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It is challenging to differentiate bacteria residing in the same habitat by direct observation. This difficulty impedes the harvest, application and manipulation of functional bacteria in environmental engineering. In this study, we developed a novel method for rapid differentiation of living denitrifying bacteria based on derivative synchronous fluorescence spectroscopy, as exemplified by three heterotrophic nitrification-aerobic denitrification bacteria having the maximum nitrogen removal efficiencies greater than 90%. The intact bacteria and their living surroundings can be analyzed as an integrated target, which eliminates the need for the complex pre-processing of samples. Under the optimal synchronous scanning parameter (Δλ = 40 nm), each bacterium possesses a unique fluorescence spectral structure and the derivative synchronous fluorescence technique can significantly improve the spectral resolution compared to other conventional fluorescence methods, which enables the rapid differentiation of different bacteria through derivative synchronous fluorescence spectra as fast as 2 min per spectrum. Additionally, the derivative synchronous fluorescence technique can extract the spectral signals contributed by bacterial extracellular substances produced in the biological nitrogen removal process. Moreover, the results obtained from our method can reflect the real-time denitrification properties of bacteria in the biological nitrogen removal process of wastewater. All these merits highlight derivative synchronous fluorescence spectroscopy as a promising analytic method in the environmental field.
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Desnitrificación , Nitrificación , Fluorescencia , Aerobiosis , Bacterias , Nitrógeno , Procesos Heterotróficos , NitritosRESUMEN
Rapid analysis of components in complex matrices has always been a major challenge in constructing sensing methods, especially concerning time and cost. The detection of pesticide residues is an important task in food safety monitoring, which needs efficient methods. Here, we constructed a machine learning-assisted synchronous fluorescence sensing approach for the rapid and simultaneous quantitative detection of two important benzimidazole pesticides, thiabendazole (TBZ) and fuberidazole (FBZ), in red wine. First, fluorescence spectra data were collected using a second derivative constant-energy synchronous fluorescence sensor. Next, we established a prediction model through the machine learning approach. With this approach, the recovery rate of TBZ and FBZ detection of pesticide residues in red wine was 101% ± 5% and 101% ± 15%, respectively, without resorting complicated pretreatment procedures. This work provides a new way for the combination of machine learning and fluorescence techniques to solve the complexity in multi-component analysis in practical applications.
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Residuos de Plaguicidas , Vino , Tiabendazol/análisis , Residuos de Plaguicidas/análisis , Vino/análisis , Fluorescencia , BencimidazolesRESUMEN
In this work, we demonstrate a unique nano-switch with triple environmental stimuli based on the action of functional copolymer brushes in a single conical nanochannel. This nanodevice flexibly and efficiently modulates ion transport properties under the influence of three environmental stimuli: light, pH and temperature. The triple factors can not only play a regulatory role independently, but their synergistic cooperation could fully activate the ionic gate and reversibly control the gating direction. In addition, the nano-switch can switch transport properties on demand in the face of complex combinations of different factors. This work promotes the development of intelligent bionic ion channels, which holds promise for biosensing, energy conversion and biomedical research.
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Polímeros , Transporte Iónico , Iones/química , Polímeros/químicaRESUMEN
Surface plasmon-coupled emission (SPCE), a novel signal enhancement technology generated by the interactions between surface plasmons and excited fluorophores in close vicinity to metallic film, has shown excellent performance in bioimaging. Variable-angle nanoplasmonic fluorescence microscopy (VANFM), based on an SPCE imaging system, can selectively modulate the imaging depth by controlling the excitation angles. In order to further improve the imaging performance, Au-Ag alloy nanoshuttles were introduced into an Au substrate to mediate the plasmonic properties. Benefiting from the strong localized plasmon effect of the modified SPCE chip, better imaging brightness, signal-to-background ratio and axial resolution for imaging of the cell membrane region were obtained, which fully displays the imaging advantages of SPCE system. Meanwhile, the imaging signal obtained from the critical angle excitation mode was also amplified, which helps to acquire a more visible image of the cell both from near- and far-field in order to comprehensively investigate the cellular interactions.
