An enzyme immunosensor for the electrochemical determination of the tumor antigen α-fetoprotein

A specific sensor for a tumor antigen, α-fetoprotein (AFP) can be prepared from a membrane with immobilized antibody and an oxygen probe with a permeable teflon membrane. Anti-AFP antibody is covalently immobilized on a membrane prepared from cellulose triacetate, 1,8-diamino-4-aminomethyloctane and glutaraldehyde. The sensor is applied to enzyme immunoassay based on competitive antigen-antibody reaction with catalase-labelled antigen. After competitive binding of free and catalase-labelled AFP, the sensor is examined for catalase activity by amperometric measurement after addition of hydrogen peroxide. AFP can be determined in the range 10^-11–10^-8 g ml^-1.     

Biofunctionalization of Multiwalled Carbon Nanotubes by Electropolymerized Poly(pyrrole‐concanavalin A) Films

The synthesis and electropolymerization of a pyrrolic concanavalin A derivative (pyrrole‐Con A) onto a multiwalled carbon nanotube (MWCNT) deposit is reported. Glucose oxidase was then immobilized onto the MWCNT‐poly(pyrrole‐Con A) coating by affinity carbohydrate interactions with the polymerized Con A protein. The resulting enzyme electrode was applied to the amperometric detection of glucose exhibiting a high sensitivity of 36 mA cm^?2 mol^?1 L and a maximum current density of 350 μA cm^?2.     

Effects of the detection window on the determination of organic copper speciation in estuarine waters

The complexation of copper by natural organic ligands in sea water was measured by cathodic stripping voltammetry with ligand (catechol and quinolin-8-ol) competition. Two methods to determine copper complexation in estuarine waters were compared, one based on a complete titration of the complexing capacity of the sample and the other on measurement of the labile and total dissolved metal concentrations only. Values for log α_CuL (ihe α-coefficient for complexation of Cu²⁺ by natural organic ligands) ranging from 3.2 to 7.7 and from 3.3 to 7.8 could be detected by varying long α_CuAL (the α-coefficient for complexation of Cu²⁺ by the added competing ligand) from 3.4 to 8.9 in samples from the Tamar estuary and from the Channel. The two methods gave comparable results and showed that the type of sites detected depends on the detection window of the technique. This effect is due to the sea and estuarine water samples containing a series of complexing ligands forming complexes of greatly varying strength, thus causing a rnage of complex stabilities to be measured as a function of the detection window of each technique. A comparison showed that lower values for α_CuL are obtained by anodic stripping voltammetry as a result of that technique having a lower detection window. A detailed study of the Tamar estuary revealed a decrease in log α_CuL from 10.8 to 8.3 with increasing salinity, demonstrating that major cations compete with copper for the complexing sites. The free Cu²⁺ concentrations were very low throughout the estuary (16.2 < pCu²⁺ < 18.2) even though the total measurements to establish potential toxic effects of copper in natural waters.     

Ion‐Selective Electrodes Based on Hydrophilic Ionophore‐Modified Nanopores

We report the synthesis and analytical application of the first Cu²⁺‐selective synthetic ion channel based on peptide‐modified gold nanopores. A Cu²⁺‐binding peptide motif (Gly‐Gly‐His) along with two additional functional thiol derivatives inferring cation‐permselectivity and hydrophobicity was self‐assembled on the surface of gold nanoporous membranes comprising of about 5 nm diameter pores. These membranes were used to construct ion‐selective electrodes (ISEs) with extraordinary Cu²⁺ selectivities, approaching six orders of magnitude over certain ions. Since all constituents are immobilized to a supporting nanoporous membrane, their leaching, that is a ubiquitous problem of conventional ionophore‐based ISEs was effectively suppressed.     

Identification of uranyl binding proteins from human kidney-2 cell extracts by immobilized uranyl affinity chromatography and mass spectrometry

To improve our knowledge on protein targets of uranyl ion (UO₂²⁺), we set up a proteomic strategy based on immobilized metal-affinity chromatography (IMAC). The successful enrichment of UO₂²⁺-interacting proteins from human kidney-2 (HK-2) soluble cell extracts was obtained using an ion-exchange chromatography followed by a dedicated IMAC process previously described and designed for the uranyl ion. By mass spectrometry analysis we identified 64 proteins displaying varied functions. The use of a computational screening algorithm along with the particular ligand-based properties of the UO₂²⁺ ion allowed the analysis and categorization of the protein collection. This profitable approach demonstrated that most of these proteins fulfill criteria which could rationalize their binding to the UO₂²⁺-loaded phase. The obtained results enable us to focus on some targets for more in-depth studies and open new insights on its toxicity mechanisms at molecular level.     

