A bifunctional molecularly imprinted polymer (MIP): analysis of binding and catalysis by a thermistor.
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Acetates↗
Biomedical subjects
Frieder W Scheller
Publications and source records attributed to Frieder W Scheller.
Engineered superoxide dismutase monomers for superoxide biosensor applications.
Because of its high reaction rate and specificity, the enzyme superoxide dismutase (SOD) offers great potential for the sensitive quantification of superoxide radicals in electrochemical biosensors. In this work, monomeric mutants of human Cu,Zn-SOD were engineered to contain one or two additional cysteine residues, which could be used to bind the protein to gold surfaces, thus making the use of promotor molecules unnecessary. Six mutants were successfully designed, expressed, and purified. All mutants bound directly to unmodified gold surfaces via the sulfur of the cysteine residues and showed a quasi-reversible, direct electron transfer to the electrode. Thermodynamic and kinetic parameters of the electron transfer were characterized and showed only slight variations between the individual mutants. For one of the mutants, the interaction with the superoxide radical was studied in more detail. For both partial reactions of the dismutation, an interaction between protein and radical could be shown. In an amperometric biosensorial approach, the SOD-mutant electrode was successfully applied for the detection of superoxide radicals. In the oxidation region, the electrode surpassed the sensitivity of the commonly used cytochrome c electrodes by approximately 1 order of magnitude while not being limited by interferences, but the electrode did not fully reach the sensitivity of dimeric Cu,Zn-SOD immobilized on MPA-modified gold.
Biosensing Techniques↗
Thirty years of haemoglobin electrochemistry.
Electrochemical investigations of the blood oxygen carrier protein include both mediated and direct electron transfer. The reaction of haemoglobin (Hb) with typical mediators, e.g., ferricyanide, can be quantified by measuring the produced ferrocyanide which is equivalent to the Hb concentration. Immobilization of the mediator within the electrode body allows reagentless electrochemical measuring of Hb. On the other hand, entrapment of the protein within layers of polyelectrolytes, lipids, nanoparticles of clay or gold leads to a fast heterogeneous electron exchange of the partially denatured Hb.
Electrochemistry↗
Affinity interactions between phenylboronic acid-carrying self-assembled monolayers and flavin adenine dinucleotide or horseradish peroxidase.
A method is provided for the recognition of glycated molecules based on their binding affinities to boronate-carrying monolayers. The affinity interaction of flavin adenine dinucleotide (FAD) and horseradish peroxidase (HRP) with phenylboronic acid monolayers on gold was investigated by using voltammetric and microgravimetric methods. Conjugates of 3-aminophenylboronic acid and 3,3'-dithiodipropionic acid di(N-hydroxysuccinimide ester) or 11-mercaptoundecanoic acid were prepared and self-assembled on gold surfaces to generate monolayers. FAD is bound to this modified surface and recognized by a pair of redox peaks with a formal potential of -0.433 V in a 0.1 M phosphate buffer solution, pH 6.5. Upon addition of a sugar to the buffer, the bound FAD could be replaced, indicating that the binding is reversible. Voltammetric, mass measurements, and photometric activity assays show that the HRP can also be bound to the interface. This binding is reversible, and HRP can be replaced by sorbitol or removed in acidic solution. The effects of pH, incubation time, and concentration of H(2)O(2) were studied by comparing the catalytic reduction of H(2)O(2) in the presence of the electron-donor thionine. The catalytic current of the HRP-loaded electrode was proportional to HRP concentrations in the incubation solution in the range between 5 microg mL(-1) and 0.1 mg mL(-1) with a linear slope of 3.34 microA mL mg(-1) and a correlation coefficient of 0.9945.
Boronic Acids↗
Cytochrome P450 biosensors-a review.
Cytochrome P450 (CYP) is a large family of enzymes containing heme as the active site. Since their discovery and the elucidation of their structure, they have attracted the interest of scientist for many years, particularly due to their catalytic abilities. Since the late 1970s attempts have concentrated on the construction and development of electrochemical sensors. Although sensors based on mediated electron transfer have also been constructed, the direct electron transfer approach has attracted most of the interest. This has enabled the investigation of the electrochemical properties of the various isoforms of CYP. Furthermore, CYP utilized to construct biosensors for the determination of substrates important in environmental monitoring, pharmaceutical industry and clinical practice.
Biosensing Techniques↗
Direct electron transfer of cytochrome P450 2B4 at electrodes modified with nonionic detergent and colloidal clay nanoparticles.
