Base4 Innovation Limited

Lightcast Discovery Ltd

United Kingdom Research and Innovation

Medical Research Council

Thomas Henry Isaac

Cameron Alexander Frayling

Barnaby Balmforth

Boris Breiner

Gareth Podd

Michele Amasio

Bruno Flavio Nogueira De Sousa Soares

David Love

Alessandra Natale

Pedro Cunha

Rebecca Palmer

Douglas J Kelly

Eoin Sheridan

Paul Dear

Maciej Sosna

Richard Ingham

Jasmin Conterio

Kerr Francis Johnson

Andreas Michael Waeber

Evangelia-Nefeli Athanasopoulou

Bruno Flavio Soares

Emma Head

Paul Loeffen

Claire Marie Murzeau

Nele Marie Godette Dieckmann

A device for manipulating microdroplets using optically-mediated electrowetting comprising: a first composite wall comprising: a first transparent substrate; a first transparent conductor layer on the substrate having a thickness of 70 to 250 nm; a photoactive layer activated by electromagnetic radiation in the wavelength range 400-1000 nm on the conductor layer having a thickness of 300-1000 nm; and a first dielectric layer on the conductor layer having a thickness of 120-160 nm; a second composite wall comprised of: a second substrate; a second conductor layer on the substrate having a thickness of 70 to 250 nm; and an A/C source to provide a voltage across the first and second composite walls connecting the first and second conductor layers; at least one source of electromagnetic radiation having an energy higher than the bandgap of the photoexcitable layer; and means for manipulating the points of impingement of the electromagnetic radiation on the photoactive layer.

Microdroplet manipulation device

A device comprising: a first zone comprising an attachment site; a first pathway; a second pathway and a means for creating a second medium comprised of aqueous microdroplets in a carrier; a microdroplet manipulation zone comprising: a first composite wall comprised of a first transparent substrate; a first transparent conductor layer on the substrate; a photoactive layer activated by electromagnetic radiation; and a first dielectric layer on the photoactive layer; a second composite wall comprised of a second substrate; a second conductor layer on the substrate; and optionally a second dielectric layer on the conductor layer; an A/C source; a source of first electromagnetic radiation; means for manipulating the points of impingement of the electromagnetic radiation on the photoactive layer; an detection zone disposed downstream of the microdroplet manipulation zone or integral therewith; and a fluorescence or Raman-scattering detection system.

Microfluidic analytical device

A device comprising: a first zone comprising an attachment site; a first pathway; a second pathway and a means for creating a second medium comprised of aqueous microdroplets in a carrier; a microdroplet manipulation zone comprising: a first composite wall comprised of a first transparent substrate; a first transparent conductor layer on the substrate; a photoactive layer activated by electromagnetic radiation; and a first dielectric layer on the conductor layer; a second composite wall comprised of a second substrate; a second conductor layer on the substrate; and optionally a second dielectric layer on the conductor layer; an A/C source; a source of first electromagnetic radiation; means for manipulating the points of impingement of the electromagnetic radiation on the photoactive layer; an detection zone disposed downstream of the microdroplet manipulation zone or integral therewith; and a fluorescence or Raman-scattering detection system.

A microfluidic sequencing device in which a stream of microdroplets at least some of which contain a single nucleotide base are made to undergo reaction with a capture system to capture and detect an ordered sequence of single nucleotide bases generated by progressive pyrophosphorolysis.

Microfluidic device for characterzing polynucleotides

A method for determining the sequence of nucleotide bases in a polynucleotide analyte is provided. It is characterised by the steps of (1) generating a stream of single nucleotide bases from the analyte by pyrophosphorolysis; (2) producing captured molecules by reacting each single nucleotide base with a capture system labelled with detectable elements in an undetectable state; (3) releasing the detectable elements from each captured molecule in a detectable state and (4) detecting the detectable elements so released and determining the sequence of nucleotide bases therefrom. The method can be used advantageously in sequencers involving the use of microdroplets.

Single nucleotide detection method

An apparatus for sequencing a nucleic acid by printing droplets at least some of which contain single nucleotides derived from the nucleic acid is provided. It is characterised by comprising; &#x2022;a planar substrate having a face with droplet-receiving locations arranged in at least one track parallel to a first axis defining the substrate; &#x22c5;a printer head comprising a plurality of droplet-dispensing nozzle groups juxtaposed above the droplet-receiving locations so that each nozzle group can in turn dispense a droplet into the droplet-receiving locations; &#x2022;a means for stepping the printer head along the first axis relative to the droplet-receiving locations; &#x22c5;a nucleotide-generating site upstream of the printer head by means of which an ordered stream of single nucleotides is created from the nucleic acid and delivered to a first droplet-dispensing nozzle group; &#x2022;at least one source of incident electromagnetic radiation adapted to illuminate the droplet-receiving locations and &#x22c5;at least one photodetector to detect adapted to detect fluorescence radiation emitted by each droplet-receiving location after illumination.