Effect of Sodium Dodecyl Sulfate on Concanavalin A-Sepharose During Affinity Chromatography of High-Affinity Binding Toxin Protein Of Bacillus thuringiensis subsp. kurstaki

Abstract

Concanavalin A- Sepharose affinity chromatography is a powerful tool for isolation or purification of peripheral or integral membrane proteins or other glycoproteins. The insecticidal crystal toxin from Bacillus thuringiensis subsp. kurstaki is a glycoprotein containing “high-mannose” or “hybrid”-type sugar chains. The protein has a high binding affinity for concanavalin A lectin and could not be eluted even with 0.5M methyl α-D-mannopyranoside. Nonspecific elution with 0.03% SDS coeluted the matrix con A with bound protein. Experimental results indicated that con A leaching is mainly because of inclusion of detergents in buffer systems and may not be directly related to the nature of the sample protein.

² Abbreviations used: Con A: concanavalin A, SDS: sodium dodecyl sulfate, SDS-PAGE: sodium dodecyl sulfate-polyacrylamide gel electrophoresis, MeG: methyl α-D-glucopyranoside, MeM: methyl α-D-mannopyranoside     

Novel affinity monolithic column modified with cuprous sulfide nanoparticles for the selective enrichment of low‐molecular‐weight electron‐rich analytes

A novel monolithic column modified with cuprous sulfide nanoparticles was developed and its affinity characteristics towards low‐molecular‐weight electron‐rich analytes were investigated. In the synthesis process, home‐made cuprous oxide nanocubes were immobilized on the surface of monolithic skeleton with the moderate thickness based on the strong interaction between imidazole groups and cuprous oxide, then the cuprous oxide layer was transformed into the more stable cuprous sulfide layer through the treatment by sodium sulfide. The resulting cuprous sulfide modified monolithic column presented good permeability and stability in a wide pH range from 2 to 10. Two kinds of typical electron‐rich analytes, kanamycin A and purine, were chosen to assess its affinity characteristics. Compared with the commercial Cu²⁺‐ and Ni²⁺‐based affinity sorbents, a larger binding capacity of cuprous sulfide modified column toward kanamycin A was obtained under basic condition and the recovery of kanamycin A in a milk sample was over 70%. Moreover, the binding capacity of cuprous sulfide modified column for purine was up to 5.57 mg/mL in frontal elution mode. These results suggested that the Cu₂S column has a promising application for the enrichment of electron‐rich analytes.     

Acetylation Rather than H50Q Mutation Impacts the Kinetics of Cu(II) Binding to α-Synuclein

The interaction between α-synuclein (αSyn) and Cu²⁺ has been suggested to be closely linked to brain copper homeostasis. Disruption of copper levels could induce misfolding and aggregation of αSyn, and thus contribute to the progression of Parkinson's disease (PD). Understanding the molecular mechanism of αSyn-Cu²⁺ interaction is important and controversies in Cu²⁺ coordination geometry with αSyn still exists. Herein, we find that the pathological H50Q mutation has no impact on the kinetics of Cu²⁺ binding to the high-affinity site of wild type αSyn (WT-αSyn), indicating the non-involvement of His50 in high-affinity Cu²⁺ binding to WT-αSyn. In contrast, the physiological N-terminally acetylated αSyn (NAc-αSyn) displays several orders of magnitude weaker Cu²⁺ binding affinity than WT-αSyn. Cu²⁺ coordination mode to NAc-αSyn has also been proposed based on EPR spectrum. In addition, we find that Cu²⁺ coordinated WT-αSyn is reduction-active in the presence of GSH, but essentially inactive towards ascorbate. Our work provides new insights into αSyn-Cu²⁺ interaction, which may help understand the multifaceted normal functions of αSyn as well as pathological consequences of αSyn aggregation.     

Effects of Divalent Metal Ions on Electrochemiluminescence Sensor with Ru(bpy)32+ Immobilized in Eastman‐AQ Membrane

Different effects of divalent metal ions on electrochemiluminescence (ECL) sensor with Ru(bpy)₃²⁺ immobilized in Eastman‐AQ membrane were investigated. Mg²⁺, Ca²⁺ and Fe²⁺ can elevate the ECL of Ru(bpy)₃²⁺/proline; while metal ions that underwent redox reactions on the electrode such as Mn²⁺ and Co²⁺ presented intensive quenching effects on Ru(bpy)₃²⁺ ECL. Also, the quenching effect of Mn²⁺ on the ECL sensor with Ru(bpy)₃²⁺ immobilized in Eastman‐AQ membrane enhanced to about 30‐folds compared with the case that Ru(bpy)₃²⁺ was dissolved in phosphate buffer, and the enhanced quenching effects of Mn²⁺ were studied.     

