Design Considerations for

Lateral Flow Test Strips

Michael A. Mansfield
OEM Diagnostics
EMD Millipore
Bedford, MA
Making a Lateral Flow Test

M P
BIOLOGICAL CHEMICAL
A R
T O
E C
R E
I S
A S
L PHYSICAL ENGINEERING E
S S

2 DESIGN CONSIDERATIONS | 24 JUNE 2015

 Test Strip Assembly
Sample Conjugate Absorbent
Membrane
Pad Pad Pad
Detector Capture
Reagent Reagents
Materials
Reagents Salts+
Processes Adhesive Cards
Top Laminate

Assembled Cards
cut

Test strips
Housing Desiccant

Pouch Packaging

FINAL PRODUCT
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Assay Formats

A
C C C C
T T T T A

Sandwich Competitive Inhibition Serum (antibody)

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The Basic Strip Design
FLOW

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What controls the flow of the sample?

 The properties of the materials

– Pore size
– Wettability
– Bed volume (% air)
 The housing
– Design features
 The make-up of the sample
– Viscosity
– Particle load

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What controls the flow of the sample?

– Alignments
– Assembly
• Contact between the materials
• Degree of overlap
• Compression within the housing
– Cutting
• Raw materials
• Finished test strip

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Chemical Complexities

• Materials are impregnated with chemistries and then dried.

– Some chemistries are mobile.
– Some chemistries are fixed.
• The liquid flow path is complex.
– Within a material
– Between materials
• The chemical environment within the test strip changes as the strip
is manufactured and then again when it is run.
• The test never reaches chemical equilibrium.
• Immunoreagents must be fully reactive when rewet with sample.

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Analyte Contribution to Signal
Analyte running
Coincident ahead of detector
reagent
Conjugate Pad

Test
Line
Analyte running
after detector
reagent

No contribution Can produce signal

to signal
Acts to clear the
background

Only the analyte that chromatographs ahead of and coincident with the detector
reagent can contribute to the signal.

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What controls signal intensity?

Concentration of the reactants

– Analyte
– Detector particle
– Capture antibody

Flow rate of the membrane

– Faster flow  lower sensitivity
– Slower flow  higher sensitivity

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Flow Rate & Signal Intensity
The effective concentration of the reactants decreases linearly as
flow rate increases.

Both the antibody fixed to the membrane and mobile

analyte/detector reagent complex are affected.

Therefore, the amount of signal formed decreases with the square

of the increase in flow rate.
 Flow rate = 1 mm/sec
s1 = ka• [Ab] • [Ag:Ab-]

 Flow rate = 2 mm/sec

s2 = ka • (0.5 • [Ab]) • (0.5 • [Ag:Ab-])

= 0.25 • ka • [Ab] • [Ag:Ab-]

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Net Effect of Flow Rate

 As the membrane’s flow rate increases, there is a disproportionate

decrease in the signal intensity.
 The test must be designed to take into account flow rate variability
of the strip.
– Membrane flow variation
– Assembly variations
– Reagent balance

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Flow Rate and Membrane Distance
 Capillary flow rate decreases as the distance from the origin increases.
Sample
front

Flow Rate
mm/sec

Distance from origin (mm)

 The rate at which the detector particle passes the test line limits the
sensitivity. (slower flow  increased sensitivity)
 Thus, the placement of the capture lines relative to the origin affects assay
sensitivity.
 Capillary flow rate is unaffected by the width of the membrane.

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Flow Rate & Detector Particle Release
FAST RELEASE
Detector
Reagent
Flow Rate

T
Before reaching
test line Position of Test Line

After reaching
test line

This is the range of flow rates Distance from origin (mm)

that controls the formation of
the immunocomplex.

