Publications by authors named "Nenad Gajovic-Eichelmann"

18 Publications

  • Page 1 of 1

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

A new one-step antigen heterologous homogeneous fluorescence immunoassay for progesterone detection in serum.

Talanta 2015 Mar 6;134:508-513. Epub 2014 Dec 6.

Fraunhofer Institute for Cell Therapy and Immunology, Am Mühlenberg 13, 14476 Potsdam-Golm, Germany.

A new homogeneous immunoassay for the detection of progesterone was developed to measure its concentration in human serum. We utilized the weak cross-reactivity of a monoclonal anti-progesterone antibody to an analog molecule (in this case β-estradiol) to create a mixture, in which the fluorescence-labeled antibody (AbF) and quencher-labeled BSA-estradiol (eBSAq) were at optimized equilibrium. At this stage, most antibodies were bound to eBSAq and the fluorescence of AbF was quenched. After adding samples containing free progesterone to the system, these would replace the eBSAq at the antigen-binding site. The fluorescence would be released. In contrast to conventional competitive immunoassays, the fluorescence signal increases with increasing progesterone concentration, greatly simplifying detection and calibration. The performance of the assay was very simple; there was only one mixing step; and other hormones like testosterone, estradiol or dehydroepiandrosterone (DHEA) do not interfere the assay. A wide linear range from 0.1 µg/L to 100 µg/L was achieved in buffer, with a LOD of 0.1 µg/L. In human serum the LOD was 5 µg/L, and the linear range was 5-500 µg/L. For this assay it is important to find the right combination of antibody and cross-reactive antigen. If such a combination could be defined, it is conceivable to apply this assay to a wide range of analytes.
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http://dx.doi.org/10.1016/j.talanta.2014.11.062DOI Listing
March 2015

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

Secretory leukocyte protease inhibitor (SLPI) might contaminate murine monoclonal antibodies after purification on protein G.

J Biotechnol 2012 Mar 20;158(1-2):34-5. Epub 2012 Jan 20.

UP Transfer GmbH, Am Neuen Palais 10, 14469 Potsdam, Germany.

The large scale production of a monoclonal anti-progesterone antibody in serum free medium followed by affinity chromatography on protein G lead to a contamination of the antibody sample with a protein of about 14 kDa. This protein was identified by mass spectrometry as secretory leukocyte protease inhibitor (SLPI). This SLPI contamination lead to a failure of the fiber-optic based competitive fluorescence assay to detect progesterone in milk. Purification of the monoclonal antibody using protein A columns circumvented this problem.
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http://dx.doi.org/10.1016/j.jbiotec.2011.12.025DOI Listing
March 2012

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

Direct detection of Δ9-tetrahydrocannabinol in aqueous samples using a homogeneous increasing fluorescence immunoassay (HiFi).

Anal Bioanal Chem 2010 Nov 26;398(5):2133-40. Epub 2010 Aug 26.

Group Biosensors, Fraunhofer Institute for Biomedical Engineering, Am Mühlenberg 13, 14476 Potsdam-Golm, Germany.

The detection of the major active component of cannabis, Δ9-tetrahydrocannabinol (THC), becomes increasingly relevant due to its widespread abuse. For control purposes, some easy-to-use, sensitive and inexpensive test methods are needed. We have developed a fluorescence immunoassay utilising THC-fluorescein conjugate as tracer. Fluorescence spectroscopy of the conjugate revealed an unusual property: The relatively weak fluorescence of a dilute tracer solution was increased by a factor of up to 5 after binding of a THC-specific antibody. Fluorescence lifetime measurements in aqueous solutions suggested two different tracer conformations both associated with quenching of fluorescein fluorescence by the intramolecular THC moiety. After antibody binding, the tracer enters a third conformation in which fluorescence quenching of fluorescein is completely suppressed. Utilising this property, we established a homogeneous competitive immunoassay (homogeneous increasing fluorescence immunoassay) with low detection limits. The test requires only two reagents, the new tracer molecule and an anti-THC antibody. A single test takes only 8 min. The dynamic detection range for THC is 0.5 to 20 ng/mL in buffer, with a limit of detection (LOD) of 0.5 ng/mL. The test also works in diluted saliva samples (1:10 dilution with buffer) with an LOD of 2 ng/mL and a dynamic range of 2-50 ng/mL.
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http://dx.doi.org/10.1007/s00216-010-4109-6DOI Listing
November 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

Direct detection of Delta9-tetrahydrocannabinol in saliva using a novel homogeneous competitive immunoassay with fluorescence quenching.

