Publications by authors named "Frieder W Scheller"

64 Publications

How Reliable Is the Electrochemical Readout of MIP Sensors?

Sensors (Basel) 2020 May 8;20(9). Epub 2020 May 8.

Institute of Biochemistry and Biology, University of Potsdam, Karl-Liebknecht-Strasse 24-25, 14476 Potsdam, Germany.

Electrochemical methods offer the simple characterization of the synthesis of molecularly imprinted polymers (MIPs) and the readouts of target binding. The binding of electroinactive analytes can be detected indirectly by their modulating effect on the diffusional permeability of a redox marker through thin MIP films. However, this process generates an overall signal, which may include nonspecific interactions with the nonimprinted surface and adsorption at the electrode surface in addition to (specific) binding to the cavities. Redox-active low-molecular-weight targets and metalloproteins enable a more specific direct quantification of their binding to MIPs by measuring the faradaic current. The in situ characterization of enzymes, MIP-based mimics of redox enzymes or enzyme-labeled targets, is based on the indication of an electroactive product. This approach allows the determination of both the activity of the bio(mimetic) catalyst and of the substrate concentration.
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http://dx.doi.org/10.3390/s20092677DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7248831PMC
May 2020

Electrochemical MIP Sensor for Butyrylcholinesterase.

Polymers (Basel) 2019 Nov 30;11(12). Epub 2019 Nov 30.

Institute of Biochemistry and Biology, University of Potsdam, Karl-Liebknecht-Strasse 24-25, 14476 Potsdam, Germany.

Molecularly imprinted polymers (MIPs) mimic the binding sites of antibodies by substituting the amino acid-scaffold of proteins by synthetic polymers. In this work, the first MIP for the recognition of the diagnostically relevant enzyme butyrylcholinesterase (BuChE) is presented. The MIP was prepared using electropolymerization of the functional monomer o-phenylenediamine and was deposited as a thin film on a glassy carbon electrode by oxidative potentiodynamic polymerization. Rebinding and removal of the template were detected by cyclic voltammetry using ferricyanide as a redox marker. Furthermore, the enzymatic activity of BuChE rebound to the MIP was measured via the anodic oxidation of thiocholine, the reaction product of butyrylthiocholine. The response was linear between 50 pM and 2 nM concentrations of BuChE with a detection limit of 14.7 pM. In addition to the high sensitivity for BuChE, the sensor responded towards pseudo-irreversible inhibitors in the lower mM range.
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http://dx.doi.org/10.3390/polym11121970DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6960762PMC
November 2019

Electrosynthesized MIPs for transferrin: Plastibodies or nano-filters?

Biosens Bioelectron 2018 May 9;105:29-35. Epub 2018 Jan 9.

Institute of Biochemistry and Biology, University of Potsdam, Karl-Liebknecht Str. 24-25, 14476 Potsdam, Germany. Electronic address:

Molecularly imprinted polymer (MIP) nanofilms for transferrin (Trf) have been synthesized on gold surfaces by electro-polymerizing the functional monomer scopoletin in the presence of the protein target or around pre-adsorbed Trf. As determined by atomic force microscopy (AFM) the film thickness was comparable with the molecular dimension of the target. The target (re)binding properties of the electro-synthesized MIP films was evaluated by cyclic voltammetry (CV) and square wave voltammetry (SWV) through the target-binding induced permeability changes of the MIP nanofilms to the ferricyanide redox marker, as well as by surface plasmon resonance (SPR) and surface enhanced infrared absorption spectroscopy (SEIRAS) of the immobilized protein molecules. For Trf a linear concentration dependence in the lower micromolar range and an imprinting factor of ~5 was obtained by SWV and SPR. Furthermore, non-target proteins including the iron-free apo-Trf were discriminated by pronounced size and shape specificity. Whilst it is generally assumed that the rebinding of the target or of cross-reacting proteins exclusively takes place at the polymer here we considered also the interaction of the protein molecules with the underlying gold transducers. We demonstrate by SWV that adsorption of proteins suppresses the signal of the redox marker even at the bare gold surface and by SEIRAS that the treatment of the MIP with proteinase K or NaOH only partially removes the target protein. Therefore, we conclude that when interpreting binding of proteins to directly MIP-covered gold electrodes the interactions between the protein and the gold surface should also be considered.
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http://dx.doi.org/10.1016/j.bios.2018.01.011DOI Listing
May 2018

Electrochemical MIP-Sensors for Drugs.

Curr Med Chem 2018 ;25(33):4007-4019

University of Potsdam, Institute of Biochemistry and Biology, Karl-Liebknecht- Strasse 25-26,14476 Potsdam, Germany.

In order to replace bio-macromolecules by stable synthetic materials in separation techniques and bioanalysis biomimetic receptors and catalysts have been developed: Functional monomers are polymerized together with the target analyte and after template removal cavities are formed in the "molecularly imprinted polymer" (MIP) which resemble the active sites of antibodies and enzymes. Starting almost 80 years ago, around 1,100 papers on MIPs were published in 2016. Electropolymerization allows to deposit MIPs directly on voltammetric electrodes or chips for quartz crystal microbalance (QCM) and surface plasmon resonance (SPR). For the readout of MIPs for drugs amperometry, differential pulse voltammetry (DPV) and impedance spectroscopy (EIS) offer higher sensitivity as compared with QCM or SPR. Application of simple electrochemical devices allows both the reproducible preparation of MIP sensors, but also the sensitive signal generation. Electrochemical MIP-sensors for the whole arsenal of drugs, e.g. the most frequently used analgesics, antibiotics and anticancer drugs have been presented in literature and tested under laboratory conditions. These biomimetic sensors typically have measuring ranges covering the lower nano- up to millimolar concentration range and they are stable under extreme pH and in organic solvents like nonaqueous extracts.
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http://dx.doi.org/10.2174/0929867324666171005103712DOI Listing
November 2018

Electrosynthesized molecularly imprinted polyscopoletin nanofilms for human serum albumin detection.

