Immunosensing of NT‐proBNP via Cu2+‐based MOFs Biolabeling and in situ Microliter‐droplet Anodic Stripping Voltammetry

Immunoassay of amino‐terminal pro‐B‐type natriuretic peptide (NT‐proBNP) was conducted using Cu²⁺‐1,3,5‐benzenetricarboxylic acid metal‐organic frameworks (HKUST‐1 MOFs) as the secondary antibody label, and in situ microliter‐droplet anodic stripping voltammetry detection of Cu²⁺ in 0.1 M HNO₃+1 M NaCl directly on the glassy carbon immunoelectrode. Electrochemical methods, quartz crystal microbalance, scanning electron microscopy and energy dispersive spectroscopy were employed for material and electrode characterizations. Under optimized conditions, the limit of detection (S/N=3) was 0.33 fg mL^?1, and the analysis of NT‐proBNP in clinical serum samples returned good results.     

A Novel Label Free Electrochemical Magnetoimmunosensor for Human Interleukin‐6 Quantification in Serum

A novel magnetoimmunosensor, designed for sensitive and selective quantification of interleukin 6, is herein reported. The experimental design involves the covalent immobilization of anti‐interleukin 6 antibody through an amidic bond formed with the carboxyl functionalities provided at the surface of protein G‐functionalized magnetic microparticles, assuring a sandwich‐type immunoassay with electrochemical label free detection. All the experimental parameters involved in the elaboration and testing protocol were optimized. A linear calibration plot between the charge transfer resistance and the logarithmic concentration of interleukin‐6 was achieved in the 1 pg mL^?1 to 1 μg mL^?1 range. A limit of quantification of 1 pg mL^?1 and a detection limit of 0.3 pg mL^?1 were obtained. The optimized magnetoimmunosensor showed an excellent selectivity against some potentially interfering proteins and has been successfully applied for the determination of target protein in human serum, proving its clinical relevance.     

An Electrochemical Immunosensor for C‐Reactive Protein Based on Multi‐Walled Carbon Nanotube‐Modified Electrodes

C‐reactive protein (CRP) is a nonspecific biomarker of inflammation and infection that can be used as a predictive risk marker of cardiovascular disease in asymptomatic individuals or as a prognostic marker of recurrent ischemia and death among patients with coronary heart disease or stroke. We developed a sensitive, disposable, and easy to operate sensor based on heterogeneous sandwich immunoassay for high sensitivity CRP (hs‐CRP) measurement. We used screen‐printed electrodes modified with multi‐walled carbon nanotubes and protein A to ensure the oriented immobilization of anti‐CRP antibodies. CRP was quantitatively measured down to a concentration of 0.5 ng mL^?1, enabling high dilution of the samples before measurement and consequently reducing interferences present in the serum. The sensor developed is suitable for point of care detection of hs‐CRP at clinically relevant concentrations for the diagnosis of both conventional and low‐grade inflammations.     

Two Photon‐Induced Electron Injection From a Nanotrigger in Native Endothelial NO‐Synthase

We have recently designed a nanotrigger (NT), a photoactive molecule addressing the NADPH sites of proteins. This nanotrigger has a 10³ times larger two‐photon cross‐section compared to the ubiquitous NADPH cofactor. In this work, we tested whether two‐photon excitation of the bound NT to NADPH sites may be used to initiate enzymatic catalysis by appropriate electron injection. To establish proof of principle, we monitored the ultrafast absorption of NT bound to the fully active endothelial NO‐Synthase (eNOS) following excitation by one and two‐photons at 405 and 810 nm, respectively. Electron injection from NT* to FAD in eNOS initiated the catalytic cycle in 15±3 ps at both exciting wavelengths. The data proved for the first time that electron transfer can be promoted by two‐photon excitation. We also show that the nanotrigger decays faster in homogeneous solvents than in the NADPH site of proteins, suggesting that hindered environments modified the natural decay of NT. The nanotrigger provides a convenient way of synchronizing an ensemble of proteins in solution with a femtosecond laser pulse. The ability of NT to initiate NOS catalysis by two‐photon excitation may be exploited for controlled and localized release of free NO in cells with enhanced spatial and temporal resolution.     

