David Kovář - Academia.edu (original) (raw)

Papers by David Kovář

Next-generation sequencing doubles genomic databases every 2.5 years. The accumulation of sequenc... more Next-generation sequencing doubles genomic databases every 2.5 years. The accumulation of sequence data provides a unique opportunity to identify interesting biocatalysts directly in the databases without tedious and time-consuming engineering. Herein, we present a pipeline integrating sequence and structural bioinformatics with microfluidic enzymology for bioprospecting of efficient and robust haloalkane dehalogenases. The bioinformatic part identified 2,905 putative dehalogenases and prioritized a “small-but-smart” set of 45 genes, yielding 40 active enzymes, 24 of which were biochemically characterized by microfluidic enzymology techniques. Combining microfluidics with modern global data analysis provided precious mechanistic insights related to the high catalytic efficiency of selected enzymes. Overall, we have doubled the dehalogenation “toolbox” characterized over three decades, yielding biocatalysts that surpass the efficiency of currently available wild-type and engineered e...

Label-free immunosensors are well suited for detection of microorganisms because of their fast re... more Label-free immunosensors are well suited for detection of microorganisms because of their fast response and reasonable sensitivity comparable to infection doses of common pathogens. Active (lever oscillator and frequency counter) and passive (impedance analyzer) modes of quartz crystal microbalance (QCM) were used and compared for rapid detection of three strains of E. coli. Different approaches for antibody immobilization were compared, the immobilization of reduced antibody using Sulfo-SMCC was most effective achieving the limit of detection (LOD) 8 × 10 4 CFU•mL −1 in 10 min. For the passive mode, software evaluating impedance characteristics in real-time was developed and used. Almost the same results were achieved using both active and passive modes confirming that the sensor properties are not limited by the frequency evaluation method but mainly by affinity of the antibody. Furthermore, reference measurements were done using surface plasmon resonance. Effect of condition of cells on signal was observed showing that cells ruptured by ultrasonication provided slightly higher signal changes than intact microbes.

Simple and rapid detection and identification of dangerous bioagents is important for preventing ... more Simple and rapid detection and identification of dangerous bioagents is important for preventing illness or even death of people due to infectious diseases and bioterrorist threats. Electrochemical detection has a large potential for development of portable instrumentation suitable for analysis in field and especially combination with specificity of immunoassays seems promising. Initially, the construction of the immunosensing layer was realised and the effect of Au nanoparticles on the response of peroxidase was studied. An amperometric immunosensor was developed using screen-printed transducer, it employed specific capture of microbes in the sensing area by formation of an immunocomplex and its subsequent labelling using the tracer- antibody conjugated to peroxidase. The obtained sandwich electrochemical immunoassay was applied for the model microbial cells of B. subtilis. The total time of analysis was 20 min and the detection of cells below 10 4 CFU/ml was feasible. The presence...

Even though microorganisms are common part of our lives, some of them are dangerous and must be m... more Even though microorganisms are common part of our lives, some of them are dangerous and must be monitored. The traditional microbiological methods are reliable but slow. In some cases, namely in military, key requirement is fast detection which can be provided by biosensors. In this work, the piezoelectric biosensors providing fast, specific and cheap way of detection are described. Three strains of E. coli (BL21, DH5 alpha and K-12) were chosen as safe model microbes and the detection using the developed piezoelectric biosensor was carried out (LOD 10^6 CFU mL-1, total analysis time 20 min). Bioaerosol chamber was constructed to allow safe dissemination of microbes in air and experiments with bioaerosol. The generated bioaerosol was sampled using the cyclone air sampler and analyzed with the on-line coupled piezoelectric biosensor. The levels of 10^6 CFU L-1 of E. coli in air were successfully detected in less than 26 min which demonstrates possible application of this system for r...

