Biomedical Engineering Reference
In-Depth Information
The HRP-Fe@C complex formation is associated with changes in IR spectra of both
nanoparticles and HRP. The observed increase in the intensity of vibrations at 1010 cm
-1
is
caused by stretching of the ADT C-C bond on the nanoparticles surface when binding with
peroxidase. The shift of the adsorption band of the COOH group on the modified
nanoparticles surface from 1346 to 1377 cm
-1
after HRP immobilization is the result of
bonding of the carboxyl group with HRP. The shift of the adsorption bands amide I from
1649 to 1638 cm
-1
and amide II from 1547 to 1540 cm
-1
in HRP-Fe@C IR spectra is caused
by HRP immobilization on the nanoparticle surface [12].
Amide I and amide II adsorption bands are the most useful markers for conformation
(secondary structure, i.e., a-helix and b-sheet) of the polypeptide backbone chain. These
adsorption bands are shifted to higher frequencies under denaturation of the HRP molecule.
However, presence of HRP-Fe@C bands in IR spectrum at 1638 cm
-1
and cm 1540 cm
-1
allows concluding immobilization does not alter the spatial structure of HRP or damage HRP
leading to a denatured state. [13]. It has been determined that HRP attached to nanoparticles,
can catalyze the oxidation of o-phenylenediamine in the presence of H
2
O
2
. (Figure 3). The
enzymatic activity of free and nanoparticle-attached HRP was 188,5±2.5 and 49,5±4.9
μmol*min
-1
*mg
-1
respectively.
The enzymatic activity of HRP-Fe@C has not significantly decreased for at least 7 days,
when stored at RT, and constituted 43.1ﾱ8.5 μmol*min
-1
*mg
-1
(Figure 4).
C
ONCLUSION
We introduce a new effective method of superparamagnetic nanoparticle
functionalization. Immobilization does not lead to disturbance of functional activity and
conformation of the protein molecule.This new method of magnetically controlled
construction formation, in which proteins are covalently attached to nanoparticles, have a
great applicability for biotechnology and biomedicine.
A
CKNOWLEDGMENTS
This research was supported by Grant of President of Russian Federation for Young
Scientists MD-1276.2010.7, RFBR № 09-04-99114, 09-03-9919, GC P1296, GC P33, GC
16.512.11.2127. We gratefully thanks to our colleagues from Institute of Physics of Metals
RAS.
R
EFERENCES
[1]
L. Merhari Hybrid Nanocomposites for Nanotechnology.
Springer Science+Business
Media
, LLC, 2009.
[2]
T. Osaka, M. Tadashi, N. Takuya, A. Atsushi et al. "Synthesis of magnetic
nanoparticles and their application in bioassays."
Anal Bioanal Chem
384: 593-600
(2006).
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