Electrochemical control of protein monolayers at indium tin oxide surfaces for the reagentless optical biosensing of nitric oxide

Cytochrome c has been immobilized onto functionalized, optically transparent indium tin oxide (ITO) electrodes by covalent and electrostatic techniques. Covalent immobilization was achieved by the formation of a disulfide bond between N-succinimidyl 3-(2-pyridyldithio)propionate-(SPDP-) modified cytochrome c and SPDP-silanized ITO. Additionally, ITO electrodes have been modified with the bifunctional reagent 1,12-dodecanedicarboxylic acid (DDCA), resulting in formation of a carboxylic acid-terminated monolayer. Covalent protein attachment to the DDCA-functionalized ITO was achieved with the cross-linker 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide hydrochloride. Electrostatic attachment of the protein involved ion-pair and hydrogen-bond interactions between the terminating carboxylic acid groups of the DDCA-functionalized ITO and the primary amine groups of the lysine residues of cytochrome c. The electrostatic interaction between the cytochrome c and the functionalized ITO resulted in greater rotational mobility of the protein at the electrode surface, leading to ca. 63% electroactivity, as compared to ca. 41% electroactivity for the covalently immobilized protein. The redox state of the electrostatically bound cytochrome c monolayers could be electrochemically switched between ferric and ferrous forms. Electrochemical control of the bound protein was used to regenerate the biosensing surface following binding of nitric oxide (NO). Ligation of NO with the cytochrome c was monitored by measurement of the change of absorbance intensity at 416 nm. Through application of a negative potential, the cytochrome c was reduced from the ferric to the ferrous form, which led to the removal of the ligated NO. Application of a positive potential regenerated the ferric cytochrome c, enabling multiple repeat measurements of NO. Such electrochemical control of proteins immobilized on transparent electrodes enables the optical biosensing of analyte targets without recourse to exogenous reagents.     

Covalent immobilization of glucose oxidase onto new modified acrylonitrile copolymer/silica gel hybrid supports

New polymer/silica gel hybrid supports were prepared by coating high surface area of silica gel with modified acrylonitrile copolymer. The concentrations of the modifying agent (NaOH) and the modified polymer were varied. GOD was covalently immobilized on these hybrid supports and the relative activity and the amount of bound protein were determined. The highest relative activity and sufficient amount of bound protein of the immobilized GOD were achieved in 10% NaOH and 2% solution of modified acrylonitrile copolymer. The influence of glutaraldehyde concentration and the storage time on enzyme efficiency were examined. Glutaraldehyde concentration of 0.5% is optimal for the immobilized GOD. It was shown that the covalently bound enzyme (using 0.5% glutaraldehyde) had higher relative activity than the activity of the adsorbed enzyme. Covalently immobilized GOD with 0.5% glutaraldehyde was more stable for four months in comparison with the one immobilized on pure silica gel, hybrid support with 10% glutaraldehyde and the free enzyme. The effect of the pore size on the enzyme efficiency was studied on four types of silica gel with different pore size. Silica with large pores (CPC-Silica carrier, 375 A) presented higher relative activity than those with smaller pore size (Silica gel with 4, 40 and 100 A). The amount of bound protein was also reduced with decreasing the pore size. The effect of particle size was studied and it was found out that the smaller the particle size was, the greater the activity and the amount of immobilized enzyme were. The obtained results proved that these new polymer/silica gel hybrid supports were suitable for GOD immobilization.     

Pulse injection analysis of the binding of serum proteins to porous polymer gels modified with formyl groups

Highly porous spherical polymer gels were modified with formyl groups by a modified Friedel-Crafts reaction and the interaction of serum proteins with the modified gels were examined by pulse injection analysis. The introduction of formyl groups into the polymer greatly increases its protein-binding capacity, and the protein bound to the gel is not eluted by washing with acid, alkali or urea solution. The effects of temperature and the percentage of formyl group substitution on the binding capacity indicate that the binding process can be interpreted as initial approach of the protein to the polymer surface, caused by the hydrophobic interaction, followed by formation of a stable Schiff base between the polymer gel and the protein. Theoretical treatment of the elution behaviour of the protein from the polymer-packed column is also examined, with the assumption that there are three kinds of binding site in the polymer gel: surface, macropore and micropore regions. These polymers are shown to be useful for the removal of proteins from biological samples in clinical assays using immobilized enzymes.     

