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Biomedical subjects

I Willner

Publications and source records attributed to I Willner.

35 records · Page 2Linked to original sources
PubMed2001

The decision-making value of magnetic resonance cholangiopancreatography in patients seen in a referral center for suspected biliary and pancreatic disease.

OBJECTIVE: To assess the ability of MRCP to alter the differential diagnosis and to prevent diagnostic and/or therapeutic ERCP. The diagnostic accuracy of magnetic resonance cholangiopancreatography (MRCP) for biliary and pancreatic disease is well documented. Some believe MRCP may prevent diagnostic ERCP or add useful information, however there are no reports of its impact on clinical management. METHODS: Consecutive patients referred for ERCP underwent clinic evaluation, then MRCP, and then ERCP. In Phase 1, the number of differential diagnoses and the perceived need for diagnostic ERCP were evaluated after each step by the endoscopist who performed the ERCP. In Phase 2, the process was repeated after presenting clinical information and MRCP results to different individual physicians: another endoscopist, a hepatologist, a radiologist, and a surgeon (all were blinded to ERCP results). RESULTS: Forty patients were enrolled. Clinical contexts were jaundice (19.7%), abnormal liver enzymes (42.6%), abdominal pain (11.5%), recurrent acute pancreatitis (11.5%), and suspected complications of chronic pancreatitis (14.7%). In Phase 1, adding MRCP information to diagnostic ERCP information did not change the mean number of differential diagnoses significantly and prevented no therapeutic ERCP. In Phase 2, adding MRCP to clinical information only (without ERCP) reduced the differential diagnosis significantly for the radiologist and the surgeon only and would have prevented < or =3% of diagnostic and therapeutic ERCP for all physicians. CONCLUSION: The value of MRCP information may be limited if patient selection is inappropriate and may differ in physicians depending on their speciality.

Biliary Tract Diseases↗
PubMed2000

Sensing of acetylcholine by a tricomponent-enzyme layered electrode using faradaic impedance spectroscopy, cyclic voltammetry, and microgravimetric quartz crystal microbalance transduction methods.

A three-enzyme layered assembly on Au electrodes or Au-quartz crystals, consisting of horseradish peroxidase, HRP, choline oxidase, ChO, and acetylcholine esterase, AChE, is used to sense acetylcholine by the HRP-mediated oxidation of 3,3',5,5'-tetramethylbenzidine, TMB (1), by H2O2, and the formation of the insoluble product (2) on the respective transducers. The analyte-substrate, acetylcholine, is hydrolyzed by AChE to choline that is oxidized by ChO and O2 to yield the respective betaine and H2O2. The amounts of generated H2O2 and the resulting insoluble product on the transducers correlate with the concentration of acetylcholine in the samples. The formation of the insoluble product (2) on electrode supports is followed by faradaic impedance spectroscopy that probes the increased interfacial electron-transfer resistance upon the formation of 2, and by cyclic voltammetry that reflects electron-transfer barriers upon the formation of the precipitate. The frequency of the Au-quartz crystal decreases as a result of the accumulation of the insoluble precipitate. The amount of insoluble product formed on the transducers is controlled by the concentration of acetylcholine and by the time interval of biocatalyzed precipitation. The generation of the insoluble product provides a means to amplify the sensing processes. Acetylcholine concentrations corresponding to 1 x 10(-5) M are easily sensed by the different transducers.

Acetylcholine↗
PubMed2000

From homoleptic to heteroleptic double stranded Copper(I) helicates: the role of self-recognition in self-assembly processes

