Glutaraldehyde Cross-linking Reaction Research Articles

Dielectrophoretic detection of electrical property changes of stored human red blood cells.

The ability to transport and store a large human blood inventory for transfusions is an essential requirement for medical institutions. Thus, there is an important need for rapid and low-cost characterization tools for analyzing the properties of human red blood cells (RBCs) while in storage. In this study, we investigate the ability to use dielectrophoresis (DEP) for measuring the storage-induced changes in RBC electrical properties. Fresh human blood was collected, suspended in K2-EDTA anticoagulant, and stored in a blood bank refrigerator for a period of 20 days. Cells were removed from storage at 5-day intervals and subjected to a glutaraldehyde crosslinking reaction to "freeze" cells at their ionic equilibrium at that point in time and prevent ion leakage during DEP analysis. The DEP behavior of RBCs was analyzed in a high permittivity DEP buffer using a three-dimensional DEP chip (3DEP) and also compared to measurements taken with a 2D quadrupole electrode array. The DEP analysis confirms that RBC electrical property changes occur during storage and are only discernable with the use of the cell crosslinking reaction above a glutaraldehyde fixation concentration of 1.0w/v%. In particular, cytoplasm conductivity was observed to decrease by more than 75% while the RBC membrane conductance was observed to increase by more than 1000% over a period of 20 days. These results show that the presented combination of chemical crosslinking and DEP can be used as rapid characterization tool for monitoring electrical properties changes of human RBCs while subjected to refrigeration in blood bank storage.

Study on determination method of substitution degree of polyvinyl alcohol glutaraldehyde

The excess hydroxylamine hydrochloride was added to react with PVA crosslink, and the HCI was titrated with NaOH standard solution. The weight percentage of PVA crosslink in the sample was calculated to characterize the degree of substitution. The results show that this method can be used to analyze the degree of substitution of PVA and glutaraldehyde crosslinking reaction effectively with high accuracy and small standard deviation.

Fabrication of dual cross-linked spherical treated waste biomass/alginate adsorbent and its potential for efficient removal of lead ions from aqueous solutions

The use of biodegradable biopolymers such as cellulose as a suitable adsorbent, is a sustainable solution for the treatment of heavy metal contaminated waters. In this study, treated biomass (relatively pure cellulose) was obtained from agricultural wastes in a completely new and applicable method using ultrasonic. Then, the treated biomass was incorporated in alginate matrix and alginate/treated biomass bead was successfully fabricated via a facile glutaraldehyde cross-linking reaction and coagulation-solidification method. The prepared bead was characterized and used as an efficient biosorbent for lead removal from aqueous solutions. The maximum experimental adsorption capacity of the adsorbent was obtained 206.75 mg/g at C0 = 210 mg/L, T =328 K, and pH = 5. The experimental kinetic and equilibrium data were well adjusted with pseudo-second-order kinetic and Freundlich isotherm models, respectively. The adsorption mechanism investigations illustrated that electrostatic interactions are responsible for metal ion adsorption and the rate-limiting step of the adsorption in the early stages was film diffusion.

Multifunctional magnetic chitosan-graphene oxide-ionic liquid ternary nanohybrid: An efficient adsorbent of alkaloids

A magnetically and recyclable multifunctional nanohybrid adsorbent (IL-Fe3O4@SiO2@CS/GO) with chitosan, graphene oxide and ionic liquid was prepared by glutaraldehyde cross-linking reaction, acylation reaction and directly grafted method. The incorporation of CS, GO and IL could collaboratively enhance adsorption for 5 alkaloids. The Box-Benhnken design was applied to optimize the experimental parameters affecting the extraction efficiency. The adsorption kinetic was further used to describe the adsorption process, revealing alkaloids adsorption was a chemisorption process, which was well represented by pseudo-second-order model. Concerning the equilibrium behavior, the adsorption was multilayer and heterogeneous adsorption that satisfactorily modeled by the Freundlich model. Under optimal conditions, good adsorption capacity for morphine, codeine, ephedrine, amphetamine and benzoylecgonine (7.2, 8.4, 9.2, 5.8 and 11.2 mg g–1, respectively) were obtained via multiple mechanisms to attain simultaneous extraction of alkaloids. Accordingly, IL-Fe3O4@SiO2@CS/GO nanohybrid adsorbent could be extended as a potential adsorbent for extraction of illicit additives in food samples.

