Design of temperature sensitive imprinted polymer hydrogels based on multiple-point hydrogen bonding

Weakly cross-linked temperature sensitive imprinted polymer hydrogels that recognize L-pyroglutamic acid (Pga) molecules via multiple-point hydrogen bonding were designed and synthesized. The amount of adsorption for Pga in imprinted hydrogels is 3-4 times higher than that in non-imprinted hydrogels. The selectivity test of imprinted polymer gels was carried out by using a series of structurally related compounds Pga, pyrrolidine, 2-pyrrolidone, L-proline as substrates. The results show that imprinted polymer gels exhibit high selectivity for Pga as compared to all the other tested substrates. The imprinted polymer hydrogels show good temperature sensitivity, special selectivity and reusability, suggesting that the polymer hydrogels would have an enormous potential for application in controlled drug release and separation field.     

Simulations of Redox Mediation within Bioactive Hydrogels of Amperometric Biosensors

Highly hydrated bioactive hydrogels containing immobilized oxidoreductase enzymes and immobilized redox mediators were simulated as the biorecognition layer of amperometric biosensors. The linear dynamic range of the amperometric response of mediated biosensors increases and moves to higher concentration brackets with an increase in the concentration of mediator. This informs the design of biosensors that target the same analyte but possesses several independently addressable electrodes modified with hydrogels that contain different concentrations of mediator. Increases in enzyme concentration increase the linear dynamic range but does not alter the sensitivity of amperometric biosensors. Both sensitivity and linear dynamic range of mediated amperometric enzyme biosensors may be “tuned” by varying the concentrations of the enzyme and the mediator. Simulations effectively guide the initial domains of study of complex systems such as implantable biosensors.     

Incorporation of single-walled carbon nanotubes into ferrocene-modified linear polyethylenimine redox polymer films

In this study, we describe the effects of incorporating single-walled carbon nanotubes (SWNTs) into redox polymer-enzyme hydrogels. The hydrogels were constructed by combining the enzyme glucose oxidase with a redox polymer (Fc-C(6)-LPEI) in which ferrocene was attached to linear poly(ethylenimine) by a six-carbon spacer. Incorporation of SWNTs into these films changed their morphology and resulted in a significant increase in the enzymatic response at saturating glucose concentrations (3 mA/cm(2)) as compared to films without SWNTs (0.6 mA/cm(2)). Likewise, the sensitivity at 5 mM glucose was significantly increased in the presence of SWNTs (74 μA/cm(2)·mM) as compared to control films (26 μA/cm(2)·mM). We demonstrate that the increase in the electrochemical and enzymatic response of these films depends on the amount of SWNTs incorporated and the method of SWNT incorporation. Furthermore, we report that the presence of SWNTs in thick films allows for more of the ferrocene redox centers to become accessible. The high current densities of the hydrogels should allow for the construction of miniature biosensors and enzymatic biofuel cells.     

Molecularly imprinted polymer-based electrochemical sensors for biopolymers

Electrochemical synthesis and signal generation dominate among the almost 1200 articles published annually on protein-imprinted polymers. Such polymers can be easily prepared directly on the electrode surface, and the polymer thickness can be precisely adjusted to the size of the target to enable its free exchange. In this architecture, the molecularly imprinted polymer (MIP) layer represents only one ‘separation plate’; thus, the selectivity does not reach the values of ‘bulk’ measurements. The binding of target proteins can be detected straightforwardly by their modulating effect on the diffusional permeability of a redox marker through the thin MIP films. However, this generates an ‘overall apparent’ signal, which may include nonspecific interactions in the polymer layer and at the electrode surface. Certain targets, such as enzymes or redox active proteins, enables a more specific direct quantification of their binding to MIPs by in situ determination of the enzyme activity or direct electron transfer, respectively.     

Wiring enzymes in nanostructures built with electrostatically self-assembled thin films.

The construction of electrostatically self-assembled intelligent nanostructures on electrodes with redox enzyme layers and redox polymer molecular wires defined in space allowed the analysis of redox charge transport from the redox enzyme to the electrode along nanometric distances. Recent results on the electrical connection of enzymes to electrodes and perspectives of generating electrical signals from molecular recognition in integrated enzyme electrodes are discussed.     