The ligands 2,9-bis[(6-methyl-2, 2'-bipyridin-6'-yl)methyleneoxymethylenyl]-1,10-phenanthroline (6), 6' ',6' "-bis[(6-methyl-2, 2'-bipyridin-6'-yl)methyleneoxymethylenyl)]-2' ',2' "-bipyridine (2), 5,5'-bis[(6-methyl-2,2'-bipyridin-6'yl)methyleneoxymethylenyl]-2, 2'-bithiophene (7), and 6,6'-bis[(6-methyl-2, 2'-bipyridin-6'-yl)methyleneoxymethylenyl]-2,2'-biphenyl (8) and their respective homo- and heteroleptic double-stranded copper(I) complexes were prepared and characterized in order to estimate the importance of self-recognition in the self-assembly processes of double-stranded copper complexes. The homoleptic double-stranded copper complexes of 2, 6, 7, and 8 were characterized by NMR, FAB-MS, and electrochemistry. It was found that 6 and 2 each form a single double-stranded helicate having the structure of [(L)(2)Cu(3)](3+) (L = 2 or 6), 7 forms two double-stranded [(7)(2)Cu(3)](3+) complexes, and 8 results in a mixture of at least two [(8)(2)Cu(2)](2+) complexes. The potential shift, DeltaE degrees (,) of the Cu(+)/Cu(2+) redox process of these complexes reflects the binding affinity of the different binding sites to the copper cation. The electrochemical data show that the central units have a higher affinity to Cu(+) as compared to the off-center binding sites. NMR was used to determine the actual complex composition obtained from different mixtures of 2, 6, or 7 with Cu(+). Interestingly, we have found that, although 6, 2, and 7 each form homoleptic double-stranded complexes, no heteroleptic double-stranded copper complexes were formed from the mixtures of 7 with either 6 or 2. However, when mixtures of 6 and 2 are used, helicate distributions seem to follow simple statistics. These results are discussed in terms of the relative importance of self-recognition in the self-assembly of double-stranded helicates.

Journal Article↗
PubMed2000

Integration of Layered Redox Proteins and Conductive Supports for Bioelectronic Applications.

Integration of redox enzymes with an electrode support and formation of an electrical contact between the biocatalysts and the electrode is the fundamental subject of bioelectronics and optobioelectronics. This review addresses the recent advances and the scientific progress in electrically contacted, layered enzyme electrodes, and discusses the future applications of the systems in various bioelectronic devices, for example, amperometric biosensors, sensoric arrays, logic gates, and optical memories. This review presents the methods for the immobilization of redox enzymes on electrodes and discusses the covalent linkage of proteins, the use of supramolecular affinity complexes, and the reconstitution of apo-redox enzymes for the nanoengineering of electrodes with protein monolayers of electrodes with protein monolayers and multilayers. Electrical contact in the layered enzyme electrode is achieved by the application of diffusional electron mediators, such as ferrocene derivatives, ferricyanide, quinones, and bipyridinium salts. Covalent tethering of electron relay units to layered enzyme electrodes, the cross-linking of affinity complexes formed between redox proteins and electrodes functionalized with relay-cofactor units, or surface reconstitution of apo-enzymes on relay-cofactor-functionalized electrodes yield bioelectrocatalytic electrodes. The application of the functionalized electrodes as biosensor devices is addressed and further application of electrically "wired" enzymes as catalytic interfaces in biofuel cells is discussed. The organization of sensor arrays, self-calibrated biosensors, or gated bioelectronic devices requires the microstructuring of biomaterials on solid supports in the form of ordered micro-patterns. For example, light-sensitive layers composed of azides, benzophenone, or diazine derivatives associated with solid supports can be irradiated through masks to enable the patterned covalent linkage of biomaterials to surfaces. Alternatively, patterning of biomaterials can be accomplished by noncovalent interactions (such as in affinity complexes between avidin and a photolabeled biotin, or between an antibody and a photoisomerizable antigen layer) to provide a means of organizing protein microstructures on surfaces. The organization of patterned hydrophilic/hydrophobic domains on surfaces, by using photolithography, stamping, or micromachining methods, allows the selective patterning of surfaces by hydrophobic, noncovalent interactions. Photoactivated layered enzyme electrodes act as light-switchable optobioelectronic systems for the amperometric transduction of recorded photonic information. These systems can act as optical memories, biomolecular amplifiers, or logic gates. The photoswitchable enzyme electrodes are generated by the tethering of photoisomerizable groups to the protein, the reconstitution of apo-enzymes with semisynthetic photoisomerizable cofactor units, or the coupling of photoisomerizable electron relay units.

Journal Article↗
PubMed2000

An integrated NAD+-dependent enzyme-functionalized field-effect transistor (ENFET) system: development of a lactate biosensor.

