Fluorescence-based multiplex protein detection using optically encoded microbeads

Potential utilization of proteins for early detection and diagnosis of various diseases has drawn considerable interest in the development of protein-based multiplex detection techniques. Among the various techniques for high-throughput protein screening, optically-encoded beads combined with fluorescence-based target monitoring have great advantages over the planar array-based multiplexing assays. This review discusses recent developments of analytical methods of screening protein molecules on microbead-based platforms. These include various strategies such as barcoded microbeads, molecular beacon-based techniques, and surface-enhanced Raman scattering-based techniques. Their applications for label-free protein detection are also addressed. Especially, the optically-encoded beads such as multilayer fluorescence beads and SERS-encoded beads are successful for generating a large number of coding.     

Stability of hydrophobic lipase derivatives immobilized on organic polymer beads

Lipase fromCandida rugosa was immobilized by attaching various hydrophobic groups to the enzyme molecule and adsorbing these hydrophobic lipase derivatives on several organic polymer beads. The immobilized enzymes were more thermostable in organic solvents compared to the native and modified Upases. Thermostability was highest with ΧAD2 beads, followed by ΧAD7 and RCOOH. Initially modifying the enzyme with hydrophobic modifiers did not have any effect on the enzyme thermostability. The best conditions for storing these enzyme preparations were at very low temperature in the lyophilized form and in a solution containing the reaction substrate. Interestingly, PEG-lipase immobilized on ΧAD7 beads showed increased operational stability when used in a stirred-tank reactor. The operational stability was further increased by a mild glutaraldehyde treatment of the enzyme preparation.     

Stimuli-responsive 2D polyelectrolyte photonic crystals for optically encoded pH sensing

A versatile photonic crystal sensing motif based on a two-dimensional (2D) inverse opal monolayer of stimuli-responsive polyelectrolyte gel with tunable optical properties is reported. The photonic membrane shows prompt response to pH and can be readily read out from either its optical spectra or interference colours.     

Preparation of optically pure cross-conjugated cyclopentadienones

The synthesis of optically pure cross-conjugated cyclopentadienones is readily achieved in two steps via a one-pot alkylcuprate addition/aldol condensation/dehydration sequence using racemic or enantioenriched endo-3a,4,7,7a-tetrahydro-1H-4,7-methano-inden-1-ones followed by microwave-mediated Lewis acid-catalysed retro Diels-Alder reaction. An alternative route involving a modified Baylis-Hillman protocol followed by conjugate addition with alkylcuprates and a retro Diels-Alder reaction was also investigated.     

Ascorbic acid biosensor using ascorbate oxidase immobilized on alkylamine glass beads

A biosensor for ascorbic acid based on enzyme kinetics of ascorbate oxidase (E.C.1.10.3.3.) was developed. The enzyme was extracted fromCucurbita maxima, or jerimun and immobilized by covalent bounding, using glutaradehyde as a bifunctional agent, on alkylamine glass beads, with and without enzyme active site protection. A low-cost, home-made oxygen electrode was applied as a transducer. The system has sensitivity from 62.5 up to 500 μM of ascorbic acid with satisfactory operation for more than 2 mo.     

Photo‐switchable spiropyran immobilized polystyrene beads using catechol chemistry

Catechol and spiropyran functional groups were conjugated to a polymer backbone, allowing immobilization on polystyrene beads (PS beads). The final product was capable of stably reproducing the optical properties of spiropyran. Through the outstanding surface adhesion properties of the catechol functional group, spiropyran was immobilized on PS beads. Switchable photoluminescence in the spiropyran coated PS bead surfaces was observed depending on irradiation with either UV or visible light. The surfaces of the PS beads were morphologically examined by field emission scanning electron microscopy and X‐ray photoelectron spectroscopy was used for characterization of the constituent atoms. Furthermore, UV–Vis and fluorescence spectroscopy were used to confirm conversion between the spiropyran (SP) and merocyanine (MC) forms through UV or visible light irradiation on SP, while fluorescent images for both SP and MC were studied using confocal laser scanning microscopy. The confocal images of the SP‐PS beads system onto MDAMB‐231 cells under UV and visible light indicate the cellular uptake by emerging color within the cytoplasm. Advancing study, the remaining catechol groups can confers adhesive properties, given by contact angle data of various coated surfaces film. These stimuli‐responsive coatings are compatible as drawing switchable photochromic material on versatile substrate shown in confocal images of propylene film. Overall, this great water solubility and biocompatibility PS beads system also showed potential as cell bio‐imaging light stimuli responsive material, and the benefits of this system can also possibly address coat able advanced material for a wide range of surface light sensor applications. Copyright © 2017 John Wiley & Sons, Ltd.     

