Ratiometric fluorescence imaging of lysosomal NO in living cells and mice brains with Alzheimer's disease.

We report an integrated ratiometric lysosomal nitric oxide (NO) nanoprobe based on engineered semiconducting polymer dots (Pdots), LyNO-Pdots, which consist of a newly designed NO-responsive dye, a fluorescent conjugated polymer and two functional polymers. The developed probe LyNO-Pdots exhibit high specificity and stability, good photostability and favorable blood-brain barrier (BBB) penetration ability. The LyNO-Pdots are successfully applied to ratiometric imaging of lysosomal NO variations in brain-derived endothelial cells, brain tissues and mice brains with Alzheimer's disease (AD). The results demonstrate that the NO content in the brains of AD mice is considerably higher than that in normal mice.

Ratiometric visualization of lysosomal pH fluctuations during autophagy by two-photon carbonized polymer dots-based probe.

Monitoring the pH variation in lysosomes is very conducive to studying the autophagy process, and fluorescent ratiometric pH nanoprobes with inherent lysosome targeting ability are highly desirable. Here, a carbonized polymer dots-based pH probe (oAB-CPDs) was developed by self-condensation of o-aminobenzaldehyde and further carbonization at low temperature. The obtained oAB-CPDs display improved performance in pH sensing, including robust photostability, intrinsic lysosome-targeting ability, self-referenced ratiometric response, desirable two-photon-sensitized fluorescence property, and high selectivity. With the suitable pKa value of 5.89, the as-prepared nanoprobe was successfully applied to monitor the variation of lysosomal pH in HeLa cells. Moreover, the occurrence that lysosomal pH decreased during both starvation-induced and rapamycin-induced autophagy was observed by using oAB-CPDs as fluorescence probe. We believe that nanoprobe oAB-CPDs can work as a useful tool for visualizing autophagy in living cells.

Monitoring of trace aquatic sulfonamides through hollow zinc-nitrogen-carbon electrocatalysts anchored on MXene architectures.

Herein, we designed and fabricated hollow N-doped carbon polyhedrons with atomically dispersed Zn species (Zn@HNCPs) through a topo-conversion strategy by utilising metal-organic frameworks as precursors. Zn@HNCPs achieved efficient electrocatalytic oxidation of sulfaguanidine (SG) and phthalyl sulfacetamide (PSA) sulfonamides through the high intrinsic catalytic activity of the Zn-N4 sites and excellent diffusion from the hollow porous nanostructures. The combination of the novel Zn@HNCPs with two-dimensional Ti3C2Tx MXene nanosheets resulted in improved synergistic electrocatalytic performance for the simultaneous monitoring of SG and PSA. Therefore, the detection limit of SG for this technique is much lower than those of other reported techniques; to the best of our knowledge, this is the first detection approach for PSA. Moreover, these electrocatalysts show promise for the quantification of SG and PSA in aquatic products. Our insights and findings can serve as guidelines for the development of highly active electrocatalysts for application in next-generation food analysis sensors.

Ratiometric and visual determination of copper ions with fluorescent nanohybrids of semiconducting polymer nanoparticles and carbon dots.

Developing nanohybrid composition based fluorescent carbon dots (CDs) for ratiometric detection of copper ions is highly appealing. Herein, a ratiometric sensing platform (GCDs@RSPN) for copper ions detection has been developed by loaded green fluorescence carbon dots (GCDs) on the surface of red emission semiconducting polymer nanoparticles (RSPN) through electrostatic adsorption. The GCDs, featuring abundant amino groups, can selectively bind copper ions to induce the photoinduced electron transfer, leading to fluorescence quenching. A good linearity within the range of 0-100 µM is obtained, and the limit of detection (LOD) is 0.577 µM by using obtained GCDs@RSPN as ratiometric probe to detect copper ion. Moreover, the paper-based sensor derived from GCDs@RSPN was successfully applied for the visual detection of Cu2+.

MXene-reinforced octahedral PtCu nanocages with boosted electrocatalytic performance towards endocrine disrupting pollutants sensing.

