Perls' Prussian Blue Stains of Lung Tissue, Bronchoalveolar Lavage, and Sputum.

Perls' Prussian blue (PPB) stain recognizes Fe3+ associated with hemosiderin. The employment of this stain in clinical medicine and research has been extensive and novel applications continue to evolve. Ferruginous bodies are intracellular structures in lung tissue, bronchoalveolar lavage (BAL), and sputum that stain with PPB. Inhaled, insoluble, biopersistent particles and fibers are phagocytosed by lung macrophages and thought to be coated, either partially or completely, with an iron-containing protein at the interface forming a ferruginous body. These structures can be categorized as ferruginous bodies having either an inorganic or a carbonaceous core (e.g., asbestos and byssinotic bodies, respectively). In lung tissue, BAL, and sputum, the only cells that stain with PPB are macrophages. These are described as iron- and hemosiderin-laden macrophages and called either siderophages or sideromacrophages. Siderophages can be observed in the lung tissue, BAL, and sputum after various exposures and can also be associated with many different pulmonary and extrapulmonary diseases.

Enhancing Enzyme-like Activities of Prussian Blue Analog Nanocages by Molybdenum Doping: Toward Cytoprotecting and Online Optical Hydrogen Sulfide Monitoring.

Artificial nanozymes have been designed to solve the problems of high cost and poor stability involving natural enzymes in analytical applications. Nevertheless, the catalytic efficiency of the nanozyme still needs to be improved so that it can meet the stability and sensitivity requirements of continuous biological detection. We presented an effective tailoring strategy to enhance the enzyme-like activities of Prussian-blue-analog-based nanozymes. Molybdenum-polysulfide-deposited nickel-iron bimetal Prussian-blue-analog-based hollow nanocages (Nanocages) with peroxidase-, catalase-, and laccase-mimicking activities were synthesized. The doping of molybdenum successfully tailored the size, morphology, composition, and complex structure of the Nanocage, and the peroxidase- and laccase-mimicking activities of the Nanocage nanozyme were enhanced by over 37 and 27 times, respectively, compared with pristine Prussian blue analogs. Moreover, in environments of harsh pH, high temperature, and high salt concentration, Nanocages exhibited much higher stability than natural enzymes. The peroxidase- and catalase-mimicking activities were applied to eliminate reactive oxygen species in cells, whereas the laccase-like activity of Nanocages was integrated with an online sensing platform for in vivo and continuous optical hydrogen sulfide monitoring in the brains of living rats. Our findings may provide possibilities for advancing the design strategy of highly active nanozymes as well as nanozyme-based in vivo detection methods and will offer unique opportunities for their involvement in bioanalytical chemistry.

An optical sensing platform based on hexacyanoferrate intercalated layered double hydroxide nanozyme for determination of chromium in water.

In this work, hexacyanoferrate intercalated Ni/Al LDH (Ni/Al-Fe(CN)6 LDH) nanozyme was synthesized by one-pot co-precipitation method and used for determination of chromium in water samples by employing its peroxidase mimicking activity. The synthesized nanozyme can effectively catalyze the oxidation of fluorometric peroxidase substrate terephthalic acid by H2O2 to produce a highly fluorescent product. It was found that Cr(VI) promotes the peroxidase-like activity of Ni/Al-Fe(CN)6 LDH and this effect was intensified by increasing the Cr(VI) concentration. Several variables affecting the fluorescence intensity including the concentration of nanoparticles and reagents as well as reaction time were investigated and optimized. Under the optimal conditions, good linearity was observed in the range of 0.067-10 µM Cr(VI), and limit of detection and quantification were found to be 0.039 and 0.131 µM, respectively. Furthermore, the developed method showed good applicability for the determination of total Cr based on the oxidation of Cr (III) to Cr (VI). The applicability of the proposed method was demonstrated by analyzing various environmental water samples. The presented nanozyme displayed superior benefits in terms of reusability, repeatability, cost and environment-friendly features. The present work aims to expand LDHs based enzyme mimics to optical sensor fields.

Electrons selective uptake of a metal-reducing bacterium Shewanella oneidensis MR-1 from ferrocyanide.