T C
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Flow Rate & Detector Particle Release

FAST RELEASE
Detector
Reagent
Flow Rate

T
Before reaching
test line
Flow Rate of Peak
at Test Line

After reaching
test line

Distance from origin (mm)

T C
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Flow Rate & Detector Particle Release

SLOW RELEASE
Detector
Reagent
Flow Rate

T
Before reaching
test line Position of Test Line

After reaching
test line

This is the range of flow rates Distance from origin (mm)

that controls the formation of
the immunocomplex.

T C
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Flow Rate & Detector Particle Release
SLOW RELEASE
Detector
Reagent
Flow Rate

T
Before reaching Flow Rate of Peak
test line at Test Line

After reaching
test line

Distance from origin (mm)

T C
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Mobile Chemistries
 Within the membrane
– Surfactant (if not washed out during blocking)
– Excess blocking agents (if used)
 Within the conjugate pad
– Detector particles
– Any buffer salts or solubilization agents
 Within the sample pad
– Any soluble additives

 Since these chemistries move in the assay, their concentration

profile is dynamic  constantly changing at any given location as
the test strip runs.

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Surfactant in the Membrane
Surfactants are applied to membranes as a coating on the surface of the
polymer. They are not covalently bound to the nitrocellulose.

sample
applied

End of End of
Start Intermediate
membrane assay

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Detector Particles vs. Chemistry
Sample pad solutes
Surfactant

T T
Flow Rate Flow Rate

Distance from origin Distance from origin

FAST RELEASE SLOW RELEASE

OF CONJUGATE OF CONJUGATE

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 Some of the solutes in my reagent buffers
are soluble. What happens to them when
I stripe my reagent buffers onto the membrane?

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Raman Confocal Microscopy

 Combines confocal microscopy with Raman spectroscopy

 Allows for scanning across and through a sample

Air side
y
Belt side

x
 Requires that the material be transparent
 Nitrocellulose can be cleared with immersion oil.

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Visualization of a Membrane

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Visualization of a Membrane
Thiocyanate anion
– behaves like a salt
– has a unique peak

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Thiocyanate Distribution on a Membrane

Air Side

Potassium
Thiocyanate

Belt Side

Air Side

Nitrocellulose

Belt Side

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Visualization of a Membrane

Distribution of Thiocyanate after Drying

 Multiple images
 Intensity correlates to concentration

140 mm

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Chemical Variations – Conclusions

 Within the signal lines, distribution of soluble chemistries is not

uniform through the depth of the membrane.
 The chemical profile of soluble components varies as the sample
travels through the test strip.
 Reactants may exhibit different levels of reactivity at the test and
control lines depending on the concentration of other chemicals at
that moment.
 Dissolution rates may vary for different chemistries.
– Salts
– Surfactants
– Blocking agents

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Configuration Effects on Flow Path
Long overlap 8 mm

FLOW

- Dead space develops.

- Serves as a reservoir for slower conjugate release

Minimal overlap
FLOW

- More uniform flow through the entire conjugate pad

- Faster release of the conjugate

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Reagents Can Be Inherently Variable
g Salts and buffers are typically not an issue.
g Detector particles may be variable.
– Are they evaluated before conjugation for size uniformity?
– After conjugation are they evaluated using a technique other than
lateral flow functionality?
g Biological reagents may have variability and stability issues that
are not readily apparent.
– Purity
– Activity
– Nature of contaminants

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Manufacturing Processes Can Be Variable
g Solution preparation
g Solution dispensing
g Treatment of pad materials
g Drying regimes
g Assembly of materials onto master cards
g Cutting of master cards into individual strips

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Realities of Test Strip Assembly

g Automation machinery can assemble lateral flow test strips rapidly

and accurately.
Corollary: Poorly designed and maintained machinery assembles
lateral flow test strips rapidly and inaccurately.

g Liquid flows through a lateral flow test strip along the path of least
resistance.
Corollary: Porous materials will fill with sample volume but may have
little or no active flow in some areas.

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 To learn more about our products and services, visit:

http://www.emdmillipore.com/diagnostics

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