Methods and Compositions for Highly Sensitive Detection of Molecules

Abstract

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 . An analyzer system kit for detecting a single protein molecule in a sample, said kit 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 comprising:
 a) an electromagnetic radiation source for stimulating said fluorescent a fluorescent moiety;   b) a capillary flow cell for passing said label;   c) a source of motive force for moving said label in said capillary flow cell;   d) an interrogation space defined within said capillary flow cell for receiving electromagnetic radiation emitted from said electromagnetic source; and   e) an electromagnetic radiation detector operably connected to said interrogation space for measuring an electromagnetic characteristic of said stimulated a stimulated fluorescent moiety;   wherein said fluorescent moiety is capable of emitting 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   a processor operatively connected to the detector, wherein the processor is configured to execute instructions stored on a non-transitory computer-readable medium, and wherein the instructions, when executed by the processor, cause the processor to:
 determine a threshold photon value corresponding to a background signal in the interrogation space, 
 determine the presence of a fluorescent moiety in the interrogation space in each of a plurality of bins by identifying bins having a photon value greater than the threshold value, and 
 compare the number of bins having a photon value greater than the threshold value to a standard curve. 
   
     
     
         2 . The analyzer system of  claim 1 , wherein said electromagnetic radiation source is a laser, and wherein said laser has a power output of at least about 3, 5, 10, or 20 mW. 
     
     
         3 . The analyzer system of  claim 1 , wherein said fluorescent moiety comprises a fluorescent molecule. 
     
     
         4 - 13 . (canceled) 
     
     
         14 . A method for determining the presence or absence of a single molecule of a protein in a biological sample, comprising labeling said molecule with a label and detecting the presence or absence of said label in a single molecule detector, wherein said label comprises a fluorescent moiety that is capable of emitting 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. 
     
     
         15 . The method of  claim 14  wherein the limit of detection of said single molecule in said sample is less than about 10, 1, 0.1, 0.01, or 0.001 femtomolar. 
     
     
         16 . The method of  claim 14  wherein the limit of detection is less than about 1 femtomolar. 
     
     
         17 . The method of  claim 14  wherein said detecting comprises detecting electromagnetic radiation emitted by said fluorescent moiety. 
     
     
         18 . The method of  claim 14  wherein said single molecule detector comprises not more than one interrogation space. 
     
     
         19 . The method of  claim 18  further comprising exposing said fluorescent moiety to electromagnetic radiation, wherein said electromagnetic radiation is provided by a laser. 
     
     
         20 - 31 . (canceled) 
     
     
         32 . The method of  claim 14  wherein said detecting the presence or absence of said label comprises:
 (i) passing a portion of said sample through an interrogation space; and 
 (ii) subjecting said interrogation space to exposure to electromagnetic radiation, said electromagnetic radiation being sufficient to stimulate said fluorescent moiety to emit photons, if said label is present; and 
 (iii) detecting photons emitted during said exposure of step (ii). 
 
     
     
         33 . The method of  claim 32 , further comprising determining a background photon level in said interrogation space, wherein said background level represents the average photon emission of the interrogation space when it is subjected to electromagnetic radiation in the same manner as in step (ii), but without label in the interrogation space. 
     
     
         34 . The method of  claim 33  further comprising comparing the amount of photons detected in step (iii) to a threshold photon level, wherein said threshold photon level is a function of said background photon level, wherein an amount of photons detected in step (iii) greater that the threshold level indicates the presence of said label, and an amount of photons detected in step (iii) equal to or less than the threshold level indicates the absence of said label. 
     
     
         35 - 49 . (canceled)