1. Silica Coating and Bioconjugation on Gold Nanoparticles
Andrea Tung, Carolyn L. Bayer, Stanislav Emelianov
Department of Biomedical Engineering
The University of Texas at Austin, Austin, TX
Ultrasound Imaging and Therapeutics
RESEARCH LABORATORY
Introduction
Results
Figure 1: Gold Nanosphere Synthesis. (a) PEGylated gold nanospheres. (b) Silica-coated with
amine functionalized groups. (c) Anti-EGFR antibodies attached to silica. (d) mPEG-silane fills
in open silica surfaces.
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Photoacoustic imaging is a nonionizing and noninvasive method that
can use metal nanoparticles to target a specific biological specimen of
interest. A pulsed laser beam is applied to the region of interest, which then
heats the nanoparticles and generates a thermoacoustic signal. 1 A ultrasound
transducer picks up the thermoacoustic signal and develops an image for
diagnosis.1
Gold nanoparticles have been shown to be optimal for photoacoustic
imaging because of their high contrast.1,2 They can then be conjugated to
antibodies that target specific biomolecules.1 In this work, a coat of silica is
grown on the gold nanoparticles to stabilize the shape of the nanoparticle and
to add an amine functional group that is needed for bioconjugation.3
The photoacoustic image contrast is dependent on the absorption and
scattering of the nanoparticle, which can be affected by the material
composition, shape, and surroundings.4 Nanorods have the necessary high
absorption cross sections for high contrast, photoacoustic imaging.
The end goal of the experiment is to use silica-coated gold nanorods
with bound antibodies to the epidermal growth factor receptor (anti-EGFR) to
promote higher contrast in photoacoustic imaging. Instead of the nanorods,
the experiment starts with the use of nanospheres due to their ease of
synthesis. Here, we demonstrate synthesis of the amine-functionalized silica
coating onto the gold nanospheres, followed by bioconjugation of an antibody
to the nanoparticle, and finally demonstrate specificity of the antibody bound
to the surface.
Silica Coating:
PEGylated gold nanospheres (AuNS), acquired from NanoHybrids, were
mixed with IPA. The silica coat consisted of 3% tetraethyl orthosilicate (TEOS,
Gelest) in anhydrous isopropyl alcohol (IPA). The TEOS solution was then added
into the AuNS reaction for the silica coating. The ammonium hydroxide in IPA
increased the pH in order to start the reaction that continued for 150 min. The silica
coat was then functionalized with amine groups trihydroxsylxilyl-3-
propylmethylphosphate (THSPMP) and 3-aminopropyldimethylethoxysilane
(APDMS) to prepare for bioconjugation. TEM images of the bare gold nanospheres
and silica-coated nanospheres were acquired.
Bioconjugation:
We modified a directional bioconjugation method, where the glycosylated
portion of the Abs attaches to the AuNS, to keep the antigen-binding site
unhindered. 5 The antiEGFR, C29.1 antibody (Ab) used in this experiment is first
labeled with AlexaFluor 594 for fluorescence imaging. To prepare for
bioconjugation, sodium periodate oxidized the glycosylated portion to form
aldehydes that then bind to the amine functional group of the AuNS. To verify the
presence of the resulting aldehydes, the Ab solution should turn purple when
mixed in Purpald solution
For attachment, the Ab solution was mixed with the AuNS and let alone to
react for 2 hours with shaking. Sodium cyanoborohydride was added to reduce the
C-N double bonded Schiff base to a stable single bond.5 An mPEG-silane coat was
later added to minimize cellular nonspecific uptake.5 After an overnight incubation
with shaking, the Ab-AuNS were cleaned and resuspended in DI water.
Au
Au
Y
Y
Y
Au
Y
Y
Y
Au
(a)
(b)
(c)
(d)
Materials & Methods
Figure 2: Transmission Electron Microscopy of Silica Coating Reaction. (a) Gold
nanosphere. (b) Silica coating on AuNS.
Figure 3: Fixed A431 cells (EGFR expressing) incubated for 3 hrs with the anti-EGFR
targeted AuNS. (a) Brightfield microscopy. (b) Darkfield microscopy. (c) Fluorescence
microscopy.
(a) (b)
(a) (b) (c)
Conclusion
The silica coating reaction is sensitive to many variables,
including the quality of the first PEG layer, the age of the TEOS, and
the timing of the centrifugation. Further studies are needed to improve
the consistency of the silica coat without compromising the thickness.
The bioconjugation process shows specificity that the Ab-AuNS are
binding to the targeted cell line.
In the future, and the gold nanospheres will be replaced with
gold nanorods, and the bioconjugated nanoparticles will be tested in
an in vivo breast cancer tumor model.
References