Liquid‐crystalline azobenzene‐containing ferrocene‐based polymers: study on synthesis and properties of main‐chain ferrocene‐based polyesters with azobenzene in the side chain

Ferrocene‐based polymers are characterized by their electrochemical activity, good redox properties, thermal, photochemical stability, and liquid crystallinity, and thus they have various applications in different fields. A comprehensive investigation on the synthesis and properties of three novel main‐chain ferrocene‐based polyesters with azobenzene in the side chain (MFPAS) was carried out. The main‐chain ferrocene‐based polyester, poly(N‐phenyldiethanolamine 1,1′‐ferrocene dicarboxylate (PPFD), was synthesized via the solution polycondensation reaction of 1,1′‐ferrocenedicarbonyl chloride with phenyldiethanolamine (PDE). The novel MFPAS were synthesized via the post‐polymerization azo‐coupling reaction of PPFD with three different 4‐substituted anilines including 4‐nitroaniline, 4‐aminobenzoic acid, and 4‐aminobenzonitrile to produce 4‐nitrophenylazo‐functionalized‐PPFD (PPFD‐NT), 4‐carboxyphenylazo‐functionalized‐PPFD (PPFD‐CA), and 4‐cyanophenylazo‐functionalized‐PPFD (PPFD‐CN), respectively. All the synthesized polymers were characterized by ¹H NMR spectroscopy, Fourier transform infrared spectroscopy, and UV–visible spectroscopy. In addition, powder X‐ray diffraction patterns were measured for the synthesized polymers. The photoisomerization of the MFPAS was studied. The thermal properties of the MFPAS were studied using thermogravimetric analysis and differential scanning calorimetry. PPFD‐CA and PPFD‐CN were found to be more thermally stable than PPFD‐NT. Finally, the liquid‐crystalline properties of PPFD and the MFPAS were examined using polarized optical microscope. It was found that all the polymers possessed nematic phases and exhibited textures with schlieren disclinations. Copyright © 2012 John Wiley & Sons, Ltd.     

Study on the Electrochemical Behavior of Dopamine and Uric Acid at a 2‐Amino‐5‐mercapto‐[1,3,4] Triazole Self‐Assembled Monolayers Electrode

Selected from a series of structurally related heteroaromatic thiols, a newly synthesized reagent 2‐amino‐5‐mercapto‐[1,3,4] triazole (MATZ) was used to fabricate self‐assembled monolayers (SAMs) on gold electrode for the first time. The MATZ/Au SAMs was characterized by electrochemical methods and scanning electronic microscopy (SEM). In 0.04 mol/L Britton–Robinson buffer solution (pH 5), the electrochemical behavior of dopamine showed a quasireversible process at the MATZ/Au SAMs with an electrode kinetic constant 0.1049 cm/s. However, the electrochemical reaction of uric acid at the SAMs electrode showed an irreversible oxidation process, the charge‐transfer kinetics of uric acid was promoted by the SAMs. By Osteryoung square‐wave voltammetry (OSWV), the simultaneous determination of dopamine and uric acid can be accomplished with an oxidation peak separation of 0.24 V, the peak current of dopamine and uric acid were linearly to its concentration in the range of 2.5×10^?6–5.0×10^?4 mol/L for dopamine and 1×10^?6–1×10^?4 mol/L for uric acid with a detection limit of 8.0×10^?7 mol/L for dopamine and 7.0×10^?7 mol/L for uric acid. The MATZ/Au SAMs electrode was used to detect the content of uric acid in real urine and serum sample with satisfactory results.     

HPLC determination of γ‐aminobutyric acid and its analogs in human serum using precolumn fluorescence labeling with 4‐(carbazole‐9‐yl)‐benzyl chloroformate

In this study, a simple analytical method for the determination of γ‐aminobutyric acid, gabapentin, and baclofen by using high‐performance liquid chromatography with fluorescence detection was developed. An amidogen‐reactive fluorescence labeling reagent, 4‐(carbazole‐9‐yl)‐benzyl chloroformate was first used to sensitively label these analytes. The completed labeling of these analytes can be finished rapidly only within 5 min at the room temperature (25°C) to form 4‐(carbazole‐9‐yl)‐benzyl chloroformate labeled fluorescence derivatives. These labeled derivatives expressed strong fluorescence property with the maximum excitation and emission wavelengths of 280 and 380 nm, respectively. The labeled derivatives were analyzed using a reversed‐phase Eclipse SB‐C18 column within 10 min with satisfactory shapes. Excellent linearity (R² > 0.995) for all analytes was achieved with the limits of detection and the limits of quantitation in the range of 0.25?0.35 and 0.70?1.10 μg/L, respectively. The proposed method was used for the simultaneous determination of γ‐aminobutyric acid and its analogs in human serum with satisfactory recoveries in the range of 94.5–97.5%.     