Pretreatment-free immunochromatographic assay for the detection of streptomycin and its application to the control of milk and dairy products

A rapid pretreatment-free immunochromatographic assay was developed for the control of the streptomycin (STR) content in milk and dairy products. The assay is based on the competition between an immobilized STR–protein conjugate and STR in a sample to be tested for the binding to monoclonal anti-STR antibodies conjugated to colloidal gold during the flow of the sample along a membrane strip with immobilized reactants. It is possible to improve the cut-off level of positive and negative samples distinguished by a change in the molar STR to protein ratio in the immobilized conjugate. The cut-off level (500 ng mL⁻¹) thus achieved corresponds to the stated MRL of STR in milk and dairy products. For STR concentrations in the range of 16–250 ng mL⁻¹ its content can be quantitatively measured based on the degree of binding of a colloidal gold label in the test strip zone with the immobilized STR–protein conjugate. The duration of the assay is 10 min. The selected sizes of membrane pores and colloidal gold particles allow the assay to be carried out at room temperature without additional reactants and pretreatment. The applicability of the assay for milk, whole milk, sour clotted milk, and kefir with different fat content (from 0.5% to 6%) was confirmed. The results of quantitative immunochromatographic assay show good correlation with traditional ELISA (r was equal to 0.935 and 0.940 for the series tested).     

Biointeraction analysis of carbamazepine binding to α1‐acid glycoprotein by high‐performance affinity chromatography

Interactions of the drug carbamazepine with the serum protein α₁‐acid glycoprotein (AGP) were examined by high‐performance affinity chromatography. Frontal analysis studies with an immobilized AGP column and control column indicated carbamazepine had both low‐affinity interactions with the support and high‐affinity interactions with AGP. When a correction was made for binding to the support, the association equilibrium constant measured at pH 7.4 and 37°C for carbamazepine with AGP was 1.0 (±0.1)×10⁵ M^?1, with values that ranged from 5.1 to 0.58×10⁵ M^?1 in going from 5 to 45°C. It was found in competition studies that these interactions were occurring at the same site that binds propranolol on AGP. Temperature studies indicated that the change in enthalpy was the main driving force for the binding of carbamazepine to AGP. These results provide a more complete picture of how carbamazepine binds to AGP in serum. This report also illustrates how high‐performance affinity chromatography can be used to examine biological interactions and drug–protein binding in situations in which significant interactions for an analyte are present with both the chromatographic support and an immobilized ligand.     

Binding Interaction Between Prazosin and Immobilized Receptor by Frontal Analysis

Binding interaction between ligand and G-protein coupled receptor plays an important role in the several steps of exertion of therapeutic effect by a drug and has become increasingly interesting to pharmacists, chemists, biologists and companies. α₁-Adrenoceptor (α₁-AR) was purified from cell line which stably expressed α₁-AR and evenly immobilized on the surface of macroporous silica gel to prepare a novel stationary phase for investigating the interaction between drug and receptor. Control drugs of α₁-AR including phentolamine, terazosin and urapidil were used to characterize the retention properties of the stationary phase containing the receptor. Further work was performed to calculate the binding sites and association constant of prazosin binding to the immobilized receptor. The results presented a value of 4.55?×?10^?7?M^?1 for binding sites and of 2.2?×?10⁴?M for the association constant during the interaction between prazosin and α₁-AR. The proposed affinity method was stable at least in seven consecutive days, as well as had the primary bioactivities of recognizing and binding the ligand, providing an alternative for representing the interaction between drug and functional protein.     

Iminodiacetic acid-modified magnetic poly(2-hydroxyethyl methacrylate)-based microspheres for phosphopeptide enrichment

Magnetic non-porous hydrophilic poly(2-hydroxyethyl methacrylate-co-glycidyl methacrylate) microspheres prepared by the dispersion polymerization and modified with iminodiacetic acid (IDA) were employed for the IMAC separation of phosphopeptides. Fe³⁺ and Ga³⁺ ions immobilized on IDA-modified magnetic microspheres were used for the enrichment of phosphopeptides from the proteolytic digests of two model proteins differing in their physico-chemical properties and phosphate group content: porcine pepsin A and bovine α-casein. The optimum conditions for phosphopeptide adsorption and desorption in both cases were investigated and compared. The phosphopeptides separated from the proteolytic digests were analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The ability of the prepared Fe³⁺- and Ga³⁺-IDA-modified magnetic microspheres to capture phosphopeptides from complex mixtures was shown on an example of bovine milk proteolytic digest.     

