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

2
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

4
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.
7
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

8
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.