An integrated NAD+-dependent enzyme field-effect transistor (ENFET) device for the biosensing of lactate is described. The aminosiloxane-functionalized gate interface is modified with pyrroloquinoline quinone (PQQ) that acts as a catalyst for the oxidation of NADH. Synthetic amino-derivative of NAD+ is covalently linked to the PQQ monolayer. An affinity complex formed between the NAD+/PQQ-assembly and the NAD+-cofactor-dependent lactate dehydrogenase (LDH) is crosslinked and yields an integrated biosensor ENFET-device for the analysis of lactate. Biocatalyzed oxidation of lactate generates NADH that is oxidized by PQQ in the presence of Ca2+-ions. The reduced catalyst, PQQH2, is oxidized by O2 in a process that constantly regenerates PQQ at the gate interface. The biocatalyzed formation of NADH and the O2-stimulated regeneration of PQQ yield a steady-state pH gradient between the gate interface and the bulk solution. The changes in the pH of the solution near the gate interface and, consequently, the gate potential are controlled by the substrate (lactate) concentration in the solution. The device reveals the detection limit of 1 x 10(-4) M for lactate and the sensitivity of 24+/-2 mV dec(-1). The response time of the device is as low as 15 s.

Biosensing Techniques↗
PubMed2000

Biomaterial engineered electrodes for bioelectronics.

A series of single-cysteine-containing cytochrome c, Cyt c, heme proteins including the wild-type Cyt c (from Saccharomyces cerevisiae) and the mutants (V33C, Q21C, R18C, G1C, K9C and K4C) exhibit direct electrical contact with Au-electrodes upon covalent attachment to a maleimide monolayer associated with the electrode. With the G1C-Cyt c mutant, which includes the cysteine residue in the polypeptide chain at position 1, the potential-induced switchable control of the interfacial electron transfer was observed. This heme protein includes a positively charged protein periphery that surrounds the attachment site and faces the electrode surface. Biasing of the electrode at a negative potential (-0.3 V vs. SCE) attracts the reduced Fe(II)-Cyt c heme protein to the electrode surface. Upon the application of a double-potential-step chronoamperometric signal onto the electrode, where the electrode potential is switched to +0.3 V and back to -0.3 V, the kinetics of the transient cathodic current, corresponding to the re-reduction of the Fe(III)-Cyt c, is controlled by the time interval between the oxidative and reductive potential steps. While a short time interval results in a rapid interfacial electron-transfer, ket1 = 20 s-1, long time intervals lead to a slow interfacial electron transfer to the Fe(III)-Cyt c, ket2 = 1.5 s-1. The fast interfacial electron-transfer rate-constant is attributed to the reduction of the surface-attracted Fe(III)-Cyt c. The slow interfacial electron-transfer rate constant is attributed to the electrostatic repulsion of the positively charged Cyt c from the electrode surface, resulting in long-range electron transfer exhibiting a lower rate constant. At intermediate time intervals between the oxidative and reductive steps, two populations of Cyt c, consisting of surface-attracted and surface-repelled heme proteins, are observed. Crosslinking of a layered affinity complex between the Cyt c and cytochrome oxidase, COx, on an Au-electrode yields an electrically-contacted, integrated, electrode for the four-electron reduction of O2 to water. Kinetic analysis reveals that the rate-limiting step in the bioelectrocatalytic reduction of O2 by the integrated Cyt c/COx electrode is the primary electron transfer from the electrode support to the Cyt c units.

Biocompatible Materials↗
PubMed1999

An Au nanoparticle/bisbipyridinium cyclophane-functionalized ion-sensitive field-effect transistor for the sensing of adrenaline.

A film consisting of polyethyleneimine (PEI), Au nanoparticles (12 +/- 1 nm) and coadsorbed cyclobis(paraquat-p-phenylene) (1) was assembled as a sensing interface on the Al2O3 insulating layer of an ion-sensitive field-effect transistor (ISFET). Adrenaline (2) was sensed by the functionalized ISFET with a detection limit of 1 x 10(-6) M. The sensing ability of the nanostructured device for the analysis of adrenaline originates from the preconcentration of the analyte in the cyclophane by pi-pi donor-acceptor interactions. Analysis of adrenaline is accomplished by the measurement of the source-drain current, Isd, or by the gate-source voltage, Vgs. The sensing device is reusable (at least 100 cycles) and exhibits high stability.

Epinephrine↗
PubMed1999

Precipitation of an insoluble product on enzyme monolayer electrodes for biosensor applications: characterization by Faradaic impedance spectroscopy, cyclic voltammetry, and microgravimetric quartz crystal microbalance analyses.

