Immobilization of trypsin on porous glycidyl methacrylate beads: effects of polymer hydrophilization

The immobilization of trypsin at porous glycidyl methacrylate (GMA-GDMA) beads was investigated. In particular, the effects of surface modification of the beads through hydrophilic polymers on the amount protein immobilized and on the extent of retained activity after immobilization were adressed. Furthermore, immobilization at unmodified and hydrophilized beads from aqueous solution was compared to that from a water-in-oil microemulsion. It was found that the amount trypsin immobilized at the unmodified GMA-GDMA beads was significantly higher than that at hydrophilized GMA-GDMA beads. However, also the extent of specific activity loss after immobilization was larger for the unmodified than for the hydrophilized beads. Despite the latter, however, the total activity displayed by the hydrophilized beads was comparable to the unmodified beads at best. On the other hand, by peforming the immobilization from the microemulsion a high immobilization yield can be reached even for the hydrophilized beads, which also results in a higher degree of retained activity in the latter case than obtained for immobilization at the unmodified beads. Using this approach therefore resulted in the highest total activity of the trypsin-activated GMA-GDMA beads.     

Immobilization of trypsin in polycation-polyanion complexes

Trypsin was immobilized in quasi-soluble polyanion-polycation complexes with retention of the enzyme activity. The activity of the immobilized enzyme strongly depends on pH of the prepared solution, composition, relative concentrations and molar masses of the polyelectrolytes. The highest activity of trypsin (about 93%) was found in the case of the complexes Na-poly(styrenesulfonate)-poly(diallyldimethylammonium chloride): trypsin = 10:1, prepared at pH 3.0, with high molar mass of the polyanion (about one million). This method can be proposed for immobilization of various serine proteases.     

Immobilization of trypsin on polysaccharides upon intense mechanical treatment

Immobilization of a ptoteolytic enzyme, trypsin, on chitosan and cellulose was accomplished in the solid state under pressure and shear strain. Trypsin immobilized in this way retains 70—80% of the catalytic activity and exhibits prolonged action. The mechanical treatment allows introduction of a much larger amount of the active protein into a polymer matrix than the equilibrium sorption from solution.     

Adamantyl-functionalized polymer monolith for capillary electrochromatography

An adamantyl (ADM)-functionalized monolithic stationary phase was newly synthesized by a single-step copolymerization of 1-adamantyl-(α-trifluoromethyl) acrylate, ethylene dimethacrylate, and 2-acrylamido-2-methyl-1-propanesulfonic acid in order to prevent the peak tailing of basic solutes in capillary electrochromatography and was compared with butyl methacrylate (BMA)-based one. The ADM structure shields the negatively charged groups on the surface of monolith from basic solutes, resulting in better peak shapes than BMA-based monolithic stationary phase. As the monomers ratio decreased, the monolithic column had lower retention and higher column efficiency which was likely due to lower phase ratio and smaller globule size of monolith, respectively. The ADM-functionalized monolithic columns exhibited a good repeatability and reproducibility of column preparation with relative standard deviation values below 9% in the studied chromatographic parameters.     

Preparation of a novel polymer monolith with functional polymer brushes by two‐step atom‐transfer radical polymerization for trypsin immobilization

Novel porous polymer monoliths grafted with poly{oligo[(ethylene glycol) methacrylate]‐co‐glycidyl methacrylate} brushes were fabricated via two‐step atom‐transfer radical polymerization and used as a trypsin‐based reactor in a continuous flow system. This is the first time that atom‐transfer radical polymerization technique was utilized to design and construct polymer monolith bioreactor. The prepared monoliths possessed excellent permeability, providing fast mass transfer for enzymatic reaction. More importantly, surface properties, which were modulated via surface‐initiated atom‐transfer radical polymerization, were found to have a great effect on bioreactor activities based on Michaelis–Menten studies. Furthermore, three model proteins were digested by the monolith bioreactor to a larger degree within dramatically reduced time (50 s), about 900 times faster than that by free trypsin (12 h). The proposed method provided a platform to prepare porous monoliths with desired surface properties for immobilizing various enzymes.     