A method for construction of biosensors with membranous cytochrome P450 isoenzymes was developed based on clay/detergent/protein mixed films. Thin films of sodium montmorillonite colloid with incorporated cytochrome P450 2B4 (CYP2B4) with nonionic detergent were prepared on glassy carbon electrodes. The modified electrodes were electrochemically characterized, and bioelectrocatalytic reactions were followed. CYP2B4 can be reduced fast on clay-modified glassy carbon electrodes in the presence of the nonionic detergent Tween 80. In anaerobic solutions, reversible oxidation and reduction is obtained with a formal potential between -0.292 and -0.305 V vs Ag/AgCl 1 M KCl depending on the preparation of the biosensor. In air-saturated solution, bioelectrocatalytic reduction currents can be obtained with the CYP2B4-modified electrode on addition of typical substrates such as aminopyrine and benzphetamine. This reaction was suppressed when methyrapone, an inhibitor of P450 reactions, was present. Measurement of product formation also indicates the bioelectrocatalysis by CYP2B4.
Aluminum Silicates↗
Molecular recognition of cocaine by acetylcholinesterases for affinity purification and bio-sensing.
Cholinesterases can be used as sensitive biorecognition elements for widely used agricultural pesticides. This requires highly purified and inhibitor-free enzyme preparations. In the present work the cocaine derivative benzoylecgonine was for the first time used as the molecular recognition element for the purification of acetylcholinesterase from Electrophorus electricus by affinity chromatography. The preparation of enriched enzyme without the contamination by an inhibitor, which is traditionally used for eluting the "affinity" bound protein, was achieved. The specific activity was 2.2-fold increased to 3100 Umg(-1). The same cocaine derivative was immobilized on the surface of a piezoelectric crystal in order to analyze the binding of acetylcholinesterases from two different species, E. electricus and Drosophila melanogaster, to the immobilized inhibitor. Evaluation of the binding curves allowed the analysis of the binding kinetics. These experiments are fundamental for the development of a (competitive) biosensor for inhibitors of cholinesterase.
Acetylcholinesterase↗
Detection of subpicomolar concentrations of human matrix metalloproteinase-2 by an optical biosensor.
We describe in this paper the development of a one-step sandwich assay for the highly sensitive and fast detection of human matrix metalloproteinase (MMP)-2 (EC 3.4.24.24), using surface plasmon resonance (SPR). For the assay, two ligands were selected: monoclonal anti-MMP-2 antibody Ab-2 and the tissue inhibitor of metalloproteinases (TIMP)-2. They were chosen on the basis of (1) their affinities to MMP-2, (2) the efficiency of immobilization to the sensor chip, (3) the efficiency of adsorption to colloidal gold, and (4) the stability of these protein-coated gold particles. The assay included mixing of MMP-2 with antibody Ab-2 adsorbed to colloidal gold with a diameter of about 20nm and injection into the flowcell of the SPR instrument containing immobilized TIMP-2. By using colloidal gold particles an amplification factor of 114 and a detection limit of 0.5pM for MMP-2 were obtained. The precision of the assay was high even at low analyte concentrations, the standard deviation being 8.3% for five determinations of 1pM MMP-2. No significant binding was observed with the structurally related MMP-9. The assay is far more sensitive and faster than commonly used methods for MMP-2 detection. As TIMP-bound MMP-2 is not detected by this method, the assay can be applied for measuring free MMP-2, reflecting the imbalance of free and inhibitor-bound enzyme in various pathological situations.
Antibodies↗
Electroactive cytochrome c multilayers within a polyelectrolyte assembly.
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Biosensing Techniques↗
A superoxide sensor based on a multilayer cytochrome c electrode.
A novel multilayer cytochrome c electrode for the quantification of superoxide radical concentrations is introduced. The electrode consists of alternating layers of cytochrome c and poly(aniline(sulfonic acid)) on a gold wire electrode. The formation of multilayer structures was proven by SPR experiments. Assemblies with 2-15 protein layers showed electrochemical communication with the gold electrode. For every additional layer, a substantial increase in electrochemically active cytochrome c (cyt. c) was found. For electrodes of more than 10 layers, the increase was more than 1 order of magnitude as compared to monolayer electrode systems. Thermodynamic and kinetic parameters of the electrodes were characterized. The mechanism of electron transfer within the multilayer assembly was studied, with results suggesting a protein-protein electron-transfer model. Electrodes of 2-15 layers were applied to the in vitro quantification of enzymatically generated superoxide, showing superior sensitivity as compared to a monolayer-based sensor. An electrode with 6 cyt. c/PASA layers showed the highest sensitivity of the systems studied, giving an increase in sensitivity of half an order of magnitude versus the that of the monolayer electrode. The stability of the system was optimized using thermal treatment, resulting in no loss in sensor signal or protein loading after 10 successive measurements or 2 days of storage.
