Biomedical Engineering Reference
In-Depth Information
Figure 13.1
[A] block-scheme of the sensor: 1- laser: 2- beamsplitter, 3 - optrode,
4 - clamp; 5 - lens, 6 - interference i lter, 7 - shutter, 8 - photoelectric cell, 9 - photon
counter, 10 - l at parallel plate, 11- light i ber, 12 - light diode, 13 - amplii er, 14 - voltmeter,
15 - recorder, 16 - casing, 17- capillary. [B]: principle of the analysis fuli llment with the
help of the optical immune biosensor based on the non-emitting energy transfer, where:
1 - optrod, 2 - sample, 3 - Ab immobilized and labeled by donor l uorochrome, 4 - Ag to be
analyzed, 5 - standard Ag labeled by acceptor l uorochrome.
the emission of which is registered. To remove artifact's caused by instabil-
ity of the light source and other factors af ecting donor l uorescence the
signal value was expressed as the ratio of the level of band-pass acceptor
l uorescence and band-pass donor excitation. It was performed by the
introducing another semi-transparent mirror (10) (at 450 angles to the
laser ray), optic i ber (11) and photodiode (12). A special device for replac-
ing interference i lters was installed in front of the photoelectric cell to
ensure it operated with two l uorescence labels.
h eoretically the idea of an immune biosensor based on energy transfer
may be expressed as follows: [Ab'] + [Ab*] = [Ab'-Ag*] and [Ab'-Ag*] +
[Ag] = [Ab'-Ag] + [Ag*], where Ab' - immobilized Ab labeled with donor
l uoroforme, Ag* - antigen labeled with acceptor l uoroforme, Ab' -Ag*
- the complex of immobilized labeled antibody with labeled antigen, Ab'
-Ag - the complex of immobilized labeled antibody with analyzed antigen
(Figure 13.1 [B]).
Since the amount of the immobilized Ab is constant the l uorescence
intensity is inversely proportional to the Ag concentration. h ere is no
need to separate the immune complex from individual components. For
the calibration it was used 20 identical optrodes at er their immersing into
the solutions containing consecutive four-fold serum dilutions (starting
from 1:20 M), standard titer of the monoclonal Ab labeled by horse radish
peroxidase (HRP). Optrodes with immobilized BSA were used as controls.
In parallel the ELISA- method was performed [2]. h e activity of HRP was
