Comparative Study of Porous Silicon Biomolecule Conjugation Procedures

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Comparative Study of Porous Silicon Biomolecule
Conjugation Procedures

• Andrea M. Rossi1,2,3, Lili Wang2, Vytas Reipa2,
  Thomas E. Murphy1
• Department of Electrical and Computer
  Engineering, University of Maryland, College
  Park, MD 20742, USA
• 2) Biochemical Science Division, National
  Institute of Standards and Technology , 100
  Bureau Drive, Stop 8312, Gaithersburg, MD 20899,
  USA
• 3) Nanotechnology departement, Istituto Nazionale
  di Ricerca Metrologica, Strada delle cacce, 91
  10135 Torino, Italy

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Outline

• Objectives
• Porous silicon formation
• Photoactivation techniques to functionalize
  porous silicon
• Surface hydrophobicity and hydrophilicity
  measurement
• Detection of antibody and antigen by fluorescence
  measurement
• Conclusions

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Objective
Realize optical sensor for MS2 virus using porous
silicon as substrate

• 26 nm diameter
• A copy of single-stranded RNA(3569 bases)

MS2 serves as a viral model system for reliable
detection of biological warfare agents (1)
safety (2) easy purification (3) training
personnel and testing antiviral and antiseptic
agents.
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Why Porous Silicon?
Anodization in HF (a few minutes)

• Easy to obtain

PS sample
Si wafer

• Large specific surface ( 200 m2/cm3)

• it is a nano-sponge

• Biocompatible
• L.T. Canham, Bioactive silicon structure
  fabrication through nanoetching techniques,
• Advanced Materials 7, 1033 (1995)

• Easily integrable with Si-based microelectronics

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Porous Silicon Fabrication
Reference electrode
Counter electrode
Power supply
Electrical contact
Silicon wafer
Total reaction Si(s) 6F 2H 2h ? SiF62
H2 (gas)
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Control of Porosity by Current
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PS surface functionalization
Method 1 Alkylation through UV assisted
hydrosilylation

• PREPARATION
• Prepare porous silicon
• Immerse in Decene solution in quartz cuvette,
  under N2 flux (15 min.)
• UV illumination (l 250 nm, 1 hr.)
• Ethanol wash and rinse under N2 flux

• BIOCONJUGATION
• Alkylated Si surface activated by
  4-benzoylbenzoic acid succinimidyl ester (BBA) in
  deaerated anhydrous carbon tetrachloride
• UV illumination (l 365 nm, 15 min.)
• Rinse with carbon tetrachloride
• Immerse in antibody solutionAlexa 488
  dye-labeled rabbit anti-MS2 antibody

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PS surface functionalization
METHOD 2 Carboxylic functionalization

• PREPARATION
• Prepare porous silicon
• Immerse in Acrylic acid solution in quartz
  cuvette, under N2 flux (15 min.)
• UV illumination (l 330 nm, 1 hr.)
• Ethanol wash and rinse under N2 flux

• BIOCONJUGATION
• Acid-derivatized Si surface immerse in antibody
  solution Alexa 488 dye-labeled rabbit anti-MS2
  antibody
• Add 1-ethyl-3-3-dimethylaminopropylcarbodiimide
  hydrochloride solution in DI water
• Reaction carried out in dark (2 hr.)

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Functionalization Process
Energy E3.75 eV
Energy E4.96 eV
Photons
Photons
Acrylic acid solut.
Hydrophobic surface
Hydrophilic surface
Organic monolayer covalently attached to the
surface through silicon-carbon bonds
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Contact angle measurement
Sessile drop method
sL
T contact angle
sS
T
sLS
Youngs Equation
sS surface free energy of solid
sL surface tension of liquid
Sketch of sessile drop apparatus
sLS interfacial tension between liquid and solid
Porous materials classic theory
Hydrophilic surface are treated like rough
surface, where apparent contact angle obeys the
Wenzel theory
Hydrophobic surface are treated as composite
surface with air hydrophobic molecules in which
the Apparent contact angle is expressed
by Cassies law
Where r is the roughness ratio
Where f is the nanorod fraction remaining dry
Contact angle measurement are an important tool
to characterize the wettability of solid surface
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Contact Angle Measurement
Time dependence of an equilibrium contact angle
Contact angle measured on 1. porous silicon as
prepared 2. Decene functionalized PS 3. Acrylic
acid functionalized PS
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Fluorescence Measurements
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Removal of Non-covalently Bound Protein
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Test of the Covalent Si-Antibody Bond
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Antigen Fluorescence Spectra
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Conclusions

• Porous silicon functionalization by two methods
• Wettability can be controlled through surface
  functionalization.
• The effective penetration of the Antibody inside
  pore is demonstrated by fluorescence
  measurement
• The selective binding of the antigen to the
  specific antibody is demonstrated

Future work

• Improve the sensitivity to the antigen
• realize optical structure like waveguide for
  label-free sensor application