Synthesis of Molecularly Imprinted Polymer for Sensitive Penicillin Determination in Milk

Abstract

Penicillin G (PG) in milk is unsafe for human and is a big problem for the foods industry. Hence, the better analytical and eliminative techniques are demanded. We investigated a PG molecular imprinted polymer (MIP), which can sensitively detect and extract the PG from milk and other samples. The MIP synthesis involved a β-lactam antibiotics PG, methacrylic acid (MAA) and dimethyl formamide (DMF). Its properties were analyzed with Scatchard plot, which showed that there were two affinity sites of the PG polymer. The first equilibrium dissociation constant of the high-affinity binding sites Kd₁ was 1.14 × 10^?4 mol/L and the binding capacity Q_max1 was 46.01 µmol/g; the second equilibrium dissociation constant of the low-affinity binding sites Kd₂ was 1.35 × 10^?3 mol/L and the binding capacity Q_max2 was 72.55 µmol/g. Furthermore, two PG determination methods using the polymer were developed. The first was carried out quantitatively with a spectrophotometer and the detection limit was 1 ppm. The other one was the combination of the MIP particles with milk fermentation for PG analysis and the sensitivity was 10 ppb.     

Affinity CE Determination of the Binding Constant of Bioactive Sulfated Polysaccharide 916 to Human Serum Albumin

Drug-protein binding is an important process in determining the activity of a pharmaceutical agent once it has entered the body. This paper developed an affinity capillary electrophoresis method to determine the binding constant between a bioactive sulfated polysaccharide 916 (916) and a potential protein, human serum albumin. This method is based on the principle that the changing analytes have different mobility shift in the zone electrophoresis. A fixed amount of protein was injected into a capillary filled with a background electrolyte containing the polysaccharide in varying concentrations. The effective mobility data of the protein were processed according to classical linearization treatments to obtain the binding constant of 916 to the HSA complex. The binding constant Ka of 916 to the human serum albumin achieved was 2.1 × 10⁴.     

Thermodynamics and molecular bases of the interaction of ampicillin and streptomycin at their binding sites of bovine serum albumin

Among the biological parameters of chemotherapeutics, serum albumin binding is a critical factor in determining drug distribution and bioavailability. In this study, the binding properties as well as the interaction of ampicillin and streptomycin at their binding sites of bovine serum albumin (BSA) were investigated. The binding constant varied from 3.2 × 10³ M^?1 at 298 K to 37.5 × 10³ M^?1 at 313 K for ampicillin, and from 10.7 × 10³ M^?1 at 298 K to 3.5 × 10³ M^?1 at 313 K for streptomycin. By increasing the temperature, from 298 to 313 K, the binding affinity decreased by about 11-fold for ampicillin. Conversely, streptomycin showed stronger binding at higher temperature, which is decreased by threefold at 298 K. Interestingly, the affinity of ampicillin with the free BSA was ~4-fold higher than the binding with BSA/streptomycin complex. In contrast, the affinity of streptomycin with the free BSA was ~6-fold lower than the binding with BSA/ampicillin complex. Mutual binding experiments indicate that ampicillin and streptomycin are sharing both of common and different binding sites on BSA. Dissection of the forces of interactions indicated that rigid-body binding was the mode of binding of ampicillin and streptomycin with BSA with minor degree of conformational changes. Both of ampicillin and streptomycin can bind with free BSA. Furthermore, the binding of ampicillin with BSA improves the binding of streptomycin, while the binding of streptomycin with BSA adversely affect the binding of ampicillin.     

Macromolecular interactions of spectinomycin with Bovine serum albumin

Among the pharmacokinetic parameters of chemotherapeutics, serum albumin binding is a critical factor in determining drug distribution and bioavailability. In this study, the binding properties as well as the interaction of spectinomycin with Bovine serum albumin was investigated. Spectinomycin showed stronger binding with BSA at higher temperatures, which diminishes by decreasing the temperature. The binding constant of spectinomycin with BSA varied from 3.1 × 10³ M^?1 at 298 K to 6.3 × 10³ M^?1 at 313 K. By increasing the temperature, from 298 to 313 K, the binding affinity was increased by twofolds. Thermodynamic analysis indicated changes in albumin conformation and partial loss of folding during spectinomycin-albumin binding. The mild-moderate binding affinity of spectinomycin with BSA will be important in determining the drug–drug interactions at the binding sites of BSA. The presence of stronger binding ligand e.g., chloramphenicol, tetracyclines or diclofenac will compete with spectinomycin for its binding sites, therefore, lowering its serum albumin binding. The result of this study will be helpful in understanding of the binding properties and mechanisms of interaction of spectinomycin with bovine serum albumin.     