Rational tailoring of hollow and porous bimetallic structures with excellent electrocatalytic performance is appealing yet challenging. Further, combining independent bimetallic nanoparticles with flexible two-dimensional substrate by forming stable heterocomplex is still highly desired for electrocatalysis. Herein, hierarchical PtCu alloy octahedrons with hollow interiors and nanosheet-assembled nanoshells were synthesized by a facile and efficient chemical transformation strategy using Cu2O as sacrificial templates. Such octahedral PtCu nanocages displayed significantly enhanced electrocatalytic activity owing to their unique hollow and porous architectures which provided easy access for analytes to the catalyst surface. Thereafter, introduction of Ti3C2Tx MXene was realized via simple incubation of Ti3C2Tx in solution containing the 3-aminopropyltriethoxysilane-capped PtCu, and their electrostatic interaction guaranteed the firm adsorption of PtCu nanocages on Ti3C2Tx nanosheets. It turned out that the sensitivity of the hybrid sensor was remarkably improved for electrochemical monitoring of endocrine disrupting pollutants in water, exhibiting ultrawide linear ranges and sub-nanomole detection limits. The eminent electrode performance is attributed to the high specific area, fast electrochemical kinetics, decent electrical catalytic ability, and the synergistic effect between Pt, Cu, and MXene.

Easy Fabrication of Amorphous Covalent Organic Nanospheres Using Schiff-Base Chemistry for Iodine Capture.

Designing a strategy for easy fabrication of amorphous porous organic polymers (POPs) with regularly nanospherical structure using common chemical raw materials is highly imperative to promote the practical application for iodine capture. Uniform covalent organic nanospheres (CONs), defined as CON-TT, were easily prepared at room temperature via a Schiff base condensation reaction of tri(4-aminophenyl) methane (TAPM) and terephthalaldehyde (TPA) catalyzed by acetic acid. The obtained CON-TT exhibits a uniform nanospherical shape, high specific surface area, effective imine sorption sites and abundant benzene rings. An excellent reversible iodine adsorption capacity of 4.80 g g-1 is achieved, which can be attributed to the hybrid of physisorption and chemisorption process. We anticipate that this work can provide general guidance for the industrial large-scale preparation of other CONs for iodine capture.

Dual-Targeting into the Mitochondria of Cancer Cells for Ratiometric Investigation of the Dynamic Fluctuation of Sulfur Dioxide and Formaldehyde with Two-Photon Integrated Semiconducting Polymer Dots.

Mitochondrial sulfur dioxide (SO2) and formaldehyde (FA) in cancer cells serve as important signal molecules in mediating multiple physiological and pathological activities. Accurate monitoring of the dynamic fluctuation of SO2 and FA in the mitochondria of cancer cells is important for insight into their relationships and functions in cancer, understanding cancer mechanism, and the role of mitochondrial homeostasis in cancer invasion and metastasis. Herein, a novel integrated two-photon semiconducting polymer dot (BF@Pdots) with dual-targeting (cancer cells and mitochondrial) and dual-emission in green and red regions, which is rationally designed through a four-step engineering strategy by using two newly synthesized functionalized polymers PFNA and FD-PSMA as precursors, has been developed for accurate tracking of the dynamic variation of SO2 and FA in the mitochondria of cancer cells. The sensing mechanism is on the basis of the fluorescence resonance energy transfer (FRET) process in BF@Pdots tuned by the reversible Michael addition reaction between the sensing-groups and SO2 (or FA). The integrated BF@Pdots nanoprobes display excellent performances in the accurate detection of the dynamic fluctuation of SO2 and FA such as precise positioning in the mitochondria of cancer cells, self-calibrating ratiometric, two-photon emission with long wavelength excitation, and fast reversible response. The BF@Pdots nanoprobes are also applied to the ratiometric detection of the dynamic fluctuation of exogenous and endogenous SO2 and FA in the mitochondria of cancer cells for the first time with satisfactory results. Taken together, this work will provide an attractive way to develop versatile integrated Pdots-based fluorescent probes through flexible molecular engineering for applications in accurate imaging of biomolecules in living systems.

General Strategy to Achieve Color-Tunable Ratiometric Two-Photon Integrated Single Semiconducting Polymer Dot for Imaging Hypochlorous Acid.

