Layer-by-layer self-assembled polyelectrolyte multilayers with embedded liposomes: immobilized submicronic reactors for mineralization

The development of chemical reactions in nanospaces is of paramount importance for the development of active nanodevices, particularly in nanofluidics. It has been shown in a previous paper that phospholipid vesicles can be incorporated without spontaneous bilayer rupture into poly-L-glutamic acid/poly(allylamine) (PGA/PAH) multilayered polyelectrolyte films. The aim of the present study was to use such a system as an "embedded submicronic reactor" able to trigger precipitation of calcium phosphates within closed spaces through an enzymatic reaction, the enzyme also being encapsulated in the vesicle interior. To this aim, large unilamellar vesicles (LUVs) were produced containing calcium ions as active ions in the mineralization process, spermine as an activator of crystal growth, and alkaline phosphatase as a catalyst to convert phosphate esters into phosphates. After stabilization by adding a layer of poly-(D-lysine), these vesicles were embedded in a (PGA-PAH)n film. A paranitrophenyl phosphate containing solution was then put in contact with this film. It is shown by means of infrared spectroscopy in the attenuated total reflection mode that, consecutively to this contact, calcium phosphates are growing inside the embedded vesicles. By using scanning near-field fluorescence microscopy, it is demonstrated that the alkaline phosphatase enzymes are most probably located inside the vesicles after their embedding. In addition, atomic force microscopy was used to show, after chemical removal of the organic top layer of the film, that the inorganic platelets produced after the precipitation reaction are localized in volumes of similar size and shape as that of the vesicles into which the phosphate ester hydrolysis and subsequent precipitation reaction did occur.     

Formation of calcium phosphates in gelatin with a novel diffusion system

The present paper demonstrated a novel and simple diffusion system to precipitate calcium phosphates in gelatin gel. In this system, a gelatin cup was specially used as the membrane separating reservoirs of calcium and phosphate ions. Relative to the conventional diffusion system, the novel one in our experiment decreased the time required for the deposition from 5-7 days to 20 h and increased the amount of the precipitated mineral phases significantly. The influence of pH values and concentrations of calcium and phosphate solutions buffered with Tris-HCl and NaOH, respectively, was investigated. The results showed that precipitation of the mineral phase at low pH values (7 for calcium and 11 for phosphate) and concentrations (200 mM for calcium and 15 mM for phosphate) resulted in the formation of plate-like octacalcium phosphate (OCP) crystals. With increasing the pH values of calcium and phosphate solutions to 8 and 12, respectively, spherical amorphous calcium phosphate (ACP) particles were obtained uniquely. Furthermore, flower-like hydroxyapatite (HAP) aggregates composed of many nano-sized needles were formed from the solutions with high pH values (8 for calcium and 12 for phosphate) and concentrations (500 mM for calcium and 37.5 mM for phosphate). The novel diffusion system is proposed to play an important role in both studying the process of biological mineralization and synthesizing calcium phosphates in different forms.     

Nematic ordered cellulose templates mediating order-patterned deposition accompanied with synthesis of calcium phosphates

In a previous study, the nematic ordered cellulose (NOC) templates successfully induced biodirected epitaxial nanodeposition of cellulose nanofibers secreted by Gluconacetobacter xylinus along the orientation of the molecular tracks (Kondo et al. 2002). As an extended concept for the NOC, this article attempts to propose a sort of biomimic mineralization using the template. It combines morphologically controlling process with synthesis of the calcium phosphate as a major component of bones. This process was initially mediated by the modified NOC template having a pair of roles of the ion supply sources and scaffolds for 3D-ordering architecture of the calcium phosphate as a biomineral in the key functions for biomineralization. The successful establishment of such an ordered deposition of the inorganic on the template was confirmed by several surface characterizations such as atomic force microscopy, X-ray photoelectron spectroscopy, scanning electron microscopy, and so on. Moreover, similarly to human bones, the obtained major assemble states of the calcium phosphates exhibited amorphous. The above process using the bifunctional cellulose template can be considered as a biomimic mineralization, which also opens pathways toward preparation of potentially versatile organic–inorganic order-patterned composites under a less energy consumption.     

