Suspension polymerization of poly(ethylene glycol) methacrylate: a route for swellable spherical gel beads with controlled hydrophilicity and functionality

 Spherical and swellable gel beads were obtained by the suspension polymerization of poly(ethylene glycol) methacrylate macromonomer (PEG-MA). The average size and size distribution properties, the equilibrium swelling behaviour and the protein adsorption characteristics of PEG-MA-based gel beads were determined. In the suspension polymerization system, the organic phase including monomer, cross-linker and diluent solution was dispersed in an aqueous medium by using poly(vinylpyrrolidone) as the stabilizer. The diluent solution was prepared by mixing cyclohexanol and octanol at different volume ratios. The suspension polymerization experiments were designed in two separate parts. In the first part, ethylene glycol dimethacrylate was selected as the cross-linker and swellable PEG-MA-based gel beads were obtained by changing the cross-linker concentration, the monomer/diluent ratio and the stirring rate. In the second part, a more hydrophobic structure, divinylbenzene (DVB) was tried as a cross-linker. In this part, PEG-MA-DVB copolymer beads were obtained by changing the DVB/PEG-MA feed ratio. Then, the hydrophicility of the resulting gel beads could be controlled by changing the feed ratio of hydrophilic macromonomer to hydrophobic cross-linker. This property was also used to control the extent of nonspecific protein adsorption onto the surface of the gel beads. The non specific albumin adsorption onto the gel beads decreased with increasing PEG-MA content. No significant nonspecific adsorption at the isoelectric point of albumin was detected onto the gel beads produced with the higher PEG-MA/DVB feed ratios. For specific albumin adsorption, a triazinyl dye (i.e., cibacron blue, CB F3G-A) was covalently attached onto the surface of the copolymer beads via terminal hydroxyl groups of PEG-MA. The results of albumin adsorption experiments with the CB F3G-A carrying beads indicated that an appreciable specific albumin adsorption capacity could be obtained with the gel beads produced with a PEG-MA/DVB feed ratio of 1.5/4.0. Received: 16 August 1999/Revised: 27 December 1999     

Application of Supermacroporous Monolithic Hydrophobic Cryogel in Capturing of Albumin

Supermacroporous poly{2-hydroxyethyl methacrylate-co-[N,N-bis(2,6-diisopropylphenyl)-perylene-3,4,9,10-tetracarboxylic diimide]} [poly(HEMA-co-DIPPER)] monolithic cryogel column was prepared by radical cryocopolymerization of HEMA with DIPPER as functional comonomer and N,N′-methylene-bisacrylamide (MBAAm) as crosslinker directly in a plastic syringe for adsorption of albumin. The monolithic cryogel contained a continuous polymeric matrix having interconnected pores of 10–50 μm size. Poly(HEMA-co-DIPPER) cryogel was characterized by swelling studies, FTIR, scanning electron microscopy, and elemental analysis. The equilibrium swelling degree of the poly(HEMA-co-DIPPER) cryogel was 14.7 g H₂O/g dry cryogel. Poly(HEMA-co-DIPPER) cryogel was used in the adsorption/desorption of albumin from aqueous solutions. The nonspecific adsorption of albumin onto plain poly(HEMA) cryogel was very low (3.36 g/g polymer). The maximum amount of albumin adsorption from aqueous solution in acetate buffer was 40.9 mg/g polymer at pH 5.0. It was observed that albumin could be repeatedly adsorbed and desorbed with the poly(HEMA-co-DIPPER) cryogel without significant loss of adsorption capacity.     

Poly(HEMA-co-NBMI) Monolithic Cryogel Columns for IgG Adsorption

Supermacroporous poly(2-hydroxyethyl methacrylate-co-1,5-naphthalene bismaleimide) [poly(HEMA-co-NBMI)] monolithic cryogel column was prepared by free radical cryo-copolymerization of HEMA with NBMI as a hydrophobic functional comonomer and N,N′-methylene-bisacrylamide as cross-linker directly in a plastic syringe for adsorption of albumin. The monolithic cryogel contained a continuous polymeric matrix which has interconnected pores of 10–100 μm size. Poly(HEMA-co-NBMI) cryogel was characterized by swelling studies, FTIR and scanning electron microscopy. The equilibrium swelling degree of the poly(HEMA-co-NBMI) cryogel was 10.5 g of H₂O/g dry cryogel. Poly(HEMA-co-NBMI) cryogel was used in the adsorption/desorption of IgG from aqueous solutions. The maximum amount of IgG adsorption from aqueous solution in phosphate buffer was 98.20 mg/g polymer at pH 7.0. The nonspecific adsorption of IgG onto plain poly(HEMA) cryogel was very low (2.79 g/g polymer). It was observed that IgG could be repeatedly adsorbed and desorbed with the poly(HEMA-co-NBMI) cryogel without significant loss of adsorption capacity.     