First photosensitized enantiodifferentiating isomerization by optically active sensitizer immobilized in zeolite supercages

Enantiodifferentiating photoisomerization of (Z)-cyclooctene sensitized by (R)- or (S)-1-methylheptyl benzoate immobilized in zeolite supercages afforded the respective enantiomer pair, (-)- and (+)-(E)-isomer (1E) in 5% enantiomeric excess, whilst racemic 1E was obtained upon homogeneous-phase photosensitization with the same antipodal sensitizer pair, thus demonstrating for the first time that chirally modified zeolites not only serve as supramolecular photosensitizing media but also enhance the original enantiodifferentiating ability of chiral photosensitizer.     

A polymeric chiral surface functionality immobilized on uniformly sized macroporous polymer beads

Uniformly sized functionalized macroporous polymer beads were prepared by either a classical copolymerization method or recently reported in situ surface modification method utilizing chiral methacrylamide as a functional modifier. To evaluate conformational and/or specific differences in their surface chiral functionality, we applied chromatographic evaluation techniques. The prepared modified beads were utilized as chiral stationary phase in high-performance liquid chromatography (HPLC). Those prepared by the in situ surface modification method tended to show higher chiral recognition ability than those by the classical copolymerization method, even if the equivalent amount of the chiral functional group was involved within polymer beads. Detailed chromatographic studies exhibited the in situ surface modification method could lead to polymeric methacrylamide functionality on the surface within relatively large pore size regions of the macroporous polymer beads, while the classical copolymerization method tended to form less polymeric surface functionality. The difference in the chiral surface functionality on both of macroporous polymer beads afforded drastic change in chromatographic chiral recognition ability. Complete resolution of a drug, thalidomide could be achieved on the chiral stationary phase with the polymeric chiral surface functionality, while no resolution was found on that with the monomeric one even if the same chiral methacrylamide was used as a modifier to prepare the chiral stationary phases. © 1997 John Wiley & Sons, Inc. J Polym Sci A: Polym Chem 35 : 2747–2757, 1997     

Preparation of optical active polydiacetylene through gelating and the control of supramolecular chirality

Achiral diacetylene 10,12-pentacosadinoic acid (PCDA) and a chiral low-molecular-weight organogelator could form co-gel in organic solvent and it could be polymerized in the presence of Zn(II) ion or in the corresponding xerogel under UV-irradiation. Optically active polydiacetylene (PDA) were subsequently obtained. Supramolecular chirality of PDA could be controlled by the chirality of gelators. Left-handed and right-handed helical fibers were obtained by using Land D-gelators in xerogels respectively, and...     

Preparation and evaluation of spore-specific affinity-augmented bio-imprinted beads

A novel, affinity-augmented, bacterial spore-imprinted, bead material was synthesized, based on a procedure developed for vegetative bacteria. The imprinted beads were intended as a front-end spore capture/concentration stage of an integrated biological detection system. Our approach involved embedding bead surfaces with Bacillus thuringiensis kurstaki (Bt) spores (as a surrogate for Bacillus anthracis) during synthesis. Subsequent steps involved lithographic deactivation using a perfluoroether; spore removal to create imprint sites; and coating imprints with the lectin, concanavalin A, to provide general affinity. The synthesis of the intended material with the desired imprints was verified by scanning electron and confocal laser-scanning microscopy. The material was evaluated using spore-binding assays with either Bt or Bacillus subtilis (Bs) spores. The binding assays indicated strong spore-binding capability and a robust imprinting effect that accounted for 25% additional binding over non-imprinted controls. The binding assay results also indicated that further refinement of the surface deactivation procedure would enhance the performance of the imprinted substrate.     