Anal Chim Acta 2017 07 5;977:1-9. Epub 2017 May 5.

MTA-BME "Lendület" Chemical Nanosensors Research Group, Department of Inorganic and Analytical Chemistry, Budapest University of Technology and Economics, Szent Gellért tér 4, H-1111 Budapest, Hungary. Electronic address:

Molecularly imprinted polymers (MIPs) rendered selective solely by the imprinting with protein templates lacking of distinctive properties to facilitate strong target-MIP interaction are likely to exhibit medium to low template binding affinities. While this prohibits the use of such MIPs for applications requiring the assessment of very low template concentrations, their implementation for the quantification of high-abundance proteins seems to have a clear niche in the analytical practice. We investigated this opportunity by developing a polyscopoletin-based MIP nanofilm for the electrochemical determination of elevated human serum albumin (HSA) in urine. As reference for a low abundance protein ferritin-MIPs were also prepared by the same procedure. Under optimal conditions, the imprinted sensors gave a linear response to HSA in the concentration range of 20-100 mg/dm, and to ferritin in the range of 120-360 mg/dm. While as expected the obtained limit of detection was not sufficient to determine endogenous ferritin in plasma, the HSA-sensor was successfully employed to analyse urine samples of patients with albuminuria. The results suggest that MIP-based sensors may be applicable for quantifying high abundance proteins in a clinical setting.
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http://dx.doi.org/10.1016/j.aca.2017.04.043DOI Listing
July 2017

MIPs and Aptamers for Recognition of Proteins in Biomimetic Sensing.

Biosensors (Basel) 2016 Jul 18;6(3). Epub 2016 Jul 18.

Fraunhofer Institute for Cell Therapy and Immunology, Branch Bioanalytics and Bioprocesses (IZI-BB), Am Mühlenberg 13, Potsdam D-14476, Germany.

Biomimetic binders and catalysts have been generated in order to substitute the biological pendants in separation techniques and bioanalysis. The two major approaches use either "evolution in the test tube" of nucleotides for the preparation of aptamers or total chemical synthesis for molecularly imprinted polymers (MIPs). The reproducible production of aptamers is a clear advantage, whilst the preparation of MIPs typically leads to a population of polymers with different binding sites. The realization of binding sites in the total bulk of the MIPs results in a higher binding capacity, however, on the expense of the accessibility and exchange rate. Furthermore, the readout of the bound analyte is easier for aptamers since the integration of signal generating labels is well established. On the other hand, the overall negative charge of the nucleotides makes aptamers prone to non-specific adsorption of positively charged constituents of the sample and the "biological" degradation of non-modified aptamers and ionic strength-dependent changes of conformation may be challenging in some application.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5039654PMC
http://dx.doi.org/10.3390/bios6030035DOI Listing
July 2016

Molecularly Imprinted Electropolymer for a Hexameric Heme Protein with Direct Electron Transfer and Peroxide Electrocatalysis.

Sensors (Basel) 2016 Feb 23;16(3):272. Epub 2016 Feb 23.

Institute of Biochemistry and Biology, Potsdam University, Karl-Liebknecht-Strasse 24-25, 14476 Potsdam, Germany.

For the first time a molecularly imprinted polymer (MIP) with direct electron transfer (DET) and bioelectrocatalytic activity of the target protein is presented. Thin films of MIPs for the recognition of a hexameric tyrosine-coordinated heme protein (HTHP) have been prepared by electropolymerization of scopoletin after oriented assembly of HTHP on a self-assembled monolayer (SAM) of mercaptoundecanoic acid (MUA) on gold electrodes. Cavities which should resemble the shape and size of HTHP were formed by template removal. Rebinding of the target protein sums up the recognition by non-covalent interactions between the protein and the MIP with the electrostatic attraction of the protein by the SAM. HTHP bound to the MIP exhibits quasi-reversible DET which is reflected by a pair of well pronounced redox peaks in the cyclic voltammograms (CVs) with a formal potential of -184.4 ± 13.7 mV vs. Ag/AgCl (1 M KCl) at pH 8.0 and it was able to catalyze the cathodic reduction of peroxide. At saturation the MIP films show a 12-fold higher electroactive surface concentration of HTHP than the non-imprinted polymer (NIP).
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http://dx.doi.org/10.3390/s16030272DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4813847PMC
February 2016

Microelectrospotting as a new method for electrosynthesis of surface-imprinted polymer microarrays for protein recognition.

Biosens Bioelectron 2015 Nov 27;73:123-129. Epub 2015 May 27.

