Trisodium Citrate as a Double-Edged Sword: Selective Etching Prussian Blue Analog Nanocubes into Orthogonal Frustums and Their Derivatives for Supercapacitors.

The construction of novel structured Prussian blue analogs (PBAs) by chemical etching has attracted the most attention to PBA derivatives with outstanding performance. In this work, the unprecedented PBA orthogonal frustums are first prepared from nanocubes through a selective chemical etching approach using trisodium citrate as an etchant. The citrate ions can chelate with nickel species from the edges/corners of NiCo-PBA nanocubes and then disintegrate NiCo-PBAs resulting in the generation of NiCo-PBA orthogonal frustums. The derived CoNi2S4/Co0.91S composites still inherit the original orthogonal frustum structure and possess outstanding supercapacitor performance. This study develops a popularized method to construct novel structured PBAs and brings inspiration for designing PBA-based electrodes with advanced electrochemical performance.

Developed rapid and simple RP-HPLC method for simultaneous separation and quantification of bovine milk protein fractions and their genetic variants.

The aim was to develop a reliable rapid reversed-phase high-performance liquid chromatography (RP-HPLC) method to simultaneously determine the main bovine milk protein fractions, including their genetic variants. Compared to the previous studies, our method is able to separate the main protein fractions within 20 min of total run time. The method validation consisted of testing repeatability, reproducibility linearity, repeatability, and accuracy. The procedure was developed using raw individual, bulk, and commercially available heat-treated cow milk samples. The RSD of peak areas ranged from 1.43 to 3.16% within analytical day and from 3.29 to 6.70% across analytical days. The method can be applied to investigate both raw and heat-treated milk samples.

Development and Troubleshooting in Lateral Flow Immunochromatography Assays.

The development of Lateral Flow Immunochromatography Assay can be divided into two levels; standardizing membrane characteristics and optimizing molecular level immunoassay reaction between analyte and detector molecules. In the preliminary phase the reaction specificity of capture and detector antibodies with the analyte has to be checked with other techniques like ELISA. Molarity and pH of conjugation buffer have prime importance in the immunoreaction among analyte and antibodies. Epitope mapping of the capture and detector antibodies is also recommended to confirm the specificity of the assay. Standardization of membrane characteristics directly relates to the sensitivity of the assay through its porosity, hydrophobicity, protein holding/releasing capacity and wicking rate. Under optimised condition a perfect Lateral Flow Immunochromatography Assay should have high on-rate (target binding efficiency), low off-rate (target releasing efficiency) and low Cross-reactivity. In this manuscript, we share our experience, especially on developmental strategies and troubleshooting, that we have experienced during Lateral Flow Immunochromatography Assay kit development.

Effects of emulsifying conditions on creaming effect, mechanical properties and microstructure of processed cheese using a rapid visco-analyzer.

The creaming effects, mechanical properties and microstructures of processed cheeses were investigated under different emulsifying conditions using a rapid visco-analyzer, and the changes in protein network related to the creaming effect and the occurrence of yielding points were discussed. The higher stirring speed affected the fat globules to be smaller, and gave the processed cheese more firmness at fixed stirring time. The longer stirring time caused the protein network to become fine-stranded. A fine-stranded structure promoted the creaming effect, and hence the formation of yielding point in the mechanical properties. The emulsifying salts had active effects on the creaming effect and mechanical properties at longer stirring time. The fine-stranded structures were shown in the cases of a binary mixture of polyphosphate and disodium phosphate (PDSP), polyphosphate (PP), and trisodium citrate (TSC). Monophosphate (MP) showed the lowest ability to alter the protein network, but was assisted at the higher stirring speed.

Continuous hemofiltration model using porcine blood for comparing filter life.