Alkyl β-D-xyloside synthesis from black liquor xylan using Aureobasidium pullulans CBS 135684 β-xylosidases immobilized on spent expanded perlite

The crude β-xylosidase from Aureobasidium pullulans CBS 135684 was immobilized on the surface of the modified spent expanded perlite via (3-aminopropyl) triethoxy-silane and glutaraldehyde cross-linking reaction. Over 80% of the initial free enzyme activity was detected on the bound enzyme. The immobilized β-xylosidase (204.7 ± 6.5 U g-1 of perlite) exhibited transxylosylation activity when sugars from black liquor xylan hydrolysate and a variety of alcohols (C1-6) were used as donor and acceptor molecules, respectively, yielding a number of alkyl xylosides. The synthesized hexyl xyloside exhibited some interesting properties comparable with those of a commercial hexyl glucoside, including an antioxidant activity (IC50 = 8.9 ± 2.0 mg mL-1), wetting time (43 s), and critical micelle concentration value (252 mmol L-1). Optimization for hexyl xyloside synthesis was performed via statistical analysis using a Box and Behnken design. The maximum predicted yield of hexyl xyloside (499.0 ± 1.1 mg g-1 xylan hydrolysate) was obtained with 10% (w/v) xylan hydrolysate, 10 U g-1 immobilized β-xylosidase, and 13% (v/v) hexanol at 70 °C, pH 6.0 for 4 h, and it was validated by the following experimental yield (498.9 ± 4.8 mg g-1 xylan hydrolysate). The maximum production yield remained relatively stable after the immobilized enzyme was reused for 12 cycles with a 471.5 ± 15.1 mg g-1 hexyl xyloside yield (94.4% of the first cycle). Thus, the immobilized β-xylosidase has a high potential as a robust catalyst for application in alkyl xyloside production.

Preparation and characterization of high-strength and water resistant lignocelluloses based composites bonded by branched polyethylenimine (PEI)

High-strength and water resistant lignocelluloses based composites (LC) were fabricated using branched polyethylenimine (PEI) as the main bonding agent combined with glutaraldehyde cross-linking reaction and grinding pre-treatment. Physical and mechanical properties of different composites prepared were measured and investigated. It is evident that PEI was efficient in endowing LC with high strength and excellent water resistance. The obtained physical and mechanical properties of LC were complied with the requirement of the Chinese national standard for medium-density fiberboard (MDF). Most notably, the glutaraldehyde cross-linking and grinding pre-treatment could further improve these properties. When 5% PEI and 2.5% glutaraldehyde were incorporated, together with 2-hour grinding treatment, the LC prepared exhibited the optimum modulus of rupture (MOR) 58.1 MPa, modulus of elasticity (MOE) 5077 MPa, internal bonding strength (IB) 2.14 MPa, and thickness swell (TS) 30.2%. The excellent properties obtained could be attributed to the cross-linking effect and Schiff's base addition reaction among lignocelluloses, PEI and glutaraldehyde, which were confirmed by the Fourier transform infrared spectroscopy (FT-IR) and X-ray photoelectron spectroscopy (XPS) analysis. The high-strength LC prepared in this study is expected to be used as load-bearing material in structural application.

