Method to detect/identify bacterial species using flow cytometry and surface enhanced Raman scattering

Patent metadata

Field	Value
Publication number	US-10132808-B2
Application number	US-201615274599-A
Country	US
Kind code	B2
Filing date	Sep 23, 2016
Priority date	Sep 23, 2016
Publication date	Nov 20, 2018
Grant date	Nov 20, 2018

Abstract

A method uses flow cytometry to prepare surface enhanced Raman scattering (SERS) substrates for obtaining SERS spectra of bacteria. The method involves using a flow cytometer to sort bacterial cells into populations of bacterial cells based upon their biophysical characteristics. The cells may then be washed with a borate buffer to remove any chemical species that degrade the SERS response. A colloid-coated bacteria suspension is then created by mixing one of the populations of bacterial cells with SERS-active colloidal particles. The colloid-coated bacteria suspension is incubated until the SERS-active colloidal particles partition through the capsule and bind to the cell wall for each bacterial cell in the colloid-coated bacteria suspension. The colloid-coated bacteria suspension is then disposed onto a filter and a SERS spectra of the colloid-coated bacteria suspension is obtained using a Raman spectrometer.

First claim

We claim:

1. A method comprising the steps of:
using a flow cytometer to sort bacterial cells into one or more populations of bacterial cells based upon their biophysical characteristics, the bacterial cells each comprising a capsule and a cell wall;
creating a colloid-coated bacteria suspension by mixing one of the populations of bacterial cells with SERS-active colloidal particles;
incubating the colloid-coated bacteria suspension until the SERS-active colloidal particles partition through the capsule and bind to the cell wall for each bacterial cell in the colloid-coated bacteria suspension;
disposing the colloid-coated bacteria suspension onto a filter; and
obtaining a surface enhanced Raman scattering (SERS) spectra of the colloid-coated bacteria suspension using a Raman spectrometer.

2. The method of claim 1 further comprising the step of washing each population of bacterial cells to remove any chemical species that degrade the SERS spectra.

3. The method of claim 2 , wherein each population of bacterial cells is washed with a borate buffer.

4. The method of claim 1 further comprising the step of, prior to the step of creating a colloid-coated bacteria suspension, measuring the optical density of the populations of bacterial cells to estimate the number of bacterial cells present.

5. The method of claim 1 , wherein the SERS-active colloidal particles comprise one of colloidal silver and colloidal gold.

6. The method of claim 5 , where the colloidal silver and the colloidal gold each comprise a capping agent to enable it to partition through the capsule and bind onto the cell wall.

7. The method of claim 6 , wherein the capping agent is selected from the group consisting of citrate, oleate, polyvinyl alcohol, and polyvinyl pyrrolidone.

8. The method of claim 1 , wherein the filter is a ceramic filter.

9. The method of claim 1 , wherein the step of obtaining a SERS spectra of the colloid-coated bacteria suspension using a Raman spectrometer is performed by rastering a laser over the surface of the colloid-coated bacteria suspension.

10. A method comprising the steps of:
using a flow cytometer to sort bacterial cells into one or more populations of bacterial cells based upon their biophysical characteristics, the bacterial cells each comprising a capsule and a cell wall;
washing each population of bacterial cells with a borate buffer to remove any chemical species that degrade the SERS spectra;
creating a colloid-coated bacteria suspension by mixing one of the washed populations of bacterial cells with SERS-active colloidal particles;
incubating the colloid-coated bacteria suspension until the SERS-active colloidal particles partition through the capsule and bind to the cell wall for each bacterial cell in the colloid-coated bacteria suspension;
disposing the colloid-coated bacteria suspension onto a filter; and
obtaining a surface enhanced Raman scattering (SERS) spectra of the colloid-coated bacteria suspension using a Raman spectrometer performed by rastering a laser over the surface of the colloid-coated bacteria suspension.

11. The method of claim 10 further comprising the step of, prior to the step of creating colloid-coated bacteria, measuring the optical density of the one or more populations of bacterial cells to estimate the number of bacterial cells present.

12. The method of claim 10 , wherein the SERS-active colloidal particles comprise one of colloidal silver and colloidal gold.

13. The method of claim 12 , where the colloidal silver and the colloidal gold each comprise a capping agent to enable it to partition through the capsule and bind onto the cell wall.

14. The method of claim 13 , wherein the capping agent is selected from the group consisting of citrate, oleate, polyvinyl alcohol, and polyvinyl pyrrolidone.

15. A method comprising the steps of:
using a flow cytometer to sort bacterial cells into one or more populations of bacterial cells based upon their biophysical characteristics, the bacterial cells each comprising a capsule and a cell wall;
creating a colloid-coated bacteria suspension by mixing one of the populations of bacterial cells with SERS-active colloidal particles, wherein the SERS-active colloidal particles comprise one of colloidal silver and colloidal gold, wherein the colloidal silver and the colloidal gold each comprise a capping agent to enable it to partition through the capsule and bind onto the cell wall;
incubating the colloid-coated bacteria suspension until the SERS-active colloidal particles partition through the capsule and bind to the cell wall for each bacterial cell in the colloid-coated bacteria suspension;
disposing the colloid-coated bacteria suspension onto a filter; and
obtaining a surface enhanced Raman scattering (SERS) spectra of the colloid-coated bacteria suspension using a Raman spectrometer by rastering a laser over the surface of the colloid-coated bacteria suspension.

16. The method of claim 15 further comprising the step of, prior to the step of creating colloid-coated bacteria, measuring the optical density of the one or more populations of bacterial cells to estimate the number of bacterial cells present.

17. The method of claim 16 , wherein the capping agent is selected from the group consisting of citrate, oleate, polyvinyl alcohol, and polyvinyl pyrrolidone.

18. The method of claim 15 , wherein the filter is a ceramic filter.

19. The method of claim 15 further comprising the step of washing each population of bacterial cells to remove any chemical species that degrade the SERS spectra.

20. The method of claim 19 , wherein each population of bacterial cells is washed with a borate buffer.

Assignees

• Us Navy
• Us Navy

Inventors

• Boss Pamela A
• Sorensen Kara C
• George Robert D
• Obraztsova Anna Y
• Oiler Jonathon K

Classifications

• G01N15/1456

  without spatial resolution of the texture or inner structure of the particle, e.g. processing of pulse signals · CPC title

• G01N33/56911 Primary

  Bacteria · CPC title

• G01N15/1459

  the analysis being performed on a sample stream · CPC title

• G01N2001/4088

  filtration · CPC title

• G01N21/658

  enhancement Raman, e.g. surface plasmons · CPC title