IMMOBILIZED CIBACRON BLUE F3G-A ON CROSSLINKED POLY (VINYL ALCOHOL) FOR AFFINITY CHROMATOGRAPHY

Immobilized triazine dye affinity chromatography has been widely used for protein purification. In this paper, cibacron Blue F3G-A was immobilized,through a spacer arm, onto a rigid hydrophilic porous polymer by reacting an epoxy-group-containing poly(vinyl alcohol) with 6-aminohexyl-N'-Cibacron Blue F3G-A,which was obtained by reacting Cibacron Blue F3G-A with excess of 1,6-diaminohexane, in a pH 8.6 buffer. The epoxy-group-containing poly(vinyl alcohol) was prepared by hydrolysis of macroporous crosslinded poly(vinyl acetate),which was synthesized by suspension copolymerization of vinyl acetate and triallyl isocyanurate in the presence of butyl acetate and n-heptane as diluents. The cibacron Blue F3G-A-immobilized poly(vinyl alcohol)was packed in a stainless steel column (250×5 mm I. D.) and the chromatographic behaviors of several proteins (cytochrome c, lysozyme, bovine serum albumin, insulin, and lactate dehydrogenase) were determined.     

Monolithic columns with immobilized monomeric avidin: preparation and application for affinity chromatography

A poly(glycidyl methacrylate-co-acrylamide-co-ethylene dimethacrylate) monolith and a poly(glycidyl methacrylate-co-ethylene dimethacrylate) monolith were prepared in fused silica capillaries (100 μm ID) and modified with monomeric avidin using the glutaraldehyde technique. The biotin binding capacity of monolithic affinity columns with immobilized monomeric avidin (MACMAs) was determined by fluorescence spectroscopy using biotin (5-fluorescein) conjugate, as well as biotin- and fluorescein-labeled bovine serum albumin (BSA). The affinity columns were able to bind 16.4 and 3.7 μmol biotin/mL, respectively. Columns prepared using the poly(glycidyl methacrylate-co-ethylene dimethacrylate) monolith retained 7.1 mg BSA/mL, almost six times more than commercially available monomeric avidin beads. Protocols based on MALDI-TOF mass spectrometry monitoring were optimized for the enrichment of biotinylated proteins and peptides. A comparison of enrichment efficiencies between MACMAs and commercially available monomeric avidin beads yielded superior results for our novel monolithic affinity columns. However, the affinity medium presented in this work suffers from a significant degree of nonspecific binding, which might hamper the analysis of more complex mixtures. Further modifications of the monolith’s surface are envisaged for the future development of monoliths with improved enrichment characteristics.     

Surface reorientation of hydrogels based on methacrylate copolymers

Surface molecular structures of two statistical copolymers, poly(2-hydroxyethyl methacrylate-co-butyl methacrylate) (HEMA-co-BMA) and poly[2-(2-ethoxyethoxy)ethyl methacrylate-co-butyl methacrylate] (EOEOEMA-co-BMA), were studied by X-ray photoelectron spectroscopy (XPS). Besides the classical “dry” XPS technique, where the polymer samples were air-dried, also “deep-freezing” technique was used, where the samples were investigated in deep-frozen hydrated state. The differences in results obtained by the two techniques are discussed from the point of view of the polymer surface chain reorientation in response to various environment. The reverse polymer chain reorientation from the hydrated towards dry state was also followed.     

Hydrazide-functionalized hollow porous poly(2-hydroxyethyl methacrylate-co-ethylene dimethacrylate) spheres for immobilization of enzyme

Hollow porous poly(2-hydroxyethyl methacrylate-co-ethylene dimethacrylate)(HEMA-co-EDMA) spheres were prepared by emulsifier-free emulsion polymerization, swelling, seed emulsion polymerization and extraction. Then the spheres activated with 2,4,6-trichloro-1,3,5-triazine were functioned with adipohydrazide (AH). After periodate oxidation of its carbohydrate moieties, horseradish peroxidase was immobilized on the hydrazide-functionalized hollow porous poly(HEMA-co-EDMA) spheres. The amount of immobilized enzyme was up to 43.4 μg of enzyme/g of support. Moreover, the immobilized horseradish peroxidase exhibited high activity and good stability.     