It is highly desired and challenging to construct integrated (all-in-one) single semiconducting-polymer-derived dot (Pdot) without any postmodification but with desired performances for bioapplications. In this work, eight hypochlorous acid (HClO)-sensitive integrated polymers and corresponding polymer-derived Pdots are designed through molecular engineering to comparatively study their analytical performances for detecting and imaging HClO. The optimized polymers-derived Pdots are obtained through regulating donor-acceptor structure, the content of HClO-sensitive units, and the position of HClO-sensitive units in the polymer backbone. The designed Pdots display distinguished characteristics including multicolours with blue, yellow, and red three primary fluorescence colors, determination mode from single-channel to dual-channel (ratiometric) quantification, ultrafast response, low detection limit, and high selectivity for ClO- sensing based on specific oxidation of ClO--sensitive unit 10-methylphenothiazine (PT) accompanied by altering the intramolecular charge transfer (ICT) and fluorescence resonance energy transfer (FRET) processes in Pdots. The prepared integrated Pdots are also applied for two-photon ClO- imaging in HeLa cells and one- and two-photon ClO- imaging produced in acute inflammation in mice with satisfactory results. We believe that the present study not only provides excellent integrated fluorescent nanoprobes for ClO- monitoring in living systems but also extends a general strategy for designing integrated semiconducting polymers and Pdots with desired performances for biological applications.

A photostable reaction-based A-A-A type two-photon fluorescent probe for rapid detection and imaging of sulfur dioxide.

In this study, a novel reaction-based A-A-A (acceptor-acceptor-acceptor) type two-photon fluorescent probe, BTC, is prepared using the benzothiadiazole (BTD) scaffold as the two-photon fluorophore and electron-accepting centre. Two ß-chlorovinyl aldehyde moieties are symmetrically connected to both ends of the BTD scaffold and act as reaction groups to recognize SO2 and quenching groups to make the dis-activated probe stay at off-state due to their weak electron-withdrawing effect. In the presence of SO2 derivatives, the aldehyde groups are consumed through aldehyde addition, resulting in the activation of intramolecular charge transfer (ICT) processes and therefore recovering the fluorescence of the probe. The designed probe shows excellent two-photon properties including large two-photon absorption cross-sections (TPA) of 91 GM and photostability. Beyond these, the BTC probe exhibits a fast response to SO2 within 30 s, high specificity without foreign interference and a broad detection range from 500 nM to 120 µM with a detection limit of 190 nM. The designed fluorescent probe is further applied to the two-photon imaging of exogenous and endogenous SO2 derivatives under different physiological processes in HeLa cells and zebrafish with satisfactory results. We believe that the proposed design strategy can be extended to fabricate versatile BTD-based two-photon fluorescent probes through molecular engineering for further applications in bioassays and two-photon imaging.

Two-photon ratiometric fluorescent probe based on NBD-amine functionalized semiconducting polymer nanoparticles for real-time imaging of hydrogen sulfide in living cells and zebrafish.

The thiolysis of 7-nitro-1,2,3-benzoxadiazole amine (NBD-A) paves the way for specific sensing of H2S over biothiols and real-time imaging in living organisms. Rational fabrication of NBD-A-based probe with ratiometric mode and two-photon excitation is highly appealing to achieve high quality bioimaging. In this work, the NBD-A molecules are assembled with poly(9,9-dioctylfluorenyl-2,7-diyl) polymer nanoparticles, defined as NBD@PFO, to construct two-photon ratiometric probes for H2S detection through the fluorescence resonance energy transfer (FRET). For the construction of NBD@PFO nanohybrids, polymer nanoparticles are employed as the NBD-A molecular vehicle, energy donor and two-photon absorber, while NBD-A is served as the response unit and energy acceptor. Taking advantages of NBD-A and polymer nanoparticles, the obtained NBD@PFO probes exhibit high selectivity, fast response (<5 s), ratiometric detection and two-photon excitation. Our results indicate that NBD@PFO nanohybrids are successfully applied for monitoring of H2S concentration in living cells and zebrafish, exhibiting great potential of polymer nanoparticles to improve the imaging capability of an organic small molecular probe.

Engineering fluorescent semiconducting polymer nanoparticles for biological applications and beyond.