Department of Inorganic and Analytical Chemistry, Budapest University of Technology and Economics, Szent Gellért tér 4, H-1111 Budapest, Hungary; MTA-BME "Lendület" Chemical Nanosensors Research Group, Department of Inorganic and Analytical Chemistry, Budapest University of Technology and Economics, Szent Gellért tér 4, H-1111 Budapest, Hungary. Electronic address:

Here we introduce microelectrospotting as a new approach for preparation of protein-selective molecularly imprinted polymer microarrays on bare gold SPR imaging chips. During electrospotting both the gold chip and the spotting tip are electrically connected to a potentiostat as working and counter electrodes, respectively. The spotting pin encloses the monomer-template protein cocktail that upon contacting the gold surface is in-situ electropolymerized resulting in surface confined polymer spots of ca. 500 µm diameter. By repeating this procedure at preprogrammed locations for various composition monomer-template mixtures microarrays of nanometer-thin surface-imprinted films are generated in a controlled manner. We show that the removal and rebinding kinetics of the template and various potential interferents to such microarrays can be monitored in real-time and multiplexed manner by SPR imaging. The proof of principle for microelectrospotting of electrically insulating surface-imprinted films is made by using scopoletin as monomer and ferritin as protein template. It is shown that microelectrospotting in combination with SPR imaging can offer a versatile platform for label-free and enhanced throughput optimization of the molecularly imprinted polymers for protein recognition and for their analytical application.
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http://dx.doi.org/10.1016/j.bios.2015.05.049DOI Listing
November 2015

Surface-tuned electron transfer and electrocatalysis of hexameric tyrosine-coordinated heme protein.

Chemistry 2015 May 30;21(20):7596-602. Epub 2015 Mar 30.

Institute of Biochemistry and Biology, Potsdam University, Karl-Liebknecht-Strasse 24-25, 14476 Potsdam (Germany), Fax: (+49) 331 977-5050.

Molecular modeling, electrochemical methods, and quartz crystal microbalance were used to characterize immobilized hexameric tyrosine-coordinated heme protein (HTHP) on bare carbon or on gold electrodes modified with positively and negatively charged self-assembled monolayers (SAMs), respectively. HTHP binds to the positively charged surface but no direct electron transfer (DET) is found due to the long distance of the active sites from the electrode surfaces. At carboxyl-terminated surfaces, the neutrally charged bottom of HTHP can bind to the SAM. For this "disc" orientation all six hemes are close to the electrode and their direct electron transfer should be efficient. HTHP on all negatively charged SAMs showed a quasi-reversible redox behavior with rate constant ks values between 0.93 and 2.86 s(-1) and apparent formal potentials ${E{{0{^{\prime }}\hfill \atop {\rm app}\hfill}}}$ between -131.1 and -249.1 mV. On the MUA/MU-modified electrode, the maximum surface concentration corresponds to a complete monolayer of the hexameric HTHP in the disc orientation. HTHP electrostatically immobilized on negatively charged SAMs shows electrocatalysis of peroxide reduction and enzymatic oxidation of NADH.
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http://dx.doi.org/10.1002/chem.201405932DOI Listing
May 2015

Carboxylated or aminated polyaniline-multiwalled carbon nanotubes nanohybrids for immobilization of cellobiose dehydrogenase on gold electrodes.

Biosensors (Basel) 2014 Dec 22;4(4):370-86. Epub 2014 Oct 22.

Fraunhofer Institute for Cell Therapy and Immunology (IZI-BB), Branch Bioanalytics and Bioprocesses Potsdam-Golm, Am Mühlenberg 13, 14476 Potsdam, Germany; E-Mail:

Polymer-multiwalled carbon nanotube (MWCNT) nanohybrids, which differ in surface charge have been synthesized to study the bioelectrocatalysis of adsorbed cellobiose dehydrogenase (CDH) from Phanerochaete sordida on gold electrodes. To obtain negatively charged nanohybrids, poly(3-amino-4-methoxybenzoic acid-co-aniline) (P(AMB-A)) was covalently linked to the surface of MWCNTs while modification with p-phenylenediamine (PDA) converted the COOH-groups to positively charged amino groups. Fourier transform infrared spectroscopy (FTIR) measurements verified the p-phenylenediamine (PDA) modification of the polymer-CNT nanohybrids. The positively charged nanohybrid MWCNT-P(AMB-A)-PDA promoted direct electron transfer (DET) of CDH to the electrode and bioelectrocatalysis of lactose was observed. Amperometric measurements gave an electrochemical response with KMapp = 8.89 mM and a current density of 410 nA/cm(2) (15 mM lactose). The catalytic response was tested at pH 3.5 and 4.5. Interference by ascorbic acid was not observed. The study proves that DET between the MWCNT-P(AMB-A)-PDA nanohybrids and CDH is efficient and allows the sensorial detection of lactose.
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http://dx.doi.org/10.3390/bios4040370DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4287708PMC
December 2014

The first electrochemical MIP sensor for tamoxifen.

Sensors (Basel) 2014 Apr 25;14(5):7647-54. Epub 2014 Apr 25.

Fraunhofer Institute for Biomedical Engineering IBMT, Am Mühlenberg 13, 14476 Potsdam, Germany.

