TY - GEN
A1 - Wessig, Pablo
A1 - Hille, Carsten
A1 - Kumke, Michael Uwe
A1 - Meiling, Till Thomas
A1 - Behrends, Nicole
A1 - Eisold, Ursula
T1 - Two-photon FRET pairs based on coumarin and DBD dyes
N2 - The synthesis and photophysical properties of two new FRET pairs based on coumarin as a donor and DBD dye as an acceptor are described. The introduction of a bromo atom dramatically increases the two-photon excitation (2PE) cross section providing a 2PE-FRET system, which is also suitable for 2PE-FLIM.
T3 - Zweitveröffentlichungen der Universität Potsdam : Mathematisch-Naturwissenschaftliche Reihe - 318
KW - resonance energy-tansfer
KW - conformational-changes
KW - microscopy
KW - proteins
KW - acid
Y1 - 2016
U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:517-opus4-394445
SP - 33510
EP - 33513
ER -
TY - GEN
A1 - Walkowiak, Jacek
A1 - Lu, Yan
A1 - Gradzielski, Michael
A1 - Zauscher, Stefan
A1 - Ballauff, Matthias
T1 - Thermodynamic analysis of the uptake of a protein in a spherical polyelectrolyte brush
T2 - Postprints der Universität Potsdam : Mathematisch-Naturwissenschaftliche Reihe
N2 - A thermodynamic study of the adsorption of Human Serum Albumin (HSA) onto spherical polyelectrolyte brushes (SPBs) by isothermal titration calorimetry (ITC) is presented. The SPBs are composed of a solid polystyrene core bearing long chains of poly(acrylic acid). ITC measurements done at different temperatures and ionic strengths lead to a full set of thermodynamicbinding constants together with the enthalpies and entropies of binding. The adsorption of HSA onto SPBs is described with a two-step model. The free energy of binding Delta Gb depends only weakly on temperature because of a marked compensation of enthalpy by entropy. Studies of the adsorbed HSA by Fourier transform infrared spectroscopy (FT-IR) demonstrate no significant disturbance in the secondary structure of the protein. The quantitative analysis demonstrates that counterion release is the major driving force for adsorption in a process where proteins become multivalent counterions of the polyelectrolyte chains upon adsorption. A comparison with the analysis of other sets of data related to the binding of HSA to polyelectrolytes demonstrates that the cancellation of enthalpy and entropy is a general phenomenon that always accompanies the binding of proteins to polyelectrolytes dominated by counterion release.
KW - ITC
KW - spherical polyelectrolyte brushes
KW - enthalpy-entropy compensation (EEC)
KW - proteins
KW - thermodynamics
Y1 - 2021
U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:517-opus4-517307
SN - 1866-8372
IS - 1
ER -
TY - JOUR
A1 - Walkowiak, Jacek
A1 - Lu, Yan
A1 - Gradzielski, Michael
A1 - Zauscher, Stefan
A1 - Ballauff, Matthias
T1 - Thermodynamic analysis of the uptake of a protein in a spherical polyelectrolyte brush
JF - Macromolecular rapid communications
N2 - A thermodynamic study of the adsorption of Human Serum Albumin (HSA) onto spherical polyelectrolyte brushes (SPBs) by isothermal titration calorimetry (ITC) is presented. The SPBs are composed of a solid polystyrene core bearing long chains of poly(acrylic acid). ITC measurements done at different temperatures and ionic strengths lead to a full set of thermodynamicbinding constants together with the enthalpies and entropies of binding. The adsorption of HSA onto SPBs is described with a two-step model. The free energy of binding Delta Gb depends only weakly on temperature because of a marked compensation of enthalpy by entropy. Studies of the adsorbed HSA by Fourier transform infrared spectroscopy (FT-IR) demonstrate no significant disturbance in the secondary structure of the protein. The quantitative analysis demonstrates that counterion release is the major driving force for adsorption in a process where proteins become multivalent counterions of the polyelectrolyte chains upon adsorption. A comparison with the analysis of other sets of data related to the binding of HSA to polyelectrolytes demonstrates that the cancellation of enthalpy and entropy is a general phenomenon that always accompanies the binding of proteins to polyelectrolytes dominated by counterion release.
