1. The effect of preconditioning strategies on the adsorption of model proteins onto screen-printed carbon electrodesTea Romih, Ivan Konjević, Lea Žibret, Ika Fazarinc, Ajda Beltram, David Majer, Matjaž Finšgar, Samo B. Hočevar, 2022, original scientific article Abstract: The preconditioning and modification of the supporting electrode surface is an essential
step in every biosensor architecture. In particular, when using screen-printed carbon electrodes (SPEs)
as inexpensive and convenient disposable sensor substrates, their somewhat lower electrochemical
(surface) reproducibility might represent a complex hurdle. Herein, we investigated the effect of
selected preconditioning strategies, such as cyclic voltammetric pretreatment, in H2SO4 and H2O2
and plasma pretreatment with a positive and negative glow discharge, which all improved the electrochemical stability of the unmodified SPEs. Furthermore, we studied the influence of preconditioning
strategies on the adsorption kinetics of the two most commonly used building blocks for biosensor
preparation, i.e., bovine serum albumin (BSA) and protein A. We observed an advantageous effect
of all the examined preconditioning strategies for the modification of SPEs with protein A, being
the most effective the negative glow discharge. On the other hand, BSA exhibited a more complex
adsorption behavior, with the negative glow discharge as the only generally beneficial preconditioning strategy providing the highest electrochemical stability. Protein A revealed a more substantial
impact on the electrochemical signal attenuation than BSA considering their same concentrations in
the modification solutions. For both BSA and protein A, we showed that the concentrations of 5 and
10 µg mL−1 already suffice for an electrochemically satisfactorily stable electrode surface after 60 min
of incubation time, except for BSA at the positive-plasma-treated electrode.
Keywords: screen-printed carbon electrode, SPE, electrochemical biosensor, adsorption, bovine serum albumin, protein A, glow discharge
Published in DKUM: 15.05.2025; Views: 0; Downloads: 0
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2. Protein stacking on the APTES-functionalized pyrochlore ▫$Bi_2Ru_2O_7$▫ clusters for ultrasensitive and selective immunosensingNikola Tasić, Nika Vranešič, Dino Metarapi, Kristina Mervič, Milan Žunić, Aleksandra Dapčević, Matjaž Finšgar, Samo B. Hočevar, 2025, original scientific article Abstract: With their unique physicochemical properties, such as metallic-like conductivity, favorable (electro)catalytic properties, electrochemical stability, and ease of functionalization, pyrochlores have found applications in various fields such as solid oxide fuel cells, batteries, thick film resistors, and temperature sensors; however, there are no reports on their application in electrochemical immunosensing. In this study, we exploited the (electro)catalytic nature and stability of the pyrochlore Bi2Ru2O7 clusters silanized with (3-aminopropyl)triethoxysilane (APTES) to demonstrate their potential for the effective stacking of functional proteins. Characterization of the clusters by XPS disclosed a dual environment of Bi, also indicating the presence of Bi2O3 alongside APTES-Bi2Ru2O7 clusters and, importantly, the predominant involvement of pyrochlore moieties in subsequent protein stacking. After stacking protein A and antibodies, the immunosensor revealed a nearly interference-free operation, high sensitivity, a detection limit of 118 fM SARS-CoV-2 spike protein, and operation in a wide examined concentration range of 10−5−10−1 μg mL−1 with an r2 of 0.98. In combination with a short incubation time of 30 min, the pyrochlore-based immunosensor provides a solid platform for future point-of-need applications.
Keywords: Bi2Ru2O7 pyrochlore, APTES, SARS-CoV-2, spike protein, immunosensors, electrochemical
Published in DKUM: 23.04.2025; Views: 0; Downloads: 1
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3. Enhancing Martini3 for protein self-interaction simulationsJonas Binder, Matja Zalar, Martin Huelsmeyer, Michael Siedler, Robin Curtis, Wolfgang Frieß, 2025, original scientific article Abstract: Coarse-grained molecular dynamics simulations are highly valuable for studying protein-protein interactions. Unfortunately, commonly used force fields often overestimate these interactions. Here, we investigate the performance of the Martini 3 force field in predicting the self-interaction behavior of lysozyme and subtilisin using Metadynamics. The original Martini 3, despite improvements over its predecessor, overestimates interaction strength. Through reparameterization of bead interactions, we achieve good agreement with experimental data of the second virial coefficient and the diffusion coefficient. The new, refined force field enables more accurate CG-MD simulations, with potential applications in understanding and prediction of protein stability, aggregation tendencies, and solubility, with the possibility to aid in the development of protein-based drugs.