Electron transfer and ligand binding to cytochrome c' immobilized on self-assembled monolayers

We have successfully immobilized Allochromatium vinosum cytochrome c' on carboxylic acid-terminated thiol monolayers on gold and have investigated its electron-transfer and ligand binding properties. Immobilization could only be achieved for pH's ranging from 3.5 to 5.5, reflecting the fact that the protein is only sufficiently positively charged below pH 5.5 (pI = 4.9). Upon immobilization, the protein retains a near-native conformation, as is suggested by the observed potential of 85 mV vs SHE for the heme FeIII/FeII transition, which is close to the value of 60 mV reported in solution. The electron-transfer rate to the immobilized protein depends on the length of the thiol spacer, displaying distance-dependent electron tunneling for long thiols and distance-independent protein reorganization for short thiols. The unique CO-induced dimer-to-monomer transition observed for cytochrome c' in solution also seems to occur for immobilized cytochrome c'. Upon saturation with CO, a new anodic peak corresponding to the oxidation of an FeII-CO adduct is observed. CO binding is accompanied by a significant decrease in protein coverage, which could be due to weaker electrostatic interactions between the self-assembled monolayer and cytochrome c' in its monomeric form as compared to those in its dimeric form. The observed CO binding rate of 24 M-1 s-1 is slightly slower than the binding rate in solution (48 M-1 s-1), which could be due to electrostatic protein-electrode interactions or could be the result of protein crowding on the surface. This study shows that the use of carboxyl acid-terminated thiol monolayers as a protein friendly method to immobilize redox proteins on gold electrodes is not restricted to cytochrome c, but can also be used for other proteins such as cytochrome c'.     

Immobilization of growing cells by polyethyleneimine-modified alginate

A unique polymer matrix that is suitable for immobilizing growing cells has been developed. Alginate was chemically modified with polyethyleneimine (PEI), and the resultant polymer aggregate was evaluated as a cell carrier. Our method of immobilization depends on reversible gelation of the PEI-modified alginate. Our hypothesis is that immobilized cells grow by dissolving the surrounding gel matrix; the dissolved polymer adduct is displaced peripherally and gelled again by the influx of calcium ion from the surrounding fermentation broth, retaining both cells and carrier polymer in the gel beads. Thus, the immobilized cells gain space for growth by expanding the carrier matrix. The PEI modification offers the following advantages: (1) improved mechanical strength; (2) improved cell retention; (3) increased catalyst life; (4) ease of pelletization; and (5) an apparent bacteriostatic capability. When immobilized yeast cells were applied to a continuous ethanol fermentation, 94% theoretical conversion of glucose to ethanol was observed, with a reactor productivity of 15–30 g/L/h in a nonsterile reactor. A 3-mo catalyst life and minimal cell washout were observed.     

Photocontrol of lactate dehydrogenase-spiropyran collagen membrane

Collagen fibrils were modified with β-l-[3,3-dimethyl-6′-nitrospiro-(indoline-2,2′-2H-benzopyran)] propionic anhydride. The spiropyran collagen membrane showed reverse photochromism. Lactate dehydrogenase (LDH, E.C. 1.1.1.27) was entrapped in the spiropyran collagen membrane. The activity of the LDH-spiropyran collagen membrane decreased under visible-light irradiation, and then increased again after incubation in the dark. The optimum pH of the LDH-spiropyran collagen membrane was displaced toward lower pH values under visible-light irradiation. The activity change of the LDH-spiropyran collagen membrane under visible-light irradiation depended on the LDH content.     

Oriented attachment and membrane reconstitution of His-tagged cytochrome c oxidase to a gold electrode: in situ monitoring by surface-enhanced infrared absorption spectroscopy

A novel concept is introduced for the oriented incorporation of membrane proteins into solid supported lipid bilayers. Recombinant cytochrome c oxidase solubilized in detergent was immobilized on a chemically modified gold surface via the affinity of its histidine-tag to a nickel-chelating nitrilo-triacetic acid (NTA) surface. The oriented protein monolayer was reconstituted into the lipid environment by detergent substitution. The individual steps of the surface modification, including (1) chemical modification of the gold support, (2) adsorption of the protein, and (3) reconstitution of the lipid bilayer, were followed in situ by means of surface-enhanced infrared absorption spectroscopy (SEIRAS) and accompanied by normal-mode analysis. The high surface sensitivity of SEIRAS allows for the identification of each chemical reaction process within the monolayer at the molecular level. Finally, full functionality of the surface-tethered cytochrome c oxidase was demonstrated by cyclic voltammetry after binding of the natural electron donor cytochrome c.     