The purpose of the present study was to establish a continuous hemofiltration model using porcine blood to compare filter life. Continuous hemofiltration (CHF) experiments were performed using an in vitro hemofilter evaluation system utilizing porcine blood containing trisodium citrate in addition to nafamostat mesilate as anticoagulants. The lifetime of the hemofilter was evaluated using the transmembrane pressure and the pressure drop across the hemofilter at varying trisodium citrate concentrations. The porcine blood used in this experiment was considered to be in a slightly hypercoagulable state because of the continuous contact with non-biological materials and calcium inflow from substitution fluid. Blood containing 7 or 8 mM of trisodium citrate and nafamostat mesilate could be effectively used to compare the lifetimes of hemofilters utilized under the same conditions. In this CHF model using porcine blood, the plugging of the hollow fibers occurred shortly after the plugging of the membrane pores. In conclusion, a CHF model using porcine blood can be established by adjusting the concentration of trisodium citrate added to the blood.

The Use of Trisodium Citrate to Improve the Textural Properties of Acid-Induced, Transglutaminase-Treated Micellar Casein Gels.

In this study, the effect of trisodium citrate on the textural properties and microstructure of acid-induced, transglutaminase-treated micellar casein gels was investigated. Various concentrations of trisodium citrate (0 mmol/L, 10 mmol/L, 20 mmol/L, and 30 mmol/L) were added to micellar casein dispersions. After being treated with microbial transglutaminase (mTGase), all dispersions were acidified with 1.3% (w/v) gluconodelta-lactone (GDL) to pH 4.4⁻4.6. As the concentration of trisodium citrate increased from 0 mmol/L to 30 mmol/L, the firmness and water-holding capacity increased significantly. The final storage modulus (G') of casein gels was positively related to the concentration of trisodium citrate prior to mTGase treatment of micellar casein dispersions. Cryo-scanning electron microscopy images indicated that more interconnected networks and smaller pores were present in the gels with higher concentrations of trisodium citrate. Overall, when micellar casein dispersions are treated with trisodium citrate prior to mTGase crosslinking, the resulted acid-induced gels are firmer and the syneresis is reduced.

Loss of antimicrobial effect of trisodium citrate due to 'lock' spillage from haemodialysis catheters.

BACKGROUND: Due to its reported antimicrobial effects, hypertonic citrate (46.7%) is a widely used catheter lock solution, but following instillation, citrate inevitably spills into the systemic circulation. This process is mainly driven by hydraulic effects during instillation and density differences between blood and lock solution. Hence, in haemodialysis catheters, intra-luminal citrate concentration ranges from 0% (at the tip in catheters with side holes), 3% (between the side holes and the highest point of the catheter) to 46.7% (at the Luer end) with possible differences in antimicrobial effects. We investigated in vitro the antimicrobial effect of pure citrate 46.7%, citrate 46.7% diluted with saline and blood to a net concentration of 3% (=citrate 3%), and of citrate-free blood, simulating in vivo conditions in different catheter sections. METHODS: Time-kill studies measuring the antimicrobial effect of citrate 46.7%, citrate 3% and citrate-free blood were performed with overnight cultures of Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus). RESULTS: Citrate 46.7% reduced the number of E. coli by 2 log units but after 24 h, 10(6) CFU/mL were still present. Citrate 3% and citrate-free blood had no antimicrobial effect on E. coli. Citrate 46.7%, citrate 3% and citrate-free blood had scarce antimicrobial effect on S. aureus within 24 h. CONCLUSIONS: Spillage of catheter lock solution leading to reduced intra-luminal citrate concentrations considerably reduces the antimicrobial effect of citrate 46.7% on E. coli. As none of the solutions tested had relevant antimicrobial effect on S. aureus, the antimicrobial effect of 46.7% citrate lock solution in vivo has to be seriously questioned.

Synthesized uniform-different sizes silver nanoparticles using TSC and SBH simultaneously for antibacterial application.

Silver nanoparticles (AgNPs) in the form of nanospheres from a few nm to 100 nm in diameter were synthesized in a controlled manner using a combination of two reducing agents: sodium borohydride (SBH) and trisodium citrate (TSC). The influence of the size of AgNPs on antibacterial activity was investigated with different concentrations of AgNPs on two types of bacteria:Pseudomonas aeruginosa(PA) andStaphylococcus aureusresistant (SA) while the positive control wasAmpicillin (Amp)50µg/ml and the negative control was water. AgNPs were investigated for morphology, size and size distribution using transmission electron microscopy (TEM) and dynamic light scattering (DLS) measurements. The optical properties of the AgNPs were investigated by recording their UV-vis absorption spectra. The antimicrobial activity of AgNPs was determined using the disc diffusion method. The results showed that the antibacterial ability of AgNPs depends on both concentration and particle size. With a particle concentration of 50µg ml-1, the antibacterial ability is the best. The smaller the particle size, the higher the antibacterial ability. The simultaneous use of two reducing agents TSC and SBH is the novelty of the article to synthesize AgNPs particles that are uniform in shape and size while controlling the particle size. On that basis, their antibacterial performance is increased.

