Rapid detection of bacterial endotoxins in ophthalmic viscosurgical device materials by direct analysis in real time mass spectrometry

Bacterial endotoxins are lipopolysaccharides bound to the bacterial cell wall and released when bacteria rupture or disintegrate. Possible contamination of endotoxin in ophthalmic devices can cause a painful eye inflammation or result in toxic anterior segment syndrome after cataract surgery. Measurement of bacterial endotoxin in medical device materials is difficult since endotoxin binds with polymer matrix and some of the materials are very viscous and non-water soluble, where traditional enzyme-based Limulus amebocyte lysate (LAL) assay cannot be applied. Here we propose a rapid and high throughput ambient ionization mass spectrometric (MS) method using direct analysis in real time (DART) for the evaluation of endotoxin contamination in medical device materials. Large and structurally complex endotoxin instantaneously breaks down into low-mass characteristic fragment ions using DART and is detected by MS in both positive and negative ion modes. This method enables the identification and separation of endotoxin from medical materials with a detection limit of 0.03 ng mL⁻¹ endotoxins in aqueous solution. Ophthalmic viscosurgical device materials including sodium hyaluronate (NaHA), non-water soluble perfluoro-n-octane (PFO) and silicone oil (SO) were spiked with different known concentrations of endotoxin and analyzed by DART MS, where the presence of endotoxin was successfully detected and featured small mass fragment ions were generated for NaHA, PFO and SO as well. Current findings showed the feasibility of measuring endotoxin contamination in medical device materials using DART-MS, which can lead to a one-step analysis of endotoxins in different matrices, avoiding any potential contamination during sample pre-treatment steps.     

Discrimination between endotoxin and (1----3)-beta-D-glucan using turbidimetric kinetic assay with Limulus amebocyte lysate

A procedure for the Limulus amebocyte lysate (LAL) test discriminating between endotoxin and (1----3)-beta-D-glucan based on the turbidimetric kinetic method was proposed. Endotoxin and (1----3)-beta-D-glucan, which are elicitors of the activation of LAL, showed different reaction courses with this lysate. To analyze the difference in the reactions, two parameters, the maximum differential coefficient of the reaction (Dmax) and the reaction time required to obtain Dmax (Tp) were defined. The logarithmic plottings of Tp versus Dmax (Tp-Dmax plot) discriminated between endotoxin and (1----3)-beta-D-glucan. Endotoxin was measured with a standard curve plotting logarithmic endotoxin concentration versus Dmax (ET-Dmax plot). The endotoxin calculated from Dmax was less influenced by (1----3)-beta-D-glucan than that calculated from the usual gelation time. A small amount of endotoxin in a sample could be concealed by the addition of polymyxin B, which inhibited the activation of LAL by endotoxin. (1----3)-beta-D-glucan was measured without being affected by the presence of a small amount of endotoxin using LAL with polymyxin B. The following procedure is proposed as a LAL test to discriminate between endotoxin and (1----3)-beta-D-glucan. (1) Identify the main substance (endotoxin or (1----3)-beta-D-glucan) triggering the activation of LAL using the Tp-Dmax plot. (2) Use the appropriate method to measure the main substance: the ET-Dmax plot for endotoxin or the LAL with polymyxin B for (1----3)-beta-D-glucan.     

Determination of nickel(II) as the nickel dimethylglyoxime complex using colorimetric solid phase extraction

Colorimetric solid phase extraction (C-SPE) is an analytical technique in which analytes in water samples are extracted onto a solid adsorbent matrix impregnated with a colorimetric reagent and then quantified directly on the adsorbent surface using diffuse reflectance spectroscopy. This paper presents a further development in C-SPE. In this case, the reagent employed to detect the analyte is not impregnated on the extraction medium. Instead, the reagent is weakly immobilized on a solid support (i.e., filter paper) and released into the sample as it flows through the support. The reagent complexes the analyte in solution, forming a highly colored precipitate that is collected on the surface of an extraction membrane. The concentration of analyte is determined using the Kubelka-Munk function calculated from the diffuse reflectance spectrum of the precipitate on the membrane surface. This precipitation-spectrophotometric platform is extensively evaluated by determining nickel(II) using dimethylglyoxime (DMG) as the precipitating reagent. The ability to optimize reaction conditions with immobilized reagents by in-line buffering is also demonstrated. Specifically, borax buffer was utilized to adjust the pH of nickel(II) samples prepared in deionized water. This combination of immobilized buffer and reagent allows C-SPE to operate in a solid-phase mode in which all the reagents requisite for optimal analyte determination are immobilized on solid supports. Using this method, nickel(II) was determined in a single processing step over the concentration range 0.50-5.0 ppm in ∼40 s with 1.0 ml sample volumes.     

