Development of a Fluorescein-Based Probe with an "Off-On" Mechanism for Selective Detection of Copper (II) Ions and Its Application in Imaging of Living Cells.

Copper is a common metallic element that plays an extremely essential role in the physiological activities of living organisms. The slightest change in copper levels in the human body can trigger various diseases. Therefore, it is important to accurately and efficiently monitor copper ion levels in the human body. Recent studies have shown that fluorescent probes have obvious advantages in bioimaging and Cu2+ detection. Therefore, a novel Cu2+ probe (N2) was designed and synthesized from fluorescein, hydrazine hydrate and 5-p-nitrophenylfurfural that is sensitive to and can detect Cu2+ within 100 s. The response mechanism of the N2 probe to Cu2+ was studied by several methods such as Job's plots and MS analysis, which showed that the Cu2+ and the N2 probe were coordinated in a complexation ratio of 1:1. In addition, compared with other cations investigated in this study, the N2 probe showed excellent selectivity and sensitivity to Cu2+, exhibiting distinct fluorescence absorption at 525 nm. Furthermore, in the equivalent range of 0.1-1.5, there is a good linear relationship between Cu2+ concentration and fluorescence intensity, and the detection limit is 0.10 µM. It is worth mentioning that the reversible reaction between the N2 probe and Cu2+, as well as the good biocompatibility shown by the probe in bioimaging, make it a promising candidate for Cu2+ biosensor applications.

The generation of empty Turnip yellow mosaic virus capsids through depletion of virion-associated divalent cations.

Turnip yellow mosaic virus (TYMV) is a well-studied icosahedral plant virus that has attractive properties for nanoscience applications. Stable empty particles devoid of viral genomic RNA have historically been generated from virions by: 1. high pressure; 2. extreme alkaline pH; and 3. freeze-thaw using liquid nitrogen. Herein we report a fourth and more convenient avenue for empty particle formation through EDTA treatment, implicating chelation of virion-associated cations. We present findings that confirm TYMV virions purified in an EDTA-based buffer are converted to 94 % empty on average during purification. Additional experimentation revealed TYMV virions purified through CsCl vs. sucrose gradients are more readily converted to empty particles after freeze thaw. These studies are novel as they show a purification method through EDTA-treatment that can generate stable empty particles devoid of viral genome. The convenience of this method should prove suitable for scientists seeking to use TYMV capsids in nanoscience-inspired applications. Importantly, these findings provide insight into historical discrepancies in creating empty particles after freeze-thaw, as the method in which TYMV virions are purified influences the downstream virion-to-empty conversion process.

A new fluorescent sensor mitoferrofluor indicates the presence of chelatable iron in polarized and depolarized mitochondria.

Mitochondrial chelatable iron contributes to the severity of several injury processes, including ischemia/reperfusion, oxidative stress, and drug toxicity. However, methods to measure this species in living cells are lacking. To measure mitochondrial chelatable iron in living cells, here we synthesized a new fluorescent indicator, mitoferrofluor (MFF). We designed cationic MFF to accumulate electrophoretically in polarized mitochondria, where a reactive group then forms covalent adducts with mitochondrial proteins to retain MFF even after subsequent depolarization. We also show in cell-free medium that Fe2+ (and Cu2+), but not Fe3+, Ca2+, or other biologically relevant divalent cations, strongly quenched MFF fluorescence. Using confocal microscopy, we demonstrate in hepatocytes that red MFF fluorescence colocalized with the green fluorescence of the mitochondrial membrane potential (ΔΨm) indicator, rhodamine 123 (Rh123), indicating selective accumulation into the mitochondria. Unlike Rh123, mitochondria retained MFF after ΔΨm collapse. Furthermore, intracellular delivery of iron with membrane-permeant Fe3+/8-hydroxyquinoline (FeHQ) quenched MFF fluorescence by ∼80% in hepatocytes and other cell lines, which was substantially restored by the membrane-permeant transition metal chelator pyridoxal isonicotinoyl hydrazone. We also show FeHQ quenched the fluorescence of cytosolically coloaded calcein, another Fe2+ indicator, confirming that Fe3+ in FeHQ undergoes intracellular reduction to Fe2+. Finally, MFF fluorescence did not change after addition of the calcium mobilizer thapsigargin, which shows MFF is insensitive to physiologically relevant increases of mitochondrial Ca2+. In conclusion, the new sensor reagent MFF fluorescence is an indicator of mitochondrial chelatable Fe2+ in normal hepatocytes with polarized mitochondria as well as in cells undergoing loss of ΔΨm.