Enzyme responsive polymer hydrogel beads

We report on a new class of enzyme responsive polymer hydrogels, the molecular accessibility of which can be changed selectively by enzymes present in a sample fluid.     

Biocatalytic hydrogels by template polymerization

Novel ionizable hydrogels were prepared from poly(acrylic acid) and dimethylaminoethyl methacrylate monomer employing template polymerization technique as an alternative to traditional physical and chemical crosslinking. The mode of interaction, as proved by Fourier Transform Infrared Spectroscopy (FTIR), was multiple H‐bonding between the tertiary amino group of the monomer and the carboxylic groups of the polymer. The hydrogels represented suitable matrices for enzyme immobilization. The effect of varying the polymer–monomer molar ratio on the swelling kinetics and parameters was investigated. The dynamic swelling isotherm exhibited a Fickian mode of penetrant sorption and a plateau that increases with the amino group content. A polymer complex of molar ratio (polymer:monomer) 0.5:0.8 had a weight swelling ratio of 10 and 7 at pHs 3 and 8, respectively. The proven pH sensitivity together with the amphoteric character of these hydrogels make them good candidates for another bioapplication such as oral delivery systems of therapeutic peptides and proteins. The structural integrity of the hydrogels was proved by their swelling reversibility. β‐Galactosidase, as an acidic model enzyme, was immobilized covalently on the synthesized hydrogels. The maximum enzyme velocity (V_max) was enhanced to 19 µmol/min/mg, for polycomplex of molar ratio 0.5:0.8, compared with 3.2 µmol/min/mg for the free enzyme. Copyright © 2008 John Wiley & Sons, Ltd.     

Evaluation of redox mediators for amperometric biosensors: Ru-complex modified carbon-paste/enzyme electrodes

The properties of reagentless amperometric biosensors are mainly governed by the interaction of the used redox enzyme and the redox mediators used to facilitate the electron-transfer reaction. Both the used redox mediators and the redox enzymes differ concerning their hydrophilicity and their properties within the matrix of a carbon-paste electrode. Since there is no general procedure which is applicable for any enzyme in combination with any redox mediator, optimisation is necessary for each possible combination. Three approaches for the development of biosensors were investigated using carbon-paste electrodes enriched with redox mediator as a base in all sensor architectures. A class of redox mediators with the common formula Ru(LL)(2)(X)(2) (where LL are 1,10-phenantroline or 2,2'-bipyridine type ligands, and X is an acido ligand) was investigated. In the first approach, enzymes were integrated into the carbon paste; in the second, the enzymes were adsorbed on the surface of the mediator-containing carbon-paste electrode and held in place by a Nafion film; and in the third approach, enzymes were entrapped in polymer films, which were electrochemically deposited onto the electrode's surface. The properties of the obtained biosensors strongly depend on the sensor architecture and the specific features of the used enzyme. Thus, our investigation using three different sensor architectures can provide valuable information about the possible interaction between a specific enzyme and a redox mediators with specific properties.     

Amperometric Detection of Catecholamine Neurotransmitters Using Electrocatalytic Substrate Recycling at a Laccase Electrode

An enzyme electrode based on the coimmobilization of an osmium redox polymer and laccase on glassy carbon electrodes has been applied to ultra sensitive amperometric detection of the catecholamine neurotransmitters dopamine, epinephrine and norepinephrine, resulting in nanomolar detection limits, as low as 4 nM for dopamine. The sensitivity of the electrode is due to signal amplification via oxidation of the catecholamine by the immobilized laccase, which is regenerated by concomitant reduction of oxygen to water, coupled to the electrocatalytic re‐reduction of the oxidized catecholamine by the osmium redox complex: electrocatalytic substrate recycling. In addition because the sensor can be operated in reductive mode at ?0.2 V (vs. Ag/AgCl), noise and interferences are diminished. Combined with its high sensitivity this enzyme electrode also exhibited excellent selectivity allowing the detection of catecholamines in the presence of ascorbic acid. However, differentiation between the current responses achieved for the three catecholamines is not possible. The effective mode of constant recycling, resulting in amplification of the current response, of the laccase enzyme electrode sensor combined with the inherent advantages of using electrochemical techniques holds great promise for the future of catecholamine detection and monitoring.     