Immobilization of trypsin on miniature incandescent bulbs for infrared-assisted proteolysis

A novel efficient proteolysis approach was developed based on trypsin-immobilized miniature incandescent bulbs and infrared (IR) radiation. Trypsin was covalently immobilized in the chitosan coating on the outer surface of miniature incandescent bulbs with the aid of glutaraldehyde. When an illuminated enzyme-immobilized bulb was immersed in protein solution, the emitted IR radiation could trigger and accelerate heterogeneous protein digestion. The feasibility and performance of the novel proteolysis approach were demonstrated by the digestion of hemoglobin (HEM), cytochrome c (Cyt-c), lysozyme (LYS), and ovalbumin (OVA) and the digestion time was significantly reduced to 5 min. The obtained digests were identified by MALDI-TOF-MS with the sequence coverages of 91%, 77%, 80%, and 52% for HEM, Cyt-c, LYS, and OVA (200 ng μL⁻¹ each), respectively. The suitability of the prepared bulb bioreactors to complex proteins was demonstrated by digesting human serum.     

Affinity monolith preconcentrators for polymer microchip capillary electrophoresis

Developments in biology are increasing demands for rapid, inexpensive, and sensitive biomolecular analysis. In this study, polymer microdevices with monolithic columns and electrophoretic channels were used for biological separations. Glycidyl methacrylate-co-ethylene dimethacrylate monolithic columns were formed within poly(methyl methacrylate) microchannels by in situ photopolymerization. Flow experiments in these columns demonstrated retention and then elution of amino acids under conditions optimized for sample preconcentration. To enhance analyte selectivity, antibodies were immobilized on monoliths, and subsequent lysozyme treatment blocked nonspecific adsorption. The enrichment capability and selectivity of these affinity monoliths were evaluated by purifying fluorescently tagged amino acids from a mixture containing green fluorescent protein (GFP). Twenty-fold enrichment and 91% recovery were achieved for the labeled amino acids, with a >25 000-fold reduction in GFP concentration, as indicated by microchip electrophoresis analysis. These devices should provide a simple, inexpensive, and effective platform for trace analysis in complex biological samples.     

Fabrication of mesoporous polymer monolith: a template-free approach

Mesoporous polyacrylonitrile (PAN) monolith has been fabricated by a template-free approach using the unique affinity of PAN towards a water/dimethyl sulfoxide (DMSO) mixture. A newly developed Thermally Induced Phase Separation Technique (TIPS) has been used to obtain the polymer monoliths and their microstructures have been controlled by optimizing the concentration and cooling temperature.     

Porous polymer monolith assisted electrospray from a glass microdevice

The coupling of a lab-on-a-chip microfluidic device to a nanoelectrospray ionization mass spectrometer has the potential to automate many routine analytical procedures and produce a powerful analytical tool. However, past coupling strategies have relied on complex manufacturing steps including drilling and etching the device to attach a capillary or building a nanospray emitter directly into the device. This study shows that a nanospray emitter can be easily fabricated using a porous polymer monolith (PPM) at the end of a glass microdevice. These devices are able to obtain a stable electrospray at a variety of flow rates (50-500 nL/min) but optimal results are obtained at lower flow rates (50-100 nL/min) compatible with electroosmotic flow processes. The PPM is photo-patterned so that it can be placed in any position within the channel of the device with no dead volume. The porous character and the hydrophobic nature of the PPM both aid in development of a stable electrospray process. Total ion current traces for the constant infusion of leucine-enkephalin and PPG show relative standard errors as low as 4%, and produce mass spectra with good signal-to-noise (S/N 43) from only 2 fmol of material. In addition, multiple experiments in a given day show good repeatability with variability as low as 13%, and the multiple flow paths inherent in the PPM limit sprayer clogging.     

