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 antimicrobial response of microbes in a sample comprising:
(a) contacting a sample comprising microbes with a reporter forming a sample mixture;
(b) dividing the sample mixture into at least two portions, wherein a first portion is treated with an antimicrobial substance forming a test portion and the second portion is a control portion;
(c) partitioning the test portion and control portion into small volumes forming small volume partitions having one microbe or a natural aggregation of microbes per small volume partition;
(d) incubating the small volume partitions over time at a specific temperature or temperatures;
(e) monitoring optical characteristics of the small volume partitions during the incubation time, wherein the optical characteristics include the amount of optical signal produced by interaction of the reporter with the microbe or environment in the small volume partition;
(f) constructing an optical signal waveform for each small volume partition resulting in a small volume partition waveform; and
(g) comparing the optical signal waveform shapes of the small volume partitions derived from the control portion to the optical signal waveform shapes of the small volume partitions derived from the test portion to determine the sensitivity of the microbes to the antimicrobial substance.
2. The method of claim 1 , wherein the small volume partition waveform is compared to a library of stored waveforms.
3. The method of claim 1 , wherein the small volume partitions are droplets in an immiscible fluid.
4. The method of claim 3 , wherein the immiscible fluid is a fluorocarbon comprising a fluorosurfactant.
5. The method of claim 1 , further comprising arranging the small volume partitions in a two-dimensional array monolayer.
6. The method of claim 5 , wherein the two-dimensional array is a static two-dimensional array.
7. The method of claim 3 , wherein partitioning the test portion and control portion is by Laplace pressure gradients.
8. The method of claim 3 , wherein partitioning the test portion and control portion is by shear stress.
9. The method of claim 1 , wherein monitoring optical characteristics of the small volume partitions is performed using a camera.
10. The method of claim 1 , wherein the reporter is a fluorescent dye.
11. The method of claim 1 , wherein the reporter is a resazurin dye.
12. The method of claim 1 , where the reporter is a tetrazolium dye.
13. The method of claim 1 , wherein the monitoring optical characteristics of the small volume partitions further comprises exciting the reporter using a light source.
14. The method of claim 13 , wherein the light source is a light emitting diode (LED).
15. The method of claim 1 , wherein the incubating of the small volume partitions is at a constant temperature.
16. The method of claim 15 , wherein the constant temperature is 37° C.