Lateral flow assays are the most mature, stable, field deployable sensor available today. They can be used in myriad applications, from medical diagnostics to veterinary testing, agriculture, bio-defense, food testing and environmental testing, to name a few. Performance of these assays has evolved to the point where they can equal that of much more highly complex laboratory based diagnostic test formats. They can be quantitative, multiplexed and highly sensitive if developed and manufactured correctly. Design and development of these high performance lateral flow systems requires a from-first-principles approach, with an eye to optimizing the system for reproduciblity and sensitivity. Employing user - centered design and development practices greatly improves the odds of successful commercialization. DCN Diagnostics (formerly Diagnostic Consulting Network) develops high performance lateral flow assay systems for use in any environment. Our concurrent design and development process, employing cross functional teams of industrial design and mechanical engineers alongside our immunoassay development teams, ensures that the right product is developed the right way to allow for most efficient regulatory approval and commercialization. Our development process is fully design controlled and operates under our ISO 9001:2008 and EN 13485 compliant quality system. With literally hundreds of assays developed, and transferred to manufacturing, DCN is the go-to supplier of contract development services in the point of care diagnostic test market. Additionally, our consulting teams can provide a deep strategic vision to our clients, assisting in all aspects of product development and commercialization, including regulatory affairs and clinical trial management. This presentation describes DCN's development process and illustrates the benefit of a user centered design process in creating the right rapid assay for your market, focusing on field deployed tests for clinical diagnostics, veterinary testing and bio-defense applications. A variety of case studies are shown, illustrating the principles discussed.

1. User centered design and development of
high performance lateral flow assay systems
for demanding applications
Brendan O’Farrell, PhD

2. 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

3. Sample collection and handling methods Device (cartridge) design
Biological recognition reagents
Signal 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

4. 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

5. Sample collection and handling methods
Design for Use
• User centric design
• Stickiness
• User friendliness
• Cost control
• Design for Manufacture
Device (cartridge) design

6. Why Focus on Device Design?

7. DCN’s Design Process

8. Ensuring Proper Function
Both strip and cartridge are engineered parts, and
must be concurrently designed for high performance applications.

9. 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

10. 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.
Integration to Readers

11. 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

12. 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.

13. 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

14. 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

15. 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

16. OD = 0.3
ELISA 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
Sensitivity
100.00 95.12 90.24
Specificity
100.00 100.00 100.00
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

17. 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

18. 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

19. Design Aesthetics and Perceived Value

20. 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
Release
Today 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……..

21. Design Aesthetics and Perceived Value

22. • 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 complexity
Assay for Visual Field-Based Protein Expression in Transgenic Plants

23. 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
AssayforVisualField-Based ProteinExpressioninTransgenicPlants

24. Design for Use
A Patented Technology facilitating multiplexing, quantification
and non-traditional result generation in rapid assays

25. 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

26. DropVol: 0.3nL
Pitch: 0.25mm
5mm Strips
Piezo electric
dispenser
Control: 0.5mg/ml
GAM
Test: 1mg/ml anti-
hCGα
Conjugate: 8ug/ml
anti-hCGβ
Negative: 100uL 0.1%
Tween-20 in 1XPBS
10mIU/mL hCG in bufferBuffer only
anti-hCGαGAM
Intuitive Results: Symbols

27. Intuitive Results: Alpha- Numeric Symbols

28. 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

29. Key Components of a High Performance Lateral Flow System
Device Design
Sample Handling and Delivery
Sample collection and handling methods

30. Sample Handling : Typical Processing Steps

31. 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

32. • 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 Example
Whole Blood Collection from Finger Stick
Capillary with integrated pipette

33. 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.

34. Custom Approach:
Plasma Separation by Vertical Filtration

35. 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

36. User – Centric Design
Step 1: User Requirement Definition

37. Critical Design Requirements

38. Process Output

39. 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

40. Cartridge with Integrated Filter
Filter Stack
Lateral Flow
Strip
Cartridge Top
Cartridge Base

41. Blood Collection/Delivery Device
Syringe Plunger
Syringe Body
Blood Collector Body
Porex – Blood Sponge
Buffer (~300ul)
Foil Piercing Needle
Foil Seal

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

43. 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.

44. 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.

45. 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

46. Signal Reagent Options
Commonly used signal reagents
(1) Visual
• Colloidal gold
• Cellulose nanobeads
• Latex
• Colloidal Carbon
• Some other enhancers

47. 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

48. Signal Reagent Options
Commonly used signal reagents
(3) Other
• Paramagnetic
• Enzymatic

49. 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

50. 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

51. Step 1: Concept Generation

52. Step 2: Line Drawings

53. Step 3: Initial Renderings

54. Step 4: Photo-realistic Renderings

55. Step 5: Working Prototype

56. Brendan O’Farrell, Ph.D.,
President,
DCN Diagnostics Inc.,
6354 Corte del Abeto,
Carlsbad, CA 92011
Tel: 760-804-3886
Mobile: 949-872-8589
Email: bofarrell@dcndx.com