Abstract:

 Synthesis of immunofluorescence probes and affinity surfaces for selective and sensitive analysis
                                of biologically important molecules


             Marcela Liskova^1, Karel Kleparnik^1, Pavel Pazdera^2, Frantisek Foret^1


    ^1Institute of Analytical Chemistry ASCR, v.v.i., Veveri 9, CZ-602 00 Brno, Czech Republic,
                                          liskova@iach.cz

  ^2Centre for Syntheses at Sustainable Conditions and Their Management Department of Chemistry,
    Faculty of Science, Masaryk University, UKB, Kamenice 753/5, CZ-625 00 Brno, Czech Republic


We will report a several methods for synthesis of immunofluorescence probes and affinity surfaces.
We used quantum dots as a fluorescence affinity surface. Quantum dots (QDs), semiconductor
inorganic nanocrystals, show a great potential as photoluminescent probes at cellular level for in
vivo immunolabelling or cellular tracking.  The main advantages of quantum dots, when compared with
the conventional organic fluorescent dyes, are practically no photobleaching, wide excitation and
narrow emission spectra and size dependent emission maximum wavelengths. Typical materials for the
preparation of quantum dots are elements of II – VI or III – V group. We have prepared water
soluble QDs in the size range from 2.8 to 4.4 nm by the chemical reaction between CdCl[2] and NaHTe
with 3-mercaptopropionic acid (MPA) as a hydrophilic surface ligand. To reduce their cytotoxicity,
QDs are coated by two epitaxial layers of CdS and ZnS.

For the preparation of conjugates, it is possible to use a lot of conjugation procedures with
variety of cross-linkers. Their role is to bind different functional groups attached to QDs with
those present in biologically important molecules. We have used the conjugation reaction via
zero-length cross-linkers such as 1-ethyl-3-(3-dimethyl-3-aminopropyl) carbodiimide hydrochloride
and N-hydroxysulfosuccinimide for bonding carboxylated QDs and proteins (especially antibodies).
The formation of conjugate is checked by capillary zone electrophoresis with laser-induced
fluorescence detection. We have found optimal conditions for the separation of the conjugate,
immunocomplex and QDs.

Non-coated and double coated QDs are conjugated with various proteins such as antibody to
proliferating cell nuclear antigen (anti-PCNA), anti-chicken egg albumin, anti-CD3 membrane protein
and annexin V. Antibodies labelled with QDs are applied as fluorescent probes for the detection of
various antigens in cancer cells and apoptotic mouse intestine and human lymphocyte cells.
Epifluorescence microscopy with excitation by using mercury arc lamp (460-550 nm) and total
internal reflection fluorescence microscopy with Nd:YAG laser (532 nm) are used as a high
sensitivity detection instrumentation.

Finally, we will report about Caspase-3 determination. Caspase 3, a cysteine-aspartic acid protease
activated in the apoptotic pathway, plays an essential role in the programmed cell death. Moreover,
failure of apoptosis is one of the main contributions to tumor development. Under normal
circumstances, caspases recognize tetra-peptide sequences Asp-x-x-Asp on their substrates and
hydrolyze peptide bonds after aspartic acid residues. It is known from literature that in one
apoptotic cell about 1.6×10^-19 mol of Caspase can be activated. Various techniques for the
determination of Caspase 3 in free cells or tissue samples are commercially available. While the
Caspase concentration is determined by Enzyme-Linked Immunosorbent Assays (ELISA) with colorimetric
detection, its activity is usually assayed by a chemiluminescence reaction. The best commercial
methods reach the limit o detection higher than 1pg of Caspase 3.

We have developed a special device for the determination of Caspase concentration or activity in
microdissected cells: (i) The concentration is determined by ELISA assay with chemiluminescence
detection. In this technique, a monoclonal antibody immobilized on the device wall interacts with a
Caspase 3 epitope. Then, a polyclonal antibody labeled by horse radish peroxidase binds another
Caspase epitope. The presence of this antigene is detected as a chemiluminescence signal after the
addition of a mixture of luminol with hydrogenperoxide. (ii) The activity of Caspase 3 is
determined by the system based on Luciferin/Luciferase chemiluminescence reaction. The luciferin
modified with tetrapeptide sequence (DEVD) specific to the recognition for Caspase-3 is cleaved to
form free luciferin, which immediately reacts with luciferase to produce light. Our detection
device consists of a microfluidic chamber held inside a housing of photomultiplier tube (Hamamatsu
R446) with spectral response 185-870 nm. The cell is accessible by a Hamilton syringe fixed in a
light-proof cassette. The sensitivity of the device proved to be by one order of magnitude better
than the commercially available technologies.