RESUMO
The design, synthesis and activity of polymodal compounds for the treatment of inflammatory bowel disease are reported. The compounds, being based on a metal-Schiff base motif, are designed to degrade during intestinal transit to release the bioactive components in the gut. The compounds have been developed sequential with the biomodal compounds combining copper or zinc with a salicylaldehyde adduct. These compounds were tested in a formalin induced colonic inflammation model in BK:A mice. From these studies a trimodal compound based on a zinc Schiff base analogue of sulfasalazine was designed. This was tested against a trinitrobenzenesulfonic acid (TNB) induced colitic model in Wistar rats. The use of two models allows us to test our compounds in both an acute and a chronic model. The trimodal compound reported is observed to provide anticolitic properties in the chronic TNB induced colitis model commensurate with that of SASP. However, the design of trimodal compound still has the capacity for further development. This the platform reported may offer a route into compounds which can markedly outperform the anti-colitic properties of SASP.
Assuntos
Colite/tratamento farmacológico , Cobre/uso terapêutico , Compostos Organometálicos/uso terapêutico , Zinco/uso terapêutico , Animais , Colite/induzido quimicamente , Cobre/administração & dosagem , Cobre/química , Edema/induzido quimicamente , Edema/tratamento farmacológico , Concentração de Íons de Hidrogênio , Inflamação/induzido quimicamente , Inflamação/tratamento farmacológico , Masculino , Camundongos , Estrutura Molecular , Compostos Organometálicos/administração & dosagem , Compostos Organometálicos/química , Ratos , Ratos Wistar , Bases de Schiff/administração & dosagem , Bases de Schiff/química , Bases de Schiff/uso terapêutico , Ácido Trinitrobenzenossulfônico , Zinco/administração & dosagem , Zinco/químicaRESUMO
A significant advantage of using surface enhanced Raman scattering (SERS) for DNA detection is the capability to detect multiple analytes simultaneously within the one sample. However, as the analytes approach the metallic surface required for SERS, they become more concentrated and previous studies have suggested that different dye labels will have different affinities for the metal surface. Here, the interaction of single stranded DNA labeled with either fluorescein (FAM) or tetramethylrhodamine (TAMRA) with a metal surface, using spermine induced aggregated silver nanoparticles as the SERS substrate, is investigated by analyzing the labels separately and in mixtures. Comparison studies were also undertaken using the dyes in their free isothiocyanate forms, fluorescein isothiocyanate (F-ITC) and tetramethylrhodamine isothiocyanate (TR-ITC). When the two dyes are premixed prior to the addition of nanoparticles, TAMRA exerts a strong masking effect over FAM due to a stronger affinity for the metal surface. When parameters such as order of analyte addition, analysis time, and analyte concentration are investigated, the masking effect of TAMRA is still observed but the extent changes depending on the experimental parameters. By using bootstrap estimation of changes in SERS peak intensity, a greater insight has been achieved into the surface affinity of the two dyes as well as how they interact with each other. It has been shown that the order of addition of the analytes is important and that specific dye related interactions occur, which could greatly affect the observed SERS spectra. SERS has been used successfully for the simultaneous detection of several analytes; however, this work has highlighted the significant factors that must be taken into consideration when planning a multiple analyte assay.
Assuntos
DNA/química , Fluoresceína/química , Corantes Fluorescentes/química , Rodaminas/química , Análise Espectral Raman/métodos , Sequência de Bases , Estrutura Molecular , Propriedades de SuperfícieRESUMO
Surface enhanced Raman scattering (SERS) can generate characteristic spectral "fingerprints" from metal complexes, thus providing the potential for the development of methods of analysis for the identification and quantitation of a range of metal ions in solution. The advantages include sensitivity and the use of one ligand for several metals without the need for a specific chromophore. Aqueous solutions of Fe(II), Ni(II), Zn(II), Cu(II), Cr(III) and Cd(II) in the presence of excess 2,2'-bipyridyl (bipy) were analysed using SERS. Specific marker bands enabled the identification of each metal ion and the limit of detection for each metal ion was estimated. Two of the ions, Zn(II) and Cu(II), could be detected below the World Health Organisation's (WHO) recommended limits for drinking water at levels of 0.22 and 0.6 mg L(-1), respectively.
