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Multiplexing and Arraying in Lateral Flow Assays

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Lateral flow assays are the most robust, mature immuno sensor available today. Performance in some applications has historically been limited by difficulties in multiplexing and quantification. Novel approaches have been developed and commercialized in recent years that allow for the development and manufacturing of highly multiplexed arrays in lateral flow assays. The patented Symbolics (tm) approach is one such methodology. Symboics (tm) allows for the creation of arrays in lateral flow fields that develop evenly, allowing in turn for the creation of highly complex features such as letters and symbols and also allows for creation of multiplex assays with advanced features such as internal controls. This presentation introduces the principles of multiplexed arraying and the Symbolics technology

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Multiplexing and Arraying in Lateral Flow Assays

  1. 1. Patented technology facilitating multiplexing, quantification and alpha-numeric result generation in rapid assays Patented www.Symbolicsdx.com
  2. 2. 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
  3. 3. 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
  4. 4. 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
  5. 5. 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
  6. 6. 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
  7. 7. 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.
  8. 8. 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.
  9. 9. 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
  10. 10. 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
  11. 11. 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
  12. 12. Offering a complete solution or a simple license *Licenses are available without using support services offered by DCN or equipment offered by BioDot
  13. 13. 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
  14. 14. Licensing A two level license is offered 1.Development License 1. Non exclusive 2. Low cost 3. Limited term 4. Patent grant-back 2.Manufacturing and Commercialization License 1. To be negotiated
  15. 15. Technical Notes Facilitating multiplexing, quantification and non-traditional result generation in rapid assays Patented
  16. 16. 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
  17. 17. 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
  18. 18. Table top equipment for R&D and batch manufacturing Reel-to-reel equipment for high volume production
  19. 19. Cutter with Leading Edge and Fiducial Sensors CARD TARGET SENSOR
  20. 20. Demonstration Applications 1. Geometric symbols 2. Alpha -numeric symbols. 3. Multiplexing. 4. Other advanced design features
  21. 21. 1. Geometric Symbols
  22. 22. 1. Geometric Symbols Sample Application: hCG 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
  23. 23. 2. Alpha – Numeric Symbols
  24. 24. 2. Alpha – Numeric Symbols Sandwich Applications Drop Vol: 7nL Pitch: 0.5mm 6mm Strips BioJet Plus CON: 0.125mg/ml protein A H1: 4mg/ml GP041 H2: 2mg/ml GP036 Conjugate: 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
  25. 25. 2. Alpha – Numeric Symbols Sandwich Applications
  26. 26. 2. Alpha – Numeric Symbols in Competitive Assays DropVol: 7nL. Pitch: 0.5mm. 6mm Strips Dispenser: BioJet Plus CON: 0.5mg/ml GAM COC: 0.5mg/ml Benzoylecgonine-BTG Conjugate AMP: 0.5mg/ml Amphetamine-BSA Conjugate Conjugates: 10ug/ml anti-Benzoylecgonine & 3.5ug/ml anti-Amphetamine Sample: 100uL PBS+ Positive Sample: 500ng/ml benzoylecgonine or amphetamine
  27. 27. Drop Vol: 0.3nL Pitch: 0.25mm 5mm Strips Piezo electric dispenser Control: 0.5mg/ml GAM C: 0.5mg/ml Benzoylecgonine-BTG Conjugate A: 0.5mg/ml Amphetamine-BSA Conjugate Conjugates: 10ug/ml anti-Benzoylecgonine & 3.5ug/ml anti-Amphetamine Negative Sample: 100uL PBS+ Positive Sample: 500ng/ml benzoylecgonine & 500ng/ml amphetamine in buffer 2. Alpha – Numeric Symbols in Competitive Assays
  28. 28. 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
  29. 29. 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
  30. 30. 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 Sample: 100uL 0.1% Tween-20 in 1XPBS 100mIU/mL hCG in buffer10mIU/mL hCG in buffer 1000mIU/mL hCG in buffer 4. Advanced Application Concepts i. Analyte depletion assays

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