The mode of protein immobilization plays a crucial role in the preparation of protein
microarrays used for a wide spectrum of applications in analytical biochemistry.
The microcontact printing technique was used to form a protein pattern using
concanavalin A (Con A) since Con A belongs to a group of proteins widely used in
analytical assays due to their selectivity as regards different kinds of carbohydrates.
Atomic force microscopy was used to image surface topography, delivering information
about the quality of the protein pattern. The force spectroscopy mode was used to verify
the functional activity of deposited proteins via determination of the forces of interaction
between Con A and carboxypeptidase Y bearing carbohydrate structure recognized by
Con A.
The calculated binding force between Con A and CaY was
105 ± 2 pN and it was compared with that measured for Con A deposited directly from the
protein solution. The similarity of the value obtained for the interaction force was
independent of the mode of protein deposition, thereby verifying that the microcontact
printing technique did not influence the carbohydrate binding activity of Con A.
The correlation between the surface topography of patterned samples and adhesion maps
obtained showed the possible use of AFM for studying the chemical properties of different
regions of the micropatterns produced.
Two analytical methods, atomic force microscopy and quartz crystal microbalance, were applied to the study of the reaction kinetics occurring between concanavalin A and carboxypeptidase Y, presenting the specific lectin-carbohydrate recognition. The dissociation rate constants for concanavalin A-carboxypeptidase Y complex obtained using both atomic force microscopy and quartz crystal microbalance were of the same order of magnitude: k diss = 0.170 ± 0.060 s −1 and k diss = 0.095 ± 0.002 s −1 , respectively. In addition, each method alone aided in determining other parameters characterizing the studied interaction. Quartz crystal microbalance permitted us to estimate the association rate (k ass = (5.6 ± 0.1) × 10 4 M −1 s −1 ) and the equilibrium (Ka = (0.59 ± 0.01) × 10 6 M −1 ) constants for the binding process occurring between concanavalin A and mannose residues of carboxypeptidase Y under given experimental conditions. Atomic force microscopy in force spectroscopy mode enabled the determination of the energy barrier position of r = 2.29 ± 0.04Å characterizing the dissociation of concanavalin Acarboxypeptidase Y molecular complex. The presented results show that both atomic force microscopy and quartz crystal microbalance can be used to determine quantitative parameters characterizing the specific molecular interaction. Both methods can be easily combined for complementary and/or alternative studies of a chosen molecular interaction. By preparing the samples in the same manner the direct comparison between the data obtained via atomic force microscopy and quartz crystal microbalance can be made.
In recent years, a single ion hit facility has been constructed at the IFJ ion microprobe. The setup is used for the precise irradiations of living cells by a controlled number of ions. Investigations of such type have two very important requirements: (1) cells must be examined in their natural state and environment (i.e. without previously being killed, preferentially neither fixed nor stained) and (2) the possibility of automatic irradiation of large number of cells (including computer recognition of cells positions) must be provided. This work presents some of the crucial features of the off-line and on-line optical systems, including self-developed software responsible for automatic cell recognition.
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