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Surface-enhanced Raman scattering (SERS) remains a significant area of research since it's discovery 50 years ago. The surface-based technique has been used in a wide variety of fields, most prominently in chemical detection, cellular imaging and medical diagnostics, offering high sensitivity and specificity when probing and quantifying a chosen analyte or monitoring nanoparticle uptake and accumulation. However, despite its promise, SERS is mostly confined to academic laboratories and is not recognised as a gold standard analytical technique. This is due to the variations that are observed in SERS measurements, mainly caused by poorly characterised SERS substrates, lack of universal calibration methods and uncorrelated results. To convince the wider scientific community that SERS should be a routinely used analytical technique, the field is now focusing on methods that will increase the reproducibility of the SERS signals and how to validate the results with more well-established techniques. This review explores the difficulties experienced by SERS users, the methods adopted to reduce variation and suggestions of best practices and strategies that should be adopted if one is to achieve absolute quantification.
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Plasmonic colloidal nanoparticles (NPs) functionalised with polymers are widely employed in diverse applications, offering advantages demonstrated over non-functionalised NPs such as enhanced colloidal stability or increased biocompatibility. However, functionalisation with polymers does not always increase the stability of the colloidal system. This work explores the intricate relationship between the functionalisation of plasmonic core@shell Au@Ag nanoparticles (NPs) with thiol-polyethylene glycol-folic acid (HS-PEG-FA) polymer chains and the resulting stability and spectral characteristics of Surface-Enhanced Raman Scattering (SERS) nanotags based on these NPs. We demonstrate that varying levels of HS-PEG-FA grafting influence nanotag stability, with a low level of grafting causing aggregation and subsequently affecting the spectral signature of Raman-reporter molecules attached to the surface of the NP. Electrostatic destabilisation is identified as the primary mechanism driving aggregation, impacting the SERS spectrum of Malachite Green isothiocyanate (MGITC) whose spectral shape is different between the aggregated and non-aggregated NPs. The findings provide valuable insights into NPs stability under different conditions, offering essential considerations for the design and optimisation of SERS nanotags in bio-analytical applications, particularly those involving data processing based on spectral shape, such as in multiplex approaches where experimental spectra are decomposed with several reference components.
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Drug-induced liver injury (DILI) is a challenge in clinical medicine and drug development. Highly sensitive novel biomarkers have been identified for detecting DILI following a paracetamol overdose. The study objective was to evaluate biomarker performance in a 14-day trial of therapeutic dose paracetamol. The PATH-BP trial was a double-blind, placebo-controlled, crossover study. Individuals (n = 110) were randomized to receive 1 g paracetamol 4× daily or matched placebo for 2 weeks followed by a 2-week washout before crossing over to the alternate treatment. Blood was collected on days 0 (baseline), 4, 7, and 14 in both arms. Alanine transaminase (ALT) activity was measured in all patients. MicroRNA-122 (miR-122), cytokeratin-18 (K18), and glutamate dehydrogenase (GLDH) were measured in patients who had an elevated ALT on paracetamol treatment (≥50% from baseline). ALT increased in 49 individuals (45%). All 3 biomarkers were increased at the time of peak ALT (K18 paracetamol arm: 18.9 ± 9.7 ng/ml, placebo arm: 11.1 ± 5.4 ng/ml, ROC-AUC = 0.80, 95% CI 0.71-0.89; miR-122: 15.1 ± 12.9fM V 4.9 ± 4.7fM, ROC-AUC = 0.83, 0.75-0.91; and GLDH: 24.6 ± 31.1U/l V 12.0 ± 11.8U/l, ROC-AUC = 0.66, 0.49-0.83). All biomarkers were correlated with ALT (K18 r = 0.68, miR-122 r = 0.67, GLDH r = 0.60). To assess sensitivity, biomarker performance was analyzed on the visit preceding peak ALT (mean 3 days earlier). K18 identified the subsequent ALT increase (K18 ROC-AUC = 0.70, 0.59-0.80; miR-122 ROC-AUC = 0.60, 0.49-0.72, ALT ROC-AUC = 0.59, 0.48-0.70; GLDH ROC-AUC = 0.70, 0.50-0.90). Variability was lowest for ALT and K18. In conclusion, K18 was more sensitive than ALT, miR-122, or GLDH and has potential significant utility in the early identification of DILI in trials and clinical practice.