KW - Spherical polyelectrolyte brushes
KW - proteins
KW - ITC
KW - thermodynamics
KW - enthalpy-entropy compensation (EEC)
Y1 - 2019
U6 - https://doi.org/10.1002/marc.201900421
SN - 1022-1336
SN - 1521-3927
VL - 41
IS - 1
PB - Wiley-VCH
CY - Weinheim
ER -
TY - JOUR
A1 - Tebaldi, Marli Luiza
A1 - Charan, Himanshu
A1 - Mavliutova, Liliia
A1 - Böker, Alexander
A1 - Glebe, Ulrich
T1 - Dual-Stimuli Sensitive Hybrid Materials: Ferritin-PDMAEMA by Grafting-From Polymerization
JF - Macromolecular chemistry and physics
N2 - The combination of stimuli-responsive polymers and proteins that can transport drugs is a promising approach for drug delivery. The formation of ferritin-poly(2-dimethylaminoethyl methacrylate) (PDMAEMA) conjugates by atom-transfer radical polymerization from the protein macroinitiator is described. PDMAEMA is a dual-stimuli-responsive polymer and the thermo- and pH-responsive properties of the resulting conjugates are studied in detail with dynamic light scattering (DLS). Additionally, it is demonstrated that the lower critical solution temperature (LCST) of the protein-polymer conjugates can be further adjusted by the ionic strength of the solution. The conjugates are also characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), matrix-assisted laser desorption ionization-time of flight (MALDI-ToF) mass spectrometry, and NMR spectroscopy. The obtained MALDI-ToF mass spectra are exceptional for protein-polymer conjugates and have not been so often reported.
KW - grafting-from
KW - MALDI-ToF MS
KW - polymerization
KW - proteins
KW - responsivity
Y1 - 2017
U6 - https://doi.org/10.1002/macp.201600529
SN - 1022-1352
SN - 1521-3935
VL - 218
PB - Wiley-VCH
CY - Weinheim
ER -
TY - JOUR
A1 - Semenyshyn, Rostyslav
A1 - Hentschel, Mario
A1 - Stanglmair, Christoph
A1 - Teutsch, Tanja
A1 - Tarin, Cristina
A1 - Pacholski, Claudia
A1 - Giessen, Harald
A1 - Neubrech, Frank
T1 - In vitro monitoring conformational changes of polypeptide monolayers using infrared plasmonic nanoantennas
JF - Nano letters : a journal dedicated to nanoscience and nanotechnology
N2 - Proteins and peptides play a predominant role in biochemical reactions of living cells. In these complex environments, not only the constitution of the molecules but also their three-dimensional configuration defines their functionality. This so-called secondary structure of proteins is crucial for understanding their function in living matter. Misfolding, for example, is suspected as the cause of neurodegenerative diseases such as Alzheimer’s and Parkinson’s disease. Ultimately, it is necessary to study a single protein and its folding dynamics. Here, we report a first step in this direction, namely ultrasensitive detection and discrimination of in vitro polypeptide folding and unfolding processes using resonant plasmonic nanoantennas for surface-enhanced vibrational spectroscopy. We utilize poly-l-lysine as a model system which has been functionalized on the gold surface. By in vitro infrared spectroscopy of a single molecular monolayer at the amide I vibrations we directly monitor the reversible conformational changes between α-helix and β-sheet states induced by controlled external chemical stimuli. Our scheme in combination with advanced positioning of the peptides and proteins and more brilliant light sources is highly promising for ultrasensitive in vitro studies down to the single protein level.
KW - Plasmonics
KW - surface-enhanced infrared absorption spectroscopy
KW - proteins
KW - conformational changes
KW - biosensing
Y1 - 2019
U6 - https://doi.org/10.1021/acs.nanolett.8b02372
SN - 1530-6984
SN - 1530-6992
VL - 19
IS - 1
SP - 1
EP - 7
PB - American Chemical Society
CY - Washington
ER -
TY - GEN
A1 - Nakamura, Moritaka
A1 - Claes, Andrea R.
