Bioanalytical chemistry

OBJECTIVES: Immunochemical assays combine the specificity of antibodies with the sensitivity of various kinds of readout methods to allow detecting target molecules. The great flexibility, provided by the possibility to use antibodies specific to desired structure, allows detecting all sorts of analytes, from small molecules, through proteins, to viruses and bacteria. The aim of this thesis is the development of advanced immunochemical approaches for use in clinical diagnostics.

FOCUS: The research will focus on clinically relevant targets, including biomarkers (prostate cancer, breast cancer, acute myocardial infarction) and pathogens (Salmonella, SARS-CoV-2). In all these cases, the highly sensitive detection with low cross-reactivity is essential for rapid diagnosis, allowing early disease treatment.

METHODS: The particular choice of the detection method will be made with respect to the target analyte and sample matrix to maximize the potential for practical applications. The first tests will be carried out using conventional enzyme immunoassays due to their simplicity and robustness. Afterward, various ways to improve the assay performance will be explored. Nanoparticle-based labels will be exploited to allow detection based on their catalytic (e.g., Prussian blue nanoparticles) or luminescence (e.g., photon-upconversion nanoparticles or quantum dots) properties. The sample preconcentration will be carried out using magnetic micro- or nanoparticles. Furthermore, the potential for point-of-care analysis will be demonstrated on lateral flow immunoassays and optical or electrochemical biosensors. The close collaboration with the Faculty of Medicine and industrial partners promises that apart from writing scientific publications, the developed methods can also find commercial applications.

LITERATURE: https://www.muni.cz/en/people/357740-zdenek-farka/publications

PLEASE NOTE: Before initiating the formal application process to doctoral studies, the interested candidates should contact Zdeněk Farka (farka@mail.muni.cz) for an informal discussion.

Supervisor

doc. Mgr. Zdeněk Farka, Ph.D.

Experimentální část práce bude zahrnovat vývoj nových mikroextrakčních technik, které jsou založeny na selektivních přechodech analytů přes semi-permeabilní fázová rozhraní [1,2]. Při přechodu analytů bude využito difuze [1] nebo bude přechod urychlen účinkem elektrického pole [2]. Výsledné mikroextrakční techniky budou spojeny off-line nebo in-line s vhodnými analytickými metodami (primárně s kapilární elektroforézou) a adekvátnost takového spojení bude demonstrována analýzami biologicky, klinicky a toxikologicky významných analytů v reálných komplexních vzorcích jako je moč, krevní sérum/plasma a plná krev. [1] Kubáň, P., Boček, P., J. Chromatogr. A 1234 (2012) 2-8. [2] Kubáň, P., Šlampová, A., Boček, P., Electrophoresis 31 (2010) 768-785.

Supervisor

RNDr. Pavel Kubáň, DSc.

OBJECTIVES: Development of instrumentation and methods for comprehensive and/or selective enrichment and analysis of nucleic acids, proteins and metabolites. The work will include electrophoretic and mass spectrometric instrumentation and will be focused on instrumental as well as methodological development of new techniques based on preliminary results obtained with an industrial partner.

FOCUS: Proof of principles of epitachophoresis for biology related samples; design and construction/modification of instrumentation for large volume sample processing. The topic is based on entirely new procedures currently in the state of patent application in the USA.

METHODS: epitachophoresis, MS, capillary electrophoresis and HPLC in combination with different detection techniques

LITERATURE: see WOS.

PLEASE NOTE: before initiating the formal application process to doctoral studies, all interested candidates should contact Ing. František Foret, CSc., e-mail: foret@iach.cz at CEITEC or Instute of nalytical Chemistry Academy of Science Czech Republic for informal discussion.

Supervisor

Ing. František Foret, DSc.

