Label-free electrochemical aptasensor for the detection of lysozyme

This work reports the advantages of a label free electrochemical aptasensor for the detection of lysozyme. The biorecognition platform was obtained by the adsorption of the aptamer on the surface of a carbon paste electrode (CPE) previously blocked with mouse immunoglobulin under controlled-potential conditions. The recognition event was detected from the decrease in the guanine and adenine electro-oxidation signals produced as a consequence of the molecular interaction between the aptamer and lysozyme. The biosensing platform demonstrated to be highly selective even in the presence of large excess (9-fold) of bovine serum albumin, cytochrome C and myoglobin. The reproducibility for 10 repetitive determinations of 10.0 mg L⁻¹ lysozyme solution was 5.1% and 6.8% for guanine and adenine electro-oxidation signals, respectively. The detection limits of the aptasensor were 36.0 nmol L⁻¹ (if considering guanine signal) and 18.0 nmol L⁻¹ (if taking adenine oxidation current). This new sensing approach represents an interesting and promising alternative for the electrochemical quantification of lysozyme.     

Adsorption of lysozyme on a hemodialysis sulfonated polyacrylonitrile membrane, with and without preadsorbed poly(ethyleneimine) on the external faces

We present the influence of pH, from pH 4 to 10, with a focus on the neutral range, on the adsorption of lysozyme (isoelectric point pI=11) on a sulphonated membrane and the same membrane pre-treated with poly(ethyleneimine) (PEI). We found a steep increase of the adsorbed amount above pH 6 in phosphate buffer. The adsorbed amount was about twice as low in Tris buffer, around the neutral pH. The difference between the two types of buffer is attributed to their different ionic composition. High interfacial concentration in phosphate buffer is especially linked to the phosphate divalent anions. In the presence of divalent sulphate anions, we measured the same level of interfacial concentration than with phosphate buffer. With the PEI pre-treated membrane, we observed, on the time scale of our experiments (15–20 h), similar adsorbed amounts than on the raw membrane, showing that the PEI layer does not constitute a true barrier to the penetration of lysozyme into the membrane core. However, its presence leads to a slower adsorption rate in a system where convection does not occur through the membrane.     

Advances in electrochemical detection for probing protein aggregation

Electrochemistry provides an array of methods to investigate protein aggregation and determine biomarkers of neurodenenerative diseases. Biosensors detecting monomeric or oligomeric biomarkers of Alzheimer's disease and Parkinson's disease evolved toward femtomolar, multiplexed detection in blood and biological fluids for less invasive diagnosis. The biosensors also serve as complementary tools in studies investigating putative biomarkers for the assessment of patient's cognitive decline. The study of protein aggregation via the direct electrochemical oxidation focused recently on enhanced sensitivity and on establishing correlations between protein structure and aggregation propensity. Departing from classic approaches, nanopore resistive pulse sensing and single-particle collision electrochemistry enable studying aggregates in solution. Growing applications converge toward accurate evaluation of aggregate populations and method adoption beyond proof of principle.     

Silica nanoparticle supported molecularly imprinted polymer layers with varied degrees of crosslinking for lysozyme recognition

Surface imprinting over nanosized support materials is particularly suitable for protein templates, considering the problems with mass transfer limitation and low binding capacity. Previously we have demonstrated a strategy for surface protein imprinting over vinyl-modified silica nanopartiles with lysozyme as a model template by polymerization in high-dilution monomer solution to prevent macrogelation. Herein, the synthesis process was further studied toward enhancement of the imprinting performance by examining the effect of several synthesis conditions. Interestingly, the feed crosslinking degree was found to have a great impact on the thickness of the formed imprinting polymer layers and the recognition properties of the resulting imprinted materials. The imprinted particles with a crosslinking degree up to 50% showed the best imprinting effect. The imprinting factor achieved 2.89 and the specific binding reached 23.3 mg g⁻¹, which are greatly increased compared to those of the lowly crosslinked imprinted materials reported previously. Moreover, the relatively high crosslinking degree led to no significant retarding of the binding kinetics to the imprinted particles, and the saturated adsorption was reached within 10 min. Therefore, this may be a promising method for protein imprinting.     