Electrochemical Redundant Microsensor Arrays for Glucose Monitoring with Patterned Polymer Films

Redundant microsensor arrays for glucose sensing were fabricated using photopolymerization of poly(ethylene glycol) diacrylate (PEG‐DA) with 2‐hydroxy‐2‐methyl phenyl‐propanone as photoinitiator to encapsulate the enzyme glucose oxidase. Silicon micro fabrication technologies were used to fabricate microelectrode sensor arrays on flexible polyimide sheets. These microarray sensors were individually addressable as observed using square‐wave voltammetry. Redox polymer, poly[4‐vinylpyridine Os(bipyridine)₂Cl]‐co‐ethylamine, was first immobilized on the electrode surface and then glucose oxidase was entrapped in PEG‐DA hydrogels. The redox polymer was found to exchange electrons with glucose oxidase in biocompatible PEG‐DA hydrogels. The entrapped glucose oxidase was found to respond linearly to glucose in solution (0–20 mM) as determined using square‐wave voltammetry.     

Bioinorganic Nanocomposite Hydrogels Formed by HRP–GOx‐Cascade‐Catalyzed Polymerization and Exfoliation of the Layered Composites

The mild preparation of multifunctional nanocomposite hydrogels is of great importance for practical applications. We report that bioinorganic nanocomposite hydrogels, with calcium niobate nanosheets as cross‐linkers, can be prepared by dual‐enzyme‐triggered polymerization and exfoliation of the layered composite. The layered HRP/calcium niobate composites (HRP=horseradish peroxidase) are formed by the assembly of the calcium niobate nanosheets with HRP. The dual‐enzyme‐triggered polymerization can induce the subsequent exfoliation of the layered composite and final gelation through the interaction between polymer chains and inorganic nanosheets. The self‐immobilized HRP‐GOx enzymes (GOx=glucose oxidase) within the nanocomposite hydrogel retain most of enzymatic activity. Evidently, their thermal stability and reusability can be improved. Notably, our strategy could be easily extended to other inorganic layered materials for the fabrication of other functional nanocomposite hydrogels.     

Long tethers binding redox centers to polymer backbones enhance electron transport in enzyme "Wiring" hydrogels

A redox hydrogel with an apparent electron diffusion coefficient (D(app)) of (5.8 +/- 0.5) x 10(-)(6) cm(2) s(-)(1) is described. The order of magnitude increase in D(app) relative to previously studied redox hydrogels results from the tethering of redox centers to the backbone of the cross-linked redox polymer backbone through 13 atom spacer arms. The long and flexible tethers allow the redox centers to sweep electrons from large-volume elements and to collect electrons of glucose oxidase efficiently. The spacer arms make the collection of electrons from glucose oxidase so efficient that glucose is electrooxidized already at -0.36 V versus Ag/AgCl, the reversible potential of the redox potential of the FAD/FADH(2) centers of the enzyme at pH 7.2. The limiting current density of 1.15 mA cm(-)(2) is reached at a potential as low as -0.1 V versus Ag/AgCl. The novel redox center of the polymer is a tris-dialkylated N,N'-biimidazole Os(2+/3+) complex. Its redox potential, -0.195 V versus Ag/AgCl, is 0.8 V reducing relative to that of Os(bpy)(2+/3+), its 2,2'-bipyridine analogue.     

Enzyme Scaffolds with Hierarchically Defined Properties via 3D Jet Writing

The immobilization of enzymes into polymer hydrogels is a versatile approach to improve their stability and utility in biotechnological and biomedical applications. However, these systems typically show limited enzyme activity, due to unfavorable pore dimensions and low enzyme accessibility. Here, 3D jet writing of water‐based bioinks, which contain preloaded enzymes, is used to prepare hydrogel scaffolds with well‐defined, tessellated micropores. After 3D jet writing, the scaffolds are chemically modified via photopolymerization to ensure mechanical stability. Enzyme loading and activity in the hydrogel scaffolds is fully retained over 3 d. Important structural parameters of the scaffolds such as pore size, pore geometry, and wall diameter are controlled with micrometer resolution to avoid mass‐transport limitations. It is demonstrated that scaffold pore sizes between 120 µm and 1 mm can be created by 3D jet writing approaching the length scales of free diffusion in the hydrogels substrates and resulting in high levels of enzyme activity (21.2% activity relative to free enzyme). With further work, a broad range of applications for enzyme‐laden hydrogel scaffolds including diagnostics and enzymatic cascade reactions is anticipated.     

