Marcin Karbarz

Thermoresponsive poly(N-isopropylacrylamide) gel for immobilization of laccase on indium tin oxide electrodes.

We report on the properties of hydrogel matrix for the immobilization of laccase on conductive supports. The poly(N-isopropylacrylamide) gel is attached firmly to the indium-tin oxide (ITO) electrode, following its silanization with dimethylethoxyvinylsilane. The enzyme entrapped in the gel structure remained active longer than in the solution, and its redox and catalytic properties could be investigated by voltammetric methods. The reduction signals of the active sites, T1 and T2, of the Cerrena unicolor laccase were determined to be 0.79 and 0.38 V, respectively. The laccase catalytic activity toward oxygen in poly(N-isopropylacrylamide) was found to depend strongly on temperature. Reversible swelling/shrinking of the matrix was studied at 30 and 35 degrees C. Shrinking of the gel at higher temperature considerably decreased the efficiency of the catalytic reaction, however, interestingly, did not lead to irreversible changes in the enzyme structure. At temperatures below that corresponding to volume phase transition, the catalytic properties of the film were fully restored. High catalytic efficiency of the gel immobilized enzyme made it possible to employ the gel covered electrode for monitoring oxygen in solutions.

Influence of polymer network-metal ion complexation on the swelling behaviour of new gels with incorporated α-amino acid groups

New gels based on N-isopropylacrylamide and amino acid L-ornithine were prepared by free radical polymerization in aqueous solutions. To make the amino acid attachable to the polymer chain the acrylic group was added to the δ-amino group of ornithine, to obtain N-δ-acrylic ornithine. After the polymerization process the α-amino acid groups were unbound. The relative content of amino acid incorporated into the polymeric network of the gels was estimated from 1HNMR spectra, then compared with the molar fraction used for the polymerization process. The presence of free α-amino acid groups attached to the polymeric network of the gels enabled the complexation of some di- and trivalent metal cations. Copper ions, which can form two complexes of different stoichiometry (1 : 1 and 1 : 2) with amino acids, were used to investigate the influence of the complexation process on the swelling behavior of the gels. The influence of amino acid content, temperature and copper ion concentration on the swelling process was also examined. The gels were found to be most sensitive to concentrations of copper ions in the range 10−6–10−5 M. As the amount of amino acid in the polymer network increases the gels gradually lose their temperature-sensitivity and become more sensitive to copper ion concentration. The volume-phase-transition temperature decreases significantly after the addition of copper ions. Analysis of the UV-Vis spectra and the swelling behavior indicates that both 1 : 1 and 1 : 2 complexes are present in the swollen state of the gels, whereas the latter complex is more dominant in the shrunken state.

Hydrogel with chains functionalized with carboxyl groups as universal 3D platform in DNA biosensors.

Application of hydrogel based on N-isopropylacrylamide with carboxyl groups grafted to the chains enabled the immobilization of DNA at an extent exceeding that for flat surfaces by at least one order of magnitude. The probe DNA strands in the 3D platform were fully available for the hybridization process. The examination of the gels containing different amounts of grafted carboxyl groups (1-10%) was done using quartz crystal microbalance, electrochemical impedance spectroscopy, chronoamperometry and ionic coupled plasma with laser ablation. The optimal carboxyl group content was determined to be 5%. A very good agreement of the data obtained with independent techniques on content of DNA in the gel was obtained. In comparison to the other methods of immobilization of DNA the new platform enabled complete removal of DNA after the measurements and analysis and, therefore, could be used many times. After a 10-fold exchange of the DNA-sensing layer the efficiency of hybridization and analytical signal did not change by more than 5%. The sensor response increased linearly with logarithm of concentration of target DNA in the range 1×10(-13)-1×10(-6) M. The obtained detection limit was circa 8×10(-13) M of target DNA in the sample which is a substantial improvement over the planar sensing layers.