Impact of divalent cations on lysozyme-induced solubilisation of waste-activated sludge: Perspectives of extracellular polymeric substances and surface electronegativity.

Lysozyme hydrolysis can accelerate waste-activated sludge (WAS) solubilisation, which can significantly shorten the process and promote the efficiency of anaerobic digestion. This study investigated the impact of divalent cations on lysozyme-induced solubilisation of WAS. The performance of lysozyme pretreatment was dramatically inhibited by Mg2+ and Ca2+. Compared to the control group, the amount of net SCOD, protein, and polysaccharides released to the supernatant were reduced by 36.6%, 44.7%, and 35.8%, respectively, in the presence of divalent cations. The extracellular polymeric substance (EPS) matrix became tightly bound, resulting in fewer proteins and polysaccharides being extracted from loosely-bound EPS (LB-EPS) with divalent cations, which was detrimental to the solubilisation of WAS. Divalent cations decreased the surface electronegativity of sludge particles and prolonged the adsorption of lysozymes by sludge flocs. More than 16.6% of total lysozymes remained in the liquid phase of WAS after 240 min Mg2+ and Ca2+ strengthened the binding among proteins and polysaccharides and promoted the intermolecular cross-linking of polysaccharides. The EPS matrix formed a dense spatial reticular structure that blocked the transfer of lysozymes from the EPS matrix to the pellet. As a result, the lysozymes accumulated in LB-EPS rather than hydrolysing the microorganism's cell wall. This study provides a new perspective on the restriction of WAS pretreatment with lysozymes and optimises the method of lysozyme-induced solubilisation of WAS.

Identification and characterization of metal-chelating bioenhancer peptide derived from fermented Citrullus lanatus seed milk.

In the present investigation, a metal-chelating bioactive peptide was derived from Citrullus lanatus seed milk fermented with Lactococcus lactis. The cationic fermented milk peptide (FMP) thus obtained was purified using the HiTrap-chelating column followed by rpHPLC. The FMP possessed the ability to chelate multiple divalent cations like Cu2+ , Ca2+ , and Fe2+ with 86.81%, 61.04%, and 24.32% of chelation respectively and further it exhibited 78.03% of DPPH free radical scavenging activity. Interaction of FMP with metal ions was assessed by change in the absorption spectra and was analyzed by ultraviolet-visible and fluorescence spectroscopy. The FMP-metal complexes were found stable at simulated gastric conditions. In vitro analysis using intestinal Caco-2 cell lines revealed that there was an increase in metal bioavailability in the presence of the FMP and was least influenced by the addition of a dietary inhibitor, phytic acid. By LC-MS analysis the molecular mass of FMP was found to be 11.6 kD and it contains oxygen-rich and nitrogen-rich amino acids that favor the metal chelation. In our study, we have found that the fermented C. lanatus seed milk can serve as a potential functional food with bioenhancer peptides that increase metal bioavailability and enhance human health. PRACTICAL APPLICATIONS: Chelated metals are preferred over non-chelated ones by most nutritionists for their better absorption rate. Chelation protects the minerals from the digestive process and increases their bioavailability. Fermentation with lactic acid bacteria produces bioactive peptides with metal-chelating and antioxidant ability which provides additional health benefits beyond supplying basic nutrients. Lactococcus lactis fermented milk acts as a probiotic product with bioenhancer peptide that increases mineral bioavailability. Consumption of metals in chelated form can reduce excess intake of metal. Fermented watermelon seed milk can be a promising probiotic drink rich in bioenhancer peptides and can enhance the bioavailability of divalent cations of a high therapeutic index.

A new fluorescence probe for detection of Cu+2 in blood samples: Circuit logic gate.

