|
|
Analysis of Thick Film Amperometrical Sensors Signal and Its Usage for Measurement and Characterization of Enzymes
Ondruch, Vít ; Kizek, René (referee) ; Masojídek,, Jiří (referee) ; Vrba, Radimír (advisor)
V práci je popsán princip synchronní detekce (SD), který byl uplatněn při měření s biosenzory. Metoda SD umožňuje dosažení výrazně lepšího poměru signálu k šumu, vyššího limitu detekce a celkové zlepšení robustnosti měření. Uplatnění SD při měření s biosenzory umožní zlepšit analýzu jeho odezvy a umožní odstranění nežádoucích interferencí nebo šumů, které mohou být způsobeny například mícháním roztoku, elektromagnetickými vlivy nebo parazitními proudy. SD také umožňuje rozložit získaný signál na odezvu stimulace a na dlouhodobý signál jiného procesu, a dále také identifikovat jevy druhého řádu. Pro identifikaci stimulačního signálu ve výstupním signálu měření byl na základě lineárního statistického modelu vyvinut specializovaný software. SD byla ověřena na modelovém případu výstupního signálu biosenzoru s aplikovaným komplexem fotosystému II (PSII) a jeho odezvě na stimulaci světlem. Odezva PSII se řídí kinetikou prvního řádu a může být také ovlivněna inhibitory. Kinetické konstanty vazby herbicidu na PSII závisí lineárně na koncentraci herbicidu. To umožňuje jejich měření také při nízkých koncentracích herbicidu.
|
|
|
Isolation and purification of nucleic acids using magnetised micro- and nanoparticles
Dostálová, Simona ; Adam, Vojtěch (referee) ; Kizek, René (advisor)
Nucleic acids are a completely unique component of every organism. They determine the appearance, behaviour or disposition to a certain disease. Despite their importance, there is still a lot of information unknown. In order to study the nucleic acids, they need to be purified from other components of the organism. The routine methods of isolation and purification are often laborious and time consuming; therefore experiments with magnetic separation are carried out. This thesis deals with magnetic micro- and nanoparticles and their application in this field. General conditions of this isolation are summarized in the thesis. Detection methods of nucleic acids are listed, focusing on electrochemical methods. A procedure for isolation and purification of nucleic acids using magnetic particles and square-wave voltammetry is described. This procedure was modified in experimental part of the thesis to achieve the maximum yield. Partial steps including washing, immobilization and elution were optimized. Results showed that the phosphate buffer was optimal for washing of beads before immobilization of nucleic acids on their surface. The optimal immobilization was carried out for 5 minutes at 20 °C and microtube needed to be firmly shaken during the process. Immobilization solution composed of 0.1M Na2HPO4 + 0.1M NaH2PO4, 0.6M guanidinium thiocyanate, 0.15M Trizma base adjusted by HCl on pH 7.5 and 2.5M CsCl. 5M NaCl was optimal for washing of the complex “beads-nucleic acid” after the immobilization. Elution was carried out for 15 minutes at 99 °C and the highest yield was obtained using elution solution composed of Tris-EDTA pH at 9.1
|
|
|
New Approaches in Cardiac Optical Mapping
Švrček, Martin ; Kizek, René (referee) ; Sovka, Pavel (referee) ; Provazník, Ivo (advisor)
This dissertation deals with new approaches in cardiac optical mapping. The principle of cardiac optical mapping as well as the current research in this field was described. The new measurement system was developed and its characteristics presented. The system design allows epicardial and endocardial mapping, employing new ratiometric techniques in 2D acquisition and simultaneous electrical and optical mapping. The measured characteristics of fluorescent dye di-4-ANEPPSS were presented. The relation between movement and consequent motion artifacts is well described. Several new approaches in signal processing were proposed, including new ratiometry technique and using image registration to suppress motion artifacts. The algorithm for elastic image registration of optical signals and innovative method for verification of registration process were presented. Application of all proposed approaches and its results are included and discussed.