In‐vitro Aluminum Determination and Preconcentration in Blood of Dialysis Patients Based on Ionic Liquid Dispersive Liquid‐Liquid Biomicroextraction by 2‐Amino‐3‐(1H‐imidazol‐4‐yl)propanoic Acid

In this study, trace amounts of aluminum in serum of dialysis patients were chelated with 2‐Amino‐3‐(1H‐imidazol‐4‐yl)propanoic acid (Histidine) and determined by electro‐thermal atomic absorption spectrometry (ETAAS). A fast and efficient method based on ionic liquid dispersive liquid‐liquid bio‐micro‐extraction (IL‐DLLBME) was developed for the determination of Al cation in human blood serum samples. In this work, a small amount of 1‐Hexyl‐3‐methylimmidazolum hexafluorophosphate ([HMIM] [PF₆]) as an extractant solvent was dissolved in acetone as a dispersant solvent and then the binary solution was rapidly injected by a syringe into the serum containing Al³⁺,Which have already in‐vitro chelated by Histidine amino acid (Al‐His) at pH = 6.5. After separation, the settled IL‐phase was dissolved in ethanol up to 200 μL and 20 μL of samples injected into the ET‐AAS by auto‐sampler. Various parameters have been studied and optimized for 10 mL of sample. Under the optimum conditions, the enrichment factor (EF), limit of detection (LOD) and working range (peak area mode) were obtained 53, 15 ng L^?1 and 0.05‐4.1 μg L^?1 respectively. In vitro Al chelation showed that His can significantly decrease aluminum concentration in serum of dialysis patients. Validation of methodology was confirmed by standard reference material (SRM).     

Validated Method for the Simultaneous Determination of Gemifloxacin and H2‐receptor Antagonists in Bulk,Pharmaceutical Formulations and Human Serum by RP‐HPLC; In‐vitro Applications

Simple, isocratic and rapid RP‐HPLC method has been developed for the simultaneous analysis of gemifloxacin and H₂‐receptor antagonists i.e. Cimetidine, Famotidine and Ranitidine, in bulk, pharmaceutical formulation and human serum. Separation was achieved on the RP‐Mediterranea column [C18 (250 × 4.6 mm, 5 μ)] at ambient temperature using mobile phase consisting of acetonitrile: methanol: water (20:28:52 v/v/v pH 2.8 adjusted by phosphoric acid). Flow rate was 1.0 mL/min with an average operating pressure of 180 kg/cm². Gatifloxacin (GATI) was used as an internal standard (IS). Quantitation was achieved with UV detection at 221, 256 and 267 nm, respectively. Linear calibration curves, at concentration ranges of 0.05‐37.5 μgmL^‐L with a correlation coefficient of ±0.9994. The detection and quantification limits were in the ranges of 0.023‐0.250 μgmL^‐L and 0.071‐0.756 μgmL^‐L, respectively. Friedman's and Student's t‐test were applied to correlate these results. Method was validated in terms of selectivity, linearity, precision, robustness, recovery, limits of detection and quantitation and is applicable to the routine analysis of GFX and H₂‐receptor antagonists, alone or in combination.     

Development of an Efficient Procedure for Determination of Copper,Zinc and Iron after Solid Phase Extraction on 3‐(1‐(1‐H‐Indol‐3‐yl)‐3‐Phenylallyl)‐1H‐Indole Loaded on Duolite XAD 761

A method for the simultaneous preconcentration of Cu²⁺,Zn²⁺ and Fe³⁺ ions, in some food samples has been reported. The method is based on the adsorption of 3‐(1‐(1‐H‐indol‐3‐yl)‐3‐phenylallyl)‐1H‐indole (IPAI) loaded on Duolite XAD 761. The metal ions adsorbed on the modified solid phase resin are eluted using 6 mL of 4 mol L^?1 nitric acid. The influences of the analytical parameters including pH and amount of ligand and solid phase and type and amount of surfactant and sample volume on the metal ions recoveries were investigated. The effects of matrix ions on the retentions of the analytes were also examined. The recoveries of analytes were generally higher than 95% with a RSD lower than 5%. The method has been successfully applied for these metals content evaluation in some real samples.     