We present an electrochemical MIP sensor for tamoxifen (TAM)-a nonsteroidal anti-estrogen-which is based on the electropolymerisation of an O-phenylenediamine‒resorcinol mixture directly on the electrode surface in the presence of the template molecule. Up to now only "bulk" MIPs for TAM have been described in literature, which are applied for separation in chromatography columns. Electro-polymerisation of the monomers in the presence of TAM generated a film which completely suppressed the reduction of ferricyanide. Removal of the template gave a markedly increased ferricyanide signal, which was again suppressed after rebinding as expected for filling of the cavities by target binding. The decrease of the ferricyanide peak of the MIP electrode depended linearly on the TAM concentration between 1 and 100 nM. The TAM-imprinted electrode showed a 2.3 times higher recognition of the template molecule itself as compared to its metabolite 4-hydroxytamoxifen and no cross-reactivity with the anticancer drug doxorubucin was found. Measurements at +1.1 V caused a fouling of the electrode surface, whilst pretreatment of TAM with peroxide in presence of HRP generated an oxidation product which was reducible at 0 mV, thus circumventing the polymer formation and electrochemical interferences.
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http://dx.doi.org/10.3390/s140507647DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4063000PMC
April 2014

Electrochemical displacement sensor based on ferrocene boronic acid tracer and immobilized glycan for saccharide binding proteins and E. coli.

Biosens Bioelectron 2014 Aug 19;58:1-8. Epub 2014 Feb 19.

Institute of Biochemistry and Biology, University of Potsdam, Karl-Liebknecht-Straße 24-25, 14476 Potsdam, Germany. Electronic address:

Pathogens such as viruses and bacteria use their envelope proteins and their adhesin lectins to recognize the glycan residues presented on the cell surface of the target tissues. This principle of recognition is used in a new electrochemical displacement sensor for the protein concanavalin A (ConA). A gold electrode was first modified with a self-assembled monolayer of a thiolated mannose/OEG conjugate and a ferrocene boroxol derivative was pre-assembled as reporter molecule onto the mannose surface. The novel tracer molecule based on a 2-hydroxymethyl phenyl boronic acid derivative binds even at neutral pH to the saccharides which could expand the application towards biological samples (i.e., urine and feces). Upon the binding of ConA, the tracer was displaced and washed away from the sensor surface leading to a decrease in the electrochemical signal. Using square wave voltammetry (SWV), the concentration of ConA in the sample solution could be determined in the dynamic concentration range established from 38nmolL(-1) to 5.76µmolL(-1) with a reproducible detection limit of 1µgmL(-1) (38nmolL(-1)) based on the signal-to-noise ratio (S/N=3) with fast response of 15min. The new reporter molecule showed a reduced non-specific displacement by BSA and ribonuclease A. The sensor was also successfully transferred to the first proof of principle for the detection of Escherichia coli exhibiting a detection limit of approximately 6×10(2)cells/mL. Specificity of the displacement by target protein ConA and E. coli was demonstrated since the control proteins (i.e., BSA and RNaseA) and the control E. coli strain, which lack of type 1 fimbriae, were ineffective.
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http://dx.doi.org/10.1016/j.bios.2014.02.028DOI Listing
August 2014

Future of biosensors: a personal view.

Adv Biochem Eng Biotechnol 2014 ;140:1-28

Fraunhofer Institute for Biomedical Engineering IBMT, 14476, Potsdam, Germany,

Biosensors representing the technological counterpart of living senses have found routine application in amperometric enzyme electrodes for decentralized blood glucose measurement, interaction analysis by surface plasmon resonance in drug development, and to some extent DNA chips for expression analysis and enzyme polymorphisms. These technologies have already reached a highly advanced level and need minor improvement at most. The dream of the "100-dollar" personal genome may come true in the next few years provided that the technological hurdles of nanopore technology or of polymerase-based single molecule sequencing can be overcome. Tailor-made recognition elements for biosensors including membrane-bound enzymes and receptors will be prepared by cell-free protein synthesis. As alternatives for biological recognition elements, molecularly imprinted polymers (MIPs) have been created. They have the potential to substitute antibodies in biosensors and biochips for the measurement of low-molecular-weight substances, proteins, viruses, and living cells. They are more stable than proteins and can be produced in large amounts by chemical synthesis. Integration of nanomaterials, especially of graphene, could lead to new miniaturized biosensors with high sensitivity and ultrafast response. In the future individual therapy will include genetic profiling of isoenzymes and polymorphic forms of drug-metabolizing enzymes especially of the cytochrome P450 family. For defining the pharmacokinetics including the clearance of a given genotype enzyme electrodes will be a useful tool. For decentralized online patient control or the integration into everyday "consumables" such as drinking water, foods, hygienic articles, clothing, or for control of air conditioners in buildings and cars and swimming pools, a new generation of "autonomous" biosensors will emerge.
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http://dx.doi.org/10.1007/10_2013_251DOI Listing
June 2014

Coupling biocatalysis with molecular imprinting in a biomimetic sensor.

Angew Chem Int Ed Engl 2013 Oct 5;52(44):11521-5. Epub 2013 Sep 5.

Fraunhofer Institute for Biomedical Engineering, Am Mühlenberg 13, 14476 Potsdam (Germany); Institute of Biochemistry and Biology, University of Potsdam, Karl-Liebknecht-Strasse 24-25, 14476 Potsdam (Germany).

Make it simple: A molecularly imprinted electropolymer was combined with an enzyme in a catalytic biomimetic sensor that enabled interference-free detection of the drug aminopyrine (AP) at submicromolar concentrations in the presence of ascorbic acid and uric acid within 15 s. The sensor functioned by the peroxide-dependent conversion of AP in a layer above a product-imprinted electropolymer on an indicator electrode.
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http://dx.doi.org/10.1002/anie.201305368DOI Listing
October 2013

Modulation of direct electron transfer of cytochrome c by use of a molecularly imprinted thin film.

