Device and method for multiple analyte detection
View Patent ↗The invention is directed to a method and device for simultaneously testing a sample for the presence, absence, and/or amounts of one or more of a plurality of selected analytes. The invention includes, in one aspect, a device for detecting or quantitating a plurality of different analytes in a liquid sample. Each chamber may include an analyte-specific reagent effective to react with a selected analyte that may be present in the sample, and detection means for detecting the signal. Also disclosed are methods utilizing the device.
1. A method of detecting or quantitating a target nucleic acid sequence in a liquid sample, comprising:
introducing a liquid sample comprising a target nucleic acid sequence through a channel in a substrate and into a chamber in the substrate which is in fluid communication with the channel, the chamber being pre-loaded with detection reagents before introduction of the liquid sample, the detection reagents comprising a first unlabeled oligonucleotide, a second unlabeled oligonucleotide, and a fluorescently-labeled oligonucleotide;
mixing the liquid sample with the detection reagents in the chamber such that the first unlabeled oligonucleotide, the second unlabeled oligonucleotide, and the fluorescently-labeled oligonucleotide are not immobilized in the chamber;
reacting the target nucleic acid sequence with both the first unlabeled oligonucleotide and the second unlabeled oligonucleotide, in the chamber, under conditions effective to selectively bind each of the first unlabeled oligonucleotide and the second unlabeled oligonucleotide to the target nucleic acid sequence, and to produce a first reaction product;
producing fluorescence in the chamber by a reaction in the chamber involving the first reaction product and the non-immobilized fluorescently-labeled oligonucleotide; and
detecting the fluorescence produced in the chamber.
2. The method of claim 1 , wherein the target nucleic acid sequence comprises a single-stranded nucleic acid sequence.
3. The method of claim 1 , wherein the first unlabeled oligonucleotide and the second unlabeled oligonucleotide, each bind to a different region of the target nucleic acid sequence.
4. The method of claim 1 , further comprising reacting to bind the fluorescently-labeled oligonucleotide to the target nucleic acid sequence such that the fluorescently labeled oligonucleotide emits a detectable fluorescent signal upon excitation thereof.
5. The method of claim 1 , wherein the reacting comprises a polymerase chain reaction.
6. The method of claim 1 , wherein the chamber comprises a plurality of chambers.
7. The method of claim 1 , wherein the chamber comprises a plurality of chambers connected to a channel.
8. The method of claim 1 , wherein the introducing a liquid sample comprises subjecting the liquid sample to a vacuum.
9. The method of claim 1 , wherein the first unlabeled oligonucleotide and the second unlabeled oligonucleotide comprise a primer pair for polymerase chain reaction amplification.
10. The method of claim 1 , wherein the fluorescently-labeled oligonucleotide is a sequence-selective oligonucleotide.
11. The method of claim 10 , wherein the sequence-selective oligonucleotide comprises a fluorescer-quencher pair.
12. The method of claim 1 , wherein the first oligonucleotide and second oligonucleotide bind selectively to adjacent, contiguous regions of the target nucleic acid.
13. The method of claim 1 , wherein the detection reagents are dried detection reagents.