Method to detect/identify bacterial species using flow cytometry and surface enhanced Raman scattering
Inventors
Boss, Pamela A. • SORENSEN, KARA C. • George, Robert D. • OBRAZTSOVA, ANNA Y. • OILER, JONATHON K.
Assignees
Publication Number
US-10132808-B2
Publication Date
2018-11-20
Expiration Date
2036-09-23
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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.
Core Innovation
The invention provides a method that uses flow cytometry combined with surface enhanced Raman scattering (SERS) to detect and identify bacterial species. The method involves sorting bacterial cells into populations based on their biophysical characteristics using a flow cytometer, washing the cells with a borate buffer to remove chemical species that degrade the SERS response, mixing the sorted cells with SERS-active colloidal particles, incubating this mixture to allow the particles to partition through the bacterial capsule and bind to the cell wall, and then depositing the mixture onto a filter to obtain SERS spectra of the bacterial sample using a Raman spectrometer.
The problem being solved is the need for a simpler and quicker method for detecting and identifying bacterial pathogens. Current conventional methods require ex situ laboratory analysis involving culturing and reagents, taking hours or days to complete. Prior methods for obtaining SERS spectra are complicated, involve multiple time-consuming steps, and require expensive Raman microscopes due to uneven sample distribution on SERS substrates.
The disclosed method simplifies the preparation of SERS substrates by ensuring homogeneous bacteria distribution on a ceramic filter substrate, thus enabling the use of portable and inexpensive Raman instrumentation to obtain bacterial SERS spectra. Additionally, the combination of flow cytometry for isolating and pre-concentrating bacterial populations and SERS for tag-less bacterial identification based on unique spectral signatures addresses the limitations of prior techniques regarding specificity, complexity, and detection speed.
Claims Coverage
The patent includes three independent claims covering distinct aspects of the method to identify bacterial species using flow cytometry and SERS. There are six main inventive features extracted from these claims.
Sorting bacterial cells by biophysical characteristics using flow cytometry
Using a flow cytometer to sort bacterial cells into one or more populations based upon their biophysical characteristics, where each bacterial cell comprises a capsule and a cell wall.
Preparation of colloid-coated bacteria suspension and incubation
Creating a colloid-coated bacteria suspension by mixing a population of bacterial cells with SERS-active colloidal particles and incubating this suspension until the particles partition through the bacterial capsule and bind to the cell wall of each bacterial cell.
Deploying the colloid-coated suspension onto a filter
Disposing the colloid-coated bacteria suspension onto a filter, wherein the filter may be a ceramic filter, such as an aluminum oxide membrane disc with an annular polypropylene ring.
Obtaining SERS spectra with laser rastering
Obtaining surface enhanced Raman scattering spectra of the colloid-coated bacteria suspension using a Raman spectrometer, with the spectra acquisition performed by rastering a laser over the surface of the bacterial sample on the filter.
Removal of chemical species that degrade SERS using washing
Washing each bacterial population, for example using a borate buffer, to remove chemical species that degrade the SERS spectra prior to mixing with colloidal particles.
Usage of colloidal silver or gold with capping agents
Use of SERS-active colloidal particles comprising colloidal silver or colloidal gold, each including a capping agent such as citrate, oleate, polyvinyl alcohol, or polyvinyl pyrrolidone, to enable partitioning through the bacterial capsule and binding onto the cell wall.
The claims collectively cover a method that combines flow cytometric sorting of bacteria, washing to enhance SERS response, preparation of a colloid-coated bacterial suspension with specific colloidal particles, deposition onto a specialized filter substrate, and SERS acquisition by laser rastering, enabling rapid and reproducible bacterial species identification.
Stated Advantages
Provides a simpler and quicker method for detecting and identifying bacteria compared to conventional culturing and reagent-based approaches.
Enables homogeneous distribution of bacteria on the substrate, allowing use of portable, inexpensive Raman instruments rather than expensive Raman microscopes.
Improves sensitivity and resolution of SERS spectra by employing laser rastering over a large sample area, avoiding damage and sampling multiple SERS hotspots.
Produces reproducible SERS samples with no significant degradation observed after one month of storage at 4°C.
Documented Applications
Detection and identification of bacterial species in surface water samples such as oceans, lakes, and rivers.
Rapid and real-time bacterial species identification in environmental or biological samples using portable Raman spectrometers combined with flow cytometry.
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