Precipitation of an insoluble, insulating product on monolayer-functionalized electrodes enables the development of new electrochemical biosensors. Faradaic impedance spectroscopy and cyclic voltammetry are used to probe the electron-transfer resistance at the conductive support upon the accumulation of the insoluble product on the electrode surface. Similarly, microgravimetric quartz crystal microbalance, QCM, analyses were used to assay the formation of the precipitate on the electrode. A horseradish peroxidase, HRP, monolayer electrode is used to analyze H2O2 via the biocatalyzed oxidation of 4-chloro-1-naphthol (1) and the precipitation of the insoluble product (2). A bienzyme-layered electrode consisting of HRP and glucose oxidase, GOx, is used to sense glucose. Biocatalyzed oxidation of glucose by O2, in the presence of GOx, yields H2O2, and the generated hydrogen peroxide effects the formation of the insoluble product (2) in the presence of HRP. The insoluble product accumulated on the electrode, and the extent of the resulting electron-transfer resistance, correlated with the amounts of H2O2 or glucose, and appropriate calibration curves are extracted.

Biosensing Techniques↗
PubMed1999

Electronic transduction of photostimulated binding interactions at photoisomerizable monolayer electrodes: novel approaches for optobioelectronic systems and reversible immunosensor devices

Photoisomerizable monolayers assembled onto electrode supports act as "command interfaces" for controlling the binding interactions of biomaterials with the functionalized surfaces. The light-induced binding and dissociation of the biomaterials to and from the electrodes, respectively, are electronically transduced. Two systems, including the photostimulated binding and dissociation of cytochrome c (Cyt c) and of anti-DNP antibody to and from functionalized surfaces, are discussed. The application of the systems as optobioelectronic devices and reversible immunosensors is addressed. A mixed monolayer consisting of pyridine and nitrospiropyran (1a) photoisomerizable units assembled on a Au-electrode acts as a command interface for the light-controlled association and dissociation of Cyt c to and from the monolayer. Cyt c binds to the pyridine/1a-monolayer electrode, resulting in electrical contact between the redox protein and the electrode. Photoisomerization of the mixed monolayer to the pyridine/protonated merocyanine state (1b) results in the electrostatic repulsion of Cyt c and its dissociation from the electrode support. This blocks the electrical contact between Cyt c and the electrode. By the cyclic photoisomerization of the mixed monolayer between the 1a and 1b states, reversible "ON"-"OFF" amperometric transduction of the affinity interactions between the redox protein and the interface is accomplished. Coupling of the photostimulated electrical contact between Cyt c and the electrode surface to the Cyt c-mediated bioelectrocatalyzed reduction of O(2) by cytochrome oxidase provides a means to amplify the transduced electronic signal. A photoisomerizable thiolated dinitrospiropyran (2a) monolayer, assembled on solid supports, acts as a light-active antigen interface that enables the photocontrolled binding and dissociation of anti-dinitrophenyl antibody (DNP-Ab) to and from the interface. The dinitrospiropyran (2a) layer acts as an antigen for the DNP-Ab, whereas the protonated dinitromerocyanine (2b) lacks antigen features for the DNP-Ab. By reversible photoisomerization of the monolayer between the 2a and 2b states, cyclic binding and dissociation of DNP-Ab to and from the monolayer interface is accomplished. The association and dissociation of the DNP-Ab to and from the 2a- and 2b-monolayer states are electronically transduced, using amperometric, Faradaic impedance and microgravimetric, quartz crystal microbalance analyses. The photostimulated binding of an antibody to a photoisomerizable antigen monolayer provides a novel method to design reversible immunosensor devices.

Journal Article↗
PubMed1998

Fully integrated biocatalytic electrodes based on bioaffinity interactions.

