| dc.description.abstracteng | Information transfer in the human brain is enabled by neurotransmitters - messenger
molecules that diffuse between cells. Static concentration measurements are useful to identify
molecules involved in neurotransmission, but so far dynamic measurements remain a
challenge. However, detecting neurotransmitters with spatiotemporal resolution is essential
to understand chemical neurotransmission and how it contributes to phenomena such as
learning or degenerative diseases. This thesis investigates various strategies to develop nearinfrared
(nIR)
fluorescent sensors and the larger goal is spatiotemporal detection of small
molecules such as neurotransmitters.
The first two sections of the thesis focus on functionalized single-walled carbon nanotubes
(SWCNTs). SWCNTs can be visualized as a graphene monolayer rolled up into a hollow
cylinder. They emit
fluorescence in the range of 850 - 1700 nm (near infrared, nIR). The
photophysics of SWCNTs is excitonic and upon excitation excitons move along the SWCNT
axis. Even small changes in their local environment affect the
fluorescence of SWCNTs, which
makes them ideal building blocks for sensors. The exact photophysical sensing mechanism
behind the
fluorescence change of SWCNT-based sensors is not fully understood. To answer
this question different possible sensing mechanisms were investigated by measuring how
small redox-active molecules with different redox potentials, such as the neurotransmitter
dopamine, ascorbic acid or cysteine, change the
fluorescence of SWCNTs functionalized with
different macromolecules, such as single stranded DNA (ssDNA) or lipids. First, a direct
electron transfer between analytes and SWCNTs was ruled out. It was further verified
that
fluorescence changes are not due to scavenging of reactive oxygen species by analyte
molecules or adsorption onto the free SWCNT surface. The results support a photophysical
sensing mechanism based on the conformational change of the macromolecule around the
SWCNT.
In the second part of the thesis, the recognition strategy was advanced by changing the
functionalization concept. Despite high sensitivity of ssDNA/SWCNT sensors, their selectivity
remains a challenge. Proteins and peptides are well-known for their specific molecular
interactions in nature, but they have only rarely been used for SWCNT functionalization
due to colloidal stability issues. Here, peptides were employed as recognition units in a modular
sensor approach. As proof of principle this strategy was tested for a well-established
cell surface receptor (integrin) ligand: Arg-Gly-Asp (RGD). The RGD peptide sequences
were synthesized and conjugated to ssDNA in different geometries: the RGD recognition
motif was either anchored between two ssDNA sequences (bridge) or attached to the ssDNA
sequence at one side (linear). Next, the inhibition of aIIbb3 integrin by different ssDNA-RGD/SWCNT hybrids was quantified by an enzyme-linked immunosorbent assay. The data suggest that all three parameters, such as ssDNA sequence, length, and geometry, modulate
the affnity of the recognition unit (RGD) to its target (integrin). IC50 values for the different
hybrids could be tuned from 20 to 309 nM. While SWCNTs serve as a confinement structure
for the recognition unit, they also add a unique nIR
fluorophore. This property was used to
label aIIbb3 integrins on human blood platelets in the nIR. These results show that it is possible
to use a small peptide recognition motif and tune binding affnities by changing a DNA
sequence, which could be highly interesting for the detection of other neurotransmitters such
as glutamate.
In the last section another nIR
fluorophore was explored: Egyptian Blue (CaCuSi4O10, em
= 930 nm). Egyptian Blue is a calcium copper silicate with high quantum yield and a
long
fluorescent lifetime in the range of microseconds. First, Egyptian Blue was exfoliated
into nanosheets and analyzed via atomic force microscopy. The nanosheets had a radius
of approximately 36 nm and a thickness of 4 - 5 nm. This height corresponds to 3 or 4
layers of Egyptian Blue unit cell layers. The mean
uorescence intensity of the nanosheets
can be described by the product of height and radius of the particles, and thus correlates
with the nanosheet volume. Therefore, the dimensionality of the nanosheets do not seem to
affect the
fluorescence quantum yield. In agreement with that,
fluorescence lifetimes of the
bulk material compared to nanosheets (tau = 124 µs) did not change. Finally, Egyptian Blue
was successfully implemented as a nIR reference signal for ratiometric dopamine detection,
which paves the way for applications of Egyptian Blue nanosheets in biomedical imaging. | de |