An introduction to bismuth film electrode for use in cathodic electrochemical detection

A new electrode surface design, the bismuth film electrode (BiFE), is presented as a promising alternative to mercury and other solid electrodes for direct cathodic electrochemical detection of organic compounds. The preparation of the BiFE, involving an ex situ electroplating of metallic bismuth onto a glassy carbon (GC) substrate electrode, was optimised. The useful negative potential windows of the BiFE in the pH range 1 (−0.2 to −0.8 V vs Ag/AgCl) to 10 (−0.2 to −1.5 V) were determined. The reproducibility of measuring 2-nitrophenol as a model compound (relative standard deviation, r.s.d., n=10) was found to be 0.5% at the same BiFE, and 1.0% at successive newly prepared BiFEs. No polishing or any other pre-treatment of the substrate GC surface was required prior to re-plating of a new Bi film. The BiFE showed similar or even favourable voltammetric behaviour when compared to mercury and bare GC electrodes, and was successfully tested for amperometric detection under hydrodynamic conditions. The results revealed that BiFE is an attractive new non-mercury metallic electrode particularly suitable for cathodic electrochemical detection in flow analytical systems.     

Tris(hydroxymethyl)aminomethane-modified magnetic microspheres for rapid affinity purification of lysozyme

A novel affinity purification method for lysozyme (LZM) based on functionalized magnetic microspheres was developed. Tris(hydroxymethyl)aminomethane (Tris)-modified magnetic microspheres with specific affinity toward LZM were prepared using Tris as ligand and silica-coated magnetic microshperes as support. Transmission electron microscopy and magnetic property measurement results showed that the Tris-modified magnetic microspheres have a very good core-shell structure and high magnetization.The maximum binding capacity of LZM was about 108.6 mg/g magnetic microspheres. LZM purified from chicken egg white had high purity and well-maintained activity of 8140 U/mg. This magnetic-mediated LZM purification strategy has advantages of high efficiency, low cost and easy operation.     

Gold nanoparticles integrated in a nanotube array for electrochemical detection of glucose

An enzyme-free amperometric glucose sensor of gold nanoparticle-constituted nanotube array electrode is presented. The resulted gold nanotube array electrode with significantly enhanced surface roughness shows prominent catalytic activity toward the electrooxidation of glucose in a pH 7.4 phosphate buffer (PBS) solution and thus can be used to individually or simultaneously determine glucose and the common interfering molecule of ascorbic acid (AA). In the case of glucose detection, the amperometric responses show a linear relationship to glucose concentration in the range of 1 mM–42.5 mM with a detection limit down to 10 μM. The present non-enzymatic glucose electrochemical biosensor shows a good stability and reproducibility.     

The electrochemical detection of Ru(II) in a methyl methacrylate solution

This article describes the voltammetric behaviour of RuCl₂(PPh₃)₃ in a methyl methacrylate (MMA) solution. Acquiring this type of information is only possible when the ohmic resistance can be kept sufficiently low. Therefore, the conductivity study of pure methyl methacrylate and a tetrabutylammonium tetrafluoroborate (TBABF₄) methyl methacrylate solution has been described as well. Impedance measurements show an increase in conductivity by adding TBABF₄, while a conductometric curve illustrates the presence of ion pairs, triple ions and quadrupoles depending on the TBABF₄ concentration. The conductivity of a 0.1 mol L⁻¹ TBABF₄-MMA solution (formation of charged triple ions) was high enough to perform electrochemical experiments and a calibration curve could be obtained. The ability of obtaining relevant electrochemical data in low conducting media opens up new perspectives, especially for electroanalytical purposes used to monitor polymer reactions, more specific atom transfer radical polymerization (ATRP) reactions. This method employs a redox process with transition metal complexes in which a halogen ion is transferred reversibly between the transition metal and the polymer chain end. The dynamic equilibrium can be monitored by measuring the ruthenium concentration.     

Indirect voltammetric detection of fluoride ions in toothpaste on a comb-shaped interdigitated microelectrode array

A novel technique based on dynamic electrochemistry for the detection of fluoride ions was developed. It is based on its strong complexation with ferric ion. Formed fluoroferric complex is cathodically inactive at the potential of the reduction of free ferric aquo ion. The voltammetric and amperometric response of platinum comb-shaped interdigitated microelectrode array is decreased after fluoride addition. This decrease serves for the quantification of fluoride ions added to the solution. The detection limit of 4.5 × 10⁻⁵ mol dm⁻³ was achieved when one of the segments of interdigitated microelectrode array (IDA) was used as an indicating electrode. The detection limit is about one order of magnitude lower than in the case of conventional platinum macroelectrode. In comparison with ISE electrodes this method is faster and also avoiding large error resulting from the antilogarithmization of ISE Nerstian response. The method was applied to the analysis of toothpaste.     