The extracellular electron transfer of Shewanella oneidensis MR-1 (MR-1) has been extensively studied due to the importance of the biosensors and energy applications of bioelectrochemical systems. However, the oxidation of metal compounds by MR-1, which represents the inward extracellular electron transfer from extracellular electron donors into the microbe, is barely understood. In this study, MR-1 immobilized on an electrode electrocatalyzes the oxidation of [Fe(CN)6]4- to [Fe(CN)6]3- efficiently and selectively. The selectivity depends on midpoint potential and overall charge(s) of redox molecules. Among 12 investigated redox molecules, the negatively charged molecules with high midpoint potentials, i.e., [Ru(CN)6]4- and [Fe(CN)6]4-, show strong electrocatalysis. Neither reference bacteria (Escherichia coli K-12 nor Streptococcus mutans) electrocatalyze the oxidation of [Fe(CN)6]4-. The electrocatalysis decays when MR-1 is covered with palladium nanoparticles presumptively involved with cytochromes c. However, cytochromes c MtrC and OmcA on MR-1 do not play an essential role in this process. The results support a model that [Fe(CN)6]4- donor electrons to MR-1 by interacting with undiscovered active sites and the electrons are subsequently transferred to the electrode through the mediating effect of [Fe(CN)6]4-/3-. The selective electron uptake by MR-1 provides valuable and fundamental insights of the applications of bioelectrochemical systems and the detection of specific redox molecules.

An in situ microenvironmental nano-regulator to inhibit the proliferation and metastasis of 4T1 tumor.

Tumor microenvironment, such as hypoxia and presence of immune cells, plays a critical role in cancer initiation, growth as well as progression, and seriously affects antitumor effect. Accordingly, we constructed a kind of multifunctional nanoparticles (NPs) with macrophage transformation and oxygen (O2) generation characteristics, to regulate the tumor microenvironment. Methods: In this study, we synthesized mesoporous Prussian blue (MPB) NPs with low molecular weight hyaluronic acid (LMWHA) surface modification (LMWHA-MPB), and discovered that LMWHA-MPB could be used as an in situ macrophages converter and O2 generator. Results:In vitro results showed after uptake by M2 macrophages, LMWHA-MPB displayed the potential in remodeling tumor-associated macrophages (TAMs) phenotype (pro-tumor M2→anti-tumor M1), and anti-metastatic effect on 4T1 cells. Furthermore, in vivo visualized near-infrared (NIR) imaging data proved IR783 labeled LMWHA-MPB NPs could selectively accumulate in tumor sites. Then plenty of O2 generated to alleviate tumor hypoxia via catalytic decomposition of endogenous hydrogen peroxide (H2O2). Based on these outstanding characteristics, LMWHA-MPB NPs were adopted as multifunctional nanocarriers to load sonosensitizer hematoporphyrin monomethyl ether (HMME) for O2 self-provided sonodynamic therapy (SDT). In vivo anti-tumor results showed LMWHA-MPB/HMME could effectively inhibit the proliferation and metastasis of 4T1 tumors by improving tumor microenvironment. Conclusion: The multifunctional NPs can be used as in situ microenvironmental nano-regulators to inhibit the proliferation and metastasis of 4T1 tumor.

Separation of cesium from wastewater with copper hexacyanoferrate film in an electrochemical system driven by microbial fuel cells.

A electrochemical adsorption system driven by microbial fuel cell (MFC-adsorption) was developed based on copper(II) hexacyanoferrate(III) (CuHCF) film for cesium (Cs) removal from wastewater. Cs uptake and elution can be simply controlled by regulating the redox states of the CuHCF films. Chemical oxygen demand (COD) removal showed little difference as MFC was connected to adsorption system. Meanwhile, power density and coulombic efficiency of MFC were dramatically reduced. The efficiencies of Cs adsorption and desorption were undesirable. MFC-adsorption technology used for actual nuclear wastewater treatment still has far to go.

Detoxification of ferrocyanide in asoil-plant system.