A three-dimensional macroporous network structured chitosan/cellulose biocomposite sponge for rapid and selective removal of mercury(II) ions from aqueous solution

A novel adsorbent, chitosan/cellulose biocomposite sponge (CCS) for removing Hg(II) ions rapidly and selectively from aqueous solution is successfully fabricated via a facile glutaraldehyde cross-linking reaction and lyophilization. The CCS can be rapidly separated and easily regenerated by simple extrusion compared with most particulate or powder absorbent. In the adsorption tests, the experimental data follows the pseudo-second-order kinetic model and Langmuir isotherm. The CCS uptakes more than 97% of the ultimate adsorption capacity in aqueous solution within 2 min, and the adsorption capacity is derived to be 495 mg/g. Furthermore, the CCS shows good selectivity for removing Hg(II) ions, and the coefficient of selectivity to Hg(II) ions is 461, 227, and 35 times higher than that to Cu(II), Pb(II), and Cd(II) ions, respectively. The mechanism for selective adsorption is elucidated via Fourier transform infrared spectrum (FTIR) and X-ray photoelectron spectroscopy (XPS) spectral analysis before and after adsorption. Density functional theory (DFT) calculations are also performed. Notably, the adsorption capacity retains 85% of its initial value (495 mg/g) after five cycles, indicating excellent reusability of the CCS. Significantly, the applicability of the CCS is evaluated in simulated Hg(II) effluent (PVC production). The results indicate that the removal efficiency of the CCS exceeds 84% of the initial value (11.45 ppm) within just 2 min. The sponge is a portable and eco-friendly composite, thereby has a great potential to decontaminate Hg(II) ions in water treatment.

Computer-aided identification of a novel pyruvate kinase M2 activator compound.

The aim of this study was to obtain antitumour molecules targeting to activate PKM2 through adequate computational methods combined with biological activity experiments. The structure-based virtual screening was utilized to screen effective activator targeting PKM2 from ZINC database. Molecular dynamics simulations were performed to evaluate the stability of the small molecule-binding PKM2 complex systems. Then, cell survival experiments, glutaraldehyde crosslinking reaction, western blot, and qPCR experiments were used to detect the effects of top hits on various cancer cells and the targeting specificity of PKM2. Two small molecules in 1,5-2H-pyrrole-dione were obtained after virtual screening. In vitro experiments demonstrated that ZINC08383544 specifically activated PKM2 and affected the expression of upstream and downstream genes of PKM2 during glycolysis, leading to the inhibition of tumour cell growth. These results indicate that ZINC08383544 conforms to the characteristics of PKM2 activator and is potential to be a novel PKM2 activator as antitumour drug. This work proves that ZINC08383544 promotes the formation of PKM2 tetramer, effectively blocks PKM2 nuclear translocation, and inhibits the growth of tumour, and ZINC08383544 may be a novel activator of PKM2. This work may provide a good choice of drug or molecular fragments for the antitumour strategy targeting PKM2. Screening of targeted drugs by combination of virtual screening and bioactivity experiments is a rapid method for drug discovery.

Development and Optimization of a Glucose Biosensor Based on Tris [5-amino-1,10-Phenanthroline]Iron(II) Polymer Films

A bilayered glucose biosensor consisting of tris[5-amino-1, 10-phenanthroline]iron(II) polymer film redox mediator and a glucose oxidase layer was prepared on glassy carbon surfaces. The polymer film of the iron complex was immobilized onto the electrode using cyclic voltammetry via electropolymerization reactions, while the enzyme layer was formed using a BSA and glutaraldehyde crosslinking reaction. The biosensors gave the largest response in the pH range of 7-8 and were evaluated with respect to storage conditions of room temperature and 4oC. There was no significant difference between the detection of glucose using the biosensor stored at room temperature versus one stored at 4°C and both bilayered films remained active for 20 days. The detection limit of the biosensors was found to be 0.30 mM which corresponds to a signal to noise ratio of 3:1.

