Method for highly sensitive detection of single protein molecules labeled with fluorescent moieties
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 method for determining the presence or absence of a single molecule of a protein in a biological sample, comprising:
a) labeling the molecule with a label, wherein the label comprises a fluorescent moiety that has low photobleaching and that is capable of emitting at least about 200 photons when stimulated by a laser emitting light at the excitation wavelength of the moiety;
b) detecting the presence or absence of the label in a single molecule detector comprising a laser, wherein the label is detected by focusing the laser on a spot not less than about 5 microns in diameter that contains the moiety and directing no more than about 5 microJoules of total energy at the spot, passing a portion of the sample through an interrogation space comprising the spot, and detecting electromagnetic radiation emitted by the fluorescent moiety, wherein the electromagnetic radiation emitted by the fluorescent moiety is such that it allows detection of a labeled molecule at a limit of detection of less than about 100 femtomolar.
2. The method of claim 1 wherein the electromagnetic radiation emitted by the fluorescent moiety is such that it allows detection of a labeled molecule at a limit of detection of less than about 10 femtomolar.
3. The method of claim 2 wherein the limit of detection is less than about 1 femtomolar.
4. The method of claim 1 wherein said single molecule detector comprises not more than one interrogation space.
5. The method of claim 1 , wherein said laser stimulates said moiety at a power output of less than about 20 mW.
6. The method of claim 5 , wherein said laser stimulates said moiety for a duration of less than about 1000 microseconds.
7. The method of claim 1 wherein said label further comprises a binding partner specific for binding said molecule.
8. The method of claim 7 wherein said binding partner is an antibody.
9. The method of claim 1 , wherein said fluorescent moiety comprises a fluorescent dye molecule.
10. The method of claim 9 , wherein said dye molecule comprises at least one substituted indolium ring system in which the substituent on the 3-carbon of the indolium ring contains a chemically reactive group or a conjugated substance.
11. The method of claim 10 wherein said dye molecule is an AlexaFluor molecule selected from the group consisting of AlexaFluor 488, AlexaFluor 532, AlexaFluor 647, AlexaFluor 680 or AlexaFluor 700.
12. The method of claim 9 , wherein said dye molecule is an AlexaFluor 647 dye molecule.
13. The method of claim 9 , wherein said fluorescent moiety comprises a plurality of AlexaFluor 647 molecules.
14. The method of claim 13 , wherein said plurality of AlexaFluor 647 molecules comprises about 2-4 AlexaFluor 647 molecules.
15. The method of claim 1 , wherein said fluorescent moiety is a quantum dot.
16. The method of claim 1 further comprising measuring the concentration of said protein in the sample.
17. The method of claim 1 , further comprising determining a background electromagnetic radiation level, wherein said background level represents the average electromagnetic radiation of the interrogation space when it is subjected to electromagnetic radiation in the same manner as a label, but without label in the interrogation space.
18. The method of claim 17 , further comprising comparing the amount of electromagnetic radiation detected in the sample to a threshold level, wherein said threshold level is a function of said background electromagnetic radiation level, wherein an amount of electromagnetic radiation detected in the sample greater that the threshold level indicates the presence of said label, and an amount of electromagnetic radiation detected in the sample equal to or less than the threshold level indicates the absence of said label.
19. The method of claim 1 wherein no more than about 3 microJoules of total energy are directed at the spot.
20. A method for determining the presence or absence of a biological state in a subject, comprising:
a) performing an immunoassay on a sample from said subject, wherein said immunoassay comprises contacting a plurality of labels with said sample, wherein said label is specific to a marker known to be indicative of the presence or absence of said biological state and wherein one label binds to one molecule of marker, and wherein said label comprises an antibody attached to a fluorescent moiety that has low photobleaching and that is capable of emitting at least about 200 photons when stimulated by a laser emitting light at the excitation wavelength of the moiety;
b) detecting said labels wherein said detecting comprises detecting single labels with a single molecule detector comprising a laser, wherein the label is detected by focusing the laser on a spot not less than about 5 microns in diameter that contains the moiety if present and directing no more than about 5 microJoules of total energy at the spot, passing a portion of said sample through an interrogation space comprising the spot, and detecting electromagnetic radiation emitted by the fluorescent moiety, wherein the electromagnetic radiation emitted by the fluorescent moiety is such that it allows detection of a labeled molecule at a limit of detection of less than about 100 femtomolar; and
c) determining the presence or absence of the marker.
21. The method of claim 20 further comprising determining a concentration for said marker in said sample based on the number of labels detected in step b) and comparing said concentration to a concentration or range of concentrations for said marker that are known to be indicative of the presence or absence of said biological state.
22. The method of claim 20 wherein no more than about 3 microJoules of total energy are directed at the spot.