A1 - Grebe, Tobias
A1 - Hermkes, Rebecca
A1 - Viotti, Corrado
A1 - Ikeda, Yoshihisa
A1 - Grebe, Markus
T1 - Auxin and ROP GTPase signaling of polar nuclear migration in root epidermal hair cells
T2 - Postprints der Universität Potsdam : Mathematisch-Naturwissenschaftliche Reihe
N2 - Polar nuclear migration is crucial during the development of diverse eukaryotes. In plants, root hair growth requires polar nuclear migration into the outgrowing hair. However, knowledge about the dynamics and the regulatory mechanisms underlying nuclear movements in root epidermal cells remains limited. Here, we show that both auxin and Rho-of-Plant (ROP) signaling modulate polar nuclear position at the inner epidermal plasma membrane domain oriented to the cortical cells during cell elongation as well as subsequent polar nuclear movement to the outer domain into the emerging hair bulge in Arabidopsis (Arabidopsis thaliana). Auxin signaling via the nuclear AUXIN RESPONSE FACTOR7 (ARF7)/ARF19 and INDOLE ACETIC ACID7 pathway ensures correct nuclear placement toward the inner membrane domain. Moreover, precise inner nuclear placement relies on SPIKE1 Rho-GEF, SUPERCENTIPEDE1 Rho-GDI, and ACTIN7 (ACT7) function and to a lesser extent on VTI11 vacuolar SNARE activity. Strikingly, the directionality and/or velocity of outer polar nuclear migration into the hair outgrowth along actin strands also are ACT7 dependent, auxin sensitive, and regulated by ROP signaling. Thus, our findings provide a founding framework revealing auxin and ROP signaling of inner polar nuclear position with some contribution by vacuolar morphology and of actin-dependent outer polar nuclear migration in root epidermal hair cells.
T3 - Zweitveröffentlichungen der Universität Potsdam : Mathematisch-Naturwissenschaftliche Reihe - 992
KW - Arabidopsis-thaliana
KW - planar polarity
KW - tip growth
KW - morphogenesis
KW - gene
KW - proteins
KW - dynamics
KW - transformation
KW - activation
KW - initiation
Y1 - 2020
U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:517-opus4-441278
SN - 1866-8372
IS - 992
SP - 378
EP - 391
ER -
TY - THES
A1 - Laux, Eva-Maria
T1 - Electric field-assisted immobilization and alignment of biomolecules
T1 - Immobilisierung und Ausrichtung von Biomolekülen mit elektrischen Wechselfeldern
N2 - In this dissertation, an electric field-assisted method was developed and applied to achieve immobilization and alignment of biomolecules on metal electrodes in a simple one-step experiment. Neither modifications of the biomolecule nor of the electrodes were needed. The two major electrokinetic effects that lead to molecule motion in the chosen electrode configurations used were identified as dielectrophoresis and AC electroosmotic flow. To minimize AC electroosmotic flow, a new 3D electrode configuration was designed. Thus, the influence of experimental parameters on the dielectrophoretic force and the associated molecule movement could be studied. Permanent immobilization of proteins was examined and quantified absolutely using an atomic force microscope. By measuring the volumes of the immobilized protein deposits, a maximal number of proteins contained therein was calculated. This was possible since the proteins adhered to the tungsten electrodes even after switching off the electric field. The permanent immobilization of functional proteins on surfaces or electrodes is one crucial prerequisite for the fabrication of biosensors.
Furthermore, the biofunctionality of the proteins must be retained after immobilization. Due to the chemical or physical modifications on the proteins caused by immobilization, their biofunctionality is sometimes hampered. The activity of dielectrophoretically immobilized proteins, however, was proven here for an enzyme for the first time. The enzyme horseradish peroxidase was used exemplarily, and its activity was demonstrated with the oxidation of dihydrorhodamine 123, a non-fluorescent precursor of the fluorescence dye rhodamine 123.
Molecular alignment and immobilization - reversible and permanent - was achieved under the influence of inhomogeneous AC electric fields. For orientational investigations, a fluorescence microscope setup, a reliable experimental procedure and an evaluation protocol were developed and validated using self-made control samples of aligned acridine orange molecules in a liquid crystal.