Initial solution pH value for the construction of a 3D hydroxyapatite via the trisodium citrate-assisted hydrothermal route.

In this study, a trisodium citrate (TSC)-assisted hydrothermal method is utilized to prepare three-dimensional hydroxyapatite (3D HA). Understanding the role of TSC in the preparation of 3D HA crystals may provide valuable methods to design advanced biomaterials. As one of the indexes of solution supersaturation, the initial pH (ipH) value can not only directly affect the nucleation rate, but also affect the growth of HA crystals. In this work, the effect of the ipH on the microstructure, particle size distribution, and specific surface area of the 3D HA is explored. Results showed that the morphology of 3D HA transformed from a bundle to a dumbbell ball and then a dumbbell with an increase in the ipH. A corresponding mechanism of such a structural evolution was proposed, providing inspiration for the fabrication of innovative 3D HA structures with enhanced biological functionality and performance.

A Comparative Evaluation of Antimicrobial and Cytotoxic Efficacy of Biosynthesized Silver Nanoparticles and Chemically Synthesized Silver Nanoparticles Against Enterococcus faecalis: An In Vitro Study.

Introduction Effective root canal cleaning and sealing are essential for a successful endodontic procedure. For the purpose of disinfecting root canals, both herbal and non-herbal medications are recommended. This study aimed to analyze the antimicrobial and cytotoxic properties of biosynthesized silver nanoparticles (AgNPs) synthesized from Azadirachta indica/neem and chemically synthesized AgNPs from trisodium citrate (TSC) against oral pathogens to be further used as an irrigant in endodontic treatment. Materials and methods To synthesize A. indica AgNPs, powdered fresh A. indica leaves were weighed, added to double distilled water, heated for 30 minutes, and then combined with silver nitrate solution. TSC was also used to create TSC AgNPs. X-ray diffraction (XRD), scanning electron microscopy (SEM), ocular observation, and the ultraviolet-visible light (UV-vis) spectrum were used to characterize the AgNPs. Studies were conducted on the extract's characteristics, including its cytotoxicity and antibacterial activity. Results The hue shift and peak on the UV-vis spectrophotometer were signs that AgNPs were forming. The XRD pattern showed that the sample included crystalline AgNPs, mostly spherical ones. By using SEM, the presence of AgNPs was also verified. AgNPs that were synthesized showed antimicrobial efficacy against Enterococcus faecalis. Compared to chemically synthesized AgNPs, A. indica AgNPs showed lower minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) values, a bigger zone of inhibition (ZOI), and less cytotoxic action. Conclusion This study demonstrates the minimal cytotoxicity and antibacterial activity of A. indica AgNPs against E. faecalis. This suggests that they might also be employed as root canal cleaners. Before experimenting with animals or cell lines in clinical trials for endodontic treatment, further research should be done.

3D Printing of Collagen Scaffold with Enhanced Resolution in a Citrate-Modulated Gellan Gum Microgel Bath.

3D printing in a microgel-based supporting bath enables the construction of complex structures with soft and watery biomaterials but the low print resolution is usually an obstacle to its practical application in tissue engineering. Herein, high-resolution printing of a 3D collagen organ scaffold is realized by using an engineered Gellan gum (GG) microgel bath containing trisodium citrate (TSC). The introduction of TSC into the bath system not only mitigates the aggregation of GG microgels, leading to a more homogeneous bath morphology but also suppresses the diffusion of the collagen ink in the bath due to the dehydration effect of TSC, both of which contribute to the improvement of print resolution. 3D collagen organ structures such as hand, ear, and heart are successfully constructed with high shape fidelity in the developed bath. After printing, the GG and TSC can be easily removed by washing with water, and the obtained collagen product exhibits good cell affinity in a tissue scaffold application. This work offers an easy-to-operate strategy for developing a microgel bath for high-resolution printing of collagen, providing an alternative path to in vitro 3D organ construction.