|
|
|
Nanotransporters for theranostics
Dostálová, Simona ; Adam,, Vojtěch (referee) ; Kizek, René (advisor)
Master thesis deals with the use of bacteriophage as a theranostic drug nanocarrier. The term theranostics is used in recent years for systems that allow connecting of diagnostics, targeted drug delivery and monitoring of patient’s response to administered treatment in a single modality. These systems are very suitable especially with heterogeneous diseases, such as cancer. Nowadays, the treatment of cancer has often severe side effects to the patient’s body, which lowers his capability to fight the disease. Theoretical part of this work is focused on the properties of viral capsids, proteins and inorganic materials as drug nanocarriers. In practical part of this work, different methods for cultivation of bacteriophage are compared, both in liquid and solid medium and with different concentrations of the maltose, trough whose receptors bacteriophage is able to enter the host cell. Optimal was cultivation with 0.2% maltose in solid medium. Practical part is focused mainly on the use of bacteriophage as a nanocarrier for cytotoxic drug doxorubicin. Bacteriophage was able to encapsulate all applied concentrations of doxorubicin (0; 12.5; 25; 50; 100 and 200 g/ml), which was proved using fluorescent detection. Different times of encapsulation (2; 4; 8 and 12 hours) were studied. Optimal time was 2 hours. Encapsulation properties of bacteriophage were compared to apoferritin. Bacteriophage was able to encapsulate 4× higher concentrations of doxorubicin and its release during rinsing with water was 10× lower compared to apoferritin. This work concludes that bacteriophage is a very suitable platform for targeted drug delivery in theranostics.
|
|
|
Study of the Regulation of NADP-Malic Enzyme (Decarboxylating) and Influence of Stress on this Enzyme
Doubnerová, Veronika ; Ryšlavá, Helena (advisor) ; Wilhelmová, Naďa (referee) ; Kizek, René (referee)
4. Conclusions l. Non-photosynthetic cytosolic isoform of NADP-mďic ilZ-oe (oxaloacetate decarboxylating) (NADp-ME; EC r.l.l.4o) was isolated from tobacco (Nicoriana tabacun L.) leaves. Specific aďiviý of the obtained enz5me preparation was 0.95pmol.miď|.mg l, relďive molecular mass of one subr'nit of tetramer approximatety 67000' pI 5'5 and pH optimum 7.r - 7.4. Kinetic constants of this isoform were determined for L-ma|ate and NADP+ in the presence of various cofactors Mď*, Mo*,co2', Ni2*, similarly the dependencies ofreaction Íates on the ooncentration ofdivalent metal ions were observed- The dependence of reaction rate on Mn2+ concentration wassigmoidal with strong positive cooperativiý and high value of Hill coefficient 7.5. -2r - J. 4. Summarv ofPh.D.Thesis 2. The kinetic meohanism of two-substate reaďion catalyzed by NADP.ME ůomtobacco leaves was determined as ordered sequmtial. The regulation of the NADP-ME purified ůomtobacco leaves occurs predominant|y in the prsence of macmergic compounds (GTP, ATP and ADP), sligbtly by metabolites of glymlysis. Biotic stress caused by potato yirus f G1;Y) (not only strain PWNN but also PWo) increased NADP.ME activiý in N. tabacum L. |eaves, stems and roots during the infeďion. Milder PVYo caused tower increase of NADP-ME aďivity than necrotic isolate of PVYNN....
|
|
|
Isolation and purification of nucleic acids using magnetised micro- and nanoparticles
Dostálová, Simona ; Adam, Vojtěch (referee) ; Kizek, René (advisor)
Nucleic acids are a completely unique component of every organism. They determine the appearance, behaviour or disposition to a certain disease. Despite their importance, there is still a lot of information unknown. In order to study the nucleic acids, they need to be purified from other components of the organism. The routine methods of isolation and purification are often laborious and time consuming; therefore experiments with magnetic separation are carried out. This thesis deals with magnetic micro- and nanoparticles and their application in this field. General conditions of this isolation are summarized in the thesis. Detection methods of nucleic acids are listed, focusing on electrochemical methods. A procedure for isolation and purification of nucleic acids using magnetic particles and square-wave voltammetry is described. This procedure was modified in experimental part of the thesis to achieve the maximum yield. Partial steps including washing, immobilization and elution were optimized. Results showed that the phosphate buffer was optimal for washing of beads before immobilization of nucleic acids on their surface. The optimal immobilization was carried out for 5 minutes at 20 °C and microtube needed to be firmly shaken during the process. Immobilization solution composed of 0.1M Na2HPO4 + 0.1M NaH2PO4, 0.6M guanidinium thiocyanate, 0.15M Trizma base adjusted by HCl on pH 7.5 and 2.5M CsCl. 5M NaCl was optimal for washing of the complex “beads-nucleic acid” after the immobilization. Elution was carried out for 15 minutes at 99 °C and the highest yield was obtained using elution solution composed of Tris-EDTA pH at 9.1
|
|
|
New Approaches in Cardiac Optical Mapping
Švrček, Martin ; Kizek, René (referee) ; Sovka, Pavel (referee) ; Provazník, Ivo (advisor)
This dissertation deals with new approaches in cardiac optical mapping. The principle of cardiac optical mapping as well as the current research in this field was described. The new measurement system was developed and its characteristics presented. The system design allows epicardial and endocardial mapping, employing new ratiometric techniques in 2D acquisition and simultaneous electrical and optical mapping. The measured characteristics of fluorescent dye di-4-ANEPPSS were presented. The relation between movement and consequent motion artifacts is well described. Several new approaches in signal processing were proposed, including new ratiometry technique and using image registration to suppress motion artifacts. The algorithm for elastic image registration of optical signals and innovative method for verification of registration process were presented. Application of all proposed approaches and its results are included and discussed.
