Laboratoire de Matériaux et de Génie Physique
Rapport de Stage de DEA
Development and Functionalisation of thin Antimony doped Tin IV oxide layer for DNA chip applications
Maître de stage : Valérie Stambouli-Sené
15/01/2004
Emmanuel DUPUY
Outlines I. INTRODUCTION II. WHAT IS A DNA BIOCHIP ? III. EXPERIMENTAL A] Biochip Assembly B] Surface Characterization Techniques
IV. A FEW RESULTS V. CONCLUSION
15/01/2004
Emmanuel DUPUY
I. INTRODUCTION
II. WHAT IS A DNA CHIP?
IV. A FEW RESULTS
V. CONCLUSION
III. EXPERIMENTS EXPERIMENTS
I. INTRODUCTION •
Multi-DisciplinaryTechnology including biology,microelectronics,
optics, instrumentations and informatics
• Huge application fields :
- medical
- industrial Quasi infinite use of biochips for all living organisms •Biochip Market Prospects : It Should reach 3 billions $ in 2004
Research stakes are huge : Need to manufacture reliable biochips with better performances.
15/01/2004
Emmanuel DUPUY
I. INTRODUCTION
II. WHAT IS A DNA CHIP?
IV. A FEW RESULTS
V. CONCLUSION
III. EXPERIMENTS EXPERIMENTS
II. What is a DNA BioChip? DEFINITION A biochip can be any device that consists of a solide substrate (Si, SiO2) onto which known biological molecules are grafted. When biological molecules are DNA sequences, it calls DNA chip A DNA chip enables to detect and quantify DNA molecules in a sample to be invastigated by hybridization process. DNA MOLECULE
Complementarity base pairs : Specific bonding occurs between base pairs : A with T and C with G This process, known as hybridization, is the biological interaction mechanism which is exploited by biochip technology. 15/01/2004
Emmanuel DUPUY
I. INTRODUCTION
II. WHAT IS A DNA CHIP?
IV. A FEW RESULTS
V. CONCLUSION
III. EXPERIMENTS EXPERIMENTS
DNA CHIP PRINCIPLE Target DNA strands in solution = molecules to be investigated Probe DNA strands fixed onto the chip
DNA Probe Grafting
Hybridization Detection
Chip
Hybridized Probe/Target DNA
C…..G A…..T
Specific Binding between complementary base pairs
•An array of known probe DNA is fixed onto the chip. • Then, unknown target DNA solution is introduced . Target DNA strands that have the complementary sequences bind with the proper grafting strands: Hybridization process. • This association can be detected and measured using a variety of electrochemical and optical techniques . Currently, fluorescence is the main technique.
15/01/2004
Emmanuel DUPUY
I. INTRODUCTION
II. WHAT IS A DNA CHIP?
IV. A FEW RESULTS
V. CONCLUSION
III. EXPERIMENTS EXPERIMENTS
DNA IMMOBILISATION
2 Main Methods • Synthesis
in-situ
By photolithography (Affymetrix)
•Grafting (DNA strands are pre-manufactured) Direct Adsorption « Electrochemical Addressing » of probe DNA molecules with a conductive polymer (CEA) Covalent Attachment on an insulated Polymer : The polymer (ATPES) deposated on substrate, acts as a biocompatible adhesive onto wich DNA molecules can be chemically bound and immobilised Advantages : - low cost, easy to carry out, reliable - But not Precise and Quantifiable 15/01/2004
Emmanuel DUPUY
I. INTRODUCTION
II. WHAT IS A DNA CHIP?
IV. A FEW RESULTS
V. CONCLUSION
III. EXPERIMENTS EXPERIMENTS
HYBRIDIZATION DETECTION Two Main Methods :
• Optical
* Fluorescence (extensively employed technique) Target DNA molecules
Label Fluorescence observed with microscope
Probe DNA strands fixed onto the chip
Hybridized Probe/Target DNA
Puce
C…G A…T
• Electronical
Advantages : High sensitivity, robustness Drawbacks : - Costly method (expensive labels), slow, no portability (external reader :laser, microscope) -Semi-quantitative method -Important background noise of solid substrate (Si and silica)
Advantages : - Analysis in real time (quick) - Direct (no fluorescente labels) - Low cost - High level of integration (lab on a chip) 15/01/2004
Necessity to use an electrical conductive layer and optically transparent to visible wave length. Emmanuel DUPUY