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Aleaciones , Resonancia por Plasmón de Superficie , Resonancia por Plasmón de Superficie/métodos , Imagen Óptica , Colorantes Fluorescentes/química , Microscopía Fluorescente/métodosRESUMEN
Early and sensitive detection of δ-aminolevulinic acid (δ-ALA) and porphobilinogen (PBG) is the cornerstone of diagnosis and effective treatment for acute porphyria. However, at present, the quantifying strategies demand multiple solvent extraction steps or chromatographic approaches to separate δ-ALA and PBG prior to quantification. These methods are both time-consuming and laborious. Otherwise, in conventional spectrofluorimetry, the overlapping spectra of the two analytes cause false diagnosis. To overcome this challenge, we present a two-step approach based on derivative matrix-isopotential synchronous fluorescence spectrometry (DMISFS) and the Hantzsch reaction, realizing the simple and simultaneous detection of δ-ALA and PBG in urine samples. The first step is chemical derivatization of the analytes by Hantzsch reaction. The second step is the determination of the target analytes by combining MISFS and the first derivative technique. The proposed approach accomplishes following advantages: 1) The MISFS technique improves the spectral resolution and resolves severe spectral overlap of the analytes, alleviating tedious and complicated pre-separation processes; 2) First derivative technique removes the background interference of δ-ALA on PBG and vice versa, ensuring high sensitivity; 3) Both the analytes can be determined simultaneously via single scanning, enabling rapid detection. The obtained detection limits for δ-ALA and PBG were 0.04 µmol L-1 and 0.3 µmol L-1, respectively. Within-run precisions (intra and inter-day CVs) for both the analytes were <5%. Further, this study would serve to enhance the availability of early and reliable quantitative diagnosis for acute porphyria in both scientific and clinical laboratories.
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We have proposed a universal label-free fluorescent nanofilm sensor based on surface plasmon coupled emission (SPCE). A metal-dye-dielectric (MDD) structure was fabricated to mediate the label-free monitoring based on SPCE. The nonfluorescent dielectric film smartly borrowed the fluorescence signal from the bottom dye layer and led to a new SPCE response through the adjacent metal film. The fluorescence emission angle and polarization strongly depended on the thickness of the nonfluorescent dielectric film on the MDD structure. As a demonstration, the growth of a two-dimensional zeolitic imidazolate framework film (ZIF-L) was in situ monitored in the liquid phase by MDD-SPCE for the first time. The label-free fluorescent sensors are facilely prepared by a spin coating technique, with the potential to be widely spread for in situ studies, especially toward nanomaterial growth processes.
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Estructuras Metalorgánicas , Nanoestructuras , Zeolitas , Colorantes Fluorescentes/química , Nanoestructuras/química , Resonancia por Plasmón de Superficie/métodosRESUMEN
Different cell membrane domains play different roles in many cell processes, and the discrimination of these domains is of considerable importance for the elucidation of cellular functions. However, the strategies available for distinguishing these cell membrane domains are limited. A novel technique called plasmon coupling enhanced micro-spectroscopy and imaging to discriminate basal and lateral membrane domains of a single cell combines the application of an additional plasmonic silver film for surface plasmon (SP) excitation to selectively excite and enhance the basal membranes in the near-field with directional enhanced microscopic imaging and spectroscopy. The SP and critical evanescent fields are induced upon excitation through a silver-coated semitransparent coverslip at the surface plasmon resonance and critical angles, respectively. The basal and lateral membrane domains located within the SP and critical evanescent fields can be selectively excited and distinguished by adjusting the incident angle of laser irradiation. Moreover, the brighter images and more intense spectra of membrane-targeting fluorescence-Raman probes under directional excitation than in conventional EPI mode allow clear identification of the membrane domains.
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Colorantes Fluorescentes , Resonancia por Plasmón de Superficie , Diagnóstico por Imagen , Plata , Análisis EspectralRESUMEN
In this article, we have demonstrated a smart pH-modulated two-way photoswitch that can reversibly switch ion transport under alternating light exposure over a wide pH range. This photoswitch was prepared by functionalizing the interior of a single conical glass nanochannel with a poly-spiropyran-linked methacrylate (P-SPMA) polymer through surface-initiated atom transfer radical polymerization. The P-SPMA polymer brushes comprise functional groups that are responsive to light and pH, which can cause configuration and charge changes to affect the properties of the nanochannel wall. The SPMA polymer-modified nanochannel not only reversibly controlled ion transport under alternating light irradiation but also efficiently and flexibly regulated the direction and extent of the ion transport based on the pH. This two-way photoswitch exhibits the considerable potential of photoresponsive polymers for the advancement of "intelligent" bionic nanochannel devices for ion screening and optical sensing in various applications.
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Surface adsorption studies play a crucial role in numerous fields from surface catalysis to molecular separation. However, investigation on adsorption mechanisms has been restricted to limited analytes and approaches, which calls for an in situ and sensitive surface analysis technique capable of revealing the mechanisms as well as discriminating different adsorbates and their geometry at different adsorption stages. In this study, we employed surface plasmon-coupled directional enhanced Raman scattering (SPCR), a novel technique developed by coupling surface plasmon-coupled emission with SERS, to study conformation-switching involved dynamic adsorption with background suppression and improved sensitivity (nearly 30-fold). We obtained the isotherms for a conformation-changing Raman model analyte, malachite green. An S-type Langmuir model was fitted from the time-resolved SPCR signals sensitively and without any interference from the bulk solution. The reorientation of the analyte from a predominantly parallel configuration to a perpendicular one was captured by the dramatic increase in the intensity ratios of the adsorption-related peaks to the adsorption-unrelated peak. We believe that this new sensitive and selective SPCR technique will be a promising tool for surface adsorption kinetics analysis.