The detoxification of iron cyanide in a soil-plant system was investigated to assess the total cyanide extracted from contaminated soil and allocated in the leaf tissue of willow trees (Salix caprea). They were grown in soil containing up to 1000 mg/kg dry weight (dw) of cyanide (CN), added as 15N-labeled potassium ferrocyanide and prepared with a new method for synthesis of labeled iron cyanides. CN content and 15N enrichment were monitored weekly over the exposure in leaf tissue of different age. The 15N enrichment in the young and old leaf tissue reached up to 15.197‰ and 9063‰, respectively; it increased significantly over the exposure and with increasing exposure concentrations (p < 0.05). Although the CN accumulation in the old leaf tissue was higher, compared to the young leaf tissue (p < 0.05), the 15N enrichment in the two tissue types did not differ statistically. This indicates a non-uniform CN accumulation but a uniform 15N allocation throughout the leaf mass. Significant differences were detected between the measured CN content and the C15N content, calculated from the 15N enrichment (p < 0.05), revealing a significant CN fraction within the leaf tissue, which could not be detected as ionic CN. The application of labeled iron CN clearly shows that CN is detoxified during uptake by the willows. However, these results do not exclude other detoxification pathways, not related to the trees. Still, they are strongly indicative of the central role the trees played in CN removal and detoxification under the experimental conditions.

Electro-addressable conductive alginate hydrogel for bacterial trapping and general toxicity determination.

In biosensors development, alginate hydrogels are a first choice for enabling stable biomolecules entrapment in biocompatible membranes obtained under soft physiological conditions. Although widely exploited, most alginate membranes are isolating and poorly repetitive, which limit their application in biosensing. Significant steps forward on improving repeatability and conductivity have been performed, but to date there is no single protocol for controlled deposition of live cells in replicable conductive alginate layers. Here, cell electrotrapping in conductive alginate hydrogels is examined in order to overcome these limitations. Conductive alginate-coated electrodes are obtained after potentiostatic electrodeposition of graphite-doped alginate samples (up to 4% graphite). The presence of graphite reduces electrode passivation and improves the electrochemical response of the sensor, although still significantly lower than that recorded with the naked electrode. Bacterial electrotrapping in the conductive matrix is highly efficient (4.4 × 107 cells per gel) and repetitive (CV < 0.5%), and does not compromise bacterial integrity or activity (cell viability = 56%). Biosensing based on ferricyanide respirometry yielded a four times increase in biosensor response with respect to non-conductive alginate membrane, providing toxicity values completely comparable to those reported. Cell electrotrapping in conductive hydrogels represents a step forward towards in high-sensitive cell-based biosensors development with important influence in environmental analysis, food and beverage industry as well as clinical diagnosis.

Catalytically Synthesized Prussian Blue Nanoparticles Defeating Natural Enzyme Peroxidase.

We synthesized Prussian Blue (PB) nanoparticles through catalytic reaction involving hydrogen peroxide (H2O2) activation. The resulting nanoparticles display the size-dependent catalytic rate constants in H2O2 reduction, which are significantly improved compared to natural enzyme peroxidase: for PB nanoparticles 200 nm in diameter, the turnover number is 300 times higher; for 570 nm diameter nanoparticles, it is 4 orders of magnitude higher. Comparing to the known peroxidase-like nanozymes, the advantages of the reported PB nanoparticles are their true enzymatic properties: (1) enzymatic specificity (an absence of oxidase-like activity) and (2) an ability to operate in physiological solutions. The ultrahigh activity and enzymatic specificity of the catalytically synthesized PB nanoparticles together with high stability and low cost, obviously peculiar to noble metal free inorganic materials, would allow the substitution of natural and recombinant peroxidases in biotechnology and analytical sciences.

An insoluble iron complex coated cathode enhances direct electron uptake by Rhodopseudomonas palustris TIE-1.

Microbial electrosynthesis (MES) is a promising bioelectrochemical approach to produce biochemicals. A previous study showed that Rhodopseudomonas palustris TIE-1 can directly use poised electrodes as electron donors for photoautotrophic growth at cathodic potentials that avoid electrolytic H2 production (photoelectroautotrophy). To make TIE-1 an effective biocatalyst for MES, we need to improve its electron uptake ability and growth under photoelectroautotrophic conditions. Because TIE-1 interacts with various forms of iron while using it as a source of electrons for photoautotrophy (photoferroautotrophy), we tested the ability of iron-based redox mediators to enhance direct electron uptake. Our data show that soluble iron cannot act as a redox mediator for electron uptake by TIE-1 from a cathode poised at +100mV vs. Standard Hydrogen electrode. We then tested whether an immobilized iron-based redox mediator Prussian blue (PB) can enhance electron uptake by TIE-1. Chronoamperometry indicates that cathodic current uptake by TIE-1 increased from 1.47±0.04 to 5.6±0.09µA/cm2 (3.8 times). Overall, our data show that immobilized PB can enhance direct electron uptake by TIE-1.