Human Saliva-Based Quantitative Monitoring of Clarithromycin by Flow Injection Chemiluminescence Analysis: A Pharmacokinetic Study

Human saliva quantitative monitoring of clarithromycin (CLA) by chemiluminescence (CL) with flow injection analysis was proposed for the first time, which was based on the quenching effect of CLA on luminol–bovine serum albumin (BSA) CL system with a linear range from 7.5?×?10^?4 to 2.0 ng/ml. This proposed approach, offering a maximum sample throughput of 100 h^?1, was successfully applied to the quantitative monitoring of CLA levels in human saliva during 24 h after a single oral dose of 250 mg intake, with recoveries of 95.2～109.0 % and relative standard deviations lower than 6.5 % (N?=?7). Results showed that CLA reached maximum concentration of 2.28?±?0.02 μg/ml at approximately 3 h, and the total elimination ratio was 99.6 % in 24 h. The pharmacokinetic parameters including absorption rate constant (0.058?±?0.006 h^?1), elimination rate constant (0.149?±?0.009 h^?1) and elimination half-life time (4.66?±?0.08 h) were obtained. A comparison of human saliva and urine monitoring was also given. The mechanism study of BSA–CLA interaction revealed the binding of CLA to BSA is an entropy driven and spontaneous process through hydrophobic interaction, with binding constant K _BSA–CLA of 4.78?×?10⁶ l/mol and the number of binding sites n of 0.82 by flow injection–chemiluminescence model. Molecular docking analysis further showed CLA might be in subdomain IIA of BSA, with K _BSA–CLA of 6.82?×?10⁵ l/mol and ΔG of ?33.28 kJ/mol.     

Kinetic studies on the interactions between glycolipid biosurfactant assembled monolayers and various classes of immunoglobulins using surface plasmon resonance

Kinetic studies on the interactions between self-assembled monolayers of mannosylerythritol lipids (MELs), which are glycolipid biosurfactants abundantly produced by microorganisms, and various classes of immunoglobulins including human IgG, IgA, and IgM were performed using surface plasmon resonance (SPR). The effect of the MEL structure on the binding behavior of HIgG was examined. Assembled monolayers of MEL-A having two acetyl groups on the headgroup gave a high affinity (K_d = 1.7 × 10⁻⁶ M) toward HIgG, while those of MEL-B or MEL-C having only one acetyl group at C-6′ or C-4′ position gave little affinity. Our kinetic analysis revealed that the binding manner of HIgG, HIgA (K_d = 2.4 × 10⁻⁷ M), and HIgM (K_d = 2.2 × 10⁻⁷ M) to the assembled monolayers of MEL-A is not the monovalent mode but the bivalent mode, and both the first and second rate association constants (k_a1, k_a2) increase with an increase in the number of antibody binding sites, while those for dissociation (k_d1, k_d2) changed little. Moreover, we succeeded in directly observing great amounts of HIgG, HIgA, and HIgM bound to MEL-A monolayers using atomic force microscopy (AFM). Finally, we found that MEL-A assembled monolayer binds toward various IgG derived from mouse, pig, rabbit, horse, goat, rat, and bovine as well as human IgG (HIgG), and the only exception was sheep IgG. These results clearly demonstrate that MEL-A assembled monolayers would be useful as noble affinity ligand system for various immunoglobulins.     