Stable and degradable microgels linked with cystine for storing and environmentally triggered release of drugs

Environmentally sensitive, degradable microgels based on poly(N-isopropylacrylamide) (pNIPA) cross-linked with the diacryloyl derivative of cystine (BISS) were synthesized by applying surfactant-free emulsion polymerization. pNIPA contributed the sensitivity to temperature to the microgels and the cross-linker made them degradable and sensitive to pH. The morphology of the microgels was investigated by using scanning and transmission electron microscopies (SEM and TEM). The gels formed spherical particles with a narrow size distribution. The influence of temperature, pH and ionic strength on the swelling behavior and the stability of new microgels with various contents of BISS (0, 1 and 3%) were investigated by dynamic light scattering (DLS). It was found that microgels with 3% content of amino acid were highly stable over a wide range of investigated temperatures, pH values and ionic strengths, including the physiological conditions (pH = 7.4, IS = 0.15 M, and 37 °C). The reduction-induced degradation of these microgels by 0.01 M solution of dithiothreitol (DTT) or glutathione (GSH) was studied by means of SEM and TEM; the obtained micrographs showed the destruction of spherical microgel particles. The microgels containing 3% of BISS could be loaded with doxorubicin (DOX) by employing the electrostatic interactions between the DOX amine group and the ionized carboxyl group from BISS. A significant increase in the cumulative release of DOX was observed after changing pH from that characteristic to blood (∼7.4) to that existing in affected cells (∼5.0) and in the presence of GSH (CGSH∼ 10 mM). The cytotoxicity tests proved that the obtained microgels are interesting as useful carriers in directed drug delivery systems.

Dual Sensing by Simple Heteroditopic Salt Receptors Containing an Anthraquinone Unit

We synthesized simple ion pair receptors consisting of a crown ether cation binding site and an anthraquinone-supported thiourea anion binding domain and studied their anion-, cation-, and salt-binding properties using spectroscopic, spectrophotometric, and electrochemical measurements in acetonitrile solution. Apart from carboxylate anions, which cause deprotonation, all the anions tested were found to associate with receptor 1 more strongly in the presence of sodium cations, whereas in the presence of potassium or ammonium cation the anion binding strength was greatly diminished. A homotopic anion receptor 3, lacking a crown ether unit, was unable to bind sodium salt more strongly than tetrabutylammonium salts. Solution and solid-state X-ray measurements revealed that strong sodium coordination with the cation-binding domain is responsible for the salt-binding enhancement. Electrochemical measurements showed that the addition of anions to the receptor 1 pretreated with sodium cations resulted in greater changes in reduction potentials compared to the addition of anions to receptor 1 in the absence of Na(+).

Oxidation of ferrocenemethanol grafted to a hydrogel network through cysteine for triggering volume phase transition

New gels based on N-isopropylacrylamide and cysteine were prepared by free radical polymerization in aqueous solutions. To attach the amino acid to the polymer chain, its α-amino group was modified with acryloyl chloride and N,N′-bisacryloylcystine was obtained. Next, the disulfide bridges were reduced to thiol groups and the attachment of ferrocenemethanol to the chains was performed. The influence of the amount of electroactive groups and their degree of oxidation on the temperature of volume phase transition was investigated. The gel formed from the polymerization solution containing 2% N,N′-bisacryloylcystine could undergo the volume phase transition triggered by changing the ferrocene state of oxidation. The temperature window of the gel sensitivity to the oxidation state of its ferrocene component was 35–40 °C. During the transition the volume of the gel could be changed even by more than an order of magnitude. Thin layers of the electroactive and thermoresponsive gel could be anchored on the surface of a glassy carbon electrode. This was achieved by electroformation of radicals at the electrode surface. The influence of the volume phase transition triggered by a temperature change on redox processes of ferrocene moieties bound to the network was examined voltammetrically.

Environmentally sensitive, quickly responding microgels with lattice channels filled with polyaniline

A conducting microcomposite composed of cross-linked poly(N-isopropylacrylamide) (pNIPA) and polyaniline (PANI) was synthesized. First, pNIPA gel microcapsules were prepared via surfactant-free emulsion polymerization (SFEP). Then by soaking the gel with a solution of an oxidizing agent (sodium persulfate) and placing it in a solution of aniline in nitrobenzene the conducting polymer was formed in pNIPA. The structure, morphology, swelling behavior and polyaniline oxidation state in the composite were investigated using SEM, TEM, DLS and Raman spectroscopy. The new microcomposite consists of micro-spheres, undergoes rapid volume phase transition and is electroactive. A result of the transition from the swollen to the collapsed state is a strong enhancement of the voltammetric peaks of polyaniline.

