基于氧化还原体系纸芯片法对血清尿酸含量的快速检测

建立了以纸芯片为分析平台的尿酸含量快速检测方法.基于尿酸的还原性,以FeCl3为氧化剂组成氧化还原体系,以邻二氮菲为显色剂对反应产物Fe2+进行显色,根据显色强度对尿酸含量进行测定.结果 表明,当FeCl3与尿酸比例为1.6∶1,反应10 min,显色剂pH 7.4时显色最佳.显色强度(RGB值)与尿酸浓度在1.19～5.95 mmol/L范围内呈良好线性关系,相关系数为0.9998.方法 的检测限为0.342 mol/L,日内和日间相对标准偏差分别为0.42％和0.47％.15种共存物质在允许误差范围内均无干扰.测得牛血清中尿酸含量为0.238 mmol/L,平均回收率为96.4％.该方法可用于血清中尿酸含量检测.     

A rapid gel electrophoretic chip for serum cholesterol determination

We present a rapid gel electrophoretic chip, composed of 2.5% (w/v) acrylamide and 1% (w/v) agarose gel, for serum cholesterol determination using a photo lithography technique. After optimizations, we determined the lipoprotein concentration of standard serum using a conventional enzyme method. The serum was diluted, stained and loaded for 15 min onto the chip. After loading, the intensities of low density lipoprotein cholesterol (LDL-C) and high density lipoprotein cholesterol (HDL-C) bands separated at the chip were estimated using an image analyzer. The intensities of these bands corresponded to concentrations obtained from a standard enzyme-based method. The detected LDL-C and HDL-C concentrations were linear up to 146 mg dL(-1) and 53 mg dL(-1) respectively. Finally, we carried out the cholesterol analysis using real biological samples obtained from nine volunteers using our electrophoretic chip. The LDL-C and HDL-C levels detected using our chip correlated well with the results obtained using the conventional enzyme-based method r(2) = 0.98 and r(2) = 0.86 for LDL-C and HDL-C, respectively. Although our sample size is small and confined only to health volunteers, we have demonstrated that this proof-of-concept gel electrophoretic chip can determine lipoproteins, simultaneously.     

A simple protocol for protein extraction of recalcitrant fruit tissues suitable for 2-DE and MS analysis

Fruit tissues are considered recalcitrant plant tissue for proteomic analysis. Three phenol-free protein extraction procedures for 2-DE were compared and evaluated on apple fruit proteins. Incorporation of hot SDS buffer, extraction with TCA/acetone precipitation was found to be the most effective protocol. The results from SDS-PAGE and 2-DE analysis showed high quality proteins. More than 500 apple polypeptides were separated on a small scale 2-DE gel. The successful protocol was further tested on banana fruit, in which 504 and 386 proteins were detected in peel and flesh tissues, respectively. To demonstrate the quality of the extracted proteins, several protein spots from apple and banana peels were cut from 2-DE gels, analyzed by MS and have been tentatively identified. The protocol described in this study is a simple procedure which could be routinely used in proteomic studies of many types of recalcitrant fruit tissues.     

Multivariate analysis of 2-DE protein patterns--practical approaches

Practical approaches to the use of multivariate data analysis of 2-DE protein patterns are demonstrated by three independent strategies for the image analysis and the multivariate analysis on the same set of 2-DE data. Four wheat varieties were selected on the basis of their baking quality. Two of the varieties were of strong baking quality and hard wheat kernel and two were of weak baking quality and soft kernel. Gliadins at different stages of grain development were analyzed by the application of multivariate data analysis on images of 2-DEs. Patterns related to the wheat varieties, harvest times and quality were detected on images of 2-DE protein patterns for all the three strategies. The use of the multivariate methods was evaluated in the alignment and matching procedures of 2-DE gels. All the three strategies were able to discriminate the samples according to quality, harvest time and variety, although different subsets of protein spots were selected. The explorative approach of using multivariate data analysis and variable selection in the analyses of 2-DEs seems to be promising as a fast, reliable and convenient way of screening and transforming many gel images into spot quantities.     

