@article{CasertaZhangYarmanetal.2021, author = {Caserta, Giorgio and Zhang, Xiaorong and Yarman, Aysu and Supala, Eszter and Wollenberger, Ulla and Gyurcs{\´a}nyi, R{\´o}bert E. and Zebger, Ingo and Scheller, Frieder W.}, title = {Insights in electrosynthesis, target binding, and stability of peptide-imprinted polymer nanofilms}, series = {Electrochimica acta : the journal of the International Society of Electrochemistry (ISE)}, volume = {381}, journal = {Electrochimica acta : the journal of the International Society of Electrochemistry (ISE)}, publisher = {Elsevier}, address = {New York, NY [u.a.]}, issn = {0013-4686}, doi = {10.1016/j.electacta.2021.138236}, pages = {8}, year = {2021}, abstract = {Molecularly imprinted polymer (MIP) nanofilms have been successfully implemented for the recognition of different target molecules: however, the underlying mechanistic details remained vague. This paper provides new insights in the preparation and binding mechanism of electrosynthesized peptide-imprinted polymer nanofilms for selective recognition of the terminal pentapeptides of the beta-chains of human adult hemoglobin, HbA, and its glycated form HbA1c. To differentiate between peptides differing solely in a glucose adduct MIP nanofilms were prepared by a two-step hierarchical electrosynthesis that involves first the chemisorption of a cysteinyl derivative of the pentapeptide followed by electropolymerization of scopoletin. This approach was compared with a random single-step electrosynthesis using scopo-letin/pentapeptide mixtures. Electrochemical monitoring of the peptide binding to the MIP nanofilms by means of redox probe gating revealed a superior affinity of the hierarchical approach with a Kd value of 64.6 nM towards the related target. Changes in the electrosynthesized non-imprinted polymer and MIP nanofilms during chemical, electrochemical template removal and rebinding were substantiated in situ by monitoring the characteristic bands of both target peptides and polymer with surface enhanced infrared absorption spectroscopy. This rational approach led to MIPs with excellent selectivity and provided key mechanistic insights with respect to electrosynthesis, rebinding and stability of the formed MIPs.}, language = {en} } @article{ZhangCasertaYarmanetal.2021, author = {Zhang, Xiaorong and Caserta, Giorgio and Yarman, Aysu and Supala, Eszter and Tadjoung Waffo, Armel Franklin and Wollenberger, Ulla and Gyurcsanyi, Robert E. and Zebger, Ingo and Scheller, Frieder W.}, title = {"Out of Pocket" protein binding}, series = {Chemosensors}, volume = {9}, journal = {Chemosensors}, number = {6}, publisher = {MDPI}, address = {Basel}, issn = {2227-9040}, doi = {10.3390/chemosensors9060128}, pages = {13}, year = {2021}, abstract = {The epitope imprinting approach applies exposed peptides as templates to synthesize Molecularly Imprinted Polymers (MIPs) for the recognition of the parent protein. While generally the template protein binding to such MIPs is considered to occur via the epitope-shaped cavities, unspecific interactions of the analyte with non-imprinted polymer as well as the detection method used may add to the complexity and interpretation of the target rebinding. To get new insights on the effects governing the rebinding of analytes, we electrosynthesized two epitope-imprinted polymers using the N-terminal pentapeptide VHLTP-amide of human hemoglobin (HbA) as the template. MIPs were prepared either by single-step electrosynthesis of scopoletin/pentapeptide mixtures or electropolymerization was performed after chemisorption of the cysteine extended VHLTP peptide. Rebinding of the target peptide and the parent HbA protein to the MIP nanofilms was quantified by square wave voltammetry using a redox probe gating, surface enhanced infrared absorption spectroscopy, and atomic force microscopy. While binding of the pentapeptide shows large influence of the amino acid sequence, all three methods revealed strong non-specific binding of HbA to both polyscopoletin-based MIPs with even higher affinities than the target peptides.