Fast and sensitive devices for monitoring of glucose are crucial in today’s medicine. Most of the... more Fast and sensitive devices for monitoring of glucose are crucial in today’s medicine. Most of the commercial glucometers are currently based on enzymatic catalysis which provides reasonable sensitivity but has a drawback in limited enzyme stability. With the development of novel surfaces, new generation of non-enzymatic sensors characterized by high sensitivity and stability is emerging. In this work, gelatin-templated nanostructured gold electrodes were developed and applied for non-enzymatic glucose determination. The analysis was done by direct electrochemical oxidation during cyclic voltammetry and amperometry with the limit of detection at 1.3 µM. The ability to detect physiological levels glucose in presence of interferents was demonstrated on deproteinized human blood serum.

Nanostructured electrodes based on gelatin-templated gold were developed for sensitive non-enzyma... more Nanostructured electrodes based on gelatin-templated gold were developed for sensitive non-enzymatic detection of glucose. The structures of specific saw tooth-like morphology were formed by electrodeposition of gold through the cavities of crosslinked gelatin. The growth of nanostructures was investigated by scanning electron microscopy (SEM) and atomic force microscopy (AFM). The electroactive surface area (ESA) and electrochemical behavior of the prepared electrodes were determined by the cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). The roughness factor (expressed as ESA divided by geometrical area) of the gelatin-templated surface was estimated to be 16.7, which represent 15.9 fold enlargement compared to the flat gold electrode. The nanoscopic surface exhibited strong electrocatalytic effect towards glucose oxidation. The detection of glucose was done by direct electrochemical oxidation during CV and amperometry. In case of CV, the sensitivity of 11...

Interactions of antibodies with the bacterium Bacillus atrophaeus were studied using piezoelectri... more Interactions of antibodies with the bacterium Bacillus atrophaeus were studied using piezoelectric (PZ) and surface plasmon resonance (SPR) based biosensors, with the aim of future development of biosensor for Bacillus species. Antibodies were covalently immobilised on gold surfaces of PZ crystal resonators and SPR chips. QCM Analyzer and Biacore 3000 were used for real-time and label-free measurements of immunocomplexes. Concentrations of B. atrophaeus below 10^5 CFU ml-1 were successfully detected.

International Journal of Electrochemical Science, 2013

Electrochemical immunosensing devices for detection of microbial agents are briefly reviewed. The... more Electrochemical immunosensing devices for detection of microbial agents are briefly reviewed. The practical results describe amperometric immunosensors based on screen-printed electrodes as a general platform for sandwich-based assay of microbes. The examples are focused on the determination of Francisella, Salmonella, Escherichia and Bacillus species. The achieved analytical parameters seem promising for real applications. In particular, combination of the immunosensor with cyclone device allowed fully automated testing of the system for detection of bioaerosols of E. coli as a model agent; the preliminary results confirm that levels below 100 CFU/L in air can be detected within 20 min.

Piezoelectric biosensors provide fast, specific and economic way of detection for a wide range of... more Piezoelectric biosensors provide fast, specific and economic way of detection for a wide range of analytes ranging from metabolites to microorganisms. Specific antibodies were immobilised on gold electrodes of piezoelectric crystal and the biosensor was applied for real-time detection of model microorganisms. Three nonpathogenic strains of Escherichia coli (BL21, DH5 alpha and K-12) were used because of easy cultivation and availability of antibodies. Several methods of antibody immobilisation were compared. The immobilisation of reduced antibody using Sulfo-SMCC was the most effective achieving the limit of detection (LOD) 10^5 CFU·mL-1. Active and passive operation modes of quartz crystal microbalance were compared concluding that for detection of microorganisms, both approaches are practically identical. The developed biosensor was connected to the cyclone air sampler allowing detection of microorganisms disseminated in the form of aerosol inside bioaerosol chamber. The achieved ...

Detection of microorganisms has a crucial role in many fields. Even though the traditional microb... more Detection of microorganisms has a crucial role in many fields. Even though the traditional microbiological methods are robust and provide low limits of detection, the analysis is usually time consuming. In some cases (e.g. monitoring of airborne pathogens), the fast detection is essential. Rapid and specific analysis can be provided by biosensors. In this work, the piezoelectric immunosensor for detection of aerosolized microorganisms was developed. Nonpathogenic E. coli K-12 was chosen as a biological warfare agents’ surrogate. The biosensing layer was prepared by immobilization of specific antibodies using bifunctional crosslinker Sulfo-SMCC. To allow safe work with aerosolized microorganisms, bioaerosol chamber was constructed. The dissemination was done using a piezoelectric aerosol generator and the samples were collected by a wetted-wall cyclone on-line coupled with the piezoelectric immunosensor. The concentration 1.45×10^4 CFU·L-1 of E. coli K-12 in air was successfully dete...