Application of chitosan beads immobilized Rhodococcus sp. NCIM 2891 cholesterol oxidase for cholestenone production

The cell-bound cholesterol oxidase from the Rhodococcus sp. NCIM 2891 was purified three fold by diethylaminoethyl–sepharose chromatography. The estimated molecular mass (SDS-PAGE) and Km of the enzyme were ∼55.0kDa and 151μM, respectively. The purified cholesterol oxidase was immobilized on chitosan beads by glutaraldehyde cross-linking reaction and immobilization was confirmed by Fourier transform infrared spectroscopy, scanning electron microscopy and energy dispersive X-ray analysis. The optimum temperature (45°C, 5min) for activity of the enzyme was increased by 5°C after immobilization. Both the free and immobilized cholesterol oxidases were found to be stable in many organic solvents except for acetone. Fe2+ and Pb2+ at 0.1mM of each acted as inhibitors, while Ag+, Ca2+, Ni2+ and Zn2+ activated the enzyme at similar concentration. The biotransformation of cholesterol (3.75mM) with the cholesterol oxidase immobilized beads (3.50U) leads to ∼88% millimolar yield of cholestenone in a reaction time of 9h at 25°C. The immobilized enzyme retains ∼67% activity even after 12 successive batches of operation. The biotransformation method thus, shows a great promise for the production of pharmaceutically important cholestenone.

Immobilized alcalase alkaline protease on the magnetic chitosan nanoparticles used for soy protein isolate hydrolysis

An efficient immobilization of Alcalase 2.4L alkaline protease has been developed by using chitosan-coated magnetic nanoparticles as support via glutaraldehyde cross-linking reaction. The Fe3O4 nanoparticles, Fe3O4-chitosan, and immobilized Alcalase 2.4L alkaline protease were characterized by X-ray diffraction, transmission electron microscope, Fourier transform infrared spectroscopy, electron spin resonance, and vibrating sample magnetometry. Results showed that the binding of chitosan and Alcalase 2.4L alkaline protease on Fe3O4 through cross-linking was successful. In addition, the Alcalase 2.4L alkaline protease immobilized with chitosan-coated magnetic nanoparticles enhanced the activity, the optimum reaction temperature and pH value for the immobilized enzyme were 55 °C and 10, respectively, compared with the free enzyme, and the optimal temperature and pH profile range were considerably broadened. Similarly, the thermal stability was enhanced by immobilization, and the kinetic parameters of free and immobilized Alcalase 2.4L alkaline protease were determined. Then, from our hydrolysis experiments, we found that immobilized Alcalase 2.4L alkaline protease uses Fe3O4-chitosan had a greatest hydrolytic activity, and the DH of soy protein isolate (SPI) can reach to 18.38 %, against 17.50 % with the free enzyme after 140 min. Furthermore, the immobilized Alcalase 2.4L alkaline protease could maintain about 86 % of its original activity after ten consecutive operations. Thus, Fe3O4-chitosan immobilized Alcalase 2.4L alkaline protease a good candidate for the continuous hydrolysis of SPI.

Thermal and hydrolytic degradation of electrospun fish gelatin membranes

The thermal and hydrolytic degradation of electrospun gelatin membranes cross-linked with glutaraldehyde in vapor phase has been studied. In vitro degradation of gelatin membranes was evaluated in phosphate buffer saline solution at 37 °C. After 15 days under these conditions, a weight loss of 68% was observed, attributed to solvation and depolymerization of the main polymeric chains. Thermal degradation kinetics of the gelatin raw material and as-spun electrospun membranes showed that the electrospinning processing conditions do not influence polymer degradation. However, for cross-linked samples a decrease in the activation energy was observed, associated with the effect of glutaraldehyde cross-linking reaction in the inter- and intra-molecular hydrogen bonds of the protein. It is also shown that the electrospinning process does not affect the formation of the helical structure of gelatin chains.

Cryogelation of molecularly imprinted nanoparticles: A macroporous structure as affinity chromatography column for removal of β-blockers from complex samples

In this work, a new macroporous molecularly imprinted cryogel (MIP composite cryogel) was synthesized by glutaraldehyde cross-linking reaction of poly(vinyl alcohol) (PVA) particles and amino-modified molecularly imprinted core–shell nanoparticles. The MIP core–shell nanoparticles were prepared using propranolol as a template by one-pot precipitation polymerization with sequential monomer addition. The characteristics of the MIP composite cryogel were studied by scanning electron microscopy (SEM) and texture analyzer. The macroporous structure of the composite (with the pore size varying from a few micrometers to 100μm) enabled high mass transfer of particulate-containing fluids. In a solid phase extraction (SPE) process, the efficiency and selectivity of the MIP composite cryogel were investigated, where the cryogel was used as an affinity matrix to remove propranolol from aqueous solution as well as from complex plasma sample without prior protein precipitation. The MIP composite cryogel maintained high selectivity and stability and could be used repeatedly after regeneration.