Droplet sequencing device

A method for determining the sequence of nucleotide bases in a polynucleotide analyte is provided. It is characterised by the steps of (1) generating a stream of single nucleotide bases from the analyte; (2) producing captured molecules by reacting each single nucleotide base with a capture system; (3) amplifying at least part of the captured molecule to produce a plurality of amplicons characteristic of the single nucleotide base; (4) labelling the amplicons with a corresponding probe having a characteristic detectable element and (5) detecting a property characteristic of the detectable element.

Disclosed is a method of identifying the contents of individual droplets in a droplet stream each droplet containing fluorophores in an initial non-fluorescing state characterized by the steps of introducing the droplets one-by-one into at least one open-ended tube to create a stack of droplets therein; activating the fluorophores within the droplets to cause them to fluoresce; releasing each droplet in the droplet stack in turn from the tube and detecting along the major axis of the tube fluorescence associated with each droplet as it emerges. Also disclosed is a method suitable for sequencing a biopolymer characterized by the steps of (1) progressively digesting the biopolymer into an ordered stream of its constituent monomers; (2) converting the stream of monomers into a corresponding stream of monomer-containing aqueous droplets each droplet additionally containing a probe capable of (a) capturing the monomer and (b) thereafter being digested to release an unqueched fluorophore characteristic of the captured monomer; (3) introducing the stream of droplets created in step (2) into an inlet end of at least one open-ended tube to create a stack of droplets therein and (4) releasing each droplet in turn from an outlet end of the tube(s) and detecting fluorophores in each droplet as each droplet emerges. The method may be used in a corresponding apparatus for sequencing a biopolymer such as a nucleic acid or protein.

Method of identifying droplets in a stack and an associated sequencer

Disclosed is a method for determining the sequence of nucleotide bases in a polynucleotide analyte characterised by the steps of: a. generating a stream of droplets at least some of which contain a single nucleotide and wherein the order of single nucleotides in the droplet stream corresponds to the sequence of nucleotides in the analyte; b. introducing into each droplet a plurality of biological probe types each type (i) comprising a different detectable element in an undetectable state and (ii) being adapted to capture a different complimentary single nucleotide from which the analyte is constituted; c. causing the single nucleotide contained in the droplet to bind to its complimentary probe to create a used probe; and d. causing the detectable element to be released from the used probe in a detectable state. Typically the biological probe employed comprises a single-stranded nucleotide region the ends of which are attached to two different oligonucleotide regions wherein at least one of the oligonucleotide regions comprises detectable elements having a characteristic detection property and wherein the detectable elements are so arranged on the oligonucleotide region that the detectable property is essentially undetectable in the probe's unused state. In a most preferred embodiment the probe is labelled with multiple fluorophores and further comprises a restriction enzyme recognition site generated by the binding of the target single nucleotide to the single-stranded nucleotide region. Suitably, step c is carried out in the presence of a polymerase and ligase and step d in the presence of a restriction enzyme and an exonuclease. Typically the flow rate of the droplets is 100 to 2000 droplets per second.

Sequencing method

A method for determining the sequence of nucleotide bases in a polynucleotide analyte is provided. It is characterized by the steps of (1) generating a stream of single nucleotide bases from the analyte by pyrophosphorolysis; (2) producing captured molecules by reacting each single nucleotide base with a capture system labelled with detectable elements in an undetectable state; (3) releasing the detectable elements from each captured molecule in a detectable state and (4) detecting the detectable elements so released and determining the sequence of nucleotide bases therefrom. The method can be used advantageously in sequencers involving the use of microdroplets.

Disclosed is a method for sequencing a polynucleotide analyte comprising: &#x2022;a. generating a stream of droplets containing a single nucleotide wherein the order of single nucleotides in the droplet stream corresponds to the sequence of nucleotides in the analyte; &#x2022;b. introducing into each droplet a plurality of biological probe types each type comprising a different label in an undetectable state and being adapted to capture a different single nucleotide; &#x2022;c. causing the single nucleotide contained in the droplet to bind to its complementary probe and &#x2022;d. causing the label to be released from the probe that has bound the nucleotide in a detectable state. The probe is a dumbbell shaped probe comprising fluorescent donor and quencher labels and a single nucleotide gap. After gap repair by a polymerase and a ligase, a restriction enzyme recognition site is cleaved by a restriction enzyme, followed by exonuclease digestion to release the labels.

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Cambridge, United Kingdom

Thomas Henry Isaac