Interaction of azide ion with hemin and cytochrome c immobilized on Au and Ag nanoparticles

This paper presents a set of investigations on the binding of a metabolic inhibitor, azide with prosthetic heme group of biomolecules, hemin chloride (Hem) and cytochrome c (Cyt c) immobilized on Au and Ag nanoparticles. A variety of spectroscopic tools have been used to understand the chemistry occurring on the nanoparticle surface. While the nature of binding of the model system, hemin has been investigated by UV-visible, fluorescence, FTIR, and Raman spectroscopies, the azide binding has been studied in detail by MALDI-TOF MS. Hemin binding on the nanoparticle surface occurs through the carboxylic acid groups. The hemin-N(3) adduct on the nanoparticle surface has been detected by mass spectrometry and its fragments have been studied by post source decay analysis. The chemistry of hemin on the nanoparticle surface has been compared with that of the protein, Cyt c. Azide binding of Cyt c requires thermal activation due to reduced accessibility of the heme center, unlike in the case of hemin. The binding chemistry is similar for free Cyt c and Cyt c bound to the nanoparticles.     

Schiff base copper(II) chelate as a tool for immobilization of protein

In our previous report a new methodology for intermolecular cross-linking or bridging of protein utilizing a spontaneous chelate formation process was proposed. In this paper the reliability of the process as a tool for protein immobilization has been further evaluated. The chromatographic behavior of tryptophan in a column packed with Sepharose coupled with salicylaldehyde residue showed that the alpha-amino acid was bound tightly to the gel in the presence of copper(II) ion and was eluted by the addition of ethylenediaminetetraacetate (EDTA). It was also proved that subtilisin modified with an alpha-amino acid residue was immobilized on the column, and this binding was reversed by the addition of EDTA as well.     

Synthesis of cation-exchange stationary phases using an adsorbed polymeric coating

We have prepared several silica-based cation-exchange materials that were suitable for the high-performance liquid chromatography of basic proteins. Two synthetic routes were examined. Central to both procedures was the adsorption of a low molecular weight polyamine. One method crosslinks the adsorbed polyamine with a multifunctional oxirane, which is then extensively derivatized with a monomeric cyclic anhydride. The second involves an adsorbed uncrosslinked polyethyleneimine layer which is reacted with polyacrylic anhydride, thereby crosslinking and imparting anionic character simultaneously. The resulting media prepared by either of these methods bound more than 40 mg of hemoglobin per gram of support depending on the reaction conditions. These cation-exchange stationary phases also exhibited good chromatographic performance, successfully resolving (horse heart) cytochrome c and lysozyme. Two of the more promising support materials were effectively used to isolate cytochrome c553 from a crude extract of cyanobacteria.     

Photoswitching of single association force between a pair of photoionizable spirobenzopyrans

We have measured the single intermolecular force of a typical photoionizable molecule, spirobenzopyran, by means of atomic force microscopy, which has proven to be useful in measuring directly single molecular forces. The spirobenzopyran moiety was immobilized covalently on both Au-coated probe tips and substrates by use of a self-assembled monolayer of a hexanethiol derivative incorporating a terminal spirobenzopyran moiety, 1'-(6'-mercaptohexyl)-3',3'-dimethylindolino-6-nitrospiro-(2H-1-benzopyran-2,2'-indoline). Force curve measurements were carried out using the spirobenzopyran-modified probe tip and substrate under dark conditions and in situ UV light irradiation. The adhesion force observed in a polar solvent (i.e., ethanol) was increased substantially under in situ UV light irradiation, which caused photoisomerization of the spirobenzopyran moiety bound to both tip and substrate from its electrically neutral spiropyran form to the corresponding zwitterionic merocyanine one. Statistical analyses of the observed force by autocorrelation technique have revealed that the photoionization enhanced by UV light caused a remarkable increase in the single intermolecular force of the photochromic compound.     

New Sol-Gel Photochromic Thin Films Made by Super-Fine Particles of Organic Photochromic Compounds

New photochromic thin gel films have been prepared by the dipping method from a sol-gel coating solution containing Malachite Green Leucocyanide soluble in sol and super-fine dispersed solution of spiropyran or spironaphthooxazine, which was effective to increase the concentration of photochromic compounds in gel film. Photochromism of these gel films is compared with that in sol under the conditions of acid catalysis. Though in sols using spiropyran and Malachite Green Leucocyanide normal photochromism was not observed, photochromism in thin gel films prepared from the same sol was confirmed. Normal photochromism of spironaphthooxazine in thin gel films was similar to that in solution. The reaction rate from the colored form to the spiro form of spiropyran and spironaphthooxazine in thin gel film is dependent on the gel matrix, which is influenced by the reaction time of the sol.     

