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Research Programmes

 

Nanobiotechnology

Doc. RNDr. Petr Skládal, CSc.
Research Group Leader

THEMATIC RESEARCH FOCUS

RESEARCH AREAS

  • Imaging of biomolecules, cells and other biological objects using scanning probe microscopies (AFM, SNOM, STM)
  • Characterisation of affi nity interactions using biosensors in real time
  • Development of biosensors using electrochemical, optical and piezoelectric transducers
  • Immobilisation, modifi cation and conjugation of biomolecules

MAIN OBJECTIVES

  • Visualisation and modifi cation of biological objects including tissues, cells, cellular structures, and biomolecules
  • Development of new methodologies for investigating the structure, interactions, and dynamics of biomolecules.
  • Investigations of the structure and interactions of biomacromolecules and their relation to the functions of living systems, disease and therapy.
  • Investigations of the behaviour of natural and chemically modifi ed biomacromolecules at electrically charged surfaces linked to the development of novel electrochemical biosensors and bioassays.

CONTENT OF RESEARCH

Nanobiotechnology represents advanced scanning probe microscopic techniques, nanolithographic machining and various types of artificial nanostructures applied for either visualisation or modifi cation of biological objects including tissues, cells, cellular structures and biomolecules. The unique opportunity to touch the single individual molecule of the protein or nucleic acid with the scanning tip provides high-resolution subnanometer and pseudo-3-dimensional images providing details of such bioobjects in their native state. These approaches are currently revolutionising many fi elds of biology, biophysics and biochemistry and provide innovative results and methodologies for application in health care – nanobiosensing systems, nanoparticles for visualisation and smart distribution of drugs (nanomedicine). Within this work package, the following research fi elds will be addressed:

Imaging of biosurfaces and biomolecules using scanning probe microscopies

The laboratory will perform AFM scanning in both dry state and in liquids in non-contact (tapping) mode. Modified tips will allow characterisation of surface hydrophobicity and specific target molecules and cellular surfaces (e.g. tips modified with antibodies) will be scanned while conductive tips with applied potential will be used for bioelectrochemical studies. Repeated scans will allow the movement and morphologic changes of cells to be studied (VideoAFM). Supplementary information on cells and cellular elements will be provided by scanning near optical field microscopy (SNOM, overcoming the diffraction limit); and scanning tunnelling microscopy (STM) will be chosen when atomic resolution is required.

Nanobiointeractions and the measurement of forces within biocomplexes

The binding of two individual complementary molecules (antibody-antigen, ligand-receptor, and hybridisation of oligonucleotides) will be studied using one partner bound to the solid support and the other linked to the scanning tip. The force data measured at the nanolevel will be correlated with the results obtained at the macrolevel using surface plasmon resonance techniques providing information about kinetics of affinity interactions in real time.

Nanomanipulation and nanolithography of biological objects

In the AFM-lithography mode, the tip of the cantilever in contact with the scanned object can be used to manipulate cells on the surface. Nano(bio)sensing arrays and other functional nanoobjects will be constructed.

Nanobiosensors and biosensing nanoarrays

Nanobiosensors will employ the cantilever as nanomechanical transducer bending due to the affinity interaction on one of its sides. Nanoarrays – biochips consisting of sets of specific recognition proteins (monoclonal or recombinant antibodies, engineered receptors and enzymes, artificial peptide folds designed by molecular modelling) will be incubated with clinical samples and then the binding pattern will be read with the help of AFM (SNOM, STM) either directly or after suitable amplification (magnetic nanoparticles, quantum dots). For the validation of these novel techniques, “larger” micrometer-sized array elements will be produced and evaluated using multichannel SPR, fluorescence scanning and scanning electrochemical microscopy (SECM).

KEY RESEARCH EQUIPMENT

PLANNED RESEARCH INFRASTRUCTURE

Core Facility

The research group will be one of the principal users of the CEITEC Nanobiotechnology and Biointeractions Core Facility.

CURRENT RESEARCH INFRASTRUCTURE

The currently available infrastructure includes AFM Ntegra Vita, SNOM Ntegra Solaris, SPR system Biacore 2000, fl uorescence microscope, cell cultivation facility, electrochemical (10), piezoelectric (5) and fi ber optic (2) detectors, light sources (4), DAD spectrophotometer, plater readers Multiskan RC and Synergy 2, autoinjectors (2), FIALab 3000, data acquisition systems (NI). The group members have programming experience in Delphi, C++ and Lab-View.

MAIN PROJECTS

  • Biomimetic sensors as new generation of biotechnological devices for food safety and quality monitoring, BIOMIMIC (230849), FP7-PEOPLE, EU, 2009-2012, P. Skladal, Masaryk University.
  • Interactions of mutant forms of cryptogein with a membrane binding site: biosensors-based characterisation (GAP501/11/1003), Czech Science Foundation, 2011-2014, P. Skladal, Masaryk University.
  • Nanobiotechnologies and biosensor for biointeraction studies – openning up the modern technology to researchers in biology (CZ.1.07/2.3.00/09.0167), Ministry of Education, Youth and Sports, 2009-2012, P. Skladal, Masaryk University.
  • Nanotechnological and bioanalytical detection of the DNA damage (2B06056), Ministry of Education, Youth and Sports, 2006-2010, J. Přibyl, Masaryk University, M. Suchanek, EXBIO Praha Inc.

SELECTED PUBLICATIONS

  • ZERAVIK, J., LACINA, K., JILEK, M., VLCEK, J., SKLADAL, P. Biosensor for determination of carboxylic acids in wines based on the inhibition of sarcosine oxidase. Microchim. Acta. 2010, 170, p. 251-256.
  • RICH, R., L., PAPALIA, G., A., FLYNN, P., J. et all. A global benchmark study using affi nity-based biosensors. Anal. Biochem. 2009, 386, p. 194-216.
  • SVOZILOVA, Z., KASPAROVSKY, T., SKLADAL, P., LOCHMAN, J. Interaction of cryptogein with its binding sites in tobacco plasma membrane studied using the piezoelectric biosensor. Anal. Biochem. 2009, 390, p. 115-120.
  • ZERAVIK, J., LACINA, K., HLAVACEK, A., SKLADAL, P. State of the Art in the Field of Electronic and Bioelectronic Tongues – Towards the Analysis of Wines. Electroanalysis. 2009, 21, p. 2509-2520.
  • MOLINKOVA, D., SKLADAL, P., CELER, V. In vitro neutralization of Equid herpesvirus 1 mediated by recombinant antibodies. J. Immunol. Meth. 2008, 333, p. 186-191.
 

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