Diagnostic Platforms

Transcription

1 Institute of Molecular Biology & Biotechnology, Foundation for Research & Technology Hellas (IMBB-FORTH) A Acoustic ti Devices D i as the th Sensing S i Element El t in i Diagnostic Platforms Electra Gizeli Dept. of Biology, University of Crete & IMBB-FORTH

2 Biomembrane studies Devices & Biochips Acoustic Devices Biomolecular studies Diagnostics Cellular studies

3 Quartz Crystal Microbalance (QCM) Quartz f o =5-75 MHz ΔFrequency (ΔF)/ΔPhase (ΔPh) Gold electrodes Sensitive to mass changes ΔDissipation (ΔD)/ΔAmplitude (ΔA) Sensitive to viscosity changes Surface Acoustic Wave (SAW) f o = MHz Gold electrode Quartz λ=32 μm 155 MHz

4 Operating principle of SAW sensors Operating principle: p AC input in IDTs piezo electricity mechanical oscillation surface perturbation change in amplitude & phase Inverse piezo electric effect AC output from IDTs δ amplitude(db): energy dissipation phase (deg): bound mass Acoustic signal ( Α or Ph) Frequency (MHz) Time

5 Biomarkers Proteins Heart disease Breast tumor Ovarian cancer Prostate cancer Diagnosis based on concentration increase DNA Breast tumor Thalassaemia Cystic fibrosis Diagnosis based on DNA detection

6 Acoustic devices for multiple analysis μ-fluidics on SAW PDMS module #1 #2 #3

7 SEM (20x) images of PDMS modules 900 μm 1100 μm 1 mm 100 μm depth 1550 μm Side view/cross-section Functional module Top view Faulty module Mitsakakis et al., JMEMS 2008, 17:

8 Phas se (deg) neu neu neu neu b-bsa b-bsa b-bsa b-bsa μf4 μf3 μf2 μf Time (s) μf4 μf3 μf2 μf1 Reproducibility better than 90% Mitsakakis et al., Microel Engin 2009, 86:

9 Detection of multiple cardiac markers CK-MB anti- CRP anti- D-dimer anti- PAPP-A anti- CKMB CRP D-dimer PAPP-A YYYYY YYYYY gold top layer PMMA waveguide YYYYY YYYYY antibody PrG Quartz substrate 1. Oriented immobilization of antibodies 2. Injection of one biomarker per microchannel 3. Specific antibody-antigen binding

10 Phase (deg) μf4:ck-mb μf3:crp μf2:d-dimer 1 μf1:papp-a Time (s) ΔPh (deg g) Multi-marker detection in 4 microchannels Phase (deg) 0,5 PAPP-A -0,5-1,5-2,5 25-3,5-4, Time (s) c (μg/ml)

11 Analytical curves - correlation to clinical values Healthy region CK-MB > 0.01 μg/ml CRP > 1 μg/ml D-dimer > 0.5 μg/ml PAPP-A > 0.05 μg/ml Pathological region ΔPh (d deg) 4 2 CK-MB CRP D-dimer Ddimer PAPP-A Logc (in nm) Mitsakakis & Gizeli, Anal. Chim. Acta 2011, 699: 1-5

12 Simulation of complex body fluids 0.5 Phase (deg) PAPP-A 5 μg/ml Time (s) PAPPA - single PAPPA - in mixture a-ckmb + PAPPA a-crp + PAPPA a-ddimer + PAPPA

13 Dip-pen nanolithography principle (+) Direct-write, maskless method (+) Compatible with many surfaces & materials (+) Upgradable to large-scale patterning

14 PDMS microfluidic module and separating walls (120 µm width) device horizontal axis acoustic c aperture: 1..6 mm..... PMMA waveguide IDTs contact pads quartz substrate Pattern lipids with functional head groups Spread under humidity 26-cantilever array Faster surface coverage

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16 PDMS microfluidic module and separating walls (120 μm width) device horizontal axis acoustic ap perture: 1.6 mm. 4a 3a 3b 3c 2a 2b 1a μm μm.... PMMA waveguide IDTs contact pads 950 μm 330 μm 580 μm 1050 μm quartz substrate DNP lipids NTA-Ni lipids biotin lipids DOPC (μf4) (μf3) (μf2) (μf1)

17 (i) (ii) (iii) (iv),(v),(vi) K. Mitsakakis, S. Sekula-Neuner, S. Lenhert, H. Fuchs, E. Gizeli Analyst, 2012, 137:3076

18 Solid-phase hybridization assays; current state-of-the-art

19 Genetic markers detection based on hybridization º * target strand Mass detection does not always provide the answer to target complementarity and presence of SNP

20 Solid-phase hybridization assay exploiting conformational changes coupled with electrochemical detection Probe strand Target strand Immoos et al., JACS 2004, 10814

21 Novel acoustic sensing of molecular conformation A Ph [ ] molecular shape and size Length variations Bending ligands Intrinsic curvature Holliday Junction 105 o 124o 118 o Tsortos et al., WO 2008/ Tsortos et al., Biophys J 2008, 94, 2706; Biosens. Bioelectron. 2008, 24, 836 Papadakis et al., Biosens Bioelectron 2008, 24, 836; Nano Letters 2010, 10, 5093

22 Our approach: Use an acoustic device/biochip for the direct probing of DNA conformation during hybridization target hybridization probe strand Single stranded, coiled DNA Double stranded, rod-shaped DNA

23 Extension of this concept to solid-phase DNA hybridization probe target

24 Conformation probing biosensor for DNA hbidi hybridization studies tdi ds-110 ds Target strand ds-21 AR surface /Hz solution Papadakis et al., Anal. Chem. 2012, 84: 1854

25 Detection of single mutation in genetic analysis (TP53 gene) 20 nts-short oligo Single-stranded structure +28full or 28mut

26 Conformation-based detection assay nt 28 full 28 mut Papadakis et al., Anal. Chem. 2012, 84: 1854 Hz -1 ) AR ΔF x10 ( / H D/F ΔD/Δ A (A) Although bound mass is the same for both targets (i.e. 80 Hz) the AR values are different due to shape differences -70 ) F (Hz) b 28full 28mut sample Fully complementary target Time (min) mutated target D (10-6 6)

27 33 nts-long oligo Single-stranded structure +28full or 28mut

28 Effect of probe-sequence to acoustic detection 33nt 28 full 28 mut z -1 ) ΔF D/F (10-10 Hz ΔD/Δ b 28full 28mut sample If a longer hairpin is used with the same TP53 targets, the SNP detection ti capability is lost Papadakis et al., Anal. Chem. 2012, 84: 1854

29 SAW devices: advantages for biosensing s applications Label-free method; biosensing based on physical parameters Novel sensing mechanism via conformation-probing; application to clinical diagnosis μf-on-saw for multi-sample analysis (reproducibility > 90%) Real-time detection (kinetics of interactions) μf-on-saw together with conformation-probing bring a μf on SAW together with conformation probing bring a paradigmatic change in traditional solid-phase hybridization assays

30 Acknowledgements Biosensors Lab Dr K K. Mitsakakis Dr. A. Tsortos Dr. G. Papadakis p Dr A. Pantazis Dr. F. Bender D K Dr K. Melzak M l k Dr M. Saitakis $ $, IMEL,, NCSR Demokritos Dr. A. Tserepi M. Vlahopoulou A. Malainou Inst. of Nanotechnology, gy KIT Dr S. Lenhert Dr H. Fuchs Dept. Biology, Univ. of Crete Dr V. Morou Prof. J. Vontas