Integrated bioelectrocatalytically active electrodes are assembled by the deposition of enzymes onto respective electrically contacted affinity matrices and further cross-linking of the enzyme monolayers. A catalyst-NAD(+)-dyad for the binding of the NAD(+)-dependent enzymes and cytochrome-like molecules for the binding of the heme-protein-dependent enzymes are used to construct integrated electrically contacted biocatalytic systems. NAD(+)-dependent lactate dehydrogenase (LDH) is assembled onto a pyrroloquinoline quinone-NAD+ monolayer. The redox-active monolayer is organized via covalent attachment of pyrroloquinoline quinone (PQQ) to a cystamine monolayer associated with a Au-electrode, followed by covalent linkage of N6-(2-aminoethyl)-NAD+ to the monolayer. The interface modified with the PQQ-NAD(+)-dyad provides temporary affinity binding for LDH and allows cross-linking of the enzyme monolayer. The cross-linked LDH is bioelectrocatalytically active towards oxidation of lactate. The bioelectrocatalyzed process involves the PQQ-mediated oxidation of the immobilized NADH. Integrated, electrically contacted bioelectrodes are produced by the affinity binding and further cross-linking of nitrate reductase (NR) (cytochrome-dependent, E.C. 1.9.6.1 from E. coli) or CoII-protoporphyrin IX reconstituted myoglobin (CoII-Mb) atop the microperoxidase-11 (MP-11) monolayer associated with a Au-electrode. The MP-11 monolayer provides an affinity interface for the temporary binding of the enzymes, that allows the cross-linkage of the enzyme molecules. The MP-11 assembly acts as electron transfer mediator for the reduction of the secondary enzyme layer. The integrated bioelectrodes consisting of NR and CoII-Mb show catalytic activities for NO3- reduction and acetylene-dicarboxylic acid hydrogenation, respectively. Two FeIII-protoporphyrin IX units are reconstituted into a four alpha-helix bundle de novo protein assembled as a monolayer on a Au-electrode. Vectorial electron transfer proceeds in the synthetic heme-protein monolayer. Cross-linking of an affinity complex generated between the FeIII-protoporphyrin IX reconstituted de novo protein monolayer and NR yields an integrated, electrically contacted enzyme electrode that stimulates the bioelectrocatalyzed reduction of nitrate.

Animals↗
PubMed1997

Piezoelectric immunosensors for urine specimens of Chlamydia trachomatis employing quartz crystal microbalance microgravimetric analyses.

The assembly of a biosensor for Chlamydia trachomatis based on the microgravimetric quartz crystal microbalance (QCM) analysis of the bacteria association to an antibody-functionalized electrode is described. The sensing interfaces consist of a primary cystamine monolayer assembled onto Au electrodes associated with the quartz crystal. The monolayer is further modified with sulfosuccinylimidyl 4-(p-maleimidophenyl)butyrate (sulfo-SMPB) and the goat IgG-anti-mouse IgG Fc-specific Ab or the fragmented F(ab')2 anti-mouse IgG Ab that act as sublayers for the association of the sensor-active anti-C. trachomatis LPS-Ab. Bacteria in the concentration range from 260 ng.mL-1 to 7.8 micrograms.mL-1 are sensed by the functionalized crystals. The association of C. trachomatis to the sensing interface can be confirmed and amplified via interaction of the crystal with various anti-C. trachomatis antibodies. Urine-pretreated functionalized quartz crystals are applied in the analysis of C. trachomatis in urine samples. The sensitivity limits of the electrodes for sensing the bacteria in urine samples corresponds to approximately 260 ng.mL-1. The functionalized crystals assembled via association of anti-C. trachomatis LPS-Ab to the fragmented F(ab')2 anti-mouse IgG Ab reveal long-term stability upon storage at 4 degrees C.

Antibodies, Bacterial↗
PubMed1997

Photochemically-activated electrodes: application in design of reversible immunosensors and antibody patterned interfaces.