Molecular insight of isotypes specific β-tubulin interaction of tubulin heterodimer with noscapinoids

Noscapine and its derivatives bind stoichiometrically to tubulin, alter its dynamic instability and thus effectively inhibit the cellular proliferation of a wide variety of cancer cells including many drug-resistant variants. The tubulin molecule is composed of α- and β-tubulin, which exist as various isotypes whose distribution and drug-binding properties are significantly different. Although the noscapinoids bind to a site overlapping with colchicine, their interaction is more biased towards β-tubulin. In fact, their precise interaction and binding affinity with specific isotypes of β-tubulin in the αβ-heterodimer has never been addressed. In this study, the binding affinity of a panel of noscapinoids with each type of tubulin was investigated computationally. We found that the binding score of a specific noscapinoid with each type of tubulin isotype is different. Specifically, amino-noscapine has the highest binding score of ?6.4, ?7.2, ?7.4 and ?7.3 kcal/mol with αβ_I, αβ_II, αβ_III and αβ_IV isotypes, respectively. Similarly 10 showed higher binding affinity of ?6.8 kcal/mol with αβ_V, whereas 8 had the highest binding affinity of ?7.2, ?7.1 and ?7.2 kcal/mol, respectively with αβ_VI, αβ_VII and αβ_VIII isotypes. More importantly, both amino-noscapine and its clinical derivative, bromo-noscapine have the highest binding affinity of ?46.2 and ?38.1 kcal/mol against αβ_III (overexpression of αβ_III has been associated with resistance to a wide range of chemotherapeutic drugs for several human malignancies) as measured using MM-PBSA. Knowledge of the isotype specificity of the noscapinoids may allow for development of novel therapeutic agents based on this class of drugs.     

Preparation and Adsorption Characteristics of Cordycepin Molecularly Imprinted Polymers

The separation of a molecularly imprinted polymer for cordycepin was investigated. The synthesis employed cordycepin as the molecular template, alpha-methylacrylic acid as the functional monomer, glycol dimethyl acrylate as the cross-linking agent, azobisisobutyronitrile as the initiator, and tetrahydrofuran as the solvent and pore-foaming agent. The interaction between cordycepin and the functional monomer was investigated by ultraviolet-visible and infrared spectroscopy. The properties of the molecularly imprinted polymer were analyzed by scanning electron microscopy, equilibrium adsorption experiments, and the Scatchard equation. Static adsorption, solid phase extraction, and high-performance liquid chromatography experiments were employed to evaluate the adsorption properties and selective recognition characteristics. The results showed that the molecularly imprinted polymer had specific adsorption with cordycepin, and the maximum absorption capacity was 1920 µg/g. Scatchard analysis suggested that high affinity and low affinity binding sites were present. For the high affinity case, the dissociation constant and apparent maximum numbers of the binding sites were 0.0089 mmol/L and 4.78 µmol/g, respectively. The dissociation constant and apparent numbers of binding sites were 0.035 mmol/L and 6.047 µmol/g for the low affinity sites. Compared with the corresponding nonimprinted polymer, the cordycepin molecularly imprinted polymer exhibited higher adsorption and selectivity for cordycepin than structural analogs.     

Immunoassay by detecting enhanced resonance light scattering signals of immunocomplex using a common spectrofluorometer

A sensitive, highly specific immunoassay method has been developed by measuring the enhanced resonance light scattering (RLS) signals of immunoreactions with simultaneously scanning both the excitation and the emission monochromators of a common spectrofluorometer. For a given content of antibody (Ab), the RLS signals of an immunoreaction follow Gaussian distribution with antigen (Ag) concentration. The central position of the Gaussian curve represents the concentration of given Ab, and the half bandwidth has proved to be a characteristic constant of a given Ab-Ag immunoreaction. With the RLS signals, the limit of detection for human immunoglobulin G (HIgG) in serum samples could reach 10 ng ml⁻¹, and the concentration of HIgG in blood serum samples could be detected with the recovery of 90.2-107.7% and R.S.D. of 0.8-2.7%. The results of determination for three human serum samples are identical to those obtained by immunoturbidimetry.     