Photo-, thermally, and pH-responsive microgels

Microgels with photo-, thermally, and pH-responsive properties in aqueous suspension have been synthesized and characterized using dynamic light scattering and UV-visible spectroscopy. The new route involved first preparing poly(N-isopropylacrylamide) (PNIPAM)-allylamine copolymer microgels and a spiropyran photochrome (SP) bearing a carboxylic acid group. Then the functionalized spiropyran was coupled to the microgel via an amide bond. The dark-equilibrated gel particles feature spiropyran molecules in the polar, merocyanine form. After irradiation of visible light, the particle size becomes smaller because spiropyran changes to the relatively nonpolar, closed spiro form. The PNIPAM-SP microgels undergo a volume phase transition in water from a swollen state to a collapsed state with increasing temperature under all light conditions. However, the transition temperature range of the PNIPAM-SP is much broader than that for the PNIPAM without SP. The PNIPAM-SP microgels are monodisperse and self-assemble into a crystalline lattice while in suspension. The UV-visible spectra of an aqueous suspension of PNIPAM-SP microgel in the dark-adapted, merocyanine form showed both an absorption peak around 512 nm due to the merocyanine (giving a reddish color to the suspension) and two sharp peaks from Bragg diffraction of colloidal crystallites. Upon visible irradiation, the 512-nm band bleached significantly due to spiropyran photoisomerization. The spiropyran photoisomerization and accompanying color changes of the suspension were reversible upon alternating dark, UV, and visible light irradiation. Due to the residues of amine groups, the swelling capability of PNIPAM-SP microgels reduces as the pH value is changed from 7 to 10.     

Preparation and properties of silica gel with immobilized formazan group

A new sorption material, silica gel with covalently immobilized formazan group, was suggested and characterized. The material was prepared by coupling the immobilized aryldiazonium salt with benzaldehyde phenylhydrazone. The equilibrium and kinetic characteristics of the sorption of Cu(II), Co(II), Ni(II), and Cd(II) from solutions onto the modified silica gel were determined. The material proved to efficiently concentrate Cu(II) from multicomponent solutions. The coordination surrounding of Cu(II) in the complex on the sorbent surface was determined by ESR.     

Synthesis of high‐molecular‐weight linear methacrylate copolymers with spiropyran side groups: Conformational changes of single molecules in solution and on surfaces

P(BMA‐co‐HEMA‐spiropyran) was synthesized by reversible addition fragmentation chain transfer (RAFT) polymerization of butyl methacrylate (BMA) and 2‐(trimethylsilyloxy)‐ethyl methacrylate (HEMA‐TMS), removal of the TMS‐protective groups, and the polymer analogous esterification of the hydroxyethyl side chains with a spiropyran containing a carboxylic acid group. UV‐induced conformational changes of the synthesized macromolecules and low‐molecular‐weight spiropyran molecules were studied. Rate constants and half‐life times of the ring closure reaction from zwitterionic merocyanine to the spiropyran species were determined in the presence and absence of mica‐dispersed particles in toluene both with the free spiropyran and the polymer‐bound spiropyran. Scanning force microscopy was used to visualize the conformation of spiropyran‐decorated single macromolecular chains and agglomerated polymer‐bound merocyanine adsorbed on mica. © 2009 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 47: 1274–1283, 2009     

Preparation and photochromic properties of oligomeric poly(dimethylsiloxane) with the spiropyran or spirooxazine moiety in the side chain

Spirobenzopyran 1 , with the 3-(diethoxymethylsilyl)-propyl group at the N atom, was synthesized. The condensation reaction of the spiropyran 1 and diethoxydimethylsilane gave oligomeric poly(dimethylsiloxane) with the spiropyran moiety in the side chain. The oligomer was photochromic; its colour changed from colorless to purple-red on uv irradiation and the color faded on visible irradiation or on standing in the dark. The half-decay time of the thermal decoloration was about twice that of monomeric spiropyran dissolved in the dimethylsiloxane oligomer. Photochromic poly(dimethylsiloxane) with the spirooxazine moiety in the side chain was also prepared.     

