The Complex Relationship between Metals and Carbonic Anhydrase: New Insights and Perspectives

Carbonic anhydrase is a ubiquitous metalloenzyme, which catalyzes the reversible hydration of CO₂ to HCO₃⁻ and H⁺. Metals play a key role in the bioactivity of this metalloenzyme, although their relationships with CA have not been completely clarified to date. The aim of this review is to explore the complexity and multi-aspect nature of these relationships, since metals can be cofactors of CA, but also inhibitors of CA activity and modulators of CA expression. Moreover, this work analyzes new insights and perspectives that allow translating new advances in basic science on the interaction between CA and metals to applications in several fields of research, ranging from biotechnology to environmental sciences.     

An Ultrasensitive Electrochemiluminescence Immunoassay for Carbohydrate Antigen 19-9 in Serum Based on Antibody Labeled Fe3O4 Nanoparticles as Capture Probes and Graphene/CdTe Quantum Dot Bionanoconjugates as Signal Amplifiers

The CdTe quantum dots (QDs), graphene nanocomposite (CdTe-G) and dextran–Fe₃O₄ magnetic nanoparticles have been synthesized for developing an ultrasensitive electrochemiluminescence (ECL) immunoassay for Carcinoembryonic antigen 19-9 (CA 19-9) in serums. Firstly, the capture probes (CA 19-9 Ab1/Fe₃O₄) for enriching CA 19-9 were synthesized by immobilizing the CA 19-9’s first antibody (CA 19-9 Ab1) on magnetic nanoparticles (dextran-Fe₃O₄). Secondly, the signal probes (CA 19-9 Ab2/CdTe-G), which can emit an ECL signal, were formed by attaching the secondary CA 19-9 antibody (CA 19-9 Ab2) to the surface of the CdTe-G. Thirdly, the above two probes were used for conjugating with a serial of CA 19-9 concentrations. Graphene can immobilize dozens of CdTe QDs on their surface, which can emit stronger ECL intensity than CdTe QDs. Based on the amplified signal, ultrasensitive antigen detection can be realized. Under the optimal conditions, the ECL signal depended linearly on the logarithm of CA 19-9 concentration from 0.005 to 100 pg/mL, and the detection limit was 0.002 pg/mL. Finally, five samples of human serum were tested, and the results were compared with a time-resolved fluorescence assay (TRFA). The novel immunoassay provides a stable, specific and highly sensitive immunoassay protocol for tumor marker detection at very low levels, which can be applied in early diagnosis of tumor.     

Capture of volatile hazardous metals using a bed of kaolinite

One of the promising technologies for reducing metals emission from the waste thermal process is the capture of vapor-phase metals through the use of solid sorbents. This study investigated the use of natural kaolinite for the removal of several volatile metals. The capture of cadmium and lead using a packed bed with porous kaolinite of the size range 300-400 Μm was effective. The capturing efficiency increased as the bed temperature increased. The ratio of the sorption reaction rate to the diffusion rate varied on the 10^-2 order of magnitude. This suggests that the resistance of the diffusion within the pores of kaolinite particles is not significant in the selection of sorbent particle size for practice. The capturing mechanism for cadmium chloride is different from that for lead chloride. Cadmium can be reactively scavenged by CdO-Al₂O₃-2SiO₂ as well as metakaolinite, suggesting that a unit mole of metakaolinite can ultimately capture two moles of cadmium.     

A Single‐Electrode,Dual‐Potential Ferrocene–PNA Biosensor for the Detection of DNA

A Fc–PNA biosensor (Fc: ferrocenyl, C₁₀H₉Fe) was designed by using two electrochemically distinguishable recognition elements with different molecular information at a single electrode. Two Fc–PNA capture probes were therefore synthesized by N‐terminal labeling different dodecamer PNA sequences with different ferrocene derivatives by click chemistry. Each of the two strands was thereby tethered with one specific ferrocene derivative. The two capture probes revealed quasi‐reversible redox processes of the Fc^0/+ redox couple with a significant difference in their electrochemical half‐wave potentials of ΔE_1/2=160 mV. A carefully designed biosensor interface, consisting of a ternary self‐assembled monolayer (SAM) of the two C‐terminal cysteine‐tethered Fc–PNA capture probes and 6‐mercaptohexanol, was electrochemically investigated by square wave (SWV) and cyclic voltammetry (CV). The biosensor properties of this interface were analyzed by studying the interaction with DNA sequences that were complementary to either of the two capture probes by SWV. Based on distinct changes in both peak current and potential, a parallel identification of these two DNA sequences was successful with one interface design. Moreover, the primary electrochemical response could be converted by a simple mathematical analysis into a clear‐cut electrochemical signal about the hybridization event. The discrimination of single‐nucleotide polymorphism (SNP) was proven with a chosen single‐mismatch DNA sequence. Furthermore, experiments with crude bacterial RNA confirm the principal suitability of this dual‐potential sensor under real‐life conditions.     

