Chemistry Reference
In-Depth Information
various glycoprotein products (e.g., antibody drugs). Another promising applica-
tion is in the differential analysis of clinical samples to investigate glycan- related
biomarkers (described later).
Nevertheless, there is a basic problem with conventional lectin microarrays,
which, as with other microarray techniques, generally require a prior washing
process to remove unbound fl uorescence probes (Figure 14.5a). However, as affi ni-
ties of lectin-glycan interactions are relatively weak (in terms of
K
d
, 10
− 4
to 10
− 7
M),
approximately two orders of magnitude lower than those for antigen-antibody
(10
− 6
to 10
− 9
M), lectin -glycan complexes are likely to be easily dissociated by the
microarray washing processes usually applied. This results in signifi cant reduction
in the signal intensity. If such a washing process could be eliminated, much
stronger signals should be obtained. Accordingly, we have recently developed a
novel lectin microarray system based on an evanescent- fi eld assisted FD principle
[10]. In this system, an evanescent wave is used to directly excite a fl uorescent
group covalently linked to a glycoconjugate such as a glycoprotein or a glycopep-
tide. When the fl uorescence-labeled glycoconjugates bind to the lectins immobi-
lized on the array, these molecules come close to the array surface. The excitation
Figure 14.5
(a) Effect of washing steps to
remove unbound glycoproteins on a lectin
microarray. Since lectin-glycan interactions are
relatively weak (in terms of
K
d
, 10
−
4
to 10
−
7
M),
washing steps to remove unbound glycopro-
teins could signifi cantly reduce binding signals
on the lectin microarray. The evanescent-fi eld
fl uorescence - assisted scanner requires no wash-
ing steps before detection and thus even weak
signals can be sustained, unlike a confocal-type
scanner used for DNA microarray that requires
washing steps to remove unbound molecules
for detection. An evanescent wave is propa-
gated only within a wavelength distance from
the sensor surface (below 200 nm) so that only
bound fl uorescently labeled glycoproteins can
be excited. (b) Binding of TMR-labeled bianten-
nary galactosylated
N
-glycan on the spots of the
plant lectin RCA120 is shown before and after
washing processes.