Characterization of mediated and non-mediated oxidase enzyme based glassy carbon electrodes

The performance and analytical characteristics of a glassy carbon glutaraldehyde immobilized glucose oxidase electrode have been established with regard to the direct detection of hydrogen peroxide produced from the reaction of glucose with oxygen. Measurements were performed at + 1.1 V vs. SCE, and selectivity was obtained by casting the surface with a cellulose acetate membrane. Results compared favorably with the classical platinum-enzyme probe. The mechanism of ascorbic acid interference in hydrogen peroxide detection is reported. Mediated detection was also investigated for oxidase enzymes (glucose oxidase and xanthine oxidase) immobilized on the bare glassy carbon electrode. The probes were characterized using a specific enzyme mediator in solution (phenazine methosulfate or dichlorophenol-indophenol) plus hexacyanoferrate(III) as an electrochemical mediator. The electrode was poised at + 0.36 V vs. SCE for the detection of hexacyanoferrate(II). The advantages of this dual mediator configuration include high stability and sensitivity of the electrochemical signal and the ability to use less positive potentials for increased selectivity. Application to other enzymes, such as hydrogenases, using such a binary redox configuration is suggested.     

Synthesis and characterization of mechanically flexible and tough cellulose nanocrystals–polyacrylamide nanocomposite hydrogels

The unique combinations of hard and soft components with core/shell structures were proposed to synthesize high strength nanocomposite hydrogels. The elastomeric hydrogels containing rod-like cellulose nanocrystals (CNCs) core and polyacrylamide shell were made from aqueous solutions via free radical polymerization in the absence of chemical cross-links. The obtained hydrogels possessed greater tensile strength and elongation ratio when compared with chemically cross-linked counterparts. Oscillatory shear experiments indicated that CNCs interacted with polymer matrix via both chemical and physical interactions and contributed to the rubbery elasticity of the hydrogels. The nanocomposite hydrogels were more viscous than the chemical hydrogels, suggesting the addition of CNC led to the increase of energy dissipating and viscoelastic properties. The network structure model was proposed and it suggested that the high extensibilities and fracture stresses were related to the well-defined network structures with low cross-linking density and lack of noncovalent interactions among polymer chains, which may promote the rearrangements of network structure at high deformations.     

Synthesis and characterization of hydrogels containing redox-responsive 2,2,6,6-tetramethylpiperidinyloxy methacrylate and thermoresponsive N-isopropylacrylamide

Smart hydrogels containing 2,2,6,6-tetramethylpiperidinoxy methacrylate (TEMPO) and N-isopropylacrylamide (NIPAM) that undergo reversible redox behavior are prepared and investigated. Several polymer networks are first prepared by free-radical copolymerization of varying amounts of TEMPO, NIPAM, and a crosslinker (diethylene glycol diacrylate) and subsequently swelled with water to lead to hydrogels. In order to investigate the effects of the redox activity of TEMPO units and of the lower critical solution temperature of NIPAM on the hydrogel properties, a study of the swelling ratio of the polymer networks in distilled water at different temperatures is performed for the two forms of TEMPO, the reduced (TEMPO) and oxidized (TEMPO⁺) one. Moreover, the rheological properties are also measured for both hydrogel forms. Finally, the encapsulation abilities of the oxidized hydrogels are demonstrated via electrostatic interactions between positively charged TEMPO⁺ units and negatively charged guest molecules, supporting future application of our system in the biomedical and environmental fields.     