Lambda-DNA strands were stretched and aligned temporarily between adjacent interdigitated electrodes, and the orientation of PicoGreen molecules, which intercalate into the DNA strands, was determined. Similarly, the aligned immobilization of enhanced Green Fluorescent Protein was demonstrated exploiting the protein's fluorescence and structural properties. For this protein, the angle of the chromophore with respect to the protein's geometrical axis was determined in good agreement with X-ray crystallographic data. Permanent immobilization with simultaneous alignment of the proteins was achieved along the edges, tips and on the surface of interdigitated electrodes. This was the first demonstration of aligned immobilization of proteins by electric fields.
Thus, the presented electric field-assisted immobilization method is promising with regard to enhanced antibody binding capacities and enzymatic activities, which is a requirement for industrial biosensor production, as well as for general interaction studies of proteins.
N2 - In dieser Doktorarbeit wurde eine Methode entwickelt, mit der Biomoleküle unter dem Einfluss von elektrischen Feldern auf Metallelektroden immobilisiert und ausgerichtet werden können. Für die Immobilisierung wurden weder Modifikationen an den Biomolekülen noch an den Elektroden benötigt. Zwei elektrokinetische Effekte, die Dielektrophorese und der AC-elektroosmotische Fluss, wurden als verantwortliche Effekte für die Molekülbewegung identifiziert. Mit einer neuen 3D Elektrodenkonfiguration wurde der AC-elektroosmotische Fluss minimiert. Damit konnte der Einfluss der experimentellen Parameter auf die Dielektrophoresekraft und deren Auswirkungen auf die Moleküle untersucht werden: Die permanente Immobilisierung von Proteinen wurde mit einem Rasterkraftmikroskop quantifiziert, indem die Volumina der immobilisierten Proteinablagerungen gemessen wurden, und daraus die maximal darin enthaltene Anzahl an Proteinen berechnet wurde. Diese Art der absoluten Quantifizierung war nur möglich, da die Proteine auch nach Abschalten des elektrischen Feldes auf den Wolframelektroden hafteten. Eine solche permanente Immobilisierung funktioneller Proteine auf Elektroden oder Oberflächen im Allgemeinen ist eine wichtige Voraussetzung für die Herstellung von Biosensoren.
Des Weiteren muss die Biofunktion der Proteine nach der Immobilisierung erhalten bleiben. Da die Proteine durch die Immobilisierung chemisch oder physikalisch verändert werden, ist auch ihre Biofunktion häufig eingeschränkt. In dieser Arbeit wurde erstmals der Erhalt der Aktivität dielektrophoretisch immobilisierter Enzyme gezeigt. Hierfür wurde das Enzym Meerrettichperoxidase exemplarisch verwendet, dessen Aktivität über die Oxidation von Dihydrorhodamin 123, einem nicht-fluoreszentem Vorläufer des Fluoreszenzfarbstoffes Rhodamin 123, nachgewiesen wurde.
Molekulare Ausrichtung und Immobilisierung – sowohl reversibel als auch permanent – wurde unter dem Einfluss inhomogener elektrischer Wechselfelder erreicht. Für die Bestimmung der Molekülausrichtung wurde mit ein Messaufbau entwickelt, der auf einem Fluoreszenzmikroskop basiert. Der Aufbau, das Messprotokoll und die Auswertungsmethode wurden mit einer selbst hergestellten Kontrollprobe, die aus ausgerichteten Acridinorangemolekülen in einem Flüssigkristall bestand, validiert.
Lambda-DNA Doppelstränge wurden zwischen benachbarten Interdigitalelektroden gestreckt und temporär ausgerichtet. Die Ausrichtung von interkalierten PicoGreen-Molekülen im rechten Winkel zur Längsachse der Doppelstränge konnte hier gezeigt werden. Zudem konnte die ausgerichtete Immobilisierung des enhanced Green Fluorescent Protein nachgewiesen werden, indem die Fluoreszenz des Proteins und seine Struktureigenschaften ausgenutzt wurden. Aus den Messungen konnte der Winkel des Chromophors relativ zur Proteinlängsachse mit guter Übereinstimmung mit Röntgenkristallstrukturdaten bestimmt werden.
Eine permanente Immobilisierung mit gleichzeitiger Ausrichtung der Proteine wurde entlang der Kanten, an den Spitzen und auf der Oberfläche von Interdigitalelektroden erzielt. Damit wurde zum ersten Mal eine ausgerichtete Immobilisierung von Proteinen mit elektrischen Wechselfeldern gezeigt.