Structural formation of hybrid siloxane-based polymer monolith in confined spaces

Structural deformation of phase-separated methylsiloxane gel under the influence of a surface has been studied. Competitive wetting of siloxane gel phase on a surface during phase formation is found to significantly affect the final morphology in a confined space. When the spinodal wavelength is sufficiently shorter than the size of the available space, a uniform bicontinuous structure forms in confined geometry. However, gel skeletons in the vicinity of a surface are elongated with decreasing size of the space, and finally when the size of the space becomes shorter than the spinodal wavelength, all the gel phase wets on a surface, showing a "wetting transition". Homogeneous bicontinuous methylsiloxane gels were successfully prepared, avoiding such structural deformation, in a long cylindrical fused silica capillary and used for capillary HPLC. The capillary gels exhibited excellent separation efficiency of nitrobenzenes and it was found that the surface character can be altered by incorporating surfactants, which will enable more advanced and extended control of surface character, depending on the analytes.     

Chip electrochromatography of polycyclic aromatic hydrocarbons on an acrylate-based UV-initiated porous polymer monolith

The first rigorous evaluation of a UV-initiated porous polymer monolith (PPM) as a stationary phase for chip electrochromatography (ChEC) is described. All channels in an offset T-injector-design-chip (25-microm deep by 50-microm wide channels) were filled by capillary action with an acrylate-based PPM precursor solution and polymerized in situ using 365 nm light for several minutes. Photodefinability of the monolith cast in the channels during the polymerization process was also demonstrated by masking off the injection arms during photoinitiation. The chromatographic performance of this chip was compared with that of chips completely filled with monolith. The detection window was photodefined after polymerization using the detection laser (257 nm doubled argon ion laser) to depolymerize the detection window. A successful ChEC separation of 10 out of 13 polycyclic aromatic hydrocarbons (PAH) was performed with on-column, off-packing laser-induced fluorescence detection at 257 nm. Van Deemter plots for early-, middle-, and late-eluting compounds showed the minimum plate height to be 5 microm. The average number of theoretical plates per meter for the PAH was 200,000. Several factors contributed to irreproducible results. Oxygen was observed to dynamically quench the fluorescence of the sample over time. Improved sealing of the reservoirs solved this problem. A within-chip variability in the retention time of 2-10% RSD was observed. These results demonstrate the feasibility and reliability of the PPM as a solid reversed-phase for electroosmotic flow-driven chip-based chromatography in microscale total analysis systems.     

Immobilization of trypsin on silica-coated fiberglass core in microchip for highly efficient proteolysis

In this report, trypsin was immobilized on silica-coated fiberglass core in microchip to form a core-changeable bioreactor for highly efficient proteolysis. To prepare the fiber core, a layer of organic-inorganic hybrid silica coating was prepared on the surface of a piece of glass fiber by a sol-gel method with tetraethoxysilane (TEOS) and 3-aminopropyltriethoxysilane (APTES) as precursors. Subsequently, trypsin was immobilized on the coating with the aid of glutaraldehyde. Prior to use, the enzyme-immobilized fiber was inserted into the channel of a microchip to form an in-channel fiber bioreactor. The novel bioreactor can be regenerated by changing its fiber core. The scanning electron microscopy images of the cross-section of a trypsin-immobilized fiber indicated that a layer of ∼1 μm thick film formed on the glass substrate. The feasibility and performance of the unique bioreactor were demonstrated by the tryptic digestion of bovine serum albumin (BSA) and cytochrome c (Cyt-c) and the digestion time was significantly reduced to less than 10 s. The digests were identified by MALDI-TOF MS with sequence coverages of 45% (BSA) and 77% (Cyt-c) that were comparable to those obtained by 12-h conventional in-solution tryptic digestion. The fiber-based microchip bioreactor provides a promising platform for the high-throughput protein identification.     