Luminescence‐Functionalized Metal–Organic Frameworks Based on a Ruthenium(II) Complex: A Signal Amplification Strategy for Electrogenerated Chemiluminescence Immunosensors

Novel luminescence‐functionalized metal–organic frameworks (MOFs) with superior electrogenerated chemiluminescence (ECL) properties were synthesized based on zinc ions as the central ions and tris(4,4′‐dicarboxylicacid‐2,2′‐bipyridyl)ruthenium(II) dichloride ([Ru(dcbpy)₃]²⁺) as the ligands. For potential applications, the synthesized MOFs were used to fabricate a “signal‐on” ECL immunosensor for the detection of N‐terminal pro‐B‐type natriuretic peptide (NT‐proBNP). As expected, enhanced ECL signals were obtained through a simple fabrication strategy because luminescence‐functionalized MOFs not only effectively increased the loading of [Ru(dcbpy)₃]²⁺, but also served as a loading platform in the ECL immunosensor. Furthermore, the proposed ECL immunosensor had a wide linear range from 5 pg mL^?1 to 25 ng mL^?1 and a relatively low detection limit of 1.67 pg mL^?1 (signal/noise=3). The results indicated that luminescence‐functionalized MOFs provided a novel amplification strategy in the construction of ECL immunosensors and might have great prospects for application in bioanalysis.     

Synthesis of a new 2‐amino‐glycan,poly‐(1→6)‐α‐D‐mannosamine,by ring‐opening polymerization of 1,6‐anhydro‐mannosamine derivatives

A stereoregular 2‐amino‐glycan composed of a mannosamine residue was prepared by ring‐opening polymerization of anhydro sugars. Two different monomers, 1,6‐anhydro‐2‐azido‐mannose derivative ( 3 ) and 1,6‐anhydro‐2‐(N, N‐dibenzylamino)‐mannose derivative ( 6 ), were synthesized and polymerized. Although 3 gave merely oligomers, 6 was promptly polymerized into high polymers of the number‐average molecular weight (M_n) of 2.3 × 10⁴ to 2.9 × 10⁴ with 1,6‐α stereoregularity. The differences of polymerizability of 3 and 6 from those of the corresponding glucose homologs were discussed. It was found that an N‐benzyl group is exceedingly suitable for protecting an amino group in the polymerization of anhydro sugars of a mannosamine type. The simultaneous removal of O‐ and N‐benzyl groups of the resulting polymers was achieved by using sodium in liquid ammonia to produce the first 2‐amino‐glycan, poly‐(1→6)‐α‐D ‐mannosamine, having high molecular weight through ring‐opening polymerization of anhydro sugars.© 2012 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem, 2012     

Efficient measurement of the sign and the magnitude of long‐range proton‐carbon coupling constants from a spin‐state‐selective HSQMBC‐COSY experiment

A spin state‐selective Heteronuclear Single‐Quantum Multiple‐Bond Connectivities (HSQMBC‐COSY) experiment is proposed to measure the sign and the magnitude of long‐range proton‐carbon coupling constants (ⁿJ(CH); n > 1) either for protonated or for non‐protonated carbons in small molecules. The simple substitution of the selective 180° ¹H pulse in the original selHSQMBC pulse scheme by a hard one allows the simultaneous evolution of both proton‐proton and proton‐carbon coupling constants during the refocusing period and enables a final COSY transfer between coupled protons. The successful implementation of the IPAP principle leads to separate mixed‐phase α/β cross‐peaks from which ⁿJ(CH) values can be easily measured by analyzing their relative frequency displacements in the detected dimension. Copyright © 2012 John Wiley & Sons, Ltd.     