Solid-phase reagent for molecular spectroscopic determination of heavy metal speciation in natural water

A solid-phase reagent based on 1-(4-adamantyl-2-thiasolylazo)-2-naphthol adsorbed onto silica gel was prepared for Co(II) recovery and preconcentration prior to its sorption-spectroscopic detection. The immobilized reagent was applied to the determination of free cobalt ions in natural water. The solid-phase reagent and chemiluminescent method coupled with membrane filtration, gel-permeation and ion-exchange chromatography were applied to the study of the speciation of iron and cobalt in water from the Dnieper reservoirs and lakes of Kyiv City; their predominant forms were complexes of Fe(III) and Co(II) with dissolved organic matter and fulvic acids play a main role in their complexation.     

Rapid detection of proteins by enzyme-linked immunofiltration assay after transfer onto nitrocellulose membranes.

Enzyme-linked immunofiltration assay (ELIFA) for labeling transferred proteins is an interesting and powerful technique for the rapid specific detection (15 min) of proteins immobilized on nitrocellulose or nylon membranes (0.20 and 0.45 micron). ELIFA does not require fastidious handling of the membranes. Saturation, specific labeling and washing procedures are achieved by filtration, controlled by a monitoring unit which regulates the flow rate and ensures excellent specificity, repetition and reproducibility. The recycling by closed circuit or by repetitive inversion of the flow direction offers the advantage of reducing the volumes of expensive reagents while simultaneously increasing the sensitivity of the technique. The detection limit is at least as low as 1-5 ng using directly or indirectly enzymatically labelled probes. ELIFA may be extended to the identification of glycoproteins using specific ligands such as lectins or to the immunocapture of an antigen using specific antibodies immobilized on an activated membrane. ELIFA complements fast separation, by e.g., isoelectric focusing, polyacrylamide gel electrophoresis, or sodium dodecyl sulfate-polyacrylamide gel electrophoresis and accelerated electrotransfer to membranes with rapid detection reducing the total time for separation transfer and detection to less than 2 h.     

亲和吸附剂对细菌内毒素吸附性能的研究

制备了以球形纤维素为载体、8种氨基酸和1种聚赖氨酸为配基的吸附剂,对质量浓度为100.0pg/mL的内毒素水溶液进行了吸附研究,绘制了吸附等温线,并初步探讨了吸附机理.结果表明,精氨酸和赖氨酸配基具有良好的吸附能力,在1.5mL100.0pg/mL内毒素溶液中吸附量分别达到182.0和160.0pg/mL;吸附等温线显示,以赖氨酸为配基的吸附剂其吸附量随溶液内毒素浓度增加而线性增加,符合Langmuir吸附方程,吸附能力强,具有一定的临床应用前景.     

Ultramicrodetermination of Amino Acids and Histidyl-Peptides by Fluorescence Measurements of Spots on Paper

Abstract

A method was developed which ensures reproducibility of fluorescence of OPT histidine and histidyl-peptides, and other amino acid-ninhydrin-phenylacetaldehyde complexes absorbed on filter paper. A linear dependence of fluorescence was found in the range 2-400 ng for histidine and histidyl-peptides with the OPT reagent, and in the range 7-Z50 ng for glycine, phenylalanine, histidine and methionine, with the ninhydrin-phenylacetaldehyde reagent.     

胶体金免疫层析法快速检测腹泻性贝毒软海绵酸的研究

腹泻性贝毒是一类分布较广的赤潮毒素,严重威胁到人类的健康和安全。本文用胶体金标记利用细胞融合技术制备的抗软海绵酸单克隆抗体,使用卵清蛋白合成高偶联比的包被抗原,以硝酸纤维素膜为载体,利用免疫层析技术原理,建立了快速检测软海绵酸的免疫层析试纸条方法。方法检出限12 ng/mL(0.96纳克/条)。     

The use of thermal lensing for the determination of pyrogens

Based on the optimized spectrophotometric determination of pyrogens (of various classes ( p-aminophenol and endotoxins), thermal lensing was applied to the determination of these substances at the submicrogram level. The limit of detection of p-aminophenol, a pyrogenic impurity in pharmaceutical formulations of paracetamol, by reaction with resorcinol in alkaline solutions is 100 ng mL(-1). Phloroglucinol was considered as an analog of resorcinol as a reagent in this reaction. The conditions of spectrophotometric determination of pyrogenic lipopolysaccharides (endotoxins) by ion-pair formation with methylene blue (the limit of detection is 100 ng mL(-1)), by ion-pair formation with Stains-All (1-ethyl-2-[3-(1-ethylnaphtho[1,2-d]thiazolin-2-ylidene)-2-methylpropenyl]naphtho[1,2-d]thiazolium bromide) (the limit of detection is 500 ng mL(-1)), and by reaction of 2-keto-3-deoxyoctonic acid with thiobarbituric acid (the limit of detection is 800 ng mL(-1)) were proposed. The optimized procedure for 2-keto-3-deoxyoctonic acid was applied for thermal lensing that provided a decrease in the limit of detection to 70 ng mL(-1) and was also used for lipopolysaccharide determination in the endotoxin standard from E. coli.     