Keywords: coarse-grained molecular dynamics, Martini 3 force field, protein-protein interactions, B22, reparameterization, NMR, diffusion coefficient
Published in DKUM: 20.03.2025; Views: 0; Downloads: 2
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4. Alginate coatings suppress unspecific protein adhesion on PVCMatej Bračič, Alenka Vesel, Lidija Fras Zemljič, 2025, original scientific article Abstract: This study involved oxygen plasma activation of PVC and its effect on alginate coating adhesion, analyzed using a quartz crystal microbalance with dissipation (QCM-D). The results demonstrated improved adhesion of alginate on plasma-activated PVC due to an increase of oxidized functional groups. Wettability measurements, conducted using optical contact angle goniometry and QCM-D, showed a significant increase in the wettability of PVC after alginate coating, with contact angles decreasing from 81.4 ± 1.1◦ to 37.3 ± 3.1◦ 3D-profilometry mapping revealed a uniform distribution of alginate on the PVC film. Real-time antifouling studies using QCM-D with two model proteins, bovine serum albumin and bovine fibrinogen, were performed to elucidate the surface interactions involved in protein inhibition. The results indicated a substantial reduction in protein adhesion on alginate-coated PVC. This reduction in protein adhesion is attributed to the high hydrophilicity of the alginate coatings and partial removal of the alginate, which together contribute to poor protein adhesion. This comprehensive approach advances the development of effective solutions for mitigating biofilm-related issues on PVC-based medical devices, offering significant implications for improving patient outcomes.
Keywords: alginate, coatings, polyvinyl chloride, biofouling, quartz crystal microbalance, protein adhesion
Published in DKUM: 10.03.2025; Views: 0; Downloads: 10
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5. Why? - Successful Pseudomonas aeruginosa clones with a focus on clone CChanghan Lee, Jens Klockgether, Sebastian Fischer, Janja Trček, Burkhard Tümmler, Ute Römling, 2020, review article Abstract: The environmental species Pseudomonas aeruginosa thrives in a variety of habitats. Within the epidemic population structure of P. aeruginosa, occassionally highly successful clones that are equally capable to succeed in the environment and the human host arise. Framed by a highly conserved core genome, individual members of successful clones are characterized by a high variability in their accessory genome. The abundance of successful clones might be funded in specific features of the core genome or, although not mutually exclusive, in the variability of the accessory genome. In clone C, one of the most predominant clones, the plasmid pKLC102 and the PACGI-1 genomic island are two ubiquitous accessory genetic elements. The conserved transmissible locus of protein quality control (TLPQC) at the border of PACGI-1 is a unique horizontally transferred compository element, which codes predominantly for stress-related cargo gene products such as involved in protein homeostasis. As a hallmark, most TLPQC xenologues possess a core genome equivalent. With elevated temperature tolerance as a characteristic of clone C strains, the unique P. aeruginosa and clone C specific disaggregase ClpG is a major contributor to tolerance. As other successful clones, such as PA14, do not encode the TLPQC locus, ubiquitous denominators of success, if existing, need to be identified.
Keywords: disaggregase, FtsH, genomic island, protein homeostasis, pulsed field gel electrophoresis, whole genome sequencing
Published in DKUM: 28.01.2025; Views: 0; Downloads: 5
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6. Določevanje vpliva multivalentnih fosfatnih in sulfonatnih pomožnih snovi na agregacijo proteinov za uporabo v biofarmacevtskih izdelkih : diplomsko delo univerzitetnega študijskega programa I. stopnjeTjaša Krepek, 2024, undergraduate thesis Abstract: V zadnjih desetletjih se je biofarmacevtska industrija osredotočila na razvoj bioloških zdravil. Večinoma gre za kompleksne beljakovinske molekule, ki so zahtevne in drage za izdelavo ter imajo omejen rok uporabe, ki je posledica nestabilnosti samih beljakovin. Na nestabilnost proteinov in agregacijo lahko vplivamo z dodatkom malih molekul in pomožnih snovi kot so površinsko aktivne snovi, pufri, sladkorji, soli itd., ki se na protein neposredno vežejo in ga tako stabilizirajo. Dosedanje raziskave so že preučile vpliv adenozin tripolifosfata in tripoli fosfata na agregacijo biofarmacevtskih proteinov, zato smo v diplomskem delu preučili še vpliv petih drugih že priznanih fosfatnih ionov in za primerjavo izbrali še deset ATP- ju podobnih sulfonatnih spojin, saj so polifosfati nagnjeni k hidrolizi in posledično niso primerni za uporabo kot pomožne snovi. Uporabili smo modelni protein lizocim kokošjega beljaka ter preverjali vpliv pomožnih snovi pri različnih koncentracijah na kinetiko agregacije proteina, ki smo jo spremljali z UV- Vis spektrometrijo. S pomočjo UV-Vis spektrometrije smo opravili tudi presejalni test pomožnih snovi na agregacijo proteina in izračunali delež monomernega proteina. Ugotovili smo, da fosfatne pomožne snovi in večina sulfonatnih učinkovito zaščitijo in stabilizirajo protein in ga tako zaščitijo pred agregacijo kot je ta podvržen temperaturnemu stresu ter ga zaščitijo pred agregacijo v daljšem časovnem obdobju, prav tako tudi zmanjšajo delež monomernega proteina v vzorcu. Preverili smo tudi vpliv pomožnih snovi na agregacijo delno razvitih beljakovin, kar smo dosegli z dodatkom ditiotreitola ali DTT, ki cepi disulfidne mostičke v beljakovini. V povprečju se je izkazalo, da dajejo boljše rezultate fosfatne pomožne snovi in ne sulfonatne. Najboljše rezultate sta med pomožnimi snovmi dala ATP in TPP, preostale pomožne snovi pa dale primerljive rezultate.