Synthesis,antimicrobial evaluation and docking study of triazole containing triaryl-1H-imidazole

An efficient route for the synthesis of triazole containing triaryl-1H-imidazole (3a–3r) was achieved involving multicomponent condensation of triazole aldehydes, ammonium acetate and 1,2-dicarbonyl compounds in glacial acetic acid. The structure of newly synthesized imidazoles was established by the FTIR, HRMS and NMR spectra. All the compounds displayed considerable antimicrobial activity against fungal and bacterial strains. The triazolyl imidazole 3p was substantially potent against P. aeruginosa (0.0113?µmol/mL), A. niger (0.0113?µmol/mL) and C. albicans (0.0056?µmol/mL) wherein triazolyl imidazoles 3i was found to be more potent against B. subtilis (0.0122?µmol/mL) & A. niger (0.0121?µmol/mL); and compound 3r was also found to be more potent against S. epidermidis (0.0117?µmol/mL) & C. albicans (0.0058?µmol/mL). As a result of docking studies, the binding affinity of the compound 3o was –9.6?kcal/mol which was even more in comparison to the binding affinity of co-crystallized ligand CBN (–9.4?kcal/mol).     

Purification of Acetylcholinesterase by 9-Amino-1,2,3,4-tetrahydroacridine from Human Erythrocytes

The acetylcholinesterase enzyme was purified from human erythrocyte membranes using a simple and effective method in a single step. Tacrine (9-amino-1,2,3,4-tetrahydroacridine) is a well-known drug for the treatment of Alzheimer's disease, which inhibits cholinesterase. We have developed a tacrine ligand affinity resin that is easy to synthesize, inexpensive and selective for acetylcholinesterase. The affinity resin was synthesized by coupling tacrine as the ligand and l-tyrosine as the spacer arm to CNBr-activated Sepharose 4B. Acetylcholinesterase was purified with a yield of 23.5 %, a specific activity of 9.22 EU/mg proteins and 658-fold purification using the affinity resin in a single step. During purification, the enzyme activity was measured using acetylthiocholine iodide as a substrate and 5,5′-dithiobis-(2-nitrobenzoicacid) as the chromogenic agent. The molecular weight of the enzyme was determined as about 70 kDa monomer upon disulphide reduction by sodium dodecyl sulphate polyacrylamide gel electrophoresis. K _m, V _max, optimum pH and optimum temperature for acetylcholinesterase were found by means of graphics for acetylthiocholine iodide as the substrate. The optimum pH and optimum temperature of the acetylcholinesterase were determined to be 7.4 and 25–35 °C. The Michaelis–Menten constant (K _m) for the hydrolysis of acetylthiocholine iodide was found to be 0.25 mM, and the V _max was 0.090 μmol/mL/min. Maximum binding was achieved at 2 °C with pH 7.4 and an ionic strength of approximately 0.1 M. The capacity for the optimum condition was 0.07 mg protein/g gel for acetylcholinesterase.     

Imidazo[1,2-a]pyridine-substituted coumarin as a selective ratiometric sensor for Cu2+ ion

A novel fluorescent chemosensor, (E)-7-(diethylamino)-3-((2-phenylimidazo[1,2-a]pyridin-3-ylimino)methyl)-2H-chromen-2-one 1a, has been synthesised and characterised. This chemosensor displayed an extreme selective fluorescence emission only with Cu²⁺ ion over all other metal ions examined. The Job’s plot experiment analysis suggested the binding ratio of the chemosensor 1a with Cu²⁺ was 1:1 metal-to-ligand ratio. The association constant for Cu²⁺ towards receptor 1a obtained from Benesi–Hildebrand plot was found to be 4.859 × 10³ M^?1 with a detection limit 4.6 × 10^?8 M. Fluorescence enhancement caused by Cu²⁺ binding with chemosensor 1a attributed to combinational effect of intramolecular charge transfer and chelation-enhanced fluorescence occurred at pH 8.0.     