Semiconducting polymer nanoparticles (SPNs), derived from conjugated polymers (CPs), have emerged as a new class of soft fluorescent nanomaterials in recent years. Owing to the distinguished properties resulting from CPs and nanosize materials including extraordinary brightness, fast emission rate, strong photostability and outstanding biocompatibility, SPNs have shown potential for application in biosensing, bioimaging and biomedical areas. More importantly, in comparison to inorganic nanomaterials, SPNs hold more flexible modification approaches. These modification approaches can be performed at any stage of the preparation process of SPNs, providing great convenience and flexibility for fabricating functionalized SPNs to expand their bioapplication in various fields. In this feature article, we summarize the recent advances in the modification approaches to fabricate functionalized SPNs for bioapplications. The challenges and further outlook for fabricating functionalized SPNs are also discussed.

Development of a novel multi-strain wheat Qu with high enzyme activities for Huangjiu fermentation.

BACKGROUND: Wheat Qu has long been used as a fermentation starter to produce Huangjiu. Wheat Qu quality depends on its microbial community structure and the hydrolytic enzymes generated by the micro-organisms. RESULTS: Strain YF1 and YF2 were successfully screened as they exhibited high acidic protease (231.9 ± 1.4 U g-1 ) and cellulase (7.1 ± 0.6 U g-1 ) activities. Based on a morphological and sequence analysis of the internal transcribed spacer (ITS) gene, YF1 and YF2 were identified as Rhizopus oryzae and Aspergillus niger, respectively. Cooked wheat Qu was produced using mixed fungal starter fermentations with Aspergillus oryzae SU-16, YF1, and YF2. For Qu-making, the optimized conditions for fermentation time, water content, and inoculum size were 47.8 h, 69.4%, and 6.1%, respectively. Under these conditions, compared with single-strain cooked wheat Qu, enzyme activities of amylase, acidic protease, and cellulase increased by 27.4%, 657.1%, and 1276.2%, respectively. Short peptides and free amino acids contents increased by 19.6% and 131.8%, respectively. This wheat Qu was used for Huangjiu brewing, and the alcohol content increased by approximately 14.6% because of the increased starch hydrolysis efficiency mainly attributed to its high enzyme activity. CONCLUSION: Using mixed fungal strains as starter cultures may be an efficient strategy to improve wheat Qu quality, with great potential for application in industrial Huangjiu production. © 2021 Society of Chemical Industry.

Dual-Emission Carbonized Polymer Dots for Ratiometric pH Sensing, pH-Dependent Generation of Singlet Oxygen, and Imaging-Guided Dynamics Monitoring of Photodynamic Therapy.

The pH environment in cancer cells has been demonstrated to display vital influences on the therapeutic effect of photodynamic therapy (PDT). It is very interesting to develop pH-responsive probes for simultaneous pH sensing and dynamics monitoring of the effects of PDT, and therefore assessing the correlation between them. In this study, a multifunctional fluorescence probe, dual-emission carbonized polymer dot (CPD) in blue and red regions, which uses ethylene imine polymer (PEI) and 4,4',4″,4‴-(porphine-5, 10, 15, 20-tetrayl) tetrakis (benzoic acid) (TCPP) as precursors through a one-step hydrothermal amide reaction, has been designed for ratiometric pH sensing, generating pH-dependent 1O2 for PDT of cancer cells, and investigating the dynamics effects of PDT through pH-guided imaging. The prepared CPDs were successfully used for ratiometric pH response, pH-dependent generation of 1O2, and dynamics monitoring PDT in HeLa cells. This study may provide an alternative strategy to prepare CPD-based theranostic integrated nanoprobes for PDT through the rational design of precursors.

Objective measures of greengage wine quality: From taste-active compound and aroma-active compound to sensory profiles.

This study is sought to identify the components in greengage wine that predict the sensory properties. Taste-active compounds and aroma-active compounds of 20 commercially available greengage wines from different regions were characterized. The relationship between these compounds, wine samples and sensory attributes was modeled by partial least squares regression. The regression analysis indicated the taste-active compounds, alanine, leucine, proline, glutamic acid, lysine, malic acid, citric acid, sucrose, glucose, gallic acid, caffeic acid and tannin made a great contribution to the characteristic taste or mouthfeel of greengage wine. Meanwhile, the aroma-active compounds, including ethyl acetate, ethyl butanoate, ethyl hexanoate, ethyl octanoate, ethyl decanoate, 3-methylbutanol, 5-hydroxymethylfurfural, octanoic acid and benzaldehyde, modeled well with the flavor characteristic of greengage wine. The study revealed new insights into the relationship between chemistry and wine sensory characters, which has implications for developing an objective measurement system for determining greengage wine quality.