Interaction of cytochrome c with 1-palmitoyl-2-azelaoyl-sn-glycero-3-phosphocholine: evidence for acyl chain reversal

The conformational dynamics of the oxidatively modified phospholipid 1-palmitoyl-2-azelaoyl-sn-glycero-3-phosphocholine (PazePC) were assessed by observing by fluorescence energy transfer the association of the water-soluble cationic protein cytochrome c with micelles composed of this lipid. In keeping with reversal of the azelaoyl chain so as to expose its carboxyl function on the micelle surface, cytochrome c bound avidly to the micelles. In contrast, the aldehyde group bearing 1-palmitoyl-2-(9'-oxo-nonanoyl)-sn-glycero-3-phosphocholine (PoxnoPC) interacted only weakly. While the physiological significance of the above interaction is uncertain, our results demonstrate that oxidative damage alters the physical properties of lipid bilayers, involving enrichment of the polar moieties of oxidatively modified lipid chains within the membrane surface.     

Orientational control of the physiological reaction of cytochrome c oxidase tethered to a gold electrode

The physiological reaction of a membrane protein is reconstituted on a solid-supported electrode by orientational control via the position of an affinity tag. Recombinant cytochrome c oxidase (CcO) from Rhodobacter sphaeroides is immobilized on a chemically modified gold surface via the affinity of a histidine tag (His-tag) to a nickel chelating nitrilotriacetic acid surface. Control of the orientation is achieved by the adsorption of CcO through the His-tag engineered into the two opposite sites of the membrane protein surface. After reconstitution into a lipid layer, the functionality of this enzyme film electrode is probed by surface-enhanced infrared absorption spectroscopy and cyclic voltammetry. We demonstrate that cytochrome c (Cc) binds and initiates the catalytic reaction of CcO only when the latter is orientated with subunit II facing the bulk aqueous phase while Cc does not interact with the oppositely orientated CcO. We infer from the observed catalytic dioxygen reduction at potentials below 240 mV (vs a normal hydrogen electrode) that reduced Cc mediates electron input into CcO in a way similar to the physiological pathway. The quantitative analysis of the IR spectra indicates the presence of an inactive population of Cc bound to CcO at equal amounts as the redox-active population. This methodological approach demonstrates that the orientation of the membrane protein can be controlled depending on the position of the affinity tag. The approach is considered to be of general applicability as the introduction of affinity tags is routine in current biochemistry.     

Direct Electrochemistry of Cytochrome c on a Silica Sol‐Gel Film Modified Electrode

Dilute silica sol‐gel was simply dropped on the surface of a basal plane graphite electrode (BPGE) to form a silica sol‐gel film modified electrode. Direct electrochemical response of cytochrome c (Cyt c) on the modified electrode was observed by cyclic voltammetry (CV). The results suggested that Cyt c could be tightly adsorbed on the surface of the silica sol‐gel film modified electrode. A couple of well‐defined and nearly reversible redox peaks can be observed in a phosphate buffer solution (pH 7.0), which anodic and cathodic peak potentials were at ?0.243 and ?0.306 V (vs. Ag/AgCl), respectively. Cyt c adsorbed on the surface of silica sol‐gel film shows a remarkable electrocatalytic activity for the reduction of oxygen. Based on these, a third‐generation biosensor could be constructed to detect the concentration of oxygen in aqueous solution.     

The effect of ionic strength on the electron-transfer rate of surface immobilized cytochrome C

Horse heart cytochrome c was immobilized on four different self-assembled monolayer (SAM) films. The electron tunneling kinetics were studied in the different assemblies as a function of the ionic strength of the buffer solution using cyclic voltammetry. When cytochrome c is electrostatically immobilized, the standard electron exchange rate constant k0 decreases with the increase of the solution's ionic strength. In contrast, the protein covalently attached or ligated has a rate constant independent of the ionic strength. The inhomogeneity of electrostatically immobilized cytochrome c increases with the increase of the solution's ionic strength whereas that of the covalently attached protein is independent of the ionic strength. A comparison of these different electron-transfer behaviors suggests that the thermodynamically stable geometry of cytochrome c in the electrostatic assemblies is also an electron transfer favorable one. It suggests that the surface charges of cytochrome c are capable of guiding it into geometries in which its front surface faces the electron-transfer partner. The inhomogeneity observed in this study indicates that a distribution of cytochrome c orientations and thus a distribution of electron transfer rate constants exists.