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Acetaminofén , Alanina Transaminasa , Biomarcadores , Enfermedad Hepática Inducida por Sustancias y Drogas , Estudios Cruzados , Queratina-18 , Humanos , Alanina Transaminasa/sangre , Biomarcadores/sangre , Masculino , Enfermedad Hepática Inducida por Sustancias y Drogas/sangre , Enfermedad Hepática Inducida por Sustancias y Drogas/etiología , Femenino , Método Doble Ciego , Queratina-18/sangre , Adulto , Persona de Mediana Edad , MicroARNs/sangre , Adulto Joven , Glutamato Deshidrogenasa/sangre , Analgésicos no NarcóticosRESUMEN
Owing to their biocompatibility, gold nanoparticles have many applications in healthcare, notably for targeted drug delivery and the photothermal therapy of tumors. The addition of a silica shell to the nanoparticles can help to minimize the aggregation of the nanoparticles upon exposure to harsh environments and protect any Raman reporters adsorbed onto the metal surface. Here, we report the effects of the addition of a silica shell on the photothermal properties of a series of gold nanostructures, including gold nanoparticle aggregates. The presence of a Raman reporter at the surface of the gold nanoparticles also allows the structures to be evaluated by surface-enhanced Raman scattering (SERS). In this work, we explore the relationship between the degree of aggregation and the position and the extinction of the near-infrared plasmon on the observed SERS intensity and in the increase in bulk temperature upon near-infrared excitation. By tailoring the concentration of the silane and the thickness of the silica shell, it is possible to improve the photothermal heating capabilities of the structures without sacrificing the SERS intensity or changing the optical properties of the gold nanoparticle aggregates.
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Glioblastoma multiforme (GBM) is a particularly aggressive and high-grade brain cancer, with poor prognosis and life expectancy, in urgent need of novel therapies. These severe outcomes are compounded by the difficulty in distinguishing between cancerous and non-cancerous tissues using conventional imaging techniques. Metallic nanoparticles (NPs) are advantageous due to their diverse optical and physical properties, such as their targeting and imaging potential. In this work, the uptake, distribution, and location of silica coated gold nanoparticles (AuNP-SHINs) within multicellular tumour spheroids (MTS) derived from U87-MG glioblastoma cells was investigated by surface enhanced Raman scattering (SERS) optical mapping. MTS are three-dimensional in vitro tumour mimics that represent a tumour in vivo much more closely than that of a two-dimensional cell culture. By using AuNP-SHIN nanotags, it is possible to readily functionalise the inner gold surface with a Raman reporter, and the outer silica surface with an antibody for tumour specific targeting. The nanotags were designed to target the biomarker tenascin-C overexpressed in U87-MG glioblastoma cells. Immunochemistry indicated that tenascin-C was upregulated within the core of the MTS, however limitations such as NP size, quiescence, and hypoxia, restricted the penetration of the nanotags to the core and they remained in the outer proliferating cells of the spheroids. Previous examples of MTS studies using SERS demonstrated the incubation of NPs on a 2D monolayer of cells, with the subsequent formation of the MTS from these pre-incubated cells. Here, we focus on the localisation of the NPs after incubation into pre-formed MTS to establish a better understanding of targeting and NP uptake. Therefore, this work highlights the importance for the investigation and translation of NP uptake into these 3D in vitro models.
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Glioblastoma , Nanopartículas del Metal , Humanos , Espectrometría Raman/métodos , Nanopartículas del Metal/química , Tenascina , Oro/química , Esferoides Celulares , Dióxido de Silicio/químicaRESUMEN
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.