A Fluorescence probe was designed based on 8-hydroxyquinoline chitosan silica precursor (HQCS) for selective detection of Al3+, Cu2+. The HQCS has no observable fluorescence signal, but after the addition of Al3+, a huge fluorescence signal appeared, and the selective quenching was absorbed after the addition of Cu2+. The effect of other different cations, including Cu2+, Mg2+, Ca2+, Pb2+, Zn2+, Hg2+, Ag+, Fe3+, and K+ was studied. The addition of Cu2+ to the probe (HQCSAL) decreased the fluorescence very repeatable, and the variation of the fluorescence vs. Cu2+ was monotonic and linear. Therefore, the prepared probe was used to determine Cu2+ ions in real samples. The mechanism of fluorescence variation by adding cations to the probe solution was studied using the Stern-Volmer equation. Under the optimum conditions, the linear range and detection limit were 3.5-31 µM and 1 µM, respectively. The probe accuracy on the copper determination in the blood and tap waters was comparable to the ICP-OES results. The circuit logic gate mimic was designed for the fluorescence behavior of the probe constituents.

Enhanced oxidase-mimicking activity of Ce4+ by complexing with nucleotides and its tunable activity for colorimetric detection of Fe2.

Complexing with adenosine-5'-monophosphate (AMP) was proven to be a facile way to enhance the oxidase-mimicking activity of Ce4+, and enabled nanoenzyme recovery and reuse. Additionally, the oxidase-mimicking activity of AMP-Ce4+ infinite coordination polymers (ICPs) could be specifically inhibited by Fe2+. Based on this finding, we developed a simple and highly selective colorimetric assay to detect Fe2+.

Effect of Ferrous Ion on Heat-Induced Aroma Deterioration of Green Tea Infusion.

Aroma deterioration is one of the biggest problems in processing tea beverages. The aroma of tea infusion deteriorates fast during heat sterilization and the presence of ferrous ion (Fe2+) aggravates it. The underlying mechanism remains unveiled. In this study, Fe2+ was verified to deteriorate the aroma quality of green tea infusion with heat treatment. Catechins were necessary for Fe2+-mediated aroma deterioration. By enhancing the degradation of catechins, Fe2+ dramatically increased the production of hydrogen peroxide (H2O2). Fe2+ and H2O2 together exacerbated the aroma of green tea infusion with heat treatment. GC-MS analysis revealed that the presence of Fe2+ enhanced the loss of green/grassy volatiles and promoted the formation of new volatiles with diversified aroma characteristics, resulting in a dull scent of green tea infusion. Our results revealed how Fe2+ induced aroma deterioration of green tea infusion with heat treatment and could help guide tea producers in attenuating the aroma deterioration of tea infusion during processing.

Simultaneous and ultra-sensitive detection of Cu2+ and Mg2+ in wine and beer based on dual DNA tweezers and entropy-driven three-dimensional DNA nanomachine.

Simultaneous and ultra-sensitive detection strategy of Cu2+ and Mg2+ in wine and beer was developed based on dual DNA tweezers and entropy-driven three-dimensional DNA nanomachine. The dual DNAzyme can simultaneously respond to two kinds of metal ions and cause two kinds of "turn-on" fluorescent signals. The working principle of this strategy was indirectly proven. In addition, some key experimental parameters were also optimized. Under the optimum conditions, the limit of detection was 10 pM for Cu2+ and 2 nM for Mg2+ respectively which was significantly improved by entropy driven amplification. This strategy also showed good selectivity and specificity. It was successfully used to detect of Cu2+ and Mg2+ in wine and beer with 5.26% to 9.12% of relative standard deviation and 90.4% to 110.5% of recoveries.

Synthesis of N2, N6-bis(5-methylthiazol-2-yl)pyridine-2,6-dicarboxamide fluoroscent sensor for detection of Cu2+ and Ni2+ Ions.

This article reports an amide based Chemosensor used for selective detection of divalent Cu+2 and Ni+2 ions via Fluorescence turn off. The selective sensing ability of Chemosensor was investigated in presence of different metal ions Mg2+, Ag+, Fe2+, K+, Cu2+, Ni2+, Hg2+, Pb2+, Mn2+, Pd2+, Cd2+ and Mn3+ as competitive ions. The receptor i. e. Chemosensor formed complexes with metal ions in 1:1 stoichiometric ratio. The detection limit and binding constant calculated as 1.92×10-4 and 1.4×10-4 M and 2.16×103 M-1 and 3.09×103 M-1 for Cu2+ and Ni2+ions respectively. The complexes were characterized by UV/visible, FT-IR, 13C NMR and 1H NMR spectroscopy. Further the structure and Crystallinity were calculated by P-XRD spectral analysis. The crystallinity found to be 65.27 and 67.87% respectively.