Animals↗
Spectroelectrochemistry of cytochrome P450cam.
The spectroelectrochemistry of camphor-bound cytochrome P450cam (P450cam) using gold electrodes is described. The electrodes were modified with either 4,4(')-dithiodipyridin or sodium dithionite. Electrolysis of P450cam was carried out when the enzyme was in solution, while at the same time UV-visible absorption spectra were recorded. Reversible oxidation and reduction could be observed with both 4,4(')-dithiodipyridin and dithionite modified electrodes. A formal potential (E(0')) of -373mV vs Ag/AgCl 1M KCl was determined. The spectra of P450cam complexed with either carbon monoxide or metyrapone, both being inhibitors of P450 catalysis, clearly indicated that the protein retained its native state in the electrochemical cell during electrolysis.
Camphor↗
Thick-film electrodes for measurement of superoxide and hydrogen peroxide based on direct protein-electrode contacts.
Cytochrome c was immobilized on screen-printed thick-film gold electrodes by a self-assembly approach using mixed monolayers of mercaptoundecanoic acid and mercaptoundecanol. Cyclic voltammetry revealed quasi-reversible electrochemical behavior of the covalently fixed protein with a formal potential of +10 mV vs. Ag/AgCl. Polarized at +150 mV vs. Ag/AgCl the electrode was found to be sensitive to superoxide radicals in the range 300-1200 nmol L(-1). Compared with metal needle electrodes sensitivity and reproducibility could be improved and combined with the easiness of preparation. This allows the fabrication of disposable sensors for nanomolar superoxide concentrations. By changing the electrode potential the sensor can be switched from response to superoxide radicals to hydrogen peroxide-another reactive oxygen species. H(2)O(2) sensitivity can be provided in the range 10-1000 micromol L(-1) which makes the electrode suitable for oxidative stress studies.
Animals↗
New principle of direct real-time monitoring of the interaction of cholinesterase and its inhibitors by piezolectric biosensor.
This paper describes a new method for the sensitive detection of cholinesterase inhibitors based on real-time monitoring using a piezoelectric biosensor. The cholinesterase inhibitor paraoxon was immobilized on the sensing surface via a chelate complex as the recognition element. At first, the conjugate of N-mercaptoundecanoic acid (MUA) with Nalpha,Nalpha-bis (carboxymethyl)-L-lysine (NTA-Lys) was chemisorbed to form a self-assembled monolayer on the surface of the gold electrode of the piezosensor. In the next step, paraoxon-spacer-hexahistidine conjugate was linked to the MUA-Lys-NTA layer via the chelate complex with Ni2+. The paraoxon-modified surface thus obtained was applied for the binding of human butyrylcholinesterase (BChE). Regeneration of the sensing surface was achieved by splitting the chelate complex with EDTA and depositing a fresh layer of Ni2+ followed by addition of the paraoxon-spacer-hexahistidine. In the presence of free inhibitors like diisopropylfluorophosphate (DFP), binding of BChE to the surface-bound paraoxon was decreased. In this way, a competitive affinity assay for organophosphorus compounds was developed. The limit of detection for DFP as a model compound was 10 nmol/l (ca. 2 microg/l). This new concept seems suitable for constructing biosensors for the group-specific detection of cholinesterase-inhibiting substances like insecticides in the field.
Biosensing Techniques↗
An amperometric bi-enzyme sensor for determination of formate using cofactor regeneration.
A biosensor for detection of formate at submicromolar concentrations has been developed by co-immobilizing formate dehydrogenase (FDH, E.C. 1.2.1.2), salicylate hydroxylase (SHL, E.C. 1.14.13.1) and NAD(+) linked to polyethylene glycol (PEG-NAD(+)) in a poly(vinyl alcohol) (PVA) matrix in front of a Clark-electrode. The principle of the bi-enzyme scheme is as follows: formate dehydrogenase converts formate into carbon dioxide using PEG-NAD(+). Corresponding PEG-NADH produced is then oxidized to PEG-NAD(+) by salicylate hydroxylase using sodium salicylate and oxygen. The oxygen consumption is monitored with the Clark-electrode. The advantages of this biosensor approach are the effective re-oxidation of PEG-NADH, and the entrapment of PEG-NAD(+) resulting in avoiding the addition of expensive cofactor to the working medium for each measurement. This bi-enzyme sensor has achieved a linear range of 1-300 microM and a detection limit of 1.98 x 10(-7) M for formate (S/N=3), with the response time of 4 min. The working stability is limited to 7 days due to the inactivation of the enzymes. Only sodium salicylate was needed in milli-molar amounts.
