Apparatus and methods for detecting target analyte

An apparatus comprising:

• a fluorescence decay detection system comprising a pulsed-light source and a digitizer;
• and
• a substrate comprising a plurality of identifiable regions each comprising a surface wherein at least one of said identifiable regions comprises a target specific probe attached to said surface, with the provision that when said probe is a polynucleotide, a fluorophore is attached to said polynucleotide;
• wherein said digitizer comprises:
• an array of memory elements that stores a representation of a time-dependent electrical signal corresponding to an analog fluorescence waveform signal from at least one of said identifiable regions as a time-series of analog voltages or charges;
• and
• at least one analog-to-digital converter that transforms the time-series of analog voltages or charges into a corresponding digitized fluorescence waveform;
• and
• wherein said fluorescence decay detection system is capable of being in optical communication with each of said regions and of measuring the fluorescence decay or lifetime of a fluorophore.

An apparatus comprising:

• a fluorescence decay detection system comprising a pulsed-light source and a digitizer;
• a substrate comprising a plurality of identifiable regions each comprising a surface wherein at least one of said identifiable regions comprises a target specific probe attached to said surface, with the provision that when said probe is a polynucleotide, a fluorophore is attached to said polynucleotide;
• wherein said digitizer comprises:
• an array of memory elements that stores a representation of a time-dependent electrical signal, corresponding to an analog fluorescence waveform signal from at least one of said identifiable regions, as a time-series of analog voltages or charges;
• at least one analog to digital converter that transforms the time-series of analog voltages or charges into a corresponding digitized fluorescence waveform;
• at least one digital signal processor for operably controlling parameters of the digitizer and for receiving the digitized fluorescence waveform;
• and
• wherein said fluorescence decay detection system is capable of being in optical communication with each of said regions and of measuring the fluorescence decay or lifetime of a fluorophore.

A method comprising

• contacting a test sample with a probe attached to an identifiable region of a substrate, wherein the probe comprises a binding domain that binds to a binding domain of a target that may be present in said test sample, wherein a fluorophore is attached to the probe and/or the target and the contacting is under conditions that allow the formation of a fluorescently labeled complex comprising said probe, said target and said fluorophore, wherein the fluorescence decay and/or lifetime of said fluorophore changes upon formation of said fluorescently labeled complex;
• and
• measuring the fluorescence decay and/or lifetime of the fluorophore at said identifiable region, as an indication of the presence or absence of said target in said test sample wherein said measuring is with an apparatus comprising:
• a fluorescence decay detection system comprising a pulsed-light source and a digitizer;
• and
• a substrate comprising a plurality of identifiable regions each comprising a surface wherein at least one of said identifiable regions comprises a target specific probe attached to said surface, with the provision that when said probe is a polynucleotide, a fluorophore is attached to said polynucleotide;
• wherein said fluorescence decay detection system is capable of being in optical communication with each of said regions and of measuring the fluorescence decay or lifetime of a fluorophore.

A method for determining the presence of heterozygous polynucleotide alleles in a test sample comprising

• contacting a fluorescent probe polynucleotide with a test sample that may contain one or more alleles of a target polynucleotide, wherein said probe polynucleotide forms a homoduplex with a first target polynucleotide allele and a heteroduplex with a second target polynucleotide allele and said contacting is under conditions that allow the formation of both the homoduplex and heteroduplex complexes, wherein the fluorophore of the probe polynucleotide has a different fluorescence decay and/or lifetime when the probe polynucleotide forms a homoduplex compared to when it forms a heteroduplex, and the fluorescence decay and/or lifetime is different among test samples that are:
• (1) homozygous for homoduplexes, (2) homozygous for heteroduplexes and (3) heterozygous for both homoduplexes and heteroduplexes;
• and
• measuring the fluorescence decay and/or lifetime of the fluorophore to determine the presence of a target polynucleotide allele (s) and the homozygous or heterozygous state in a test sample.