Patented technology facilitating multiplexing, quantification and alpha-numeric result generation in rapid assays

Patented

www.Symbolicsdx.com

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

Background

• Symbolics LLC was founded in 2010 as a joint venture between DCN Inc and BioDot Inc

• Wholly owned by DCN and BioDot

• Key management and joint development team in place

• Initial patent allowed, January 2013

• Subsequent filings in progress

The Problem

• Lateral flow assays are limited to linear results, created by the placement of lines of capture reagents across the width of a flow path

• User Error: Linear results are non-intuitive and can be difficult to interpret

• Multiplexing and quantification are hard:– Lines are stacked behind one another– Line bleed occurs– Cross reactivity issues are worsened– Flow rate decreases non-linearly with distance from origin: quantification of multiplexed lines is difficult and slow

The Problem with Lines

The Problem

Traditional manufacturing methods create large structures in flow paths

Large, discreet linear features cause flow pattern disturbances, preventing formation of features behind them over relatively large distances in the direction of flow.

High AffinityLow AffinityNo Affinity

Creating other types of features using standard production methods results in imperfect feature geometry

Strong leading edge binding due to high affinity binding reagents is extreme in drop sizes used in standard approaches. Reagents that are useful for lateral flow (with decent affinity) will not form complete features if spotted on a membrane in standard spot sizes

In Other Words….

• The industry has stuck with lines because they are the only way to produce even, reproducible features in a lateral flow path.

• Alternatives currently in market, such as the “+” symbol rely on pre-printed ink features to create part of the symbol

• Digital devices returning easily interpreted alpha-numeric results such as “Pregnant” require expensive, patented reader technologies in the cassettes, resulting in complexity and margin hits for manufacturers

Pre-printed ink line

The Unseen Impact of Linear Results

• Limited success of lateral flow systems in multiplexed or quantitative applications

• Limitations on user-friendliness of consumer assays• Limitation in overall market growth

Average 7% CAGR does not account for the growing potential in consumer markets, for multiplexed applications such as Traumatic Brain Injury, Kidney, Cardiac or Dehydration panels, or for other novel quantitative applications: There is a lot of untapped potential.

The Solution: Pixelation

• The problems of incomplete feature formation can be overcome through the

use of appropriately sized features (“pixels”) spaced at appropriate distances in

the flow path.

• This makes the formation of larger, complex features possible.

• Pixels are created using precise methods for dispensing and spacing discrete

spots of protein in the lateral flow field. Volumes range from the picoliter range

to the low nanoliter range at carefully controlled pitch (spacing).

• Dispensing is done using a variety of technologies including piezo-electric or

solenoid-based dispensers.

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

The Opportunity

• Revolutionize entire rapid testing market segments by facilitating the development and manufacturing of novel lateral flow assays with a range of advantages:

– A new generation of user-centered consumer assays that are intuitive, simple to interpret but do not require digital interpretation

– Better quantification

– Higher dynamic range

– Internal controls

– Multiplexing of analytes, at virtually any density required for diagnostics or testing in most market segments (high or low)

– Lower reagent usage

Where we are

• 1 issued US patent: 13/343,681: “Lateral flow assays using two dimensional features”

• 1 published PCT: PCT/US2012/021586

• Patent portfolio expansion underway with multiple US and international filings

• Licensing strategy in place

• Licenses and development support available

Offering a complete solution or a simple license

*Licenses are available without using support services offered by DCN or equipment offered by BioDot

Getting Started

• Symbolics technology is available for license and integration into

your assay development programs

• DCN and BioDot are available if so desired to support your assay

development or re-formatting program

Licensing

A two level license is offered

1.Development License1. Non exclusive

2. Low cost

3. Limited term

4. Patent grant-back

2.Manufacturing and Commercialization License1. To be negotiated

Technical Notes

Facilitating multiplexing, quantification and non-traditional result generation in rapid assays

Patented

Assay Development Strategy

• Assay development follows essentially the same process as standard lateral flow

• Development requires higher fidelity dispensing equipment

• All of the same labels, reagents and many of the same materials can be applied

• For higher fidelity quantitative applications, some alternative material choices may yield better results

• An understanding of the affinity of the reagents being used is useful given the interaction between reagent affinity and feature spacing and size

• Assistance can be provided by DCN Inc if required

Manufacturing

• Manufacturing process design is similar to standard lateral flow

• Dispensing processes require higher fidelity in terms of

placement and volume

• Dispensing processes must be robust to ensure fidelity of

feature formation

• Consideration must be given to feature position during cutting,

so tracking features (fiducials) must be included in the card

design

• Virtually all other processes will stay the same as in standard

lateral flow

• Equipment can be provided by BioDot Inc if required

Table top equipment for R&D and batch manufacturingReel-to-reel equipment for high volume production

Cutter with Leading Edge and Fiducial Sensors

CARD TARGET SENSOR

Demonstration Applications

1. Geometric symbols

2. Alpha -numeric symbols.

3. Multiplexing.

4. Other advanced design features

1. Geometric Symbols

1. Geometric SymbolsSample Application: hCG

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

2. Alpha – Numeric Symbols

2. Alpha – Numeric SymbolsSandwich Applications

Drop Vol: 7nL Pitch: 0.5mm 6mm Strips BioJet Plus CON: 0.125mg/ml protein AH1: 4mg/ml GP041H2: 2mg/ml GP036Conjugate: 10 uL of 8ug/ml protein A

Sample: 5uL human plasma positive for HIV-1, 100uL HIV Running Buffer

Sample: 5uL human plasma positive for HIV-2, 100uL HIV Running Buffer

2. Alpha – Numeric SymbolsSandwich Applications

2. Alpha – Numeric Symbols in Competitive Assays

DropVol: 7nL. Pitch: 0.5mm. 6mm StripsDispenser: BioJet Plus CON: 0.5mg/ml GAMCOC: 0.5mg/ml Benzoylecgonine-BTG ConjugateAMP: 0.5mg/ml Amphetamine-BSA Conjugate Conjugates: 10ug/ml anti-Benzoylecgonine & 3.5ug/ml anti-AmphetamineSample: 100uL PBS+Positive Sample: 500ng/ml benzoylecgonine or amphetamine

Drop Vol: 0.3nL Pitch: 0.25mm 5mm StripsPiezo electric dispenserControl: 0.5mg/ml GAMC: 0.5mg/ml Benzoylecgonine-BTG ConjugateA: 0.5mg/ml Amphetamine-BSA Conjugate Conjugates: 10ug/ml anti-Benzoylecgonine & 3.5ug/ml anti-AmphetamineNegative Sample: 100uL PBS+ Positive Sample: 500ng/ml benzoylecgonine & 500ng/ml amphetamine in buffer

2. Alpha – Numeric Symbols in Competitive Assays

3. 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

4. Advanced Application Concepts

• Advanced internal control features• Flow controls for quantification• Internal cross reactivity controls and true negatives• Reader calibration features

• Large scale multiplexing through lateral flow arrays

• Novel quantification approaches - “thermometer” quantification- large dynamic range assays

DropVol: 0.3nL Pitch: 0.25mm 5mm StripsPiezo electric DispenserControl: 0.5mg/ml GAMTest: 1mg/ml anti-hCGαConjugate: 8ug/ml anti-hCGβSample: 100uL 0.1% Tween-20 in 1XPBS

100mIU/mL hCG in buffer10mIU/mL hCG in buffer 1000mIU/mL hCG in buffer

4. Advanced Application Conceptsi. Analyte depletion assays