I. INTRODUCTION
II. WHAT IS A DNA CHIP?
IV. A FEW RESULTS
V. CONCLUSION
III. EXPERIMENTS EXPERIMENTS
OBJECTIVES of the Project ¾To evaluate the feasibility of a conductive ATO layer for biochip applications To develop a new antimony doped Tin Oxide electrode substrate biochip To use charactrisation techniques to study and control each step of the biochip development pathway
¾ HOW ? To Find Key Physical Characteristics of the Electrode Biochip for optimising DNA immobilisation and detection Layer Electrical Resistivity : the lowest as possible to maximise DNA electrochemical immobilisation and electrical hybridation detection. * depends of : - Sb doping level - Layer thickness and layer structure. Resistivity measurements 15/01/2004
Emmanuel DUPUY
I. INTRODUCTION
II. WHAT IS A DNA CHIP?
IV. A FEW RESULTS
V. CONCLUSION
III. EXPERIMENTS EXPERIMENTS
Surface Topography and Morphology : a highly smooth surface (RoughnessÆ 0) for a flat and homogenous polymerisation, so that DNA can be more easily immobilised. * depend of growth mode of ATO layer Æ deposition technique parameters:Aerosol Pyrolysis AFM SEM Wettability (with contact angle measurements)
Difficulty to characterise silane functionalisation surface on SnO2 layer in relation to smooth SiO2 and Si surfaces. - To Quantify the influence of SnO2 layer polymerisation by contact angle measurements.
roughness
on
silane
- To Invastigate if wettability measurements are reliable to characterise surfaces.
Biochip development pathway 15/01/2004
Emmanuel DUPUY
I. INTRODUCTION
II. WHAT IS A DNA CHIP?
IV. A FEW RESULTS
V. CONCLUSION
III. EXPERIMENTS EXPERIMENTS
IV. A FEW RESULTS 1) Resistivity measurements
Resistivity (10e-3 ohm cm)
10
8
6
1% Sb/Sn 2%
The lowest resistivity, 1.8×10-3 Ωcm, was found to be for a 930nm, 2% Sb deposition . T=298K
5%
4
2
0 0
250
500
750
1000
1250
1500
Compared to Si doped semi-conductor (~ 2 Ωcm ), 2% ATO layer is a very good conductor.
Epaisseur (nm)
Resistivity against layer thickness for three different dopages.
15/01/2004
Emmanuel DUPUY
I. INTRODUCTION
II. WHAT IS A DNA CHIP?
IV. A FEW RESULTS
V. CONCLUSION
III. EXPERIMENTS EXPERIMENTS
2) Characterization of SnO2 doped with 2% Sb Evolution of SnO2 roughness with deposition time ( = Thickness) 200 nm
AFM pictures of thin SnO2 doped Sb (2%) layer for a deposition time of 1 minute (A) et de 20 minutes (B).
0 250nm
A] 1 min (118nm)
250nm
B] 20 min (1.06µm)
Roughness (Rms) for 2 time of deposition : - 1 minute : 5.5 nm
20 18
- 20 minutes : 17.8 nm
16 14 12 10 8
- Roughness
6 4 2
Thickness
- Size of Grains
0 0
200
400
600
800
1000
1200
E p a is s e u r S n O 2 ( n m )
15/01/2004
Emmanuel DUPUY
I. INTRODUCTION
II. WHAT IS A DNA CHIP?
IV. A FEW RESULTS
V. CONCLUSION
III. EXPERIMENTS EXPERIMENTS
3)Wettability of surfaces Evolution of contact angle during functionalisation steps 50 45 40 35 30 25 20 15 10 5 0
- Wettability of surfaces is a good qualitative indicator of chemical composition changement But It does not inform how silane polymer is deposed on SnO2 layer. Contact angles are not influenced by roughness
Si
SiO2
SnO2
Silane
- Detection of measurements?
silane
layer
by
wettability
According to graph we can conclude that there is a big variation of contact angle between SnO2 and Silane proving his presence. θ = 0° for SnO2 , then θ = 50° for Silane
15/01/2004
Emmanuel DUPUY
I. INTRODUCTION
II. QU’EST CE QU’UNE BIOPUCE à ADN
IV. RESULTATS ET DISCUSSION
III. EXPERIMENTATION
V. CONCLUSION
Fluorescence Hybridization détection Encouraging results - We observed fluorescent area for a few samples tested. It shows that hybridization took place on this antimony doped Tin Oxide electrode substrate biochip - It seems that low values of roughness are required to optimise DNA Grafting. Others results are expected to confirm.
V. CONCLUSION We can use antimony doped Tin Oxide layer as substrate and active element for DNA biochip applications. Global Process of biochip development is checked. Silane layer is confirmed (a few nanometers).
Research Prospects These Encouraging results could lead to develop a device based on electrical hybridization
detection which would be easy to use, reliable, low cost, and portable. It could be integrated into microelectronic systems (Lab on a chip). 15/01/2004
Emmanuel DUPUY