鲎试剂检测乌司他丁中细菌内毒素探讨

根据中国药典2000版二部内毒素检查法,对不同批号的乌司他丁进行了细菌内毒素检测,并与家兔法进行了比较。     

Structure and sorption characteristics of bioaffinity membranes

The new bioaffinity membranes comprise mammalian blood and tissue cells immobilized in the polymer matrix. The method of immobilization does not assume the retention of physiological and enzymatic activity of immobilized cells, but it ensures the safety of cellular membrane receptors that are used as specific ligands. Macroporous polymer carriers based on polyacrylonitrile maintain the accessibility of the cellular receptors for all blood plasma components including immunoglobulins and viral particles. The sorption capacity of membranes with respect to model substances in a batchwise technique is evaluated. Although the results are of a preliminary nature, the membranes may be used in crossflow modules for selective blood plasma correction of endogenous substances.     

A MutS protein-immobilized au electrode for detecting single-base mismatch of DNA.

A novel electrochemical biosensor was developed to detect gene mutation by using a DNA-mismatch binding protein: MutS from Escherichia coli. The MutS protein was immobilized onto an Au-electrode surface via complex formation between a histidine tag of the MutS protein and a thiol-modified nitrilotriacetic acid chemically adsorbed on the Au-electrode surface. When a target double-stranded DNA having a single-base mismatch was captured by the MutS protein on the electrode, some electrostatic repulsion arose between polyanionic DNA strands and anionic redox couple ions. Consequently, their redox peak currents on a cyclic voltammogram with the Au electrode drastically decreased, depending on the concentration of the target DNA, according to the redox couple-mediated artificial ion-channel principle. By using this assay, one can detect all types of single-base mismatch and single-base deletion.     

Development and evaluation of a rapid lateral flow immunochromatographic strip assay for screening 19-nortestosterone

The lateral flow strip test for 19-nortestosterone is one kind of immunochromatographic assay. Nitrocellulose membrane was separately immobilized with goat anti-rabbit IgG (control line) and 19-NT-OVA conjugate (test line). Anti-19-NT polyclonal antibody labeled with colloidal gold particles acted as the detector reagent. The assay is qualitatively, not quantitatively, judged with positive or negative result. We tested the sensitivity of the strip using spiked swine urine, and each specimen was independently measured by LC/MS/MS. The sensitivity, measure by eye, was determined to be 200 ng/mL. The assay time was less than 15 min, and so suitable for on-site rapid test.     

Bismuth Film Electrode as Sensing Platform for IgE–anti‐IgE Interactions

In this study, a bismuth film was used as a sensing platform for immunoreaction assay which includes the interactions between IgE and anti‐IgE molecules. For preparing this assay, IgE was deposited on a carbon paste electrode via bismuth(III) cations without using any membrane or functional reagent. The immobilized reagents and interaction of anti‐IgE on this formation were monitored by using atomic force microscopy technique and electrochemical impedance spectroscopy. Then some experimental parameters like the effect of IgE amount and the IgE–anti‐IgE interaction time were optimized. Under the optimal experimental conditions, the binding of anti‐IgE was also examined by following current decrease in differential pulse voltammograms of neutral red.     

Rapid polyelectrolyte-based immunofiltration technique for testosterone detection in serum samples

A new immunofiltration assay for testosterone is proposed. During the first step of the assay, testosterone molecules in serum samples compete in solution with the testosterone-peroxidase conjugate for interaction with anti-testosterone antibodies pre-bound to the conjugate between staphylococcal protein A and polymethacrylate polyanion. The reaction mixture is then filtered through a membrane charged with immobilized poly(N-ethyl-4-vinylpyridinium) polycation. The filtration is accompanied by a rapid separation of the polyanion containing complexes due to high-affinity electrostatic interactions. Following removal of unbound compounds the immobilized peroxidase is detected using a substrate that produces an insoluble coloured product. The proposed assay has been shown to combine high speed (20 min) and sensitivity (0.1 ng ml(-1)), and to be applicable for out-of-laboratory conditions. Based on densitometric measurements, the RSD of the assay is calculated to be 3.2-5.1% (n = 4). The proposed assay is 4 times faster than the microplate enzyme immunoassay (ELISA) based on the same immunoreagents. Pre-incubation of the antibody and the polyanion-protein A conjugate at a certain ratio excludes the influence of immunoglobulins from the tested serum samples on the assay results. The polyanion-protein A conjugate can be used as a universal reagent, eliminating the necessity to modify specific antibodies for each immunoassay.     