}, language = {en} } @article{TadjoungWaffoMitrovaTiedemannetal.2021, author = {Tadjoung Waffo, Armel Franklin and Mitrova, Biljana and Tiedemann, Kim and Iobbi-Nivol, Chantal and Leimk{\"u}hler, Silke and Wollenberger, Ulla}, title = {Electrochemical trimethylamine n-oxide biosensor with enzyme-based oxygen-scavenging membrane for long-term operation under ambient air}, series = {Biosensors : open access journal}, volume = {11}, journal = {Biosensors : open access journal}, number = {4}, publisher = {MDPI}, address = {Basel}, issn = {2079-6374}, doi = {10.3390/bios11040098}, pages = {17}, year = {2021}, abstract = {An amperometric trimethylamine N-oxide (TMAO) biosensor is reported, where TMAO reductase (TorA) and glucose oxidase (GOD) and catalase (Cat) were immobilized on the electrode surface, enabling measurements of mediated enzymatic TMAO reduction at low potential under ambient air conditions. The oxygen anti-interference membrane composed of GOD, Cat and polyvinyl alcohol (PVA) hydrogel, together with glucose concentration, was optimized until the O-2 reduction current of a Clark-type electrode was completely suppressed for at least 3 h. For the preparation of the TMAO biosensor, Escherichia coli TorA was purified under anaerobic conditions and immobilized on the surface of a carbon electrode and covered by the optimized O-2 scavenging membrane. The TMAO sensor operates at a potential of -0.8 V vs. Ag/AgCl (1 M KCl), where the reduction of methylviologen (MV) is recorded. The sensor signal depends linearly on TMAO concentrations between 2 mu M and 15 mM, with a sensitivity of 2.75 +/- 1.7 mu A/mM. The developed biosensor is characterized by a response time of about 33 s and an operational stability over 3 weeks. Furthermore, measurements of TMAO concentration were performed in 10\% human serum, where the lowest detectable concentration is of 10 mu M TMAO.}, language = {en} } @article{YanFrokjarEngelbrektetal.2021, author = {Yan, Jiawei and Fr{\o}kj{\ae}r, Emil Egede and Engelbrekt, Christian and Leimk{\"u}hler, Silke and Ulstrup, Jens and Wollenberger, Ulla and Xiao, Xinxin and Zhang, Jingdong}, title = {Voltammetry and single-molecule in situ scanning tunnelling microscopy of the redox metalloenzyme human sulfite oxidase}, series = {ChemElectroChem}, volume = {8}, journal = {ChemElectroChem}, number = {1}, publisher = {Wiley-VCH}, address = {Weinheim}, issn = {2196-0216}, doi = {10.1002/celc.202001258}, pages = {164 -- 171}, year = {2021}, abstract = {Human sulfite oxidase (hSO) is a homodimeric two-domain enzyme central in the biological sulfur cycle. A pyranopterin molybdenum cofactor (Moco) is the catalytic site and a heme b(5) group located in the N-terminal domain. The two domains are connected by a flexible linker region. Electrons produced at the Moco in sulfite oxidation, are relayed via heme b(5) to electron acceptors or an electrode surface. Inter-domain conformational changes between an open and a closed enzyme conformation, allowing "gated" electron transfer has been suggested. We first recorded cyclic voltammetry (CV) of hSO on single-crystal Au(111)-electrode surfaces modified by self-assembled monolayers (SAMs) both of a short rigid thiol, cysteamine and of a longer structurally flexible thiol, omega-amino-octanethiol (AOT). hSO on cysteamine SAMs displays a well-defined pair of voltammetric peaks around -0.207 V vs. SCE in the absence of sulfite substrate, but no electrocatalysis. hSO on AOT SAMs displays well-defined electrocatalysis, but only "fair" quality voltammetry in the absence of sulfite. We recorded next in situ scanning tunnelling spectroscopy (STS) of hSO on AOT modified Au(111)-electrodes, disclosing, a 2-5 \% surface coverage of strong molecular scale contrasts, assigned to single hSO molecules, notably with no contrast difference in the absence and presence of sulfite. In situ STS corroborated this observation with a sigmoidal tunnelling current/overpotential correlation.}, language = {en} } @article{NeumannWollenberger2020, author = {Neumann, Bettina and Wollenberger, Ulla}, title = {Electrochemical biosensors employing natural and artificial heme peroxidases on semiconductors}, series = {Sensors}, volume = {20}, journal = {Sensors}, number = {13}, publisher = {MDPI}, address = {Basel}, issn = {1424-8220}, doi = {10.3390/s20133692}, pages = {24}, year = {2020}, abstract = {Heme peroxidases are widely used as biological recognition elements in electrochemical biosensors for hydrogen peroxide and phenolic compounds. Various nature-derived and fully synthetic heme peroxidase mimics have been designed and their potential for replacing the natural enzymes in biosensors has been investigated. The use of semiconducting materials as transducers can thereby offer new opportunities with respect to catalyst immobilization, reaction stimulation, or read-out. This review focuses on approaches for the construction of electrochemical biosensors employing natural heme peroxidases as well as various mimics immobilized on semiconducting electrode surfaces. It will outline important advances made so far as well as the novel applications resulting thereof.