Evaluation and optimization of the metal‐binding properties of a complex ligand for immobilized metal affinity chromatography

The simultaneous determination of two binding parameters for metal ions on an immobilized metal affinity chromatography column was performed by frontal chromatography. In this study, the binding parameters of Cu²⁺ to l ‐glutamic acid were measured, the metal ion‐binding characteristics of the complex ligand were evaluated. The linear correlation coefficients were all greater than 99%, and the relative standard deviations of two binding parameters were 0.58 and 0.059%, respectively. The experiments proved that the frontal chromatography method was accurate, reproducible, and could be used to determine the metal‐binding parameters of the affinity column. The effects of buffer pH, type, and concentration on binding parameters were explored by uniform design experiment. Regression, matching and residual analyses of the models were performed. Meanwhile, the optimum‐binding conditions of Cu²⁺ on the l ‐glutamic acid‐silica column were obtained. Under these binding conditions, observations and regression values of two parameters were similar, and the observation values were the best. The results demonstrated that high intensity metal affinity column could be effectively prepared by measuring and evaluating binding parameters using frontal chromatography combined with a uniform design experiment. The present work provided a new mode for evaluating and preparing immobilized metal affinity column with good metal‐binding behaviors.     

Interactions between an amphipathic di‐histidine peptide and a metal affinity chromatographic resin derived from a bis(tacn)butane chelating ligand

The interactive behavior of an amphipathic peptide with the Cu²⁺, Ni²⁺, and Zn²⁺ complexes of 1,4‐bis(triazacyclonon‐1‐yl)butane), bis(tacn)^but, immobilized onto Sepharose CL‐4B, has been investigated. The effects of incubation time, as well as the incubation buffer pH and ionic strength, have been examined. The binding data have been interrogated using Langmuir, Langmuir‐Freundlich, bi‐Langmuir, and Temkin isothermal models and Scatchard plots. These results confirm that this amphipathic peptide binds with relatively high capacities to the immobilized Cu²⁺‐ and Ni²⁺‐1,4‐bis(triazacyclonon‐1‐yl)butane)‐Sepharose CL‐4B sorbents via at least two discrete sites. However, the corresponding immobilized Zn²⁺‐sorbent had low binding capacity. Moreover, the magnitude of the binding capacities of these sorbents was dependent on the pH and ionic strength of the incubation buffer. These results are relevant to the isolation of E. coli expressed recombinant proteins that incorporate this and related amphipathic peptide tags, containing two or more histidine residues, located at the N‐ or C‐terminus of the recombinant protein, and the co‐purification of low abundance host cell proteins of diverse structure, by immobilized metal ion affinity chromatographic methods.     

Phospholipase C-catalyzed sphingomyelin hydrolysis in a membrane reactor for ceramide production

A membrane reactor for the production of ceramide through sphingomyelin hydrolysis with phospholipase C from Clostridium perfringens was studied for the first time. Ceramide has raised a large interest as an active component in both pharmaceutical and cosmetic industry. The enzymatic hydrolysis of sphingomyelin has been proven to be a feasible method to produce ceramide. In the membrane reactor constructed, the aqueous phase and the organic phase were separated by a membrane containing the immobilized enzyme, while the organic phase was continuously circulated. Among the 10 selected membranes, the enzyme immobilized in membrane RC 70PP had low immobilization efficiency, but retained the highest catalytic activity. Three immobilization methods, i.e. filtration (adsorption/entrapment), covalent binding, and cross-linking, were compared. The enzyme immobilized by filtration had the highest activity even under the low fixation level (9.4%). The optimal flow rate of the organic phase was 5 ml/min. High initial enzyme amount in the immobilization led to the decrease in the fixation level. Both the initial reaction rate and the specific activity of the enzyme increased with increasing enzyme loading, and slightly decreased after the immobilized enzyme amount over 50 μg in 9.6 cm² membrane area. The immobilized enzyme retained 16% of the original activity after five cycles. Finally, the liquid enzyme, the enzyme immobilized on particle carriers, and the enzyme immobilized in the membrane were compared. The study demonstrated the improved enzyme reusability, the fast immobilization process, the straightforward up-scaling and the combination of the hydrolysis with the product separation in the membrane reactor developed.     