International Journal of Electrochemical Science, 2011

Simple and rapid detection and identification of dangerous bioagents is important for preventing ... more Simple and rapid detection and identification of dangerous bioagents is important for preventing illness or even death of people due to infectious diseases and bioterrorist threats. Electrochemical detection has a large potential for development of portable instrumentation suitable for analysis in field and especially combination with specificity of immunoassays seems promising. Initially, the construction of the immunosensing layer was realised and the effect of Au nanoparticles on the response of peroxidase was studied. An amperometric immunosensor was developed using screen-printed transducer, it employed specific capture of microbes in the sensing area by formation of an immunocomplex and its subsequent labelling using the tracer - antibody conjugated to peroxidase. The obtained sandwich electrochemical immunoassay was applied for the model microbial cells of B. subtilis. The total time of analysis was 20 min and the detection of cells below 104 CFU/ml was feasible. The presence...

The instrumentation and services available at the Core Facility of Nanobiotechnology in CEITEC MU... more The instrumentation and services available at the Core Facility of Nanobiotechnology in CEITEC MU will be introduced. The research possibilities will be demonstrated on experiments with proteins, nucleic acids, their affinity complexes and cells. The interaction of ssDNA binding protein with oligonucleotides was imaged using bare tips (Fig. 1), the binding forces in the affinity complex were studied using the protein ligand-modified tip and the ForceRobot AFM head for automated recording of force-distance curves. Similar experiments characterised immunoreactions between antibody and serum albumin and microbial cells as antigens and hybridisation of nucleic acids; interactions were confirmed using surface plasmon resonance and electrochemical measurements. Properties of mast cells were characterised in relation to the changes accompanying biotransformation events (Fig. 2). Simultaneous recording of contractions and electric activity of cardiomyocytes was followed using the AFM cantil...

The instrumentation and services available at the Core Facility of Nanobiotechnology, CEITEC Masa... more The instrumentation and services available at the Core Facility of Nanobiotechnology, CEITEC Masaryk University, will be introduced. The research possibilities will be demonstrated on experiments with proteins, nucleic acids and their affinity complexes - interaction of ssDNA binding protein with oligonucleotides and immunoreaction between antibody and antigen. Properties of cellular membrane will be characterised in relation to the changes accompanying malignant transformation. Simultaneous recording of contractions and electric activity of cardiomyocytes will be presented using AFM cantilever with conductive tip functioning as nanomechanical transducer for cellular biosensor suitable for evaluation of fyziologically active compounds in real time.

A precise monitoring of glucose level in blood is of high importance in clinical medicine, thus, ... more A precise monitoring of glucose level in blood is of high importance in clinical medicine, thus, sensitive, rapid and reliable methods for its detection are required. In this work, gelatin templated gold nanostructures were fabricated in order to improve the sensitivity of glucose analysis. SEM and AFM were used for characterization of the created surfaces. Glucose was detected by a direct electrochemical oxidation during cyclic voltammetry in alkaline solution. Limit of detection of 10 uM was achieved in aqueous samples. The sensor was also able to detect real concentration of glucose in deproteinised human serum with negligible effect of interferents. All results were verified by commercial glucometer and the standard kit for photometric detection of glucose.

Due to the importance of glucose in physiological processes, sensitive, rapid and reliable method... more Due to the importance of glucose in physiological processes, sensitive, rapid and reliable methods for its detection are required. The biocatalytic sensors provide low limits of detection but the enzymatic layer is volatile. Sensors that are not based on enzymes are very stable in all conditions, however worse detection limits are typically obtained. One of the ways to achieve an improved sensitivity of the detection is by developing of a sensor with optimized surface morphology. In this work gelatin templated gold nanostructures were fabricated in order to improve the sensitivity of glucose detection. The 3D cavities were made by crosslinking of gelatine layer on the gold surface and nanostructured surface was formed in these cavities by electrodeposition of gold from the electroplating solution. Gel layers on the electrodes were cleaned by KOH and chromic acid. Scanning electron microscopy and atomic force microscopy were used for characterisation of the created surfaces. The gluc...