Miniaturized electrochemical biosensor based on whole-cell for heavy metal ions detection in water.

The heavy metals pollution represents one of the important issues in the environmental field since it is involved in many pathologies from cancer, neurodegenerative, and metabolic diseases. We propose an innovative portable biosensor for the determination of traces of trivalent arsenic (As(III)) and bivalent mercury (Hg(II)) in water. The system implements a strategy combining two advanced sensing modules consisting in (a) a whole cell based on engineered Escherichia coli as selective sensing element towards the metals and (b) an electrochemical miniaturised silicon device with three microelectrodes and a portable reading system. The sensing mechanism relies on the selective recognition from the bacterium of given metals producing the 4-aminophenol redox active mediator detected through a cyclic voltammetry analysis. The miniaturized biosensor is able to operate a portable, robust, and high-sensitivity detection of As(III) with a sensitivity of 0.122 µA ppb-1 , LoD of 1.5 ppb, and a LoQ of 5 ppb. The LoD value is one order of magnitude below of the value indicated to WHO to be dangerous (10 µg/L). The system was proved to be fully versatile being effective in the detection of Hg(II) as well. A first study on Hg(II) showed sensitivity value of 2.11 µA/ppb a LOD value of 0.1 ppb and LoQ value of 0.34 ppb. Also in this case, the detected LOD was 10 times lower than that indicated by WHO (1 ppb). These results pave the way for advanced sensing strategies suitable for the environmental monitoring and the public safety.

Revealing differences in the chemical form of zinc in brain tissue using K-edge X-ray absorption near-edge structure spectroscopy.

Zinc is a prominent trace metal required for normal memory function. Memory loss and cognitive decline during natural ageing and neurodegenerative disease have been associated with altered brain-Zn homeostasis. Yet, the exact chemical pathways through which Zn influences memory function during health, natural ageing, or neurodegenerative disease remain unknown. The gap in the literature may in part be due to the difficulty to simultaneously image, and therefore, study the different chemical forms of Zn within the brain (or biological samples in general). To this extent, we have begun developing and optimising protocols that incorporate X-ray absorption near-edge structure (XANES) spectroscopic analysis of tissue at the Zn K-edge as an analytical tool to study Zn speciation in the brain. XANES is ideally suited for this task as all chemical forms of Zn are detected, the technique requires minimal sample preparation that may otherwise redistribute or alter the chemical form of Zn, and the Zn K-edge has known sensitivity to coordination geometry and ligand type. Herein, we report our initial results where we fit K-edge spectra collected from micro-dissected flash-frozen brain tissue, to a spectral library prepared from standard solutions, to demonstrate differences in the chemical form of Zn that exist between two brain regions, the hippocampus and cerebellum. Lastly, we have used an X-ray microprobe to demonstrate differences in Zn speciation within sub-regions of thin air-dried sections of the murine hippocampus; but, the corresponding results highlight that the chemical form of Zn is easily perturbed by sample preparation such as tissue sectioning or air-drying, which must be a critical consideration for future work.

One-pot fabrication of dual-emission and single-emission biomass carbon dots for Cu2+ and tetracycline sensing and multicolor cellular imaging.

Dual-emission and single-emission carbon dots (DCDs and SCDs) have been simultaneously synthesized by one-pot solvothermal treatment of leek. Different graphitization and surface functionalization were responsible for their distinction in fluorescence characteristics. DCDs with an average size of 5.6 nm exhibited two emissions at 489 and 676 nm under 420-nm excitation. Complexation between DCDs' surface porphyrins and Cu2+ led to quenching of the 676-nm emission, which resulted in the ratiometric determination of Cu2+ with a limit of detection (LOD) of 0.085 µM. SCDs, containing additional sulfur element (0.50%) with an average size of 7.7 nm, presented a single emission at 440 nm under 365-nm excitation. The static quenching and inner filter effects between SCDs and tetracyclines (TCs) made SCDs a fluorescence nanoprobe for TCs' determination with LODs of 0.26-0.48 µM. Applications of DCDs and SCDs for respective determination of Cu2+ and TCs in milk and pig liver samples were successfully demonstrated. Moreover, good photostability, low toxicity, and outstanding biocompatibility made DCDs and SCDs suitable for multicolor cellular imaging. Results indicate that natural products are excellent raw materials to controllably synthesize CDs with prominent physicochemical and fluorescence properties.Graphical abstract.