Diese Methode ist vielversprechend für die Immobilisierung von Antikörpern oder Enzymen mit einheitlicher Ausrichtung und dadurch verbessertem Zugang zu den aktiven Zentren, was nicht nur für die industrielle Biosensorherstellung von Interesse ist, sondern genauso für allgemeine Wechselwirkungsstudien von Proteinen.
KW - dielectrophoresis
KW - electrokinetics
KW - proteins
KW - immobilization
KW - alignment
KW - Dielektrophorese
KW - elektrokinetische Effekte
KW - Proteine
KW - Immobilisierung
KW - Ausrichtung
Y1 - 2016
U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:517-opus4-90271
ER -
TY - JOUR
A1 - Fichtner, Franziska
A1 - Olas, Justyna Jadwiga
A1 - Feil, Regina
A1 - Watanabe, Mutsumi
A1 - Krause, Ursula
A1 - Hoefgen, Rainer
A1 - Stitt, Mark
A1 - Lunn, John Edward
T1 - Functional features of Trehalose-6-Phosphate Synthase 1
BT - an essential enzyme in Arabidopsis
JF - The Plant Cell
N2 - Tre6P synthesis by TPS1 is essential for embryogenesis and postembryonic growth in Arabidopsis, and appropriate Suc signaling by Tre6P is dependent on the noncatalytic domains of TPS1. In Arabidopsis (Arabidopsis thaliana), TREHALOSE-6-PHOSPHATE SYNTHASE1 (TPS1) catalyzes the synthesis of the sucrose-signaling metabolite trehalose 6-phosphate (Tre6P) and is essential for embryogenesis and normal postembryonic growth and development. To understand its molecular functions, we transformed the embryo-lethal tps1-1 null mutant with various forms of TPS1 and with a heterologous TPS (OtsA) from Escherichia coli, under the control of the TPS1 promoter, and tested for complementation. TPS1 protein localized predominantly in the phloem-loading zone and guard cells in leaves, root vasculature, and shoot apical meristem, implicating it in both local and systemic signaling of Suc status. The protein is targeted mainly to the nucleus. Restoring Tre6P synthesis was both necessary and sufficient to rescue the tps1-1 mutant through embryogenesis. However, postembryonic growth and the sucrose-Tre6P relationship were disrupted in some complementation lines. A point mutation (A119W) in the catalytic domain or truncating the C-terminal domain of TPS1 severely compromised growth. Despite having high Tre6P levels, these plants never flowered, possibly because Tre6P signaling was disrupted by two unidentified disaccharide-monophosphates that appeared in these plants. The noncatalytic domains of TPS1 ensure its targeting to the correct subcellular compartment and its catalytic fidelity and are required for appropriate signaling of Suc status by Tre6P.
KW - cyanobacterial sucrose-phosphatase
KW - trehalose 6-phosphate
KW - vegetative growth
KW - crystal-structure
KW - gene-expression
KW - thaliana
KW - metabolism
KW - phosphorylation
KW - reveals
KW - proteins
Y1 - 2020
U6 - https://doi.org/10.1105/tpc.19.00837
SN - 0032-0781
SN - 1471-9053
VL - 32
IS - 6
SP - 1949
EP - 1972
PB - Oxford University Press
CY - Oxford
ER -
TY - GEN
A1 - Fichtner, Franziska
A1 - Olas, Justyna Jadwiga
A1 - Feil, Regina
A1 - Watanabe, Mutsumi
A1 - Krause, Ursula
A1 - Hoefgen, Rainer
A1 - Stitt, Mark
A1 - Lunn, John Edward
T1 - Functional features of Trehalose-6-Phosphate Synthase 1
BT - an essential enzyme in Arabidopsis
T2 - Zweitveröffentlichungen der Universität Potsdam : Mathematisch-Naturwissenschaftliche Reihe
N2 - Tre6P synthesis by TPS1 is essential for embryogenesis and postembryonic growth in Arabidopsis, and appropriate Suc signaling by Tre6P is dependent on the noncatalytic domains of TPS1. In Arabidopsis (Arabidopsis thaliana), TREHALOSE-6-PHOSPHATE SYNTHASE1 (TPS1) catalyzes the synthesis of the sucrose-signaling metabolite trehalose 6-phosphate (Tre6P) and is essential for embryogenesis and normal postembryonic growth and development. To understand its molecular functions, we transformed the embryo-lethal tps1-1 null mutant with various forms of TPS1 and with a heterologous TPS (OtsA) from Escherichia coli, under the control of the TPS1 promoter, and tested for complementation. TPS1 protein localized predominantly in the phloem-loading zone and guard cells in leaves, root vasculature, and shoot apical meristem, implicating it in both local and systemic signaling of Suc status. The protein is targeted mainly to the nucleus. Restoring Tre6P synthesis was both necessary and sufficient to rescue the tps1-1 mutant through embryogenesis. However, postembryonic growth and the sucrose-Tre6P relationship were disrupted in some complementation lines. A point mutation (A119W) in the catalytic domain or truncating the C-terminal domain of TPS1 severely compromised growth. Despite having high Tre6P levels, these plants never flowered, possibly because Tre6P signaling was disrupted by two unidentified disaccharide-monophosphates that appeared in these plants. The noncatalytic domains of TPS1 ensure its targeting to the correct subcellular compartment and its catalytic fidelity and are required for appropriate signaling of Suc status by Tre6P.