Cheap, rapid and user friendly tools for detection of microorganisms were intensively discussed d... more Cheap, rapid and user friendly tools for detection of microorganisms were intensively discussed during last decades. The use of immunospecific biosensors seems advantageous for samples originating from clinical, environmental and agricultural area, food processing and military. The piezoelectric (quartz crystal microbalance, QCM) and optical (surface plasmon resonance, SPR) biosensors represented by QCM Analyzer and Biacore 3000 were compared for future development of real-time and label-free microbial biosensors. The spores of Bacillus atrophaeus were used as a non-pathogenic surrogate for B. anthracis. Seven commercially available antibodies were immobilised on the sensor surfaces to select the most promising one. Various immobilisation techniques were used. The total time of analysis did not exceed 30 min and the estimated limits of detection (LOD) were 10^6 CFU mL-1 and 10^5 CFU mL-1 in case of QCM and SPR, respectively.

Cilem prace je studium biointerakci molekul kombinaci piezoelektrickeho (PZ) a optickeho systemu.... more Cilem prace je studium biointerakci molekul kombinaci piezoelektrickeho (PZ) a optickeho systemu. Originalita prace spociva v paralelnim měřicim uspořadani těchto dvou systemů. Sledovane interakce působi změnu rezonancni frekvence piezoelektrickeho sensoru. Použitim sekundarně znacených protilatek, nebo přimo znaceneho ligandu, je možno sledovat flourimetrický signal uměrný zachycenemu ligandu v aktivnim povrchu PZ sensoru. Jako modelove antigeny jsou použity E. coli DH5a (v kombinaci se sekundarně znacenou anti E. coli). Dale 3 typy znaceneho HSA: HSA–FITC, HSA–Fluo, HSA–HRP.

Next-generation sequencing doubles genomic databases every 2.5 years. The accumulation of sequenc... more Next-generation sequencing doubles genomic databases every 2.5 years. The accumulation of sequence data provides a unique opportunity to identify interesting biocatalysts directly in the databases without tedious and time-consuming engineering. Herein, we present a pipeline integrating sequence and structural bioinformatics with microfluidic enzymology for bioprospecting of efficient and robust haloalkane dehalogenases. The bioinformatic part identified 2,905 putative dehalogenases and prioritized a “small-but-smart” set of 45 genes, yielding 40 active enzymes, 24 of which were biochemically characterized by microfluidic enzymology techniques. Combining microfluidics with modern global data analysis provided precious mechanistic insights related to the high catalytic efficiency of selected enzymes. Overall, we have doubled the dehalogenation “toolbox” characterized over three decades, yielding biocatalysts that surpass the efficiency of currently available wild-type and engineered e...

Label-free immunosensors are well suited for detection of microorganisms because of their fast re... more Label-free immunosensors are well suited for detection of microorganisms because of their fast response and reasonable sensitivity comparable to infection doses of common pathogens. Active (lever oscillator and frequency counter) and passive (impedance analyzer) modes of quartz crystal microbalance (QCM) were used and compared for rapid detection of three strains of E. coli. Different approaches for antibody immobilization were compared, the immobilization of reduced antibody using Sulfo-SMCC was most effective achieving the limit of detection (LOD) 8 × 10 4 CFU•mL −1 in 10 min. For the passive mode, software evaluating impedance characteristics in real-time was developed and used. Almost the same results were achieved using both active and passive modes confirming that the sensor properties are not limited by the frequency evaluation method but mainly by affinity of the antibody. Furthermore, reference measurements were done using surface plasmon resonance. Effect of condition of cells on signal was observed showing that cells ruptured by ultrasonication provided slightly higher signal changes than intact microbes.

