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1.
Nat Commun ; 11(1): 2508, 2020 May 19.
Artigo em Inglês | MEDLINE | ID: mdl-32427840

RESUMO

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.

2.
Anal Chem ; 92(5): 4053-4064, 2020 Mar 03.
Artigo em Inglês | MEDLINE | ID: mdl-32045217

RESUMO

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.

3.
Chemistry ; 26(26): 5789-5793, 2020 May 07.
Artigo em Inglês | MEDLINE | ID: mdl-32059067

RESUMO

Microbial surface attachment negatively impacts a wide range of devices from water purification membranes to biomedical implants. Mimics of antimicrobial peptides (AMPs) constituted from poly(N-substituted glycine) "peptoids" are of great interest as they resist proteolysis and can inhibit a wide spectrum of microbes. We investigate how terminal modification of a peptoid AMP-mimic and its surface immobilization affect antimicrobial activity. We also demonstrate a convenient surface modification strategy for enabling alkyne-azide "click" coupling on amino-functionalized surfaces. Our results verified that the N- and C-terminal peptoid structures are not required for antimicrobial activity. Moreover, our peptoid immobilization density and choice of PEG tether resulted in a "volumetric" spatial separation between AMPs that, compared to past studies, enabled the highest AMP surface activity relative to bacterial attachment. Our analysis suggests the importance of spatial flexibility for membrane activity and that AMP separation may be a controlling parameter for optimizing surface anti-biofouling.

4.
Analyst ; 145(5): 1871-1877, 2020 Mar 02.
Artigo em Inglês | MEDLINE | ID: mdl-31970342

RESUMO

The use of surface enhanced Raman scattering (SERS) for the detection of DNA has significant potential in terms of sensitivity, multiplex target detection and robust signal detection from nanoparticles. Current methods are usually performed with short chain DNA fragments or require additional separation steps for detection of longer chain fragments. We present an integrated method for the rapid and sensitive detection of extended (≥100-base) nucleic acids with reduced preparation and sample separation steps. Key to this is the DNA sequence-specific assembly of silver nanoparticles labelled with a Raman tag to provide an enhanced signal from the tag and hence molecular recognition of the target DNA. DNA probe orientation and hybridisation procedures are critical for the success of this assay. Effective detection of extended nucleic acids was achieved with head-to-head probes and by adding polyethylene glycol 10 000 (PEG 10 000) to the hybridisation buffer. This gave a 34-fold discriminatory enhancement factor when applied to a synthetic target. A structured approach toward maximising hybridisation procedures and SERS response is described, followed by an initial demonstration of SERS detection of single-stranded DNA target amplified by asymmetric PCR which was used without further separation. This has implications for future developments in using SERS for DNA detection due to the new-found ability to integrate SERS with asymmetric PCR.

5.
Anal Chem ; 92(4): 3253-3261, 2020 02 18.
Artigo em Inglês | MEDLINE | ID: mdl-31927940

RESUMO

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.

6.
ACS Nano ; 14(1): 28-117, 2020 Jan 28.
Artigo em Inglês | MEDLINE | ID: mdl-31478375

RESUMO

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.

7.
Analyst ; 145(3): 983-991, 2020 Feb 03.
Artigo em Inglês | MEDLINE | ID: mdl-31829323

RESUMO

The development of viable point-of-care diagnostic formats is integral to achieving better patient care and improved outcomes. The need for robust and low-cost tests is especially important in under-resourced and rural settings. Perhaps the greatest challenge is ensuring that an untrained individual is capable of operating and interpreting the test, out with a care facility. Here we present a paper-based diagnostic device capable of sensing miR-29a using both colorimetric and surface enhanced Raman scattering (SERS) analysis. Rather, than carry out the two types of analyses in tandem, we envisage that the colorimetric output is easy enough to be interpreted by the untrained-individual administering the test to provide them with qualitative feedback. If deemed positive, the test can be further validated at a centralized care facility using a handheld-Raman spectrometer to provide a semi-quantitative result. Detection of miR-29a, a microRNA associated with myocardial infarction, was achieved at a level of pg µL-1 through the combination of three-dimensional paper-based microfluidics, colorimetric detection, and surface enhanced Raman scattering (SERS) analysis. RGB analysis of the colorimetric output generated from samples containing miR-29a at different concentrations (18-360 pg µL-1) showed differentiation from the control sample, however significant repeat variability indicated that it could not be used for quantifying miR-29a levels. However, the SERS analysis exhibited greater reproducibility at varying concentrations, achieving an LoD of 47 pg µL-1. The union of the paper-based device and the two analysis methods resulted in the production of a sensitive, reproducible and facile, point of care test (POCT), which paves the way for future implementation in the diagnosis of a range of diseases.

8.
Biofabrication ; 11(4): 045018, 2019 09 13.
Artigo em Inglês | MEDLINE | ID: mdl-31370051

RESUMO

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 (>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 BaCl2 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.