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Fluorescence imaging technology has been extensively applied in chemical and biological research profiting from its high sensitivity and specificity. Much attention has been devoted to breaking the light diffraction-limited spatial resolution. However, it remains a great challenge to improve the axial resolution in a way that is accessible in general laboratories. Surface plasmon-coupled emission (SPCE), generated by the interactions between surface plasmons and excited fluorophores in close vicinity of the thin metal film, offers an opportunity for optical imaging with potential application in analysis of molecular and biological systems. Benefiting from the highly directional and distance-dependent properties, SPCE imaging (SPCEi) has displayed excellent performance in bioimaging with improved sensitivity and axial confinement. Herein, we give a brief overview of the development of SPCEi. We describe the unique optical characteristics and constructions of SPCEi systems and highlight recent advances in the use of SPCEi for biological applications. We hope this review provides readers with both the insights and future prospects of SPCEi as a new promising imaging platform for potentially widespread applications in biological research and medical diagnostics. Graphical abstract.
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Espectrometría de Fluorescencia/métodos , Resonancia por Plasmón de Superficie/métodos , Fluorescencia , Colorantes Fluorescentes/química , Espectrometría de Fluorescencia/instrumentación , Resonancia por Plasmón de Superficie/instrumentaciónRESUMEN
Simple, stable, easily-fabricated smooth metallic nanofilm can improve the imaging intensity and imaging contrast. However, its application in micrometer-scale cells has not been popularized due to the lack of full understanding of their related fluorescence properties. In this study, fluorescence enhancement of cell imaging on smooth Au nanofilm was investigated over a micrometer-scale range via employment of the optical sectioning method available with a laser scanning confocal fluorescence microscope. The fluorescence enhancement reduced with the distance away from the surface of metallic nanofilm, and this distance dependence was determined by the factors of numerical aperture, dye-substrate distance, and emission wavelength. In addition, distance-dependent fluorescence lifetime images of cells were also measured to study the interaction between fluorophores and metallic film. The enhancement effect of Au nanofilm on fluorescence cell imaging can be induced not only by the standing wave formed by the reflected light and exciting light but also by the interaction between fluorophore and surface plasmons on the metallic nanofilm. Our study on smooth metallic nanofilm should pave the way for utilizing its uniform fluorescence enhancement characteristic for biological imaging.
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Colorantes Fluorescentes , Microscopía , Rayos Láser , Espectrometría de FluorescenciaRESUMEN
Light-matter interactions have always been a fundamentally significant topic that has attracted much attention. It is important to reveal a fluorophore-plasmon interaction on the nanoscale. However, as a powerful investigative tool, fluorescence spectroscopy still suffers from a limited spectral resolution and the susceptibility to interfering substances. In this work, excitation-emission synchronization-mediated surface-plasmon-coupled emission (EES-SPCE) is proposed to break the bottleneck. By actively screening the energy transitions for observation, an improved spectral resolution has been achieved, which is advantageous to the investigation of the fluorophore-plasmon interactions under different coupling modes. The spectral information related to the plasmonic interactions through tuning vibrational energy levels is clearly distinguished at directional emission angles. EES-SPCE is demonstrated to selectively and efficiently extract the coupled emission from the vibrational resolution, which would open up the opportunity to improve the capability of spectral feature identification and signal collection for practical applications of plasmonic fluorescence spectroscopy.
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Antracenos/análisis , Pirenos/análisis , Antracenos/química , Fluorescencia , Prueba de Estudio Conceptual , Pirenos/química , Espectrometría de Fluorescencia , Resonancia por Plasmón de Superficie , VibraciónRESUMEN
The study of endocytosis, which encompasses diverse mechanisms in biology, requires the utilization of high axial resolution to monitor molecular behavior on both the cell surface and interior of the cell. We have designed a novel axially resolved fluorescence microscopic technique, termed variable-angle nanoplasmonic fluorescence microscopy. The proof-of-principle of this approach is achieved by selectively following the events in the vicinity of a cell membrane or in a cell. We use a 30 nm Au-coated semitransparent coverslip as the nanoplasmonic chip to achieve both surface plasmon resonance excitation and critical angle excitation by tuning the incident angles. This approach leads to improved axial resolution compared to total internal reflection fluorescence microscopy, which is a common imaging technique in cell biology. It offers a unique opportunity to semiquantitatively determine fluorophore axial distributions in the cell. Observing the epidermal growth factor receptor-mediated endocytosis in Caski cells clearly demonstrates the potential application of this new method for cell biology studies.