RESUMO
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.
Assuntos
DNA/análise , Nanopartículas de Magnetita/química , Prata/química , Análise Espectral Raman/métodos , Candida/isolamento & purificação , Candidíase/microbiologia , DNA Fúngico/análise , Modelos MolecularesRESUMO
Here, we report the first use of resonance Raman scattering for the detection of miniaturized microscale arrays fabricated by dip-pen nanolithography. Antibody arrays for prostate-specific antigen (PSA) were printed, and a sandwich immunoassay was carried out. An enzyme-linked detection antibody was used to provide an insoluble and stable colored microdot in the recommended size range for microarray readers, which could be read with resonance Raman scattering. This gives quantitative detection as well as an improved detection limit and a larger dynamic range than that previously achieved by direct fluorescent detection methods. By Raman mapping across the arrayed area, the microdots were easily detected with very little background signal from surrounding areas. Levels of PSA as low as 25 pg/mL were detected using this method, which could be extended to a large number of useful biomarkers.
Assuntos
Nanotecnologia/métodos , Antígeno Prostático Específico/análise , Antígeno Prostático Específico/imunologia , Análise Espectral Raman/métodos , Animais , Bovinos , Humanos , Imunoensaio/métodos , MasculinoRESUMO
A highly SERS-active substrate was fabricated by trapping gold "nanoworms" on commercially available filter membranes providing significant enhancement of the Raman signal as a result of the remarkable electromagnetic couplings induced by the dense packing. The resultant substrate provides a simple and cost-effective porous SERS surface for use and quantitative analytical procedures.
RESUMO
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.
Assuntos
Biotecnologia , Nanotecnologia , Análise Espectral Raman , Ressonância de Plasmônio de Superfície , Técnicas de Diagnóstico Molecular , NanoestruturasRESUMO
Optimisation of colloidal properties allows Surface Enhanced Raman Scattering (SERS) to be recorded from a range of analytes at 1546 nm, demonstrating the potential of SERS for use in a wavelength region of particular value for applications such as homeland security.
Assuntos
Coloides/química , Ouro/química , Análise Espectral Raman/métodosRESUMO
The chemisorption of the soft scorpionate Li[PhTm(Me)] onto silver and gold surfaces is reported. Surface enhanced Raman spectroscopy in combination with the Raman analysis of suitable structural models, namely, [Cu(kappa(3)-S,S,S-PhTm(Me))(PCy(3))], [Ag(kappa(3)-S,S,S-PhTm(Me))(PCy(3))], [Ag(kappa(2)-S,S-PhTm(Me))(PEt(3))], and [Au(kappa(1)-S-PhTm(Me))(PCy(3))], are employed to identify the manner in which this potentially tridentate ligand binds to these surfaces. On colloidal silver surface-enhanced Raman spectroscopy (SERS) spectra are consistent with PhTm(Me) binding in a didentate fashion to the surface, holding the aryl group in close proximity to the surface. In contrast, on gold colloid, we observe that the species prefers a monodentate coordination in which the aryl group is not in close proximity to the surface.
Assuntos
Ouro/química , Conformação Proteica , Escorpiões/química , Análise Espectral Raman/métodos , Propriedades de Superfície , Adsorção , Animais , Eletroquímica/métodos , Humanos , Masculino , Nanopartículas Metálicas/química , Nanotecnologia , Ressonância de Plasmônio de Superfície/métodosRESUMO
A novel wide-field approach for the real-time Surface Enhanced Raman Scattering (SERS) imaging of multiple silver nanoparticle clusters suspended in solution is described. This method enables direct correlation of the SERS activity of a single nanoparticle aggregate and its size through measurement of the cluster diffusion coefficient and can also be performed in a high-throughput basis. As a first demonstration, we investigate the salt-induced aggregation of silver nanoparticles in the presence of a reporter tag molecule, which has a high affinity for the nanoparticle surface. In addition to tracking individual particles, direct comparison of Rayleigh and SERS videos of the same colloid solution enabled measurement of the fraction of individual clusters that are SERS active and the dependence of this value on the relative concentration of the tag molecule. Furthermore, given the ability to also rapidly profile any nonuniformity in particle size distributions, we expect this approach will not only provide a new tool for the fundamental understanding of SERS but also significantly contribute to the development of an array of emerging nanoparticle-enhanced biomolecule and imaging detection platforms.