Anal Chim Acta 2010 Jan 14;658(2):187-92. Epub 2009 Nov 14.

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

For the detection of the major active component of cannabis, Delta9-tetrahydrocannabinol (THC) in aqueous samples, a homogeneous competitive immunoassay based on fluorescence quenching induced by fluorescence resonance energy transfer (FRET) has been developed. The fluorescence of anti-THC-antibody, labeled with fluorescence dye DY-481XL, can be quenched after its binding to THC-BSA-quencher conjugate (bovine serum albumin coupled with THC and another fluorescence dye, DYQ-661, as quencher). This quenching effect is inhibited when the antibodies bind to free THC in aqueous sample, thus competing for binding sites with the THC-BSA-quencher conjugate. The extent of the inhibition corresponds to the concentration of THC in the samples. The assay principle is simple and the test duration is within 10 min. The detection limit for THC in buffer was 2 ng mL(-1). In pooled saliva samples a detection limit of 50 ng mL(-1) was achieved.
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http://dx.doi.org/10.1016/j.aca.2009.11.012DOI Listing
January 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

Serodiagnosis of Lyme borreliosis infection using surface plasmon resonance.

Clin Chim Acta 2008 Aug 12;394(1-2):110-3. Epub 2008 Apr 12.

Fraunhofer Institute for Biomedical Engineering, Branch Potsdam-Golm, Department of Molecular Bioanalytics & Bioelectronics, Am Mühlenberg 13, Potsdam, Germany.

Background: Label-free biosensors are ideally suited for the direct monitoring of binding events without the need for additional labeling substances; however, their application in the field of serodiagnosis is not trivial. The major problem is the unspecific adsorption of blood serum components to the sensor surface.

Methods: A surface plasmon resonance (SPR) sensor has been used for the direct detection of Lyme borreliosis specific antibodies in blood serum. The combination of an optimal dilution factor with the addition of suitable detergents minimizes the unspecific adsorption. Serum samples from healthy donors and infected patients have been analyzed and the results were compared with a certified immunoassay and a western blot.

Results: A serum dilution of 1:20 in HBS-buffer with 0.05% Tween20 and 1 mg/mL carboxymethyl dextran reduces unspecific adsorption significantly and enables the identification of antibodies against the OspC/pepC10 antigen pair with a sensitivity of 92% and that against the VlsE/C6 pair with 81% sensitivity; the specificities are 82% and 86% respectively. Positive hits in the western blot could also be determined in the SPR-assay with a correlation of 96.5%.

Conclusion: The presented optical label-free technique has the potential for a precise and fast identification of pathogen-specific antibodies without the need for a secondary labeling antibody.
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http://dx.doi.org/10.1016/j.cca.2008.04.009DOI Listing
August 2008

Cohort analysis of a single nucleotide polymorphism on DNA chips.

Biosens Bioelectron 2004 Nov;20(5):956-66

Department of Molecular Bioanalytics and Bioelectronics, Fraunhofer Institute for Biomedical Engineering, Arthur-Scheunert-Allee 114-116, D-14558 Nuthetal, Germany.

A method has been developed to determine SNPs on DNA chips by applying a flow-through bioscanner. As a practical application we demonstrated the fast and simple SNP analysis of 24 genotypes in an array of 96 spots with a single hybridisation and dissociation experiment. The main advantage of this methodical concept is the parallel and fast analysis without any need of enzymatic digestion. Additionally, the DNA chip format used is appropriate for parallel analysis up to 400 spots. The polymorphism in the gene of the human phenol sulfotransferase SULT1A1 was studied as a model SNP. Biotinylated PCR products containing the SNP (The SNP summary web site: ) (mutant) and those containing no mutation (wild-type) were brought onto the chips coated with NeutrAvidin using non-contact spotting. This was followed by an analysis which was carried out in a flow-through biochip scanner while constantly rinsing with buffer. After removing the non-biotinylated strand a fluorescent probe was hybridised, which is complementary to the wild-type sequence. If this probe binds to a mutant sequence, then one single base is not fully matching. Thereby, the mismatched hybrid (mutant) is less stable than the full-matched hybrid (wild-type). The final step after hybridisation on the chip involves rinsing with a buffer to start dissociation of the fluorescent probe from the immobilised DNA strand. The online measurement of the fluorescence intensity by the biochip scanner provides the possibility to follow the kinetics of the hybridisation and dissociation processes. According to the different stability of the full-match and the mismatch, either visual discrimination or kinetic analysis is possible to distinguish SNP-containing sequence from the wild-type sequence.
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http://dx.doi.org/10.1016/j.bios.2004.06.012DOI Listing
November 2004

Directed immobilization of nucleic acids at ultramicroelectrodes using a novel electro-deposited polymer.