Simple and rapid detection and identification of dangerous bioagents is important for preventing ... more Simple and rapid detection and identification of dangerous bioagents is important for preventing illness or even death of people due to infectious diseases and bioterrorist threats. Electrochemical detection has a large potential for development of portable instrumentation suitable for analysis in field and especially combination with specificity of immunoassays seems promising. Initially, the construction of the immunosensing layer was realised and the effect of Au nanoparticles on the response of peroxidase was studied. An amperometric immunosensor was developed using screen-printed transducer, it employed specific capture of microbes in the sensing area by formation of an immunocomplex and its subsequent labelling using the tracer- antibody conjugated to peroxidase. The obtained sandwich electrochemical immunoassay was applied for the model microbial cells of B. subtilis. The total time of analysis was 20 min and the detection of cells below 10 4 CFU/ml was feasible. The presence...

Even though microorganisms are common part of our lives, some of them are dangerous and must be m... more Even though microorganisms are common part of our lives, some of them are dangerous and must be monitored. The traditional microbiological methods are reliable but slow. In some cases, namely in military, key requirement is fast detection which can be provided by biosensors. In this work, the piezoelectric biosensors providing fast, specific and cheap way of detection are described. Three strains of E. coli (BL21, DH5 alpha and K-12) were chosen as safe model microbes and the detection using the developed piezoelectric biosensor was carried out (LOD 10^6 CFU mL-1, total analysis time 20 min). Bioaerosol chamber was constructed to allow safe dissemination of microbes in air and experiments with bioaerosol. The generated bioaerosol was sampled using the cyclone air sampler and analyzed with the on-line coupled piezoelectric biosensor. The levels of 10^6 CFU L-1 of E. coli in air were successfully detected in less than 26 min which demonstrates possible application of this system for r...

Fast and sensitive devices for monitoring of glucose are crucial in today’s medicine. Most of the... more Fast and sensitive devices for monitoring of glucose are crucial in today’s medicine. Most of the commercial glucometers are currently based on enzymatic catalysis which provides reasonable sensitivity but has a drawback in limited enzyme stability. With the development of novel surfaces, new generation of non-enzymatic sensors characterized by high sensitivity and stability is emerging. In this work, gelatin-templated nanostructured gold electrodes were developed and applied for non-enzymatic glucose determination. The analysis was done by direct electrochemical oxidation during cyclic voltammetry and amperometry with the limit of detection at 1.3 µM. The ability to detect physiological levels glucose in presence of interferents was demonstrated on deproteinized human blood serum.

Nanostructured electrodes based on gelatin-templated gold were developed for sensitive non-enzyma... more Nanostructured electrodes based on gelatin-templated gold were developed for sensitive non-enzymatic detection of glucose. The structures of specific saw tooth-like morphology were formed by electrodeposition of gold through the cavities of crosslinked gelatin. The growth of nanostructures was investigated by scanning electron microscopy (SEM) and atomic force microscopy (AFM). The electroactive surface area (ESA) and electrochemical behavior of the prepared electrodes were determined by the cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). The roughness factor (expressed as ESA divided by geometrical area) of the gelatin-templated surface was estimated to be 16.7, which represent 15.9 fold enlargement compared to the flat gold electrode. The nanoscopic surface exhibited strong electrocatalytic effect towards glucose oxidation. The detection of glucose was done by direct electrochemical oxidation during CV and amperometry. In case of CV, the sensitivity of 11...

Interactions of antibodies with the bacterium Bacillus atrophaeus were studied using piezoelectri... more Interactions of antibodies with the bacterium Bacillus atrophaeus were studied using piezoelectric (PZ) and surface plasmon resonance (SPR) based biosensors, with the aim of future development of biosensor for Bacillus species. Antibodies were covalently immobilised on gold surfaces of PZ crystal resonators and SPR chips. QCM Analyzer and Biacore 3000 were used for real-time and label-free measurements of immunocomplexes. Concentrations of B. atrophaeus below 10^5 CFU ml-1 were successfully detected.