Lactic acid‐ and carbonate‐based crosslinked polymeric micelles for drug delivery

Our objective was to synthesize and evaluate lactic acid‐ and carbonate‐based biodegradable core‐ and core‐corona crosslinkable copolymers for anticancer drug delivery. Methoxy poly(ethylene glycol)‐b‐poly(carbonate‐co‐lactide‐co‐5‐methyl‐5‐allyloxycarbonyl‐1,3‐dioxane‐2‐one) [mPEG‐b‐P(CB‐co‐LA‐co‐MAC)] and methoxy poly(ethylene glycol)‐b‐poly(acryloyl carbonate)‐b‐poly(carbonate‐co‐lactide) [mPEG‐b‐PMAC‐b‐P(CB‐co‐LA)] copolymers were synthesized by ring‐opening polymerization of LA, CB, and MAC using mPEG as an macroinitiator and 1,8‐diazabicycloundec‐7‐ene as a catalyst. These amphiphilic copolymers which exhibited low polydispersity and critical micelle concentration values (0.8–1 mg/L) were used to prepare micelles with or without drug and stabilized by crosslinking via radical polymerization of double bonds introduced in the core and interface to improve stability. mPEG₁₁₄‐b‐P(CB₈‐co‐LA₃₅‐co‐MAC_2.5) had a higher drug encapsulation efficiency (78.72% ± 0.15%) compared to mPEG₁₁₄‐b‐PMAC_2.5‐b‐P(CB₉‐co‐LA₃₉) (20.29% ± 0.11%).¹H NMR and IR spectroscopy confirmed successful crosslinking (～70%) while light scattering and transmission electron microscopy were used to determine micelle size and morphology. Crosslinked micelles demonstrated enhanced stability against extensive dilution with aqueous solvents and in the presence of physiological simulating serum concentration. Furthermore, bicalutamide‐loaded crosslinked micelles were more potent compared to non‐crosslinked micelles in inhibiting LNCaP cell proliferation irrespective of polymer type. Finally, these results suggest crosslinked micelles to be promising drug delivery vehicles for chemotherapy. © 2012 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem, 2013     

Chromatography of functional polymers: A new approach to the characterization of reactive polymers obtained by chemical modification

High-performance liquid chromatography (HPLC) has been used to complement size-exclusion (gel permeation) chromatography (SEC) for the characterization of functional polymers. Whereas SEC is unable to detect compositional changes, HPLC in an appropriate interacting medium can provide detailed information on compositional changes occurring during chemical modification of a polymer. The method has been demonstrated using a normal-phase column consisting of porous monodisperse 10 μm poly(2,3-dihydroxypropyl methacrylate-co-ethylene dimethacrylate) beads that have a homogeneous coverage of aliphatic hydroxyl groups for the analysis of brominated poly(isobutylene-co-4-methylstyrene). Differences of well below 1 mol % of bromomethylstyrene units are easily detected and quantified. © 1997 John Wiley & Sons, Inc. J Polym Sci A: Polym Chem 35: 1173–1180, 1997     

Preparation of Immobilized Metal Affinity Chromatographic Packings Based on Monodisperse Hydrophilic Non‐porous Beads and Their Application

Three hydrophilic immobilized metal affinity chromatographic packings for HPLC have been synthesized by chemical modification of 3.0 µm monodisperse non‐porous poly(glycidyl methacrylate‐co‐ethylenedimethacrylate) (P_GMA/EDMA) beads. The retention behavior of proteins on the metal ion chelated columns loaded with copper(II), nickel(II) and zin(II) ion was studied. The effect of pH on the protein retention was investigated on both the naked and metal ion chelated columns in the range from 4.0 to 9.0. Four proteins were quickly separated in 3.0 min with linear gradient elution at a flow rate of 3.0 mL/min by using the synthesized Ni²⁺‐IDA (iminodiacetic acid) packings. The separation time was shorter than other immobilized metal affinity chromatography reported in the literature. Purification of lysozyme from egg white and trypsin on the commercially available trypsin was performed on the naked‐IDA and Cu²⁺‐IDA columns, respectively. The purities of the purified trypsin and lysozyme were more than 92% and 95%, respectively.     

