Case Studies of Micro-Biosensors

Transcription

1 Case Studies of Micro-Biosensors Lecture April 18 Jeff T.H.Wang website: New course : BioMEMS and BioSensing (Spring 04 )

2 Advantages of Micro Biosensors Bench process Miniaturization µ-tas Parallel Processing Integration Automation

3 Micro Total Analysis System (µ-tas) Sample input Sample pretreatment Micro fluidics Sensors Output signal Electronic control/signal processing

4 Conventional DNA Hybridization Based Detection 1 Mixing Detection probe Target DNA Anchoring probe 2 Immobilization Separation 3 Washing Detection relies on a solid surface for Separation Surface contamination and non-specific binding

5 Surface-to-Volume Ratio vs. Scaling L L Surface 1 L Volume L (1/m) Surface-to-volume ratio L (m) Drawbacks for detection basing on separation for a microsystem High drag force, high power consumption Low washing efficiency, time consuming process High noise level

6 Molecular Beacon (MB) Simplified the Detection Procedures Molecular Beacon Fluorescence Resonance Energy Transfer(FRET) Loop (Probe sequence) Stem (GC rich) Dipole-Dipole Interaction 1 Distance dependent (6th power) E = r 1+ ( Heat R (Fluorophore) Light D r 1 A (Quencher) Fluorescence 0 ) 6 Fluorophore Quencher (Fluorophore) Light D r 2 A (Quencher) Molecular Beacon Target DNA Fluorophore Hybrid Target DNA MB Quencher Immobilization and Washing Steps Can be Eliminated

7 Fluorescence Excitation and Emission S S Absorption Internal Conversion Intersystem crossing hν A hν E hν P Emission Emission (Fluorescence) (Phosphorescence) S

8 0 N.A. : Numerical Apterure Micro-reactors Integrated with Mirrors Transparent substrate (Glass, Plastic or Polymer) Objective (N.A.) Φ Opaque Substrate (Silicon) Objective (N.A.) Φ I n Glass molecule Glass Light collection efficiency(ε C ) ε ε C (%) 100 C Φ 2sin = = 2π 1 ( N. A./ n) 2π N.A.=0.2 N.A.=0.5 N.A.=1.0 ε C ε C (%) 100 N.A.= = Φ I 0 All R 0 Glass Au Mirror Al Mirror (Reflectivity: Au(0.6), Al(0.92)) I 0 n *Patent pending Perfect Mirror

9 Using Si as the Substrate for the Biosensors Metal(Al) Glass Si Objective Photon Collection Efficiency 1.00 Si/SiO2 (R=0.18) Al Coating(R=0.92) Au Coating(R=0.38) Glass Φ I n R 3 R I R R 4 1 R 5 Glass Si Aspect Ratio (D/W) W D

10 V-groove Fabrication Rhombus Si PR SiO 2 Si 3 N 4 SiO 2 1. Pattern & Fluorine RIE etch SiO 2 and Si 3 N 4 4. Deposit Al layer using e-bean evaporator 1. Deposit SiO 2, Si4 3 N 4 & pattern the pre-trench 5. Remove Si3N4 mask & wet oxidation for a dielectric layer 2. KOH etch the channel 5. Pattern Al using Lift-Off technique 2. Fluorine RIE etch SiO 2 and Si 3 N 4 6. Sputtering Al layer PR 3. DRIE etch the pre-trench 7. Pattern & direct etch Al layer 3. Remove Si3N4 mask & wet oxidation for a dielectric layer 6. Anodic bonding with glass Glass 4. KOH etch the channel 8. Anodic bonding with glass

11 Control of sensor opening Fabrication W 1 D W 2 D Aspect 1 = D 1 /W Aspect 2 = D 2 /W 2 Anodic Bonding (a) Cr/Au 2200Å Pyrex Glass (b) (a) Au SiO 2 (b) SiO Å Air Water Air (a) (b) Detection channel 100 µm 100 µm Reservoir (c) (d) Detection Mirror Electrode 100 µm 100 µm

12 Sensitivity Enhancement Normalized Signal Level Al Coating Au Coating No Coating Background x x x x Concentration (M)

13 Cantilever Beam Biosensor Principles Internal molecular forces arising from adsorption of small molecules induces surface stress Waston-Crick base pairing between unlabeled oligo-nucleotides and their surface-immobilized partner The surface stress results in beam bending that is measured by the optical beam deflection technique Procedues Fabrication of cantilever beam, coating with Au Immobilization of binding molecules Introduce target molecules Measure the bending and subtract common background bending

14 Fabrication Material of the beam Releasing of the beam Etching stop

15 Calculations of Cantilever Beam Bending due to Surface Stress (1) Calculations of Cantilever bending (2) Surface stress induced bending Stoney formula

16 Optical Beam Deflection Measurement

17 DNA Array High density arrays of oligonucleotide probes Used for gene sequencing, gene identification Importance New targets for drugs or other therapeutic intervention Diagnostic markers for disease Development of genetic database Genetic Database Function must be assigned to gene (discovery) Location of gene determined (mapping) How often is gene used (expression) How do these genes differ between individuals (genetic variations)

18 Manufacturing G11 G21 G31 G12 G22 G32 G13 G23 G33 Photolithography Inkjet printing Stamping Inject printing

19 Fabrication of DNA Array Basing Photolithography

20 Fabrication of DNA Array

21 Fabrication of Affymetrix DNA Array