Appending zinc tetraphenylporphyrin with an amine receptor at β-pyrrolic carbon for designing a selective histamine chemosensor

Adopting the rarely used β-functionalisation strategy in porphyrin-based sensor design, an amine receptive site is appended onto the zinc(II) porphyrin molecular framework affording a ditopic chemosensor 4. The assembled chemosensor interacts selectively with histamine in toluene via a ‘two-site’ binding mode. Association constant of the complex evaluated from the respective UV–vis spectra is found to be (2.32 ± 0.57) × 10⁶, which is approximately 4-fold greater than those complexes derived from 4 and nicotine/histidine. On the basis of a combined spectroscopic method and molecular modelling, the binding model of the porphyrin host and biogenic guest molecules is established. Our results clearly demonstrate the viability of the design and development of the porphyrin-based chemosensor by appending a receptor at the β-pyrrolic carbon of the porphyrin scaffold.     

A new lawsone azo-dye for optical sensing of Fe3+ and Cu2+ and their DFT study

A new lawsone-based azo-dye 2-hydroxy-3-((pyridin-2-ylmethyl)diazenyl)naphthalene-1,4-dione (1) was synthesized and applied for sensing of metal ions. Receptor 1 showed selective fluorescent and colorimetric response for the detection of Cu²⁺ and Fe³⁺ over other tested metal ions. The fluorescence intensity of 1 was significantly quenched allowing detection of Fe³⁺ and Cu²⁺ down to 0.61 and 6.06 μM, respectively. The binding has been established by fluorescence spectroscopic method. Receptor 1 provided a 1?:?1 binding scaffold for recognition of Fe³⁺ and Cu²⁺ ions with the association constant of 3.33 × 10⁶ and 3.33 × 10⁵ M^?1, respectively. The B3LYP/6-31G/LANL2DZ method was employed for the optimization of 1 and 1·Fe³⁺ and 1·Cu²⁺.     

Polarographic and Voltammetric Study of the Sc(III)-Acid Chrome Blue K Complex and Determination of Trace Scandium

Abstract

A new system of polarographic adsorptive wave for determining trace scandium was proposed. In 0.2 mol/L NH₄OAc, the Sc(III)- ACBK [1,8- dihydroxy- 2- (2- hydroxy- 5- sulfo- 1- phenylazo)- 3,6- disulfo- naphthalene, called acid chrome blue K] complex emerged a sensitive adsorptive complex wave(Ep′ = -0.67V). The molar ratio of Sc(III) to ACBK in the complex was established as 1: 2 and the apparent stability constant β₂ = 2.7 × 10¹⁵. But for Y(III), the molar ratio was 1: 1 and β = 1.5 × 10⁵. Because of the particularity of Sc complex, the sensitivity and the selectivity of determination Sc are much better than that of other rare earth ions. The detection limit is 1.1 × 10^?7 mol/L for oscillopolarography and 2.0 × 10^?8 mol/L for adsorptive stripping voltammetry.     

Bis-thiourea macrocycles incorporating fused [n]polynorbornanes: binding of flexible versus rigid dicarboxylates

Two macrocyclic bis-thiourea hosts 5 and 6 were constructed and their interactions with two dicarboxylates of similar size (pimelate = flexible and terephthalate = rigid) were evaluated using ¹H NMR titration techniques. In contrast to previous work with thiourea functionalised [n]polynorbornanes (where a notable increase in H:G affinity was noted for the rigid guest), the new macrocyclic hosts, in particular host 6, bind pimelate more strongly than terephthalate (for 6 binding pimelate log K_a = 4.7, terephthalate log K_a = 3.7). A binding arrangement in which the flexible dicarboxylate is ‘perched’ above the macrocycle is proposed to justify these results.     

Binding of anticancer drug Ru(η 6 -C6H5(CH2)2OH)Cl2(DAPTA) to DNA purine bases and amino acid residues: a theoretical study

