Digital molecular assays

Abstract

Provided herein are systems, devices and methods for the rapid and accurate measurement of analytes by assay of binding events, by direct, digital measurement of individually resolved analyte/reporter binding events. The digital molecular assay systems, devices and methods disclosed herein are capable of particle-by-particle readout using optical reporter molecules that detect and report the binding of a single analyte molecule, and report each such binding in binary format. Such digital molecular assay systems, devices and methods are useful in a variety of applications, such as on mobile electronic devices for use in the field.

1 - 43 . (canceled) 
     
     
         44 . A digital assay system for determining a concentration of analyte in a sample, comprising:
 a microprocessor;   memory;   image analysis software stored in the memory and comprising instructions being executable by the microprocessor to perform a method, the method comprising:
 analyzing an image of a plurality of signals emitted by at least one type of optical reporter molecules incubated with at least one type of analyte molecules, and 
 digitally classifying the plurality of signals as active or null; and 
   optionally, a communication interface.   
     
     
         45 - 65 . (canceled) 
     
     
         66 . The digital assay system of  claim 44 , wherein the image of a plurality of signals is sent to a virtual server in a computer cloud. 
     
     
         67 . The digital assay system of  claim 44 , wherein each of the optical reporter molecules is spatially resolvable. 
     
     
         68 . The digital assay system of  claim 44 , wherein the optical reporter molecules comprise plasmonic nanoparticles functionalized with a capture element. 
     
     
         69 . The digital assay system of  claim 68 , wherein the capture element is chosen from:
 one or more nucleotide sequences that binds the analyte; and   an antibody or a fragment thereof that binds the analyte.   
     
     
         70 . The digital assay system of  claim 69 , wherein the analyte is chosen from an antigen and a nucleotide sequence. 
     
     
         71 . The digital assay system of  claim 70 , wherein the capture element is chosen from an antibody, or a fragment thereof, and one or more nucleotide sequences complimentary to the analyte nucleotide sequence. 
     
     
         72 . The digital assay system of  claim 66 , comprising a communication interface. 
     
     
         73 . The digital assay system of  claim 72 , wherein the communication interface is wireless. 
     
     
         74 . The digital assay system of  claim 73 , further comprising an image sensor. 
     
     
         75 . The digital assay system of  claim 74 , wherein the image sensor is configured to operating as part of a dark-field microscope. 
     
     
         76 . The digital assay system of  claim 74 , wherein the image sensor is chosen from a megapixel camera and a complementary metal-oxide semiconductor camera. 
     
     
         77 . The digital assay system of  claim 74 , further comprising a source of light or other electromagnetic radiation. 
     
     
         78 . The digital assay system of  claim 77 , wherein the source of light or other electromagnetic radiation is a light-emitting diode. 
     
     
         79 . The digital assay system of  claim 78 , further comprising a sample chamber that is optionally removable. 
     
     
         80 . The digital assay system of  claim 79 , additionally comprising:
 a reporter surface made of glass or polymer, to one side of which optical reporter molecules comprising plasmonic nanoparticles functionalized with capture elements have been affixed, optionally in a grid or an approximation thereof; and   a waveguide that is suitable for dark-field microscopy in contact with the opposite side of the reporter surface.   
     
     
         81 . The digital assay system of  claim 80 , wherein each affixed optical reporter molecule is resolvable as one pixel of a recording device. 
     
     
         82 . The digital assay system of  claim 81 , wherein the microprocessor, memory, image sensor, software, screen capable of displaying an image, and communication interface are all comprised within a single, portable device. 
     
     
         83 . The digital assay system of  claim 82 , wherein the single, portable device is chosen from a smartphone and a tablet computer. 
     
     
         84 . The digital assay system of  claim 83 , additionally comprising a case for positioning the portable device, sample chamber, and light source in close and stable proximity to each other.