Optimization of Quantum Dot – Nerve Cell Interfaces
Jessica Winter1*, Christine Schmidt2, Brian Korgel1
1
Department of Chemical Engineering and 2Department of Biomedical Engineering
University of Texas at Austin, MC0400, Austin, TX, 78712, *Speaker
ABSTRACT
Nerve cells communicate by passing electrical signals through extensions from their cell
bodies. Micron-scale devices such as microelectrode arrays and field effect transistors have
already been used to externally manipulate these signals. However, these devices are almost as
large as the cell body of the neuron. In order to make functional electrical connections to nerve
extensions or their component ion channels that propagate signals, it will be necessary to utilize
increasingly smaller components. We propose the use of semiconductor quantum dots, which can
be optically activated, as a potential means of perturbing the nerve membrane potentials. As a
first step, we have created qdot-neuron interfaces with cadmium sulfide quantum dots. The qdots
may be attached to cells either non-specifically or through selected interactions exploiting
biorecognition molecules attached to the qdot particle surface. We have investigated the effect of
altered synthesis conditions including pH, concentration, reactant ratio, ligand length, ligand R
group, and ligand concentration on the nature and quality of qdot-cell binding. We discuss the
effect of these altered synthesis conditions on particle fluorescence intensity and color.
Additionally, we studied the interaction of these particles with cells and determined that larger
particles are more likely to bind non-specifically than smaller particles produced with the same
amount of passivating ligand. It is possible this results from reduced surface area coverage of
passivating chemical. Finally, we have produced particles passivated with the biorecognition
peptide CGGGRGDS in the absence of other chemical stabilizers and characterized their
surfaces using NMR and IR.
INTRODUCTION
Nanometer-sized colloidal particles, or quantum dots (qdots), exhibit sizes between that
of bulk material and molecular clusters. This results in a unique mix of material properties. For
example, qdots are large enough to retain semiconductor characteristics, yet small enough so that
quantum confinement of electron-hole pairs1 results in distinct spectroscopic traits. Optical
excitation can produce dipole moments,2 heat production,3 fluorescent emission,4 and electron
transfer.5,6 Further; many of these properties are size-dependant,2,7 allowing for the development
of devices with tunable characteristics. Several applications utilizing qdots have been explored,
including LEDs,8 solar cells,9 and biological labels.10-12 Although the latter application has led to
the development of commercially available qdot dyes, it fails to take advantage of the potential
for direct interaction between qdots and cells. Because cells communicate through nanometersized components (e.g.., biomolecules, receptors), the ability to interface nanomaterials and
biological components affords many new opportunities to alter cell behavior. This technology
may lead to the development of new prosthetic devices and bio-inorganic electronics.
We are designing a system to utilize qdot material properties (e.g., dipole moment,
electron transfer, magnetism) to both visualize and interact with cellular receptors. For example,
the dipole moment produced by qdots is much larger than that of bulk material.3 Attachment of
biorecognition molecules to the qdot surface can facilitate intimate contact between the dipole
and neuronal ion channels. If the separation distance is small enough, it might be possible to
induce a membrane potential change, producing an action potential. However, this will require
optimization of the qdot-cell labeling system. Labeling efficiency needs to be high to increase
signal, while qdot-cell separation distance must be minimized to reduce charge screening. Also,
it may be necessary to decrease non-specific binding so that labeled tissue may be distinguished
from surrounding tissue. Here, we present the initial development of this system, including the
optimization of aqueous qdot syntheses, studies of qdot non-specific binding, and the
development of bioconjugated labels. Additionally, we are currently using whole-cell clamping
to examine the effect of qdot labeling on nerve cell membrane potential.
EXPERIMENTAL DETIALS
Aqueous arrested precipitation of cadmium sulfide qdots was examined using a model
ligand, mercaptoacetic acid (Fluka, Cat. No. 88652), as described previously.12 Several synthesis
conditions were systematically altered, including pH, reactant ratios and concentrations, and
ligand concentrations, carbon chain length, and R group. A second type of particle was also
created, using the peptide CGGGRGDS as a capping ligand with no additional chemical ligands.
The presence of the peptide was confirmed using FTIR and NMR. All particles were
manufactured at least four hours before analysis, which consisted of measuring the resultant
spectroscopic properties. A Beckman DU500 spectrophotometer (Beckman Coulter, Fullerton,
CA) was used to measure particle absorbance, and PL spectroscopy was performed using a
Quanta Master Model C Cuvette-based scanning spectrofluorometer (Photon Technology
International, Lawrenceville, NJ).
Additionally, particle non-specific binding to biological substrates was investigated using
SK-N-SH neuroblastoma cells (ATCC, HTB-11). Cells were cultured following ATCC
recommendations in EMEM with 10% FBS and 2% Penicillin-Streptomycin. Then, cells were
incubated with 2% BSA in Dulbecco’s PBS for one hour to block inherent protein binding. The
cells were rinsed with Dulbecco’s PBS. Next the cells were incubated for 30 minutes with
quantum dots which had been isolated using 1-propanol and centrifugation, resuspended in
Dulbecco’s PBS, and sterile filtered. The cells were then rinsed with Dulbecco’s PBS three
times, and stored in Dulbecco’s PBS for observation.