Enzymatic activity of trypsin adsorbed on temperature-sensitive composite polymer particles

Two kinds of temperature-sensitive composite polymer particles were prepared by seeded emulsion copolymerizations of (dimethylamino)ethyl methacrylate and ethylene glycol dimethacrylate with 0.14 μm-sized polystyrene and 0.26 μm-sized poly(methylmethacrylate) seed particles. To evaluate the usefulness as a carrier for biomolecules, the enzymatic activities of trypsin adsorbed on these two composite polymer particles were measured at temperatures above and below each lower critical solution temperature (LCST). In both cases, adsorbed trypsin retained its enzymatic activity during repeated adsorption/desorption measurements. © 1998 John Wiley & Sons, Inc. J Polym Sci A: Polym Chem 36: 883–888, 1998     

An evaluation of the performance of porous polymer monolith assisted electrospray

A novel electrospray interface, which has distinct advantages over conventional pulled silica emitters, has been developed. This novel interface can be easily fabricated by forming a porous polymer monolith (PPM) at the end of a fused-silica capillary that facilitates a stable electrospray over a wide range of flow rates with only a modest increase in back-pressure. A comparison was made between the PPM-assisted electrospray and a commercial nanosprayer in terms of sensitivity, stability and robustness. A PPM-filled electrospray tip produced a day-to-day signal variation of 23% relative standard deviation (RSD) over a 3-day period when spraying a 1.0 microM test peptide solution. Furthermore, three different capillaries fabricated by the same process produced a signal variation of 17% RSD, indicating that the fabrication process shows good reproducibility. The multiple flow paths of the PPM function to split the flow and reduce clogging. Even following the accumulation of debris after prolonged use, a stable spray could still be generated with the PPM-filled capillary while the commercial nanosprayer ceased to function properly. In terms of sensitivity, PPM-assisted electrospray showed an enhancement in sensitivity at infusion flow rates between 100 to 1000 nL/min while commercial nanosprayers performed slightly better at flow rates below 100 nL/min. A sample purification step can be combined with the PPM-assisted sprayer, using the PPM as a stationary phase to desalt and preconcentrate samples prior to mass spectrometric detection.     

Immobilization of proteolytic enzymes trypsin and α-chymotrypsin to cellulose matrix

Trypsin and α-chymotrypsin were immobilized on a microcrystalline cellulose matrix by adsorption interaction with cellulose. The adsorption of enzymes was determined as a function of their concentration and the pH of solutions. Mechanisms of enzyme binding to functional groups of cellulose were suggested.     

Immobilization of trypsin on graphene oxide for microwave-assisted on-plate proteolysis combined with MALDI-MS analysis

With an ultra-high surface area and abundant functional groups, graphene oxide (GO) provides an ideal substrate for the immobilization of trypsin. We demonstrated that trypsin could be immobilized on GO sheets assisted by polymers as molecular spacers to maintain the activity of the enzyme. And with the trypsin-linked GO as the enzyme immobilization probe, a novel microwave-assisted on-plate digestion method has been developed with subsequent analysis by MALDI-MS. The feasibility and performance of the digestion approach were demonstrated by the proteolysis of standard proteins. The results show that this novel approach substantially accelerated proteolysis and reduced the time required for traditional procedures involving on-plate enzymatic digestion and sample preparation prior to MALDI-MS analysis. The novel digestion approach is simple and efficient, offering great promise for high throughput protein identification.     

Chiral separation of binaphthol enantiomers on molecularly imprinted polymer monolith by capillary electrochromatography.

A novel enantioseparational monolithic stationary phase for binaphthol based on a molecular imprinting method was introduced and evaluated in capillary electrochromatography (CEC). The monolithic stationary was prepared by the in situ copolymerization of methacrylic acid and ethylene glycol dimethacrylate in a porogenic solvent (toluene or toluene-isooctane) in the presence of an imprinting molecule, (R)-1,1'-bi-2,2'-naphthol. Such stationary phases could separate the enantiomers of binaphthol. The influence of several parameters on the column permeability was investigated. These parameters included the polymerization time, the molar ratio of the functional monomer to the imprinting molecule and the content of porogen. The influence of the polymerization condition and the electrochromatographic parameters on the enantiomer separation was also studied. Initial studies showed that a higher molecular ratio of the imprinted molecule to the functional monomer, a higher content of porogen, a higher content of acetonitrile, a higher pH, as well as the addition of Tween 20, gave a higher enantiomer selectivity.     