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Condensados Biomoleculares , Proteínas Intrínsecamente Desordenadas , Aminoácidos Aromáticos , Espectroscopía de Resonancia Magnética , Péptidos/análisis , Proteínas Intrínsecamente Desordenadas/metabolismo , Orgánulos/metabolismoRESUMEN
A fundamental question crucial to surface-enhanced spatially offset Raman spectroscopy (SESORS) imaging and implementing it in a clinical setting for in vivo diagnostic purposes is whether a SESORS image can be used to determine the exact location of an object within tissue? To address this question, multiple experimental factors pertaining to the optical setup in imaging experiments using an in-house-built point-collection-based spatially offset Raman spectroscopy (SORS) system were investigated to determine those critical to the three-dimensional (3D) positioning capability of SESORS. Here, we report the effects of the spatial offset magnitude and geometry on locating nanoparticles (NPs) mixed with silica powder as an imaging target through tissue and outline experimental techniques to allow for the correct interpretation of SESORS images to ascertain the correct location of NPs in the two-dimensional x, y-imaging plane at depth. More specifically, the effect of "linear offset-induced image drag" is presented, which refers to a spatial distortion in SESORS images caused by the magnitude and direction of the linear offset and highlight the need for an annular SORS collection geometry during imaging to neutralize these asymmetric effects. Additionally, building on these principles, the concept of "ratiometric SESORS imaging" is introduced for the location of buried inclusions in three dimensions. Together these principles are vital in developing a methodology for the location of surface-enhanced Raman scattering-active inclusions in three dimensions. This approach utilizes the relationship between the magnitude of the spatial offset, the probed depth, and ratiometric analysis of the NP and tissue Raman intensities to ultimately image and spatially discriminate between two distinct NP flavors buried at different depths within a 3D model for the first time. This research demonstrates how to accurately identify multiple objects at depth in tissue and their location using SESORS which addresses a key capability in moving SESORS closer to use in biomedical applications.
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Nanopartículas , Espectrometría Raman , Nanopartículas/química , Espectrometría Raman/métodosRESUMEN
Folate receptor α (FRα) is a high affinity folate membrane receptor that is overexpressed in a wide variety of cancers. Detecting the overexpression of this receptor is important for cancer cells identification and to potentially guide the choice of treatment since several FRα-targeted drugs are currently in clinical trials. In this work, we built SERS nanotags based on core@shell Au@Ag nanoparticles labelled with resonant Raman-reporter and functionalised with a thiolated PEG linker bearing folic acid at the chain end. Using SERS mapping on single cells, we showed that the nanotags (FR-nanotags) could specifically target FRα on overexpressing HeLa cells and could measure the gradual blocking of FRα by free folic acid introduced in the media along the nanotags. With a control nanotag, we showed that the SERS response was 10-fold higher on HeLa cells when folic acid is present on the PEG linker compared to PEG chains without folic acid. Non-specific binding of the FR-nanotags was demonstrated to be low and mainly caused by the folic acid molecule at the PEG chain end. When comparing cancer cells with different expression levels of FRα, we obtained 4-fold higher SERS response on overexpressing HeLa cells compared to non-overexpressing A549 cells, allowing the discrimination of both cell lines with a high contrast. Owing to the biocompatibility of the developed nanotags, we demonstrated that measurements of FRα on live HeLa cells were also possible and gave similar results to measurements on fixed cells, indicating the versatility of the developed nanotags for detecting FRα under various experimental conditions.
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Receptor 1 de Folato , Nanopartículas del Metal , Receptor 1 de Folato/metabolismo , Ácido Fólico/química , Células HeLa , Humanos , Nanopartículas del Metal/química , Plata/químicaRESUMEN
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.
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Nanopartículas del Metal , Sistemas de Atención de Punto , Oro/química , Inmunoensayo/métodos , Nanopartículas del Metal/química , Espectrometría Raman/métodosRESUMEN
Raman spectroscopy is a non-invasive technique that allows specific chemical information to be obtained from various types of sample. The detailed molecular information that is present in Raman spectra permits monitoring of biochemical changes that occur in diseases, such as cancer, and can be used for the early detection and diagnosis of the disease, for monitoring treatment, and to distinguish between cancerous and non-cancerous biological samples. Several techniques have been developed to enhance the capabilities of Raman spectroscopy by improving detection sensitivity, reducing imaging times and increasing the potential applicability for in vivo analysis. The different Raman techniques each have their own advantages that can accommodate the alternative detection formats, allowing the techniques to be applied in several ways for the detection and diagnosis of cancer. This feature article discusses the various forms of Raman spectroscopy, how they have been applied for cancer detection, and the adaptation of the techniques towards their use for in vivo cancer detection and in clinical diagnostics. Despite the advances in Raman spectroscopy, the clinical application of the technique is still limited and certain challenges must be overcome to enable clinical translation. We provide an outlook on the future of the techniques in this area and what we believe is required to allow the potential of Raman spectroscopy to be achieved for clinical cancer diagnostics.