Antigen monolayers assembled onto Au electrodes associated with a quartz crystal act as electrochemical or microgravimetric quartz-crystal-microbalance (QCM) sensing interfaces for the complementary antibody. Electrochemical analysis of the antibody (Ab) is based on the insulation of the antigen monolayer electrode by the associated Ab towards a redox probe in the electrolyte solution. Ferrocene-modified glucose oxidase (Fc-GOx) and glucose are employed as redox probes for the amperometric transduction of the Ab association to the electrode. Bioelectrocatalyzed oxidation of glucose provides an electrochemical route to amplify the antigen-Ab complex formation. Electrochemical analysis of the dinitrophenyl antibody, DNP-Ab, by a dinitrophenyl-lysine monolayer electrode is presented. QCM analysis of the Ab is based on the frequency changes of the quartz crystal resulting from the association of the Ab to the crystal assembly. This method is discussed with the analysis of the fluorescein antibody, Flc-Ab, using a fluorescein monolayer-modified quartz crystal. A novel method to tailor reversible immunosensor devices by the application of photoisomerizable antigen monolayers on electrodes is presented. The antigen is modified by photoactive units exhibiting reversible photoisomerizable properties. In one photoisomer state, the antigen exhibits affinity for the Ab and enables its electrochemical or QCM analysis. Photoisomerization to the complementary state perturbs the antigen structure and the monolayer lacks affinity for the Ab. This enables the washing-off of the Ab and the regeneration of the actively sensing interface by a second illumination process that restores the antigen monolayer-modified surface. This method is exemplified by the development of a reversible DNP-Ab sensing electrode. N-Mercaptobutyl dinitrospiropyran was assembled as a photoisomerizable monolayer on a Au electrode. The dinitrospiropyran monolayer, SP-state, exhibits affinity for the DNP-Ab and enables the amperometric detection of the Ab using Fc-GOx and glucose as redox probe. The complementary photoisomerized protonated dinitromerocyanine monolayer, MRH(+)-state, lacks affinity for the DNP-Ab. By photoisomerization of the DNP-Ab associated with the SP-monolayer electrode to the MRH(+)-monolayer state, the DNP-Ab is washed-off, and by a second illumination process, the MRH(+)-monolayer is re-isomerized to the SP-monolayer assembly, which is the active interface for further analysis of the DNP-Ab. Cyclic amperometric detection of the DNP-Ab by the photoisomerizable dinitrospiropyran monolayer is demonstrated. The association of the DNP-Ab to the SP-monolayer electrode and the dissociation of the Ab from the MRH(+)-monolayer electrode are confirmed by QCM experiments using a dinitrospiropyran monolayer-modified quartz crystal. The insulating features of an antigen-Ab complex on a conductive surface and the photochemically controlled association of an antibody to a photoisomerizable monolayer assembled onto the surface were used to develop means for micropatterning of surfaces by the antibody. A dinitrospiropyran antigen monolayer was assembled onto conductive ITO glass. A DNP-Ab solution was used as 'ink solution' to pattern the surface. The Ab-pattern was imaged by electrochemical copper deposition onto the Ab-lacking surface domains. The dinitrospiropyran monolayer assembled onto ITO or Pyrex glass surfaces was employed as an active interface for the photolithographic patterning of the surface with the DNP-Ab. (ABSTRACT TRUNCATED)

Antigen-Antibody Reactions↗
PubMed1996

Application of redox enzymes for probing the antigen-antibody association at monolayer interfaces: development of amperometric immunosensor electrodes.

Insulation of the electrical contact between a redox protein and an electrode surface upon association of an antibody to an antigen monolayer assembled on the electrode is used to develop immunosensor devices. In one configuration, a mixed monolayer consisting of the N epsilon-(2,4-dinitrophenyl)lysine antigen and ferrocene units acting as electron transfer mediators is applied to sense the dinitrophenyl antibody (DNP-Ab) in the presence of glucose oxidase (GOx) and glucose. In the absence of DNP-Ab, the mixed monolayer electrode stimulates the mediated electrocatalyzed oxidation of glucose that results in an amplified amperometric response. Association of the DNP-Ab to the modified electrode blocks the electrocatalytic transformation. The extent of the electrode insulation by the DNP-Ab is controlled by the Ab concentration in the sample. In the second configuration, a N epsilon-(2,4-dinitrophenyl)lysine antigen monolayer assembled on a Au electrode is applied to sense the DNP-Ab in the presence of a redox-modified GOx, exhibiting electrical communication with the electrode surface. Two kinds of redox-modified "electrically wired" GOx are applied: GOx modified by N-(ferrocenylmethyl)caproic acid, Fc-GOx, and a novel electrobiocatalyst generated by reconstitution of apo-GOx with a ferrocene-modified FAD semisynthetic cofactor. Electrocatalytic oxidation of glucose by the electrically wired biocatalysts proceeds in the presence of the antigen monolayer electrode, giving rise to an amplified amperometric signal. The electrocatalytic transformation is blocked upon association of the DNP-Ab to the monolayer electrode. The extent of electrode insulation toward the bioelectrocatalytic oxidation of glucose is controlled by the DNP-Ab concentrations in the samples. The application of biocatalysts for amperometric sensing of antigen-antibody interactions at the electrode surface makes the electrode insensitive to microscopic pinhole defects in the monolayer assembly. The antigen monolayer electrode is applied to sense the DNP-Ab in the concentration range 1-50 micrograms mL-1.

Antigen-Antibody Reactions↗
PubMed1995

Improving enzyme-electrode contacts by redox modification of cofactors.

