User centered design and development of high performance lateral flow assay systems

for demanding applications

Brendan O’Farrell, PhD

We have to approach the design and

development of these sensors – and the

Products they are a part of – differently to

how it has historically been done

Sample collection and handling methods Device (cartridge) design

Biological recognition reagentsSignal generation technologies

Manufacturing Process Design

Assay Design and Components

Design and Development of high performance assay systems must be approached from first principles

Interpretation and signal transduction technologies

Good technology does not necessarily make for a successful product

1. Performance

2. Manufacturability

3. Quality

4. User – centric design

5. Market desire

6. Regulation

7. Intellectual property

8. Channels to market

9. Competitive environment

Sample collection and handling methods

Design for Use

• User centric design• Stickiness• User friendliness• Cost control• Design for Manufacture

Device (cartridge) design

Why Focus on Device Design?

DCN’s Design Process

Ensuring Proper Function

Both strip and cartridge are engineered parts, and

must be concurrently designed for high performance applications.

Sample Well Interface w/Strip

Conjugate/Capture Interface Feature

Recessed sample area to stop capillary flow & reduce flooding.

Keys to ensuring assay performance

Control of flow: Fit of test strip into cartridge – tolerance of materials and laminations relative to pressure points in cassettes

Interpretation/Reader integration issues: Positioning, shadowing

Ensuring Proper Function

Closure pins: Design is critical to performance

Device Design for Reader Integration. Key issues:1.Tolerance of Line Position relative to optics.2.Shadowing of optical region.3.Locating and registration features.4.Material Selection (auto-fluorescence)5.Features to allow for intuitive insertion into instrument.6.Reader companies such as LRE have established clear requirements.

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Integration to Readers

Many lateral flow assays require the use of a running buffer, diluent and/or a chase buffer in the system.

This typically imposes a requirement for a separate buffer bottle and possibly the use of a volumetric or disposable pipette.

This can lead to operator error as well as inconvenience and effects on the ability to CLIA waive a product.

One alternative is to design features for onboard storage of buffers into the system using:

Added Functionality: On board reagent storage

Added Functionality: On board reagent storage

Integrated unit-dose buffers/reagents can minimize user errors and enhance ease of use.

Options Include:– Plastic blister with

puncturable foil lid.– Foil pouch.– Plastic pouch.– Glass Ampoules.– Inside device chamber.

Considerations include:

- Volume loss during shelf life (MVTR).

- Material compatibility and stability.

- Volume storage and release accuracy.

- Manufacturability

- User steps/ease of use.

- Cost

Added Functionality: On board reagent storage

A reader based semi quantitative serological assay for anti-PF4:Heparin in human plasma (HIT)

Challenge:•Semi quantitative, threshold assay•Reader based•Minimal operator steps: Competitive assays are complex•High sensitivity, high correlation required to commercial ELISA•No known standards•Highly charged molecule resulting in high NSB in traditional labeling system•Total immunoglobulin assay imparts formatting requirement – sample and conjugate cannot mix prior to interaction with test line•Lot specific information to be transmitted via integrated RFID tag in cassette•Intended for the professional clinical laboratory market•Will be labeled as an IVD :developed under full design controls•Assay and device developed for large scale manufacturing

Solution

• A fluorescent assay, utilizing an ESEQuant reader from Qiagen Lake

Constance

• Integrated RFID tag

• Direct labeled fluorescent molecule

• Multiple steps simplified through labeling, flexible timing and

cassetted design

• Generation 2 cassette designed to include all buffers, with simple

actuation

OD = 0.3ELISA TL/CL TL

True positives 41 39 37

False Negative 0 2 4

True Negatives 42 42 42

False Positive 0 0 0

n 83 83 83

PPV 100.00 100.00 100.00

NPV 100.00 95.45 91.30

Sensitivity100.00 95.12 90.24

Specificity100.00 100.00 100.00

Collated Testing Results: ELISA vs LFIA

• A panel of 83 samples, characterized by predicate ELISA• All samples came from patients who had received heparin• Testing in triplicate / quadruplicate if adequate volume

Lot specific information is typically encoded in 2-D bar codes or RFID tags

Device design considerations including the area available for printing, should 2-D bar codes, logos or patient identification areas be required can have impacts on device design.

RFID tags can require significant space in a device (typically up to 25mm diameter) and the cassette must be designed with the labeling process in mind.