2-DE and MALDI-TOF-MS analysis of therapeutic fusion protein abatacept

2-DE and MALDI-TOF MS are useful techniques for the quality evaluation of medicinal products derived from recombinant DNA technology. The principal objective of this study has been to evaluate the suitability of 2-DE in combination with MALDI-TOF MS for the quality study of the therapeutic recombinant protein, abatacept. 1-DE SDS-PAGE, under reducing and nonreducing conditions, and 2-DE analysis were used for the assessment of M(r) , pI, and enzymatic deglycosylation efficiency of abatacept. 2-DE allowed the assessment of product identity, purity, charge heterogeneity, isoform pattern, and post-translational modifications. Furthermore, optimization of the deglycosylation procedure, charge heterogeneity, and sample preparation for the subsequent MALDI-TOF MS analysis has been addressed. PMF analysis allowed rapid identity confirmation of abatacept.     

2-DE proteomic analysis of the model cyanobacterium Anabaena variabilis

Cyanobacteria are photosynthetic bacteria capable of producing hydrogen and secondary metabolites with potential pharmaceutical applications. A limited number of cyanobacterial 2-DE proteomic studies have been published, most of which are based on Synechocystis sp. PCC 6803. Here, we report the use of 2-DE, ESI-MS/MS and protein bioinformatics tools to characterize the proteome of Anabaena variabilis ATCC 29413, a heterocystous nitrogen-fixing cyanobacterium that is a model organism for the study of nitrogen fixation. Using a 2-DE workflow that included the use of a detergent-based extraction buffer and 3-10 nonlinear IPG strips resulted in the identification of 254 unique proteins, with significantly better coverage of basic and low-abundance proteins that has been reported in 2-DE analyses of Synechocystis sp. A set of protein bioinformatics tools was employed to provide estimates of protein localization, hydrophobicity, abundance and other properties. The characteristics of the A. variabilis proteins identified in this study were compared against the theoretical proteome for this organism, and more generally within the cyanobacteria, to identify opportunities for further development of 2-DE-based cyanobacterial proteomics.     

L-cysteine modified gold nanoparticles / paper chip system for rapid detection of chiral dopaEI北大核心CSCD

A new detection method for L-Dopa based on paper chips was established. The L-Cys-AuNPs were characterized by their size, zeta potential, and UV-visible absorption spectra. The system had the high selectivity for the colorimetric detection of L-Dopa with the color changing from red to blue. The results were recorded using a common cell phone and subsequently analyzed using Photoshop software. A ratiometric color intensity method was designed for the quantification analysis. The ratio of color intensity at red channel and blue channel (R / B) increases linearly with L-Dopa concentration in the range of 5 to 80 μmol / L (R = 0. 9944), with the limit of detection of 4 μmol / L. The spiked recoveries of samples were 98%-102%. The RSDs of inter-day and intraday were 3. 3% and 3. 8%, respectively. Real samples were detected, and the error between the determination and the labeled value was within 5%. The method can be used to detect L-Dopa in real samples. © 2022, Youke Publishing Co.,Ltd. All rights reserved.     

An efficient protein preparation for proteomic analysis of developing cotton fibers by 2-DE

Preparation of high-quality proteins from cotton fiber tissues is difficult due to high endogenous levels of polysaccharides, polyphenols, and other interfering compounds. To establish a routine procedure for the application of proteomic analysis to cotton fiber tissues, a new protocol for protein extraction was developed by optimizing a phenol extraction method combined with methanol/ammonium acetate precipitation. The protein extraction for 2-DE was remarkably improved by the combination of chemically and physically modified processes including polyvinylpolypyrrolidone (PVPP) addition, acetone cleaning, and SDS replacement. The protocol gave a higher protein yield and vastly greater resolution and spot intensity. The efficiency of this protocol and its feasibility in fiber proteomic study were demonstrated by comparison of the cotton fiber proteomes at two growth stages. Furthermore, ten protein spots changed significantly were identified by MS/tandem MS and their potential relationships to fiber development were discussed. To the best of our knowledge, this is the first time that a protocol for protein extraction from cotton fiber tissues appears to give satisfactory and reproductive 2-D protein profiles. The protocol is expected to accelerate the process of the proteomic study of cotton fibers and also to be applicable to other recalcitrant plant tissues.     