Characterisation of Photoaffinity‐Based Chemical Probes by Fluorescence Imaging and Native‐State Mass Spectrometry

Chemical probes are small‐molecule reagents used by researchers for labelling and detection of biomolecules. We present the design, synthesis, and characterisation of a panel of 11 structurally diverse photoaffinity labelling (PAL) probes as research tools for labelling the model enzyme carbonic anhydrase (CA) in challenging environments, including in protein mixtures and cell lysates. We targeted the ubiquitous CA II as well as the two cancer‐associated CAs (CA IX and CA XII) that are of high priority as potential biomarkers of aggressive and/or multidrug‐resistant cancer. We utilise an atypical biophysical approach, native state mass spectrometry, to monitor the initial protein–probe binding and subsequent UV crosslinking efficiency of the protein:probe complex. This mass spectrometry methodology represents a new approach for chemical probe optimisation and development that might have broader applications to chemical probe characterisation beyond this study. This also represents one of the first studies, to the best of our knowledge, in which a comprehensive set of PAL probes has been used to establish the relationship between probe structure, noncovalent protein–probe binding, and covalent protein–probe crosslinking efficiency. Our results demonstrate the benefits of a comprehensive analysis of chemical probe structure–activity relationships to support the development of optimum chemical probes.     

Inhibitors of CA IX Enzyme Based on Polyhedral Boron Compounds

This review describes recent progress in the design and development of inhibitors of human carbonic anhydrase IX (CA IX) based on space-filling carborane and cobalt bis(dicarbollide) clusters. CA IX enzyme is known to play a crucial role in cancer cell proliferation and metastases. The new class of potent and selective CA IX inhibitors combines the structural motif of a bulky inorganic cluster with an alkylsulfamido or alkylsulfonamido anchor group for Zn²⁺ ion in the enzyme active site. Detailed structure-activity relationship (SAR) studies of a large series containing 50 compounds uncovered structural features of the cluster-containing inhibitors that are important for efficient and selective inhibition of CA IX activity. Preclinical evaluation of selected compounds revealed low toxicity, favorable pharmacokinetics and ability to reduce tumor growth. Cluster-containing inhibitors of CA IX can thus be considered as promising candidates for drug development and/or for combination therapy in boron neutron capture therapy (BNCT).     

Facile modification of polysulfone hollow-fiber membranes via the incorporation of well-dispersed iron oxide nanoparticles for protein purification

Protein existence in wastewater is an important issue in wastewater management because proteins are generally present as contaminants and foulants. Hence, in this study, we focused on designing a polysulfone (PSf) hollow-fiber membrane embedded with hydrophilic iron oxide nanoparticles (IONPs) for protein purification by means of ultrafiltration. Before membrane fabrication, the dispersion stability of the IONPs was enhanced by the addition of a stabilizer, namely, citric acid (CA). Next, PSf–IONP–CA nanocomposite hollow-fiber membranes were prepared via a dry–wet spinning process and then characterized in terms of their hydrophilicity and morphology. Ultrafiltration and adsorption experiments were then conducted with bovine serum albumin as a model protein. The results that an IONP/CA weight ratio of 1:20 contributed to the most stable IONP dispersion. It was also revealed that the membrane incorporated with IONP–CA at a weight ratio of 1:20 exhibited the highest pure water permeability (58.6 L m⁻² h⁻¹ bar⁻¹) and protein rejection (98.5%) while maintaining a low protein adsorption (3.3 μg/cm²). The addition of well-dispersed IONPs enhanced the separation features of the PSf hollow-fiber membrane for protein purification. © 2019 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2019 , 136, 47502.     

