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ABSTRACT
This paper describes studies of surface-patterned nanohydrogels and their use as possible
substrates for high-density and high-sensitivity protein arrays. Nanohydrogels, approximately
200 nm in diameter, were created by locally crosslinking dry amine-terminated poly(ethylene
glycol) [PEG] (5000 Da) thin films using a focused electron beam. These gels then had a dry
height of 50 nm and a swell ratio of about five. They were patterned into arrays with
approximately 1 m inter-gel spacing. These arrayed gels were functionalized with Zinc Finger 9
(ZNF9), a nucleic-acid binding protein. As part of ongoing research, we are interested in how the
performance of the functionalized gels compares to that of current commercial substrates. We
show, using an assay that binds -GST antibody to mediate attachment of GST-tagged ZNF9,
that nanohydrogels have a consistently higher combination of fluorescent signal and signal-to-
noise (SNR) ratio than the comparable commercial substrates. We further show that the SNR
characteristic of our assay degrades slower on the nanohydrogel array than on a comparable
microarray printed on an epoxide substrate, and we speculate that the difference is in part
attributable to the fact that the nanohydrogel array presents a softer and more hydrophilic
surface.
INTRODUCTION
immobilized proteins are less likely to denature [5-8], and the nanoscale feature size brings an
approximately 10,000-fold increase in the areal density of spots on a chip.
EXPERIMENTAL DETAILS
Thin films (~100 nm) of PEG-NH2 (5000 Da) were prepared on glass slides as in [10].
The PEG arrays were arranged to yield 100 m diameter spots each made up of ~7500 discrete
individual nanohydrogels. This format enabled us to use the same microarray scanning and
analysis methods used to characterize conventional microarrays spotted onto commercially-
available substrates. Within a given 100 m diameter spot all of the nanohydrogels were
functionalized identically with -GST crosslinked to gel amine end-groups via thioether bonding
using Sulfo-SMCC and Trauts Reagent. The gels were then exposed to 1.25 g/ml of ZNF9-
GST in PBS. Once functionalized with ZNF9, the nanohydrogels were probed with an
oligonucleotide called universal substrate [US] whose structure and details of hybridization are
given in [11]. All characterization was done using a GenePix 4000B scanner and Genepix 5.1
software.
The nanohydrogel format was compared to microarrays produced using four different
commercially available slide chemistries: nitrocellulose (FAST) slides (Schleicher & Schuell),
3D hydrogel slides (Perkin Elmer), epoxide slides (Corning), and Ultra-GAPS (amino-silane)
slides (Corning). All four slide types were spotted using a GeneMachines Omnigrid 100 Arrayer
(Genomic Solutions) with SMP3 print pins (Telechem) yielding spots of approximately 100 m
in diameter. In order to compare microarrays on these slides to the nanohydrogel slides all
proteins were printed at a concentration of 0.5 mg/ml.
The fact that nanoscale spots can be created using functional nanohydrogels is illustrated
by Figure 1. It shows an image of a 5 m x 5 m portion of a 100 m diameter spot of arrayed
0897-J06-07.3
Fluorescence
Substrate Signal Intensity
Nanohydrogel 22089 +/- 880
Nitrocellulose 20988 +/- 844
3D Hydrogel 16307 +/- 136
Epoxide 5241 +/- 223
Amino-silane 4951 +/- 83
Table 1: Fluorescent intensities characteristic of the -GST antibody mediated ZNF9 nanohydrogel
compared to the four microarray formats.
nanohydrogels. Because the GenePix scanner has insufficient resolution to detect individual
nanohydrogels, this image was collected using a Nikon E1000 fluoresence optical microscope at
a magnification of 1000x. -GST coupling and ZNF9 immobilization were performed as above
and hybridized using a Cy3-labled universal substrate.
Figure 1: Individual nanohydrogels visualized at a magnification of 1000X. Shows optical and fluorescent
(FITC) micrographs of three set of nanohydrogels functionalized with ZNF9, GST and BSA respectively
0897-J06-07.4
by Table 1, the positive signals in these assays are well above the noise and can be distinguished
in a statistically significant way.
While the e-beam patterning process can be easily modified to decrease the inter-gel
spacing between adjacent nanohydrogels and increase the total surface area of a 100 m
diameter spot of nanohydrogels, the higher signal characteristic of the nanohydrogel arrays
observed here can not be attributed merely to differences in exposed surface. We speculate that
the differences are in part due to the highly hydrated nature of the nanohydrogel arrays. Because
these gels are principally composed of water, they are better able to maintain the ZNF9
conformation in its natural conformation thus preserving its ability to bind to a nucleic acid such
as the universal substrate. In contrast, the nitrocellulose, epoxide, and amino-silane microarray
substrates provide harder surfaces to which the ZNF must bind and increase the possibility that
ZNF9 on the array surfaces is in a conformation or orientation whose binding sites are less
accessible to the universal substrate.
In order to shed further light on our above speculation, we took the nanohydrogel and its
analogous SNR performer the epoxide substrate and analyzed its performance over time
when stored in a dessicator. As the SNR results in Figure 3 show, the signal from the epoxide
microarray decays substantially when compared with the hydrogel nanoarrays over a time period
of 24 hours. We would posit that this downward trend would continue over time till the signal
from the epoxide microarray can be questioned for its statistical significance. In addition to our
discussion above, we further speculate that this decrease in SNR may be attributed to the
expulsion of water from the hard and flat surface of the epoxide substrate. Such a loss of water
would cause conformational stress on the immobilized protein that would further decrease its
functional ability of binding its target nucleic acid. Contrastingly, the nanohydrogels, being
hydrophilic, would tend to retain a higher amount of water thereby maintaining a more natural
20
15
SNR
10
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Figure 2: The SNR characteristic of the -GST antibody mediated ZNF9 nanoarray compared to the four
microarray formats
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conformation. We are currently studying whether this phenomena holds for proteins of diverse
molecular weights and charges.
CONCLUSIONS
PEG-NH2 nanohydrogels can be created using the energy from a focused electron beam
to locally crosslink thin films of dry homopolymer and bind them to a substrate such as glass or
silicon. Gels can be made with lateral dimensions on the order of 200 nm and can be patterned
in arrays at inter-gel intervals of 1 m. Utilizing the amine end-groups on the nanohydrogels we
can chemically functionalize them with various biological reagents.
For our model assay involving the interaction between a specific nucleic acid binding
protein and a well-characterized 55-mer oligonucleotide, we find that the arrayed nanohydrogel
assay provides correct positive and negative signals and achieves signal intensity and signal-to-
noise ratio consistently higher than each of four comparable microarray formats used as controls.
Nanoarray
20 Epoxide
15
SNR
10
0
0 8 16 24
Dessication Time (Hours)
Figure 3: SNR of Nanoarray and Epoxide substrates stored in a dessicator over time
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Together with the fact that a fluorescence optical microscope can image individual
nanohydrogels, these results indicate that chemically functional nanohydrogels can serve as an
attractive new substrate platform for a next-generation protein-array technology.
ACKNOWLEDGEMENTS
This research has been supported by the Army Research Office (ARO grant DAAD19-
03-1-0271) and uses instrumentation supported by the National Science Foundation and the New
Jersey Commission on Science and Technology.
REFERENCES