The hydrolyzed Ru(η ⁶ -C₆H₅(CH₂)₂OH)Cl₂(DAPTA) (DAPTA = 3,7-diacetyl-1,3,7-triaza-5-phosphabicyclo[3.3.1]nonane) binding to guanine(G), adenine (A), cytosine(C), cysteine (Cys), and histidine (His) residues were explored using the B3LYP hybrid functional and IEF-PCM solvation models. The computed activation barriers for the reactions of diaqua complex were lower than those of chloroaqua complex except for binding to cytosine. For the chloroaqua complex, the activation free energy was lowest when binding to cytosine (10.5 kcal/mol). Whereas, the substitution reaction of diaqua complex binding to cysteine showed the lowest activation free energy with 10.1 kcal/mol, closely followed by histidine (15.8 kcal/mol), adenine (20.1 kcal/mol), cytosine (20.7 kcal/mol), and guanine (24.4 kcal/mol) by turns. It could be deduced that the completely hydrolyzed Ru(η ⁶ -C₆H₅(CH₂)₂OH)Cl₂(DAPTA) compounds might preferentially bind to amino acids residues in vivo. In addition, to simulate the protein and DNA environment in vivo, a detailed investigation of the activation free energies for the substitution reactions in dependence of the dielectric constant ε (4, 24, and 78.39) was systematically performed as well. The calculated results demonstrated that the environmental effect had a little impact on these substitution reactions.     

Investigations on Adsorption Potentiometry Part V Derivative Adsorption Chronopotentiometry of Beryllium(II)-4-[(4-diethylamino-2-hydroxypheanyl)-azo]-5-hydroxy-naphthalene-2,7-disurphonic Acid System

Abstract

An electroanalytical methood, based on derivative chronopotentiometry of the complex of beryllium(II) with 4-[(4-diethylamino-2-hydroxy-phenyl)-azo]-5-hydroxy-naphthalene-2,7-disurphonic acid (Beryllon II) accumulated adsorptively on the surface of a hanging mercury drop electrode, has been developed for determining trace beryllium in food. The dependences of the peak height on the dt/dE vs. E curve on the pre-concentration time, preconcentration potential and the constant reducing current are discussed. In 0.15 mol/1 NH_s+0.05 mol/1 NH₄Cl, 4×10^?7 mol/l Beryllon II, and at a preconcentration potential of -0.30 V (ve. SCE), the limit of detection and linear range are 1 × l0^?10 mol/l and 3 × 10^?10 -2 × l0^?7 mol/l, Iwpectively. The relative standard error of the method is 2.3% for 6 × 10^?8 mol/l Be(II). The method WBB applied to samples of food. The electrode procees hae been diecueeed.     

Electrochemical Behavior of Hydrogen Peroxide at a Glassy Carbon Electrode Modified with Nickel Hydroxide–Decorated Multiwalled Carbon Nanotubes

Abstract

The multiwalled carbon nanotube–nickel hydroxide composite film used to modify glassy carbon electrode was prepared and confirmed by transmission electron microscopy and cyclic voltammetry. The process and mechanism of film formation were discussed in detail. The electrode modified with the composite film exhibited good catalytic activity toward electrochemical oxidation of hydrogen peroxide in 0.1 mol/L sodium hydroxide solution. Various factors affecting the electrocatalytic activity of nickel hydroxide film were investigated. The anodic peak current increased with the increased concentration of hydrogen peroxide. The linear range for the determination of hydrogen peroxide was from 1.5 × 10^?6 mol/L to 2.5 × 10^?3 mol/L with the detection limit 6.1 × 10^?7 mol/L (S/N = 3). And the proposed method was applied to the determination of hydrogen peroxide in disinfector with higher sensitivity and lower detection limit.     

Electrochemical Investigation of Ascorbic Acid Interacting with Bovine Serum Albumin

Abstract

Electrochemical investigation of the interaction of Ascorbic acid (AA) with bovine serum albumin (BSA) on a glassy carbon electrode is reported for the first time. In a 0.1 mol/l, pH 7.3 NaCl solution, AA had a well‐defined voltammetric oxidative peak at +0.2086 V (versus Ag/AgCl) on a GC electrode. After the addition of BSA into the AA solution, the oxidative peak current decreased significantly without a shift of the peak potential, and no new peak appeared. The experimental results showed that a new nonelectrochemical supramolecular complex was formed after the interaction of BSA with AA, which resulted in a decrease of the diffusion coefficient, and then a decrease of the oxidative peak current. The interaction conditions and the electrochemical detection conditions were carefully investigated. The stoichiometry of this supramolecular complex was calculated based on voltammetric data with a binding number of two and a binding constant of 1.38×10⁷.