Anal Bioanal Chem 2013 Aug 10;405(20):6437-44. Epub 2013 May 10.

Institute of Biochemistry and Biology, University of Potsdam, Golm, Germany.

We describe the preparation of a molecularly imprinted polymer film (MIP) on top of a self-assembled monolayer (SAM) of mercaptoundecanoic acid (MUA) on gold, where the template cytochrome c (cyt c) participates in direct electron transfer (DET) with the underlying electrode. To enable DET, a non-conductive polymer film is electrodeposited from an aqueous solution of scopoletin and cyt c on to the surface of a gold electrode previously modified with MUA. The electroactive surface concentration of cyt c was 0.5 pmol cm(-2). In the absence of the MUA layer, no cyt c DET was observed and the pseudo-peroxidatic activity of the scopoletin-entrapped protein, assessed via oxidation of Ampliflu red in the presence of hydrogen peroxide, was only 30% of that for the MIP on MUA. This result indicates that electrostatic adsorption of cyt c by the MUA-SAM substantially increases the surface concentration of cyt c during the electrodeposition step, and is a prerequisite for the productive orientation required for DET. After template removal by treatment with sulfuric acid, rebinding of cyt c to the MUA-MIP-modified electrode occurred with an affinity constant of 100,000 mol(-1) L, a value three times higher than that determined by use of fluorescence titration for the interaction between scopoletin and cyt c in solution. The DET of cyt c in the presence of myoglobin, lysozyme, and bovine serum albumin (BSA) reveals that the MIP layer suppresses the effect of competing proteins.
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http://dx.doi.org/10.1007/s00216-013-7009-8DOI Listing
August 2013

Peroxide-dependent analyte conversion by the heme prosthetic group, the heme Peptide "microperoxidase-11" and cytochrome C on chitosan capped gold nanoparticles modified electrodes.

Biosensors (Basel) 2012 May 14;2(2):189-204. Epub 2012 May 14.

Fraunhofer Institute for Biomedical Engineering, IBMT, D-14476 Potsdam, Germany.

In view of the role ascribed to the peroxidatic activity of degradation products of cytochrome c (cyt c) in the processes of apoptosis, we investigate the catalytic potential of heme and of the cyt c derived heme peptide MP-11 to catalyse the cathodic reduction of hydrogen peroxide and to oxidize aromatic compounds. In order to check whether cyt c has an enzymatic activity in the native state where the protein matrix should suppress the inherent peroxidatic activity of its heme prosthetic group, we applied a biocompatible immobilization matrix and very low concentrations of the co-substrate H2O2. The biocatalysts were entrapped on the surface of a glassy carbon electrode in a biocompatible chitosan layer which contained gold nanoparticles. The electrochemical signal for the peroxide reduction is generated by the redox conversion of the heme group, whilst a reaction product of the substrate oxidation is cathodically reduced in the substrate indication. The catalytic efficiency of microperoxidase-11 is sufficient for sensors indicating HRP substrates, e.g., p-aminophenol, paracetamol and catechol, but also the hydroxylation of aniline and dehalogenation of 4-fluoroaniline. The lower limit of detection for p-aminophenol is comparable to previously published papers with different enzyme systems. The peroxidatic activity of cyt c immobilized in the chitosan layer for catechol was found to be below 1 per mill and for p-aminophenol about 3% as compared with that of heme or MP-11.
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http://dx.doi.org/10.3390/bios2020189DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4263574PMC
May 2012

Label-free detection of enhanced saccharide binding at pH 7.4 to nanoparticulate benzoboroxole based receptor units.

J Mol Recognit 2011 Nov-Dec;24(6):953-9

Fraunhofer Institute for Biomedical Engineering, Am Mühlenberg 13, 14476, Potsdam, Germany.

Nanoparticles modified with either 6-amino-1-hydroxy-2,1-benzoxaborolane (3-aminobenzoboroxole) or 3-aminophenylboronic acid were prepared by nucleophilic substitution of a styrene-co-DVB-co-vinylbenzylchloride latex (25 nm). Isothermal titration calorimetry (ITC) was used as a label-free detection method for the analysis of the binding between monosaccharides and these two differently derivatized nanoparticle systems at pH 7.4. Because ITC reveals, thermodynamical parameters such as the changes in enthalpy ΔH, free energy ΔG, and entropy ΔS, possible explanations for the higher binding constants can be derived in terms of entropy and enthalpy changes. In case of the modified nanoparticles, the free energy of binding is dominated by the entropy term. This shows that interfacial effects, besides the intrinsic affinity, lead to a higher binding constant compared with the free ligand. The highest binding constant was found for fructose binding to the benzoboroxole modified nanoparticles: Its value of 1150 M(-1) is twice as high as for the free benzoboroxole and five times as high as with phenylboronic acid or 3-aminophenylboronic acid. In contrast to the binding of fructose to free boronic acids, which is an enthalpically driven process, the binding of fructose to the modified nanoparticles is dominated by the positive entropy term.
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http://dx.doi.org/10.1002/jmr.1142DOI Listing
February 2012

The aromatic peroxygenase from Marasmius rutola--a new enzyme for biosensor applications.

Anal Bioanal Chem 2012 Jan 25;402(1):405-12. Epub 2011 Oct 25.