Immobilization of laccase on polymer grafted polytetrafluoroethylene membranes for biosensor construction

In this study, Trametes versicolor laccase was immobilized on polytetrafluoroethylene (PTFE) membranes using two different techniques, entrapment to gelatin and covalent immobilization to the surface. For surface immobilization, functional groups were formed on PTFE surface by radiofrequency (RF) plasma treatment followed by polymer grafting. Two different polymers, polyacrylamide (pAAm) and polyacrylic acid (pAAc) were tried. For polyacrylamide grafted PTFE, a two-step polymerization process was used. The membranes were first treated with hydrogen plasma and pAAm grafted PTFE (pAAm-g-PTFE) was then formed by argon plasma treatment. To produce pAAc grafted PTFE (pAAc-g-PTFE), the surface was first treated with argon plasma and AAc was then attached to the surface by heat treatment (70 °C, 6 h). For both cases, an optimized carbodiimide coupling reaction was used for laccase immobilization. Enzyme activity was measured by an oxygen electrode using guaiacol as substrate. All three biosensing membranes were characterized and compared in terms of optimum working conditions, storage stability and reusability. Our study concluded that although a higher activity was obtained by gelatin entrapped laccase, its mechanical instability and poor storage life makes the gelatin biosensor unattractive for multiple usages and for field measurements. pAAc-g-PTFE biosensor was found to be more stable and highly reusable (ca. 50 times) when compared with the other two biosensors. In addition, its sensitivity was suitable for field applications. Therefore, the pAAc-g-PTFE biosensor could be proposed as an alternative on-site detection tool for phenolic compound monitoring.     

Immobilization of imidazolium cation based ionic liquids on thin polymer films

Methods of gravimetry, optical microscopy, FTIR spectroscopy, and conductometry were applied to the study of the adsorption of the following ionic liquids: 1-butyl-3-methylimidazolium chloride, bistrifluoromethylsulfonylimide, and trifluoroacetate on thin-layer films of polymers of different nature including polypropylene, polyethylene terephthalate, polytetrafluoroethylene, poly(vinyl chloride), and hydrated cellulose. It was established that the hydrated cellulose film can serve as polymer matrices for the ionconducting 1-butyl-3-methylimidazolium halide salts. The hydrated cellulose additive in the ionic liquids promotes their immobilization on the poly(vinyl chloride) film.     

Immobilization of amyloglucosidase using two forms of polyurethane polymer

Amyloglucosidase was covalently immobilized using two hydrophilic prepolymers: Hypol FHP 2002 (creates foams) and Hypol FHP 8190H (creates gels). The foamable prepolymer was superior as a support for enzyme immobilization. The percent activity immobilized in the polyurethane foams was 25 +/- 1.5%. Large substrates (greater than 200,000 daltons in mol wt) were hydrolyzed as effectively as smaller ones by the immobilized enzyme. The Km value of the foam-immobilized enzyme increased from 0.76 mg/mL (free) to 0.86 mg/mL (immobilized), whereas the Vmax dropped from 90.9 (free) to 12.4 nmol glucose/min/mL (immobilized). The long-term (2 mo) storage stability of amyloglucosidase was enhanced by immobilization in foams (70% activity retained; free enzyme only retained 50%). Immobilization also improved the enzyme stability to various denaturing agents (sodium chloride, urea, and ethanol). The immobilized enzyme exhibited increased stability compared to the free enzyme at high temperatures (95 degrees C). Both glycogen and starch could be utilized by the immobilized enzyme, indicating that this technique could prove useful for starch hydrolysis.