Keywords: protein, agregacija, pomožne snovi, fosfati, sulfonati, fazni diagram
Published in DKUM: 01.10.2024; Views: 0; Downloads: 22
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7. Preučevanje interakcij med tanini in lizocimom : diplomsko delo univerzitetnega študijskega programa I. stopnjeAnja Zaverla, 2024, undergraduate thesis Abstract: Razvoj odpornosti bakterij na antibiotike je eden izmed večjih izzivov človeštva, ki zahtevajo tako iskanje novih protibakterijskih učinkovin, kot tudi izboljšanih načinov uporabe protibakterijskih sredstev. Raziskovalci nove protibakterijske učinkovine iščejo tudi med naravnimi spojinami s protimikrobnimi in protibakterijskimi učinki. Rastline vsebujejo tanine, ki so polifenolni sekundarni metaboliti, ki jih raziskujemo zaradi njihovih številnih potencialnih za zdravje ugodnih učinkov. Glede na njihovo kemijsko strukturo jih delimo na proantocianidine (kondenzirane tanine) in hidrolizirajoče tanine. Slednji se nadalje razdelijo na dve glavni podskupini (galotanin in elagitanini).
V diplomskem delu smo proučevali vezavo elagitaninov na lizocim – modelni protein. Po dializi v pufru s pH = 5 smo lizocim titrirali z galno kislino, metil galatom in veskalaginom. Opazovali smo, kako prisotnost elagitaninov vpliva na fluorescenco lizocima. Vpliv dodatka taninov na sekundarno strukturo lizocima smo opazovali s cirkularnim dikroizmom (CD-spektroskopija), medtem ko smo termodinamiko vezave opazovali z izotermno titracijsko kalorimetrijo (ITC). Z opazovanjem temperaturne odvisnosti fluorescenčnega spektra lizocima smo ugotovili, da prisotnost taninov ne vpliva na temperaturo tališča proteina. S pomočjo Stern-Volmerjeve enačbe ob upoštevanju učinka notranjega filtra smo poskusili pridobiti konstante vezave taninov na lizocim. S CD-spektroskopijo smo ugotovili, da ima veskalagin večji vpliv na denaturacijo proteina, kot metil galat in galna kislina. Ugotovili smo, da je interakcija veskalagina in lizocima mnogo bolj eksotermna, kot interakcija metil galata ali galne kisline z lizocimom. Poleg tega smo z ITC-jem potrdili, da gre pri interakcijah taninov z lizocimom za razmeroma šibke interakcije.