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Neoplasias/diagnóstico , Espectrometría Raman/métodos , Alquinos/química , Biomarcadores de Tumor/análisis , Biomarcadores de Tumor/química , Humanos , Concentración de Iones de Hidrógeno , Lípidos/química , Nanopartículas/química , Neoplasias/terapiaRESUMEN
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.
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Enzimas/metabolismo , Melaninas/química , Oligopéptidos/química , Protones , Oxidación-ReducciónRESUMEN
Plasmon-enhanced Raman scattering is a powerful approach to detecting and characterizing proteins in live and dynamic biological systems. However, the selective detection/enhancement of specific residues as well as spectral diffusion and fluctuations have complicated the interpretation of enhanced Raman spectra and images of biological matter. In this work, we demonstrate that the amino acid tryptophan (Trp) can capture an electron from an excited plasmon, which generates a radical anion that is resonantly enhanced: a visible excited electronic state slides into resonance upon charging. This surface enhanced resonance Raman scattering (SERRS) mechanism explains the persistence of Trp signatures in the SERS and TERS spectra of proteins. Evidence for this picture includes the observation of visible resonances in the UV-Vis extinction spectrum, changes in the ground state vibrational spectrum, and plasmon-resonance dependent behavior. DFT calculations support the experimental observations. The behavior observed from the free Trp molecule is shown to explain the SERS spectrum of the Trp-cage protein. In effect, resonant Raman scattering from radicals formed through plasmonic excitation represents an under-investigated mechanism that may be exploited for chemical sensing applications.
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Chemical signals are conveyed to cells through ligand-receptor binding, triggering cascades of biochemical reactions and resulting in pivotal cellular functions. These binding events are important in understanding membrane signaling and drug interactions. To probe ligand-receptor binding, surface enhanced Raman scattering (SERS) tags are a promising tool. SERS tags are plasmonic nanostructures functionalized with a protective coating, a Raman reporter molecule, and a biorecognition element. In biological fluids, native proteins have affinity for bare nanoparticles and form a protein corona. SERS tags have a protective shell which eliminates this complication. It is important to analyze ligand-receptor binding with SERS tags in live cells since cell fixatives alter protein structure, leading to spectral changes and data misinterpretation. In this study, we synthesized a novel SERS tag by creating a mixed monolayer of the small cyclic arginine-glycine-aspartic acid-phenylalanine-cysteine (RGDFC) peptide and 4-mercaptobenzonitrile (MBN) on the surface of spherical gold nanoparticles (Au NP). Au-RGDFC-MBN NP showed resistance to PC formation and were successfully detected in both fixed and living human metastatic colon cancer cells.
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Integrina alfaVbeta3/metabolismo , Corona de Proteínas/química , Espectrometría Raman/métodos , Línea Celular Tumoral , Supervivencia Celular , Oro/química , Humanos , Nanopartículas del Metal/química , Nitrilos/química , Oligopéptidos/químicaRESUMEN
Protein-ligand recognition is a key activity where chemical signals are communicated to cells to activate various biochemical pathways, which are important for understanding membrane signaling and drug interactions. Gold nanostars are highly attractive for biological applications due to their readily modified surface chemistry, facile synthesis and optical properties. The increase in electromagnetic field at their branches increases the surface enhanced Raman scattering (SERS) making them ideal candidates as label free in vitro probes that can be used to detect a variety of cellular activities. However, the use of particles in vitro is complicated by the adsorption of proteins, which forms the protein corona. In this paper we demonstrate gold nanostars as label free in vitro probes to study the interaction between αvß3 integrin and RGD. Nanostars functionalized with cyclic-RDGFC reduced the formation of the protein corona, due to its zwitterionic nature, indicating a small peptide approach to minimizing protein absorption. Additionally, the functionalized nanostars evince a SERS response from their interaction with αvß3 integrin representative of the amino acids present at the binding site which is also retained in a complex biological matrix. The nanostars were used in vitro to selectively detect αvß3 integrin on the membrane of human metastatic colon cancer cells. By exploiting the intense SERS and tunable plasmon resonance properties of gold nanostars functionalized with cyclic RGDFC, we have demonstrated a label free approach to investigate the chemical interactions associated with protein-ligand binding from both purified proteins and membrane bound receptors in cells.
