Imagination at work.

Nano Bio Manufacturing Consortium WorkshopBlood, Sweat and Tears III

Ralf LenigkSenior Microfluidics Researcher, MicroSystems Lab

GE Global Research Center, Niskayuna, NY USA

November 2, 2016

Lateral Flow Assays and Point of Care Devices –Detecting Infectious Diseases in Low Resource Settings

See tutorial regarding confidentiality disclosures. Delete if not needed.

GE Global Research: Market-focused R&DThe cornerstone of GE’s commitment to technology

• First U.S. industrial lab.

• ~2000 scientists/engineers, nearly two-thirds PhDs.

• ~3615 U.S. patents filed by GE in 2011 alone.

• One of the world’s most diversified industrial research organizations, providing innovative technology for all of GE’s businesses.

© 2013, General Electric Company

See tutorial regarding confidentiality disclosures. Delete if not needed.

Expanding our global presence

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Global Software Center San Ramon, CA

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Brazil Technology Center Rio de Janeiro, Brazil

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China Technology Center Shanghai, China

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India Technology Center Bangalore, India

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Global Research Europe Munich, Germany

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Global Research Headquarters Niskayuna, NY

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AMSTCAnn Arbor, MI

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November 2, 2016

Agenda

1. Lateral flow assays - membranes and papers

2. Towards Precision Diagnostics - Tailoring Component Properties

3. Modifying Membrane & Paper Surfaces & Transport Characteristics

4. Materials Applications into Advanced IVD Devices for POC detection

5. Photothermal spectroscopy reader for enhanced sensitivity read-out

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November 2, 2016

Introduction

• LFA is low-cost test of choice for immuno-assays, protein detection

• Lessons learned from LFA & POC development are applicable to Wearables

• Fluidics and volumes are comparable to Wearable devices

• Efficient supply chain and manufacturing processes

• Many materials, such as membranes, are usable for Wearables

Membranes & Papers

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November 2, 2016

Polymeric: most commonInorganic: more stable

What is the Difference: Membrane vs Paper?

Cellulular

e.g. Lungs

Biological

Glassy Rubbery

Organic

(polymeric)

Glass

Ceramic

Metallic

Zeolitic

Inorganic

Synthetic

Membrane

Materials

Membrane: Layer(s) of material which act as a selective barrier between components

hybrid

Paper: Semi-permeable material, typically derived from mineral fibers or lignocellulosic pulps

Lignocellulosic

Glass Fiber

Quartz

Mineral Fibers

Paper

Materials

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November 2, 2016

Micrometers(Log scale) Ionic Range Molecular Range Macro Molecular Range Particle Range

Membranes & Papers

0.001 0.01 0.1 1

Membranes & Paper: Across the Separation Range

Membranes

Paper

10

Relative Size of Common Materials

Separation Processes

Reverse Osmosis(Hyperfiltration)

Nanofiltration (NF)

Ultrafiltration (UF)

Microfiltration (MF)

particleFiltration

BacteriaVirusAqueous Salt

SugarAlgae

Albumin ProteinMetal IonPaint Pigment

Markets

Brackish water

Sea water

Tough-to-Treat Produced Waters – oily waste, high suspended solids

Boiler feed – especially power

Cooling tower - blow down

Industrial process fluid separations

Surface water treatment

Dairy, food , pharma

Diagnostics - Lateral flow assays Biopharmaceutical processing

Non-porous

Porous

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November 2, 2016

1 - Sample Pad2 - Conjugate Release Pad3 – Test Line4 – Reaction Membrane5 – Control Line6 – Absorbent Pad (Wick)7 – Backing Layer

Reverse Osmosis

Pervaporation

Nanofiltration

Ultrafiltration

Microfiltration

Dense Thin Film

Gross Filtration

Membranes & PapersAcross the Separation Range

Depth FiltersLab Filtration

Forensics/Human IDDried Blood Spots

Biospecimen Stabilization

Diagnostics Components

Glass Fiber

NitrocelluloseParticle Size(m)

0.0001

0.001

0.01

0.1

1

10

100

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November 2, 2016

Membrane vs. Paper: Morphology

Composite Membrane

NanofibersTrack Etched

Nonwoven fabric

Expanded film

Pore Filled

Phase Inversion

Membrane Paper

Lignocellulosics

FTA™ – DNA storage

Mineral Fibers

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November 2, 2016

Membranes & Papers: Substrate to DeviceM

em

bra

ne

Pa

pe

r

Substrate Format

Module/ Cartridge

System/ Device

Hollow Fiber

Flat Sheet

EasiCollect™

Towards Precision Diagnostics: Tailoring Component Properties

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November 2, 2016

Membranes/Papers: Discovery to Manufacturing

Paper/Membrane Development

Advanced Materials Design & Membrane Fabrication Surface Activation & Modification

