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1.
2.
The use of probe beads for lab-on-chip affinity assays is very interesting from a practical point of view. It is easier to handle and trap beads than molecules in microfluidic systems. We present a method for the immobilization of probe beads at defined areas on a chip using dielectrophoresis (DEP)-controlled adhesion. The method is fast, i.e., it takes between 10 and 120 s--depending on the protocol--to functionalize a chip surface at defined areas. The method is versatile, i.e., it works for beads with different types of probe molecule coatings. The immobilization is irreversible, i.e., the retained beads are able to withstand high flow velocities in a flow-through device even after the DEP voltage is turned off, thus allowing the use of conventional high-conductivity analyte buffers in the following assay procedure. We demonstrate the on-chip immobilization of fluorescent beads coated with biotin, protein A, and goat-antimouse immunoglobulin G (IgG). The number of immobilized beads at an electrode array can be determined from their fluorescence signal. Further, we use this method to demonstrate the detection of streptavidin and mouse IgG. Finally, we demonstrate the feasibility of the parallel detection of different analyte molecules on the same chip.  相似文献   

3.
Kaneta T  Inoue J  Koizumi M  Imasaka T 《Electrophoresis》2006,27(16):3218-3223
A method for capturing specific molecules separated by CE has been explored. To demonstrate on-column capture of migrating analyte molecules, two detection windows were fabricated on a capillary. Magnetic beads containing immobilized molecules that react with the specific molecules under study were placed between the detection windows in the capillary using magnets. Molecules in a sample solution injected into the capillary were separated and detected at the first detection window. After passing through the first detection window, the separated molecules encountered the magnetic beads, where the specific analyte was captured. As a result, the peak area for those analyte molecules decreased or disappeared completely at the second detection window. Rabbit IgG and carbonic anhydrase were employed to demonstrate on-column capture of a specific molecule. For rabbit IgG, magnetic beads containing the immobilized antibody (anti-rabbit IgG) were used. Rabbit IgG molecules were captured on the magnetic beads during CE migration. Furthermore, the capture of carbonic anhydrase was demonstrated by the reaction between magnetic beads (containing immobilized anti-rabbit IgG) and anti-carbonic anhydrase (rabbit IgG), before the beads were packed in the capillary. After packing the magnetic beads in the capillary, a mixture of two proteins was injected into the capillary. Two proteins were detected at the first detection window, while the peak corresponding to carbonic anhydrase disappeared at the second detection window. The results show that using an appropriate antibody, the present technique would be applicable to any proteins.  相似文献   

4.
The thrombin-binding DNA aptamer was used with thrombin as a model system to investigate protein capture using aptamer stationary phases in affinity capillary chromatography. The aptamer was covalently attached to the inner surface of a bare fused-silica glass capillary to serve as the stationary phase. Proteins were loaded onto the capillary via an applied pressure. The capillary was then washed to remove unbound and non-specifically associated proteins. Finally, the bound protein was released and eluted using 20 mM Tris buffer containing 8 M urea, pH 7.3, at 50 degrees C. Eluate was collected after each step (load, wash and elute) and relative amounts of protein each were compared using fluorescence spectroscopy. The identity of the protein in the collections was confirmed using matrix assisted laser desorption ionization-time-of-flight (MALDI-TOF) mass spectrometry. The experiment was repeated for thrombin on a bare (unmodified) capillary and a capillary coated with a scrambled-sequence, non-G-quartet forming oligonucleotide that does not bind with thrombin. The results show that the aptamer stationary phase captures approximately three times as much thrombin as the control columns. The experiment was also repeated using human serum albumin (HSA) alone and in an equimolar mixture with thrombin. HSA was not retained on the aptamer capillary, nor did it affect the capture of thrombin from the mixture.  相似文献   