Electrochemical Oxidation of Sanguinarine and of Its Oxidation Products at a Glassy Carbon Electrode – Relevance to Intracellular Effects

The electrochemical behavior of sanguinarine, a quaternary benzophenanthridine glycoside alkaloid with antimicrobial, anti‐inflammatory, antioxidant and/or immune‐stimulatory activities, was studied at a glassy carbon electrode using cyclic, differential pulse, and square wave voltammetry. The oxidation of sanguinarine is a quasireversible, diffusion‐controlled process and occurred in a cascade mechanism with the formation of several oxidation products which adsorbed at the electrode surface. The oxidation of sanguinarine is pH dependent and involves the transfer of the same number of electrons and protons. The adsorbed sanguinarine oxidation products are reversibly oxidized at the glassy carbon electrode surface and their oxidation for a wide range of pHs was also studied by differential pulse and square wave voltammetry. A mechanism for the oxidation of sanguinarine at glassy carbon electrode is proposed.     

Serum-protein effects on the detection of the β-blocker propranolol by ion-transfer voltammetry at a micro-ITIES array

In this work, the effect of the serum protein, bovine serum albumin (BSA), on the detection of propranolol in artificial serum by ion-transfer voltammetry at an array of micro-interfaces between two immiscible electrolyte solutions (μITIES) is presented. Cyclic voltammetry (CV), differential pulse voltammetry (DPV), and differential pulse stripping voltammetry (DPSV) were examined for the detection of low concentrations of propranolol. Both CV and DPV had an interference effect from BSA, manifested as lower currents in the presence of the protein. DPSV proved to be the most effective technique, enabling the detection of 0.05 μM propranolol in the presence of BSA. The DPSV method employed a preconditioning step as well as a preconcentration step followed by the analytical signal generation step. The latter was based on the back-transfer of the drug across the μITIES. The preconcentration step was crucial to prevention of the adverse effects of BSA on the voltammetric detection. These results demonstrate that serum-protein effects on drug detection at low concentrations can be eliminated by use of DPSV at arrays of μITIES. CVs of propranolol with increasing concentrations of BSA revealed the influence of the drug-protein binding interaction, with decreases in current but no change in transfer potential. Therapeutic concentrations of propranolol were detected, demonstrating the viability of this approach for bioanalytical investigations.     

基于多肽的电化学传感器在蛋白质检测中应用的研究进展

生物体内的细胞通常会分泌各种各样的蛋白质,这些蛋白质在生物体中发挥着重要作用,尤其是可被用于诊断各种疾病的发生和发展。多肽具有良好选择性、空间适应能力和识别灵活的特点,可与不同类型的蛋白分子形成非共价键,用于蛋白质的生物检测。将多肽与电化学生物传感器结合用于蛋白质的广谱检测具有良好的发展前景。本文介绍了多肽修饰的电化学传感器在不同蛋白质检测方面的研究进展,分析了待测蛋白质的不同对多肽修饰的电化学传感器分类的影响及其优缺点,提出了基于多肽的电化学传感器在不同蛋白质检测中存在的问题,并展望了其未来发展。     

Preparation and application of hollow molecularly imprinted polymers with a super‐high selectivity to the template protein

Protein‐imprinted polymers with hollow cores that have a super‐high imprinting factor were prepared by etching the core of the surface‐imprinted polymers that used silica particles as the support. Lysozyme as template was modified onto the surface of silica particles by a covalent method, and after polymerization and the removal of template molecules, channels through the polymer layer were formed, which allowed a single‐protein molecule to come into the hollow core and attach to the binding sites inside the polymer layer. The adsorption experiments demonstrated that the hollow imprinted polymers had an extremely high binding capacity and selectivity, and thus a super‐high imprinting factor was obtained. The as‐prepared imprinted polymers were used to separate the template lysozyme from egg white successfully, indicating its high selectivity and potential application in the field of separation of protein from real samples.     

Rheological properties of a surfactant-induced gel for the lysozyme–sodium dodecyl sulfate–water system

Rheological properties of isotropic solutions and gel structures of lysozyme–sodium dodecyl sulfate mixtures in water are investigated. Isotropic solutions behave as Newtonian fluids with very low viscosity values. For the lysozyme solutions the intrinsic viscosity and the Huggins coefficient were calculated on the basis of the Mooney equation. Above a certain yield stress value, the viscosity of the gel samples decreases continuously in the whole range of the shear rate. Dynamic rheological experiments show weak gel behavior where the storage modulus and the loss modulus are almost parallel and are frequency-dependent. A belated gel stage with very slow kinetics has been characterized. There is a substantial enhancement of the gel strength by ageing since the belated gel stage manifests a higher yield stress value and a higher storage modulus than the initial gel stage. The gels are stable in the temperature range between 10 and 32 °C.     