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Endocitosis , Microscopía Fluorescente/métodos , Células Cultivadas , Colorantes Fluorescentes/química , Humanos , Fracciones Subcelulares/metabolismo , Resonancia por Plasmón de SuperficieRESUMEN
Herein, we report the ultrasensitive DNA detection through designing an elegant nanopore biosensor as the first case to realize the reversal of current rectification direction for sensing. Attributed to the unique asymmetric structure, the glass conical nanopore exhibits the sensitive response to the surface charge, which can be facilely monitored by ion current rectification curves. In our design, an enzymatic cleavage reaction was employed to alter the surface charge of the nanopore for DNA sensing. The measured ion current rectification was strongly responsive to DNA concentrations, even reaching to the reversed status from the negative ratio (-6.5) to the positive ratio (+16.1). The detectable concentration for DNA was as low as 0.1 fM. This is an ultrasensitive and label-free DNA sensing approach, based on the rectification direction-reversed amplification in a single glass conical nanopore.
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Técnicas Biosensibles/métodos , ADN/análisis , Nanoporos , Conductividad Eléctrica , Diseño de Equipo , Equipo Reutilizado , Sensibilidad y EspecificidadRESUMEN
The enhancement of surface plasmon-coupled emission (SPCE) by the synergistic effect of silver nanocubes (AgNCs) and graphene oxide (GO) on gold film has been observed with the enhancement factor over 30. The enhancement mechanisms were investigated through simulating the electromagnetic (EM) field patterns of near field and testing different concentration of AgNCs and thickness of dye layer. The enhancement was mainly triggered by the high electromagnetic field of AgNCs, the interaction between localized surface plasmons (LSP) and propagating surface plasmons (PSP) and the assistance of GO. This synergistic enhancement strategy provides a simple way to increase SPCE signal and enable develop a new fluorescence-based detection system.
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Integrating probes and a substrate together, a fluorescence-enhanced interfacial "molecular beacon" (FEIMB) is demonstrated, based on directional surface plasmon coupled emission. Through this simple yet efficient interfacial modulation engineering to create an interfacial quencher (graphene oxide)-enhancer (gold nanofilm) pair, the quenching-to-enhancing region of FEIMB can be actively tuned. Therefore, it provides a spatial match between signal transduction and interface-mediated biorecognition switching. Via combination of strong quenching and efficient plasmonic coupling, a synergistically amplified signal-to-background ratio of >1000-fold has been achieved. FEIMBs have been employed in protein and DNA detection, creating a high-performance and universal chip-based plasmon-mediated fluorescence sensing platform.
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Fluorescent polymers have attracted interest in many fields such as sensing, diagnostics, imaging, and organic electronic devices. Real-time techniques to monitor and understand the polymerization process are important for obtaining controllable fluorescence polymers. We present a new technique to in situ monitor the growth process of fluorescent polymer brushes by using angle-scanning based surface plasmon coupled emission (AS-SPCE) approach during electrochemically mediated atom-transfer radical polymerization. The polymer thickness was determined by modeling the location of SPCE emission angle(s) with theoretical calculation. The advantages of unique angle distribution patterns, thickness dependence and effective background rejection of AS-SPCE guarantee the success in the real-time investigation for controllable fabrication of fluorescent polymers.
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In this work, we report studies that focus on correlating fluorescence enhancement with crystal structures, using Pt nanoparticles as a demonstration. Both experimental and theoretical calculation results provide evidence to support an interesting phenomenon that high-index structures, especially step atoms, contribute in enhancing fluorescence signals.
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We demonstrate that the propagating surface plasmon coupled fluorescent thin film can be utilized as a fluorescence modulator to mimic multiple representative Boolean logic operations. Surface plasmon mediated fluorescence presents characteristic properties including directional and polarized emission, which hold the feasibility in creating a universal optical modulator. In this work, through constructing the thin layer with the specific thickness, surface plasmon mediated fluorescence can be modulated with an ON-OFF ratio by more than 5-fold, under a series of coupling configurations.
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In this article, we have demonstrated for the first time a triple stimuli-responsive nanofluidic diode that can rectify ionic current under multiple external stimuli including temperature, pH, and sugar. This diode was fabricated by immobilizing poly[2-(dimethylamino)ethyl methacrylate]-co-[4-vinyl phenylboronic acid] (P(DMAEMA-co-VPBA)) onto the wall of a single glass conical nanopore channel via surface-initiator atom transfer radical polymerization (SI-ATRP). The copolymer brushes contain functional groups sensitive to pH, temperature and sugar that can induce charge and configuration change to affect the status of the pore wall. The experimental results confirmed that the P(DMAEMA-co-VPBA) brush modified nanochannel regulated the ionic current rectification successfully under three different external stimuli. This biomimetically inspired research simulates the complex biological multi-functions of ion channels and promotes the development of "smart" biomimetic nanochannel systems for actuating and sensing applications.