Electrochemical System for the Study of Trans-Plasma Membrane Electron Transport in Whole Eukaryotic Cells.

The study of trans-plasma membrane electron transport (tPMET) in oncogenic systems is paramount to the further understanding of cancer biology. The current literature provides methodology to study these systems that hinges upon mitochondrial knockout genotypes in conjunction with cell surface oxygen consumption, or the detection of an electron acceptor using colorimetric methods. However, when using an iron redox based system to probe tPMET, there is yet to be a method that allows for the simultaneous quantification of iron redox states while providing an exceptional level of sensitivity. Developing a method to simultaneously analyze the redox state of a reporter molecule would give advantages in probing the underlying biology. Herein, we present an electrochemical based method that allows for the quantification of both ferricyanide and ferrocyanide redox states to a highly sensitive degree. We have applied this system to a novel application of assessing oncogenic cell-driven iron reduction and have shown that it can effectively quantitate and identify differences in iron reduction capability of three lung epithelial cell lines.

Interaction of Prussian blue nanoparticles with bovine serum albumin: a multi-spectroscopic approach.

Owning to their exceptional properties, Prussian blue nanoparticles (PBNPs) are promising in a variety of biomedical applications. In this scenario, understanding of how PBNPs interact and behave in biological systems is essential. Herein, the interaction of PBNPs with protein was investigated. Specifically, the citric acid stabilized PBNPs with a size of 10 nm were synthesized and characterized. The interactions of these PBNPs with the model protein, bovine serum albumin (BSA), were then probed by spectroscopic methods. It was found that the BSA intrinsic fluorescence was quenched upon addition of PBNPs due to the static interaction, suggesting the binding of PBNPs with BSA. Moreover, the synchronous fluorescence and circular dichroism spectra indicated the conformational change of BSA due to the presence of PBNPs.

Biosynthesis regulation of natamycin production from Streptomyces natalensis HDMNTE-01 enhanced by response surface methodology.

Natamycin has been widely applied in medical treatments and food protection widely due to its effective inhibition to the growth of yeast and mold. As polyene macrolide antibiotic, the biosynthesis pathway of natamycin is relatively clear. To regulate the biosynthesis of natamycin, additions of precursors affecting cell growth and natamycin production were investigated. The results showed that 0.003% (w/v) potassium ferrocyanide and sodium propionate: n-butanol at a ratio of 4:1 was added into the broth at 0 and 24 hr, respectively, and they contributed to yield natamycin, reaching 1.62 g L-1 (174.6% higher than control). Furthermore, response surface methodology was undertaken to enhance natamycin production by Streptomyces natalensis HDMNTE-01 (a wild strain). The optimum conditions determined were: glucose 3.97%; soya peptone 2%; yeast extract 0.5%; original pH 7.0; inoculum volume 6%; growth in a 250-mL flask containing 24.68 mL of medium; shaken (220 rpm) at 28°C for 4 days. Under the optimized conditions, the yield was 2.81 g L-1 natamycin in 5-L fermentor when the fermentation was processed.

Oscillatory Reaction Induced Periodic C-Quadruplex DNA Gating of Artificial Ion Channels.

Many biological ion channels controlled by biochemical reactions have autonomous and periodic gating functions, which play important roles in continuous mass transport and signal transmission in living systems. Inspired by these functional biological ion channel systems, here we report an artificial self-oscillating nanochannel system that can autonomously and periodically control its gating process under constant conditions. The system is constructed by integrating a chemical oscillator, consisting of BrO3-, Fe(CN)64-, H+, and SO32-, into a synthetic proton-sensitive nanochannel modified with C-quadruplex (C4) DNA motors. The chemical oscillator, containing H+-producing and H+-consuming reactions, can cyclically drive conformational changes of the C4-DNA motors on the channel wall between random coil and folded i-motif structures, thus leading to autonomous gating of the nanochannel between open and closed states. The autonomous gating processes are confirmed by periodic high-low ionic current oscillations of the channel monitored under constant reaction conditions. The utilization of a chemical oscillator integrated with DNA molecules represents a method to directly convert chemical energy of oscillating reactions to kinetic energy of conformational changes of the artificial nanochannels and even to achieve diverse autonomous gating functions in artificial nanofluidic devices.