Hyaluronan(HA) je lineární polysacharid z disacharidických jednotek kyseliny D-glukuronové a D-N-acetylglukosaminem. HA je štěpen na kratší fragmenty dvěma typy enzymů. Savčí hyaluronidázy hydrolyticky štěpí řetězce za vzniku menších fragmentů z nezměněných monosacharidů. Mikroorganismy produkují hyaluronan lyázy, které rovněž štěpí řetězce hyaluronanu beta–eliminační reakcí, při které na neredukujícím konci fragmentu vzniká kyselina glukuronová s dvojnou vazbu mezi C4 a C5. Houba Talaromyces stipitatus produkuje hyaluronidázu (TsHr), která štěpí hydrolyticky 1,4 glykosidickou vazbu. Ačkoli svojí primární strukturou je savčím hyaluronidázám podobná jen vzdáleně, patří do rodiny hydroláz glykosidických vazeb GH16, konečnými produkty jsou krátké fragmenty ze 4 až 6 monosacharidů. Předběžné výsledky s rekombinantní TsHr ukazují, že dokáže štěpit deriváty HA savčím enzymem neštěpitelné. TsHr je strukturně příbuzný s transglykosylázami, které patří do stejné rodiny. To by mohlo být využitelná v chemoenzymatických syntézách derivátů HA s definovanou polohou substituentů.
Cílem první části studia bude přispět k poznání TsHr po stránce kinetiky i mechanismu enzymatické reakce a porovnat získané výsledky se savčí bovinní testikulární hyaluronidázou. Bude studována inhibice TsHr různě dlouhými a modifikovanými fragmenty hyaluronanu. Poté by mělo být rozhodnuto, zda enzym TsHr spolu s dalšími enzymy bude využitelný při stanovování rozložení substituentů na řetězci derivátů HA. U savčích hydroláz byla prokázána transglykosylační aktivita - spojování kratších fragmentů HA na delší řetězce. Nikdo však neprokázal, že je to možné u fragmentů substituovaných vybranými substituenty v určitých polohách. Pokud se prokáže, že enzym TsHr není tak citlivý na substituci hyaluronanu jako savčí enzymy, bude zkoumána transglykosylační aktivita a případně hledány podmínky pro využití při syntéze derivátů HA.
Enzym štěpí vysokomolekulární hyaluronan na kratší fragmenty, čímž vytváří koktejl kratších fragmentů hyaluronanu v reakční směsi. Tyto nově vzniklé fragmenty jsou rovněž štěpeny hyaluronidázou, avšak pravděpodobně s jinými kinetickými parametry. Ve výsledku by se tento stav mohl jevit jako inhibice substrátem/produktem. V savčích organismech by toto mohla být cesta regulující rychlost degradace hyaluronanu ve tkáních. Toto by mohlo přispět jednak k potvrzení potenciální regulační role fragmentů a k poznání rozdílů mezi houbovým a savčím enzymem. Navíc v literatuře se popisují interakce různých fragmentů HA se savčím enzymem, které se odehrávají mimo aktivní centrum - projevuje se změnou kinetických parametrů.

Literatura:
L. Bobková, D. Smirnou, M. Krčmář, J. Kulhák, M. Hermannová, L. Franke, V. Velebný (2018) Discovery and characteristic of hyaluronidases from filamentous fungi. Current Biotechnology 7: 2.

Supervisor

prof. RNDr. Petr Skládal, CSc.

OBJECTIVE: Neurodegenerative foldopathies represent a group of human protein-misfolding disorders that are characterized by a pathological alteration in conformation of a native protein which makes it resistant to degradation and leads to pathological gain and loss of function. These are followed by aggregation of the misfolded proteins into insoluble deposits. One of the most prominent protein-misfolding disorders is AD. The number of patients suffering dementia in the Czech Republic is estimated at 160 thousand. Up to two-thirds of cases are due to Alzheimer's disease, others include, for example, vascular dementia, Parkinson's disease or other degenerative brain disorders.

FOCUS: In neurodegenerative disorders (including Alzheimer's disease, AD) astrocytes/astroglia undergo complex changes that range from atrophy with loss of function to accumulation of reactive cells around disease-specific lesions (senile plaques in the case of AD). The cellular pathology of astrocytes in the context of human AD remains enigmatic; mainly because of severe limitations of animal models, which, although reproducing some pathological features of the disease, do not mimic its progression in full. The human induced pluripotent stem cells (hiPSCs) technology creates a novel and potentially revolutionizing platform for studying fundamental mechanisms of the disease and for screening to identify new therapeutic compounds.

EXAMPLE of a potential doctoral project - the student will focus on: Suitable procedures for studying early pathology of Alzheimer's disease (AD) are currently being sought. The main aim of the project is to create a model of cell cultures suitable for monitoring changes in the biology of nerve cells (neurons and astrocytes) in patients with AD. Using the induced pluripotent cell method (iPSC), the role of nerve cells in the formation of sporadic form of AD will be studied.

The specific aims are following:
• Analysis of DEGs (differently expressed genes) between patients and controls and measured physiological parameters in and between groups.
• Analysis of DEGs (differently expressed genes) between patients and controls and measured physiological parameters in and between groups.
• Verification of selected differences in genes expression by qPCR analysis.
• Verification of very promising candidate genes expression by Western Blot.
• Analysis of selected neurotransmitters by LC-MS/MS.

PLEASE NOTE: Before initiating the formal application process to doctoral studies, the candidate is required to contact Assoc. Prof. Jan Lochman for an informal discussion.