Synthesis and characterization of well‐defined poly(2‐hydroxyethyl methacrylate‐co‐styrene)‐graft‐poly(ε‐caprolactone) by sequential controlled polymerization

A new graft copolymer, poly(2‐hydroxyethyl methacrylate‐co‐styrene) ‐graft‐poly(?‐caprolactone), was prepared by combination of reversible addition‐fragmentation chain transfer polymerization (RAFT) with coordination‐insertion ring‐opening polymerization (ROP). The copolymerization of styrene (St) and 2‐hydroxyethyl methacrylate (HEMA) was carried out at 60 °C in the presence of 2‐phenylprop‐2‐yl dithiobenzoate (PPDTB) using AIBN as initiator. The molecular weight of poly (2‐hydroxyethyl methacrylate‐co‐styrene) [poly(HEMA‐co‐St)] increased with the monomer conversion, and the molecular weight distribution was in the range of 1.09 ～ 1.39. The ring‐opening polymerization (ROP) of ?‐caprolactone was then initiated by the hydroxyl groups of the poly(HEMA‐co‐St) precursors in the presence of stannous octoate (Sn(Oct)₂). GPC and ¹H‐NMR data demonstrated the polymerization courses are under control, and nearly all hydroxyl groups took part in the initiation. The efficiency of grafting was very high. © 2004 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 42: 5523–5529, 2004     

Ti (IV) attached‐phosphonic acid functionalized capillary monolith as a stationary phase for in‐syringe‐type fast and robust enrichment of phosphopeptides

In this study, poly(vinylphosphonic acid‐co‐ethylene dimethacrylate), poly(VPA‐co‐EDMA) capillary monolith was synthesized as a starting material for obtaining a stationary phase for microscale enrichment of phosphopeptides. The chelation of active phosphonate groups with Ti (IV) ions gave a macroporous monolithic column with a mean pore size of 5.4 μm. The phosphopeptides from different sources were enriched on Ti (IV)‐attached poly(VPA‐co‐EDMA) monolith using a syringe‐pump. The monolithic capillary columns exhibited highly sensitive/selective enrichment performance with phosphoprotein concentrations as low as 1.0 fmol/mL. Six different phosphopeptides were detected with high intensity by the treatment of β‐casein digest with the concentration of 1.0 fmol/mL, using Ti (IV)@poly(VPA‐co‐EDMA) monolith. Highly selective enrichment of phosphopeptides was also successfully carried out even at trace amounts, in a complex mixture of digested proteins (molar ratio of β‐casein to bovine serum albumin, 1:1500) and three phosphopeptides were successfully detected. Four highly intense signals of phosphopeptides in human serum were also observed with high signal‐to‐noise ratio and a clear background after enrichment with Ti (IV)@poly(VPA‐co‐EDMA) monolith. It was concluded that the capillary microextraction system enabled fast, efficient and robust enrichment of phosphopeptides from microscale complex samples. The whole enrichment process was completed within 20 min, which was shorter than in the previously reported studies.     

Highly Sensitive Functionalized Conducting Copolypyrrole Film for DNA Sensing and Protein‐resistant

In order to exploit the applications of polypyrrole (PPy) derivatives in biosensors and bioelectronics, the different immobilization mechanisms of biomolecules onto differently functionalized conducting PPy films are investigated. Pyrrole and pyrrole derivatives with carboxyl and amino groups were copolymerized with ω‐(N‐pyrrolyl)‐octylthiol self‐assembled on Au surface by the method of the chemical polymerization to form a layer of the copolymer film, i.e., poly[pyrrole‐co‐(N‐pyrrolyl)‐caproic acid] (poly(Py‐co‐PyCA)) and poly[pyrrole‐co‐(N‐pyrrolyl)‐hexylamine] (poly(Py‐co‐PyHA)), in which the carboxyl groups in poly(Py‐co‐PyCA) were activated to the ester groups. Based on the structure characteristics, the immobilization/hybridization of DNA molecules on PPy, poly(Py‐co‐PyCA) and poly(Py‐co‐PyHA) were surveyed by cyclic voltammograms measurements. For differently functionalized copolymers, the immobilization mechanisms of DNA are various. Besides the electrochemical properties of the composite electrodes of PPy and its copolymers being detected before and after bovine serum albumin (BSA) adsorption, the kinetic process of protein binding was determined by surface plasmon resonance of spectroscopy. Since few BSA molecules could anchor onto the PPy and its copolymers surfaces, it suggests this kind of conducting polymers can be applied as the protein‐resistant material.     