}, language = {en} } @article{OthmanWollenberger2020, author = {Othman, Abdelmageed M. and Wollenberger, Ulla}, title = {Amperometric biosensor based on coupling aminated laccase to functionalized carbon nanotubes for phenolics detection}, series = {International journal of biological macromolecules}, volume = {153}, journal = {International journal of biological macromolecules}, publisher = {Elsevier}, address = {New York, NY [u.a.]}, issn = {0141-8130}, doi = {10.1016/j.ijbiomac.2020.03.049}, pages = {855 -- 864}, year = {2020}, abstract = {A biosensor for phenolic compounds based on a chemically modified laccase from Coriolus hirsula immobilized on functionalized screen-printed carbon electrodes (SPCEs) was achieved. Different enzyme modifications and immobilization strategies were analyzed. The electrochemical response of the immobilized laccase on SPCEs modified with carboxyl functionalized multi-walled carbon nanotubes (COOH-MWCNT) was the highest when laccase was aminated prior to the adsorption onto the working electrode. The developed lactase biosensor sensitivity toward different phenolic compounds was assessed to determine the biosensor response with several phenolic compounds. The highest response was obtained for ABTS with a saturation value of I-max = 27.94 mu A. The electrocatalytic efficiency (I-max/K-m(app)) was the highest for ABTS (5588 mu A mu M-1) followed by syringaldazine (3014 mu A.mu M-1). The sensors were considerably stable, whereby 99.5, 82 and 77\% of the catalytic response using catechol as substrate was retained after 4, 8 and 10 successive cycles of reuse respectively, with response time average of 5 s for 12 cycles. No loss of activity was observed after 20 days of storage.}, language = {en} } @article{WerchmeisterTangXiaoetal.2019, author = {Werchmeister, Rebecka Maria Larsen and Tang, Jing and Xiao, Xinxin and Wollenberger, Ulla and Hjuler, Hans Aage and Ulstrup, Jens and Zhang, Jingdong}, title = {Three-Dimensional Bioelectrodes Utilizing Graphene Based Bioink}, series = {Journal of The Electrochemical Society}, volume = {166}, journal = {Journal of The Electrochemical Society}, number = {16}, publisher = {The Electrochemical Society}, address = {Pennington}, issn = {0013-4651}, doi = {10.1149/2.0261916jes}, pages = {G170 -- G177}, year = {2019}, abstract = {Enzyme immobilization using nanomaterials offers new approaches to enhanced bioelectrochemical performance and is essential for the preparation of bioelectrodes with high reproducibility and low cost. In this report, we describe the development of new three-dimensional (3D) bioelectrodes by immobilizing a "bioink" of glucose oxidase (GOD) in a matrix of reduced graphene oxides (RGOs), polyethylenimine (PEI), and ferrocene carboxylic acid (FcCOOH) on carbon paper (CP). CP with 3D interwoven carbon fibers serves as a solid porous and electronically conducting skeleton, providing large surface areas and space for loading the bioink and diffusion of substrate molecules, respectively. RGO enhances contact between the GOD-matrix and CP, maintaining high conductivity. The composition of the bioink has been systematically optimized. The GOD bioelectrodes show linearly increasing electrocatalytic oxidation current toward glucose concentration up to 48 mM. A hybrid enzymatic biofuel cell equipped with the GOD bioelectrode as a bioanode and a platinum cathode furthermore registers a maximum power density of 5.1 mu W cm(-2) and an open circuit voltage of 0.40 V at 25 degrees C. The new method reported of preparing a bioelectrode by drop-casting the bioink onto the substrate electrode is facile and versatile, with the potential of application also for other enzymatic bioelectrodes.