Digital nucleic acid amplification using encoded particles

A method of performing a digital assay, the method comprising:

• providing a plurality of compartmentalized volumes, wherein:
• each compartmentalized volume in the plurality of compartmentalized volumes comprises a probe, wherein each probe comprises an encoded particle comprising a plurality of distinct chromophores;
• and a plurality of binding nucleic acid molecules configured to bind to a target molecule or to a molecule that is correlated with the presence of the target molecule, and a plurality of quenchers, wherein each quencher of the plurality of quenchers is coupled to a quencher nucleic acid molecule configured to hybridize with the target molecule, an amplification product, or the binding nucleic acid molecule, wherein each quencher of the plurality of quenchers is configured to quench fluorescence from a chromophore of the plurality of distinct chromophores, and wherein the encoded particle has at least one dimension that is greater than 3 nm;
• and
• at least some of the compartmentalized volumes in the plurality of compartmentalized volumes comprise the target molecule;
• amplifying a molecule that is correlated with the presence of the target molecule;
• and
• detecting an optically detectable code emitted by an encoded particle in the compartmentalized volume, wherein the detection of the optically detectable code indicates that the target molecule is present in the compartmentalized volume.

A method of performing a digital melt-curve assay, the method comprising:

• providing a plurality of compartmentalized volumes distributed into a plurality of containers, wherein each compartmentalized volume in the plurality of compartmentalized volumes comprises:
• a probe comprising an encoded particle comprising:
• a plurality of distinct chromophores;
• and
• a plurality of binding nucleic acid molecules configured to bind to a target molecule or to a molecule that is correlated with the presence of the target molecule, and
• a plurality of quenchers, wherein each quencher of the plurality of quenchers is coupled to a quencher nucleic acid molecule configured to hybridize with the target molecule, an amplification product, or the binding nucleic acid molecule, wherein each quencher of the plurality of quenchers is configured to quench fluorescence from a chromophore of the plurality of distinct chromophores;
• wherein at least some of the compartmentalized volumes in the plurality of the compartmentalized volumes comprise a target molecule;
• applying a thermal energy gradient to the plurality of compartmentalized volumes to yield a plurality of assay temperatures varying over an area;
• and
• determining the melting temperature of the target molecule or of a molecule that is correlated with the presence of the target molecule by performing the digital melt-curve assay of the target molecule or of the molecule that is correlated with the presence of the target molecule in the presence of a temperature gradient.

A method of performing a digital melt-curve assay, the method comprising:

• providing a plurality of compartmentalized volumes, wherein:
• each compartmentalized volume in the plurality of compartmentalized volumes comprises:
• a probe comprising an encoded particle comprising:
• a plurality of distinct chromophores;
• and
• a plurality of binding nucleic acid molecules configured to bind to a target molecule or to a molecule that is correlated with the presence of the target molecule, and
• a plurality of quenchers, wherein each quencher of the plurality of quenchers is coupled to a quencher nucleic acid molecule configured to hybridize with the target molecule, an amplification product, or the binding nucleic acid molecule, wherein each quencher of the plurality of quenchers is configured to quench fluorescence from a chromophore of the plurality of distinct chromophores;
• and
• at least some of the compartmentalized volumes in the plurality of the compartmentalized volumes comprise the target molecule;
• amplifying the target molecule to produce an amplified molecule;
• applying a thermal energy gradient to the plurality of compartmentalized volumes to yield a plurality of assay temperatures varying over an area, such that when the assay temperature in each compartmentalized volume is:
• below a target molecule or amplified molecule melting temperature, at least 50% of the target molecule or of the amplified molecule is hybridized;
• and
• above the target molecule or amplified molecule melting temperature, less than 50% of the target molecule or of the amplified molecule is hybridized;
• detecting an optically detectable signal produced by a chromophore of the plurality of distinct chromophores associated with the hybridized target molecule or the amplified molecule, wherein the optically detectable signal is detected when the chromophore is associated with the hybridized target molecule or the amplified molecule;
• and
• determining the melting temperature for the target molecule or amplified molecule based on the presence or absence or magnitude of the optically detectable signal at each of the assay temperatures of the plurality of assay temperatures.