초록 ▼

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 det

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.

대표청구항 ▼

1. A system for detecting a single protein molecule in a sample, the system comprising an analyzer and at least one label comprising a fluorescent moiety and a binding partner for said protein molecule, and wherein said analyzer comprises: a) an electromagnetic radiation source for stimulating said

1. A system for detecting a single protein molecule in a sample, the system comprising an analyzer and at least one label comprising a fluorescent moiety and a binding partner for said protein molecule, and wherein said analyzer comprises: a) an electromagnetic radiation source for stimulating said fluorescent moiety,b) a capillary flow cell for passing said label;c) a source of motive force of moving said label in said capillary flow cell;d) an interrogation space within said capillary flow cell for receiving electromagnetic radiation emitted from said electromagnetic radiation source, wherein the interrogation space is defined by the focused spot of a beam from the electromagnetic radiation source wherein the spot has a diameter of 1-10 microns; ande) an electromagnetic radiation detector operably connected to said interrogation space for measuring an electromagnetic characteristic of said stimulated fluorescent moiety;wherein said fluorescent moiety emits at least about 200 photons when stimulated at the excitation wavelength of the moiety and the total energy directed at the interrogation space by the electromagnetic radiation source is no more than about 15 micro Joules. 2. The system of claim 1, wherein the electromagnetic radiation source has a power output greater than 0.5 mW. 3. The system of claim 1, wherein the analyzer comprises not more than one interrogation space. 4. The system of claim 1, wherein the fluorescent moiety comprises a fluorescent molecule. 5. The system of claim 4, wherein the fluorescent molecule comprises a dye molecule. 6. The system of claim 5, wherein the 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 group. 7. The system of claim 1, wherein the fluorescent moiety is a quantum dot. 8. The system of claim 1, wherein the fluorescent moiety comprises a dye selected from the group consisting of AlexaFluor 488, AlexaFluor 532, AlexaFluor 647, AlexaFluor 680 or AlexaFluor 700. 9. The system of claim 1, wherein the electromagnetic radiation source is a continuous wave electromagnetic radiation source. 10. The system of claim 9, wherein the continuous wave electromagnetic radiation source is a light-emitting diode or a continuous wave laser. 11. The system of claim 1, wherein the electromagnetic radiation source is a laser having a power output of at least about 3, 5, 10, or 20 mW. 12. The system of claim 1, wherein the source of motive force comprises a pressure source. 13. The system of claim 12, wherein the pressure source comprises a pump, a vacuum source, a centrifuge, or a combination thereof. 14. The system of claim 1, wherein a limit of detection of the protein molecule in the sample is less than about 100 femtomolar. 15. The system of claim 1, wherein the limit of detection of the protein molecule in the sample is less than about 10 femtomolar. 16. The system of claim 1, wherein the interrogation space has a volume between about 0.05 pL and about 50 pL. 17. The system of claim 1, wherein the interrogation space has a volume between about 0.1 pL and about 25 pL. 18. The system of claim 1, wherein the volume of the interrogation space is adjustable. 19. The system of claim 1, wherein the electromagnetic radiation detector is an avalanche photodiode. 20. The system of claim 1, further comprising a data analysis system that analyzes the electromagnetic characteristic and reports the results of the analysis. 21. The system of claim 1, wherein the analyzer is capable of detecting a difference of less than 20% in concentration of an analyte between a first sample and a second sample, when the first sample and the second sample are introduced into the analyzer, the volume of the first sample and the second sample introduced into the analyzer is less than 5 μl, and wherein the analyte is present at a concentration of less than 50 femtomolar in the first and second samples. 22. A system for detecting a single protein molecule in a sample, comprising: a label comprising a fluorescent moiety and a binding partner for the protein molecule and an analyzer comprising: (a) an electromagnetic radiation source for emitting electromagnetic radiation, wherein said fluorescent moiety emits at least about 200 photons when stimulated at the excitation wavelength of the moiety and the total energy directed at the interrogation space by the electromagnetic radiation source is no more than about 15 micro Joules;(b) a first interrogation space positioned to receive electromagnetic radiation emitted from the electromagnetic radiation source wherein the interrogation space is defined by the focused spot of a beam from the electromagnetic radiation source wherein the spot has a diameter of 1-10 microns;(c) a second interrogation space positioned to receive electromagnetic radiation emitted from the electromagnetic radiation source, wherein the second interrogation space is in fluid communication with the first interrogation space and wherein a motive source provides a motive force between the first interrogation space and the second interrogation space such that a particle comprising at least the label can be moved between the first interrogation space and the second interrogation space;(d) a first electromagnetic radiation detector operably connected to the first interrogation space to measure a first electromagnetic characteristic of the particle in the first interrogation space;(e) a second electromagnetic radiation detector operably connected to the second interrogation space to measure at least one of a second electromagnetic characteristic of the particle in the second interrogation space and the first electromagnetic characteristic of the particle. 23. A system for detecting a single protein molecule in a sample, comprising: (a) a sampling system providing a fluid communication between a sample container and a first interrogation space, wherein the interrogation space is defined by the focused spot of a beam from an electromagnetic radiation source wherein the spot has a diameter of 1-10 microns and the total energy directed at the interrogation space by an electromagnetic radiation source is no more than about 15 micro Joules;(b) a single particle analyzer comprising the first interrogation space and a second interrogation space wherein the second interrogation space is in fluid communication with the first interrogation space and wherein a pressure source provides a motive force between the first interrogation space and the second interrogation space such that a particle can be moved between the first interrogation space and the second interrogation space;(c) a detector operably connected to at least one of the first or the second interrogation spaces, wherein the detector is located for detecting a detectable characteristic of the particle, if present, and wherein the interrogation spaces are defined by the field of view of the detector;(d) a sample recovery system comprising a means for moving the sample whereby the sample can move from the sample container to the interrogation spaces and back to the sample container without contacting other components of the analyzer and with no substantial contact with clean buffer within the analyzer; and(e) a data analyzer that receives input from the detector, analyzes the presence or absence of the particle, and reports a result based on the presence or absence.