Biosens Bioelectron 2003 Dec;19(5):417-22

Fraunhofer Institute for Biomedical Engineering, A-Scheunert-Allee 114-116, 14558 Bergholz-Rehbrucke, Germany.

A two-step method for the directed immobilization of nucleic acids at ultramicroelectrodes with micron-size dimensions is described. The approach is based on the immobilization of streptavidin at the surface of carbon or noble metal electrodes within a novel electro-deposited polymer, formed by electropolymerization of the natural compound scopoletin (7-hydroxy-6-methoxy-coumarin) at potentials between 0.4 and 0.7 V vs. Ag/AgCl. Biotin-tagged nucleic acids or proteins are immobilized on top of the modified electrodes in a second step. The new method has some advantages compared to classical electropolymerization approaches (e.g. polypyrrole, polyphenol), because the growing polymer is highly hydrophilic, resulting in efficient incorporation of streptavidin and a high biotin binding capacity of 6 pmol cm(-2). The polymer film seems to be non-conductive but shows good swelling properties in aqueous solutions. The feasibility of the method for the electro-directed biochemical modification of individual microelectrodes has been demonstrated by sequential immobilization of two different single strand oligonucleotides onto interdigitated ultramicroelectrodes. The resulting miniature DNA probe was used for single base mutation detection with two synthetic targets (fluorescence-labeled 17-mer oligomers) by evaluating the fluorescence patterns after hybridisation with the immobilised DNA probes. The new method is useful for the production of microelectrode based DNA chips and for the electro-directed immobilisation of biomolecules at microelectrode structures with high spatial resolution and yield.
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http://dx.doi.org/10.1016/s0956-5663(03)00224-0DOI Listing
December 2003

Oriented and vectorial immobilization of linear M13 dsDNA between interdigitated electrodes--towards single molecule DNA nanostructures.

Biosens Bioelectron 2003 May;18(5-6):555-64

Department of Molecular Bioanalytics and Bioelectronics, Fraunhofer Institute for Biomedical Engineering, A.-Scheunert-Allee 114, 14558, Bergholz-Rehbrücke, Germany.

The ability to control molecules at a resolution well below that offered by photolithography has gained much interest recently. DNA is a promising candidate for this task since it offers excellent specificity in base-pairing combined with addressability at the nanometer scale. New applications in biosensing, e.g. interaction analysis at the single molecule level, or nanobiotechnology, e.g. ultradense DNA microarrays, have been devised that rely on stretched DNA bridges. The basic technology required is the ability to deposit spatially defined, stretched DNA-bridges between anchoring structures on surfaces. In this paper we present two techniques for spanning 2 microm long dsDNA bridges between neighboring interdigitated electrodes (IDEs). The extended DNA used was linearized M13 dsDNA (M13mp18 7231 bp, ca. 2.5 microm length), either unmodified, or with chemical modifications at both ends. The first approach is based on the dielectrophoretic (DEP) concentration and alignment of linearized wild-type dsDNA. IDEs with 1.7 microm spacing are driven with an AC voltage around 1 MHz leading to field strengths in the order of 1 MV m(-1). The dsDNA is polarized and linearized by the force field and accumulates in the gap between two neighboring electrodes. This process is reversible and was visualized by fluorescence staining of M13 DNA using PicoGreen, as intercalating dye. The resulting dsDNA bridges and their orientation are discernible under the fluorescence microscope using fluorescent particles of different color. The particles are tagged with sequence specific peptide nucleic acid (PNA) probes that bind to the DNA double strand at specific sites. The second approach is based on asymmetric electrochemical modification of a gold IDE with 2.0 microm spacings followed by spontaneous or stimulated deposition of a chemically modified M13-DNA. One side of the IDE was selectively coated with streptavidin by electropolymerization of a novel hydrophilic conductive polymer in the presence of the binding protein. The second side was modified with gold nanoparticles by reductive plating from aqueous gold chloride solution. An asymmetric double stranded (ds) M13 DNA carrying a 5'-thiol group at one end and a 5'-biotin at the other end was obtained by polymerase chain reaction (PCR) using two differently labeled primers. For DNA bridges to form spontaneously the modified IDE was incubated over night with a 50 nM solution of the modified M13 DNA. Potential applications of DNA-bridge formation in biosensing and biotechnology are discussed.
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http://dx.doi.org/10.1016/s0956-5663(03)00024-1DOI Listing
May 2003
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