A new approach of magnetic field application in miniaturized pipette-tip extraction for trace analysis of four synthetic hormones in breast milk samples.

Herein, a novel homemade electrical device was designed, including two pieces of external neodymium magnets, providing a reciprocating magnetic field to introduce a magnetic-assisted dispersive pipette-tip micro solid-phase extraction. To evaluate the performance efficiency of the proposed method, a novel magnetic calcined GO/SiO2@Co-Fe nanocube sorbent was synthesized, filled into the pipette-tip, exposed to the reciprocating magnetic field, and applied for the preconcentration of some hormone therapy drugs in human biological matrices. The effective adsorption and desorption parameters were optimized using a rotatable central composite design and one-variable-at-a-time approaches. Under the optimized conditions, the target analytes' detection limits were found to be below 0.02 ng mL-1. Moreover, the calibration curves were linear in the range of 0.03-500.00 ng mL-1 (R2 > 0.9966), with RSDs% less than 7.8 %. Eventually, the established method was applied to extract the analytes from breast milk samples, followed by LC-ESI-MS/MS analysis.

Investigation of Aggregation and Disaggregation of Self-Assembling Nano-Sized Clusters Consisting of Individual Iron Oxide Nanoparticles upon Interaction with HEWL Protein Molecules.

In this paper, iron oxide nanoparticles coated with trisodium citrate were obtained. Nanoparticles self-assembling stable clusters were ~10 and 50-80 nm in size, consisting of NPs 3 nm in size. The stability was controlled by using multi-angle dynamic light scattering and the zeta potential, which was -32 ± 2 mV. Clusters from TSC-IONPs can be destroyed when interacting with a hen egg-white lysozyme. After the destruction of the nanoparticles and proteins, aggregates are formed quickly, within 5-10 min. Their sizes depend on the concentration of the lysozyme and nanoparticles and can reach micron sizes. It is shown that individual protein molecules can be isolated from the formed aggregates under shaking. Such aggregation was observed by several methods: multi-angle dynamic light scattering, optical absorption, fluorescence spectroscopy, TEM, and optical microscopy. It is important to note that the concentrations of NPs at which the protein aggregation took place were also toxic to cells. There was a sharp decrease in the survival of mouse fibroblasts (Fe concentration ~75-100 µM), while the ratio of apoptotic to all dead cells increased. Additionally, at low concentrations of NPs, an increase in cell size was observed.

Surface-modified magnetite nanoparticles affect lysozyme amyloid fibrillization.

BACKGROUND: The surface of nanoparticles (NPs) is an important factor affecting the process of poly/peptides' amyloid aggregation. We have investigated the in vitro effect of trisodium citrate (TC), gum arabic (GA) and citric acid (CA) surface-modified magnetite nanoparticles (COAT-MNPs) on hen egg-white lysozyme (HEWL) amyloid fibrillization and mature HEWL fibrils. METHODS: Dynamic light scattering (DLS) was used to characterize the physico-chemical properties of studied COAT-MNPs and determine the adsorption potential of their surface towards HEWL. The anti-amyloid properties were studied using thioflavin T (ThT) and tryptophan (Trp) intrinsic fluorescence assays, and atomic force microscopy (AFM). The morphology of amyloid aggregates was analyzed using Gwyddion software. The cytotoxicity of COAT-MNPs was determined utilizing Trypan blue (TB) assay. RESULTS: Agents used for surface modification affect the COAT-MNPs physico-chemical properties and modulate their anti-amyloid potential. The results from ThT and intrinsic fluorescence showed that the inhibitory activities result from the more favorable interactions of COAT-MNPs with early pre-amyloid species, presumably reducing nuclei and oligomers formation necessary for amyloid fibrillization. COAT-MNPs also possess destroying potential, which is presumably caused by the interaction with hydrophobic residues of the fibrils, resulting in the interruption of an interface between ß-sheets stabilizing the amyloid fibrils. CONCLUSION: COAT-MNPs were able to inhibit HEWL fibrillization and destroy mature fibrils with different efficacy depending on their properties, TC-MNPs being the most potent nanoparticles. GENERAL SIGNIFICANCE: The study reports findings regarding the general impact of nanoparticles' surface modifications on the amyloid aggregation of proteins.