Added Functionality: Labeling, Bar Codes and RFID

Features for Intuitive and Easy Use

• Product identification by color

• Shape ensures easy operation

• Ports shaped differently for easy identification

• Easy to hold

• Shape ensures that insertion into reader can only be in one direction

• Clear product labeling

• Areas for user to write information

Design Aesthetics and Perceived Value

U.S. Food and Drug Administration

FDA News Release

FDA allows marketing of the first test to assess risk of developing acute kidney injury

For Immediate Release

September 5, 2014

ReleaseToday the U.S. Food and Drug Administration allowed marketing of the NephroCheck test, a first-of-a-kind laboratory test to help determine if certain critically ill hospitalized patients are at risk of developing moderate to severe acute kidney injury (AKI) in the 12 hours following the administration of the test. Early knowledge that a patient is likely to develop AKI may prompt closer patient monitoring and help prevent permanent kidney damage or death……..

Design Aesthetics and Perceived Value

• Challenges:

– Visual read

– Dipstick (no cassette)

– No electronics

– Untrained users

– Single step

– No extraction

– Particulate sample

– High sensitivity: pg/ml levels required

– <10 minutes

– Thermal stability

Good design for use does not equal complexityAssay for Visual Field-Based Protein Expression in Transgenic Plants

Solution: Simple dipstick, plant material extracted in water with non-instrumented grinding. Single step. 10 minute test. Extensive optimization was required to reach sensitivity level.

Test Strip # Sample TL CL

1 zero 0 10

2 50pg 1 10

3 100pg 2 10

4 500pg 6.5 10

5 1000pg 7.5 10

1000pg 500pg 100pg 50pg 0

Assay for Visual Field-Based Protein Expression in Transgenic Plants

Design for Use

A Patented Technology facilitating multiplexing, quantification and non-traditional result generation in rapid assays

Intuitive Results, Better Results

SymbolicsTM pixilation technology allows for new approaches to

result generation in lateral flow, including:

1. Geometric symbols

2. Alpha -numeric symbols.

3. Multiplexing (spot arrays or other formats)

4. Other advanced design features

Contrls

MOR

AMP

DropVol: 0.3nLPitch: 0.25mm5mm StripsPiezo electric dispenserControl: 0.5mg/ml GAMTest: 1mg/ml anti-hCGαConjugate: 8ug/ml anti-hCGβNegative: 100uL 0.1% Tween-20 in 1XPBS

10mIU/mL hCG in bufferBuffer only

anti-hCGαGAM

Intuitive Results: Symbols

Intuitive Results: Alpha- Numeric Symbols

Multiplexed Lateral Flow Arrays

• Dispenser: BioDot AD2000 Piezo

• Drop Size: 2nL

• Pitch: 1mm

• Capture reagents:

• Control: Goat anti-Mouse

• Test: Amphetamine-BSA, Morphine-BSA

• Detectors:

• Mouse anti-Morphine Colloidal Gold Conjugate

• Mouse anti-Amphetamine Colloidal Gold Conjugate

• Running Buffer:

• 1XPBS, 0.1% BSA, 0.01% Tween-20

• Positive Controls:

• 100ng/ml Morpine in running buffer

• 10ug/ml Ampetamine in running buffer

Key Components of a High Performance Lateral Flow System

Device Design

Sample Handling and Delivery

Sample collection and handling methods

Sample Handling : Typical Processing Steps

Example: Whole Blood Processing Steps

Metering of quantitative or semi quantitative

amounts of blood from the tube or fingerstick

Separation of plasma from the sample

without significant hemolysis

Delivery of the sample, either neat, neat with a chase buffer, or pre-diluted, to the device

• Common methods– Off the shelf capillaries

– Low cost, disposable semi quantitative pipettes.

• More novel approaches – Capillaries with integrated dispense pipettes

– Collection using sponges

– Custom collection and vertical filtration devices

Sample Handling ExampleWhole Blood Collection from Finger Stick

Capillary with integrated pipette

33

Plasma Separation on Lateral Flow Devices

• Method has been successfully employed for decades

• Depth filters such as Vivid or Cytosep (Pall), Fusion 5, MF1 or VF2 (GE), commonly used

• Requires capillarity to transfer volume out of pad.

• Cartridge and test strip lamination must be designed specifically to ensure no red blood cell leakage or occlusion.