Enzyme sensor for the electrochemical detection of the marine toxin okadaic acid

An enzyme sensor for the electrochemical detection of the marine toxin okadaic acid (OA) has been developed. The strategy was based on the inhibition of immobilised protein phosphatase (PP2A) by this toxin and the electrochemical measurement of the enzyme activity by the use of appropriate enzyme substrates, electrochemically active after dephosphorylation by the enzyme. Colorimetric inhibition assays have demonstrated the PP2A from human red blood cells to be more sensitive and to provide a wider linear range than the one produced by genetic engineering. Catechyl monophosphate (CMP) and p-aminophenyl phosphate (p-APP) have been tested as enzyme substrates, the former providing higher electrochemical currents at convenient working potentials (+450 mV vs. Ag/AgCl). Biosensors with 19.1 and 5.0 U of immobilised enzyme have been applied to the OA detection. Whereas the 19.1-U biosensor has provided higher electrochemical currents and more reliable determinations, the 5.0-U one has attained a lower 50% inhibition coefficient (IC₅₀) value (22.19 in front of 154.84 μg L⁻¹) and a larger working range (2.69-171.87 in front of 42.97-171.87 μg L⁻¹). The analysis of toxicogenic dinoflagellate extracts with both biosensors and the comparison with the colorimetric assay and liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) have demonstrated the applicability of the developed electrochemical devices as screening biotools for the assessment of the toxicity of a sample.     

Preparation of a novel hybrid organic-inorganic monolith for the separation of lysozyme by high performance liquid chromatography

A novel hybrid organic-inorganic monolith for high performance liquid chromatography (HPLC) was firstly developed by atom transfer radical polymerization (ATRP) by a simple and rapid method, in which vinyl ester resin was used as the monomer, natrium bisulfurosum was used both as organic adjunct and coadunate initiator to alter the activity of the free radical in the process of polymerization and then to control the molecular mass. The conditions of polymerization were optimized. The chemical group of the monolith was assayed by infrared spectra method, the morphology of monolithic material was studied by scanning electron microscopy (SEM) and the pore size distribution was determined by a mercury porosimeter. Finally, the monolith was used to separate lysozyme (Lys) from chicken egg white with good resolution and reproducibility that were obtained in a short time (10 min) by HPLC. In addition, the influences of buffer concentration and pH value on elution have been investigated and the hybrid monolith was used to separate benzene and its homologs from the mixture.     

Fabrication of a nanostructure-based electrochemical sensor for simultaneous determination of N-acetylcysteine and acetaminophen

A carbon-paste electrode modified with 2,7-bis(ferrocenyl ethyl)fluoren-9-one (2,7-BF) and carbon nanotubes (CNTs) was used for the sensitive and selective voltammetric determination of N-acetylcysteine (NAC). The mediated oxidation of NAC at the modified electrode was investigated by cyclic voltammetry (CV). Also, the values of catalytic rate constant (k), and diffusion coefficient (D) for NAC were calculated. Differential pulse voltammetry (DPV) of NAC at the modified electrode exhibited two linear dynamic ranges with a detection limit (3σ) of 52.0 nmol L⁻¹. DPV was used for simultaneous determination of NAC and acetaminophen (AC) at the modified electrode, and quantitation of NAC and AC in some real samples by the standard addition method.     

Enzyme inhibition-based biosensor for the electrochemical detection of microcystins in natural blooms of cyanobacteria

An electrochemical biosensor for the detection of microcystin has been developed based on the inhibition of the protein phosphatase 2A (PP2A) by this cyanobacterial toxin. The enzyme has been immobilised by entrapment using a poly(vinyl alcohol) azide-unit pendant water-soluble photopolymer (PVA-AWP). Electrode supports and immobilisation conditions have been optimised by colorimetric assays, the highest immobilisation yields being obtained with screen-printed graphite electrodes and the 1:2 PP2A:PVA ratio. Catechyl monophosphate (CMP), α-naphthyl phosphate (α-NP) and 4-methylumbelliferyl phosphate (4-MUP) have been used as phosphorylated substrates to monitor the protein phosphatase activity by electrochemical methods, the former providing the highest chronoamperometric currents at appropriate working potentials (+450 mV versus Ag/AgCl). Incubation with standard microcystin solutions has demonstrated the inhibition of the immobilised enzyme, proportional to the toxin concentration. The standard inhibition curve has provided a 50% inhibition coefficient (IC₅₀) of 83 μg L⁻¹, a limit of detection (LOD; 35% inhibition) of 37 μg L⁻¹, and 100% inhibition at about 1000 μg L⁻¹. Real samples of cyanobacterial blooms from the Tarn River (Midi-Pyrénées, France) have been analysed using the developed amperometric biosensor and the toxin contents have been compared to those obtained by a conventional colorimetric protein phosphatase inhibition (PPI) assay and high-performance liquid chromatography (HPLC). The results clearly justify the use of the developed amperometric biosensor as screening method for microcystin detection.