}, language = {en} } @article{OzcelikayKurbanogluZhangetal.2019, author = {Ozcelikay, Goksu and Kurbanoglu, Sevinc and Zhang, Xiaorong and S{\"o}z, {\c{C}}ağla Kosak and Wollenberger, Ulla and Ozkan, Sibel A. and Yarman, Aysu and Scheller, Frieder W.}, title = {Electrochemical MIP Sensor for Butyrylcholinesterase}, series = {Polymers}, volume = {11}, journal = {Polymers}, number = {12}, publisher = {MDPI}, address = {Basel}, issn = {2073-4360}, doi = {10.3390/polym11121970}, pages = {11}, year = {2019}, abstract = {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.}, language = {en} } @misc{SchellerZhangYarmanetal.2019, author = {Scheller, Frieder W. and Zhang, Xiaorong and Yarman, Aysu and Wollenberger, Ulla and Gyurcs{\´a}nyi, R{\´o}bert E.}, title = {Molecularly imprinted polymer-based electrochemical sensors for biopolymers}, series = {Current opinion in electrochemistry}, volume = {14}, journal = {Current opinion in electrochemistry}, publisher = {Elsevier}, address = {Amsterdam}, issn = {2451-9103}, doi = {10.1016/j.coelec.2018.12.005}, pages = {53 -- 59}, year = {2019}, abstract = {Electrochemical synthesis and signal generation dominate among the almost 1200 articles published annually on protein-imprinted polymers. Such polymers can be easily prepared directly on the electrode surface, and the polymer thickness can be precisely adjusted to the size of the target to enable its free exchange. In this architecture, the molecularly imprinted polymer (MIP) layer represents only one 'separation plate'; thus, the selectivity does not reach the values of 'bulk' measurements. The binding of target proteins can be detected straightforwardly by their modulating effect on the diffusional permeability of a redox marker through the thin MIP films. However, this generates an 'overall apparent' signal, which may include nonspecific interactions in the polymer layer and at the electrode surface. Certain targets, such as enzymes or redox active proteins, enables a more specific direct quantification of their binding to MIPs by in situ determination of the enzyme activity or direct electron transfer, respectively.}, language = {en} } @article{TangWerchmeisterPredaetal.2019, author = {Tang, Jing and Werchmeister, Rebecka Maria Larsen and Preda, Loredana and Huang, Wei and Zheng, Zhiyong and Leimk{\"u}hler, Silke and Wollenberger, Ulla and Xiao, Xinxin and Engelbrekt, Christian and Ulstrup, Jens and Zhang, Jingdong}, title = {Three-dimensional sulfite oxidase bioanodes based on graphene functionalized carbon paper for sulfite/O-2 biofuel cells}, series = {ACS catalysis}, volume = {9}, journal = {ACS catalysis}, number = {7}, publisher = {American Chemical Society}, address = {Washington}, issn = {2155-5435}, doi = {10.1021/acscatal.9b01715}, pages = {6543 -- 6554}, year = {2019}, abstract = {We have developed a three-dimensional (3D) graphene electrode suitable for the immobilization of human sulfite oxidase (hSO), which catalyzes the electrochemical oxidation of sulfite via direct electron transfer (DET). The electrode is fabricated by drop-casting graphene-polyethylenimine (G-P) composites on carbon papers (CPs) precoated with graphene oxide (GO). The negatively charged hSO can be adsorbed electrostatically on the positively charged matrix (G-P) on CP electrodes coated with GO (CPG), with a proper orientation for accelerated DET. Notably, further electrochemical reduction of G-P on CPG electrodes leads to a 9-fold increase of the saturation catalytic current density (j(m)) for sulfite oxidation reaching 24.4 +/- 0.3 mu A to cm(-2), the highest value among reported DET-based hSO bioelectrodes. The increased electron transfer rate plays a dominating role in the enhancement of direct enzymatic current because of the improved electric contact of hSO with the electrode, The optimized hSO bioelectrode shows a significant catalytic rate (k(cat): 25.6 +/- 0.3 s(-1)) and efficiency (k(cat)/K-m: 0.231 +/- 0.003 s(-1) mu M-1) compared to the reported hSO bioelectrodes. The assembly of the hSO bioanode and a commercial platinum biocathode allows the construction of sulfite/O-2 enzymatic biofuel cells (EBFCs) with flowing fuels. The optimized EBFC displays an open-circuit voltage (OCV) of 0.64 +/- 0.01 V and a maximum power density of 61 +/- 6 mu W cm(-2) (122 +/- 12 mW m(-3)) at 30 degrees C, which exceeds the best reported value by more than 6 times.}, language = {en} }