A biased solid-phase radioimmunoassay for thyroxine (T4) using T4-125I presaturated antibodies

An assay for thyroxine (T₄) is demonstrated using specific antibodies covalently coupled to controlled pore glass (CPG). In this assay immobilized antibodies saturated with labeled T₄ are employed in a preloaded unit tube configuration. These complexes are stable for long periods of time when stored in buffer. This concept results in a highly reproducible and sensitive assay for T₄ that requires a single pipetting step.     

Recyclable fluorescent gold nanocluster membrane for visual sensing of copper(II) ion in aqueous solution

Recently, metal-selective fluorescent chemosensors have attracted intense attention for their simple and real-time tracking of metal ions in environmental samples. However, most of the existing fluorescent sensors are one-off sensors and thus suffer from large amount of reagent consumption, significant experimental cost and raising the risk of environmental pollution. In this paper, we developed a green (low reagent consumption, low-toxicity reagent use), recyclable, and visual sensor for Cu(2+) in aqueous solution by using a fluorescent gold nanoclusters membrane (FGM) as the sensing unit, basing on our findings on gold nanoclusters (Au NCs) that the bovine serum albumin (BSA)-coated Au NCs exhibit excellent membrane-forming ability under the isoelectric point of BSA, and thus enable us to obtain a new type of sensing membrane (i.e. FGM) by denaturing Au NCs; the fluorescence of FGM can be significantly quenched by Cu(2+) ion, and the quenched fluorescence can be totally recovered by histidine; the as-prepared FGM is very stable and recyclable, which makes it an ideal sensing material.     

An electrokinetic study on a synthetic adsorbent of crystalline calcium silicate hydrate and its mechanism of endotoxin removal

A synthetic, disposable adsorbent of crystalline calcium silicate hydrate, LRA product by Advanced Minerals Corp., has been found highly effective for endotoxin removal from aqueous solutions. Endotoxin removal by this adsorbent is greatly enhanced by the addition of an electrolyte, such as NaCl or Tris-HCl. The electrophoretic method has been used to study the mechanism of endotoxin adsorption. In many cases, adding the electrolyte increases the magnitude of negative zeta potential of the adsorbent in water, while endotoxin adsorption reduces the magnitude. It is hypothesized that ion-exchange between monovalent cations from the aqueous phase and Ca2+ ions near the surface of the adsorbent shift zeta potential of the adsorbent to the more negative direction. It is further hypothesized that endotoxins form cationic species through binding between its phosphate groups and Ca2+ ions dissolved from the adsorbent. The adsorption of endotoxins in the form of cationic species is enhanced by the increased negative zeta potential of the adsorbent when an electrolyte is added.     

CE to monitor endotoxins by protein complexation

A new CE method for fast and efficient analysis of bacterial endotoxins (lipopolysaccharides) is described. It is based on the strong interaction between proteins and endotoxins. The UV absorption of the protein component in the complex is used for the detection. The electrophoretic mobility of the complex hemoglobin/endotoxin can be employed for qualitative analysis of the endotoxin. For instance, the structural differences between "smooth" and "rough" lipopolysaccharides from Salmonella minnesota (wild-type), Salmonella minnesota R595 and Shigella sonnei R562H are reflected in the electrophoretic mobilities of their hemoglobin complex.     

Biomimetic membranes with aqueous nano channels but without proteins: impedance of impregnated cellulose ester filters

Earlier we have shown that many important properties of ionic aqueous channels in biological membranes can be imitated using simple biomimetic membranes. These membranes are composed of mixed cellulose ester-based filters, impregnated with isopropyl myristate or other esters of fatty acids, and can be used for high-throughput drug screening. If the membrane separates two aqueous solutions, combination of relatively hydrophilic polymer support with immobilized carboxylic groups results in the formation of thin aqueous layers covering inner surface of the pores, while the pore volume is filled by lipid-like substances. Because of these aqueous layers biomimetic membranes even without proteins have a cation/anion ion selectivity and specific (per unit of thickness) electrical properties, which are similar to typical properties of biological membranes. Here we describe frequency-dependent impedance of the isopropyl myristate-impregnated biomimetic membranes in the 4-electrode arrangement and present the results as Bode and Nyquist diagrams. When the membranes are placed in deionized water, it is possible to observe three different dispersion processes in the frequency range 0.1 Hz to 30 kHz. Only one dispersion is observed in 5 mM KH(2)PO(4) solution. It is suggested that these three dispersion features are determined by (a) conductivity in aqueous structures/channels, formed near the internal walls of the filter pores at high frequencies, (b) dielectric properties of the whole membrane at medium frequencies, determined by polymer support, aqueous layers and impregnating oil, and, finally, (c) by the processes in hydrated liquid crystal structures formed in pores by impregnating oil in contact with water at low frequencies.