Material Characterization & TestingStructure-Property-Performance Evaluation

Substrate Modified

substrate

Membrane Pilot Manufacturing Process Optimization & LifingMaterials and Process Cost Analysis

Transfer Function for Performance Balance

Biological Performance

Biomolecule Stabilization (DNA, RNA, proteins)Dried Blood Spot (Small Molecule Collection & Testing)Nucleic Acid Amplification & TransportDetection - Immunoassay/Nucleic Acid Testing

Chemically Modifying Membrane & Paper Surfaces

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November 2, 2016

Platform Approach to Modifying Membrane/Paper

Membrane/Paper

Activated Substrate

1) Activation 2) Modification

ModifiedSubstrate

Modified Membranes - Nitrocellulose Substrates:• Activated Nitrocellulose• Hydrophilic Nitrocellulose

Bing Li

Cathryn Olsen

Bill Alberts

Li, B.; Moore, D. R.; Olsen, C. O.; “Porous Membranes Having a Polymeric Coating and Methods for Their Preparation and Use.” U.S. Pat. Appl. 13/339,960; 13/339,996; 13/340,052 .

Li, B.; Moore, D. R.; Olsen, C. O.; “Porous Membranes Having a Hydrophilic Coating and Methods for Their Preparation and Use.” U.S. Pat. Appl. 13/340,793; 13/362,793.

Substrate Breadth – Cellulosics to Synthetics

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November 2, 2016

Materials Enabling Diagnostic PerformanceReducing Non-Specific Binding

[DARPA contract HR0011-11-2-0007]

Isothermal NA Amplification in Porous Substrates

NCHydrophilic

NC

Improved Biomolecule Transport in Porous Matrices

HydrophilicNC

UnmodifiedNC

Time

Running buffer: 40 nm gold nanoparticle coated bovine serum

albumin (BSA)

Controlling Membrane/Paper Transport - Hydrophilicity

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November 2, 2016

Physical Properties… HydrophilicityResults for qcf of (un)modified membranes

J. Nichols

J. Davis

Sample qcf

° (s)*Cap. Rise

s*

Nitrocellulose-1 34(2) 101

Nitrocellulose-2 51 134

Nitrocellulose-3 67(1) 2000

Studies performed on membranes from GE Healthcare or modified membranes; *Average of >3 replicates

Membrane Contact Angle via Capillary Flow Porometry

Hydrophilicity/contact angle is a critical (& often oversimplified) control parameter to enable precision diagnostics

𝒅𝑽 =𝜸𝝅𝑫𝟑

𝟑𝟐𝝁𝑳𝒄𝒐𝒔𝜽𝒅𝒕

Materials Applications into Advanced IVD Devices

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November 2, 2016

http://www.fujifilmusa.com/

http://www.biodot.com/

Printing Methods at GE GRC-Lab scale at GE GRC

Gravure

Printing

Screen

Printing

Inkjet

Printing

BioDot XYZ-

3000

Printing FormEngraved

cylinder

Stencil and

meshDigital Digital

Ink Viscosity (Pas) 0.01-0.2 0.1-50 0.002-0.1 0.01-1

SubstratesPaper,

polymersAll All

Paper,

polymers

Line Width (μm) 10-50 50-150 1-20 500

Registration (μm) >10 >25 <5 <15

Throughput

(m²/sec)10 <10 0.01-0.1 <1

Adapted and updated from Cagler 2009

http://www.dek.com/

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November 2, 2016

Patterned Membranes and Fluid Paths

Flow demonstration

Time

Patterned Membrane

Pattern Printed on to Nitrocellulose

Create Fluidic Structures in Membranes

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November 2, 2016

DARPA Diagnostics on Demand Limited Resource Settings“Multiplexable Autonomous Disposables for Nucleic Acid Amplification Test (MAD NAAT)”

Overview

• Instrument‐free, single-use, paper-baseddisposable device

• Unitized, self-contained nucleic acid test

• Administered by untrained user (CLIA waived) w/ sample to read-out in < 1 hr

• Low Cost to Enable Broad Use

Potential end-use scenarios

• Nurse or physician in hospital, ER, ICU

• Physician’s office

• Retail clinics

• Home use (prescribed by physicians)

• Telemedicine

PI = Paul Yager

The Team:

Image provided by Paul Yager,

U. Wash

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November 2, 2016

Sample

Flow

1 - Sample Pad2 - Conjugate Release Pad3 – Test Line4 – Reaction Membrane5 – Control Line6 – Absorbent Pad (Wick)7 – Backing Layer

Overview

Sample Prep

Nucleic Acid Amplification & Detection - PCR

Immunoassay - Lateral Flow IVD Test

Instrumented Workflow

MAD NAAT Device:Sample Collection (Swab/Blood)

Sample PrepIsothermal NA Amplification

DetectionSample Archiving

The Vision: A platform device as simple as a pregnancy test with capability for detecting multiple infectious diseases

within a single biospecimen

Paul Yager, U. Wash

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November 2, 2016

Target Indication: integrated, paper-based MAD NAAT device that is Superior to current hospital workflows for MRSA DNA detection.

