Next Generation Lateral flow

Quality

Innovation

Partnership

Results

Next Generation Lateral flow Steven Lamont Technical Manager Version 1 Final

Next Generation

The next development or version of a

product / service / technology

What does this statement mean when it comes to Lateral Flow?

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Overview Key areas this talk will focus on: 

End user experience

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Molecular  Formats  Aptamers  Platforms

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Antibody replacements

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Alternative detection  SERS

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End User Experience

How can this be improved?

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Improving User Experience 

Increasing the usability of lateral flow devices and their interpretation is key to gaining access to certain markets

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Increase in perceived quality and value of the assay

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Clever design can reduce the complexity and provide greater control to overcome common user errors

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Incorporating new design / functionality can give you a USP in the market

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AtomoRapid™ Platform – What is it? Lateral flow cassette with an integrated lancet and blood capillary

AtomoRapid™ Platform

Collect blood sample

Deliver sample to test

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AtomoRapid™ Platform

Addition of buffer

Reading the result

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Next generation reader solution

The use of mobile technology in the healthcare industry is spreading rapidly It is transforming the way in which providers deliver and patients access care. Mobile enabled diagnostics is facilitating: 

The migration from lab testing to a range of environments

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A faster more accurate result

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Increased primary care in under developed countries

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The production of quantitative lab quality test results immediately in the field

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End user process

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Ease of Use

Focus

Strip recognised Control line in range

Guide to help user correctly align the test cassette

Illumination

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Connectivity

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How is Novarum revolutionising lateral flow? 

Remove ambiguity in testing  More accurate than a visual reading

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Take your qualitative test and quantify it  Provide an exact numerical value

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Connect end users and professionals to test results immediately

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Smartphone app – users already familiar with technology

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A low cost reading with no additional hardware

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Better for patients, better for doctors, better for healthcare  Patients – privacy, convenience, immediacy  Doctors – efficiency, better diagnosis, accuracy  Healthcare – centralized data, cost savings, effective treatment

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Molecular Diagnostics

Molecular lateral flow tests – next generation

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Molecular Diagnostics “Molecular Diagnostics” is a broad term that normally describes tests detecting and measuring specific genetic sequences in DNA, RNA or the proteins they

express.  Took over 10 years and $3 billion to 1st sequence the human genome  To date over 228,000 genomes have now been sequenced and this number is predicted to double every year  Cost is now ~$1000 to sequence a single genome

Nucleic Acid Lateral Flow – NALF Molecular Diagnostics can be applied to Lateral Flow in two main ways:  Detection of molecular targets  Use of oligonucleotides to detect proteins / antigens Sectors in which molecular lateral flows have published:  Human diagnostics  Food safety  Veterinary  Biodefense  Agriculture

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Conventional vs Molecular Conventional antibody-antigen techniques rely on:

NALF relies on:

 Specificity of the antibody  Abundance of the antigen

 Specificity of the target DNA sequence  Abundance of the target sequence.

Au chromosome

Target DNA

Thus for a single DNA target per cell there maybe 100−1000s of protein targets. However nucleic acids are amenable to amplification allowing a single copy of the target sequence to be (theoretically) amplified to millions of copies Version 1 Final

Amplification PCR is the standard amplification technique, however isothermal techniques are better suited for inclusion into lateral flow assay platforms. Two techniques that are commonly considered are:

Recombinase Polymerase Amplification (RPA)

Loop mediated isothermal amplification (LAMP)

Temp

37-42C

60-65C

Time

10-15 min

60 min – amplification can be seen after 20min

Primers Amplicons

References

2 Consistent in size Piepenburg, Olaf, et al. PLoS biology 4.7 (2006).

4-6 Variable – can be very large Multiple gene copies per amplicon

Kiatpathomchai, Wansika, et al. Journal of Virological Methods 153.2 (2008): 214-217.