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Our lab has shown that nanoparticles functionalized with short peptides can selectively bind to receptor proteins in vitro. Our results indicate that the Raman signals observed from purified receptors in surface enhanced Raman scattering (SERS) experiments match those observed with tip-enhanced Raman scattering (TERS) experiments performed on membrane receptors in intact cell membranes. Analysis of the observed Raman signals suggest the signals arise from the amino-acids in the protein receptor responsible for binding and recognition of the ligand attached to the nanoparticle probe. Further experiments show the variance in the data correlates with affinity of the nanoparticle probe with a specific receptor. This result illustrates a new approach to studying membrane receptors.
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Nanozymes are metal nanoparticles with catalytic properties that can be used to oxidise peroxidase substrates giving a colorimetric response which can be detected using UV-vis, and recently, Raman spectroscopy. Due to their ease of synthesis and increased stability, nanozymes are being increasing investigated to replace conventional enzymes for the detection of biomolecules. Here we exploit the catalytic activity of iron oxide (Fe2O3) nanoparticles combined with the substrate 2,2-azinobis(3-ethyl-benzothiazoline-6-sulfonic acid) (ABTS) in a decolourisation assay for the detection of antioxidants. Fe2O3 nanoparticles were used to catalyse the oxidation of ABTS to its green radical cation which, upon the addition of an antioxidant, resulted in a decolourisation due to the reduction of the radical cation caused by the hydrogen donating antioxidant. The assay was applied for the detection of multiple antioxidants (glutathione, chlorogenic acid and ascorbic acid), and was followed by monitoring the resonance Raman scattering from the ABTS solution using a portable Raman system with 785 nm laser excitation. This novel assay has the potential to be optimised to detect antioxidant activity in body fluid with low limits of detection with point of use monitoring.
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Antioxidantes/análisis , Compuestos Férricos , Nanopartículas , Ácido Ascórbico/análisis , Benzotiazoles , Ácido Clorogénico/análisis , Glutatión/análisis , Ácidos SulfónicosRESUMEN
Correction for 'A novel nanozyme assay utilising the catalytic activity of silver nanoparticles and SERRS' by Sian Sloan-Dennison et al., Analyst, 2017, 142, 2484-2490.
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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. An important area of this research involves using metal nanoparticles as artificial enzymes, known as nanozymes, which exhibit peroxidase-like activity enabling them to catalyse the oxidation of substrates such as 3,3',5,5'-tetramethylbenzidine (TMB) in the presence of hydrogen peroxide (H2O2), giving a colorimetric response. Here we exploit the catalytic activity of silver nanoparticles (Ag NPs) in a surface based silver-linked immunosorbent assay (SLISA) to detect human C-reactive protein (CRP), an inflammatory marker. Ag NPs were conjugated to antibodies with specific recognition for the corresponding target antigenic molecule, CRP, and the Ag NPs were used to catalyse the oxidation of TMB by H2O2. The resulting coloured oxidation product was detected using SERRS. We demonstrate that Ag NPs can replace the enzymes used in a conventional ELISA and a detection limit of 1.09 ng mL-1 of CRP can be achieved. It indicates the promise for SLISAs for biomarker detection and opens the way for further assays of this nature to be created. This novel assay has the potential to be optimised to detect lower levels of CRP and can be further extended for the sensitive and specific detection of other relevant biomarkers.
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Proteína C-Reactiva/análisis , Colorimetría , Inmunoensayo , Nanopartículas del Metal/química , Plata/química , Biomarcadores/sangre , Humanos , Peróxido de HidrógenoRESUMEN
Investigation into the use of artificial enzymes has become an increasingly popular area of research due to the numerous advantages offered in comparison to protein enzymes. One particular area of research interest involves the use of metal nanoparticles as artificial enzymes. The peroxidase-like activity of a variety of nanoparticles has recently been shown and their use in a range of assay formats for the detection of various analytes has been explored. Herein the enzyme mimicking activity of silver nanoparticles is investigated using the peroxidase substrate 3,3',5',5'-tetramethylbenzidine (TMB). The peroxidase-like nature of these nanoparticles can be used in combination with surface enhanced resonance Raman scattering (SERRS) to provide a novel spectroscopic method of analysis. Negatively charged silver nanoparticles were investigated in combination with TMB using SERRS and it was found that upon formation of the oxidation intermediate of TMB, small clusters of positively charged nanoparticles were formed. The enzyme like behaviour of silver nanoparticles along with their use as a SERRS substrate is combined to demonstrate a simple and rapid method for the direct detection of hydrogen peroxide with a detection limit of 100 nM.