Inhibitory effect of trisodium citrate on biofilms formed by Klebsiella pneumoniae.

OBJECTIVES: Klebsiella pneumoniae is a significant nosocomial pathogen related to ventilator-associated pneumonia owing to biofilm formation. Trisodium citrate (TSC) has antibacterial activity, but there is little research on the effect of TSC on biofilm formed by K. pneumoniae. The aims of this study were to evaluate the inhibitory effect of 4% TSC on K. pneumoniae biofilm formation and to determine the best time of TSC addition for biofilm inhibition. METHODS: A total of 45 K. pneumoniae strains isolated from tracheal tip and sputum specimens were included. Modified Congo red agar was used to screen for biofilm production. Biofilm-positive strains were cultured for 4 days. TSC (4%) was added either initially or 3 days later. Crystal violet staining was used to quantify biofilm mass by measuring the optical density at 570 nm (OD570). Scanning electron microscopy (SEM) was used to observe biofilm morphology. RESULTS: The OD570 was significantly lower in the 4% TSC group than that in the no-TSC group during the 4-day experiment. Compared with addition of TSC after 3 days, initial TSC addition resulted in a significant absorbance decrease (Day 4, 0.63 ± 0.11 later-TSC group vs. 0.41 ± 0.16 initial-TSC group). As observed by SEM, bacteria were stacked most densely in the no-TSC group on Day 4. In contrast, few bacteria were observed when TSC was added initially, whilst bacteria were obviously dispersed when TSC was added after 3 days. CONCLUSION: TSC can inhibit K. pneumoniae biofilm formation and has the best effect when added initially.

Anticoagulants Interfere With the Angiogenic and Regenerative Responses Mediated by Platelets.

BACKGROUND AND AIMS: Platelet rich plasma (PRP) obtained from blood anticoagulated with acid-citrate-dextrose (ACD) or sodium-citrate (SC) is used for regenerative medicine as source of platelet-derived growth factors. Allergic reactions against citrate were reported in patients after local injection of PRP allowing us to hypothesize that anticoagulants exert a harmful and local effect that interferes with the regenerative proprieties of platelets. Herein we test this hypothesis by analyzing the effect of ACD and SC on angiogenic and regenerative responses mediated by platelets. METHODS: PRP was obtained from SC- or ACD-anticoagulated blood; platelets were lysed to release growth factors; and PRP releasates (PRPr) were used to induce in vitro endothelial proliferation and 2D-migration, and regeneration of mouse skin wounds. RESULTS: We first compared proliferation and migration of endothelial cells mediated by anticoagulated-PRPr supplemented or not with CaCl2. Alteration of endothelial adhesion and impediment of proliferation and migration was observed without CaCl2. Although endothelial morphology was normalized in SC- and ACD-PRPr after calcium restitution, angiogenic responses were only markedly induced by SC-PRPr. In vivo studies revealed a delay in mouse skin regeneration after treatment with anticoagulated-PRPr without CaCl2. Healing was only induced after calcium restitution in SC- but ACD-PRPr. Moreover, the development of inflammatory intradermal papules was evidenced after injection of ACD-PRPr. Supplementation of SC-PRPr with the equivalent concentration of dextrose (D-Glucose, 18 mM) present in ACD-PRPr resulted in reduction of endothelial proliferation and migration, delay of mouse skin regeneration and development of intradermal papules. Finally, collecting blood with half amount of SC significantly improved all the angiogenic and regenerative responses mediated by PRPr. In contrast, the delay of skin regeneration and the development of inflammatory papules remained stable after dilution of ACD. CONCLUSION: Our findings indicate that (1) calcium restitution is required to impair the cellular and tissue alterations induced by citrated-anticoagulants contained in PRP; (2) ACD-derived dextrose confers anti-angiogenic, anti-regenerative and pro-inflammatory proprieties to PRP; and (3) half concentration of SC improves the angiogenesis and regeneration mediated by PRP.