Fraunhofer Institute for Biomedical Engineering IBMT, Potsdam, Germany.

The aromatic peroxygenase (APO; EC 1.11.2.1) from the agraric basidomycete Marasmius rotula (MroAPO) immobilized at the chitosan-capped gold-nanoparticle-modified glassy carbon electrode displayed a pair of redox peaks with a midpoint potential of -278.5 mV vs. AgCl/AgCl (1 M KCl) for the Fe(2+)/Fe(3+) redox couple of the heme-thiolate-containing protein. MroAPO oxidizes aromatic substrates such as aniline, p-aminophenol, hydroquinone, resorcinol, catechol, and paracetamol by means of hydrogen peroxide. The substrate spectrum overlaps with those of cytochrome P450s and plant peroxidases which are relevant in environmental analysis and drug monitoring. In M. rotula peroxygenase-based enzyme electrodes, the signal is generated by the reduction of electrode-active reaction products (e.g., p-benzoquinone and p-quinoneimine) with electro-enzymatic recycling of the analyte. In these enzyme electrodes, the signal reflects the conversion of all substrates thus representing an overall parameter in complex media. The performance of these sensors and their further development are discussed.
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http://dx.doi.org/10.1007/s00216-011-5497-yDOI Listing
January 2012

Enzyme electrode for aromatic compounds exploiting the catalytic activities of microperoxidase-11.

Biosens Bioelectron 2011 Dec 16;30(1):320-3. Epub 2011 Sep 16.

Fraunhofer Institute for Biomedical Engineering IBMT, D-14476 Potsdam, Germany.

Microperoxidase-11 (MP-11) which has been immobilised in a matrix of chitosan-embedded gold nanoparticles on the surface of a glassy carbon electrode catalyzes the conversion of aromatic substances. This peroxide-dependent catalysis of microperoxidase has been applied in an enzyme electrode for the first time to indicate aromatic compounds such as aniline, 4-fluoroaniline, catechol and p-aminophenol. The electrode signal is generated by the cathodic reduction of the quinone or quinoneimine which is formed in the presence of both MP-11 and peroxide from the substrate. The same sensor principle will be extended to aromatic drugs.
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http://dx.doi.org/10.1016/j.bios.2011.09.004DOI Listing
December 2011

Thermometric sensing of nitrofurantoin by noncovalently imprinted polymers containing two complementary functional monomers.

Anal Chem 2011 Oct 29;83(20):7704-11. Epub 2011 Sep 29.

Fraunhofer Institute for Biomedical Engineering, Am Muehlenberg 13, Potsdam 14476, Germany.

Molecularly imprinted polymers (MIPs) for nitrofurantoin (NFT) recognition addressing in parallel of two complementary functional groups were created using a noncovalent imprinting approach. Specific tailor-made functional monomers were synthesized: a diaminopyridine derivative as the receptor for the imide residue and three (thio)urea derivatives for the interaction with the nitro group of NFT. A significantly improved binding of NFT to the new MIPs was revealed from the imprinting factor, efficiency of binding, affinity constants and maximum binding number as compared to previously reported MIPs, which addressed either the imide or the nitro residue. Substances possessing only one functionality (either the imide group or nitro group) showed significantly weaker binding to the new imprinted polymers than NFT. However, the compounds lacking both functionalities binds extremely weak to all imprinted polymers. The new imprinted polymers were applied in a flow-through thermistor in organic solvent for the first time. The MIP-thermistor allows the detection of NFT down to a concentration of 5 μM in acetonitrile + 0.2% dimethyl sulfoxide (DMSO). The imprinting factor of 3.91 at 0.1 mM of NFT as obtained by thermistor measurements is well comparable to the value obtained by batch binding experiments.
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http://dx.doi.org/10.1021/ac201099hDOI Listing
October 2011

Preparation and characterization of novel molecularly imprinted polymers based on thiourea receptors for nitrocompounds recognition.

Talanta 2011 Apr 13;84(2):274-9. Epub 2011 Jan 13.

Fraunhofer Institute for Biomedical Engineering, Am Muehlenberg 13, Potsdam 14476, Germany.

Molecularly imprinted polymers (MIPs) for the recognition of nitro derivatives are prepared from three different (thio)urea-bearing functional monomers. The binding capability of the polymers is characterized by a batch binding experiment. The imprinting factors and affinity constants (K) of the imprinted polymers exhibit the same tendency as the binding constants (K(a)) of the functional monomers to the target substance in solution. Not only nitrofurantoin is efficiently bound by these MIPs but also a broad spectrum of other nitro compounds is bound with at the intermediate level, addressing that these (thio)urea-based monomers can be utilized to prepare a family of MIPs for various nitro compounds, which can be applied as recognition elements in separation and analytical application.
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http://dx.doi.org/10.1016/j.talanta.2010.12.049DOI Listing
April 2011

Focus on bioanalysis.

Anal Bioanal Chem 2010 Nov;398(6):2337-9

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http://dx.doi.org/10.1007/s00216-010-4203-9DOI Listing
November 2010

Peroxygenase based sensor for aromatic compounds.

Biosens Bioelectron 2010 Dec 29;26(4):1432-6. Epub 2010 Jul 29.

Institute of Biochemistry and Biology, University of Potsdam, Karl-Liebknecht-Str. 24-25, 14476 Golm, Germany.

