Biomedical Engineering Reference
In-Depth Information
13.2.1.1
Construction, Fluorescent Labels, Used Reagents and
Measurements [1]
An argon laser was served as the source of light. h e optrodes (diame-
ter: 1, length: 50 mm) were made from mono i ber light conductors. h ey
were pretreated with a mixture containing concentrated hydrochloric acid
and dichloroethane in equal doses. Before immobilization the surface of
optrodes was activated with cyanogen bromide at -150
0
C in the presence
of triethylamine. h en they were immersed into the antigen (Ag) solution
for about 30 min and in 0.1 M glycine solution for 40 min to block the
remaining free functional groups. For the immobilization of Ab to human
IgG the optrodes were aminosilanised and activated with glutaraldehyde
(GA). At er such treatment they were immersed into the conjugate solu-
tion (1:10 titer) prepared in phosphate buf er (PhB), pH 7.2 for 1 h at room
temperature. h e remaining free functional groups were blocked as men-
tioned before. h e prepared optrodes were stored dry and sterile at 4
0
C.
To measure the level of non-specii c IgG using the i ber optic l uorescent
biosensor based on the principle non-emitting energy transfer, one half of
the prepared optrodes was placed in the solution containing human IgG
(1:20 conjugate titer). h is IgG were labeled by tetramethylrhodamine iso-
thiocyanates (TRIC). h e other ones were introduced in the same solu-
tion but with the B-phycoerythryn (B-Phyco)-labeled IgG. Both labeled
conjugates were dissolved in PBS, pH 7.2 containing tween-20 and bovine
serum albumin (BSA) in i nal concentration of 0.05 and l%, respectively
(buf er A). h e degree of antibody (Ab) saturation with Ag was estimated
by measuring the value of the signal B = I
f
,/I
fd
, where I
f
, is maximum emis-
sion intensity at band-pass acceptor l uorescence (TRIC, 560 and B-Phy,
575 nm); I
fd
is a maximum emission intensity at band-pass donor excita-
tion (TRIC, 488 nm). Every other 3 min during the incubation one of the
optrodes was taken out to measure the l uorescence signal. h e minimum
saturation period was 21 min. For sensor calibration the optrodes were
immersed one by one into solutions containing a number of consecutive
dilutions of human IgG in buf er A starting from a concentration of 500
ng/ml. While being measured the optrode was placed into a black Tel on
microcell.
h e block-scheme of such an immunosensor is given in Figure 13.1 [A].
h e optrode was connected to the beam splitter by a special joint. h e laser
emission spread through one beam splitter branch and the l uorescence
emission to the photo detector through the other one. When the complex
of immobilized labeled Ab (donor) -1abelled Ag (acceptor) is formed, the
l uorescent labels approach each other thus provoking an energy transfer,