Capillary electrochromatography of proteins with polymer-based strong-cation-exchanger microspheres

Monodisperse poly(glycidyl methacrylate-divinylbenzene) microspheres were functionalized with propyl sulfonic acid moieties to obtain beads negatively charged in a wide pH range. They were packed into fused-silica capillary of 50 micro, I.D. in order to separate proteins by capillary electrochromatography (CEC). Baseline separation of four basic proteins as well as three cytochrome c variants with an average column efficiency of 60,000 theoretical plates was obtained under isocratic elution conditions. The high efficiency is attributed to the uniformity of the column packing and the hydrophilic surface coverage of the polymer beads derived from the functionalization process. The effect of pH and salt concentration on protein separations was investigated and the results showed that the CEC separation mechanism is the combination of chromatographic retention and electrophoretic migration. Moreover, the column packed with the strongly acidic poly(glycidyl methacrylate-divinylbenzene) beads was also suitable for protein separations by micro-HPLC with a salt gradient. The comparison between the two kinds of elution modes shows that the column described here exhibited higher peak efficiency with isocratic elution in CEC than with gradient elution in micro-HPLC.     

Poly(N‐isopropylacrylamide)‐based comb‐type grafted hydrogel with rapid response to blood glucose concentration change at physiological temperature

A new type of glucose‐responsive hydrogel with rapid response to blood glucose concentration change at physiological temperature has been successfully developed. The polymeric hydrogel contains phenylboronic acid (PBA) groups as glucose sensors and thermo‐responsive poly (N‐isopropylacrylamide) (PNIPAM) groups as actuators. The response rate of the hydrogel to environmental glucose concentration change was significantly enhanced by introducing grafted poly(N‐isopropylacrylamide‐co‐3‐acrylamidophenylboronic acid) [poly(NIPAM‐co‐AAPBA)] side chains onto crosslinked poly(NIPAM‐co‐AAPBA) networks for the first time. The synthesized comb‐type grafted poly(NIPAM‐co‐AAPBA) hydrogels showed satisfactory equilibrium glucose‐responsive properties, and exhibited much faster response rate to glucose concentration change than normal type crosslinked poly(NIPAM‐co‐AAPBA) hydrogels at physiological temperature. Such glucose‐responsive hydrogels with rapid response rate are highly attractive in the fields of developing glucose‐responsive sensors and self‐regulated drug delivery systems. Copyright © 2008 John Wiley & Sons, Ltd.     

Synthesis and properties of organic-inorganic hybrid P(NIPAM-<Emphasis Type="Italic">co</Emphasis>-AM-<Emphasis Type="Italic">co</Emphasis>-TMSPMA) microgels

Utilizing the hydrolysis and condensation of the methoxysilyl moieties, organic-inorganic hybrid poly(N-isopropylacrylamide-co-acrylamide-co-3-(trimethoxysilyl)propylmethacrylate) P(NIPAM-co-AM-co-TMSPMA) microgels were prepared via two different methods. The first method was that the microgels were post-fabricated from the crosslinkable linear P(NIPAM-co-AM-co-TMSPMA) terpolymer aqueous solutions above the lower critical solution temperature (LCST) of the terpolymer. For the second method, the microgels were directly synthesized by conventional surfactant free emulsion copolymerization of NIPAM, AM, and TMSPMA. The hydrodynamic diameter and stability of the resultant P(NIPAM-co-AM-co-TMSPMA) microgels strongly depend on the pH and temperature of the microgel aqueous solution. The hydrodynamic diameters of the microgels decreased with increasing the measuring temperature. The phase transition temperature of the microgels was found to be around 34°C, which was independent of the initial terpolymer concentration and shifted to lower temperature with increasing the preparation temperature. Increasing the initial amount of AM will enhance the instability of the microgels at high pH values. Moreover, the P(NIPAM-co-AM-co-TMSPMA) microgels obtained from the linear terpolymer had more homogeneous microstructures as compared with the corresponding NIPAM/AM/TMSPMA microgels prepared by one step emulsion copolymerization as revealed by light scattering measurements.     