RESUMO
We demonstrate the use of a scanning transmission electron microscope (STEM) equipped with a monochromator and an electron energy loss (EEL) spectrometer as a powerful tool to study localized surface plasmons in metallic nanoparticles. We find that plasmon modes can be influenced by changes in nanostructure geometry and electron beam damage and show that it is possible to delineate the two effects through optimization of specimen preparation techniques and acquisition parameters. The results from the experimental mapping of bright and dark plasmon energies are in excellent agreement with the results from theoretical modeling.
RESUMO
Bacillus megaterium flavocytochrome P450 BM3 is a catalytically self-sufficient fatty acid hydroxylase formed by fusion of soluble NADPH-cytochrome P450 reductase and P450 domains. Selected mutations at residue 264 in the haem (P450) domain of the enzyme lead to novel amino acid sixth (distal) co-ordination ligands to the haem iron. The catalytic, spectroscopic and thermodynamic properties of the A264M, A264Q and A264C variants were determined in both the intact flavocytochromes and haem domains of P450 BM3. Crystal structures of the mutant haem domains demonstrate axial ligation of P450 haem iron by methionine and glutamine ligands trans to the cysteine thiolate, creating novel haem iron ligand sets in the A264M/Q variants. In contrast, the crystal structure of the A264C variant reveals no direct interaction between the introduced cysteine side chain and the haem, although EPR data indicate Cys(264) interactions with haem iron in solution. The A264M haem potential is elevated by comparison with wild-type haem domain, and substrate binding to the A264Q haem domain results in a approximately 360 mV increase in potential. All mutant haem domains occupy the conformation adopted by the substrate-bound form of wild-type BM3, despite the absence of added substrate. The A264M mutant (which has higher dodecanoate affinity than wild-type BM3) co-purifies with a structurally resolved lipid. These data demonstrate that a single mutation at Ala(264) is enough to perturb the conformational equilibrium between substrate-free and substrate-bound P450 BM3, and provide firm structural and spectroscopic data for novel haem iron ligand sets unprecedented in nature.
Assuntos
Proteínas de Bactérias/metabolismo , Sistema Enzimático do Citocromo P-450/metabolismo , Heme/metabolismo , Mutação , NADPH-Ferri-Hemoproteína Redutase/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Dicroísmo Circular , Sistema Enzimático do Citocromo P-450/química , Sistema Enzimático do Citocromo P-450/genética , Espectroscopia de Ressonância de Spin Eletrônica , Ácidos Graxos/química , Ácidos Graxos/metabolismo , Glutamina/química , Glutamina/genética , Glutamina/metabolismo , Heme/química , Cinética , Metionina/química , Metionina/genética , Metionina/metabolismo , Mutagênese Sítio-Dirigida , NADPH-Ferri-Hemoproteína Redutase/química , NADPH-Ferri-Hemoproteína Redutase/genética , Ligação Proteica , TermodinâmicaRESUMO
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).
Assuntos
Impressões Digitais de DNA/métodos , Interpretação Estatística de Dados , Análise Espectral Raman/métodos , Biometria , DNA Bacteriano/análise , Escherichia coli/genética , Sensibilidade e Especificidade , Especificidade da EspécieRESUMO
Enhanced Raman scattering from metal surfaces has been investigated for over 30 years. Silver surfaces are known to produce a large effect, and this can be maximized by producing a roughened surface, which can be achieved by the aggregation of silver nanoparticles. However, an approach to control this aggregation, in particular through the interaction of biological molecules such as DNA, has not been reported. Here we show the selective turning on of the surface enhanced resonance Raman scattering effect on dye-coded, DNA-functionalized, silver nanoparticles through a target-dependent, sequence-specific DNA hybridization assembly that exploits the electromagnetic enhancement mechanism for the scattering. Dye-coded nanoparticles that do not undergo hybridization experience no enhancement and hence do not give surface enhanced resonance Raman scattering. This is due to the massive difference in enhancement from nanoparticle assemblies compared with individual nanoparticles. The electromagnetic enhancement is the dominant effect and, coupled with an understanding of the surface chemistry, allows surface enhanced resonance Raman scattering nanosensors to be designed based on a natural biological recognition process.