Quartz Crystal Microbalance Analysis of DNA-Templated Calcium Phosphate Mineralization

A quartz crystal microbalance (QCM) sensor was developed for the quantitation of calcium phosphate mineralization and the assessment of DNA as a template molecule. Inherent advantages of QCM, such as nanogram sensitivity, temporal resolution, surface-based measurements, and flow capabilities, were leveraged in the design of this sensor, and in-line fluidic mixing was used to control precursor reaction. This research shows that DNA, a highly programmable anionic polymer, is able to template and control mineralization of calcium phosphate, with nucleation occurring in less than 15 min and initial rates ranging from 4 to 8 ng/min. FT-IR measurements show mineralized material to be calcium phosphate resembling hydroxyapatite (HAP) when a DNA template is used. DNA is a promising mineralization template, and the QCM proves to be a dynamic technique for a broad range of heterogeneous mineralization experiments in complement to classic, diffusion-limited, end-point analysis techniques.     

Rare earth ions-enhanced gold nanoclusters as fluorescent sensor array for the detection and discrimination of phosphate anions

The discrimination and detection of phosphate anions have attracted extensive attention due to their important roles in various biological processes. Compared with sensors to detect one individual phosphate at a time, sensor arrays are able to discriminate multiple phosphates simultaneously. In this study, we developed a rare earth ions enhanced AuNCs-based sensor array to achieve facile and rapid identification of phosphate anions (PPi, ADP and ATP). The rare earth ions (i. e., Ce³⁺, Gd³⁺, Tm³⁺ and Yb³⁺) can significantly enhance the fluorescence of AuNCs through aggregation-induced emission effect. And the subsequent addition of phosphate anions can recover the fluorescence of the AuNCs-rare earth ions assembly. Thanks to the different numbers of phosphate group and different steric hindrance effects of phosphate anions, the recovery fluorescence of AuNCs-rare earth ions assembly induced by PPi, ADP or ATP are respectively distinct. Thus the sensor array composed of AuNCs and different rare earth ions is able to distinguish those phosphate anions. Finally, the sensor array was successfully demonstrated to identify the phosphates in blind samples.     

Reaction of calcium and phosphate ions with titanium,zirconium, niobium,and tantalum

To understand the bone formation ability of constituent metal elements of new titanium alloys, titanium, zirconium, niobium, and tantalum, these metals were immersed in various electrolytes containing calcium and/or phosphate ions and characterized using X‐ray photoelectron spectroscopy. In addition, cathodic polarization of the metals in the electrolytes was performed to evaluate the stability of the surface oxide films on the metals in the electrolyte. The calcium phosphate layer formed on Ti in electrolytes containing calcium and phosphate ions is relatively protective against mass transfer throughout the layer. However, the zirconium phosphate layer formed on Zr is much more protective and stable than that on Ti. Therefore, calcium ions were not incorporated. Nb and Ta formed calcium phosphate, but the amount was smaller than that in Ti, because phosphates formed on Nb and Ta are somewhat protective and the incorporation of the calcium ion is inhibited. Titanium played the most important role in forming calcium phosphate, while zirconium inhibited the formation of calcium phosphate on titanium alloys. The control of bone formation is feasible by the design of titanium alloys. Copyright © 2015 John Wiley & Sons, Ltd.     

Mineralization mechanism of calcium phosphates under three kinds of Langmuir monolayers

Three kinds of Langmuir monolayers formed by dipalmitoylphosphatidylcholine (DPPC), arachidic acid (AA), and octadecylamine (ODA) were used as templates to study the initial stage of nucleation and crystallization of calcium phosphates. It was demonstrated that the combination of calcium ions (or phosphates) to the monolayer/subphase interface is a prerequisite for subsequent nucleation. It was found that calcium phosphate dihydrate (DPCD) formed at 25.0 degrees C for 12 h has a biphasic structure containing both amorphous and crystalline phases. These results showed that calcium phosphates were formed through a multistage assembly process, during which an initial amorphous phase DPCD was followed by a phase transformation into a crystalline phase and then the most stable hydroxyapatite (HAp). This provided new insights into the template-biomineral interaction and a mechanism for biomineralization.     

