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1.
J Colloid Interface Sci ; 649: 750-760, 2023 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-37385040

RESUMO

The accurate design of labelled oligo probes for the detection of miRNA biomarkers by Surface Enhanced Raman Scattering (SERS) may improve the exploitation of the plasmonic enhancement. This work, thus, critically investigates the role of probe labelling configuration on the performance of SERS-based bioassays for miRNA quantitation. To this aim, highly efficient SERS substrates based on Ag-decorated porous silicon/PDMS membranes are functionalized according to bioassays relying on a one-step or two-step hybridization of the target miRNA with DNA probes. Then, the detection configuration is varied to evaluate the impact of different Raman reporters and their labelling position along the oligo sequence on bioassay sensitivity. At high miRNA concentration (100-10 nM), a significantly increased SERS intensity is detected when the reporters are located closer to the plasmonic surface compared to farther probe labelling positions. Counterintuitively, a levelling-off of the SERS intensity from the different configurations is recorded at low miRNA concentration. Such effect is attributed to the increased relative contribution of Raman hot-spots to the whole SERS signal, in line with the electric near field distribution simulated for a simplified model of the Ag nanostructures. However, the beneficial effect of reducing the reporter-to-surface distance is partially retained for a two-step hybridization assay thanks to the less sterically hindered environment in which the second hybridization occurs. The study thus demonstrates an improvement of the detection limit of the two-step assay by tuning the probe labelling position, but sheds at the same time light on the multiple factors affecting the sensitivity of SERS-based bioassays.


Assuntos
Técnicas Biossensoriais , Nanopartículas Metálicas , MicroRNAs , Análise Espectral Raman , Nanopartículas Metálicas/química , Bioensaio
2.
Biosensors (Basel) ; 11(12)2021 Dec 16.
Artigo em Inglês | MEDLINE | ID: mdl-34940277

RESUMO

A sharpened control over the parameters affecting the synthesis of plasmonic nanostructures is often crucial for their application in biosensing, which, if based on surface-enhanced Raman spectroscopy (SERS), requires well-defined optical properties of the substrate. In this work, a method for the microfluidic synthesis of Ag nanoparticles (NPs) on porous silicon (pSi) was developed, focusing on achieving a fine control over the morphological characteristics and spatial distribution of the produced nanostructures to be used as SERS substrates. To this end, a pSi membrane was integrated in a microfluidic chamber in which the silver precursor solution was injected, allowing for the real-time monitoring of the reaction by UV-Vis spectroscopy. The synthesis parameters, such as the concentration of the silver precursor, the temperature, and the flow rate, were varied in order to study their effects on the final silver NPs' morphology. Variations in the flow rate affected the size distribution of the NPs, whereas both the temperature and the concentration of the silver precursor strongly influenced the rate of the reaction and the particle size. Consistently with the described trends, SERS tests using 4-MBA as a probe showed how the flow rate variation affected the SERS enhancement uniformity, and how the production of larger NPs, as a result of an increase in temperature or of the concentration of the Ag precursor, led to an increased SERS efficiency.


Assuntos
Nanopartículas Metálicas , Microfluídica , Prata , Tamanho da Partícula , Silício , Análise Espectral Raman
3.
Spectrochim Acta A Mol Biomol Spectrosc ; 223: 117299, 2019 Dec 05.
Artigo em Inglês | MEDLINE | ID: mdl-31277027

RESUMO

Gram-negative bacteria represent a substantial fraction of pathogens responsible for periprosthetic infections. Given the increasing resistance of such bacteria to antibiotics, significant efforts are nowadays paid in developing new biomaterial surfaces, which offer resistance against bacterial adhesion and/or possess inherent antibacterial effects. Non-oxide silicon nitride (Si3N4) bioceramic in its polycrystalline form is a biomaterial with inherent antibacterial properties. Building upon previous phenomenological findings, the present study focuses on vibrational analyses of the metabolic response of Escherichia coli at the molecular level. A time-lapse study is conducted upon exposing the bacteria in vitro to Si3N4 bioceramic surfaces. A comparison is carried out with the as-cultured bacterial strain and with bacteria exposed to other commercially available biomaterials, namely, Ti-alloy (Ti6Al4V-ELI) and zirconia-toughened alumina (ZTA) oxide bioceramic tested under exactly the same experimental conditions. The metabolic pathways before and after exposure to different substrates were monitored by means of Raman and FTIR spectroscopies. Results indicated the development of significant osmotic stress in the bacterial strain and constant concentration decreases of its cellular compounds markers over time upon exposure to Si3N4. This ultimately led to bacterial lysis (also confirmed by conventional fluorescence microscopy assays). The main antibacterial effect was of chemical origin and driven by the elution of nitrogen ions from the Si3N4 surface, successively converted into ammonia (NH3) or ammonium (NH4)+ in aqueous solution, depending on environmental pH. The presence of these nitrogen species created osmotic stress in the cytoplasmic space. In answer to the osmotic stress, metabolic rates changed rapidly, the bacterial membrane was damaged, and lysis occurred almost completely within 48 h exposure. The antibacterial behavior exerted by the Si3N4 substrate on E. coli was more effective than that observed on the biomedical Ti6Al4V alloy. Conversely, no lysis but bacterial proliferation was recorded for E. coli exposed to ZTA bioceramic oxide substrates.