New method for characterizing electron mediators in microbial systems using a thin-layer twin-working electrode cell.

Microbial biofilms are significant ecosystems where the existence of redox gradients drive electron transfer often via soluble electron mediators. This study describes the use of two interfacing working electrodes (WEs) to simulate redox gradients within close proximity (250µm) for the detection and quantification of electron mediators. By using a common counter and reference electrode, the potentials of the two WEs were independently controlled to maintain a suitable "voltage window", which enabled simultaneous oxidation and reduction of electron mediators as evidenced by the concurrent anodic and cathodic currents, respectively. To validate the method, the electrochemical properties of different mediators (hexacyanoferrate, HCF, riboflavin, RF) were characterized by stepwise shifting the "voltage window" (ranging between 25 and 200mV) within a range of potentials after steady equilibrium current of both WEs was established. The resulting differences in electrical currents between the two WEs were recorded across a defined potential spectrum (between -1V and +0.5V vs. Ag/AgCl). Results indicated that the technique enabled identification (by the distinct peak locations at the potential scale) and quantification (by the peak of current) of the mediators for individual species as well as in an aqueous mixture. It enabled a precise determination of mid-potentials of the externally added mediators (HCF, RF) and mediators produced by pyocyanin-producing Pseudomonas aeruginosa (WACC 91) culture. The twin working electrode described is particularly suitable for studying mediator-dependent microbial electron transfer processes or simulating redox gradients as they exist in microbial biofilms.

Thiamine biosensor based on oxidative trapping of enzyme-substrate intermediate.

In the present work, we describe a new thiamine amperometric biosensor based on thiamine pyrophosphate (ThDP)-dependent transketolase (TK)-catalyzed reaction, followed by the oxidative trapping of TK intermediate α,ß-dihydroxyethylthiamine diphosphate (DHEThDP) within the enzymatic active site. For the biosensor design purpose, TK from Escherichia coli (TKec) was immobilized in Mg2Al-NO3 Layered Double Hydroxides (LDH) and the electrochemical detection was achieved with the TKec/LDH modified glassy carbon electrode (GCE). The transduction process was based on the ability of Fe(CN)63- to oxidize DHEThDP to glycolic acid along with ThDP regeneration. The released Fe(CN)64- was re-oxidized at +0.5V vs Ag-AgCl and the reaction was followed by chronoamperometry. The TKec/LDH/GCE biosensor was optimized using the best TK donor substrates, namely l-erythrulose and d-fructose-6-phosphate. ThDP was assayed with great sensitivity (3831mAM-1cm-2) over 20-400nM linear range.

LA-ICP-MS Allows Quantitative Microscopy of Europium-Doped Iron Oxide Nanoparticles and is a Possible Alternative to Ambiguous Prussian Blue Iron Staining.

The development of iron oxide nanoparticles for biomedical applications requires accurate histological evaluation. Prussian blue iron staining is widely used but may be unspecific when tissues contain substantial endogenous iron. Here we tested whether microscopy by laser ablation coupled to inductively coupled plasma mass spectrometry (LA-ICP-MS) is sensitive enough to analyze accumulation of very small iron oxide particles (VSOP) doped with europium in tissue sections. For synthesis of VSOP, a fraction of Fe3+ (5 wt%) was replaced by Eu3+, resulting in particles with 0.66 mol% europium relative to iron (Eu-VSOP) but with otherwise similar properties as VSOP. Eu-VSOP or VSOP was intravenously injected into ApoE-/- mice on Western cholesterol diet and accumulated in atherosclerotic plaques of these animals. Prussian blue staining was positive for ApoE-/- mice with particle injection but also for controls. LA-ICP-MS microscopy resulted in sensitive and specific detection of the europium of Eu-VSOP in liver and atherosclerotic plaques. Furthermore, calibration with Eu-VSOP allowed calculation of iron and particle concentrations in tissue sections. The combination of europium-doped iron oxide particles and LA-ICP-MS microscopy provides a new tool for specific and quantitative analysis of particle distribution at the tissue level and allows correlation with other elements such as endogenous iron.