Redox electrodeposition polymers: adaptation of the redox potential of polymer-bound Os complexes for bioanalytical applications

The design of polymers carrying suitable ligands for coordinating Os complexes in ligand exchange reactions against labile chloro ligands is a strategy for the synthesis of redox polymers with bound Os centers which exhibit a wide variation in their redox potential. This strategy is applied to polymers with an additional variation of the properties of the polymer backbone with respect to pH-dependent solubility, monomer composition, hydrophilicity etc. A library of Os-complex-modified electrodeposition polymers was synthesized and initially tested with respect to their electron-transfer ability in combination with enzymes such as glucose oxidase, cellobiose dehydrogenase, and PQQ-dependent glucose dehydrogenase entrapped during the pH-induced deposition process. The different polymer-bound Os complexes in a library containing 50 different redox polymers allowed the statistical evaluation of the impact of an individual ligand to the overall redox potential of an Os complex. Using a simple linear regression algorithm prediction of the redox potential of Os complexes becomes feasible. Thus, a redox polymer can now be designed to optimally interact in electron-transfer reactions with a selected enzyme.     

Synthesis and characterization of a multi-sensitive crosslinked injectable hydrogel based on Pluronic

This paper reports the construction of a novel multi-sensitive chemically crosslinked injectable hydrogel with strong mechanical strength by modifying Pluronic F127 responsive against temperature, pH and redox potential. Crosslinked polymer between benzaldehyde grafted Pluronic (P-A) and amine end capped Pluronic having disulfide linkage (P-B) have been synthesized and characterized with 1H NMR spectroscopy and GPC. The hydrogel under physiological conditions significantly altered sol-gel transition behaviors with much lower critical gelation concentrations and temperatures, compared to Pluronic hydrogels. The rheological characterization demonstrated that the moduli of the hydrogels were able to be tuned depending on molecular weight as well as pH, redox and temperature conditions.     

A pH‐sensitive,strong double‐network hydrogel: Poly(ethylene glycol) methyl ether methacrylates–poly(acrylic acid)

We here describe new double network (DN) hydrogels with excellent mechanical strength and high sensitivity to pH changes. The first polymer network has a bottle brush structure and is formed from oligo‐monomers of poly(ethylene glycol) methyl ether methacrylate (PEGMA). Poly(acrylic acid) (PAA) is used as the second network. This double network features strong intermolecular interactions between the neutral poly(ethylene glycol) (PEG) side chains of PPEGMA and the non‐ionized carboxylic acid groups of the PAA second network. When immersed in solutions with a pH below ～4 the DN hydrogels have a low swelling ratio and are opaque as a result of solvent‐polymer phase separation driven by the formation of dense hydrogen‐bonded clusters. The compression strength (～8 MPa) is at least 14 times higher than the analogous single networks. When immersed in solutions with a pH >4, the hydrogels are transparent and exhibit a high swelling ratio with a compression strength of ～1 MPa. The PEG side chain length can be readily controlled without greatly altering the overall DN topology by choosing PEGMA monomers having different PEG side chain lengths. Longer PEG side branches give higher compression and tensile strengths at pH <4 when hydrogen bonded clusters form. The robust nature of these DN gels over a wide pH range may be useful for applications such as artificial muscles and controlled release devices. © 2011 Wiley Periodicals, Inc. J Polym Sci Part B: Polym Phys, 2012     

Spherical phospholipid polymer hydrogels for cell encapsulation prepared with a flow-focusing microfluidic channel device

To prepare spherical polymer hydrogels, we used a flow-focusing microfluidic channel device for mixing aqueous solutions of two water-soluble polymers. Continuous encapsulation of cells in the hydrogels was also examined. The polymers were bioinspired 2-methacryloyloxyethyl phosphorylcholine polymer bearing phenyl boronic acid groups (PMBV) and poly(vinyl alcohol) (PVA), which spontaneously form a hydrogel in aqueous medium via specific molecular complexation upon mixing, even when they were in cell culture medium. The microfluidic device was prepared with polydimethylsiloxan, and the surface of the channel was treated with fluoroalkyl compound to prevent sticking of the polymers on the surface. The microfluidic channel process could control the diameter of the spherical hydrogels in the range of 30-90 μm and generated highly monodispersed diameter spherical hydrogels. We found that the polymer distribution in the hydrogel was influenced by the PVA concentration and that the hydrogel could be dissociated by the addition of d-sorbitol to the suspension. The single cells could be encapsulated and remain viable in the hydrogels. The localized distribution of polymers in the hydrogel may provide an environment for modulating cell function. It is concluded that the spontaneous hydrogel formation between PMBV and PVA in the flow-focusing microfluidic channel device is applicable for continuous preparation of a spherical hydrogel-encapsulating living cell.