A Multi‐electrochemical Competitive Immunosensor for Sensitive Cocaine Determination in Biological Samples

A multi‐electrochemical competitive immunosensor for the rapid determination of unmetabolized cocaine (COC) in urine, saliva and human serum matrices is reported. Anti‐cocaine polyclonal antibodies were immobilized in an oriented way onto protein‐G functionalized magnetic beads. The immunosensor is based on an array of eight carbon‐based screen‐printed electrodes for simultaneous electrochemical determinations. The treatments of the biological samples were simplified and optimized for avoiding matrix interferences. The immunosensor was sensitive (EC₅₀≈2.92–3.88 ng mL^?1 COC), required a very small volume of sample (200 µL), was reproducible (%RSD was lesser than about 18 %), and accurate (recovery percentages ranged 88–117 %).     

Development of a novel hollow‐fiber liquid‐phase microextraction based on oil‐in‐salt and its comparison with conventional one

A novel hollow‐fiber liquid‐phase microextraction based on oil‐in‐salt was proposed and introduced for the simultaneous extraction and enrichment of the main active compounds of hesperidin, honokiol, shikonin, magnolol, emodin, and β,β′‐dimethylacrylshikonin in a formula of Zi‐Cao‐Cheng‐Qi decoction and the single herb, Fructus Aurantii Immaturus , Cortex Magnoliae Officinalis , Radix et Rhizoma , and Lithospermum erythrorhizon , composing the formula prior to their analysis by high‐performance liquid chromatography. The results obtained by the proposed procedure were compared with those obtained by conventional hollow‐fiber liquid‐phase microextraction, and the proposed procedure mechanism was described. In the procedure, a hollow‐fiber segment was first immersed in organic solvent to fill the solvent in the fiber lumen and wall pore, and then the fiber was again immersed into sodium chloride solution to cover a thin salt membrane on the fiber wall pore filling organic solvent. Under the optimum conditions, the enrichment factors of the analytes were 0.6–109.4, linearities were 0.002–12 μg/mL with r ² ≥ 0.9950, detection limits were 0.6–12 ng/mL, respectively. The results showed that oil‐in‐salt hollow‐fiber liquid‐phase microextraction is a simple and effective sample pretreatment procedure and suitable for the simultaneous extraction and concentration of trace‐level active compounds in traditional Chinese medicine.     

Development and validation of a LC method for the separation and determination of the anticancer‐active FeIII(4‐methoxy‐salophene) using the new second‐generation monolith

LC method with the newly introduced second‐generation monolithic silica RP‐18e column has been developed for the separation of Fe^III(salophene) and four methoxy‐substituted Fe^III(salophene) complexes. The method has been validated for the quantitation of Fe^III(4‐OMe‐salophene), a highly active anticancer substance in vitro, bound to serum albumin. Our routinely used high‐resolution continuum‐source atomic absorption spectroscopy method based on the determination of the central iron atom was unsuitable in this case because serum originally contains significant amounts of iron as revealed by a blank sample of serum albumin. The developed LC method depends on detecting the whole complex rather than the bound iron. Two morphologically different first‐ and second‐generation HPLC monolithic columns have been compared for this purpose. The newly introduced second‐generation monolithic silica column Chromolith® HighResolution RP‐18e column (100 × 4.6 mm, Merck) separated the mixture successful within 13 min. A mobile phase consisting of 25 mM phosphate buffer pH 3/methanol (60:40, v/v) was used at a flow rate of 1 mL/min. The dynamic linear working range of the calibration curve for Fe^III(4‐OMe‐salophene) was found to be between 1 and 200 μg/mL. Detection and quantitation limits were 0.3 and 1 μg/mL, respectively.     

Interaction of Bovine Serum Albumin with N‐(4‐Ethoxyphenyl)‐N′‐(4‐Antipyrinyl)Thiourea (EPAT) Using Spectroscopies

The interaction between N‐(4‐ethoxyphenyl)‐N′‐(4‐antipyrinyl)thiourea (EPAT) and bovine serum albumin (BSA) was studied by fluorescence spectroscopy in combination with UV absorption spectroscopy. The intrinsic fluorescence of bovine serum albumin was quenched by EPAT through a static quenching procedure. The binding constants of EPAT with BSA were estimated according to the fluorescence quenching results at different temperatures. The thermodynamic parameters: enthalpy change (ΔH) and entropy change (ΔS) were calculated to be ?10.69 kJ/mol and 42.64 J·mol^?1·K^?1 according to thermodynamic equations, respectively, and indicating that the binding force was suggested to be mainly a hydrophobic force. The effect of common ions on the binding constant was also investigated. A new fluorescence spectroscopy assay of the proteins was presented in this paper. The determination results of the proteins in bovine serum by means of this method were very close to those obtained using Coomassie Brilliant Blue G‐250 colorimetry.     