Identification of colloidal haze protein in Chinese rice wine (Shaoxing Huangjiu) mainly by matrix-assisted laser ionization time-of-flight mass spectrometry.

As one of the three most famous brewed wines in the world, Chinese rice wine is made from rice and husked millet, containing 14 percent to 20 percent alcohol. Highly original, yellow wine brewing techniques are regarded as the model of the wine brewing industry in Asia. Shaoxing Huangjiu is produced in Zhejiang province and remains the oldest and most representative Chinese rice wine. During storage, Shaoxing Huangjiu is susceptible to environmental disturbance and produces colloidal haze to result in turbidity. In this study, the main composition and source of colloidal haze protein in Shaoxing Huangjiu were analyzed by two-dimensional electrophoresis and matrix-assisted laser ionization time-of-flight tandem mass spectrometry (MALDI-TOF/TOF MS). The results showed that the proteins in colloidal haze mainly consisted of oat protein b1, oat-like protein, di-amylase inhibitor, pathogenesis-related protein, pathogenesis-related protein-4, chitinase II derived from wheat and oat-like protein, and beta-amylase derived from rice. The amino acid composition and secondary structure of haze protein and supernatant protein in Huangjiu were further explored by high-performance liquid chromatography and Fourier transform infrared spectroscopy. The study has broadened knowledge of the main composition and source of colloidal haze protein in Shaoxing Huangjiu. The corresponding results indicated that the amino acid composition from colloidal haze had the main characteristics of high hydrophobicity and low water solubility.

Trichromatic-emission and dual-ratio semiconducting polymer dots as fluorescent probe for simultaneous quantification of Cu2+ and pH in vitro and in vivo.

Novel dual-ratio semiconducting polymer dots (Pdots) with trichromatic-emission in the red, green and blue primary-color regions, are designed as lysosome-targeting nanoprobes for the simultaneous detection and multicolor imaging of pH and Cu2+ in HeLa cells and zebrafish without cross-interference.

Uncovering mechanisms of greengage wine fermentation against acidic stress via genomic, transcriptomic, and metabolic analyses of Saccharomyces cerevisiae.

Acid stress is one of the most common adverse conditions during fermentation of fruit wines, and the acid tolerance of yeasts is, therefore, critical for fruit wine production. However, the biological mechanism underlying the acquired tolerance of yeasts against acid stress is poorly understood. We have previously obtained an evolved Saccharomyces cerevisiae strain ET008-c54 with increased tolerance against acid stress, and potentially, it serves as a promising yeast strain for greengage wine fermentation. In the current study, we further revealed the alterations responsible for the adaptation of ET008-c54 to low pH by whole-genome re-sequencing, transcriptomic, and metabolic analyses. Results confirmed the outstanding fermenting performance of ET008-c54 at low pH as compared with the parental ET008. More specifically, the growth rate of ET008-c54 at low pH was increased by 6.24 times and the fermentation time was shortened by 70%. Differences were also observed in the physiology of the strains through ergosterol, H+-ATPase activity, and aroma determinations. By integrating both RNA-seq and whole-genome re-sequencing data, we demonstrated some metabolic pathways in ET008-c54, namely ergosterol synthesis and ferrous iron uptake, in which several acid-responsive genes were involved being upregulated. Also, upregulation of the pathways responsible for aroma compound formation, including fatty acid ethyl ester synthesis and aromatic amino acid biosynthesis, was identified. Thus, the enhanced fermentation ability of ET008-c54 at low pH should be, at least partly, contributed by the altered gene expressions associated with the aforementioned pathways. By elucidating the biological mechanism of yeasts against acid stress, this current study allows better-defined targets for future studies of genetic improvement of wine yeasts and enhancement of the fermentation processes. KEY POINTS: • Metabolic analysis confirmed the excellent fermentation performance of ET008-c54. • Acid tolerance genes for ergosterol synthesis and ferrous iron uptake were upregulated. • Aroma genes for fatty acid ethyl ester and aromatic amino acid synthesis were upregulated.

