Improvements for methods in digital counting
AssigneeSelma Diagnostics Aps
InventorAndreas Hjarne Kunding
Life of this patent
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Abstract
The present invention relates to methods and systems for testing for the presence of a material such as one or more analyte types within a sample and more particularly, for improved single enzyme-linked immunosorbent assay (sELISA) testing as well as other variants of single-enzyme linked molecular analysis (SELMA). Background and false positives are reduced due to the presence of at least two detection cycles where each detection cycle comprises the steps of a) triggering a signal from captured and labelled analyte(s), b) recording of the number and positions of capture sites exhibiting a signal from the captured and labelled analyte(s), c) and before a further detection cycle is performed, deactivation of signal(s).
Claims41
A method for digital counting analysis of a sample potentially containing at least one analyte, wherein the sample has been contacted with a solid phase having a plurality of discrete capture sites, each site being capable of capturing the at least one analyte, which method comprises at least two detection cycles, where each detection cycle comprises the steps of
- a) triggering a signal from captured and labelled analyte(s),
- b) recordation of the number and positions of capture sites exhibiting a signal from the captured and labelled analyte(s),
- c) and before a further detection cycle is performed, deactivation of signal(s).
The method according to any of the previous claims, where the sample contains or potentially contains a target analyte and a non-target compound, where the target analyte is captured by the capture site with capture efficiency C1, where the non-target compound is captured by the capture site with capture efficiency C2 and C1≥C2, where the target analyte is labelled by a first labelling agent with labelling efficiency L1, where the non-target compound is labelled by the first labelling agent with labelling efficiency L2 and L1≥L2, where the number of detection cycles NC is adjusted such that the ratio α=C1N1NC/C2N2NC is between 1-10, preferably between 10-100, preferably between 100-1000, preferably between 1,000-10,000, preferably between 10,000-100,000, preferably greater than 100,000, and where each detection cycle applies the first labelling agent in the labelling step.
The method according to any one of the previous claims, which includes a false-positive detection cycle, wherein the method does not comprise any labelling steps.
The method according to any one of the previous claims, wherein the labelling agent comprises a detection modality, and where the step of triggering a signal(s) is by delivering detection agents to the detection modality.
The method according to any one of the previous claims, wherein the step of deactivation of signal(s) is selected from
- a) detaching the labelling agent from the captured analyte,
- b) deactivating the ability of the labelling agent to facilitate a signal or
- c) the combination of a) and b),
- and wherein the step of deactivation of signal(s) is optionally followed by a rinsing step.
The method according to any one of the previous claims, wherein the capturing of the at least one analyte from the sample is by immobilization on the solid phase.
The method according to any one of the previous claims, wherein the capturing of the at least one analyte from the sample is by using one or more capture probes specific to the analyte and where the capture probes are attached to the solid phase.
The method according to any one of the previous claims, wherein the labeling agent is deactivated by detachment from the captured analyte and removed by flushing.
The method according to any one of the previous claims, wherein the captured and labelled analyte is compartmentalized to produce liquid compartments hosting the captured and labelled analyte by introducing and withdrawing a hydrophilic liquid on the plurality of discrete capture sites, where each discrete capture site is rendered hydrophilic and where the plurality of discrete capture sites is placed on a hydrophobic substrate, such that upon withdrawing of the hydrophilic liquid a plurality of liquid droplets is formed and each droplet occupies one discrete capture site.
The method according to any one of the previous claims, wherein the positions of liquid compartments exhibiting a signal in each detection cycle are compared to the positions of liquid compartments exhibiting a signal in the other detection cycles, such that the number of consecutive detection cycles a liquid compartment exhibits a signal is counted, and where liquid compartments are classified in at least two categories, the first category of liquid compartments exhibiting a greater count than the second category.
The method according to any of the previous claims, where the discrete capture sites are circular or spherical and where the diameter of the individual discrete site is less than 1 mm, preferably less than 100 μm, preferably less than 10 μm, preferably less than 1 μm.
The method according to any of the previous claims, where the discrete capture sites are circular or spherical and where the diameter of the discrete sites is between 0.5-5 μm, between 0.5-10 μm, between 0.5-50 μm, between 0.5-100 μm, between 10-1000 μm, between 50-1000 μm, between 100-1000 μm.
The method according to any of the previous claims, where the solid phase is
- a) a solid substrate,
- b) a colloid bead, or
- c) a collection of colloid beads.
The method according to any one of the previous claims, wherein the liquid compartments are in the form of a plurality of liquid nano-to-attoliter droplets under a gas phase seal.
The method according to any one of the previous claims, wherein the digital counting is performed in a flow system (10) for digital counting of one or more analyte types in a sample, the flow system comprising a support (12) having a pattern of hydrophilic features (14) in or on a hydrophobic substrate (16), the hydrophobic substrate (16) being embedded in a flow compartment (18) comprising at least one opening (20), the hydrophilic features (14) configured to support a plurality of liquid nano-to-attoliter droplets each having a maximum droplet volume, and the flow compartment (18) configured to support a gas phase seal reducing evaporation of each nano-to-attoliter droplet.
The method according to any one of the previous claims, wherein the capture probes (22) are selected from the following group of probes:
- oligonucleotides, aptamers, proteins, antibodies, peptides or synthetic variants thereof.
The method according to any one of the previous claims, wherein the analyte is selected from the following group of analytes:
- single-stranded oligonucleotides, double-stranded oligonucleotide complexes, proteins, protein/oligonucleotide complexes, protein/lipid complexes, peptides, exosomes, virus particles, virus like particles, nanoparticles, cell fragments or cells.
The method according to any one of the previous claims, wherein the sample is selected from the following group of samples:
- blood, plasma, serum, urine, saliva, cerebrospinal fluid, tears fluid, or tissue.
The method according to any one of the previous claims, wherein the sample is selected from laboratory-processed samples of the following sample group:
- blood, plasma, serum, urine, saliva, cerebrospinal fluid, tears fluid or tissue.
The method according to any one of the previous claims, wherein the captured analytes become covalently coupled to the capture probe(s) (22) subsequent to capture.
The method according to any one of the previous claims, wherein the digital counting measurement comprises a single-enzyme linked molecular analysis (SELMA), digital polymerase chain reaction (dPCR), single enzyme-linked immunosorbent assay (sELISA) or digital single-enzyme linked immunosorbent assay (dELISA).
The method according to any of the previous claims for reduction of false-positive detections and/or background noise in single molecule digital counting analysis of a sample.
Use of a solid phase having a plurality of discrete capture sites, each site being capable of capturing the at least one analyte in a method according to any one of the preceding claims.
Use of a solid phase having a plurality of discrete capture sites, each site being capable of capturing the at least one analyte in a method according to any one of the preceding claims for reducing counting error in a digital counting analysis such as reduction of false-positive detections and/or reducing background noise.
Dependencies are inferred from the claim text.