骨及生物材料中的纳米磷酸钙

纳米磷酸钙在自然界骨组织的形成过程中起到了关键作用。尽管骨的类型有所不同,但在其初级结构中的无机成分都是纳米磷酸钙。纳米磷酸钙结构能够给予骨良好的机械性能和生物学活性。在生物体中,无机纳米磷酸钙在有机基质的调控下能定向自组装成特定的生物矿物。体外细胞实验显示小尺寸纳米羟基磷灰石更能促进骨髓基质干细胞的增殖,而同尺寸的结晶型纳米磷酸钙则比无定形磷酸钙更能利于干细胞分化。鉴于纳米磷酸钙具有很好的生物相容性和骨诱导性,可以发展成为理想的生物材料常用于骨组织工程和生物医学。     

骨及生物材料中的纳米磷酸钙

纳米磷酸钙在自然界骨组织的形成过程中起到了关键作用。尽管骨的类型有所不同,但在其初级结构中的无机成分都是纳米磷酸钙。纳米磷酸钙结构能够给予骨良好的机械性能和生物学活性。在生物体中,无机纳米磷酸钙在有机基质的调控下能定向自组装成特定的生物矿物。体外细胞实验显示小尺寸纳米羟基磷灰石更能促进骨髓基质干细胞的增殖,而同尺寸的结晶型纳米磷酸钙则比无定形磷酸钙更能利于干细胞分化。鉴于纳米磷酸钙具有很好的生物相容性和骨诱导性,可以发展成为理想的生物材料常用于骨组织工程和生物医学。     

Biological and medical significance of calcium phosphates

The inorganic part of hard tissues (bones and teeth) of mammals consists of calcium phosphate, mainly of apatitic structure. Similarly, most undesired calcifications (i.e. those appearing as a result of various diseases) of mammals also contain calcium phosphate. For example, atherosclerosis results in blood-vessel blockage caused by a solid composite of cholesterol with calcium phosphate. Dental caries result in a replacement of less soluble and hard apatite by more soluble and softer calcium hydrogenphosphates. Osteoporosis is a demineralization of bone. Therefore, from a chemical point of view, processes of normal (bone and teeth formation and growth) and pathological (atherosclerosis and dental calculus) calcifications are just an in vivo crystallization of calcium phosphate. Similarly, dental caries and osteoporosis can be considered to be in vivo dissolution of calcium phosphates. On the other hand, because of the chemical similarity with biological calcified tissues, all calcium phosphates are remarkably biocompatible. This property is widely used in medicine for biomaterials that are either entirely made of or coated with calcium phosphate. For example, self-setting bone cements made of calcium phosphates are helpful in bone repair and titanium substitutes covered with a surface layer of calcium phosphates are used for hip-joint endoprostheses and tooth substitutes, to facilitate the growth of bone and thereby raise the mechanical stability. Calcium phosphates have a great biological and medical significance and in this review we give an overview of the current knowledge in this subject.     

Generality of solvation effects on the hydrolysis rates of phosphate monoesters and their possible relevance to enzymatic catalysis

Previous work by Kirby and co-workers revealed a significant acceleration of the rate of hydrolysis of p-nitrophenyl phosphate by added dipolar solvents such as DMSO. Activation parameters and kinetic isotope effects have been measured to ascertain the origin of this effect. The generality of this phenomenon was examined with a series of esters with more basic leaving groups. Computational analyses of the effects of desolvation of dianionic phosphate monoesters were carried out, and the possible effect of the transfer from water to the active site of alkaline phosphatase was modeled. The results are consistent with a desolvation-induced weakening of the P-O ester bond in the ground state. Other aryl phosphate esters show similar rate accelerations at high fractions of DMSO, but phenyl and methyl phosphates do not, and their hydrolysis reactions are actually slowed by these conditions.     

Preliminary characterization of calcium chemical environment in apatitic and non-apatitic calcium phosphates of biological interest by X-ray absorption spectroscopy

Several reports have mentioned the existence of non-apatitic environments of phosphate and carbonate ions in synthetic and biological poorly crystalline apatites. However there were no direct spectroscopic evidences for the existence of non-apatitic environment of calcium ions. X-ray Absorption Spectroscopy, at the K-edge of calcium, allows the discrimination between different calcium phosphates of biological interest despite great spectral similarities. A primary analysis of the spectra reveals the existence, in synthetic poorly crystalline apatites, of variable features related to the maturation stage of the sample and corresponding to the existence of non-apatitic environments of calcium ions. Although these features can also be found in several other calcium phosphate salts, and do not allow a clear identification of the ionic environments of calcium ions, they give a possibility to directly determine the maturity of poorly crystalline apatite from calcium X-ray Absorption Near Edge Structure spectra.     