International Journal of Electrochemical Science, 2013

Electrochemical immunosensing devices for detection of microbial agents are briefly reviewed. The... more Electrochemical immunosensing devices for detection of microbial agents are briefly reviewed. The practical results describe amperometric immunosensors based on screen-printed electrodes as a general platform for sandwich-based assay of microbes. The examples are focused on the determination of Francisella, Salmonella, Escherichia and Bacillus species. The achieved analytical parameters seem promising for real applications. In particular, combination of the immunosensor with cyclone device allowed fully automated testing of the system for detection of bioaerosols of E. coli as a model agent; the preliminary results confirm that levels below 100 CFU/L in air can be detected within 20 min.

Piezoelectric biosensors provide fast, specific and economic way of detection for a wide range of... more Piezoelectric biosensors provide fast, specific and economic way of detection for a wide range of analytes ranging from metabolites to microorganisms. Specific antibodies were immobilised on gold electrodes of piezoelectric crystal and the biosensor was applied for real-time detection of model microorganisms. Three nonpathogenic strains of Escherichia coli (BL21, DH5 alpha and K-12) were used because of easy cultivation and availability of antibodies. Several methods of antibody immobilisation were compared. The immobilisation of reduced antibody using Sulfo-SMCC was the most effective achieving the limit of detection (LOD) 10^5 CFU·mL-1. Active and passive operation modes of quartz crystal microbalance were compared concluding that for detection of microorganisms, both approaches are practically identical. The developed biosensor was connected to the cyclone air sampler allowing detection of microorganisms disseminated in the form of aerosol inside bioaerosol chamber. The achieved ...

Detection of microorganisms has a crucial role in many fields. Even though the traditional microb... more Detection of microorganisms has a crucial role in many fields. Even though the traditional microbiological methods are robust and provide low limits of detection, the analysis is usually time consuming. In some cases (e.g. monitoring of airborne pathogens), the fast detection is essential. Rapid and specific analysis can be provided by biosensors. In this work, the piezoelectric immunosensor for detection of aerosolized microorganisms was developed. Nonpathogenic E. coli K-12 was chosen as a biological warfare agents’ surrogate. The biosensing layer was prepared by immobilization of specific antibodies using bifunctional crosslinker Sulfo-SMCC. To allow safe work with aerosolized microorganisms, bioaerosol chamber was constructed. The dissemination was done using a piezoelectric aerosol generator and the samples were collected by a wetted-wall cyclone on-line coupled with the piezoelectric immunosensor. The concentration 1.45×10^4 CFU·L-1 of E. coli K-12 in air was successfully dete...

International Journal of Electrochemical Science, 2011

Simple and rapid detection and identification of dangerous bioagents is important for preventing ... more Simple and rapid detection and identification of dangerous bioagents is important for preventing illness or even death of people due to infectious diseases and bioterrorist threats. Electrochemical detection has a large potential for development of portable instrumentation suitable for analysis in field and especially combination with specificity of immunoassays seems promising. Initially, the construction of the immunosensing layer was realised and the effect of Au nanoparticles on the response of peroxidase was studied. An amperometric immunosensor was developed using screen-printed transducer, it employed specific capture of microbes in the sensing area by formation of an immunocomplex and its subsequent labelling using the tracer - antibody conjugated to peroxidase. The obtained sandwich electrochemical immunoassay was applied for the model microbial cells of B. subtilis. The total time of analysis was 20 min and the detection of cells below 104 CFU/ml was feasible. The presence...

The instrumentation and services available at the Core Facility of Nanobiotechnology in CEITEC MU... more The instrumentation and services available at the Core Facility of Nanobiotechnology in CEITEC MU will be introduced. The research possibilities will be demonstrated on experiments with proteins, nucleic acids, their affinity complexes and cells. The interaction of ssDNA binding protein with oligonucleotides was imaged using bare tips (Fig. 1), the binding forces in the affinity complex were studied using the protein ligand-modified tip and the ForceRobot AFM head for automated recording of force-distance curves. Similar experiments characterised immunoreactions between antibody and serum albumin and microbial cells as antigens and hybridisation of nucleic acids; interactions were confirmed using surface plasmon resonance and electrochemical measurements. Properties of mast cells were characterised in relation to the changes accompanying biotransformation events (Fig. 2). Simultaneous recording of contractions and electric activity of cardiomyocytes was followed using the AFM cantil...