5.
康丁  张洪斌  西成胜好 《化学进展》2014,26(7):1172-1189
结冷胶是一种线型聚阴离子微生物多糖,具有独特的凝胶特性和溶液流变学性质,自发现起即被应用于食品和化妆品中。近年来,随着生物医学学科的发展,天然高分子结冷胶及其水凝胶,在药物传递系统和组织工程材料等领域展现出了广阔的应用前景。结冷胶无毒,具有生物相容性和可生物降解性,所形成的水凝胶透明且稳定性好,并在一定条件下凝胶的力学性质与人体普通组织相近。结冷胶的这些优势使其成为一种良好的生物医用材料的制备来源。但是这种基于结冷胶的水凝胶也有其自身的缺点,如作为组织工程材料缺乏一定的韧性和组织负载能力等。这些不足在很大程度上限制了其在生物医学领域的应用。为了解决上述问题,许多研究者对结冷胶进行了化学和物理的改性。改性后的结冷胶材料在生物医学领域展现出更有发展的应用前景。本文综述了结冷胶凝胶的形成机理以及结冷胶的改性方法,重点详述了结冷胶及其改性材料在生物医学领域中的应用,并指出了结冷胶基组织工程材料在应用上应解决的一些挑战性问题。  相似文献   

6.
In this study we describe the activation with chloroformates of Trisacryl-GF-2000, a new synthetic gel support that is stable, hydrophilic, and contains large amounts of hydroxyl groups available for activation. Of all the reagents tested, the activation withN-hydroxysuccinimide-chloroformate andp-nitrophenylchloroformate in organic solvents provides the best activation yield and subsequent coupling. When Trisacryl was activated in acetone with the chloroformates in the presence of 4-dimethylaminopyridine as base and catalyst, up to 30% of the hydroxyl groups, (i.e., 1/repeating unit) could be activated. Amino-containing ligands and proteins could be coupled to these carriers at pH 8 or higher. For better results in affinitychromatographic applications, spacers of ε-amino caproic acid or diaminohexane were introduced. The efficacy of these columns was demonstrated by purification of enzymes, antibodies, and antigens. The performance of these new columns were compared with that of Sepharose columns activated in various ways. In every case, the properties of the Trisacryl support proved superior with particular reference to the purity of the product obtained.  相似文献   

7.
To improve general understanding of biochemical mechanisms in the field of uranium toxicology, the identification of protein targets needs to be intensified. Immobilized metal affinity chromatography (IMAC) has been widely developed as a powerful tool for capturing metal binding proteins from biological extracts. However uranyl cations (UO2(2+)) have particular physico-chemical characteristics which prevent them from being immobilized on classical metal chelating supports. We report here on the first development of an immobilized uranyl affinity chromatography method, based on the cation-exchange properties of aminophosphonate groups for uranyl binding. The cation distribution coefficient and loading capacity on the support were determined. Then the stability of the uranyl-bonded phase under our chromatographic conditions was optimized to promote affinity mechanisms. The successful enrichment of uranyl binding proteins from human serum was then proven using proteomic and mass spectral analysis.  相似文献   

8.
A surface for the capture of biotin-tagged proteins on matrix-assisted laser desorption/ionisation (MALDI) targets has been investigated. Binding of a poly-L-lysine poly(ethylene glycol)-biotin polymer to glass and gold surfaces has been demonstrated using dual wavelength interferometry. Biotinylated proteins were captured onto this surface using tetrameric neutravidin as a multivalent bridging molecule. Biotin tagging of proteins was achieved by chemical biotinylation or by expressing a protein with a biotinylation consensus sequence in E. coli. The specificity of the surface for biotin-tagged proteins allowed the purification of biotin-tagged glutathione-S-transferase from a bacterial lysate directly onto a MALDI target. Subsequently, the protein was digested on the MALDI target and a protein fingerprint analysis confirmed its presence directly, but no E. coli proteins were detected. Therefore, we conclude that this surface is highly specific for the capture of biotin-labelled proteins and has low non-specific binding properties for non-biotinylated proteins. Furthermore, protein-protein interactions using biotinylated lectins were investigated, and the selective capture of the glycoprotein fetuin with wheat germ agglutinin was demonstrated. Also, immobilised Arachis hypogea agglutinin recognised a minor asialo component of this glycoprotein on the array. The high affinity immobilisation of proteins onto this surface allowed effective desalting procedures to be used which improved the desorption of high molecular weight proteins. Another aspect of this surface is that a highly ordered coupling of the analyte can be achieved which eliminates the search for the sweet spot and allows the creation of densely packed protein microarrays for use in mass spectrometry.  相似文献   