We report on the redox behaviour of the peroxygenase from Agrocybe aegerita (AaeAPO) which has been electrostatically immobilized in a matrix of chitosan-embedded gold nanoparticles on the surface of a glassy carbon electrode. AaeAPO contains a covalently bound heme-thiolate as the redox active group that exchanges directly electrons with the electrode via the gold nanoparticles. The formal potential E°' of AaeAPO in the gold nanoparticles-chitosan film was estimated to be -(286±9) mV at pH 7.0. The heterogeneous electron transfer rate constant (k(s)) increases from 3.7 in the scan rate range from 0.2 to 3.0 V s(-1) and level off at 63.7 s(-1). Furthermore, the peroxide-dependent hydroxylation of aromatic compounds was applied to develop a sensor for naphthalene and nitrophenol. The amperometric measurements of naphthalene are based on the indication of H(2)O(2) consumption. For the chitosan-embedded gold nanoparticle system, the linear range extends from 4 to 40 μM naphthalene with a detection limit of 4.0 μM (S/N=3) and repeatability of 5.7% for 40 μM naphthalene.
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http://dx.doi.org/10.1016/j.bios.2010.07.075DOI Listing
December 2010

Ionic topochemical tuned biosensor interface.

Langmuir 2010 Jun;26(11):9088-93

Fraunhofer Institute for Biomedical Engineering, Am Muhlenberg 13, 14476 Potsdam, Germany.

Two new hydrophilic, poly(ethylene glycol) (PEG)-based redox copolymers bearing electrochemically active ferrocene (Fc) and thiol/disulfide anchoring functionalities were synthesized. These copolymers are shown to adsorb on gold surfaces causing polymeric self-assembled monolayers (pSAMs) that possess triple functions: "redox-active", "ionic-tunable", and "bio-inert". Both immobilized polymers showed redox potentials at +400 mV (Ag|AgCl), and facilitate the electrocatalytical oxidation of NADH. Additionally, interfacial architecture of the polymers is affected by an increase in Ca(2+) concentration, which leads to an amplification of the electrochemical response. The electrode current, measured for NADH-oxidation, increased by 80% after addition of 10 mM Ca(2+) ions. Considering the Ca(2+) influence on the heterogeneous electron transfer a structural model of the immobilized polymers is proposed based on the strong chelating ability of noncyclic PEG moieties.
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http://dx.doi.org/10.1021/la9047215DOI Listing
June 2010

Development of molecularly imprinted polymers for the binding of nitrofurantoin.

Biosens Bioelectron 2009 Sep 10;25(1):82-7. Epub 2009 Jun 10.

Fraunhofer Institute for Biomedical Engineering, Am Muehlenberg 13, Potsdam 14476, Germany.

Novel molecularly imprinted polymers (MIPs) for the recognition of nitrofurantoin (NFT) were prepared by photoinitiated polymerisation in polar solvent using 2,6-bis(methacrylamido) pyridine (BMP) as the functional monomer and carboxyphenyl aminohydantoin (CPAH) as the analogue of the template. The binding constants of the complex between BMP and nitrofurantoin or CPAH in DMSO were determined with 1H NMR titration to be 630+/-104 and 830+/-146 M(-1), respectively. To study the influence of the functional monomer, two polymer compositions were prepared containing the template, the functional monomer and the crosslinker in the molar ratio 1:1:12 for MIP1 and 1:4:20 for MIP2, respectively. The imprinting factor at saturation concentration of nitrofurantoin, which is the ratio of the amount bound to the MIP and the non-imprinted control polymer (NIP), was determined to be 2.47 for MIP1 and 2.49 for MIP2. The cross reactivity of the imprinted polymers seems to be determined by the ability to form hydrogen bonds to the functional monomer while the shape of the molecule has no real influence.
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http://dx.doi.org/10.1016/j.bios.2009.06.003DOI Listing
September 2009

Direct electrochemistry and spectroelectrochemistry of osmium substituted horseradish peroxidase.

Bioelectrochemistry 2009 Sep 7;76(1-2):28-33. Epub 2009 Apr 7.

Department of Analytical Biochemistry, Institute of Biochemistry and Biology, University of Potsdam, Karl-Liebknecht-Strasse 24-25, 14476 Potsdam, Germany.

In this contribution the substitution of the central protoporphyrin IX iron complex of horseradish peroxidase by the respective osmium porphyrin complex is described. The direct electrochemical reduction of the Os containing horseradish peroxidase (OsHRP) was achieved at ITO and modified glassy carbon electrodes and in combination with spectroscopy revealed the three redox couples Os(III)HRP/Os(IV)HRP, Os(IV)HRP/Os(V)HRP and Os(V)HRP/Os(VI)HRP. The midpoint potentials differ dependent on the electrode material used with E(1/2) (Os(III/IV)) of -0.4 V (ITO) and -0.25 V (GC), E(1/2) (Os(IV)/(V)) of -0.16 V (ITO) and +0.10 V (GC), and E(1/2) (Os(V/VI))of +0.18 V (ITO), respectively. Moreover, with immobilised OsHRP the direct electrocatalytic reduction of hydrogen peroxide and tert-butyl hydroperoxide was observed. In comparison to electrodes modified with native HRP the sensitivity of the OsHRP-electrode for tert-butyl hydroperoxide is higher.
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http://dx.doi.org/10.1016/j.bioelechem.2009.03.015DOI Listing
September 2009

Self-assembly of electro-active protein architectures on electrodes for the construction of biomimetic signal chains.