Paper-based Device Workflow

DetectionCollection &

Swab TransferLysis NA Purification Amplification

Swab CapacityTransfer Efficiency

Lysis TimeLysis Efficiency

NA CaptureNA Release

Amplification RateLimit of Amplification

Limit of DetectionRate of DetectionDynamic Range

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November 2, 2016

IVD Development Across Specimen Testing Workflow:Materials That Matter

Chemical & Biological Reagents

IVD Materials

Fluidics

System Integration

Device

+

+

DetectionCollection &

Swab TransferLysis NA Purification Amplification

Cellulose (FTATM) Glass fiber Nitrocellulose

• Excellent wicking power

• Supports efficient lysis methods

• Compatibility with amplification, lysis and reagent storage

• Fastest flow• High fluid capacity

• Compatibility with amplification (when chemically modified)

• Predictable flow • High binding capacity

(for detection)

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November 2, 2016

Point-of-Care In Vitro Diagnostics

Typical assembly of a lateral

flow assay strip.

1 = Sample Pad

2 = Conjugate Release Pad

3 = Test Line

4 = Reaction Membrane

5 = Control Line

6 = Absorbent Pad (wick)

7 = Backing Layer

GE Healthcare WhatmanTM catalogue. “Whatman A Guide to Diagnostic Rapid Test Development.”

Sample Pad

Sample

Flow

Conjugate Release Pad

Test Line

Backing Layer

Absorbent Pad (Wick)

Reaction MembraneControl Line

Lateral Flow Assay Strip Components

How does a Pregnancy Test Work?

Analyte / antigen

Bioconjugate with reporting tag

Capture antibody

Control Line antibody

Positive

Negative

Phase I Objectives: producing a TRL-5 prototype of the complete MRSA device

2/5/2014 MAD NAAT Project Open Talk 27

Integrated device that performs DNA test for MRSA (atTRL-5 level)Nasal swab sample captureBacterial lysis(DNA purification)Isothermal amplificationVisible lateral flow readoutCell-phone image capture and data transmission of result Staged expansion of targets to include:Broader bacterial panel (Gram + and –species using DNA)Viruses (RSV by RNA using RT step)Blood sample capabilitySample archiving capability for sequencing

Valve 2

Valve 1

Inner Workings I: naming the parts

Detection reagents dried on pads

Amplification reagents dried on pads

Chase buffer

Swab

Wicks

Lysis buffer and denaturation heater

Heater

Protective film (pulled back by opening case)

Fluid source and distributor (glass fiber)

Amplification zones (glass fiber)

Detection strips (nitrocellulose)

2/5/2014 MAD NAAT Project Open Talk 28

Estimated minutes elapsed for TRL-5 version

Sa Sa IC 5000 copiescopies

• Perform Sa/IC biplex amplification in a tube• Followed by lateral flow detection•Not yet optimized

Lateral Flow Detection of a S. aureus gene -Internal Control-biplex iSA Amplicon Mixture

2/5/2014 MAD NAAT Project Open Talk 29

Photothermal SpectroscopyEnhanced Sensitivity LFA Reader for POC

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Development of a Photothermal Spectroscopy Reader that Enhances Sensitivity 5-10X

Improved RDT

+

Advanced Reader

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1. Focused light

2. Conjugated Anti-Ag

3. Ag

4. Anti-Ag

5. Nitrocellulose

Photothermal Spectroscopy Response

33

1. Focused light

2. Conjugated Anti-Ag

3. Ag

4. Anti-Ag

5. Nitrocellulose

Photothermal Spectroscopy Response

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November 2, 2016

Conclusions & Outlook

• Complex fluidics functions can be achieved in paper-based devices

• Paper & membranes used for LFA, POC can be employed in Wearables

• Material properties can be fine-tuned for parameters like flow speed etc.

• Sensitivity of LFAs can be significantly enhanced with new technologies

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FTA, EasiCollect, and Whatman are trademarks of General Electric Company or one of its subsidiaries.

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