9.
The lock-and-key (LAK) motif, a common structural moiety found in subunit interfaces of glutathione S-transferases (GSTs), plays an important role in biomolecular recognition and quaternary structure integrity. Inspection of the key structural features of the LAK motif prompted the de novo design and combinatorial synthesis of a 13-membered solid-phase ligand library, employing as a lead ligand the Phe-Trz-X structure, mimicking the LAK motif. 1,3,5-Triazine (Trz) was used as the scaffold for assembly, substituted with different LAK-mimetic amino acids. De novo ligand design was effected using bioinformatics and molecular modeling and based on mimicking the interactions of the LAK motif. The library of affinity adsorbents was assessed for binding corn and human serum proteomes and purified proteins of different structure and ligand binding specificity. The results showed remarkable differences in the binding specificity of LAK-mimetic adsorbents for a wide range of proteins, as a consequence of minor changes in ligand structure. One LAK-mimetic adsorbent was integrated in a single-step purification protocol for human monoclonal anti-human immunodeficiency virus 2F5 antibody (mAb 2F5) from spiked corn extract, affording high recovery and purity. The results demonstrate that the principle of natural recognition found in the lock-and-key motif, in combination with de novo combinatorial design, may lead to synthetic affinity ligands, useful in downstream processing and proteomic research.  相似文献   

10.
Palecek E  Fojta M 《Talanta》2007,74(3):276-290
Magnetic beads (MBs) are versatile tools in the separation of nucleic acids, proteins and other biomacromolecules, their complexes and cells. In this article recent application of MBs in electrochemical biosensing and particularly in the development of DNA hybridization sensors is reviewed. In these sensors MBs serve not only for separation but also as a platform for optimized DNA hybridization. A hybridization event is detected separately at another surface, which is an electrode. The detection is based either on the intrinsic DNA electroactivity or on various kinds of DNA labeling, including chemical modification, enzyme tags, nanoparticles, electroactive beads, etc., greatly amplifying the signals measured. In addition to DNA hybridization, other kinds of biosensing in combination with MBs, such as DNA-protein interactions, are reviewed.  相似文献   

11.
The on-column capture of a specific protein using magnetic beads was applied to SDS-CGE. In a preliminary study, an immunological reaction in the presence of SDS, using a batch method, was attempted. Carbonic anhydrase (CA), alpha-lactalbumin (LA), and HSA were denatured by heating in the presence of SDS and 2-mercaptoethanol, and then reacted with anti-CA that had been immobilized on magnetic beads. Not only native CA, but also the denatured CA reacted with anti-CA, even in the presence of SDS. Therefore, the on-column capture of denatured CA separated by SDS-CGE was attempted using a two-point detection technique. A mixture of proteins, containing LA, CA, and HSA, were separated by SDS-CGE according to their Mr. The CA was then specifically captured on anti-CA-immobilized magnetic beads, which were packed between two detection windows in the capillary column, during the electrophoresis. The results show that the technique leads to information similar to that obtained by Western blotting, i.e., a protein can be identified by its Mr and reaction with its antibody.  相似文献   