Assuntos
Antibacterianos/farmacologia , Cerâmica/farmacologia , Escherichia coli/efeitos dos fármacos , Compostos de Silício/farmacologia , Vibração , Ligas/farmacologia , Escherichia coli/metabolismo , Testes de Sensibilidade Microbiana , Modelos Biológicos , Análise Espectral Raman , Titânio/farmacologia
4.
Biosensors (Basel) ; 8(4)2018 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-30558342

RESUMO

Surface enhanced Raman spectroscopy (SERS) has been proven suitable for identifying and characterizing different bacterial species, and to fully understand the chemically driven metabolic variations that occur during their evolution. In this study, SERS was exploited to identify the cellular composition of Gram-positive and Gram-negative bacteria by using mesoporous silicon-based substrates decorated with silver nanoparticles. The main differences between the investigated bacterial strains reside in the structure of the cell walls and plasmatic membranes, as well as their biofilm matrix, as clearly noticed in the corresponding SERS spectrum. A complete characterization of the spectra was provided in order to understand the contribution of each vibrational signal collected from the bacterial culture at different times, allowing the analysis of the bacterial populations after 12, 24, and 48 h. The results show clear features in terms of vibrational bands in line with the bacterial growth curve, including an increasing intensity of the signals during the first 24 h and their subsequent decrease in the late stationary phase after 48 h of culture. The evolution of the bacterial culture was also confirmed by fluorescence microscope images.


Assuntos
Escherichia coli/crescimento & desenvolvimento , Estágios do Ciclo de Vida , Análise Espectral Raman/métodos , Staphylococcus epidermidis/crescimento & desenvolvimento , Técnicas Bacteriológicas , Nanopartículas Metálicas/química , Microscopia de Fluorescência , Porosidade , Silício/química , Prata/química , Propriedades de Superfície
5.
Faraday Discuss ; 205: 271-289, 2017 12 04.
Artigo em Inglês | MEDLINE | ID: mdl-28884170

RESUMO

In this work, SERS-based microfluidic PDMS chips integrating silver-coated porous silicon membranes were used for the detection and quantitation of microRNAs (miRNAs), which consist of short regulatory non-coding RNA sequences typically over- or under-expressed in connection with several diseases such as oncogenesis. In detail, metal-dielectric nanostructures which provide noticeable Raman enhancements were functionalized according to a biological protocol, adapted and optimized from an enzyme-linked immunosorbent assay (ELISA), for the detection of miR-222. Two sets of experiments based on different approaches were designed and performed, yielding a critical comparison. In the first one, the labelled target miRNA is revealed through hybridization to a complementary thiolated DNA probe, immobilized on the silver nanoparticles. In the second one, the probe is halved into shorter strands (half1 and half2) that interact with the complementary miRNA in two steps of hybridization. Such an approach, taking advantage of the Raman labelling of half2, provides a label-free analysis of the target. After suitable optimisation of the procedures, two calibration curves allowing quantitative measurements were obtained and compared on the basis of the SERS maps acquired on the samples loaded with several miRNA concentrations. The selectivity of the two-step assay was confirmed by the detection of target miR-222 mixed with different synthetic oligos, simulating the hybridization interference coming from similar sequences in real biological samples. Finally, that protocol was applied to the analysis of miR-222 in cellular extracts using an optofluidic multichamber biosensor, confirming the potentialities of SERS-based microfluidics for early-cancer diagnosis.


Assuntos
MicroRNAs/análise , Nanoestruturas/química , Análise Espectral Raman/métodos , Linhagem Celular Tumoral , Humanos , Dispositivos Lab-On-A-Chip , Limite de Detecção , Nanopartículas Metálicas/química , Hibridização de Ácido Nucleico , Prata/química
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