Scanning analyzer for single molecule detection and methods of use
The invention encompasses analyzers and analyzer systems that include a single molecule analyzer, methods of using the analyzer and analyzer systems to analyze samples, either for single molecules or for molecular complexes. The single molecule uses electromagnetic radiation that is translated through the sample to detect the presence or absence of a single molecule. The single molecule analyzer provided herein is useful for diagnostics because the analyzer detects single molecules with zero carryover between samples.
1. An analyzer comprising:
(a) an electromagnetic radiation source;
(b) a system that directs the electromagnetic radiation to an interrogation space in a sample;
(b) a system for moving the interrogation space through at least a portion of the sample; and
(d) a detector for detecting electromagnetic radiation emitted from a single molecule in the interrogation space when a single molecule is present in the interrogation space.
2. The analyzer of claim 1 , wherein the electromagnetic radiation source emits between about 1-15 microJoules of electromagnetic radiation.
3. The analyzer of claim 1 , wherein the electromagnetic radiation source directs electromagnetic radiation at the interrogation space for a time period of from 100 microseconds to 10 millisecond.
4. The analyzer of claim 1 , wherein the total energy directed at the interrogation space is between about 100 microWatts to about 150 milliWatts.
5. The analyzer of claim 1 , wherein the single molecule comprises a single type of label and analyzer determines the presence or amount of an analyte in the sample by detecting the electromagnetic radiation emitted from the single type of label.
6. The analyzer of claim 1 , wherein the detector determines a background photon level in the interrogation space in the absence of a single molecule and the analyzer compares the background photon level to an amount of photons detected from the interrogation space when a single molecule comprising a single type of label is present.
7. The analyzer of claim 1 , wherein the sample is contained in a stationary sample container.
8. The analyzer of claim 1 , wherein the system for moving the interrogation space moves a sample container containing the sample.
9. The analyzer of claim 1 , further comprising a microscope objective wherein a depth of field of the microscope objective and a diameter of an aperture imaged to the microscope objective together define the interrogation space.
10. The analyzer of claim 1 , further comprising a microscope objective wherein a depth of field of the microscope objective and a lateral extent of an electromagnetic radiation beam together define the interrogation space.
11. The analyzer of claim 1 , wherein the analyzer discriminates signal from a single type of label and background noise.
12. The analyzer of claim 11 , wherein the background noise comprises intrinsic fluorescence of non-labelled particles present in the sample, Raman scattering or electronic noise.
13. The analyzer of claim 1 , wherein the interrogation space is of a volume between about 15 μm 3 and about 11000 μm 3 .
14. The analyzer of claim 1 , wherein the interrogation space is of a volume between about 200 μm 3 and about 3000 μm 3 .
15. The analyzer of claim 1 , wherein the interrogation space is of a volume between about 500 μm 3 and about 600 μm 3 .
16. The analyzer of claim 1 , wherein the system for moving the interrogation space is configured so that the sample remains substantially stationary relative to the electromagnetic radiation directed to the interrogation space.
17. The analyzer of claim 1 , wherein the system for moving the interrogation space is configured so that the interrogation space is moved through the first position of sample more than one time.
18. The analyzer of claim 1 , wherein the system for moving the interrogation space is configured to move the interrogation space through the sample at a sufficiently slow speed as to allow a single molecule of interest, if present, to substantially diffuse out of the first position after a first time the single molecule is detected and to further allow a subsequent single molecule of interest, if present, to substantially diffuse into the first position before the subsequent single molecule of interest is detected in the interrogation space.
19. The analyzer of claim 1 , wherein the system for moving the interrogation space is configured to move the interrogation space such that the interrogation space returns to the first position of sample after sufficient time has passed so that a single molecule detected in the first position can diffuse out of the first position, and another molecule can diffuse into the first position.
20. The analyzer of claim 1 , wherein the analyzer is configured to sequentially detect the presence or absence of a single molecule of a particular type in the sample, and detect the presence or absence of a single molecule of the same type in a second sample, wherein there is no carryover between the first and the second sample.
21. A method for detecting an analyte, the method comprising:
(a) specifically binding an analyte analog comprising a label or a first binding partner for the analyte comprising a label on a solid phase as a result of the presence or absence of the analyte in a sample;
(b) removing unbound analyte analog or binding partner from the sample;
(c) eluting at least the label from the solid phase;
(d) directing electromagnetic radiation from an electromagnetic radiation source to an interrogation space in a first position in the sample;
(e) detecting the presence or absence of a single molecule of the label in the interrogation space in the first position;
(f) moving the interrogation space to a subsequent position in the sample,
(g) directing electromagnetic radiation from the electromagnetic radiation source to the interrogation space in the subsequent position;
(h) detecting the presence or absence of a subsequent single molecule of the label in the interrogation space in the subsequent position;
(i) determining the presence or amount of the analyte in the sample as a result of the presence of single molecules in the interrogation space.
22. The method of claim 21 , further comprising repeating steps (f)-(h).
23. The method of claim 21 , wherein the solid phase comprises a second binding partner for the analyte.
24. The method of claim 21 , wherein the solid phase comprises an analyte analog.
25. The method of claim 21 , wherein the electromagnetic radiation source emits between about 1-15 microJoules of electromagnetic radiation.
26. The method of claim 21 , wherein the electromagnetic radiation source directs electromagnetic radiation at the interrogation space for a time period of from 100 microseconds to 10 millisecond.
27. The method of claim 21 , wherein the total energy directed at the interrogation space is between about 100 micro Watts to about 150 milliWatts.
28. The method of claim 21 , wherein the label is a single type of label.
29. The method of claim 21 , wherein the detector determines a background photon level in the interrogation space in the absence of the single molecule of the label and the analyzer compares the background photon level to amount of photons detected from a single type of label.
30. The method of claim 21 , wherein the interrogation space is of a volume between about 15 μm 3 and about 11000 μm 3 .
31. The method of claim 21 , wherein the interrogation space is of a volume between about 200 1 μm 3 and about 3000 μm 3 .
32. The method of claim 21 , wherein the interrogation space is of a volume between about 500 μm 3 and about 600 μm 3 .
33. The method of claim 21 , further comprising discriminating signal and background noise, wherein the signal is detected from a single type of label.
34. The method of claim 33 , wherein the background noise comprises intrinsic fluorescence of non-labelled particles present in the sample, Raman scattering or electronic noise.
35. The method of claim 21 , wherein the sample remains substantially stationary relative to the electromagnetic radiation directed to the interrogation space.
36. The method of claim 21 , wherein the interrogation space is moved through the first position of sample more than one time.
37. The method of claim 36 , wherein the interrogation space is moved through the sample at a sufficiently slow speed as to allow a single molecule of interest, if present, to substantially diffuse out of the first position after a first time the single molecule is detected and to further allow a subsequent single molecule of interest, if present, to substantially diffuse into the first position before the subsequent single molecule of interest is detected in the interrogation space.
38. The method of claim 36 , wherein the interrogation space is moved such that the interrogation space returns to the first position of sample after sufficient time has passed so that a single molecule detected in the first position can diffuse out of the first position, and another molecule can diffuse into the first position.
39. The method of claim 21 , further comprising the steps of sequentially detecting the presence or absence of a single molecule of a particular type in the sample, then detecting the presence or absence of a single molecule of the same type in a second sample, wherein there is no carryover between the first and the second sample.