RESULTS AND DISCUSSION
Previously, particles were examined while systematically varying the pH, reactant
concentrations and ratios, and ligand concentration, carbon chain length, and R group.13
Regardless of the variable examined, we observed a fluorescence emission intensity maximum at
intermediate particle sizes (~ 2 nm). We theorized that fluorescent maxima result from
thermodynamically stable surfaces, perhaps because of the formation of Cd-thiol precursor
complexes. These qdots displayed an emission wavelength of ~ 530 nm (green-yellow) with a
quantum yield of ~ 15%. This wavelength is easily distinguishable from blue autofluorescence
produced by the cell. Next, the particles were examined for non-specific binding. Larger particles
demonstrated the most non-specific binding, in some cases forming a thin film over both the
cells and substrate (Figure 1A). Minimal non-specific binding appeared to occur at intermediate
qdot sizes (Figure 1B). However, non-specific binding for small qdots was difficult to measure
because the emission wavelength is similar to that of cellular autofluorescence (Figure 1C).
Given the high quantum yield and low non-specific binding shown at intermediate sizes, it was
decided to develop biolabels in this size range.
B
A
C
20 μm
Figure 1: Non-Specific Binding Produced by (A) Large (~ 5 nm) Qdots, (B) Intermediate
Size (~ 2nm) Qdots, and (C) Small (~ 1 nm) Qdots. Qdots appear orange/yellow, cells
autofluoresce blue. For large qdots (A), a film is formed on the surface of the qdots and
substrate. Intermediate sizes (B) produce minimal non-specific binding. Smaller sizes (C)
produce limited binding that is difficult to distinguish from background autofluorescence.
Thus, we attempted to construct particles passivated with peptide ligands (i.e.,
CGGGRGDS). These ligands are substantially larger than the model ligand examined
previously. From our model experiments, we knew that increasing ligand length would blue shift
emission fluorescence and decrease particle size (Figure 2A). However, we had also discovered
that increasing reaction pH can compensate for this effect. Therefore, using altered synthesis
conditions, we were able to produce qdots with sizes near 2 nm. However, the amount of peptide
required for such syntheses is large. Given cost limitations, we also experimented with
composite systems, incorporating mercaptoacetic acid as a “filler” ligand in addition to the
peptide. The resultant qdots were studied using both IR and NMR to verify the presence of
peptide, evaluate qdot-peptide binding characteristics, and roughly determine the amount of
peptide present. Finally, the peptide-conjugated qdots were used to label cells (Figure 2B).12
Ligands recognized and bound model cell surface receptors (i.e., integrins), allowing for
nanometer scale (i.e., 3-30 nm) separation distances.12 Currently, we are attempting to measure
changes in the nerve cell membrane potential produced by optically excited qdots.
1
Intensity
0.8
0.6
0.4
0.2
A
0
250
350
450
550
Wavelength (nm)
650
10 μm
B
Figure 2: (A) Absorbance and PL Emission Spectra of MAA (Red) and Peptide (Blue)
Passivated Nanocrystals. (A) Peptide conjugation blue shifts the absorbance (i.e., 370 nm vs.
350 nm) and emission peaks (i.e., 520 nm vs. 550 nm) when compared to the MAA control. (B)
Cells were incubated with peptide passivated qdots, and bound to integrins on the cell surface.
Qdots appear green-yellow, the cell autofluorescence is blue.
CONCLUSIONS
The goal of our work is to demonstrate the ability of qdots to serve not only as labels, but
also to manipulate cell function. In pursuit of this goal, we have examined the effect of synthesis
conditions on the spectroscopic properties of the qdots produced. In particular, we optimized
qdots for maximum quantum yield and minimum non-specific binding. Further, we produced
peptide conjugated qdots that are capable of binding cell surface receptors. These qdots will
serve as a starting point for future work, where we hope to demonstrate the ability of optically
excited qdots to produce nerve membrane potential changes. Although our goal was to create
biological labels, the ability to tune spectroscopic properties is relevant to several applications
(e.g., LEDs). Ultimately, this work will improve the ability of nanometer-scale colloids to
interact with cells and biomolecules. As the emphasis in materials science shifts from the
micron-scale to nanometer-scale applications, the ability to interface technologies at this level
becomes increasingly important. Additionally, cells communicate using nanometer-sized
biomolecules, and the capability for integration of biological components and nanomaterials will
provide intriguing opportunities to influence cell behavior. This work highlights many of the
issues that may inhibit the construction of such devices, and describes some of the first steps
taken to build novel nanoscale biomaterials.
ACKNOWLEDGEMENTS
The authors gratefully acknowledge discussions with Fred Mikulec and David Jurbergs.
Funding for this work was provided by the Texas Higher Education Coordinating Board through
their ARP program (CES, BAK), an NSF graduate research fellowship (JOW), and the NSF
IGERT program (JOW). FTIR facilities were provided by the center for nano- and molecular
materials sponsored by the Robert A. Welch foundation.
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