Highly sensitive system and method for analysis of troponin
The invention provides methods, compositions, kits, and systems for the sensitive detection of cardiac troponin, Such methods, compositions, kits, and systems are useful in diagnosis, prognosis, and determination of methods of treatment in conditions that involve release of cardiac troponin.
1. A method for determining the presence or absence of cardiac troponin selected from cardiac I (cTnI) or cardiac troponin T (cTnT) or complex thereof at a concentration of less than 3 pg/mL in a biological sample from a healthy human patient, comprising
i) labeling said troponin or complex thereof, if present, with a label comprising a binding partner for the troponin and a detectable moiety; and
ii) detecting troponin in the sample at concentrations of less than 3 pg/mL by determining the presence or absence of said label in a single molecule counting assay, wherein detection of the presence of said label indicates the presence of said troponin or complex in said sample, wherein said single molecule counting assay has a limit of detection of less than about 3 pg/mL with a coefficient of variation of less than about 10%.
2. The method of claim 1 wherein the troponin is cTnI.
3. The method of claim 1 wherein said single molecule counting assay has a limit of detection of less than about 1 pg/mL.
4. The method of claim 1 , wherein said label comprises a fluorescent moiety that emits at least about 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 about 5 microns in diameter that contains the moiety, and wherein the total energy directed at the spot by the laser is no more than about 3 microJoules.
5. The method of claim 1 wherein said label comprises a molecule that 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.
6. The method of claim 1 wherein said binding partner comprises an antibody specific to said troponin or complex.
7. The method of claim 1 wherein said sample is a blood, serum, or plasma sample.
8. The method of claim 1 wherein step ii) comprises passing said label through a single molecule detector comprising an interrogation space defined by the focus of a beam from an electromagnetic radiation source.
9. The method of claim 8 wherein said label is a fluorescent moiety and the single molecule detector further comprises
(a) an electromagnetic radiation detector operably connected to said interrogation space for measuring an electromagnetic characteristic of said stimulated fluorescent moiety; and
(b) a microscope objective lens situated between said interrogation space and said detector.