Custom Approach: Plasma Separation by Vertical Filtration

Example: Assay for human cardiac marker in whole blood, serum and plasma

Original Client Technical Inputs:

•Assay for human cardiac FABP in whole blood from a fingerstick sample•Threshold assay required – some level of analyte is present in the entire population•High sensitivity requirement (target cutoff low ng/ml)•High reproducibility requirement (>90% of positives detectable at cutoff level). Virtually no false negatives allowed•No hook effect allowed over three logs of range•Time from finger stick to completion of assay must be less than 4 mins•Visual interpretation (gold based)•No instrumentation•Single step desired•CLIA waivable

User – Centric Design Step 1: User Requirement Definition

Critical Design Requirements

Process Output

Design Approach

1. Device Design: Designed and developed a quantitative

collection device for fingerstick blood, that could quantitatively

dilute the sample and deliver plasma in a single step to the strip

2. Test Design: Designed the strip for high sensitivity, high

accuracy and reproducibility around a cutoff and selected

materials that would allow the test to run in less than 2 minutes.

Images courtesy of FABPulous BV

Cartridge with Integrated Filter

Filter Stack

Lateral FlowStrip

Cartridge Top

Cartridge Base

Blood Collection/Delivery Device

Syringe Plunger

Syringe Body

Blood Collector Body

Porex – Blood Sponge

Buffer (~300ul)

Foil Piercing Needle

Foil Seal

The solution: A whole blood collection and plasma delivery device integrated to a lateral flow cassette

System Performance

h-FABP conc. (ng/ml)

2.0 3.0 3.5 4.0 4.5 5.0 6.0 10.0

# pos 0 0 0 14 15 15 15 10

# neg 10 10 15 1 0 0 0 0

% pos 0.0% 0.0% 0.0% 93.3% 100.0% 100.0% 100.0% 100.0%

Assay specifications require a >90% positive rate at 4.0 ng/ml of HFABP and <10% positive rate at 3.0 ng/ml of HFABP. Below are the results from the release of the verification lot of product.

Key Points

1. The key to the development of this system so that it serves its

intended purpose was to understand the usage environment,

the workflow of the users and the critical performance

parameters in the field

2. Going through a User-Centered Design process allowed DCN to

identify the parameters that were actually key to delivering a

useful device

3. The output of the User Centered Design process uncovered

unrecognized market needs, completely re-focused the

development process and resulted in the generation of highly

valuable intellectual property for the client.

Performance Considerations: Impact of Readers and Labels on Assay Design

• Label Choice will affect overall assay performance in a

number of ways, all of which are inter-related

1. Sensitivity

2. Speed

3. Need for multiple steps (eg washing)

4. Available chemistry options (covalent/passive)

5. User experience

6. Need for a reader

7. Manufacturing processes and QC requirements

Signal Reagent Options

Commonly used signal reagents

(1) Visual

• Colloidal gold

• Cellulose nanobeads

• Latex

• Colloidal Carbon

• Some other enhancers

Signal Reagent Options

Commonly used signal reagents

(2) Fluorescent

• organic dyes

• metal-ligand complexes

• fluorescent proteins

• semiconductor quantum dots

• lanthanide complexes

• dye-doped polymer nanoparticles

• fluorescent silica nanoparticles

Signal Reagent Options

Commonly used signal reagents

(3) Other

• Paramagnetic

• Enzymatic

Conclusion

• A focus on appropriate device design can is critical to success– Performance– Perceived value– User friendliness– Regulatory ease

• A carefully conceived user-centric design process is critical to extracting maximum value

• Design control should be followed to ensure that the product is made right and the right product for your users is made

Case Study: Designing and Developing a Low Cost Hand Held Fluorescent Lateral Flow Test Strip Reader

Summary Brief:•Design and develop a reader for fluorescent lateral flow test strips and PCR tubes•Low cost: Bill of Materials <$20USD•Qualitative•Robust•Long life•Operates off 9V battery•Easy to operate

Step 1: Concept Generation

Step 2: Line Drawings

Step 3: Initial Renderings

Step 4: Photo-realistic Renderings

Step 5: Working Prototype

Brendan O’Farrell, Ph.D.,President,DCN Diagnostics Inc.,6354 Corte del Abeto,Carlsbad, CA 92011

Tel: 760-804-3886Mobile: 949-872-8589Email: bofarrell@dcndx.com