Supervisor

doc. Mgr. Jan Lochman, Ph.D.

OBJECTIVES: Use metal nanoparticles as labels for sensitive mass spectrometry imaging. The method allows detection of a single molecule on a tissue section.

EXAMPLES of doctoral projects:

- Development of sample preparation protocol for specific detection of selected markers on sections of 3D cell aggregates or other tissues.

- Optimization of specific labelling with nanoparticles. The specificity will be based on antibody-antigen and avidin-biotin interactions, aptamer bindings etc.

- Development of nanoparticle detection schemes using inductively coupled plasma (ICP) and matrix-assisted laser desorption/ionization (MALDI) techniques.

- Study of nanoparticle transport efficiency in ICP MS. Confocal fluorescence or electron microscopy will be used as a reference method.

MORE INFORMATION: bart.chemi.muni.cz

Supervisor

prof. Mgr. Jan Preisler, Ph.D.

OBJECTIVES: The dissertation projects focus on matrix-assisted laser desorption ionization mass spectrometry imaging (MALDI MSI) for visualization of spatial distribution of biologically important compounds without chemical labelling.

EXAMPLES of doctoral projects:

- Development of sample preparation protocols for MALDI MSI. Samples may include 3D cell aggregates or other biological tissues.

- MALDI MSI of perifosine and other antitumor agents in 3D cell aggregates.

- Optimization of reactions on tissue sections. The aim will be, e.g., determination of double bond position in fatty acid chains in lipids.

MORE INFORMATION: bart.chemi.muni.cz

Supervisor

prof. Mgr. Jan Preisler, Ph.D.

OBJECTIVES: The advanced nanomaterials as quantum dots (QD), photon up-converting nanoparticles (UCNP) and coloid metal nanoparticles (Au, Ag, Cu NP) will be combined with sensing systems as waveguides, optical and Raman microscopic set-ups, surface plasmon resonance transducers and other options in order to develop biosensing devices with enhanced sensitivity. Both static evaluation - analog response and scanning / tracking / discriminating approaches - digital response for single-molecules (object, cell, nanoparticle) detection will be evaluated.

FOCUS: The target analytes will include metabolites and clinical markers in body fluids (serum, saliva), food and drinks (allergens, contaminants, toxins) and infectious microbes (honey bee pathogens, Salmonella, Enterobacteria).

METHODS: Bioconjugation and immobilization of biomolecules, characterization of bioconjugates and biointerfaces, ink-jet based microarrays, scanning probe microscopies, design of biosensing devices, advanced optical techniques, scanning probe microscopy, signal acquisition and processing, chemometrics, software development.

LITERATURE: https://www.muni.cz/en/people/2202-petr-skladal/publications

PLEASE NOTE: before initiating the formal application process to doctoral studies, all interested candidates should contact Petr Skladal (skladal@chemi.muni.cz) for informal discussion.

Supervisor

prof. RNDr. Petr Skládal, CSc.

Bioortogonální aktivace pro-léčiv je nový přístup v terapii nádorových onemocnění. Spočívá v katalytické aktivaci léčiva se zablokovanou funkční skupinou přímo v nádorové tkáni. V naší laboratoři se zabýváme aktivitou organokovových komplexu paladia a ruthenia v biologicky relevantních podmínkách. Pro studium katalytické aktivity jsou často využívány fluorescenční sondy, které lze také využít pro detekci paládiové kontaminace ve farmaceutických přípravcích. Z různých důvodů (buněčná toxicita, buněčný efflux) je mnoho standardních fluorescenčních látek nepoužitelných pro dlouhodobé studium katalytické aktivity organokovových sloučenin. Úkolem studenta bude podílet se na přípravě modifikovaných fluorescenčních sond se sníženou toxicitou a zvýšenou retenci v buňkách. Bude studovat citlivost vyvinutých sond k paladiu a dalším kovovým komplexům. Dále se bude podílet na syntéze a charakterizaci nových organokovových katalytických komplexů a evaluaci jejich katalytické aktivity v buňkách. Práce bude metodologicky obšírná a bude zahrnovat techniky organické syntézy, purifikace látek, biochemické analýzy a techniky tkáňových kultur.

Supervisor

Mgr. Vladimír Pekařík, Dr.