An improved LC-MS method to profile molecular diversity and quantify the six main bovine milk proteins, including genetic and splicing variants as well as post-translationally modified isoforms.

Here we describe a method based on Liquid Chromatography coupled with Mass Spectrometry (LC-MS) that provides an accurate determination of the six main bovine milk proteins, including allelic and splicing variants, as well as isoforms resulting from post-translational modifications, with an unprecedented level of resolution. Proteins are identified from observed molecular masses in comparison with theoretical masses of intact proteins indexed in an "in-house" database that includes nearly 3000 entries. Quantification was performed either from UV (214 nm) or mass signals. Thus, up to one hundred molecules, derived from the six major milk proteins, can be identified and quantified from an individual milk sample. This powerful and reliable method, initially developed as an anchoring method to estimate the composition of the six main bovine milk proteins from MIR spectra, is transferable to several mammalian species, including small ruminants, camels, equines, rabbits, etc., for which specific mass databases are available.

A Density-Changing Centrifugation Method for Efficient Separation of Free Drugs from Drug-Loaded Particulate Delivery Systems.

Commonly used separation techniques, such as ultracentrifugation, chromatography, and membrane separation, have inherent drawbacks that limit their usage. Herein, we introduced a new separation method, density-changing centrifugation (DCC), which is based on trisodium citrate (TC) and ultracentrifugation. Paclitaxel-loaded cationic solid lipid nanoparticles (SLNs/PTX) and doxorubicin-loaded PEGylated liposomes (Lipo/Dox) were prepared as model drug delivery particulates. After optimizing TC concentration and centrifugal conditions, DCC showed superior separation efficiency and accuracy over common ultracentrifugation and ultrafiltration methods and displayed comparable or even better separation efficiency compared with size-exclusion chromatography, as demonstrated by the determination of encapsulation efficiency, Tyndall effect, transmittance, and drug recovery. DCC was also proven to minimally impact the size distribution, surface morphology, and thermal properties of the nanoparticles and liposomes, and moreover, it did not affect the determination of drug concentrations. Together, DCC has been demonstrated as a neat and effective method for the separation of free drugs from drug-loaded SLNs and liposomes, which shall be of great benefit for the development of particulate based delivery systems.

The application of forward osmosis for simulated surface water treatment by using trisodium citrate as draw solute.

In this study, trisodium citrate was used as draw solute in forward osmosis (FO) due to its biodegradability and easy reuse after FO dilution. The effect of operating conditions on FO performance was investigated. The study focused on the long-term flux performance and membrane fouling when surface water was used as feed solution. A water flux of 9.8 LMH was observed using 0.5 M trisodium citrate as draw solution in PRO mode. In the long-term FO process, trisodium citrate showed a slight decrease in total flux loss (13.06%) after 20 h of operation. The membrane fouling was significantly reduced after a two-step physical cleaning. A considerable flux recovery (> 95%) of the fouled membrane was finally obtained. Therefore, this study proves the superiority of trisodium citrate as draw solution and paves a new way in applying FO directly for surface water reclamation.

Phase Separation and Collection of Annular Flow by Phase Transformation.

A polyethylene glycol/citrate mixed solution was fed into a single channel of a Y-type micro-channel on a microchip as an aqueous two-phase system. A phase separation multi-phase flow with a liquid-liquid interface was generated due to a phase transformation. An annular flow, one of the flow types in the phase separation multi-phase flow, was observed through bright-field microscopy. The flow consisted of citrate-rich inner and polyethylene glycol-rich outer phases. We attempted to separate and collect the two phases in the single channel into two separate Y-type channels. When the pressure losses for the separated channels were not very different, we observed symmetric flow in the Y-type channel. When the pressure losses were quite different, the polyethylene glycol-rich phase with higher viscosity was selectively distributed to the separated channel with lower pressure loss. Thus, the polyethylene glycol-rich phase was successfully and intentionally collected from the chosen Y-type channel via the creation of annular flow in the single channel.