Compositions and methods for disease diagnosis using single cell analysis
Certain embodiments of the invention are directed to evaluating and identifying cells by recording and interpreting a time-dependent signal produced by unique cell respiration and permeability attributes of isolated viable cells.
1. A method for detecting and characterizing microbes in a sample, the method comprising:
(a) contacting a sample containing microbes with at least one reporter forming a sample mixture, wherein optical emissions of the reporter indicate microbial cell viability;
(b) dividing the sample mixture into at least two portions, wherein a first portion is treated with an antimicrobial drug forming a test portion and the second portion is a control portion;
(c) partitioning the test portion and the control portion into two-dimensional arrays of sub-nanoliter droplets, wherein a portion of the droplets have one microbe or one natural microbe aggregate per droplet;
(d) incubating the sub-nanoliter droplets for a period of time at a specified temperature or series of temperatures;
(e) monitoring optical emissions of the sub-nanoliter droplets during the incubation time;
(f) constructing an optical emission waveform for each sub-nanoliter droplet based on the optical emissions of the sub-nanoliter droplet over time resulting in a sub-nanoliter droplet waveform;
(g) identifying the microbes in the sample using the sub-nanoliter droplet waveforms; and
(h) determining susceptibility of the microbes in the sample to the antimicrobial drug by comparing waveforms for sub-nanoliter droplets of the treated portion to waveforms of the sub-nanoliter droplets of the control portion, wherein differences in the sub-nanoliter droplet waveforms between the droplets of the treated portion to droplets of the control portion indicates susceptibility to the antimicrobial.
2. The method of claim 1 , wherein the sub-nanoliter droplets are formed in an immiscible fluid.
3. The method of claim 2 , wherein the immiscible fluid is a fluorocarbon comprising a fluorosurfactant.
4. The method of claim 1 , wherein partitioning of the sample into sub-nanoliter droplets is done by Laplace pressure gradients.
5. The method of claim 1 , wherein monitoring the optical emissions of the sub-nanoliter droplets is performed using a camera.
6. The method of claim 1 , wherein the at least one reporter is a fluorescent dye.
7. The method of claim 6 , where the reporter is resazurin or tetrazolium.
8. The method of claim 6 , wherein the monitoring the optical emissions of the sub-nanoliter droplets further comprises exciting the reporter using a light emitting diode (LED).
9. The method of claim 1 , wherein the incubating of the sub-nanoliter droplets is at a constant temperature.
10. The method of claim 9 , wherein the temperature is 37° C.
11. The method of claim 1 , wherein the microbes are bacteria.
12. The method of claim 11 , wherein the bacteria present in sub-nanoliter droplets are identified by gram-stain group based on the sub-nanoliter waveform.
13. The method of claim 1 wherein the sub-nanoliter droplet waveform is compared to a library of waveforms.
14. The method of claim 11 , wherein the bacteria present in sub-nanoliter droplets are identified by species, genus, family, or order based on the sub-nanoliter waveform.