Microwaves make hydroformylation a rapid and easy process

[reaction: see text] Hydroformylation of alkenes can be carried out in a few minutes under microwave activation at a relatively low pressure (40 psi) using commercially available catalysts and ligands. The 80 mL vial of a Discover microwave oven was connected to a cylinder of CO and H2, and after filling the reactor at 40 psi, a mixture of an alkene, the Wilkinson catalyst, and XANTPHOS was submitted to microwave irradiation giving, after 4 min, high conversion into the corresponding aldehyde without formation of the isomerized alkene.     

Tris interference in IEF and 2-DE

When dried IPGs are hydrated with protein solutions, the concentration of protein and other ionic constituents is constant throughout the strip. Tris, initially present at a very low concentration, focuses during IEF and accumulates in the gradient at a pH corresponding to its pK(a) at the operative temperature of electrophoresis. Tris focuses more rapidly than many basic proteins, and concentrates into a localized zone of increased conductivity which coincides with a precipitous voltage drop in that vicinity. Basic proteins, already near their pI, are frequently observed to align at the periphery of this zone. Acidic proteins imbibed at the basic end of the gradient must traverse this region before this ionic boundary is formed, or otherwise may fail to migrate to their proper positions in the pH gradient.     

A novel method of protein extraction from perennial Bupleurum root for 2-DE

The perennial Bupleurum root is thick and woody and contains high levels of interfering compounds. Common protein extraction methods have proved refractory towards the isolation of proteins suitable for 2-DE, due to the presence of interfering compounds. A novel method for extracting proteins suitable for 2-DE was established to overcome these problems. The main characteristic of this protocol is the partitioning of the proteins into the aqueous (fraction A-2), chloroform and isoamyl alcohol phases (A-3), and the interphase (A-1). The proteins are then extracted from each of these phases. From A-1, 85% (extracted protein against total proteins) proteins could be extracted and purified. For fraction A-2, a novel phenol extraction step is employed for the extraction of proteins. Based on the well-resolved 2-DE patterns, our protein preparation is free of interfering compounds. Using these methods (A-1, A-2, and A-3-3), a total of 3662 (1526 + 1128 + 1008) spots could be separated, and a protein yield of about 1.41 mg per 1.0 g fresh root material was obtained. To our knowledge, this is the first time that a protocol for protein extraction from perennial Bupleurum root has been reported that gives reproducible results. The protocol is expected to be applicable to other recalcitrant plant tissues as well.     

A novel polymeric stabilizing system for modified lyocell solutions

A novel polymeric stabilizer consisting of iminodiacetic acid sodium salt (ISDB) and benzyl amine (BSDB) covalently bound to a styrene/divinyl benzene copolymer were studied. Calorimetric, spectroscopic, rheological, and high performance liquid chromatography analysis revealed distinctive improvements of the thermal stability of modified Lyocell solutions compared to the unstabilized solution and to that with conventional NaOH/propyl gallate stabilizer. Segregation processes of the system cellulose/N‐methylmorpholine‐N‐oxide and autocatalytic reactions caused by carboxyl group‐containing additives are suppressed by ISDB/BSDB. Concerning to surface‐active additives, enhanced thermal stability is only received for a weakly reactive charcoal. In the case of nanoscaled carbon black modifier, autocatalytic reactions indicated by isoperibolic measures are prevented by the new polymeric stabilizer system. © 2006 Wiley Periodicals, Inc. J Polym Sci Part B: Polym Phys 44: 1702–1713, 2006     

A rapid feedback characterization technique for polymeric hollow fiber membranes using disperse dyes

The long time lag between fiber spinning and the availability of characteristic data for process evaluation currently complicates research in hollow fiber membrane formation. This lag time is due to the down-stream processing required before traditional gas based permeation measurements can be made on the spun fiber. A rapid feedback characterization technique, based on commercially available disperse dyes, is described here for polymeric hollow fiber membrane spinning applications. This technique involves dyeing wet hollow fibers, immediately after spinning, in an aqueous dye bath. In the present work, polysulfone fibers are shown to be characterizable using this method before lengthy down-stream processing (i.e. solvent exchange, drying, and post-treatment). Dye uptake in the hollow fibers is a function of skin porosity, thereby allowing quick evaluation of permeation characteristics. Dye uptake was measured using UV-visible spectrophotometry. Examples of fibers characterized using this technique and relationships between dye uptake and post-treated permselectivity are shown and discussed.     

