a weaker, yet observable, signal from HGNs with a LSPR of 775 nm. However, no obvious signals were observed in the case of standard citrate reduced gold, silver or HGNs with a LSPR of 1080 nm. The absorption properties of HGNs were investigated by monitoring their photothermal activity. In this case, different nanoparticle suspensions including citrate reduced gold, silver, and HGNs were illuminated by a continuous laser at 785 nm excitation wavelength and the absorption efficiency of HGNs with a LSPR of 775 nm was calculated to be 0.81% which is more than 5 times higher than the absorption efficiency of citrate reduced gold nanoparticles under similar conditions.

SERS primers have been used to directly detect specific PCR products utilizing the difference in adsorption of single-stranded and double-stranded DNA onto nanoparticle surfaces. Herein, seven parameters important for improved positive SERS assays for real applications were investigated. First, we applied a model system for optimization experiments, followed by a PCR assay to detect pathogen DNA, and then the introduction of a new assay that utilizes the 5′→3′ exonuclease activity of Taq DNA polymerase to partly digest the SERS probe, generating dye-labeled single-stranded DNA increasing the SERS signals for detection of pathogen DNA. Applying the model system, it was found that uni-molecular SERS primers perform better than bi-molecular SERS primers. However, within the PCR assays, it was found that uni- and bi-molecular SERS primers performed very similarly, and the most reproducible results were obtained using the 5′→3′ exonuclease digestion assay. These SERS-based assays offer new routes over conventional fluorescence-based techniques without compromising sensitivity or selectivity.

A novel DNA assay is demonstrated which relies on the production of a surface enhanced resonance Raman scattering (SERS) signal following sequence specific hybridisation and enzyme hydrolysis. This method achieves high sensitivity and specificity that can be applied to the detection of unlabelled duplex DNA and is the first step towards a SERS based signal amplification approach.

Abstract Single‐molecule detection by surface‐enhanced resonance Raman scattering (SERRS) spectroscopy has been demonstrated for a variety of molecules. The detection of single molecules that do not have a resonance contribution, SERS, has been shown in the case of adenine. However, when colloidal particles isolated on planar substrates are used as the enhancing medium, the presence of anomalous signals significantly complicates the analysis of the spectra. Selection of a silver colloid that minimizes these spurious signals should improve the ultra‐sensitive detection of non‐resonant single molecules by SERS. A range of silver colloids, prepared by different methods, were investigated with respect to their activity and stability. Minimal anomalous signals were obtained from hydroxylamine‐reduced silver colloids, which suggests that this colloid will be better for ultra‐sensitive SE(R)RS experiments compared to the more common citrate‐ and borohydride‐reduced silver colloids. Copyright © 2011 John Wiley &amp; Sons, Ltd.

Traditional methods for the construction of nanoparticle arrays and lattices exploit Watson–Crick base pairing of single-stranded DNA sequences as a proxy for self-assembly. Although this approach has been utilized in a variety of applications in nanoassembly, diagnostics, and biomedicine, the diversity of this recognition lexicon could be considerably increased by developing strategies that recognize the base-pairing landscape of double-stranded DNA (dsDNA) sequences. Herein we describe the first report of programmed gold nanoparticle (GNP) aggregation directed by the recognition of dsDNA sequences using pyrrole–imidazole polyamide–GNP (PA–GNP) conjugates. We demonstrate the reversibility and selectivity of this strategy for forming GNP aggregates in the presence of fully matched dsDNA sequences relative to dsDNA sequences containing one- and two-base-pair mismatches.

Spherical and monodispersed silver nanoparticles (AgNPs) are ideal for fundamental research as the contribution from size and shape can be accounted for in the experimental design. In this paper a seeded growth method is presented, whereby varying the concentration of sodium borohydride-reduced silver nanoparticle seeds, different sizes of stable spherical nanoparticles with a low polydispersity nanoparticles are produced using hydroquinone as a selective reducing agent. The surface-enhanced Raman scattering (SERS) enhancement factor for each nanoparticle size produced (17, 26, 50, and 65 nm) was then assessed using three different analytes, rhodamine 6G (R6G), malachite green oxalate (MGO) and thiophenol (TP). The enhancement factor gives an indication of the Raman enhancement effect by the nanoparticle. Using non-aggregated conditions and two different laser excitation wavelength (633 nm and 785 nm) it is shown that an increase in particle size results in an increased enhancement for each analyte used.

An on/off SERS aggregation system has been designed to investigate carbohydrate–lectin interactions. Detection of the lectin ConA was achieved at pico molar levels. Discrimination was also demonstrated between two different carbohydrate moieties demonstrating the specific interaction between the carbohydrate and specific protein present.