From RNA Extraction to Microarray Based Readout · 2016. 12. 20. · From RNA Extraction to...
Transcript of From RNA Extraction to Microarray Based Readout · 2016. 12. 20. · From RNA Extraction to...
-
From RNA Extraction to Microarray Based Readout:
Biofunctionalization of a Microfluidic Cartridge
for Fully Integrated microRNA Analysis
Andreas Mader
SCIENION AG
INTERNATIONAL MICRO NANO CONFERENCE 13./14. December 2016 Amsterdam 1
-
SCIENION AG:
Excellence in low volume liquid handling
KEY FACTS
• Founded in Berlin in 2001
• Spin-off from the Max Planck Institute for Molecular Genetics
• Life science company focused on ultra-low volume liquid
handling systems and solutions
• Four locations: Berlin, Dortmund, Monmouth Junction, NJ (USA;
since December 2011), Lyon (France, since May 2016)
• 56 people
• ISO 9001 certified
2
-
SCIENION AG:
Excellence in low volume liquid handling
3
TECHNOLGY
• Core competence: Handling and dispensing pico- and
nanoliters
Biofunctionalization of microfluidic chips, biosensor
surfaces, membranes, microplates
Array/Spot based analytics
• Lead product sciFLEXARRAYER: system for automated ultra-
low volume liquid handling of biological samples in
diagnostics, genomics and proteomics
• Non-contact dispensing technologies:
sciDROP PICO, sciDROP NANO
• Consumables leveraging instrument performance
• Integrated portfolio comprising array printer, reader,
consumables and services
-
Small droplets are all around us –
typically microliters
4
-
Small droplets with big impact in (Bio)Science
5
Fertilization
Bead handling
Drug delievery
POCT
Single cell handling
Biosensors
Microarrays
PCR /
Library preparation
Sample Prep
-
Spots on Slides, Wafers, Biosensors, …
6
Microplates
CCD Images of the loaded
electrodes of a CMOS sensor
Slides
Microfluidic Cartridges
Wafer
Microarrays
arrays of spots in a predefind area
Biosensors
arrays of spots exactly at a predefined position
-
Application:
Microfluidic Cartridge for miRNA Analysis
7
Polymer-based microarrays
universal
NASBA
mRNA
+
miRNA
Clinical Background: Breast cancer
diagnostics
Assay: RNA
amplification
System integration
-
Biofunctionalization of polymer materials
sciPOLY3D immobilization technology
• Enables covalent immobilization of
biomolecules on non-activated surfaces –
such as microfluidic chips
• No wet chemistry processes
• water-soluble and simply added to the
printing media
8
Probe/sciPOLY3D mix
UV light
-
Application:
Microfluidic Cartridge for miRNA Analysis
9
Polymer-based microarrays
universal
NASBA
mRNA
+
miRNA
Clinical Background: Breast cancer
diagnostics
Assay: RNA
amplification
System integration
-
Background:
Personalized healthcare
Tailor-made therapy
• Efficacy
• Side effects
• Cost-efficiency
More detailed diagnostic
• Genetic predisposition
• Therapeutic response
10
one treatment for
all patients
individual treatment
Breast Care (Basel), 2011. 6(2): p. 136-141. Clin Transl Oncol, 2009. 11(10): p. 634-42.
Nature, 2012. 490(7418): p. 61-70. Roche PHC Brochure, 2011.
-
Background:
Tumor diagnostics
Example: breast cancer
• Heterogeneous disease
• Therapy depends on subtyping
– Phenotypical features
– Immunohistochemistry
• Estimated ~80 % of patients are treated suboptimal
11
Tumor tissue IHC of HER2 protein
(200x magnification)
surgery cytostatic drugs
radiation hormonotherapy
immunotherapy
Journal of Clinical Investigation, 2011. 121(10): p. 3789-3796. Modern Pathology, 2008. 21: p. S8-S15.
Clin Transl Oncol, 2009. 11(10): p. 634-42.
-
Background:
Tumor diagnostics
Example: breast cancer
• Heterogeneous disease
• Therapy depends on subtyping
– Phenotypical features
– Immunohistochemistry
• Estimated ~80 % of patients are treated suboptimal
More detailed diagnostics
based on nucleic acids
12
Tumor tissue IHC of HER2 protein
(200x magnification)
Journal of Clinical Investigation, 2011. 121(10): p. 3789-3796. Modern Pathology, 2008. 21: p. S8-S15.
Clin Transl Oncol, 2009. 11(10): p. 634-42.
