Biomedical Engineering Reference
In-Depth Information
negligible. This obvious difference between the control and positively labeled
samples is highly important for DMR assay in reducing false positives in biosensing.
9.4.2
New Labeling Method for High MNP Loading
Besides improving the physical properties of MNPs, equally important for the
DMR assay is to develop an efficient method for MNP labeling on target cells. A
conventional way is to use MNPs preconjugated with target-specific affinity ligands
[
22
], which often requires extensive optimization of the affinity ligands and the
conjugation method for each new target.
We have developed a new targeting strategy, BOND (bioorthogonal nanoparticle
detection), that is modular and broadly applicable and can amplify MNP binding to
biological objects [
27
]. The BOND is based on Œ4
2 Diels-Alder cycloaddition,
especially between tetrazine (Tz) and
trans
-cyclooctene (TCO; Fig.
9.5
a) [
50
].
The reaction is fast and irreversible (covalent) and can be performed at room
temperature without using any catalyst (copper). We have adapted the chemistry
for MNP labeling of cells (Fig.
9.5
b), wherein cells are pretargeted with TCO-
modified antibodies and subsequently incubated with Tz-loaded MNPs (Tz-MNPs).
Multiple TCO tags (usually
C
20) could be incorporated onto an antibody, and
such modified antibodies were found to maintain their affinity. Consequently, the
antibodies functioned as a scaffold to promote multiple attachments of Tz-MNPs.
Indeed, flow cytometry measurements showed that the BOND yielded
15-fold
improvement in MNP loading on cells, compared to labeling with antibody-MNP
direct conjugates (Fig.
9.5
c). The trend was further confirmed in NMR-based cell
detection (Fig.
9.5
d); the BOND method yielded more pronounced T
2
changes and
improved the cellular detection limit.
BOND has been successfully adapted for DMR molecular profiling of
experimental cellular samples [
27
] and clinical fine-needle aspirate samples [
26
].
Recently, the BOND technology was further generalized by developing newer
two-step detection schemes based on complementary oligonucleotide approaches
[
29
],
alternative
cycloaddition
chemistries
[
51
],
and
cyclodextrin/adamantine
supramolecular interactions [
52
].
9.5
Miniaturized NMR System
The development of miniaturized nuclear magnetic resonance (NMR) systems
[
22
] represents a key milestone in sensitive detection in DMR. Device miniatur-
ization brings several distinctive advantages for sensitive detection and clinical
translation. First, it provides a promising way to improve the detection sensitivity.
Smaller NMR systems reduce the detection volume, which in turn effectively
increases the concentration of MNP-targeted cells for large R
2
changes (Eq.
9.3
)
