A novel derivative synchronous fluorescence method for the rapid, non-destructive and intuitive differentiation of denitrifying bacteria.

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.

Label-Free Fluorescent Nanofilm Sensor Based on Surface Plasmon Coupled Emission: In Situ Monitoring the Growth of Metal-Organic Frameworks.

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.

Machine Learning-Assisted Synchronous Fluorescence Sensing Approach for Rapid and Simultaneous Quantification of Thiabendazole and Fuberidazole in Red Wine.

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.

Plasmon Coupling Enhanced Micro-Spectroscopy and Imaging for Sensitive Discrimination of Membrane Domains of a Single Cell.

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.

Surface plasmon-coupled emission imaging for biological applications.

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.

In situ and sensitive monitoring of configuration-switching involved dynamic adsorption by surface plasmon-coupled directional enhanced Raman scattering.

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.

Variable-Angle Nanoplasmonic Fluorescence Microscopy: An Axially Resolved Method for Tracking the Endocytic Pathway.

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.

Reversing current rectification to improve DNA-sensing sensitivity in conical nanopores.

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.

Label-free aptasensor based on ultrathin-linker-mediated hot-spot assembly to induce strong directional fluorescence.

We have demonstrated the proof-of-concept of a label-free biosensor based on emission induced by an extreme hot-spot plasmonic assembly. In this work, an ultrathin linking layer composed of cationic polymers and aptamers was fabricated to mediate the assembly of a silver nanoparticles (AgNPs)-dyes-gold film with a strongly coupled architecture through sensing a target protein. Generation of directional surface plasmon coupled emission (SPCE) was thus stimulated as a means of reporting biorecognition. Both the biomolecules and the nanoparticles were totally free of labeling, thereby ensuring the activity of biomolecules and allowing the use of freshly prepared metallic nanoparticles with large dimensions. This sensor smartly prevents the plasmonic assembly in the absence of targets, thus maintaining no signal through quenching fluorophores loaded onto a gold film. In the presence of targets, the ultrathin layer is activated to link NPs-film junctions. The small gap of the junction (no greater than 2 nm) and the large diameter of the nanoparticles (~100 nm) ensure that ultrastrong coupling is achieved to generate intense SPCE. A >500-fold enhancement of the signal was observed in the biosensing. This strategy provides a simple, reliable, and effective way to apply plasmonic nanostructures in the development of biosensing.

[Determination of benzo(alpha)pyrene in food with microwave-assisted extraction].

Coupling derivative technique and constant-energy synchronous fluorescence scanning technique, a method of determining benzo[alpha] pyrene in foods by second derivative constant-energy synchronous spectrofluorimetry after microwave-assisted treatment of samples was established using domestic microwave oven. The main factors of influencing the efficiency of microwave extraction were discussed, including the extraction solvent types and amounts, the microwave extraction time, microwave radiation power and cooling time. And the comparison with ultrasonic extraction was made. Low-fat food samples, which were just microwave-extracted with mixed-solvents, could be analyzed immediately by the spectrofluorimetric technique. For high-fat food samples, microwave-assisted saponification and extraction were made at the same time, thus simplifying operation steps and reducing sample analysis time. So the whole sample analysis process could be completed within one hour. This method was simple, rapid and inexpensive. In consequence, it was applied to determine benzo(a)pyrene in food with good reproducibility and the recoveries of benzo(alpha) pyrene ranged from 90.0% to 105.0% for the low fat samples and 83.3% to 94.6% for high-fat samples.

Prism-based surface plasmon coupled emission imaging.

A prism-based surface plasmon coupled emission (SPCE) imaging apparatus with a reverse Kretschmann (RK) configuration was developed and applied to dye-doped polymer films. Highly polarized, directional and enhanced fluorescence images were obtained. The angular distribution of the SPCE images was in accordance with the validated theoretical calculation performed using Fresnel equation. Prism-based SPCE imaging combined with microarray technology appears to be a promising platform for rapid and high-throughput analysis, especially for high-density arrays. We believe that prism-based SPCE imaging has potential applications in biochemical research.

Au-Ag Alloy Nanoshuttle Mediated Surface Plasmon Coupling for Enhanced Fluorescence Imaging.

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.

Derivative Matrix-Isopotential Synchronous Spectrofluorimetry and Hantzsch Reaction: A Direct Route to Simultaneous Determination of Urinary δ-Aminolevulinic Acid and Porphobilinogen.

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.

Smart Nano-switch with Flexible Modulation of Ion Transport Using Multiple Environmental Stimuli.

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.

Electric field assisted surface plasmon-coupled directional emission: an active strategy on enhancing sensitivity for DNA sensing and efficient discrimination of single base mutation.