Assuntos
Antibacterianos/farmacologia , Bactérias/efeitos dos fármacos , Biofilmes/efeitos dos fármacos , Bioimpressão , Farmacorresistência Bacteriana/efeitos dos fármacos , Testes de Sensibilidade Microbiana/métodos , Impressão Tridimensional , Alginatos/química , Anaerobiose , Reagentes para Ligações Cruzadas/química , Hidrogéis/química
9.
Analyst ; 143(22): 5358-5363, 2018 Nov 05.
Artigo em Inglês | MEDLINE | ID: mdl-30325368

RESUMO

Resonant chalcogenpyrylium nanotags demonstrate an exceptional surface enhanced Raman scattering (SERS) performance for use in SORS applications. Using surface enhanced spatially offset Raman spectroscopy (SESORS), nanotags modified with a chalcogenpyrylium dye were observed at concentrations as low as 1 pM through 5 mm of tissue. Calculated limits of detection suggest that these SERS nanotags can be detected at 104 fM using surface enhanced spatially offset resonance Raman scattering (SESORRS) demonstrating their potential for in vivo applications.


Assuntos
Compostos Heterocíclicos com 1 Anel/química , Nanopartículas/química , Compostos Organosselênicos/química , Animais , Limite de Detecção , Análise Espectral Raman/métodos , Suínos
10.
Analyst ; 143(24): 5965-5973, 2018 Dec 03.
Artigo em Inglês | MEDLINE | ID: mdl-30225477

RESUMO

The ability to probe through barriers and tissue non-invasively is an urgent unmet need in both the security and biomedical imaging fields. Surface enhanced Raman spectroscopy (SERS) has been shown to yield superior enhancement in signal over conventional Raman techniques. Furthermore, by utilising a resonant Raman reporter to produce surface enhanced resonance Raman spectroscopy (SERRS), even greater enhancement in chemical signal can be generated. Here we show the benefit of using red-shifted chalcogenpyrylium based Raman reporters for probing through large thicknesses of plastic and tissue barriers using a conventional Raman instrument. In addition, the benefit of using a resonant Raman reporter for superior levels of through barrier detection is demonstrated, and we aim to show the advantage of using resonant nanotags in combination with conventional Raman spectroscopy to probe through plastic and tissue barriers. Raman signals were collected from SERRS active nanotags through plastic thicknesses of up to 20 mm, as well as the detection of the same SERRS nanotags through up to 10 mm of tissue sections using a handheld conventional Raman spectrometer. The ability to detect SERRS-active nanotags taken up into ex vivo tumour models known as multicellular tumour spheroids (MTS), through depths of 5 mm of tissue is also shown. The advantages of applying multivariate analysis for through barrier detection when discriminating analytes with similar spectral features as the barrier is also clearly demonstrated. To the best of our knowledge, this is the first report of the assessment of the maximum level of through barrier detection using a conventional handheld Raman instrument for SERS applications as well as demonstration of the power of resonant nanotags for probing through barriers using conventional Raman spectroscopy.


Assuntos
Músculos/química , Plásticos/química , Análise Espectral Raman/métodos , Animais , Corantes/análise , Ouro/química , Humanos , Células MCF-7 , Nanopartículas Metálicas/química , Polietilenotereftalatos/química , Polipropilenos/química , Análise Espectral Raman/instrumentação , Esferoides Celulares/química , Suínos
11.
Chem Sci ; 9(34): 6935-6943, 2018 Sep 14.
Artigo em Inglês | MEDLINE | ID: mdl-30258563

RESUMO

De novo lipid synthesis is upregulated in cancer cells and inhibiting these pathways has displayed anti-tumour activity. Here we use Raman spectroscopy, focusing solely on high wavenumber spectra, to detect changes in lipid composition in single cells in response to drugs targeting de novo lipid synthesis. Unexpectedly, the beta-blocker propranolol showed selectively towards cancerous PC3 compared to non-cancerous PNT2 prostate cells, demonstrating the potential of this approach to identify new anti-cancer drug leads. A unique and simple ratiometric approach for intracellular lipid investigation is reported using statistical analysis to create phenotypic 'barcodes', a globally applicable strategy for Raman drug-cell studies. High wavenumber spectral analysis is compatible with low cost glass substrates, easily translatable into the cytological work stream. The analytical strength of this technique could have a significant impact on cancer treatment through vastly improved understanding of cancer cell metabolism, and thus guide drug design and enhance personalised medicine strategies.