Immobilization of carbonic anhydrase on polyethylenimine/dopamine codeposited membranes

Carbonic anhydrase (CA) catalyzing CO₂ hydration has an important application in carbon capture, and its immobilization is very significant. Here, CA was covalently linked by glutaraldehyde (GA) to the surface of poly(vinylidene fluoride) (PVDF) and polyethylene (PE) membranes, which were previously modified via a simple codeposition of polyethyleneimine (PEI) and dopamine (DA). The effects of the modification conditions were investigated, and the membranes were characterized by Fourier transform infrared spectroscopy and scanning electron microscopy. The immobilization process was optimized, and the catalytic properties of immobilized CA were studied. The results show that the optimal mass ratio of PEI and DA was 1:1 and the deposition time was 10–12 h, at which the surface amino group density could reach 1.278 × 10⁻⁷ and 1.397 × 10⁻⁷ mol/cm² for PVDF and PE, respectively. For enzyme immobilization, the optimal CA and GA concentrations were 0.2 mg/mL and 0.1 wt %, and a maximum activity recovery of about 53% and 76% could be achieved for PVDF-attached CA and PE-attached CA, respectively. Their K_m values were 10.62 mM and 8.6 mM, and the corresponding K_cat/K_m values were 132.2 M⁻¹ s⁻¹ and 312.9 M⁻¹ s⁻¹. After immobilization, the storage stability and reusability of CA were much improved. © 2019 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2019 , 136, 47784.     

Quantitative Analysis of High Temperature Toxic Metal Sorption Rates Using Aerosol Fractionation

The airborne emission of toxic metals, such as lead, arsenic, and cadmium, from coal-fired boilers and incinerators is a major problem. These metals, which are considered to be semivolatile, are vaporized in the flame and subsequently form submicron particles. Since submicron particles are difficult to remove, one strategy to prevent emission is to inhibit ultra-fine particle formation within the combustor. Recent work has demonstrated that the sorbents such as kaolinite and lime are able to capture semivolatile metal vapors at high temperatures, thus preventing nucleation and condensation at lower temperatures. However, to develop practical scale technology, the rate of metal sorption by sorbents must be quantified. In this paper, an aerosol size fractionation approach is proposed to determine the extent of metal vapor-sorbent interactions above the metal dew point. The applicability of this methodology is illustrated by determining the rate of capture of cadmium by powdered, kaolinite sorbent at high combustion temperatures. An estimate of the rate of reaction was found to be 0.8 10⁹cm³ gas/mol sorbent s. A simple competitive reaction model was constructed to investigate the competition between sodium and cadmium vapor sorption on a dispersed kaolinite substrate. Previous work had demonstrated that the presence of sodium hindered the sorption of cadmium by kaolinite. Results of the competition model suggest that sodium not only competes for available sorbent but also inhibits the sorption of cadmium when both metals are present.     

柠檬酸和谷氨酸N，N-二乙酸优化处理污泥重金属

采用柠檬酸（CA）和谷氨酸N,N-二乙酸（GLDA）处理污泥重金属,研究了试剂浓度、pH和反应时间对重金属去除效果的影响,并以重金属去除率为表征,通过正交试验获得CA和GLDA处理重金属的最佳条件。结果表明,增加试剂用量、降低体系pH均有利于重金属的去除,但延长反应时间对重金属的去除效果影响不明显。CA和GLDA处理污泥重金属的最佳条件分别为：CA 0.3 mol·L^-1、pH 4、反应时间2 h和GLDA 0.05 mol·L^-1、pH 4、反应时间3 h。最佳反应条件下,对于CA和GLDA,重金属Cd、Cu、Pb和Ni的去除率可分别达80.25%、77.75%、64.66%和75.16%,及78.57%、78.48%、64.84%和76.71%。CA和GLDA对重金属的去除效果大小顺序均为：Cd>Cu> Ni>Pb,但GLDA的处理效果优于CA。污泥经CA和GLDA处理后,污泥固相酸溶态重金属含量下降幅度最大,平均达81%,残渣态重金属含量下降52.1%,而污泥液相中重金属含量则增加了17.54倍,说明重金属从污泥固相向液相转移。SEM镜检发现,污泥由处理前表面分散的絮状结构,变成更为明显的团块结构和片层结构,污泥的吸附能力下降,且体积缩小。研究结果表明,CA和GLDA处理污泥能有效降低污泥重金属含量并提高污泥固相重金属的化学稳定性,有利于污泥脱水及脱水后的进一步处理及其资源化。     