Biosensing Techniques↗
Detection of progesterone in whole blood samples.
The progesterone concentration in blood samples can be utilised as a marker for the diagnosis of early pregnancy, endocrinopathy and virilism. Here, we describe a method for progesterone detection and measurement in whole blood samples by a surface sensitive biosensor used in conjunction with an integrated optical grating coupler. This device determines refractive index changes near the biosensor's surface. Hence, biological species bound to a surface layer can be measured in real-time without any label. For the measurements, we have modified the indirect competitive immunoassay principle. The concentration of the progesterone antibody was kept at 1 microg/ml. Progesterone concentration was determined in buffer solution and whole blood in a range between 0.005 and 10 ng/ml. The detection limit was determined to be 3 pM. The relative standard deviation was calculated to be 3.5%.
Antigen-Antibody Complex↗
Activation of cellulose membranes with 1,1'-carbonyldiimidazole or 1-cyano-4-dimethylaminopyridinium tetrafluoroborate as a basis for the development of immunosensors.
Methods for the activation of a cellulose dialysis membrane for immunosensor applications have been developed. For activation two reagents, 1,1'-carbonyldiimidazole (CDI) and 1-cyano-4-dimethylaminopyridinium tetrafluoroborate (CDAP), were compared with respect to the coupling efficiency for glucose oxidase (GOx) and 1,8-diamino-2,6-dioxaoctane. The maximum level of activation was 2.4 micromol cm(-2) for CDI and 0.2 micromol cm(-2) for CDAP activation. We observed 1.5 microg cm(-2) and 0.4 x 10(-4) U cm(-2) GOx with CDI-activated membranes whereas 1.7 microg cm(-2) and 7.2 x 10(-4) U cm(-2) GOx were observed with CDAP-activated membranes. With 1,8-diamino-2,6-dioxaoctane amino group densities of 0.165 and 0.09 micromol cm(-2) were observed via CDI and CDAP activation, respectively. An amino-modified membrane was used for coupling a ligand (pentapeptide) and an immunoenzymometric assay for hemoglobin A1c was carried out.
Affinity Labels↗
In vivo measurement of oxygen-derived free radicals during reperfusion injury.
By use of an optimized cytochrome c-based biosensor, superoxide radical production was measured continuously in vivo. The aim of this study was the online detection of superoxide concentration during reperfusion after a variable time of ischemia. Measurements were performed by placing the detecting sensor into gastrocnemius muscle tissue. Ischemia was induced by clamping the vena and arteria femoralis. Current response of the sensor was recorded continuously as an equivalent for superoxide concentration. Ischemia times varied from 5 to 120 minutes. The minimum ischemia time to record superoxide production was 10 minutes. By inducing longer periods of ischemia, an increase in superoxide concentration reached its highest levels at 2 hours. Furthermore, the total time of superoxide production after reperfusion depended on the total time of ischemia.
Animals↗
Highly sensitive detection of cocaine using a piezoelectric immunosensor.
This paper describes the development of a highly sensitive competitive immunoassay with the piezoelectric sensor. The immobilized derivative of cocaine was benzoylecgonine-1,8-diamino-3,4-dioxaoctane (BZE-DADOO). For the immobilization of BZE-DADOO, the conjugate BZE-DADOO with 11-mercaptomonoundecanoic acid (MUA) was synthesized via 2-(5-norbornen-2,3-dicarboximide)-1,1,3,3-tetramethyluronium-tetrafluoroborate (TNTU), followed by the creation of the conjugate monolayer on the piezosensor electrodes. For the optimization of the competitive assay we used electrodes with rough or smooth gold areas and for the interaction with immobilized antigen different anti-cocaine sheep polyclonal (pAb, either whole IgG or Fab fragment) and mouse monoclonal (mAb, whole IgG) antibodies. The assay of cocaine developed achieved a detection limit (LOD) of 100 pmol/l (34 ng/l) using the sheep antibody (IgG) and piezoelectric sensors with a smooth gold surface. The total time of one analysis was 15 min and the measuring area of the sensor could be used more than 40 times without losing its sensitivity.
Antibodies, Monoclonal↗