Assuntos
DNA/química , Corantes Fluorescentes/química , Nanopartículas Metálicas/química , Prata/química , Análise Espectral Raman , Campos Eletromagnéticos , Hibridização de Ácido Nucleico/métodos , Análise Espectral Raman/métodosRESUMO
Mycobacterium tuberculosis (Mtb) cytochrome P450 gene CYP121 is shown to be essential for viability of the bacterium in vitro by gene knock-out with complementation. Production of CYP121 protein in Mtb cells is demonstrated. Minimum inhibitory concentration values for azole drugs against Mtb H37Rv were determined, the rank order of which correlated well with Kd values for their binding to CYP121. Solution-state spectroscopic, kinetic, and thermodynamic studies and crystal structure determination for a series of CYP121 active site mutants provide further insights into structure and biophysical features of the enzyme. Pro346 was shown to control heme cofactor conformation, whereas Arg386 is a critical determinant of heme potential, with an unprecedented 280-mV increase in heme iron redox potential in a R386L mutant. A homologous Mtb redox partner system was reconstituted and transported electrons faster to CYP121 R386L than to wild type CYP121. Heme potential was not perturbed in a F338H mutant, suggesting that a proposed P450 superfamily-wide role for the phylogenetically conserved phenylalanine in heme thermodynamic regulation is unlikely. Collectively, data point to an important cellular role for CYP121 and highlight its potential as a novel Mtb drug target.
Assuntos
Antituberculosos/química , Azóis/química , Proteínas de Bactérias/química , Domínio Catalítico/fisiologia , Sistema Enzimático do Citocromo P-450/química , Mycobacterium tuberculosis/enzimologia , Proteínas de Bactérias/genética , Coenzimas/química , Coenzimas/genética , Cristalografia por Raios X , Inibidores das Enzimas do Citocromo P-450 , Sistema Enzimático do Citocromo P-450/genética , Farmacorresistência Bacteriana/genética , Teste de Complementação Genética , Heme/química , Heme/genética , Ferro/química , Mutação , Mycobacterium tuberculosis/genética , Oxirredução , TermodinâmicaRESUMO
A new class of SERRS-active macromolecule designed to protect silver nanoparticle surfaces against salt corrosion whilst retaining colloidal stability of the particles is reported.
RESUMO
A micro-bead sandwich assay for P38 mitogen-activated protein kinase using surface enhanced resonance Raman spectroscopy (SERRS) detection is reported. Monoclonal capture antibodies were immobilised on a solid phase of magnetic micro-beads with secondary detection using a rhodamine-labelled antibody. Quantitative SERRS detection of the secondary antibody was possible with a limit of detection of 9.5 x 10(-12) mol dm(-3). The sandwich assay was quantitative and sensitive to 6 ng ml(-1). The mechanism of the SERRS detection in the immunoassay was investigated. The addition of SERRS aggregating agents causes the dissociation of the immuno-complex from the magnetic beads. Scanning electron microscopy images indicate that the colloidal suspension rather than adsorbed silver nanoparticles on the beads provide the SERRS signals, that the aggregate size is partially controlled and that there is some inhomogeneity in the distribution of organic matter on the nanoscale.
Assuntos
Imunoensaio/métodos , Análise Espectral Raman , Proteínas Quinases p38 Ativadas por Mitógeno/análise , Coloides , Humanos , Magnetismo , Nanopartículas Metálicas , Microscopia Eletrônica de Varredura , Microesferas , Proteínas Recombinantes/análise , Rodaminas , Sensibilidade e Especificidade , PrataRESUMO
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.
Assuntos
Ácido Fólico/sangue , Análise Espectral Raman/métodos , Água/química , Humanos , Nanopartículas/química , Nanopartículas/ultraestrutura , Sensibilidade e Especificidade , Prata/química , Propriedades de SuperfícieRESUMO
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 x 10-11 mol.dm-3) than that obtained using fluorescence (3.46 x 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 x 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.
Assuntos
Anticorpos/análise , Anticorpos/imunologia , Espectrometria de Fluorescência/métodos , Análise Espectral Raman/métodos , Anticorpos/química , Estrutura Molecular , Propriedades de SuperfícieRESUMO
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.