A label-free lead(II) ion sensor based on surface plasmon resonance and DNAzyme-gold nanoparticle conjugates.

Detection of lead(II) (Pb2+) ions in water is important for the protection of human health and environment. The growing demand for onsite detection still faces challenges for sensitive and easy-to-use methods. In this work, a novel surface plasmon resonance (SPR) biosensor based on GR-5 DNAzyme and gold nanoparticles (AuNPs) was developed. Thiolated DNAzyme was immobilized on the gold surface of the sensor chip followed by anchoring the substrate-functionalized AuNPs through the DNAzyme-substrate hybridization. The coupling between the localized surface plasmon (LSP) of AuNPs and the surface plasmon polaritons (SPP) on the gold sensor surface was used to improve the sensitivity. The substrate cleavage in the presence of Pb2+ ions was catalyzed by DNAzyme, leading to the removal of AuNPs and the diminished LSP-SPP coupling. The optimal detection limit was 80 pM for the sensor fabricated with 1 µM DNAzyme, corresponding to two or three orders of magnitude lower than the toxicity levels of Pb2+ in drinking water defined by WHO and USEPA. By tuning the surface coverage of DNAzyme, the sensitivity and dynamic range could be controlled. This sensor also featured high selectivity to Pb2+ ions and simple detection procedure. Successful detection of Pb2+ ions in groundwater indicates that this method has the prospect in the onsite detection of Pb2+ ions in water. Given the variety of AuNPs and metal-specific DNAzymes, this detection strategy would lead to the development of more sensitive and versatile heavy metal sensors. Graphical abstract.

A colorimetric aptamer-based method for detection of cadmium using the enhanced peroxidase-like activity of Au-MoS2 nanocomposites.

In this work, a colorimetric aptamer-based method for detection of cadmium using gold nanoparticles modified MoS2 nanocomposites as enzyme mimic is established. In short, biotinylated Cd2+ aptamers are immobilized by biotin-avidin binding on the bottoms of the microplate, the complementary strands of Cd2+ aptamers are connected to the Au-MoS2 nanocomposites which have the function of enhanced peroxidase-like activity. The csDNA-Au-MoS2 signal probe and target Cd2+ compete for binding Cd2+ aptamer, the color change can be observed by addition of chromogenic substrate, thereby realizing visual detection of Cd2+. The absorbance of the solution at 450 nm has a clear linear relationship with the Cd2+ concentration. The linear range is 1-500 ng/mL, and the limit of detection is 0.7 ng/mL. The assay was used to test white wine samples, the results are consistent with those of atomic absorption spectrometry; which prove that this method can be used for detection of Cd2+ in real samples.

Sensitive photoelectrochemical assay of Pb2+ based on DNAzyme-induced disassembly of the "Z-scheme" TiO2/Au/CdS QDs system.

Herein, based on DNAzyme-induced disassembly of the "Z-scheme" TiO2/Au/CdS QDs system, a facile and sensitive photoelectrochemical biosensor was developed for lead ion assay and a low detection limit of 0.13 pM was obtained.

Colorimetric detection of mercury ions from environmental water sample by using 3-(Trimethoxysilyl)propyl methacrylate functionalized Ag NPs-tryptophan nanoconjugate.

