Membrane-based lateral flow assay devices that utilize phosphorescent detection
View Patent ↗A lateral flow, membrane-based assay device for detecting the presence or quantity of an analyte residing in a test sample is provided. The device utilizes phosphorescence to detect the signals generated by excited phosphorescent labels. The labels may have a long emission lifetime so that background interference from many sources, such as scattered light and autofluorescence, is practically eliminated during detection. In addition, the phosphorescent labels may be encapsulated within particles to shield the labels from quenchers, such as oxygen or water, which might disrupt the phosphorescent signal.
1. A method for detecting an analyte within a test sample, the method comprising:
i) providing a lateral flow assay device that comprises a porous membrane in fluid communication with detection probes, the detection probes comprising a phosphorescent particle, the phosphorescent particle comprising a phosphorescent metal complex encapsulated within a barrier matrix comprising polymethylmethacrylate, the detection probes being conjugated with a specific binding member configured to bind with the analyte and form an analyte complex, wherein the porous membrane defines a conjugate pad, the conjugate pad including the detection probes; a detection zone positioned downstream of the conjugate pad within which is immobilized a capture reagent that is configured to bind to the analyte complex to generate a detection signal; and a calibration zone positioned downstream of the detection zone within which a capture reagent is immobilized, the capture reagent being capable of binding to uncaptured detection probes to generate a calibration signal;
ii) contacting the lateral flow assay device with the test sample;
iii) subjecting the detection zone to illumination pulses to generate the detection signal;
iv) subjecting the calibration zone to illumination pulses to generate the calibration signal; and
v) thereafter, measuring the intensity of the detection signal and the calibration signal, wherein the amount of the analyte within the test sample is proportional to a ratio of the intensity of the detection signal to the intensity of the calibration signal.
2. A method as in claim 1 , wherein the metal complex comprises a metal selected from the group consisting of ruthenium, osmium, rhenium, iridium, rhodium, platinum, indium, palladium, molybdenum, technetium, copper, iron, chromium, tungsten, zinc, and combinations thereof.
3. A method as in claim 1 , wherein the metal complex comprises a ligand selected from the group consisting of pyridine, pyrazine, isonicotinamide, imidazole, bipyridine, terpyridine, phenanthroline, dipyridophenazine, porphyrin, porphine, derivatives thereof, and combinations thereof.
4. A method as in claim 1 , wherein the metal complex comprises a porphine complex.
5. A method as in claim 1 , wherein the metal complex is selected from the group consisting of platinum(II) tetra-meso-fluorophenylporphine and palladium(II) tetra-meso-fluorophenylporphine.
6. A method as in claim 1 , wherein the particle comprises a nanoparticle.
7. A method as in claim 6 , wherein the nanoparticle has a diameter of from about 1 nanometer to about 10 microns.
8. A method as in claim 1 , wherein the barrier matrix shields the encapsulated phosphorescent label from phosphorescence quenchers.
9. A method as in claim 1 , wherein the specific binding member is selected from the group consisting of antigens, haptens, aptamers, primary or secondary antibodies, biotin, and combinations thereof.
10. The method as in claim 1 , wherein the metal complex comprises platinum(II) tetra-meso-fluorophenylporphine.
11. The method as in claim 1 , wherein the metal complex comprises palladium(II) tetra-meso-fluorophenylporphine.
12. The method as in claim 1 , wherein the phosphorescent metal complex comprises a phosphorescent label that has a phosphorescent lifetime of greater than about 1 microsecond.
13. The method as in claim 1 , wherein the phosphorescent metal complex comprises a phosphorescent label that has a Stokes shift of greater than about 100 nm.
14. The method as in claim 1 , further comprising a sampling pad located upstream of the conjugate pad.
15. The method as in claim 1 , wherein the capture reagent in the calibration zone is a polyelectrolyte capture reagent.
16. The method as in claim 15 , wherein the polyelectrolyte capture reagent is a non-biological reagent.
17. The method as in claim 1 , wherein the detection zone and the calibration zone are simultaneously subjected to one or more pulses of light.
18. The method as in claim 1 , wherein the intensity of the detection signal and the intensity of the calibration signal are measured simultaneously.
19. The method as in claim 1 , wherein the intensity of the detection signal is measured after about 1 to about 100 microseconds.