12.
Casein is well known as a good protein emulsifier and beta-casein is the major component of casein and commercial sodium caseinate. Dye affinity adsorption is increasingly used for protein separation. beta-Casein adsorption onto Reactive Red 120 attached magnetic poly(2-hydroxyethyl methacrylate) (m-PHEMA) beads was investigated in this work. m-PHEMA beads (80-120 microm in diameter) were produced by dispersion polymerization. The dichlorotriazine dye Reactive Red 120 was attached covalently as a ligand. The dye attached beads, having a swelling ratio of 55% (w/w) and carrying different amounts of Reactive Red 120 (9.2 micromol . g(-1)-39.8 micromol . g(-1)), were used in beta-casein adsorption studies. The effects of the initial concentration, pH, ionic strength and temperature on the adsorption efficiency of dye attached beads were studied in a batch reactor. The non-specific adsorption on the m-PHEMA beads was 1.4 mg . g(-1). Reactive Red 120 attachment significantly increased the beta-casein adsorption up to 37.3 mg . g(-1). More than 95.4% of the adsorbed beta-casein was desorbed in 1 h in a desorption medium containing 1.0 M KSCN at pH 8.0. We concluded that Reactive Red 120 attached m-PHEMA beads can be applied for beta-casein adsorption without significant losses in the adsorption capacities.  相似文献   

13.
In our previous report a new methodology for intermolecular cross-linking or bridging of protein utilizing a spontaneous chelate formation process was proposed. In this paper the reliability of the process as a tool for protein immobilization has been further evaluated. The chromatographic behavior of tryptophan in a column packed with Sepharose coupled with salicylaldehyde residue showed that the alpha-amino acid was bound tightly to the gel in the presence of copper(II) ion and was eluted by the addition of ethylenediaminetetraacetate (EDTA). It was also proved that subtilisin modified with an alpha-amino acid residue was immobilized on the column, and this binding was reversed by the addition of EDTA as well.  相似文献   

14.
A novel strategy for preparation of a boronate affinity hybrid monolith was developed using a Cu(I)-catalyzed 1,3-dipolar azide–alkyne cycloaddition (CuAAC) reaction of an alkyne–boronate ligand with an azide-functionalized monolithic intermediate. An azide-functionalized hybrid monolith was first synthesized via a single-step procedure to provide reactive sites for click chemistry; then the alkyne–boronate ligands were covalently immobilized on the azide-functionalized hybrid monolith via an in-column CuAAC reaction to form a boronate affinity hybrid monolith under mild conditions. The boronate affinity monolith was characterized and evaluated by means of elemental analysis, Fourier transform infrared spectroscopy, and scanning electron microscopy. The boronate affinity hybrid monolith exhibited excellent specificity toward nucleosides and glycoproteins, which were chosen as test cis-diol-containing compounds under neutral conditions. The binding capacity of the monolith for the glycoprotein ovalbumin was 2.36 mg?·?g-1 at pH 7.0. The practicability of the boronate affinity hybrid monolithic material was demonstrated by specific capture of the glycoproteins ovalbumin and ovotransferrin from an egg sample.
Figure
A novel strategy for preparation of boronate affinity hybrid monolith was developed by utilizing Cu(I)-catalyzed 1,3-dipolar azide-alkyne cycloaddition reaction (CuAAC). The obtained boronate affinity hybrid monolith exhibited excellent performance for isolation and enrichment of nucleosides and glycoproteins and was successfully employed to specific capture of glycoproteins from the egg sample  相似文献   

15.
16.
Yang F  Lin Z  He X  Chen L  Zhang Y 《Journal of chromatography. A》2011,1218(51):9194-9201
A macroporous boronate affinity monolithic column was prepared and applied to specifically capture glycoproteins using metal-organic gels (MOGs) as a porogenic template. This newly explored application of MOGs has proven to be a more convenient method for the formation of macropores in contrast to traditional porogenic methods. The poly (3-acrylamidophenylboronic acid-co-ethylene dimethacrylate) monolithic columns were synthesized in stainless columns by in situ polymerization. To fabricate the macroporous formation with a uniformed open-channel network, the preparation conditions, such as reaction temperature, the concentration of the MOGs and the ratio of monomers were systematically investigated. The prepared macroporous monoliths were characterized by scanning electron microscope (SEM) and mercury intrusion porosimetry. Furthermore, horseradish peroxidase (HRP) and transferrin (TF) were chosen as test glycoproteins, and the chromatographic analysis demonstrated that the macroporous boronate affinity monoliths exhibited a higher selectivity and better dynamic binding capacity toward glycoproteins compared with non-glycoproteins. The resulted affinity monolithic column was successfully employed to specifically capture TF from a bovine serum sample.  相似文献   