12.
Beilstein J Org Chem ; 14: 2186-2189, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30202470

RESUMO

A novel methodology towards fabrication of multilayer organic devices, employing electrochemical polymer growth to form PEDOT and PEDTT layers, is successfully demonstrated. Moreover, careful control of the electrochemical conditions allows the degree of doping to be effectively altered for one of the polymer layers. Raman spectroscopy confirmed the formation and doped states of the PEDOT/PEDTT bilayer. The electrochemical deposition of a bilayer containing a de-doped PEDTT layer on top of doped PEDOT is analogous to a solution-processed organic semiconductor layer deposited on top of a PEDOT:PSS layer without the acidic PSS polymer. However, the poor solubility of electrochemically deposited PEDTT (or other electropolymerised potential candidates) raises the possibility of depositing a subsequent layer via solution-processing.

13.
Chem Commun (Camb) ; 54(61): 8530-8533, 2018 Jul 26.
Artigo em Inglês | MEDLINE | ID: mdl-30010164

RESUMO

Through utilizing the depth penetration capabilities of SESORS, multiplexed imaging and classification of three singleplex nanotags and a triplex of nanotags within breast cancer tumour models is reported for the first time through depths of 10 mm using a handheld SORS instrument.


Assuntos
Neoplasias da Mama/diagnóstico por imagem , Neoplasias da Mama/diagnóstico , Feminino , Humanos , Células MCF-7 , Modelos Moleculares , Estrutura Molecular , Análise Espectral Raman , Propriedades de Superfície
14.
Chem Sci ; 9(15): 3788-3792, 2018 Apr 21.
Artigo em Inglês | MEDLINE | ID: mdl-29780511

RESUMO

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.

15.
Artigo em Inglês | MEDLINE | ID: mdl-29525355

RESUMO

Intracellular uptake, distribution and metabolism of lipids are tightly regulated characteristics in healthy cells. An analytical technique capable of understanding these characteristics with a high level of species specificity in a minimally invasive manner is highly desirable in order to understand better how these become disrupted during disease. In this study, the uptake and distribution of three different alkyne tagged fatty acids in single cells were monitored and compared, highlighting the ability of Raman spectroscopy combined with alkyne tags for better understanding of the fine details with regard to uptake, distribution and metabolism of very chemically specific lipid species. This indicates the promise of using Raman spectroscopy directly with alkyne tagged lipids for cellular studies as opposed to subsequently clicking of a fluorophore onto the alkyne for fluorescence imaging.


Assuntos
Alquinos/química , Ácidos Graxos/metabolismo , Corantes Fluorescentes/química , Lipídeos/análise , Análise Espectral Raman/métodos , Transporte Biológico , Humanos
16.
R Soc Open Sci ; 5(12): 181483, 2018 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-30662753

RESUMO

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.

17.
Theranostics ; 8(22): 6195-6209, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30613292

RESUMO

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.


Assuntos
Vasos Coronários/diagnóstico por imagem , Vasos Coronários/imunologia , Células Endoteliais da Veia Umbilical Humana/química , Imagem Molecular/métodos , Análise Espectral Raman/métodos , Animais , Feminino , Ouro/química , Células Endoteliais da Veia Umbilical Humana/imunologia , Humanos , Molécula 1 de Adesão Intercelular/genética , Molécula 1 de Adesão Intercelular/imunologia , Masculino , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Imagem Molecular/instrumentação , Nanopartículas/química , Selectina-P/genética , Selectina-P/imunologia , Molécula 1 de Adesão de Célula Vascular/genética , Molécula 1 de Adesão de Célula Vascular/imunologia
20.
Analyst ; 143(1): 157-163, 2017 Dec 18.
Artigo em Inglês | MEDLINE | ID: mdl-29143837

RESUMO

During malarial infection, the host uses the spleen to clear the malaria parasites, however, the parasites have evolved the ability to bind to endothelial receptors in blood vessels of tissues to avoid removal, known as sequestration, and this is largely responsible for the symptoms and severity of infection. So a technique which could non-invasively diagnose tissue burden could be utilised as an aid for localised malaria diagnosis within tissue. Raman spectroscopy is a label-free imaging technique and can provide unique and chemically specific Raman 'fingerprint' spectrum of biological samples such as tissue. Within this study, Raman imaging was used to observe the changes to the molecular composition of mice spleen tissue under malarial infection, compared with non-infected samples. From analysis of the Raman imaging data, both tissue types showed very similar spectral profiles, which highlighted that their biochemical compositions were closely linked. Principal component analysis showed very clear separation of the two sample groups, with an associated increase in concentration of heme-based Raman vibrations within the infected dataset. This was indicative of the presence of hemozoin, the malaria pigment, being detected within the infected spleen. Separation also showed that as the hemozoin content within the tissue increased, there was a corresponding change to hemoglobin and some lipid/nucleic acid vibrations. These results demonstrate that Raman spectroscopy can be used to easily discriminate the subtle changes in tissue burden upon malarial infection.


Assuntos
Malária/diagnóstico , Plasmodium/isolamento & purificação , Análise Espectral Raman , Baço/parasitologia , Animais , Feminino , Heme , Hemoglobinas , Camundongos , Camundongos Endogâmicos BALB C
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