Fluorescence Resonance Energy Transfer Assay Based on Modified Solid Surface
First Claim
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1. A method of detecting an analyte in a fluid sample, the method comprising:
- contacting a binding partner with a fluid sample, the binding partner being connected to a first surface of a solid support that also has a first member of a fluorescence resonance energy transfer (FRET) pair connected to the first surface;
forming a binding complex connected to the first surface via the binding partner and including a second member of the FRET pair, formation of the binding complex being affected by an analyte, if any, in the sample;
exposing the FRET pair to excitation light via a second surface of the solid support, with the second surface spaced from the sample;
measuring a FRET response of the FRET pair to the step of exposing by detecting emitted light received from the second surface, wherein excitation light reaches the FRET pair, and emitted light is received from the FRET pair, without passing through a substantial portion of the sample, thereby minimizing optical interference from the sample; and
correlating the FRET response with an amount of the analyte in the sample.
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Abstract
System, including methods, apparatus, and kits, for fluorescence resonance transfer (FRET) binding assays that are surface-based.
34 Citations
20 Claims
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1. A method of detecting an analyte in a fluid sample, the method comprising:
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contacting a binding partner with a fluid sample, the binding partner being connected to a first surface of a solid support that also has a first member of a fluorescence resonance energy transfer (FRET) pair connected to the first surface; forming a binding complex connected to the first surface via the binding partner and including a second member of the FRET pair, formation of the binding complex being affected by an analyte, if any, in the sample; exposing the FRET pair to excitation light via a second surface of the solid support, with the second surface spaced from the sample; measuring a FRET response of the FRET pair to the step of exposing by detecting emitted light received from the second surface, wherein excitation light reaches the FRET pair, and emitted light is received from the FRET pair, without passing through a substantial portion of the sample, thereby minimizing optical interference from the sample; and correlating the FRET response with an amount of the analyte in the sample. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16)
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17. A method of detecting an analyte in a fluid sample, the method comprising:
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adding to a fluid sample a binding partner connected to a member of a fluorescence resonance energy transfer (FRET) pair; contacting a surface of a solid support with the sample, the solid support being substantially transparent and having an other member of the FRET pair substantially immobilized with respect to the surface; forming a binding complex connected to the surface via the binding partner, formation of the binding complex being affected by an analyte, if any, in the sample; passing excitation light through the solid support towards the surface to expose the FRET pair to the excitation light; measuring a FRET response of the FRET pair to exposing by detecting emitted light that has traveled through the solid support away from the surface, wherein excitation light reaches the FRET pair, and emitted light is received from the FRET pair, without passing through a substantial portion of the sample, thereby minimizing optical interference from the sample; and correlating the FRET response with an amount of the analyte in the sample.
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18. A device for assaying an analyte in fluid samples, comprising:
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a microplate forming a plurality of wells for holding fluid samples and each having a bottom wall that is substantially transparent such that optical detection can be performed from below the bottom wall, the bottom wall including upper and lower surfaces; and a fluorescent lanthanide connected to the upper surface of each bottom wall, with about a same amount of the fluorescent lanthanide being connected to each upper surface, thereby providing a similar environment in each of the wells for surface-based fluorescence resonance energy transfer (FRET) assay of fluid samples. - View Dependent Claims (19, 20)
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Specification