Immuno-pillar chip: a new platform for rapid and easy-to-use immunoassay

We present a new rapid and easy-to-use immunoassay chip which we have named the immuno-pillar chip. It has hydrogel pillars, fabricated inside a microchannel, with many antibody molecules immobilized onto 1 μm diameter polystyrene beads. To evaluate the chip performance, we applied it to the sandwich assay of C-reactive protein (CRP), α-fetoprotein (AFP) and prostate-specific antigen (PSA), a cardiac and inflammation marker, tumors and prostate cancer markers, respectively. For detection of disease markers, we confirmed the chip provides rapid analysis (total assay time of about 4 min) with high sensitivity, it is easy-to-use (no special skills are needed), and it uses small volumes of the sample and reagent (0.25 μL each). Moreover, multiplex assay for three biomarkers was also possible. Additionally, the immuno-pillar chip has a big advantage of having hardly any influence on the assay results even if the introduction quantities of the sample or reagents are different.     

A low-cost system for rapid automatic neutron activation analysis at small research reactors

A system for rapid automatic neutron activation analysis is governed by software performing irradiation control, neutron flux monitoring and gamma-spectrometry with real-time correction of counting losses as well as spectra evaluation, nuclide identification and calculation of concentrations in a fully automatic flow of operations.^1,2 Elemental concentrations are derived from a list of experimentally determined specific saturation activities. To expand this list, the “k0_IAEA” software³ is presently under evaluation and will be reported on in this paper. At a Triga reactor, reactor pulse activation may enhance the sensitivity for very short half-lives,⁴ and will be presented in our paper.     

A parylene-based dual channel micro-electrophoresis system for rapid mutation detection via heteroduplex analysis

A new dual channel micro-electrophoresis system for rapid mutation detection based on heteroduplex analysis was designed and implemented. Mutation detection was successfully achieved in a total separation length of 250 microm in less than 3 min for a 590 bp DNA sample harboring a 3 bp mutation causing an amino acid change. Parylene-C was used as the structural material for fabricating the micro-channels as it provides conformal deposition, transparency, biocompatibility, and low background fluorescence without any surface treatment. A new dual channel architecture was derived from the traditional cross-channel layout by forming two identical channels with independent sample loading and waste reservoirs. The control of injected sample volume was accomplished by a new u-turn injection technique with pull-back method. The use of heteroduplex analysis as a mutation detection method on a cross-linked polyacrylamide medium provided accurate mutation detection in an extremely short length and time. The presence of two channels on the microchip offers the opportunity of comparing the sample to be tested with a desired control sample rapidly, which is very critical for the accuracy and reliability of the mutation analyses, especially for clinical and research purposes.     

Layer-by-layer assembly for rapid fabrication of thick polymeric films

In the past two decades, layer-by-layer (LbL) assembly has been proven to be a convenient and versatile method to fabricate functional films. However, using traditional dipping LbL assembly to fabricate micrometer-thick films is time consuming. Compared with ultrathin films, micrometer-thick films prepared by LbL assembly possess enhanced mechanical stability, and allow deposition of a significantly increased amount of materials and the integration of multiple functions. These merits of thick films produced by LbL assembly can result in new functions and allow the functions of ultrathin films fabricated by LbL assembly to be optimized. In this tutorial review, the methods for rapid fabrication of thick polymeric films involving LbL assembly are reviewed. The functions of such films that are relevant to their micrometer thickness are discussed.     

A rapid and simple approach for glycoform analysis

Fast glycoform analysis is important for quality control of glycoproteins that account for over 40% of the approved biopharmaceuticals. Herein, we realized an Au nanoparticle-based lectin affinity chromatography (LAC) using simple standard laboratory equipment for fast glycoform analysis. Pisum sativum agglutinin (PA), a lectin derived from P. sativum, was covalently conjugated to Au nanoparticles via naturally formed carboxylic groups onto the surface of Au nanoparticles and amino groups of PA. Each model glycoprotein was separated into several fractions including the unbound, weakly bound, modestly bound, and strongly bound glycoforms based on affinity strength of the glycoform toward PA. A single run of Au nanoparticle-based LAC was finished within 18 min, which could be further decreased by centrifuging the mixture of the PA functionalized Au nanoparticles and the glycoproteins at a higher speed. To our knowledge, we are the first to use Au nanoparticles as LAC matrix.