|
|
|
Analysis of Thick Film Amperometrical Sensors Signal and Its Usage for Measurement and Characterization of Enzymes
Ondruch, Vít ; Kizek, René (referee) ; Masojídek,, Jiří (referee) ; Vrba, Radimír (advisor)
V práci je popsán princip synchronní detekce (SD), který byl uplatněn při měření s biosenzory. Metoda SD umožňuje dosažení výrazně lepšího poměru signálu k šumu, vyššího limitu detekce a celkové zlepšení robustnosti měření. Uplatnění SD při měření s biosenzory umožní zlepšit analýzu jeho odezvy a umožní odstranění nežádoucích interferencí nebo šumů, které mohou být způsobeny například mícháním roztoku, elektromagnetickými vlivy nebo parazitními proudy. SD také umožňuje rozložit získaný signál na odezvu stimulace a na dlouhodobý signál jiného procesu, a dále také identifikovat jevy druhého řádu. Pro identifikaci stimulačního signálu ve výstupním signálu měření byl na základě lineárního statistického modelu vyvinut specializovaný software. SD byla ověřena na modelovém případu výstupního signálu biosenzoru s aplikovaným komplexem fotosystému II (PSII) a jeho odezvě na stimulaci světlem. Odezva PSII se řídí kinetikou prvního řádu a může být také ovlivněna inhibitory. Kinetické konstanty vazby herbicidu na PSII závisí lineárně na koncentraci herbicidu. To umožňuje jejich měření také při nízkých koncentracích herbicidu.
|
|
|
Nanotransporters for theranostics
Dostálová, Simona ; Adam,, Vojtěch (referee) ; Kizek, René (advisor)
Master thesis deals with the use of bacteriophage as a theranostic drug nanocarrier. The term theranostics is used in recent years for systems that allow connecting of diagnostics, targeted drug delivery and monitoring of patient’s response to administered treatment in a single modality. These systems are very suitable especially with heterogeneous diseases, such as cancer. Nowadays, the treatment of cancer has often severe side effects to the patient’s body, which lowers his capability to fight the disease. Theoretical part of this work is focused on the properties of viral capsids, proteins and inorganic materials as drug nanocarriers. In practical part of this work, different methods for cultivation of bacteriophage are compared, both in liquid and solid medium and with different concentrations of the maltose, trough whose receptors bacteriophage is able to enter the host cell. Optimal was cultivation with 0.2% maltose in solid medium. Practical part is focused mainly on the use of bacteriophage as a nanocarrier for cytotoxic drug doxorubicin. Bacteriophage was able to encapsulate all applied concentrations of doxorubicin (0; 12.5; 25; 50; 100 and 200 g/ml), which was proved using fluorescent detection. Different times of encapsulation (2; 4; 8 and 12 hours) were studied. Optimal time was 2 hours. Encapsulation properties of bacteriophage were compared to apoferritin. Bacteriophage was able to encapsulate 4× higher concentrations of doxorubicin and its release during rinsing with water was 10× lower compared to apoferritin. This work concludes that bacteriophage is a very suitable platform for targeted drug delivery in theranostics.
|
|
|
Multichannel instrumentation for electrochemical measurement from sensor array
Žák, Jaromír ; Kizek, René (referee) ; Hubálek, Jaromír (advisor)
The work deals with n-channel system enabling many sample analysis at sort time from sensor array using electrochemical methods. The 8x12 sensor arrays are formed from 3 electrodes system created on PCB which can be used for heavy metal analysis and toxic substances determination. The control unit switches each 3 electrode sensor to 8 channel precise potentiostat which was designed for electrochemical analysis with current sensitivity below 10 pA. The potentiostat can synchronize with control unit. Developed system is able to measure up to eight single inputs and may be simply converted to measure other nonchemical values. Measured data will be sent to user-friendly application in computer and analyzed or saved consequently.
|
guest ::
RSS feed.