Synthesis, transport and ionophore properties of α,ω-diphosphorylated aza podands: III. Liquid ion-selective electrodes based on phosphorylated aza podands

Aza podands having side α-aminomethylphosphine oxide groups were prepared and used as electrode-active agents in liquid membrane ion-selective electrodes. A series of liqiud membrane electrodes sensitive to Cu²⁺ and Hg²⁺ cations were prepared on the basis of N,N-bis[di(n-hexyl)phosphorylmethyl]piperazine. The electrodes containing copper complexes with aza podands as ionophores exhibit anionic function toward lipophilic anions. Potentiometric and argentometric determination of iodide ions was performed with an iodide-selective electrode based on the mercury complex.     

Efficient fabrication of high‐capacity immobilized metal ion affinity chromatographic media: The role of the dextran‐grafting process and its manipulation

Novel high‐capacity Ni²⁺ immobilized metal ion affinity chromatographic media were prepared through the dextran‐grafting process. Dextran was grafted to an allyl‐activated agarose‐based matrix followed by functionalization for the immobilized metal ion affinity chromatographic media. With elaborate regulation of the allylation degree, dextran was completely or partly grafted to agarose microspheres, namely, completely dextran‐grafted agarose microspheres and partly dextran‐grafted ones, respectively. Confocal laser scanning microscope results demonstrated that a good adjustment of dextran‐grafting degree was achieved, and dextran was distributed uniformly in whole completely dextran‐grafted microspheres, while just distributed around the outside of the partly dextran‐grafted ones. Flow hydrodynamic properties were improved greatly after the dextran‐grafting process, and the flow velocity increased by about 30% compared with that of a commercial chromatographic medium (Ni Sepharose FF). A significant improvement of protein binding performance was also achieved by the dextran‐grafting process, and partly dextran‐grafted Ni²⁺ chelating medium had a maximum binding capacity for His‐tagged lactate dehydrogenase about 2.5 times higher than that of Ni Sepharose FF. The results indicated that this novel chromatographic medium is promising for applications in high‐efficiency and large‐scale protein purification.     

Electrochemistry of complex formation of carbaryl with ds-DNA using [Ru(bpy)2dppz]2+ as probe

An electrochemical competition method was used to study the interaction of carbaryl with natural double-stranded DNA (ds-DNA). Layer-by-layer films of negatively charged natural ds-DNA and polycationic poly (diallyldimethylammonium chloride) were assembled on the surface of a glassy carbon electrode. The DNA intercalator [Ru(bpy)₂(dppz)]²⁺ (bpy?=?2, 2′-bipyridine, dppz?=?dipyrido [3, 2-a: 2′,3′-c] phenazine) was chosen as an electrochemical probe. Tripropylamine was used as an electron donor to chemically amplify the oxidation current of the probe. In order to examine the effects of substituting group on the binding interaction of carbaryl with DNA, the interaction of naphthalene or α-naphthol with DNA was also studied by square wave voltammetry (SWV). The values of binding constant K _b of the three compounds to DNA are determined, which fall in the range of (0.2?×?10⁵) to (1.3?×?10⁵)?M^?1. The correlation suggests that the functional groups may play an important role in the DNA/analyte competition binding interaction. We demonstrated that it is conducive to the combination of small molecules and DNA when the functional groups are hydrophobic and have the lone-pair electrons as the electron donor. Furthermore, UV-absorption and fluorescence intensity of Ru-dppz decreases in the presence of carbaryl. These characteristics strongly support the intercalation of carbaryl into double-stranded DNA.     

Acrolein and Copper as Competitive Effectors of α-Synuclein

Mounting evidence supports the role of amyloidogenesis, oxidative stress, and metal dyshomeostasis in the development of neurodegenerative disorders. Parkinson's Disease is characterized by α-synuclein (αSyn) accumulation and aggregation in brain regions, also promoted by Cu²⁺. αSyn is modified by reactive carbonyl species, including acrolein (ACR). Notwithstanding these findings, the interplay between ACR, copper, and αSyn has never been investigated. Therefore, we explored more thoroughly the effects of ACR on αSyn using an approach based on LC-MS/MS analysis. We also evaluated the influence of Cu²⁺ on the protein carbonylation and how the ACR modification impacts the Cu²⁺ binding and the production of Reactive Oxygen Species (ROS). Finally, we investigated the effects of ACR and Cu²⁺ ions on the αSyn aggregation by dynamic light scattering and fluorescence assays. Cu²⁺ regioselectively inhibits the modification of His50 by ACR, the carbonylation lowers the affinity of His50 for Cu²⁺ and ACR inhibits αSyn aggregation both in the presence and in the absence of Cu²⁺.