Nanodiamond bioconjugate probes and their collection by electrophoresis

The application of detonation nanodiamonds (NDs) as probes for protein capture and electrophoretic collection was investigated. NDs were chemically modified in a series of reactions to produce a ND-NH₂ product that had increased chemical homogeneity. The product was characterized by X-ray photoelectron spectroscopy (XPS) and Fourier transform infrared spectroscopy (FTIR). FTIR spectra were taken using an IR vacuum cuvette and the samples were dehydrated at different temperatures. The ND-NH₂ product was capable of conjugating to N-hydroxysuccinimide derivatives of TAMRA and biotin. We calculated that the number of chemically attached TAMRA molecules on ND-NH₂ was ∼ 1 molecule/nm². The singly conjugated TAMRA-ND (T-ND) and doubly conjugated TAMRA-ND-Biotin (T-ND-B) products formed stable aqueous colloidal suspensions. T-ND and T-ND-B were collected on planar electrodes and silicon field tip arrays using a field of 10 V/cm. The rate of collection for the aminated ND is dependent upon field strength and an exponential decrease in current was observed as a function of time. Streptavidin was captured by the T-ND-B bioconjugate probe and this nanoparticle–protein complex was collected from solution by electrophoresis.     

Photocontrollable Probes for Mitochondrial Protein Profiling

The mitochondrion is the core site of cell signaling, energy metabolism and biosynthesis. Here, taking advantage of activity-based probes, we synthesized two photocontrollable probes ( YGH-1 and YGH-2 ), composed of a mitochondrial localization moiety “triphenylphosphonium”, a photo-triggered group to achieve spatially and temporally controlled protein capture, and an alkyne group to enrich the labeled protein. Proteomic validation was further carried out to facilitate identification of the mitochondrial proteome in HeLa cells. The results show that half of the identified protein hits (∼300) labeled by YGH-1 and YGH-2 belong to mitochondria, and are mostly localized in the mitochondrial matrix and inner mitochondrial membrane. Our results provide a new tool for spatial and temporal analysis of subcellular proteomes.     

Electrochemical immunoassay for CA125 based on cellulose acetate stabilized antigen/colloidal gold nanoparticles membrane

A novel separation-free electrochemical immunosensor for carcinoma antigen-125 (CA125) was proposed based on the immobilization of CA125 antigen on colloidal gold nanoparticles that was stabilized with cellulose acetate membrane on a glassy carbon electrode. A competitive immunoassay format was employed to detect CA125 antigen with horseradish peroxidase (HRP) labeled CA125 antibody as tracer, o-phenylenediamine and hydrogen peroxide as enzyme substrates. After the immunosensor was incubated with a mixture of HRP labeled CA125 antibody and CA125 sample at 35 °C for 50 min, the amperometric response decreased with an increasing CA125 concentration in the sample solution. The decreased percentage of the electrocatalytic current was proportional to CA125 concentration ranging from 0 to 30 U ml⁻¹ with a detection limit of 1.73 U ml⁻¹ (S/N = 3). The proposed immunosensor showed good stability, acceptable accuracy, and would be applicable to clinical immunoassay of CA125.     

Long Residence Times Revealed by Aurora A Kinase‐Targeting Fluorescent Probes Derived from Inhibitors MLN8237 and VX‐689

We report the development of three fluorescent probes for protein kinase Aurora A that are derived from the well‐known inhibitors MLN8237 and VX‐689 (MK‐5108). Two of these probes target the ATP site of Aurora A, and one targets simultaneously the ATP and substrate sites of the kinase. The probes were tested in an assay with fluorescence polarisation/anisotropy readout, and we demonstrated slow association kinetics and long residence time of the probes (k_on 10⁵–10⁷ M ^?1 s^?1, k_off 10^?3–10^?4 s^?1; residence time 500–3000 s). The presence of the Aurora A activator TPX2 caused a significant reduction in the on‐rate and increase in the off‐rate of fluorescent probes targeting ATP site. These observations were supported by Aurora A inhibition assays with MLN8237 and VX‐689. Overall, our results emphasise the importance of rational design of experiments with these compounds and correct interpretation of the obtained data.     

Design,Synthesis, and Biological Evaluation of Novel Fluorescent Probes Targeting the 18-kDa Translocator Protein

A series of fluorescent probes from the 6-chloro-2-phenylimidazo[1,2-a]pyridine-3-yl acetamides ligands featuring the 7-nitro-2-oxa-1,3-diazol-4-yl (NBD) moiety has been synthesized and biologically evaluated for their fluorescence properties and for their binding affinity to the 18-kDa translocator protein (TSPO). Spectroscopic studies including UV/Vis absorption and fluorescence measurements showed that the synthesized fluorescent probes exhibit favorable spectroscopic properties, especially in nonpolar environments. In vitro fluorescence staining in brain sections from lipopolysaccharide (LPS)-injected mice revealed partial colocalization of the probes with the TSPO. The TSPO binding affinity of the probes was measured on crude mitochondrial fractions separated from rat brain homogenates in a [¹¹C]PK11195 radioligand binding assay. All the new fluorescent probes demonstrated moderate to high binding affinity to the TSPO, with affinity (K_i) values ranging from 0.58 nM to 3.28 μM. Taking these data together, we propose that the new fluorescent probes could be used to visualize the TSPO.     

