Methods and compositions for highly sensitive detection of molecules
Disclosed are methods, kits, and compositions for the highly sensitive detection of molecules. The methods, kits, and compositions are useful in determining concentrations of molecules in samples to levels of 1 femtomolar, 1 attomolar, or lower. The methods, kits, and compositions also allow the determination of concentration over a wide range, e.g., 7-log range, without need for sample dilution.
1. A system comprising:
(a) an electromagnetic radiation source that emits electromagnetic radiation that defines an interrogation space comprising a focused spot of a beam from the electromagnetic radiation source;
(b) a capillary flow cell for passing a sample through the interrogation space;
(c) an electromagnetic radiation detector operatively connected to the interrogation space that detects photons emitted from the interrogation space during each of a plurality of bins comprising bin times; and
(d) a processor operatively connected to the detector and configured to determine the presence or amount of an analyte in a sample by determining a threshold photon value corresponding to a background signal in the interrogation space, and determining the presence of a photon emitting species comprising or corresponding to the analyte in the interrogation space in each bin of the plurality of bins by identifying bins having a photon value greater than the threshold value.
2. The system of claim 1 , wherein the interrogation space has a volume between about 0.05 pL and about 50 pL.
3. The system of claim 1 , wherein the interrogation space has a volume between about 0.1 pL and about 25 pL.
4. The system of claim 1 , wherein the interrogation space has a volume between about 0.2 pL to about 2 pL.
5. The system of claim 1 , wherein the species comprises a label that emits photons when stimulated by electromagnetic energy, the label comprising a binding partner specific for the analyte.
6. The system of claim 5 , further comprising a confocal optical arrangement for deflecting a laser beam onto said interrogation space and for imaging said stimulated dye molecule, wherein said confocal optical arrangement comprises an objective lens having a numerical aperture of at least 0.8.
7. The system of claim 5 , wherein the label further comprises a fluorescent moiety.
8. The system of claim 7 , wherein the fluorescent moiety is low photobleaching.
9. The system of claim 1 , wherein the bin times have a duration of about 1 to about 1000 microseconds.
10. The system of claim 1 , wherein the bin times have a duration of about 100 to about 2000 microseconds.
11. The system of claim 1 , wherein the bin times have a duration of about 10 to about 100 microseconds.
12. The system of claim 1 , wherein the electromagnetic radiation source has a power output of less than about 20 mW.
13. The system of claim 1 , wherein the electromagnetic radiation source is a continuous wave electromagnetic radiation source.
14. The system of claim 13 , wherein the continuous wave electromagnetic radiation source is a light-emitting diode or a continuous wave laser.
15. The system of claim 1 , wherein the electromagnetic radiation source is a laser having a power output of at least about 3 mW.
16. The system of claim 1 , further comprising an attenuator operatively connected between the interrogation space and the detector and configured to receive electromagnetic radiation emitted from the interrogation space.
17. The system of claim 16 , wherein the processor is further configured to output instructions to the attenuator to attenuate the electromagnetic radiation from the interrogation space.
18. The system of claim 16 , wherein the attenuator is configured to attenuate the electromagnetic radiation when a number of photons detected in one or more bins exceeds a saturation threshold.
19. The system of claim 1 , wherein the processor is further configured to determine the presence or amount of the analyte in a sample by measuring a total number of photons per bin.
20. The system of claim 1 , wherein the total energy received by the interrogation from the electromagnetic radiation source during each bin is 1-10 microJoules.
21. The system of claim 1 , further comprising a pressure source for passing the sample through the capillary flow cell.
22. The system of claim 21 , wherein pressure source passes the sample through the flow cell at a rate of 1 to 20 microliters per minute.
23. The system of claim 21 , wherein the pressure source passes the species through the interrogation space so that the species is exposed to the electromagnetic radiation for 10 to 500 microseconds.
24. The system of claim 1 , wherein the electromagnetic radiation source stimulates the species for a duration of less than 1000 microseconds.
25. The system of claim 1 , wherein the bin time is longer than the time the species passes through the interrogation space.
26. The system of claim 1 , wherein the photon emitting species comprises a fluorescent moiety that is capable of emitting at least 200 photons when simulated by a laser emitting light at the excitation wavelength of the moiety, wherein the laser is focused on a spot not less than 5 microns in diameter that contains the moiety, and wherein the total energy directed at the spot by the laser is no more than 3 microJoules.
27. The system of claim 1 , wherein the interrogation space has a diameter of 1-10 microns.
28. The system of claim 1 , wherein the interrogation space has a diameter of 1-5 microns.
29. The system of claim 1 , wherein the threshold photon value is a function of the background photon level.
30. The system of claim 29 , wherein the threshold photon value is a fixed number of standard deviations above the background photon level.
31. The system of claim 1 , wherein the processor determines detection events representing photon bin counts above a threshold photon value as a single molecule so that each bin is analyzed as a “yes” or “no” for the presence of the molecule.
32. The system of claim 1 , wherein the cross sectional area of the interrogation space is smaller than the cross sectional area of the capillary flow cell.