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Design of UltraHigh Density DNALaden Polylysinemodified Silicon Surfaces
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Presented by Michael D. Soriano. Graduate Mentor: Yuli Wang. Faculty Mentor: ... Zhang, L.; Strother, T.; Cai, W.; Cao, X.; Smith, L. M.; Hamers, R. J. Langmuir ... – PowerPoint PPT presentation
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Title: Design of UltraHigh Density DNALaden Polylysinemodified Silicon Surfaces
1
Design of Ultra-High Density DNA-Laden
Polylysine-modified Silicon Surfaces
- Presented by Michael D. Soriano
- Graduate Mentor Yuli Wang
- Faculty Mentor Dr. Ying C. Chang
- UC LEADS Program
- August 15, 2002
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Abstract
- Single-stranded DNA was attached to
polylysine-modified silicon (PLS) surfaces.
Initially, the PLS substrate was protected.
Using a mixture of HBr/CH3COOH, the surface was
deprotected, ready for the coupling reaction with
the natural DNA, 1-ethyl-3-(3-dimethylaminopropyl)
-carbodiimide hydrochloride (EDAC), and
N-hydroxysuccinimide (NHS). Measurements of the
characterization of each surface were taken the
contact angle, refractive index, and thickness
before and after deprotection thickness and
refractive index after the coupling reactions.
The coupling reactions were optimized by
modifying the surface density of the PLS.
Binding specificity of complementary DNA was
tested for using fluorescence hybridization.
Preliminary results showed increases in substrate
thickness from 20-80 after the coupling
reactions.
3
Introduction
- The current technology of DNA microarrays
attaches the nucleotides via bi-hetero-functional
linkers1 or heat/light2, e.g. ultraviolet (UV)
light. Protocols for microarray design call for
coating silicon or glass slides with polylysine
(PLL), then DNA attachment is mediated. The
resulting microarrays may effectively only be
used once and have a superficial coating of DNA
on the surfaces. Lenigk et al.3 have created
microarrays for repeated use by using
(3-mercaptopropyl)trimethoxysilane. They have
successfully shown that their design may be used
repeatedly up to five times. - Our design implements attachment of DNA strands
to polylysine substrates. We have effectively
increased the thickness of the substrate from 20
80 after coupling reactions. Background
fluorescence contaminated the sample
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Materials/MethodsDeprotection of Substrates
- Protected substrates according to Wang4
- Substrates were suspended in a layer of benzene.
- The aqueous layer was denser, containing acetic
acid and bromic acid. - System was sonicated for one hour
- Benzene layer facilitated passage of HBr, thereby
deprotecting the polylysine - Deprotected substrates washed with deionized
water and acetone
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Materials/MethodsCoupling Reactions
- The coupling reactions were carried out according
to Sehgal5. - Reaction time lasts from 20 to 24 hours
- Covalent (amide) bond formed between carboxyl
group of modified DNA and e-amino group on
polylysine monomers - Fluorescent scans were taken of the substrate
products
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Results/DiscussionCoupling Reactions
Table below shows percentage increase of
substrate thickness ranges from 20 to 80.
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Results/DiscussionWashing with 8.5 M urea
solution
- Both images are fluorescent scans of a 1.0
surface density substrate after the coupling
reaction - Top image is the coupling reaction product before
washing the with 8.5 M urea solution - Bottom image is after washing for one minute
- Result after washing
- 47.9 intensity decrease
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Results/Discussion Washing with pH 11.5 MES
solution
- Both images are fluorescent scans of a 1.0
surface density substrate after the coupling
reaction - Top image is the coupling reaction product before
washing the with pH 11.5 MES solution - Bottom image is after washing for 30 seconds
- Result after washing
- 26.7 intensity decrease
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Conclusions
- Preliminary results from coupling reactions
reveal that microarray size can potentially
double - Controlled by length of polylysine chains
- Large percentage increase in thickness shows that
efficient attachment of coupling reactions were
achieved - pH 11.5 MES solution best method for the removal
of unreacted DNA and other molecules causing
additional background intensity signals - Though smaller percentage change, larger absolute
change
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Future Work
- Hybridization of fluorescence-tagged DNA
complement - Analysis of kinetics and hybridization efficiency
- Testing different geometric patterns on silicon
surfaces (i.e. circles, squares) - Selective hybridization
- Lifetime of the microarray reusability
- Application using other biopolymers
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Acknowledgements
- I would like to give thanks to
- Dr. Chang, Yuli, and the Polymer Lab Group
- Ilona Pak, Lisa Gauf, the Office of Graduate
Studies - Summer Research Program Colleagues
- UCLEADS program
- UC Irvine and UC Davis
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References
- Strother, T Cai, W Zhao, X. Hamers, R. Smith,
L. M. J. Am. Chem. Soc. 2000, 122, 1205-1209 - Zhang, L. Strother, T. Cai, W. Cao, X. Smith,
L. M. Hamers, R. J. Langmuir 2002, 18,
788-796. - Lenigk, R. Carles, M. Ip, N. Y. Sucher, N. J.
Langmuir 2001, 17, 2497-2501. - Wang, Y Chang, Y. C. Submitted for review on
August 1, 2002. - Sehgal, D. Vijay, I. K. Anal. Biochem. 1994,
218, 87-91.
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