T3 - Zweitveröffentlichungen der Universität Potsdam : Mathematisch-Naturwissenschaftliche Reihe - 1432
KW - cyanobacterial sucrose-phosphatase
KW - trehalose 6-phosphate
KW - vegetative growth
KW - crystal-structure
KW - gene-expression
KW - thaliana
KW - metabolism
KW - phosphorylation
KW - reveals
KW - proteins
Y1 - 2020
U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:517-opus4-516532
SN - 1866-8372
IS - 6
ER -
TY - JOUR
A1 - Deutschmann, Claudia
A1 - Roggenbuck, Dirk
A1 - Schierack, Peter
A1 - Rödiger, Stefan
T1 - Autoantibody testing by enzyme-linked immunosorbent assay-a case in which the solid phase decides on success and failure
JF - Heliyon
N2 - Background: The enzyme-linked immunosorbent assay (ELISA) is an indispensable tool for clinical diagnostics to identify or differentiate diseases such as autoimmune illnesses, but also to monitor their progression or control the efficacy of drugs. One use case of ELISA is to differentiate between different states (e.g. healthy vs. diseased). Another goal is to quantitatively assess the biomarker in question, like autoantibodies. Thus, the ELISA technology is used for the discovery and verification of new autoantibodies, too. Of key interest, however, is the development of immunoassays for the sensitive and specific detection of such biomarkers at early disease stages. Therefore, users have to deal with many parameters, such as buffer systems or antigen-autoantibody interactions, to successfully establish an ELISA. Often, fine-tuning like testing of several blocking substances is performed to yield high signal-to-noise ratios.
Methods: We developed an ELISA to detect IgA and IgG autoantibodies against chitinase-3-like protein 1 (CHI3L1), a newly identified autoantigen in inflammatory bowel disease (IBD), in the serum of control and disease groups (n = 23, respectively). Microwell plates with different surface modifications (PolySorp and MaxiSorp coating) were tested to detect reproducibility problems.
Results: We found a significant impact of the surface properties of the microwell plates. IgA antibody reactivity was significantly lower, since it was in the range of background noise, when measured on MaxiSorp coated plates (p < 0.0001). The IgG antibody reactivity did not differ on the diverse plates, but the plate surface had a significant influence on the test result (p = 0.0005).
Conclusion: With this report, we want to draw readers' attention to the properties of solid phases and their effects on the detection of autoantibodies by ELISA. We want to sensitize the reader to the fact that the choice of the wrong plate can lead to a false negative test result, which in turn has serious consequences for the discovery of autoantibodies.
KW - biochemistry
KW - coatings
KW - surface chemistry
KW - immunology
KW - proteins
KW - laboratory medicine
KW - clinical research
KW - enzyme-linked immunosorbent
KW - assay
KW - biomarker discovery
KW - reproducibility
KW - solid-phase
KW - autoantibody
Y1 - 2020
U6 - https://doi.org/10.1016/j.heliyon.2020.e03270
SN - 2405-8440
VL - 6
IS - 1
PB - Elsevier
CY - London [u.a.]
ER -