DNA-microarray, Agendia MammaPrint®
-
Background:
Tumor diagnostics
Nucleic acids biomarker
• SNP (mutation)
• mRNA (gene expression)
• microRNA (regulation of gene
expression)
• Risk of disease
• Prediction
• Prognosis
13 Clin Pharmacol Ther, 2001. 69(3): p. 89-95.
Cell and Molecular Biology: Concepts and Experiments. 6th edition. Karp G. Wiley 2009.
-
Project Aim & Strategy
Aim
Parallel measurement of multiple biomarker classes in a single test
Strategy
14 Oncology (Williston Park), 2012. 26(4): p. 364-5, 369. Nucleic Acids Research, 2013. 41(D1): p. D977-82
Expert Review of Anticancer Therapy, 2012. 12(3): p. 323-330.
sensitivity and specificity of
in-vitro nucleic acid amplification
multiplex capacity of microarrays
Combination of
with
miRNA
SNP
mRNA
-
Application:
Microfluidic Cartridge for miRNA Analysis
15
Polymer-based microarrays
universal
NASBA
mRNA
+
miRNA
Clinical Background: Breast cancer
diagnostics
Assay: RNA
amplification
System integration
-
RNA Amplification Assay
NASBA: Nucleic Acid Sequence-Based Amplification
• Transcription-based
• 3 enzymes (AMV Reverse Transcriptase,
RNase H, T7 RNA Polymerase)
Alternative for PCR
• Isothermal (41 °C)
• Direct amplification of RNA
• Tolerates gDNA
• Produces single stranded RNA
16
mRNA
cDNA
Primer 1 mit T7 Promotor
Primer 2
Nature, 1991. 350(6313): p. 91-92. Lab on a Chip, 2012. 12(14): p. 2469-2486.
Molecular Biotechnology, 2002. 20(2): p. 163-179.
-
RNA Amplification Assay
Reverse Transcription universal NASBA (RT-uNASBA)
• Introducing universal sequences
• Integration of miRNA in
RT-uNASBA
17
univ
erse
lle
NA
SB
A
m
ult
iple
x R
T
mRNA miRNA
un
ive
rsa
l N
AS
BA
m
ultip
lex R
T
-
RNA Amplification and Detection
Parallel amplification and detection of mRNA and miRNA
• Up to 14-plex mRNA / miRNA tested
18
4-plex miRNA 7-plex miRNA
1 2 3 4 5 6 7
8 9 10 11 12 13 14
1 2 3 4 5 6 7
8 9 10 11 12 13 14
5-plex mRNA 7-plex mRNA
1 2 3 4 5 6 7
8 9 10 11 12 13 14
1 2 3 4 5 6 7
8 9 10 11 12 13 14
mR
NA
m
iRN
A
mR
NA
+ m
iRN
A
9-plex miRNA + mRNA
Negativkontrolle
1 2 3 4 5 6 7
8 9 10 11 12 13 14
1 2 3 4 5 6 7
8 9 10 11 12 13 14
1 2 3 4 5 6 7
8 9 10 11 12 13 14
1 2 3 4 5 6 7
8 9 10 11 12 13 14
6-plex miRNA + mRNA
14-plex miRNA + mRNA Negative control
-
Application:
Microfluidic Cartridge for miRNA Analysis
19
Polymer-based microarrays
universal
NASBA
mRNA
+
miRNA
Clinical Background: Breast cancer
diagnostics
Assay: RNA
amplification
System integration
-
Integration in fully automated µTAS
• Isothermal assay procedure
• Functionalization of unmodified plastics
• Combination with on-chip
RNA extraction
20 Lab Chip, 2010, 10, 610-616
EPC4_7_fluidics4.wmv
-
Integration in fully automated µTAS
21
-
Integration in fully automated µTAS
All-in-one system
Actuation
• Sample transport
• Blister control
• Turning valves
Temperature control
Detection
• 2-channel
fluorescence
22
-
Summary
Microarray fabrication with sciPOLY3D
Biofunctionalization of unmodified polymer substrates
Isothermal assay for multiplex RNA analyses
Parallel amplification of multiple miRNA and mRNA sequences
Integration in fully automated µTAS
23
universal
NASBA
mRNA
+
miRNA
41 °C
-
Thank you very much for your attention
Dr. Andreas Mader
Scienion AG, Volmerstr. 7b, 12489 Berlin
Tel. +49 (0)30 63 92 1700 Fax - 1701
[email protected] www.scienion.de
24
-
Tools and Service
for Microarray Based Application
Key Technologies
Contact Free Spotting
Surface Functionalization
Microarray Applications
Products sciFLEXARRAYER
sciREADER
sciPLEXPLATE
sciCHIP
sciBUFFER
Services Product /
Process
Development &
Manufacturing
http://www.mpikg.mpg.de/index.html