Chem Commun (Camb) 2009 Jan 12(3):274-83. Epub 2008 Nov 12.

Wildau University of Applied Sciences, Biosystems Technology, Bahnhofstr. 1, 15745, Wildau, Germany.

The layer-by-layer adsorption technique based on the consecutive deposition of oppositely charged species is suitable for the preparation of protein multilayers with fully electro-active protein molecules. The methodology was established with cytochrome c and the polyelectrolyte sulfonated polyaniline (PASA). The technique is also useful for the construction of bi-protein architectures confining protein-protein communication to an electrode. Following natural examples of protein complexes with defined signal transfer, cytochrome c was arranged with enzymes such as xanthine oxidase, bilirubin oxidase, laccase, and sulfite oxidase in self-assembled multilayer architectures. Thus, biomimetic signal chains from the enzyme substrate via the enzyme and cytochrome c towards the electrode can be established. Communication between proteins immobilised in multiple layers on the electrode can be achieved by in situ generation of small shuttle molecules or more advantageously by direct interprotein electron transfer. This allows the construction of new sensing electrodes, the properties of which can be tuned by the number of deposited protein layers. The mechanism of electron transfer within such protein assemblies on gold electrodes will be discussed.
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http://dx.doi.org/10.1039/b813559bDOI Listing
January 2009

A set of piezoelectric biosensors using cholinesterases.

Methods Mol Biol 2009 ;504:3-22

Department of Analytical Biochemistry, University of Potsdam, Potsdam, Germany.

Piezoelectric sensors have become a versatile tool in biosensorics to study protein-protein and protein-small molecule interactions. Here we present theoretical background on piezoelectric sensors and instructions, how to modify their surface with various recognition elements for cholinesterases. These recognition elements comprise an organophosphate (paraoxon), a cocaine derivative (BZE-DADOO), and a tricyclic, aromatic compound (propidium). Additionally, a guide to the kinetic evaluation of the obtained binding curves is given in this chapter.
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http://dx.doi.org/10.1007/978-1-60327-569-9_1DOI Listing
March 2009

Piezoelectric affinity sensors for cocaine and cholinesterase inhibitors.

Talanta 2005 Jan;65(2):337-42

Department of Analytical Biochemistry, Institute of Biochemistry and Biology, University of Potsdam, Karl-Liebknecht-Str. 24-25, 14476 Golm, Germany.

We report here the development of piezoelectric affinity sensors for cocaine and cholinesterase inhibitors based on the formation of affinity complexes between an immobilized cocaine derivative and an anti-cocaine antibody or cholinesterase. For both binding reactions benzoylecgonine-1,8-diamino-3,4-dioxaoctane (BZE-DADOO) was immobilized on the surface of the sensor. For immobilization, pre-conjugated BZE-DADOO with 11-mercaptomonoundecanoic acid (MUA) via 2-(5-norbornen-2,3-dicarboximide)-1,1,3,3-tetramethyluronium-tetrafluoroborate (TNTU) allowed the formation of a chemisorbed monolayer on the piezosensor surface. The detection of cocaine was based on a competitive assay. The change of frequency measured after 300s of the binding reaction was used as the signal. The maximum binding of the antibody resulted in a frequency decrease of 35Hz (with an imprecision 3%, n = 3) while the presence of 100pmoll(-1) cocaine decreased the binding by 11%. The limit of detection was consequently below 100pmoll(-1) for cocaine. The total time of one analysis was 15min. This BZE-DADOO-modified sensor was adapted for the detection of organophosphates. BZE-DADOO - a competitive inhibitor - served as binding element for cholinesterase in a competitive assay.
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http://dx.doi.org/10.1016/j.talanta.2004.07.008DOI Listing
January 2005

Electrocatalytic sulfite biosensor with human sulfite oxidase co-immobilized with cytochrome c in a polyelectrolyte-containing multilayer.

Anal Bioanal Chem 2009 Jan 21;393(1):225-33. Epub 2008 Oct 21.

Institute of Biochemistry and Biology, University of Potsdam, Karl-Liebknecht Strasse 24-25, 14476, Golm, Germany.

An efficient electrocatalytic biosensor for sulfite detection was developed by co-immobilizing sulfite oxidase and cytochrome c with polyaniline sulfonic acid in a layer-by-layer assembly. QCM, UV-Vis spectroscopy and cyclic voltammetry revealed increasing loading of electrochemically active protein with the formation of multilayers. The sensor operates reagentless at low working potential. A catalytic oxidation current was detected in the presence of sulfite at the modified gold electrode, polarized at +0.1 V (vs. Ag/AgCl 1 M KCl). The stability of the biosensor performance was characterized and optimized. A 17-bilayer electrode has a linear range between 1 and 60 microM sulfite with a sensitivity of 2.19 mA M(-1) sulfite and a response time of 2 min. The electrode retained a stable response for 3 days with a serial reproducibility of 3.8% and lost 20% of sensitivity after 5 days of operation. It is possible to store the sensor in a dry state for more than 2 months. The multilayer electrode was used for determination of sulfite in unspiked and spiked samples of red and white wine. The recovery and the specificity of the signals were evaluated for each sample.
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http://dx.doi.org/10.1007/s00216-008-2432-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2755739PMC
January 2009
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