New ampholytic microgels based on N-isopropylacrylamide and α-amino acid: changes in swelling behavior as a function of temperature, pH and divalent cation concentration

Several new microgels based on N-isopropylacrylamide and the amino acid L-ornithine were synthesized by means of surfactant free emulsion polymerization. N-δ-Acryloyl ornithine was copolymerized with N-isopropylacrylamide and N,N′-methylenebisacrylamide, leading to the synthesis of a new ampholytic microgel. The swelling behavior of the obtained microgels with respect to the amount of amino acid incorporated in the polymer network, temperature, concentration of ions and pH was investigated. The pH dependence of the microgel size, measured at a constant temperature, was found to exhibit a minimum; the pH range where the minimum appeared corresponded well with the pH range where zwitterions dominated. The temperature dependence of the swelling process obtained for different pH values showed that for the pH region where zwitterions dominated, the polymer networks collapsed more efficiently. The presence of free α-amino acid groups attached to the polymeric network of the microgels also enabled the complexation of some metal cations. The influence of the presence of two metal ions (Cu2+ and Ca2+), which differ significantly in their ability to form complexes with the α-amino-acid, on the swelling behavior was investigated. It was found that the presence of copper ions that form stable complexes with the α-amino acid strongly influenced the swelling behavior of the investigated microgels.

An environmentally sensitive three-component hybrid microgel

A new multifunctional microcomposite has been synthesized. It is sensitive to several medium parameters, such as temperature, pH and ionic strength, is electroactive and conductive and can be easily attached to gold surfaces. All these properties were cumulated in one microcomposite by combining three components: poly(N-isopropylacrylamide) crosslinked with N,N′-bisacryloylcystine (p(NIPA–BISS) microgel), polyaniline nanofibers and gold nanoparticles. The presence of the polyaniline nanofibers and gold nanoparticles in the composite particles led to a substantial increase in conductivity and electroactivity. Additionally, the transition from the swollen to the collapsed state further enhanced the electroactivity and conductivity of the microcomposite. The structure, morphology and swelling behavior of the microgels were investigated using SEM, TEM, TGA, DLS and Raman spectroscopy. The electrocatalytic properties of the microcomposite towards oxidation of ethanol were also found.

Quartz crystal microbalance electrode modified with thermoresponsive crosslinked and non-crosslinked N-isopropylacrylamide polymers. Response to changes in temperature

Electrochemical quartz crystal microbalance (EQCM) electrode was modified with environmentally sensitive polymers. The polymer layers on the electrode were composed of either crosslinked or non-crosslinked thermoresponsive poly(N-isopropylacrylamide). For anchoring thin gel films on the electrode surface, the electrochemically induced free radical polymerization (EIFRP) was employed. The electroreduction of the peroxydisulfate anion led to generation of free radical. This radical initiated the free radical polymerization process that led to the formation of a thin gel layer attached to the electrode surface. To monitor in situ the growth of the polymer film the changes in resonant frequency of the quartz crystal were recorded. A significant decrease in the frequency (growth of the layer) was seen in the potential range where the reduction of peroxydisulfate anion took place. The morphology of the layers was examined with a scanning electron microscopy (SEM). The phenomenon of volume phase transition (shrinking/swelling process) in the gel layers initiated by a change in temperature was investigated. Unexpected big, sharp, and of negative sign minima appeared at the change-in-frequency vs. temperature plots. These minima were not seen in the plots obtained for the polymers without linker. That situation should be useful in the investigation of chemical interactions proceeding under the conditions of volume phase transition. The influence of volume phase transition on the transport of a member of a model red-ox system, ferro- and ferricyanide couple, and therefore on height of its voltammetric response was examined. The changes in electrochemical properties of the layers induced by volume phase transition were monitored with electrochemical impedance spectroscopy.