Keywords: elagitanini, protein, konstanta vezave, sekundarna struktura, spektroskopija, termodinamični parametri vezave
Published in DKUM: 17.09.2024; Views: 0; Downloads: 45
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8. Study of V[sub]2CT[sub]x-MXene Based Immunosensor for Sensitive Label-Free Impedimetric Detection of SARS-CoV-2 Spike ProteinNikola Tasić, Ivan Konjević, Alnilan Cristina Barros Lobato, Dino Metarapi, Matjaž Finšgar, Filipa M. Oliveira, Zdeněk Sofer, Rui Gusmão, Xueji Zhang, Samo B. Hočevar, 2024, original scientific article Abstract: Rapid and reliable immunosensing is undoubtedly one of the priorities in the efficient management and combat against a pandemic, as society has experienced with the SARS-CoV-2 outbreak; simple and cost-effective sensing strategies are at the forefront of these efforts. In this regard, 2D-layered MXenes hold great potential for electrochemical biosensing due to their attractive physicochemical properties. Herein, we present a V$_2$CT$_x$ MXene-based sensing layer as an integral part of a label-free immunosensor for sensitive and selective detection of the SARS-CoV-2 spike protein. The sensor was fabricated on a supporting screen-printed carbon electrode using Nafion as an immobilizing agent for MXene and glutaraldehyde, the latter enabling effective binding of protein A for further site-oriented immobilization of anti-SARS-CoV-2 antibodies. A thorough structural analysis of the sensor architecture was carried out, and several key parameters affecting the fabrication and analytical performance of the immunosensor were investigated and optimized. The immunosensor showed excellent electroanalytical performance in combination with an impedimetric approach and exhibited a low detection limit of only 45 fM SARS-CoV-2 spike protein. Its practical applicability was successfully demonstrated by measuring the spike protein in a spiked artificial nasopharyngeal fluid sample.
Keywords: imunosenzorji, polprevodniške nanostrukture, proteini, spike protein, immunosensors, SARS-CoV-2, V2CTxMXene
Published in DKUM: 29.08.2024; Views: 42; Downloads: 4
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9. Novel magnetic iron oxide-dextran sulphate nanocomposites as potential anticoagulants: Investigating interactions with blood components and assessing cytotoxicityOlivija Plohl, Lidija Fras Zemljič, Boštjan Vihar, Alenka Vesel, Sašo Gyergyek, Uroš Maver, Irena Ban, Matej Bračič, 2024, original scientific article Abstract: Examining the critical role of anticoagulants in medical practice, particularly their central function in preventing abnormal blood clotting, is of the utmost importance. However, the study of interactions between blood proteins and alternative anticoagulant nano-surfaces is still understood poorly. In this study, novel approach involving direct functionalisation of magnetic iron oxide nanoparticles (MNPs) as carriers with sulphated dextran (s-dext) is presented, with the aim of evaluating the potential of magnetically-responsive MNPs@s-dext as anticoagulants. The physicochemical characterisation of the synthesised MNPs@s-dext includes crystal structure analysis, morphology study, surface and electrokinetic properties, thermogravimetric analysis and magnetic properties` evaluation, which confirms the successful preparation of the nanocomposite with sulfonate groups. The anticoagulant potential of MNPs@s-dext was investigated using a standardised activated partial thromboplastin time (APTT) test and a modified APTT test with a quartz crystal microbalance with dissipation (QCM-D) which confirmed the anticoagulant effect. Time-resolved solid-liquid interactions between the MNPs@s-dext and model blood proteins bovine serum albumin and fibrinogen were also investigated, to gain insight into their hemocompatibility, and revealed protein-repellence of MNPs@s-dext against blood proteins. The study also addressed comprehensive cytotoxicity studies of prepared nanocomposites, and provided valuable insights into potential applicability of MNPs@s-dext as a promising magnetic anticoagulant in biomedical contexts.
Keywords: dextran sulphate, magnetic nanoparticles, blood protein interactions, clot formation, anticoagulants, cytotoxicity studies
Published in DKUM: 25.07.2024; Views: 107; Downloads: 28
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10. Droplet-based screening of phosphate transfer catalysis reveals how epistasis shapes MAP kinase interactions with substratesRemkes A. Scheele, Laurens H. Lindenburg, Maya Petek, Markus Schober, Kevin N. Dalby, Florian Hollfelder, 2022, original scientific article Abstract: The combination of ultrahigh-throughput screening and sequencing informs on function and intragenic epistasis within combinatorial protein mutant libraries. Establishing a droplet-based, in vitro compartmentalised approach for robust expression and screening of protein kinase cascades (>107 variants/day) allowed us to dissect the intrinsic molecular features of the MKK-ERK signalling pathway, without interference from endogenous cellular components. In a six-residue combinatorial library of the MKK1 docking domain, we identified 29,563 sequence permutations that allow MKK1 to efficiently phosphorylate and activate its downstream target kinase ERK2. A flexibly placed hydrophobic sequence motif emerges which is defined by higher order epistatic interactions between six residues, suggesting synergy that enables high connectivity in the sequence landscape. Through positive epistasis, MKK1 maintains function during mutagenesis, establishing the importance of co-dependent residues in mammalian protein kinase-substrate interactions, and creating a scenario for the evolution of diverse human signalling networks.
Keywords: protein, epistasis, catalysis, phosphate transfer, MAP kinase
Published in DKUM: 04.07.2024; Views: 104; Downloads: 13
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