Long-term ferrocyanide application via deicing salts promotes the establishment of Actinomycetales assimilating ferrocyanide-derived carbon in soil.

Cyanides are highly toxic and produced by various microorganisms as defence strategy or to increase their competitiveness. As degradation is the most efficient way of detoxification, some microbes developed the capability to use cyanides as carbon and nitrogen source. However, it is not clear if this potential also helps to lower cyanide concentrations in roadside soils where deicing salt application leads to significant inputs of ferrocyanide. The question remains if biodegradation in soils can occur without previous photolysis. By conducting a microcosm experiment using soils with/without pre-exposition to road salts spiked with (13) C-labelled ferrocyanide, we were able to confirm biodegradation and in parallel to identify bacteria using ferrocyanide as C source via DNA stable isotope probing (DNA-SIP), TRFLP fingerprinting and pyrosequencing. Bacteria assimilating (13) C were highly similar in the pre-exposed soils, belonging mostly to Actinomycetales (Kineosporia, Mycobacterium, Micromonosporaceae). In the soil without pre-exposition, bacteria belonging to Acidobacteria (Gp3, Gp4, Gp6), Gemmatimonadetes (Gemmatimonas) and Gammaproteobacteria (Thermomonas, Xanthomonadaceae) used ferrocyanide as C source but not the present Actinomycetales. This indicated that (i) various bacteria are able to assimilate ferrocyanide-derived C and (ii) long-term exposition to ferrocyanide applied with deicing salts leads to Actinomycetales outcompeting other microorganisms for the use of ferrocyanide as C source.

Colorimetric method to detect ε-poly-l-lysine using glucose oxidase.

We describe a new colorimetric assay method using glucose oxidase (GOx) to detect ε-poly-l-lysine (εPL). This method uses εPL's remarkable effect of promoting the enzymatic reaction of GOx with ferricyanide ion. This reaction reduces ferricyanide ion to ferrocyanide ion, accompanied by a color change from yellow to colorless. In this colorimetric assay, the detection limit of εPL was estimated to be approximately 0.5 mg/L when purified εPL samples were used. εPL has usually been produced by a fermentation process using Streptomyces albulus species. The components of the culture broth showed interference effects against the assay method. However, due to the high sensitivity of the assay method for εPL, εPL could be detected in the culture broth without any pretreatment. The detectable concentration of εPL in the culture broth, cPL,ac, was estimated to be approximately 20 mg/L. By combining the Berlin blue reaction with this method, the cPL,ac was reduced to 10 mg/L. In light of the proposed method's simplicity and sensitivity, it could be useful for screening εPL synthetic enzymes and microorganisms.

A Multifunctional Theranostic Nanoagent for Dual-Mode Image-Guided HIFU/Chemo- Synergistic Cancer Therapy.

High-intensity focused ultrasound (HIFU) is deemed to be a promising noninvasive therapeutic modality for cancers as well as non-neoplastic diseases. However, the accuracy of the technique in the diagnosis and treatment of tumors remains unsatisfactory. HIFU, when combined with multifunctional synergistic agents (SAs), has the potential to be of greater diagnostic and therapeutic efficacy. Here we describe a smart and multifunctional hollow mesoporous Prussian blue (HMPBs) theranostic nanoplatform, the hollow structure of which is capable of encapsulating doxorubicin (DOX) and perfluorohexane (HMPBs-DOX/PFH). In vitro and in vivo studies validated that HMPBs-DOX/PFH can be used as an amplifiable dual-mode imaging contrast agent, which can simultaneously enhance ultrasound (US) and photoacoustic (PA) imaging for guiding and monitoring tumor therapy. When exposed to HIFU, this versatile HMPBs-DOX/PFH agent could increase the cavitation effect and use lower HIFU intensity to achieve coagulative necrosis. Furthermore, it significantly accelerated the release of DOX thereby enhancing chemotherapeutic efficacy and avoiding systemic side effects of the drug. Such a novel theranostic nanoplatform is expected to integrate dual-mode guided imaging with greater therapeutic efficacy and fewer side effects and is very promising for the noninvasive synergistic tumor therapy.