Biomimetic peptide bond formation in water with aminoacyl phosphate esters

Aminoacyl phosphates, biomimetic analogues of aminoacyl adenylates, react efficiently with amino acid esters to form dipeptides with retention of stereochemical integrity. The reactions are selective and occur readily in the presence of nucleophiles other than amino groups on their side chains. Aminoacyl phosphate esters that lack an amino-protecting group are also suitable for peptide bond formation, leading to a simplified overall process.     

Probing isomeric differences of phosphorylated carbohydrates through the use of ion/molecule reactions and FT-ICR MS

Through the use of ion/molecule reactions and tandem mass spectrometry, phosphate position is assigned in both phosphorylated monosaccharides and oligosaccharides. In previous work phosphate moieties of monosaccharides were stabilized under collisional activation, by first derivatizing the deprotonated monosaccharide with trimethyl borate through an ion/molecule reaction, and the phosphate position determined through marker ions generated in tandem mass spectra. In this work, the methodology is extended to larger phosphorylated oligomers employing chlorotrimethylsilane (TMSCl) as the ion/molecule reagent. Phosphorylated monosaccharides were first investigated to determine diagnostic ions for phosphate linkage in monomeric standards. It was observed that the diagnostic ions showed both linkage and some monosaccharide stereochemical information. Furthermore, it was observed that TMS addition stabilized the phosphate moiety under collisionally activated conditions. Upon identification of the diagnostic ions, the methodology was applied to lactose-1-phosphate. It was found that TMSCl, stabilized the phosphate moiety upon collisional activation, and furthermore, the phosphate linkage could be determined through tandem mass spectrometric analysis. As a further extrapolation to biologically relevant problems, the methodology was applied to a lipophosphoglycan analog from the protozoan parasite Leishmania. This sample contains bridging phosphates which were converted to terminal phosphates through collision induced dissociation. The sample was then analyzed in the same manner as lactose-1-phosphate, yielding phosphate linkage information and stereochemical information. This study showed that, using the developed methodology, phosphate linkage can be determined from both monosaccharides and larger oligosaccharides; furthermore it is applicable to samples in which the phosphates are either terminating or bridging.     

Mineralization of Organophosphorous Pesticides by Electro-generated Oxidants

In this study we have investigated organophosphorous pesticides destruction by oxidizing system, which was synthesized in the polluted aqueous sulfuric acidic solutions by constant current conducting. Bulk electrolyses with lead electrode were performed in an undivided cell under amperestatic conditions to study the oxidative cleavage of commercial pesticides and pure active ingredients. The pesticides were completely destroyed following pseudofirst-order kinetics estimated according to chemical oxygen demand (COD) data and phosphate ions accumulation. The assessment of decay rate constants shows the high mineralization degree. Moreover, the general current efficiency corresponds to high impact of chemical reactions in the bulk along with electrochemical interactions and defines the reasonable treatment process time as 120 min. The decay rate constants obtained by phosphate formation are higher for glyphosate than rate constants estimated by COD. It may be explained by P–C bond breaking and phosphate ions cleavage. For further study of organophosphorous pesticides oxidation pathways in detail by described process, the conditions of electrolysis should be milder.     

Biomimetic gelatin-octacalcium phosphate core-shell microspheres

Calcium phosphate/polymeric microparticles synthesized through a biomimetic approach are regarded with increasing interest for their various potential applications, including tissue engineering and regenerative medicine. Herein we report the synthesis and characterization of gelatin/octacalcium phosphate core/shell microspheres. Deposition of the calcium phosphate shell on the polymeric microspheres was obtained through bio-inspired mineralization on the surface of functionalized gelatin microparticles. Gelatin microspheres stabilized by alginate dialdehyde were prepared using an inverse microemulsion. Functionalization was achieved by enriching the microspheres composition with calcium ions or, alternatively, with alendronate, a bisphosphonate widely employed for the treatment of bone diseases. Functionalization and synthesis of the inorganic phase in the microemulsion environment were key factors for the achievement of a complete coating of the microspheres with calcium phosphate. The inorganic shell is constituted of small crystals of octacalcium phosphate, which control gelatin and alendronate release.     