Recent trend in the nanotechnology made an interest to make nano based system to detect the environmental pollutant including heavy metals, pesticides and antibiotics. Mercury is toxic heavy metals which causes hazardous effect to human and environmental organisms. They usually reach to the environment by mining, petrol refining and coal burning, which can change to its ionic forms according to the environmental condition. The present study was concentrated on the effective detection of Hg (II) ion from environmental sample colorimetrically by developing Ag NPs - tryptophan nanoconjugate functionalized with 3-(Trimethoxysilyl)propyl methacrylate (TMPM). The characterization of prepared particles was performed by UV-visible spectrophotometer, transmission electron microscopy (HRTEM), Fourier-transform infrared spectroscopy (FTIR), X-ray diffractometer (XRD), particle size analyzer and zeta sizer. The size of prepared NPs was 9 ± 1.10 nm and it possess the surface charge of -37.41 ± 4 mV. Upon the interaction of Ag NPs - tryptophan nanoconjugate and Hg (II) ion, the colour of the conjugate disappeared. The effect of environmental factors (Temperature, pH and saline concentration) on Hg (II) ion detection was also investigated. The probe indicated that Ag NPs - tryptophan nanoconjugate functionalized with TMPM was found to be an efficient tool for mercury detection from various environmental water samples.

Magnetically separable and recyclable bamboo-like carbon nanotube-based FRET assay for sensitive and selective detection of Hg2.

The global occurrence of toxic hazards in aquatic ecosystems has aroused concern about the potential impacts on the ecological environment and human health in recent decades. Mercury(II) ions that originate from widespread sources including the mining industry, fossil fuel consumption, and industrial wastes are now well known as a highly toxic pollutant. Despite various detection methods which have been reported to sense Hg2+, it still poses a great challenge for us to develop a new effective sensing platform to replenish current fluorescent detection techniques. Here, we report a novel fluorescent biosensor using bamboo-like magnetic carbon nanotubes (BMCNTs) and FAM-labeled T-rich ssDNA for efficient detection of Hg2+ in aqueous solution. The proposed biosensor shows a good response toward Hg2+ detection over a linear response range of 0.05~1 µM (R2 = 0.98) with a detection limit of 20 nM. It also exhibits the capability to discriminate Hg2+ ions with negligible response to other metal ions, such as Ca2+, Cd2+, Cu2+, Mg2+, Mn2+, Ni2+, Pb2+, and Zn2+. Interestingly, the BMCNTs could be separated and recycled easily by using an external magnet, which means a much more cost-effective, easy-to-operate, and eco-friendly method for Hg2+ ion detection.

Astrocytes in Atp1a2-deficient heterozygous mice exhibit hyperactivity after induction of cortical spreading depression.

The ATP1A2 coding α2 subunit of Na,K-ATPase, which is predominantly located in astrocytes, is a causative gene of familial hemiplegic migraine type 2 (FHM2). FHM2 model mice (Atp1a2tmCKwk/+ ) are susceptible to cortical spreading depression (CSD), which is profoundly related to migraine aura and headache. However, astrocytic properties during CSD have not been examined in FHM2 model mice. Using Atp1a2tmCKwk/+ crossed with transgenic mice expressing G-CaMP7 in cortical neurons and astrocytes (Atp1a2+/- ), we analyzed the changes in Ca2+ concentrations during CSD. The propagation speed of Ca2+ waves and the percentages of astrocytes with elevated Ca2+ concentrations in Atp1a2+/- were higher than those in wild-type mice. Increased percentages of astrocytes with elevated Ca2+ concentrations in Atp1a2+/- may contribute to FHM2 pathophysiology.

A near-infrared fluorescent probe based on a novel rectilinearly π-extended rhodamine derivative and its applications.

We designed and synthesized a novel near-infrared (NIR) mitochondria-targeted fluorescent probe RQNA for the specific detection of mitochondrial Cu2+ because mitochondria are important reservoirs of intracellular copper. For the preparation of this probe, a novel π-extended fluorescent xanthene dye RQN was firstly synthesized via an intramolecular nucleophilic substitution of aromatic hydrogen (SNArH) strategy. Then, probe RQNA was prepared by the reaction of RQN and hydrazine hydrate, followed by treatment with acetone. RQNA exhibited selectivity, sensitivity (22 nM), and fast response time (20 s) for the detection of Cu2+via a specific Cu2+-triggered ring-opening and hydrolysis cascade reaction. RQNA is cell-membrane permeable and mitochondria-targetable, and can be used for monitoring mitochondrial Cu2+ in living cells.