Multishell Au@Ag@SiO2 nanorods embedded into a molecularly imprinted polymer as electrochemical sensing platform for quantification of theobromine.

A highly uniform and monodisperse silica-encapsulated Au@Ag multilayered core-shell nanorods (~ 80 nm in length) has been prepared with excellent electrocatalytic properties. Using the Au@Ag@SiO2 nanoassemblies to substantially enhance the sensitivity and the sol-gel molecularly imprinted polymer (MIP) with imprinted cavities to present special molecular recognition sites, a novel electrochemical sensing platform was rationally designed, fabricated, and tested for efficient theobromine (THB) quantification. The formation of final Au@Ag@SiO2@MIP was characterized by transmission electron microscopy, X-ray photoelectron spectroscopy, Fourier transform infrared spectroscopy, cyclic voltammetry, and electrochemical impedance spectroscopy. The performance of the Au@Ag@SiO2@MIP modified electrode was evaluated by differential pulse voltammetry with the changes in peak current of hexacyanoferrate redox probe measured at a working potential of 0.2 V (vs. saturated calomel electrode) as determination signal. Under optimal conditions, the quantitation of THB was attained in a broad linear range from 10 nM to 100 µM with a detection limit of 8.0 nM. The selectivity of Au@Ag@SiO2@MIP was examined according to its recognition to THB and the interferents. Finally, the sensing platform was successfully applied to extract and determine THB from food, biological, and environmental samples with acceptable recoveries (92.20-107.1%) and relative standard deviation < 4%. The propsed sensor provides a robust means for monitoring alkaloids in complex matrices and a promising opportunity to develop sensitive and selective electrode materials with good reusability. Graphical abstract.

Barley α-amylase/subtilisin inhibitor shows inhibitory activity against endogenous xylanase isozyme I of malted barley: A novel protein function.

Mashing process had little influence on the arabinoxylan content in the finished wort. In this paper, a protein with inhibitory activity against the endogenous xylanase isozyme I (X-I) of malted barley was extracted and purified using a combination of ion-exchange and size-exclusion chromatography. The protein was identified as barley α-amylase/subtilisin inhibitor (BASI). According to the amino acid sequence analysis, BASI was completely different from the previous reported xylanase inhibitors. BASI showed dosage-dependent inhibitory activity. BASI exhibited a maximum inhibitory activity at 50°C and pH 6.0. BASI inhibited X-I as a competitive manner. PRACTICAL APPLICATIONS: A protein with inhibitory activity against the major endogenous xylanase isozyme I (X-I) of malted barley was extracted, purified, and characterized, which was identified as barley α-amylase/subtilisin inhibitor (BASI). The results help brewers to achieve a better understanding of the mechanism of arabinoxylan degradation during mashing. BASI can be used as an indicator to screen microbial xylanases. The microbial xylanases insensitive to BASI would have obvious advantages in the degradation of arabinoxylan polymers and filterability improvement during mashing.

A Glycoside Hydrolase Family 62 A-L-Arabinofuranosidase from Trichoderma Reesei and Its Applicable Potential during Mashing.

Arabinoxylan is the second most abundant component in the endosperm cell wall of barley and it has been shown to have negative effects on the viscosity and filtration rate of wort and beer. In this study, a glycoside hydrolase (GH) family 62 α-L-arabinofuranosidase (AFase), termed as TrAbf62A, was purified from the culture filtrate of Trichoderma reesei CICC 41495 by a combined chromatographic method. The preferred substrates of the purified TrAbf62A were soluble, highly substituted arabinoxylan oligosaccharides and polymers, similar to the type found in barley grain. TrAbf62A exhibited activity towards oligomeric and polymeric arabinoxylans, as well as colorimetric arabinose-based substrates, thus meeting the criteria to be classified as a type B AFase. TrAbf62A released mainly arabinose and xylose from soluble wheat arabinoxylan, thus indicating a dual lytic enzyme activity. Supplementation of TrAbf62A during mashing, with a loading of 12 mU/g malt, resulted in a 36.3% decrease in arabinoxylan polymer content, a 5.6% reduction in viscosity, and finally, a 22.1% increase in filtration rate. These results revealed that TrAbf62A has a high potential value in improving lautering performance during mashing.