Efficient electron transfer of redox proteins to and from their environment is essential for the use of such proteins in biotechnological applications such as amperometric biosensors and photosynthetic biocatalysts. But most redox enzymes lack pathways that can transport an electron from their embedded redox site to an electrode or a diffusing photoexcited species. Electrical communication between redox proteins and electrode surfaces has been improved by aligning proteins on chemically modified electrodes, by attaching electron-transporting groups and by immobilizing proteins in polymer matrices tethered by redox groups. Generally these methods involve contacting the enzymes at random with electron relay units. Here we report an approach that allows site-specific positioning of electron-mediating units in redox proteins. We strip glucose oxidase of its flavin adenine dinucleotide (FAD) cofactors, modify the latter with redox-active ferrocene-containing groups, and then reconstitute the apoprotein with these modified cofactors. In this way, electrical contact between an electrode and the resulting enzyme in solution is greatly enhanced in a controlled and reproducible way.

Apoproteins↗
PubMed1994

Photoregulation of alpha-chymotrypsin activity in organic media: effects of bioimprinting.

alpha-Chymotrypsin exhibits photoswitchable activities in an organic solvent after covalent modification of the protein backbone with thiophenefulgide active ester (2). The thiophenefulgide-modified alpha-chymotrypsin exhibits reversible photoisomerizable properties between states (3)-E and (3)-C. The modified alpha-chymotrypsin, where nine lysine residues are substituted by thiophenefulgide units, retains 60% of the activity of the native enzyme. The activities of thiophenefulgide-modified alpha-chymotrypsin toward esterification of N-acetyl-L-phenylalanine (4) by ethanol in cyclohexane are controlled by the configuration of the attached photoisomerizable component and by prior bioimprinting of the protein backbone with the reaction substrate (4). The esterification of (4) in cyclohexane using bioimprinted (3)-C is two-fold faster than in the presence of (3)-E. In the presence of a nonbioimprinted enzyme, esterification of (4) by (3)-C is five-fold faster than with (3)-E. The activity of bioimprinted (3)-E toward esterification of (4) is 4.5-fold higher than that of nonbioimprinted (3)-E. Switchable cyclic esterification of (4) is accomplished by sequential photoisomerization of the thiophenefulgide-modified alpha-chymotrypsin between states (3)-C and (3)-E.

Chymotrypsin↗
PubMed1981

Photosensitized electron transfer processes in SiO(2) colloids and sodium lauryl sulfate micellar systems: Correlation of quantum yields with interfacial surface potentials.

The effectiveness of negatively charged colloidal SiO(2) particles in controlling photosensitized electron transfer reactions has been studied and compared with that of the negatively charged sodium lauryl sulfate (NaLauSO(4)) micellar system. In particular, the photosensitized reduction of the zwitterionic electron acceptor propylviologen sulfonate (PVS(0)) with tris(2,2'-bipyridinium)ruthenium(II) [Ru(bipy)(3) (2+)] as the sensitizer and triethanolamine as the electron donor is found to have a quantum yield of 0.033 for formation of the radical anion (PVS([unk])) in the SiO(2) colloid compared with 0.005 in the homogeneous system and 0.0086 in a NaLauSO(4) micellar solution. The higher quantum yields obtained with the SiO(2) colloidal system are attributed to substantial stabilization against back reaction of the intermediate photoproducts-i.e., Ru(bipy)(3) (3+) and PVS([unk])-by electrostatic repulsion of the reduced electron acceptor from the negatively charged particle surface. The binding properties of the SiO(2) particles and NaLauSO(4) micelles were investigated by flow dialysis. The results show that the sensitizer binds to both interfaces and that the SiO(2) interface is characterized by a much higher surface potential than the micellar interface ( approximately -170 mV vs. -85 mV). The effect of ionic strength on the surface potential was estimated from the Gouy-Chapman theory, and the measured quantum yields of photosensitized electron transfer were correlated with surface potential at different ionic strengths. This correlation shows that the quantum yield is not affected by surface potentials smaller than approximately -40 mV. At larger potentials, the quantum yield increases rapidly. The quantum yield obtained in the micellar system at different strengths fits nicely on the correlation curve for the colloid SiO(2) system. These results indicate that the surface potential is the dominant factor in the quantum yield improvement for PVS(0) reduction.

Journal Article↗