Competition for Sodium and Toxic Metals Capture on Sorbents

The removal of toxic heavy metals by sorbents has been the focus of much recent research. In single metal/single sorbent systems, laboratory experiments have demonstrated capture of lead and cadmium by kaolinite and lime at high temperatures. However, practical systems (i.e., pulverized coal fired boilers) generally involve sodium as well as toxic metals and the amount of sodium usually exceeds that of the toxic metals. The purpose of this paper is to explore the effect of sodium on the capture of lead and cadmium by sorbents. Experiments were conducted in a 16 kW, 0.15 m ID, 6 m tall laboratory downflow furnace. Aqueous solutions of toxic metals and metal/sodium pairs were introduced into the furnace through a natural gas flame via atomization and subse quent vaporization. For all experiments, the total metal/sorbent equivalence ratio was maintained at 0.7. Approximately 0.5g/min of lime or kaolinite powder (mean particle diameter congruent 1.4 mu m) was injected along the furnace centerline in the post flame at a temperature of 1500 K. Further downstream, at 1200 K, particulate samples were isokinetically extracted and size segregated in a Berner low pressure impactor. Experimental results showed that cadmium and sodium were captured by lime. Furthermore, cadmium was captured by lime preferentially over sodium. Lead capture by lime occurred at a much slower rate than that for cadmium or sodium. Kaolinite, on the other hand, showed a preference for lead and sodium capture over cadmium. Consequently, for capture of multiple metals, a sorbent mixture of lime and kaolinite might be more effective than a single sorbent.     

A New Postprocessing Strategy for Secondary Pollution: Synthesis of CdS Crystals

Eichhornia crassipes (E.C.) is a special aquatic plant, which exhibits excellent tolerance to heavy metals and can be used for the large scale removal of heavy metals from waste water. Here the E.C was used to capture Cd²⁺, then as an in situ bio-template to successfully synthesize photoelectric CdS microcrystals. The products were characterized by X-ray diffraction, scanning electron microscopy, transmission electron microscopy, and fluorescence spectrometer. The results indicated that the peanut-like microcrystals with a length of 1.5–1.8 µm were oriented along (002) plane. The main organic groups in E.C including hydroxyl, primary amide and carboxylate played inducing and controlling roles during the formation of CdS microcrystals in the solution. The PL spectrum showed an emission peak at 450 nm and indicated that the product may have applications in an electronic light device. This approach provided an inspiration on the post-processing of the secondary pollution.     

Effect of additives concentration on performance of cellulose acetate and polyethersulfone blend membranes

Asymmetric micro porous membranes have been prepared successfully from blending of cellulose acetate (CA) and polyethersulfone (PES) by the phase inversion method with N, N-dimethylformamide (DMF) as solvent. Two additives were selected in this study, including polyethylene glycol 600 (PEG 600) and polyvinylpyrrolidone (PVP). The effects of concentration of additives on CA/PES blend membrane performance and cross-section morphology were investigated in detail. CA/PES membranes were compared with CA/PES/PEG and CA/PES/PVP membranes in the performance such as pure water flux, membrane resistance, porosity and cross-section morphology. The resulting blend membranes were also carried out the rejection and permeate flux of Egg Albumin (EA) proteins with molecular weight of 45 Da. The membranes thus obtained with an additive concentration of 5 wt% of both PEG and PVP exhibited superior properties than the 80/20% blend composition of CA and PES membranes. The permeate flux of protein was increased from 44 to 134 lm² h with increase in concentrations of both PVP and PEG in 80/20% blend composition of CA and PES membranes. Cross-sectional images from scanning electron microscopy showed larger macropores in the bottom layer of the membranes with increasing additives content. Observations from scanning electron microscopy provided qualitative evidence for the trends obtained for permeability and porosity results.     

镁质浇注料中纯铝酸钙水泥加入量对钢液总氧含量的影响

在实验室内研究了镁质浇注料中纯铝酸钙水泥加入量对钢液总氧含量的影响。结果表明 :当纯铝酸钙水泥加入量在 6 % (质量分数 ,下同 )以下时 ,随着纯铝酸钙水泥加入量的增加 ,钢样中的总氧含量随之增加 ;当纯铝酸钙水泥加入量超过6 %时 ,钢样中的总氧含量下降