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, …

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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

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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

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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

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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

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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

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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

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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

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Integration in fully automated µTAS

All-in-one system

Actuation

• Sample transport

• Blister control

• Turning valves

Temperature control

Detection

• 2-channel

fluorescence

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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

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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

a.mader@scienion.de www.scienion.de

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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