Membrane-Based Assay Devices that Utilize Time-Resolved Fluorescence
A membrane-based assay device for detecting the presence or quantity of an analyte residing in a test sample is provided. The device utilizes time-resolved fluorescence to detect the signals generated by excited fluorescent labels. Because the labels can have relatively long emission lifetime, short-lived background interference can be practically eliminated through delayed fluorescence detection. In addition, the resulting fluorescent reader can have a simple and inexpensive design. For instance, in one embodiment, the reader can utilize a silicon photodiode and a pulsed light-emitting diode (LED) to accurately excite labels and detect fluorescence on a membrane-based assay device without requiring the use of expensive components, such as monochromators or narrow emission band width optical filters.
1 - 42 . (canceled)
43 . A system for detecting the presence or quantity of analyte residing in a test sample, the system comprising:
a flow-through assay device, the flow-through assay device comprising a porous membrane in fluid communication with a conjugate pad, the conjugate pad including probes comprising particles modified with a specific binding member configured to bind with the analyte and containing a fluorescent label, said fluorescent label having a fluorescence emission lifetime of greater than about 1 microsecond, said porous membrane defining a detection zone within which is immobilized a capture reagent configured to bind with the analyte, said porous membrane defining a calibration zone positioned downstream from the detection zone within which is immobilized a capture reagent configured to bind with the probes;
a pulsed excitation source configured to subject the detection zone to pulses of illumination to generate a detection signal;
a time-gated detector configured to measure the intensity of the detection signal after a certain period of time has elapsed following a pulse of illumination, wherein the amount of the analyte within the test sample is proportional to the intensity of the detection signal as calibrated by a calibration signal.
44 . The system of claim 43 , the pulsed excitation source being configured to subject the calibration zone of the flow-through assay device to pulses of illumination to generate the calibration signal.
45 . The system of claim 44 , the time-gated detector being configured to measure the intensity of the calibration signal after a certain amount of time has elapsed following a pulse of illumination at the calibration zone.
46 . The system of claim 44 , the time-gated detector being configured to measure the intensity of the calibration signal after a certain amount of time has elapsed following each pulse of illumination at the calibration zone.
47 . The system of claim 44 , the pulsed excitation source being configured to simultaneously subject the detection zone and the calibration zone to pulses of illumination.
48 . The system of claim 47 , the time-gated detector being configured to simultaneously measure the intensity of the detection signal and the intensity of the calibration signal.
49 . The system of claim 43 , further comprising a second pulsed excitation source configured to subject a calibration zone of the flow-through assay device to pulses of illumination to generate the calibration signal.
50 . The system of claim 49 , further comprising a second time-gated detector configured to measure the intensity of the calibration signal after a certain amount of time has elapsed following a pulse of illumination at the calibration zone.
51 . The system of claim 50 , the second time-gated detector being configured to measure the intensity of the calibration signal after a certain amount of time has elapsed following each pulse of illumination at the calibration zone.
52 . The system of claim 43 , wherein said fluorescent label has an emission lifetime of greater than about 10 microseconds.
53 . The system of claim 43 , wherein said fluorescent label has an emission lifetime of from about 100 to about 1000 microseconds.
54 . The system of claim 43 , wherein said fluorescent label has a Stokes shift greater than about 50 nanometers.
55 . The system of claim 43 , wherein said fluorescent label has a Stokes shift of greater than about 100 nanometers.
56 . The system of claim 43 , wherein said fluorescent label has a Stokes shift of from about 250 to about 350 nanometers.
57 . The system of claim 43 , wherein said fluorescent label includes a lanthanide chelate of samarium, dysprosium, europium, terbium, or combinations thereof.
58 . The system of claim 43 , wherein said fluorescent label is europium chelate.
59 . The system of claim 43 , wherein the capture reagent of the detection zone is an antigen or antibody.
60 . The system of claim 59 , wherein the specific binding member is an antigen or antibody.
61 . The system of claim 43 , wherein the capture reagent of the calibration zone is a polyelectrolyte.
62 . The system of claim 43 , wherein the polyelectrolyte is configured to bind to the particles.
63 . The system of claim 43 , wherein the amount of particles exceeds the amount of available binding sites in the detection zone.
64 . The system of claim 43 , wherein the capture reagents of the detection zone and the calibration zone are substantially non-diffusively immobilized on the porous membrane.
65 . The system of claim 43 , wherein the particles are diffusively immobilized on the conjugate pad.
66 . The system of claim 43 , wherein the particles are latex particles.
67 . The system of claim 43 , wherein the pulsed excitation source is configured to subject multiple detection regions of the detection zone of the flow-through assay device to pulses of illumination.
68 . The system of claim 43 , wherein the pulsed excitation source is a light-emitting diode.
69 . The system of claim 43 , wherein the time-gated detector is a silicon photodiode.
70 . The system of claim 43 , further comprising an optical filter positioned adjacent to the pulsed excitation source, the time-gated detector, or combinations thereof.
71 . The system of claim 43 , further comprising a fluorescence reader, the fluorescence reader comprising timing circuitry in communication with the pulsed excitation source and the time-gated detector.
72 . The system of claim 43 , wherein the certain period of time is between about 100 and about 200 microseconds.
73 . The system of claim 43 , wherein the time-gated detector is configured to measure the detection signal after the certain period of time has elapsed following each pulse of illumination at the detection zone.