Detection of Analytes in Samples Using Liposome-Amplified Luminescence and Magnetic Separation
First Claim
1. A method for detecting an analyte comprising the steps of:
- a) obtaining a sample potentially comprising an analyte;
b) providing liposomes comprising a luminescence-related amplificant encapsulated within said liposomes, a buffer and paramagnetic beads;
c) incubating said sample potentially comprising an analyte, said liposomes, and said paramagnetic beads to provide a complex of said paramagnetic beads, said analyte and said liposomes;
d) separating said complex from non-complexed paramagnetic beads and non-complexed liposomes;
e) treating said complex with a liposome extractant to release the contents of said liposomes to form an assay sample; and
f) measuring light via a luminescent means;
wherein said liposomes of step b) comprise at least one reporter probe;
wherein said paramagnetic beads of step b) comprise at least one capture probe, and wherein the presence of said analyte is determined by an amount of light emitted from said assay sample.
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Abstract
The invention relates to the encapsulation of luminescence-related molecules, including but not limited to, adenosine triphosphate (ATP), adenylate kinase (AK), alkaline phosphatase (ALP), luminol and luciferin/luciferase cocktails, within liposomes. These liposomes can be employed to enhance the luminescence detection of microorganisms and compounds in various products and samples. The liposomes containing the luminescence-related molecules can bear a probe which has a specific sequence or structure that, in turn can be used to hybridize to, or couple with, a portion of the target analyte. Within the same assay, paramagnetic beads can bear a probe having a specific sequence or structure that, can hybridize to, or couple with, a second portion of the target analyte to create a complex of analyte bound to paramagnetic beads and liposomes. This type of assay can be often referred to as a ‘sandwich’ assay. Once the probes hybridize to, or couple with, their targets, a complex can be formed of the paramagnetic beads, the analyte, or portion thereof, and the liposomes. This complex can then be washed to remove those components that are non-hybridized or non-coupled. Then, the paramagnetic bead-analyte-liposome complexes can be isolated from the sample using magnetic separation techniques and can be treated so as to release their encapsulated ATP, AK or other luminescence-related compounds. The resulting luminescence can then be determined in a chemical assay. This determination can be qualitative (i.e., an absence/presence assay) or quantitative (i.e., which can measure a specific amount of analyte present). Through the use of a cocktail of probe types, the assay can also qualitatively or quantitatively measure the presence of more than one analyte simultaneously. This type of assay can be of commercial importance in clinical and forensic applications, the personal care, pharmaceutical, food and beverage markets, as well as in environmental sample assays.
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Citations
37 Claims
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1. A method for detecting an analyte comprising the steps of:
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a) obtaining a sample potentially comprising an analyte; b) providing liposomes comprising a luminescence-related amplificant encapsulated within said liposomes, a buffer and paramagnetic beads; c) incubating said sample potentially comprising an analyte, said liposomes, and said paramagnetic beads to provide a complex of said paramagnetic beads, said analyte and said liposomes; d) separating said complex from non-complexed paramagnetic beads and non-complexed liposomes; e) treating said complex with a liposome extractant to release the contents of said liposomes to form an assay sample; and f) measuring light via a luminescent means; wherein said liposomes of step b) comprise at least one reporter probe;
wherein said paramagnetic beads of step b) comprise at least one capture probe, and wherein the presence of said analyte is determined by an amount of light emitted from said assay sample. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22)
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23. A kit for the detection of analytes in a sample comprising
a) at least one buffer; -
b) liposomes, wherein said liposomes comprise an encapsulated amplificant and comprise at least one reporter probe on the surface of said liposomes; c) at least one probe; d) paramagnetic beads, wherein said paramagnetic beads comprise at least one capture probe; e) a liposome extractant; f) at least one luminescence reagent. - View Dependent Claims (24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37)
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Specification