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 for detecting a single molecule in a sample, comprising:
(a) an electromagnetic radiation source;
(b) a system for directing the electromagnetic radiation from the electromagnetic radiation source to define an interrogation space in a sample container for retaining a sample;
(c) a translating system for moving the interrogation space through at least a portion of a sample when a sample is retained in the container; and
(d) a detector for detecting electromagnetic radiation emitted from a single molecule in the interrogation space in a sample if the molecule is present in the interrogation space.
2. The analyzer of claim 1 wherein the translating system can translate the interrogation space in one or more of a linear path and a non-linear path.
3. The analyzer of claim 2 wherein the non-linear path comprises a substantially circular path.
4. The analyzer of claim 2 wherein the non-linear path comprises a helical path.
5. The analyzer of claim 2 wherein the non-linear path comprises a raster pattern.
6. The analyzer of claim 1 , the sample container is a surface that confines at least one sample on the surface and the translating system translates the interrogation space through a sample on the surface.
7. The analyzer of claim 1 wherein the sample container is well.
8. The analyzer of claim 7 wherein the well is associated with a microtiter plate.
9. The analyzer of claim 1 wherein the interrogation space is of a volume between about 15 μm 3 and about 11000 μm 3 .
10. The analyzer of claim 1 wherein the interrogation space is of a volume between about 200 μm 3 and about 3000 μm 3 .
11. The analyzer of claim 1 wherein the interrogation space is of a volume between about 500 μm 3 and about 600 μm 3 .
12. The analyzer of claim 1 wherein the molecule is attached to the surface of the container.
13. The analyzer of claim 12 wherein a noncovalent bond attaches the molecule to the surface of the container.
14. The analyzer of claim 13 wherein the noncovalent bonds are formed between the molecule and one or more antibodies covalently or non-covalently bound to the surface of the container.
15. 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.
16. 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.
17. The analyzer of claim 1 wherein the translating system is constructed and arranged to translate the interrogation space through the portion of sample more than one time.
18. The analyzer of claim 17 wherein the translating system is constructed and arranged to translate through a same portion of sample a first time and a second time at a sufficiently slow speed as to allow a molecule of interest, if present, detected the first time the interrogation space is translated through the portion of sample to substantially diffuse out of the portion of sample after the first time the portion of sample is interrogated by the interrogation space and to further allow a subsequent molecule of interest, if present, to substantially diffuse into the portion of sample the second time the portion of sample is interrogated by the interrogation space.
19. The analyzer of claim 18 wherein the translating system is constructed and arranged to translate the interrogation space in a substantially circular pattern, wherein the system is capable of translating the interrogation space at a speed of between about 100 and about 1000 RPM.
20. The analyzer of claim 17 wherein the translating system is constructed and arranged to translate the interrogation space such that the detection spot returns to the portion of sample after sufficient time has passed so that the molecule detected in the first pass can diffuse out of the portion, and other molecules can diffuse into the portion.
21. The analyzer of claim 1 wherein the analyzer is adapted and configured to sequentially detect the presence or absence of a single molecule of a particular type in a first sample, and detect the presence or absence of a single molecule of the type in a second sample, wherein there is no carryover between the first and the second sample.
22. The analyzer of claim 1 , wherein the analyzer determines a background photon level in the interrogation space in the absence of the single molecule and the apparatus compares the background photon level to amount of photons detected from the single molecule.
23. The analyzer of claim 22 , wherein single molecules are detected by a photon bin count greater than a threshold level.
24. The analyzer of claim 22 , wherein a value is assigned to the background photon level that is calculated as the average background signal detected in a plurality of bins.
25. The apparatus of claim 22 , wherein the background photon level comprises intrinsic fluorescence of non-labeled particles present in the sample, Raman scattering or electronic noise.
26. The analyzer of claim 1 , wherein the electromagnetic radiation source emits between about 1 to about 15 microJoules of electromagnetic radiation.
27. 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.
28. The analyzer of claim 1 , wherein the total energy defining the interrogation space is between about 100 microWatts to about 150 milliWatts.
29. 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 a sample by detecting the electromagnetic radiation emitted from the single type of label.
30. The analyzer of claim 1 , wherein the sample container is stationary.
31. The analyzer of claim 1 , wherein the system for moving the interrogation space moves the sample container.