Preparation of optically active poly-β-propiolactones—II. Preparation of optically active poly-α-methyl-α-ethyl-β-propiolactones

Optically active poly-α-methyl-α-ethyl-β-propiolactones were prepared by stereoelective polymerization of the racemic monomer and by anionic polymerization of optically active monomers. Important differences in properties were observed between racemic and optically pure polymers, the latter having a much higher melting point. Chiroptical and structural studies of polymers of different enantiomeric composition are reported.     

固定化过氧化氢酶的制备及其抗氧化作用

以烟用醋酸纤维的生物化学改性为目标,研究了以壳聚糖为载体时,吸附交联固定化过氧化氢酶的条件,并考察了固定化酶的性质。结果表明,固定化的最佳条件为:加酶量(酶活2×104C IU/m l)6m l,3%壳聚糖20m l,乙二醛浓度6%(w/v),交联剂用量100m l,吸附时间0.5 h,交联时间2.5h,酶活收率可达42.9%。过氧化氢酶固定化后,动学参数Km值为61.7mmol/L;对活性氧具有较好清除作用。     

Preparation of immobilized tannins for protein adsorption

Preparation and adsorption specificity of tannins immobilized by covalent binding on water-insoluble matrices were investigated. Immobilized tannins were prepared by condensing cyanogen bromide activated tannins with aminohexyl derivatives of several kinds of matrices. The most suitable matrix for the immobilization of tannin was alkali-treated cellulose powder. The concentration of sodium hydroxide solution for alkali treatment influenced the adsorption capacity of immobilized tannin for a protein, but temperature and time for alkali treatment did not. Immobilized tannins having spacers of long chain length exhibited high adsorption capacity for a protein. Chinese gallotannin was the most favorable ligand for protein adsorption. The immobilization of tannin on aminohexyl matrices was also possible by using epichlorohydrin instead of cyanogen bromide. The maximum adsorption capacity of the immobilized tannin for a protein was about 50 mg/ml of the absorbent. Immobilized tannin adsorbed proteins specifically but did not absorb low molecular weight compounds.     

Preparation and properties of immobilized diphtheria toxin

Commercially available diphtheria toxin was immobilized on nylon stocking material by a covalent activation of the polymeric material. Toxin was bound both directly to nylon as well as to nylon-polyethyleneimine copolymer. The immobilized toxin retained some of its catalytic activity as evidenced by ADP-ribosylation of elongation factor II. Kinetics of this reaction are reported. After repeated use for several times the immobilized toxin retains 75% of its activity.     

新型亲水聚合物修饰硅胶颗粒固定化酶的制备及在蛋白质组鉴定中的应用

采用原子转移自由基聚合法制备了亲水聚合物修饰的硅胶颗粒作为一种新型固定化酶载体,在实现胰蛋白酶高密度固定的同时,显著降低了载体材料非特异性吸附导致的样品损失。因此,该固定化酶材料兼具高酶解效率和高回收率的特性。以标准蛋白质牛血清白蛋白(BSA)为样本,使用该固定化酶1 min即可完成酶解,鉴定到肽段对BSA的氨基酸序列覆盖率可达90%以上。该固定化酶材料成功应用于酵母菌全蛋白质复杂样本的酶解,从3 min酶解产物中鉴定到666个蛋白质,超过同样条件下溶液酶解12 h的鉴定结果。     

Synthesis of optically active fluorinated materials by use of immobilized enzymes for asymmetric hydrolysis

A synthetic approach to optically active fluorinated compounds was based on the enantiotopic specificity of asymmetric hydrolysis by an immobilized enzyme.     

Preparation of immobilized albumin and its properties

An immobilized form of human albumin on a polyamide support has been obtained. The complex-forming properties of the immobilized albumin with egg yolk phospholipids and essential phospholipids have been studied. Translated from Khimiya Prirodnykh Soedinenii, No. 4, pp. 541–544, July–August, 1998.