The instrumentation and services available at the Core Facility of Nanobiotechnology, CEITEC Masa... more The instrumentation and services available at the Core Facility of Nanobiotechnology, CEITEC Masaryk University, will be introduced. The research possibilities will be demonstrated on experiments with proteins, nucleic acids and their affinity complexes - interaction of ssDNA binding protein with oligonucleotides and immunoreaction between antibody and antigen. Properties of cellular membrane will be characterised in relation to the changes accompanying malignant transformation. Simultaneous recording of contractions and electric activity of cardiomyocytes will be presented using AFM cantilever with conductive tip functioning as nanomechanical transducer for cellular biosensor suitable for evaluation of fyziologically active compounds in real time.

A precise monitoring of glucose level in blood is of high importance in clinical medicine, thus, ... more A precise monitoring of glucose level in blood is of high importance in clinical medicine, thus, sensitive, rapid and reliable methods for its detection are required. In this work, gelatin templated gold nanostructures were fabricated in order to improve the sensitivity of glucose analysis. SEM and AFM were used for characterization of the created surfaces. Glucose was detected by a direct electrochemical oxidation during cyclic voltammetry in alkaline solution. Limit of detection of 10 uM was achieved in aqueous samples. The sensor was also able to detect real concentration of glucose in deproteinised human serum with negligible effect of interferents. All results were verified by commercial glucometer and the standard kit for photometric detection of glucose.

Due to the importance of glucose in physiological processes, sensitive, rapid and reliable method... more Due to the importance of glucose in physiological processes, sensitive, rapid and reliable methods for its detection are required. The biocatalytic sensors provide low limits of detection but the enzymatic layer is volatile. Sensors that are not based on enzymes are very stable in all conditions, however worse detection limits are typically obtained. One of the ways to achieve an improved sensitivity of the detection is by developing of a sensor with optimized surface morphology. In this work gelatin templated gold nanostructures were fabricated in order to improve the sensitivity of glucose detection. The 3D cavities were made by crosslinking of gelatine layer on the gold surface and nanostructured surface was formed in these cavities by electrodeposition of gold from the electroplating solution. Gel layers on the electrodes were cleaned by KOH and chromic acid. Scanning electron microscopy and atomic force microscopy were used for characterisation of the created surfaces. The gluc...

Cheap, rapid and user friendly tools for detection of microorganisms were intensively discussed d... more Cheap, rapid and user friendly tools for detection of microorganisms were intensively discussed during last decades. The use of immunospecific biosensors seems advantageous for samples originating from clinical, environmental and agricultural area, food processing and military. The piezoelectric (quartz crystal microbalance, QCM) and optical (surface plasmon resonance, SPR) biosensors represented by QCM Analyzer and Biacore 3000 were compared for future development of real-time and label-free microbial biosensors. The spores of Bacillus atrophaeus were used as a non-pathogenic surrogate for B. anthracis. Seven commercially available antibodies were immobilised on the sensor surfaces to select the most promising one. Various immobilisation techniques were used. The total time of analysis did not exceed 30 min and the estimated limits of detection (LOD) were 10^6 CFU mL-1 and 10^5 CFU mL-1 in case of QCM and SPR, respectively.

Cilem prace je studium biointerakci molekul kombinaci piezoelektrickeho (PZ) a optickeho systemu.... more Cilem prace je studium biointerakci molekul kombinaci piezoelektrickeho (PZ) a optickeho systemu. Originalita prace spociva v paralelnim měřicim uspořadani těchto dvou systemů. Sledovane interakce působi změnu rezonancni frekvence piezoelektrickeho sensoru. Použitim sekundarně znacených protilatek, nebo přimo znaceneho ligandu, je možno sledovat flourimetrický signal uměrný zachycenemu ligandu v aktivnim povrchu PZ sensoru. Jako modelove antigeny jsou použity E. coli DH5a (v kombinaci se sekundarně znacenou anti E. coli). Dale 3 typy znaceneho HSA: HSA–FITC, HSA–Fluo, HSA–HRP.