Studies of Fe3O4-chitosan nanoparticles prepared by co-precipitation under the magnetic field for lipase immobilization

Abstract A simple method was introduced to prepare magnetic Fe3O4-chitosan nanoparticles by co-precipitation with 0.45 T static magnetic field via glutaraldehyde cross-linking reaction, and the nanoparticles were used to immobilize lipase. The influence of magnetic field on the properties of magnetic nanoparticles was studied by SEM, XRD and VSM. The results showed that no obvious difference of the nanoparticles structure was found, while the morphology varied from random sphere-like to rod-like and magnetic character was significantly changed. Furthermore, the lipase immobilized with nanoparticles prepared under the magnetic field enhanced the activity, operational and storage stability compared with the control sample.

High Enzyme-Adsorptive Activity of a Cup-Stacked Carbon Nanotube and Its Application to Flow-Injection Analysis

A cup-stacked carbon nanotube (CSNT) was formed by stacking cup-shaped carbon units with a hollow open channel of 80 to 100 nm in diameter, in contrast to the structure of a conventional multi-walled carbon nanotube (MWNT) made up of some seamless cylinders. The side of the cup is hydrophobic and the edge is hydrophilic due to the presence of carboxyl groups. Thus, a CSNT has a large hollow space in which one can incorporate enzyme molecules based on a physical adsorption. In this work, CSNT and other nanosize supports [MWNT, graphite, activated carbon and aminopropyl CPG (APCPG)] were packed into similar stainless-steel columns (4 × 3 mm), and glucose oxidase (GOD) was adsorbed by circulating an enzyme solution into each column. The CSNT adsorbed GOD both strongly and rapidly. The adsorbed amount was significantly larger than those onto MWNT, graphite, and activated carbon, and equivalent to that immobilized onto APCPG by a glutaraldehyde crosslinking reaction. As a result, it was found that CSNT has a high adsorptive activity for GOD. The flow-injection system with a GOD adsorbed CSNT packed column responded linearly to the concentration of glucose over a dynamic range of 5 × 10−7∼1 × 10−3 M.

Elucidation of the mechanism and end products of glutaraldehyde crosslinking reaction by X‐ray structure analysis

Glutaraldehyde has been used for several decades as an effective crosslinking agent for many applications including sample fixation for microscopy, enzyme and cell immobilization, and stabilization of protein crystals. Despite of its common use as a crosslinking agent, the mechanism and chemistry involved in glutaraldehyde crosslinking reaction is not yet fully understood. Here we describe feasibility study and results obtained from a new approach to investigate the process of protein crystals stabilization by glutaraldehyde crosslinking. It involves exposure of a model protein crystal (Lysozyme) to glutaraldehyde in alkaline or acidic pH for different incubation periods and reaction arrest by medium exchange with crystallization medium to remove unbound glutaraldehyde. The crystals were subsequently incubated in diluted buffer affecting dissolution of un-crosslinked crystals. Samples from the resulting solution were subjected to protein composition analysis by gel electrophoresis and mass spectroscopy while crosslinked, dissolution resistant crystals were subjected to high resolution X-ray structural analysis. Data from gel electrophoresis indicated that the crosslinking process starts at specific preferable crosslinking site by lysozyme dimer formation, for both acidic and alkaline pH values. These dimer formations were followed by trimer and tetramer formations leading eventually to dissolution resistant crystals. The crosslinking initiation site and the end products obtained from glutaraldehyde crosslinking in both pH ranges resulted from reactions between lysine residues of neighboring protein molecules and the polymeric form of glutaraldehyde. Reaction rate was much faster at alkaline pH. Different reaction end products, indicating different reaction mechanisms, were identified for crosslinking taking place under alkaline or acidic conditions.