Polymer-supported reagents. II. Kinetics of esterification of acrylic acid with n-butanol using polymer supported titanium tetrachloride as catalyst

Crosslinked poly(4-vinylpyridine-co-styrene) was prepared and functionalized with titanium tetrachloride to afford the corresponding poly(4-vinylpyridine-co-styrene)-titanium tetrachloride complex. This insoluble functionalized polymer-supported catalyst shows good catalytic activity for esterification reactions. In this article, the kinetics of esterification of acrylic acid with n-butanol is reported. The rate of formation of product depends on many experimental parameters, viz., stirring speed, concentration of acrylic acid, catalyst amount, temperature, percent active site, percent crosslinking, and mesh size of the polymer catalyst. The reaction rates were found to increase with increase in the stirring speed, concentration of acrylic acid, catalyst amount, and temperature, and decreases with increasing percentage crosslinking and mesh size of the polymer beads. © 1997 John Wiley & Sons, Inc. J Polym Sci A: Polym Chem 35: 727–733, 1997     

Preparation and characterization of crosslinked poly(N,N‐diethylacrylamide‐co‐2‐hydroxyethyl methacrylate) resins and their application as support in solid‐phase peptide synthesis

Novel hydrophilic and thermosensitive poly(N,N‐diethylacrylamide‐co‐2‐hydroxyethyl methacrylate) resins were prepared by inverse suspension polymerization with N,N′‐methylenebis(acrylamide) as a crosslinker. The effects of chemical composition and degree of crosslinking on the polymerization were investigated. The polymer resins were characterized by elemental analysis, infrared spectroscopy, differential scanning calorimetry, and scanning electron microscopy. The thermosensitivity of the crosslinked resins was demonstrated by their lower critical swelling temperatures. The swelling and deswelling volume of the beads in water varied depending on the molar fraction of the N,N‐diethylacrylamide. These beads swelled extensively in a variety of common solvents. They had high loadings of functional hydroxyl groups and were used as supports in the solid‐phase synthesis of an oligopeptide. © 2003 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 41: 1681–1690, 2003     

An implantable biochip to influence patient outcomes following trauma-induced hemorrhage

Following hemorrhage-causing injury, lactate levels rise and correlate with the severity of injury and are a surrogate of oxygen debt. Posttraumatic injury also includes hyperglycemia, with continuously elevated glucose levels leading to extensive tissue damage, septicemia, and multiple organ dysfunction syndrome. A temporary, implantable, integrated glucose and lactate biosensor and communications biochip for physiological status monitoring during hemorrhage and for intensive care unit stays has been developed. The dual responsive, amperometric biotransducer uses the microdisc electrode array format upon which were separately immobilized glucose oxidase and lactate oxidase within biorecognition layers, 1.0–5.0 μm thick, of 3 mol% tetraethyleneglycol diacrylate cross-linked p(HEMA-co-PEGMA-co-HMMA-co-SPA)-p(Py-co-PyBA) electroconductive hydrogels. The device was then coated with a bioactive hydrogel layer containing phosphoryl choline and polyethylene glycol pendant moieties [p(HEMA-co-PEGMA-co-HMMA-co-MPC)] for indwelling biocompatibility. In vitro cell proliferation and viability studies confirmed both polymers to be non-cytotoxic; however, PPy-based electroconductive hydrogels showed greater RMS 13 and PC12 proliferation compared to controls. The glucose and lactate biotransducers exhibited linear dynamic ranges of 0.10–13.0 mM glucose and 1.0–7.0 mM and response times (t ₉₅) of 50 and 35–40 s, respectively. Operational stability gave 80% of the initial biosensor response after 5 days of continuous operation at 37 °C. Preliminary in vivo studies in a Sprague–Dawley hemorrhage model showed tissue lactate levels to rise more rapidly than systematic lactate. The potential for an implantable biochip that supports telemetric reporting of intramuscular lactate and glucose levels allows the refinement of resuscitation approaches for civilian and combat trauma victims.     

Purification of neutral protease by dye affinity chromatography

Twenty triazinic dyes were assayed as ligands for the chromatographic affinity purification of a neutral protease from Flavourzyme^™, a commercial preparation. Screening at pH 4.0 allowed the selection of eight dyes on the basis of their high protease adsorption. When the pH was set to 5.0 in order to increase selectivity, only Yellow HE-4R, Red HE-3B, and Cibacron Blue F3G-A maintained protease adsorption at high values. Neither maximum capacities nor dissociation constants calculated from isotherms measured at 8 and 25°C showed great differences. By contrast, a strong temperature effect was evidenced in the elution step: elution at 8°C allowed 70, 81, and 98% recovery of adsorbed protease with Yellow HE-4R, Red HE-3B, and Cibacron Blue F3G-A, respectively, whereas only 20% recovery was attained at 25°C. Based on the results obtained, a purification process for the neutral protease contained in Flavourzyme with Cibacron Blue F3G-A as the affinity ligand was developed, yielding 96% of electrophoretically pure enzyme in a single step, the specific activity rising from 850 to 3650 U/mg.