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Molecular – Antibody dependent Detection of amplified DNA-hapten labelled products

Anti-biotin gold conjugate Nucleic acid amplification target

Biotin

FITC

T

T

Anti-FITC nitrocellulose

C

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Molecular – Antibody independent 2 options for the test line capture reagent with this format:  Streptavidin  Oligonucleotide

T

C

T

C

Key Streptavidin

Oligo – biotin labelled

Gold – Oligo conjugate

Oligo – specific to antigen

Antigen (e.g. BNP)

Gold – Oligo conjugate

Aptamers Named from the latin word aptus meaning ‘to fit’ they are synthetic bio recognition molecules made from DNA, RNA or peptides

 DNA & RNA aptamers are the most common type of aptamers  Typically 15-60 nucleotide bases in length  Peptide aptamers are small, simple peptides with a single variable loop region tied to a protein on both ends  They are engineered through repeated rounds of in-vitro selection (SELEX) and can be targeted against small molecules, toxins, peptides, proteins, viruses, bacteria, and even whole cells

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Molecular - Platforms  Integrated Multiplexed Assay Sampling System  

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Based upon holding 8 test strips The strips are mounted in pairs within 4 totally separated chambers. Each chamber can hold 500µl of amplified and diluted sample which is used to run the two test strips. Excess sample is stored within a separate foam pad mounted in each chamber at the bottom of the pair of strips. This pad controls the flow into the strips

Molecular - Platforms 

Amplification technology and power are all integrated within the device

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The drive nut is turned 1 complete turn

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This compresses the sponge and the diluent is added into the 4 chambers.

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The sample travels up into the 8 strips.

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Read the results on the 8 test strips.

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Points to Consider Buffer formulations  Certain buffer constituents are required for the amplification techniques  To promote hybridisation when flowing up the nitrocellulose  These are not always ideal for lateral flow assays Most published work is using a purified amplification product  POC assays are likely to require an all-in-one platform approach  Lyophilisation of reagents required for amplification  Transfer of sample to amplification step and then transfer to lateral flow strip Apatmers  Form tertiary structures – orientation could be important  Use of any linkers  Size of nucleotide sequence

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Antibody replacements

Affimers in lateral flow

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Affimers  An Affimer is a peptide based aptamer  Fixed core structure with 2 variable loops  They are small, highly stable proteins engineered to display peptide loops which provide a high affinity binding surface for a specific target protein  Affimers routinely display binding affinities in the nM region  Can be easily functionalised  Benefit over aptamers:  DNA/RNA aptamers can be easily sequenced / copied

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Raman lateral flows

SERS detection technology

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SERS and Lateral Flow Surface Enhanced Raman Scattering When light, usually from a laser, is shined on a sample, the target molecule vibrates and scatters back in its own unique light, often referred to as the Raman scatter. However, this Raman response is extremely weak. When the target molecule is coupled with a metal nanoparticle or nanostructure, the Raman response is greatly enhanced by the SERS effect Cost and lack of portable readers have always been a barrier to using this technology in conjunction with lateral flow:  Handheld readers are now available  Cost has significantly reduced

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SERS and Lateral flow Gold conjugates prepared that include a SERS tag in conjunction with an antibody against the relevant target Au and Ag particles used as effective SERS substrates for years

Laser

Detector

Key T

C

Antibody Antigen SERS tag

Gold colloid

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SERS - Nanostars 

Nanostars offer an alternative substrate for SERS that promises greater amplification

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Research has been published that demonstrates, compared to other colloid shapes, nanostars can dramatically enhance the reflected light

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Technology within lateral flow is in its infancy but offers great potential to increase sensitivity

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Summary 

The term “Next Generation” encompasses a number of areas  User experience  New applications of Lateral Flow  New reader systems

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Technology advancements outwith of Lateral Flow have the potential to create new opportunities for Lateral Flow assays for many years to come

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Raw Materials

Contract Development & Manufacture

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Antibodies Antigens Enzymes Gold Nanoparticles Serum & Plasma Cell Culture Reagents

www.bbisolutions.com

Lateral Flow Test Development Lateral Flow Test Manufacture Full Regulatory & Post Market Support Sensitivity Enhancement Technology Platforms Readers

[email protected]

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