Bioremediation of Heavy Metal Ions by Phosphate‐mineralization Bacteria and Its Mechanism

Bioremediation of heavy metal ions by phosphate‐mineralization bacteria (PMB), as a new green and en‐ vironmental method, relies on microbe‐inducing phosphate precipitation and can prevent heavy metal ions from transferring. The growth of PMB was investigated via four aspects respectively — the control of incubation time, pH value, environmental conditions, and heavy metal ions. At the same time, phosphate? mineralization precipitations and mechanism of four common kinds of heavy metal ions were analyzed. The experimental results indicated that PMB didn’t grow immediately in the first 5 hours, and they reached to the fastest reproduction rate after 13 hours. The pH value of PMB solution increased gradually from 7.0 to 8.6 when PM₀B grew, which plays an important role in the mineralization process. PMB could grow most rapidly at 30 °C, pH of 8 and low concentration of heavy metal ions. It showed that too high or too low temperature and pH, as well as high concentration of heavy metal ions, could inhibit the reproduction of PMB. Stable and large particles phosphate ‐ mineralization precipitation, whose particle size could be more than 10 microns, were obtained by the process that PMB induced substrate to decompose and thus mineralized heavy metal ions effectively.     

Intercalation of n-alkyltrimethylammonium ions into layered calcium octyl phosphates thermally treated in vacuo

Layered calcium octyl phosphate (CH3(CH2)7OPO3Ca.1.6H2O: CaOP), which is composed of a multilayer alternating bilayer of octyl phosphates and a dicalcium phosphate dihydrate (DCPD)-like phase, was thermally treated in vacuo and the intercalation of n-alkyltrimethylammonium ions into the materials was examined. The octyl groups in the layer were eliminated by outgassing above 250 degrees C to give the amorphous calcium phosphates. Further, the specific surface area was steeply increased and mesopores with a diameter of ca. 2.0 nm were formed. IR results indicated that the surface P-OH groups were generated by outgassing at 250 degrees C. When the CaOP outgassed at 250 degrees C was treated with n-alkyltrimethylammonium ion solutions (carbon number of alkyl group, n=14-18), three XRD peaks reappeared below 2theta=15 degrees and the d-spacing ratio of these peaks was 1:1/2:1/3. These facts indicate that the n-alkyltrimethylammonium ions were intercalated into the amorphous calcium phosphate phases.     

Constant composition dissolution of mixed phases. II. Selective dissolution of calcium phosphates

Characterization of the dissolution kinetics of individual synthetic and biological calcium phosphates is of considerable importance since these phases often coexist in biological minerals. The constant composition method has been used to study the dissolution kinetics of a series of synthetic calcium phosphates, brushite (DCPD), beta-tricalcium phosphate (TCP), octacalcium phosphate (OCP), hydroxyapatite (HAP), and carbonated apatite (CAP) in the presence and absence of citric acid, as a function of pH and thermodynamic driving force. While citric acid markedly accelerates the dissolution of TCP, HAP dissolution is significantly inhibited. Moreover, this additive has almost no influence on the dissolution of DCPD, OCP, and CAP. Dual constant composition dissolution studies of mixed calcium phosphates in the presence of citric acid have also been made. Another factor, pH, also plays an important role in the dissolution of these calcium phosphates. In suspensions of calcium phosphate mixtures, specific phases can be selectively dissolved by changing experimental parameters such as pH and the presence of rate modifiers. This result has important applications for the dissolution control of dental hard tissues such as dentin, enamel, and calculus.     

Determination of phosphate based on inhibition of crystal growth of calcite

The growth of calcium carbonate seed crystals (calcite) is strongly inhibited by the presence of phosphate ions. The kinetics of crystal growth were followed potentiometrically using a calcium ion-selective electrode or through the use of a pH electrode. The study of different variables on such a process was carried out with the aim of developing kinetic methods to determine phosphate ions (50–400 ng ml^?1). The selectivity and sensitivity of these processes allow the application of crystallization reactions to the determination of phosphate in human urine.