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 small volume partitions, wherein some of the small volume partitions have one microbe or one natural microbe aggregate per small volume partitions;
(d) incubating the small volume partitions for a period of time at a specified temperature or series of temperatures;
(e) monitoring optical emissions of the small volume partitions during the incubation time;
(f) constructing an optical emission waveform for each small volume partitions based on the optical emissions of the small volume partitions over time resulting in a small volume partitions waveform;
(g) identifying the microbes in the sample using the small volume partitions waveforms; and
(h) determining susceptibility of the microbes in the sample to the antimicrobial drug by comparing waveforms for small volume partitions of the treated portion to waveforms of the small volume partitions of the control portion, wherein differences in the small volume partitions waveforms between the small volume partitions of the treated portion to small volume partitions of the control portion indicates susceptibility to the antimicrobial.
2. The method of claim 1 , wherein the small volume partitions are droplets 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 2 , wherein partitioning of the sample into small volume partitions is done by Laplace pressure gradients.
5. The method of claim 1 , wherein monitoring the optical emissions of small volume partitions 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 small volume partitions further comprises exciting the reporter using a light emitting diode (LED).
9. The method of claim 1 , wherein the incubating of the small volume partitions 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 or fungi.
12. The method of claim 11 , wherein the bacteria present in small volume partitions are identified by gram-stain group based on the small volume partitions waveform.
13. The method of claim 11 , wherein the bacteria present in small volume partitions are identified by species, genus, family, or order based on the sub-nanoliter waveform.
14. The method of claim 1 , wherein the small volume partitions waveform is compared to a library of waveforms.