17.
A simple and novel approach was developed to detect non-covalent interactions. It is based on combination of solid-phase affinity capture with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS). One of the interacting molecules is bound to magnetic beads and is incubated with the target molecules in solution. The complex bound on the solid support is removed from the solution and transferred for MALDI analysis. Mass spectrometry is used only to detect the target compound, which is far more straightforward than detecting the intact non-covalent complex. To demonstrate the applicability of the method, an AT-rich oligonucleotide (5'-CCCCCAATTCCCCC-3') and its complementary biotinylated sequence (5'-biotin-GGGGGAATTGGGGG-3') were hybridized and immobilized to paramagnetic particles by streptavidin-biotin interaction. The immobilized duplex oligonucleotide was reacted with minor groove binding drugs, Netropsin, Distamycin A, Hoechst 33258 and 4',6-diamidino-2-phenylindole. The resulting DNA-drug complex bound to the particles was separated and analyzed by linear MALDI-TOFMS after washing. Drugs were selectively detected in the spectra. Relative binding strengths were also estimated using competitive complexation.  相似文献   

18.
A novel DNA biosensor was fabricated through a facile sulfamide coupling reaction. First, the versatile sulfonic dye molecule of 1-amino-2-naphthol-4-sulfonate (AN-SO3) was electrodeposited on the surface of a glassy carbon electrode (GCE) to form a steady and ordered AN-SO3 layer. Then the amino-terminated capture probe was covalently grafted to the surface of SO3-AN deposited GCE through the sulfamide coupling reaction between the amino groups in the probe DNA and the sulfonic groups in the AN-SO3. The step-by-step modification process was characterized by electrochemistry and attenuated total reflectance Fourier transform infrared (ATR-FTIR) spectroscopy. Using Ru(NH3)63+ as probe, the probe density and the hybridization efficiency of the biosensor were determined to be 3.18 × 1013 strands cm−2 and 86.5%, respectively. The hybridization performance of the biosensor was examined by differential pulse voltammetry using Co(phen)33+/2+ (phen = 1,10-phenanthroline) as the indicator. The selectivity experiments showed that the biosensor presented distinguishable response after hybridization with the three-base mismatched, non-complementary and complementary sequences. Under the optimal conditions, the oxidation peak currents of Co(phen)33+/2+ increased linearly with the logarithm values of the concentration of the complementary sequences in the range from 1.0 × 10−13 M to 1.0 × 10−8 M with a regression coefficient of 0.9961. The detection limit was estimated to be 7.2 × 10−14 M based on 3σ.  相似文献   

19.
Qiao  Liangzhi  Liao  Yuxin  Wang  Xiawen  Wang  Shanshan  Du  Kaifeng 《Cellulose (London, England)》2022,29(13):7263-7276
Cellulose - Cellulose microspheres are commonly chromatographic media yet seriously limited in biomacromolecules separation and purification due to the slow mass transfer kinetics resulting from...  相似文献   

20.
An artificial polypeptide scaffold composed of surface anchor and protein capture domains was designed and expressed in vivo. By using a mutant E. coli phenylalanyl-tRNA synthetase, the photoreactive amino acid para-azidophenylalanine was incorporated into the surface anchor domain. Octyltrichlorosilane-treated surfaces were functionalized with this polypeptide by spin coating and photocrosslinking. The resulting protein films were shown to immobilize recombinant proteins through association of coiled coil heterodimer.  相似文献   

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