We have demonstrated the proof-of-principle of electric field assisted surface plasmon-coupled directional emission (E-SPCDE). The combination of SPCDE and electric field control produced a significant synergistic effect to amplify the right signal and suppress the wrong signal intelligently in an active strategy. A novel hairpin structured DNA biosensor based on the quenching and enhancing of fluorescence in SPCDE has been designed. With modulation of the fluorescence coupling efficiency, a high discrimination ratio up to more than 20-fold has been achieved by enhancing the signal of match and suppressing that of mismatch. E-SPCDE has shown a successful application in DNA sensing, eliminating false positives and false negatives in the detection. E-SPCDE should provide an opportunity to create a new generation of miniaturized high-performance sensing platforms especially in chip-based microarrays and to make the manipulation of the nanometer-scale processes more accessible and detectable.

A novel baseline-correction method for standard addition based derivative spectra and its application to quantitative analysis of benzo(a)pyrene in vegetable oil samples.

In the present work, a baseline-correction method based on peak-to-derivative baseline measurement was proposed for the elimination of complex matrix interference that was mainly caused by unknown components and/or background in the analysis of derivative spectra. This novel method was applicable particularly when the matrix interfering components showed a broad spectral band, which was common in practical analysis. The derivative baseline was established by connecting two crossing points of the spectral curves obtained with a standard addition method (SAM). The applicability and reliability of the proposed method was demonstrated through both theoretical simulation and practical application. Firstly, Gaussian bands were used to simulate 'interfering' and 'analyte' bands to investigate the effect of different parameters of interfering band on the derivative baseline. This simulation analysis verified that the accuracy of the proposed method was remarkably better than other conventional methods such as peak-to-zero, tangent, and peak-to-peak measurements. Then the above proposed baseline-correction method was applied to the determination of benzo(a)pyrene (BaP) in vegetable oil samples by second-derivative synchronous fluorescence spectroscopy. The satisfactory results were obtained by using this new method to analyze a certified reference material (coconut oil, BCR(®)-458) with a relative error of -3.2% from the certified BaP concentration. Potentially, the proposed method can be applied to various types of derivative spectra in different fields such as UV-visible absorption spectroscopy, fluorescence spectroscopy and infrared spectroscopy.

Observations of the effect of confined space on fluorescence and diffusion properties of molecules in single conical nanopore channels.

In this work, we investigated the fluorescence emission spectra and diffusion properties of dye molecules confined in different positions of conical nanopore channels using a laser scanning confocal fluorescence microscope. The results showed that a red shift of the emission spectra is observed from the tip section to the bottom section and the diffusion rate is slower in the channel than that in bulk solution, indicating a single conical nanopore channel can be used as a convenient tool for investigating the effect of confined space on the behaviors of molecules.

Highly sensitive and selective spectrofluorimetric approach for the rapid determination of trace benzo[α]pyrene in drinking water and in solutions leached from disposable paper cups.

Based on the excellent band narrowing and background suppressing features of second-derivative constant-energy synchronous spectrofluorimetry with a Δ⁻υ value of 1400 cm(-1) , the strong fluorescence intensity for benzo[α]pyrene (BaP) obtained in dichloromethane and the use of standard addition method, a highly sensitive and selective approach for the quantitative determination of trace amount of BaP in drinking water has been established in this study. The detection and quantification limits were 0.11 and 0.37 ng L(-1) , respectively, and the recoveries obtained from spiked Milli-Q water, bottled natural spring water, tank-purified water and tap water at different concentrations, ranged from 86.0 to 104.0%. This method has been applied for the determination of trace BaP in solution leached from disposable paper cups. The experimental results indicated that BaP was leached from paper cups when filled with hot water, but it was not detected when cool (unheated) water was used.

[Determination of migration level of benzo(a) pyrene from paper cups into food simulants].

This paper quantitatively determined the migration level of benzo(a)pyrene (BaP) from paper cups into aqueous food simulants or fatty stimulants, under different conditions by second derivative constant-energy synchronous fluorescence spectrometry (DCESFS), which proved to be a method with high sensitivity and spectral resolution. The influences of food simulants, time and temperature on the relative migration percent(%) of benzo(a) pyrene were discussed. Results showed that the recoveries of this method ranged from 89.2% to 104.3%. The migration percent(%) of fatty stimulants was higher than that of aqueous simulants. Migration into hot water was also observed, while the migration into cool water was almost negligible. The relative migration percent(%) increased with the increase of time and temperature.

Smart pH-Modulated Two-Way Photoswitch Based on a Polymer-Modified Single Nanochannel.

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.