Preparative separation of anti‐oxidative constituents from Rubia cordifolia by column‐switching counter‐current chromatography

An effective column‐switching counter‐current chromatography (CCC) protocol combining stepwise elution mode was successfully developed for simultaneous and preparative separation of anti‐oxidative components from ethyl acetate extract of traditional Chinese herbal medicine Rubia cordifolia. The column‐switching CCC system was interfaced by a commercial low‐pressure six‐port switching valve equipped with a sample loop, allowing large volume introduction from the first dimension (1^st‐D) to the second dimension (2^nd‐D). Moreover, to extend the polarity window, three biphasic liquid systems composed of n‐hexane/ethyl acetate/methanol/water (1:2:1:2, 2:3:2:3, 5:6:5:6 v/v) were employed using stepwise elution mode in the 1^st‐D. By valve switching technique the whole interested region of 1^st‐D could be introduced to second dimension for further separation with the solvent system 5:5:4:6 v/v. Using the present column‐switching CCC protocol, 500 mg of crude R. cordifolia extract were separated, producing milligram‐amounts of four anti‐oxidative components over 90% pure. Structures of purified compounds were identified by ¹H and ¹³C NMR.     

Simultaneous Determination of Dopamine and Ascorbic Acid Using the Nano‐Gold Self‐Assembled Glassy Carbon Electrode

Electrochemical behavior of dopamine (DA) was investigated at the gold nanoparticles self‐assembled glassy carbon electrode (GNP/LC/GCE), which was fabricated by self‐assembling gold nanoparticles on the surface of L ‐cysteine (LC) modified glassy carbon electrode (GCE) via successive cyclic voltammetry (CV). A pair of well‐defined redox peaks of DA on the GNP/LC/GCE was obtained at E_pa=0.197 V and E_pc=0.146 V, respectively. And the peak separation between DA and AA is about 0.2 V, which is enough for simultaneous determination of DA and AA. The peak currents of DA and AA were proportional with their concentrations in the range of 6.0×10^?8–8.5×10^?5 mol L^?1 and 1.0×10^?6–2.5×10^?3 mol L^?1, with the detection limit of 2.0×10^?8 mol L^?1 and 3.0×10^?7 mol L^?1 (S/N=3), respectively. The modified electrode exhibits an excellent reproducibility, sensibility and stability for simultaneous determination of DA and AA in human serum with satisfactory result.     

Simultaneous determination of cotinine and trans‐3‐hydroxycotinine in urine by automated solid‐phase extraction using gas chromatography–mass spectrometry

A gas chromatography–mass spectrometry method was developed and validated for the simultaneous automated solid‐phase extraction and quantification of cotinine and trans‐3‐hydroxycotinine in human urine. Good linearity was observed over the concentration ranges studied (R² > 0.99). The limit of quantification was 10 ng/mL for both analytes. The limits of detection were 0.06 ng/mL for cotinine (COT) and 0.02 ng/mL for trans‐3‐hydroxycotinine (OH‐COT). Accuracy for COT ranged from 0.98 to 5.28% and the precision ranged from 1.24 to 8.78%. Accuracy for OH‐COT ranged from ?2.66 to 3.72% and the precision ranged from 3.15 to 7.07%. Mean recoveries for cotinine and trans‐3‐hydroxycotinine ranged from 77.7 to 89.1%, and from 75.4 to 90.2%, respectively. This analytical method for the simultaneous measurement of cotinine and trans‐3‐hydroxycotinine in urine will be used to monitor tobacco smoking in pregnant women and will permit the usefulness of trans‐3‐hydroxycotinine as a specific biomarker of tobacco exposure to be determined. © 2014 The Authors. Biomedical Chromatography published by John Wiley & Sons Ltd.