Biomedical Engineering Reference
In-Depth Information
as significantly increase sensitivity of analysis. Due to superparamagnetic properties of
nanoparticles it is possible to control movements of these constructions and detect them when
the external magnetic field overlaps [2, 3]. However, creation of constructions based on
nanoparticles requires development of effective methods for immobilization of biomolecules
which will not lead to disturbance of their structure and function. It has been shown that
aryldiazonium chlorides ArN
2
+
Cl
-
interact with the surface of certain carbon-coated
nanoparticles in the presence of nitrogen molecules; as a result of this the aryl (Ar) radical
bonded with the surface [4]. However, these reagents were characterized by a series of
significant disadvantages related to their low stability and explosion hazard and required
special reaction environment. Relatively stable diazonium salts (aryldiazonium
tetrafluoroborates ArN
2
+
BF
4
-
) are known, however, their application is restricted by their low
stability in water and organic solvents. Recently we have obtained a new type of stable
diazonium salts: aryldiazonium tosylates ArN
2
+-
OTs (ADT) [5]. ADTs are characterized by
higher applicability in comparison with the known diazonium salts: stability in water and
organic solvents, high storage stability, and explosion safety (confirmed by DSC data). We
demonstrated the possibilities of ADT to modify the surface of carbon-coated iron
nanoparticles [6, 7]. In this study, we investigated enzymatic activity of horseradish
peroxidise (HRP) immobilized onto carbon-coated iron nanoparticles, using arenediazonium
tosylates.
M
ATERIALS AND
M
ETHODS
The carbon-coated iron nanoparticles were used as a core of the developed construction.
The Fe@C nanoparticles (7-10 nm) were obtained by gassphase synthesis [6]. The 4-
carboxybenzenediazonium tosylates were obtained according to the described procedures [5].
The surface of nanoparticles was modified by 4-carboxybenzenediazonium tosylate [7]. The
method of surface functionalization and biomolecule immobilisation is shown on Figure 1.
Figure 1. Scheme of the method of HRP immobilisation onto carbon-coated iron nanoparticles.
Horseradish peroxidase (HRP) (Merck, Germany) was diluted in water to the final
concentration 2.8*10
-5
mol/L. The immobilization was carried out in a borate buffer (рН 8.6)
containing 0.84*10
-5
mol/L of HRP and 3 mg of the modified Fe@C nanoparticles. For
coupling of HRP with COOH-groups on the surface the EDC/NHS chemistry was applied.
The coupling procedure was carried out according to the protocol [8]. After immobilisation
the obtained complex was separated from the solution by magnetic separation using a
permanent magnet (0.2 T) and washed consecutively with the borate buffer, twice with H
2
O
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