Our research is focused on the development of new experimental methods in bioanalytical chemistry that enable highly sensitive measurements. We employ surface-modified photon-up-converting nanoparticles, which can be detected under near-infrared excitation light without any background interference even at the single nanoparticle level. With our broad repertoire of methods, we detect pathogens, cancer markers and environmental toxins and analyze the function of enzymes.
Objectives: We will use UCNPs as a detection label for single-molecule (digital) immunoassays. While UCNPs enable the detection of analytes without any optical background noise, non-specific binding is still a challenge because it lowers the sensitivity of the immunoassays. Non-specific binding has conventionally been reduced by optimizing the surface architecture of UCNP labels and coating of microtiter plates. In this project, we will investigate an entirely new approach for the reduction of non-specific binding, which is only possible by using single-molecule immunoassays: If we use two labels showing different colors for the detection of one analyte, the presence of the analyte can be verified by two-color detection under the microscope; however, if there is only one color detectable, the signal can be attributed to non-specific binding. In this way, we aim to achieve the highest possible sensitivity for the detection of cancer markers and virus particles such as Covid-19.

Profile: High motivation and joy in working on new experimental approaches in bioanalytical chemistry. Willingness to use English communication in an international team.

We offer: Supportive environment for Interdisciplinary work in an international team. Work on state-of-the-art projects with high societal relevance. Publications in high-impact journals. Building an international network with various European groups.

Supervisor

Dr. rer. nat. habil. Hans-Heiner Gorris

The first aim of this project is to establish a new wide-field upocnversion microscope setup that enables the detection of Förster resonance energy transfer (FRET) between upconversion nanoparticles (UCNPs) and fluorescent acceptor dyes. This new detection scheme eliminates optical background interference and thus is well suitable for investigationg molecular interactions at the single molecule level. This new microscope technique lays the foundation for the second aim of the project - the exploration of several bioanalytical applications such as single-molecule DNA sequencing as well as as digital (single-molecule) immunoassays.

Notes

This project is supported by the Czech grant agency (GAČR) within the binational (Czech-Polish) project: https://gacr.cz/dalsich-sest-cesko-polskych-projektu-se-zacne-resit-pristi-mesic/

Supervisor

Dr. rer. nat. habil. Hans-Heiner Gorris

OBJECTIVES: Ambient mass spectrometry (MS) offers the versatile group of techniques providing analysis of solid sample surfaces and liquids in an open atmospheric pressure environment. It provides fast, direct analysis of objects without any sample pretreatment with the use of the mass spectrometer. Direct Analysis in Real Time (DART) and Desorption electrospray ionization (DESI) will be used in this project for the important clinical and pharmacological studies. As a comparative methods Direct Injection (DI) and MS combination with the separation methods HPLC a CE will be used.

FOCUS: The target analytes will include drugs and their metabolites and clinical markers in body fluids (blood, serum, saliva).

METHODS: DART-MS and DESI-MS, capillary electrophoresis and HPLC in combination with different detection techniques – MS, UV-VIS, LIF, C4CD. A new sampling technique based on the dry blood spot will be used.

LITERATURE: https://www.sci.muni.cz/en/about-us/faculty-staff/1865-zdenek-glatz/publications

GRANT PROJECTS: https://www.sci.muni.cz/en/about-us/faculty-staff/1865-zdenek-glatz/projects

PLEASE NOTE: before initiating the formal application process to doctoral studies, all interested candidates should contact Zdenek Glatz(glatz@chemi.muni.cz) for informal discussion.

Notes

Only one of the proposed theme will be finally occupied.

Supervisor

prof. RNDr. Zdeněk Glatz, CSc.

OBJECTIVES: Droplet microfluidics offers significant advantages for performing high-throughput screens and sensitive assays. Droplets allow sample volumes to be significantly reduced, leading to concomitant reductions in cost and increasing of assay sensitivity. It combines these powerful features to enable currently inaccessible high-throughput screening applications, including single-cell and single-molecule assays.

FOCUS: The subjects of the study will be medicinally and pharmacologically important enzymes and the screening of their enzymes.

METHODS: droplet microfluidic system with the LIF detection, MS, UV-VIS, LIF, C4CD. A new sampling technique based on the dry blood spot will be used. Capillary electrophoresis and HPLC in combination with different detection techniques – MS, UV-VIS, LIF, C4CD will be used as supporting methods.

LITERATURE: https://www.sci.muni.cz/en/about-us/faculty-staff/1865-zdenek-glatz/publications

GRANT PROJECTS: https://www.sci.muni.cz/en/about-us/faculty-staff/1865-zdenek-glatz/projects

PLEASE NOTE: before initiating the formal application process to doctoral studies, all interested candidates should contact Zdenek Glatz (glatz@chemi.muni.cz) for informal discussion.

Notes

Only one of the proposed theme will be finally occupied.

Supervisor

prof. RNDr. Zdeněk Glatz, CSc.