Spectroscopic methods to detect and characterize microorganisms
Inventors
Assignees
Publication Number
US-11105747-B2
Publication Date
2021-08-31
Expiration Date
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Abstract
Methods and systems for Resonant Raman spectroscopy are provided. Methods according to certain embodiments include irradiating a sample with a monochromatic light source at a first irradiation intensity and a second irradiation intensity, determining the intensity of one or more of the Resonant Raman scattering and fluorescence scattering at the first irradiation intensity and second irradiation intensity, calculating a rate of change of one or more of the intensity of Resonant Raman scattering and fluorescence in response to the change in irradiation intensity from the first irradiation intensity to the second irradiation intensity and comparing one or more of the rate of change in the intensity of Resonant Raman scattering and the rate of change in the intensity of fluorescence scattering with the rate of change in the irradiation intensity by the monochromatic light source to determine the Resonant Raman response of the sample. Methods also include determining the presence or absence of a microorganism in a sample and correcting for variations associated with measurement instrumentation (e.g., monochromatic light source) and variations associated with the sample (e.g., fluorescence from non-target compounds). Also provided are methods for determining the antimicrobial susceptibility of a microorganism to an antimicrobial agent as well as methods for characterizing a phenotype of an unknown microorganism in a sample. Systems for practicing the subject methods are also provided.
Core Innovation
Aspects of the present disclosure include methods and systems for Resonant Raman spectroscopy. Methods according to certain embodiments include irradiating a sample with a monochromatic light source at a first irradiation intensity and a second irradiation intensity, determining the intensity of one or more of the Resonant Raman scattering and fluorescence scattering at the first irradiation intensity and second irradiation intensity, calculating a rate of change of one or more of the intensity of Resonant Raman scattering and fluorescence scattering in response to the change in irradiation intensity from the first irradiation intensity to the second irradiation intensity and comparing one or more of the rate of change in the intensity of Resonant Raman scattering and the rate of change in the intensity of fluorescence scattering with the rate of change in the irradiation intensity by the monochromatic light source to determine the spectroscopic response of the sample. Systems for practicing the subject methods are also provided.
The subject methods include determining the presence or absence of a microorganism in a sample and correcting for variations associated with measurement instrumentation and variations associated with the sample. Also provided are methods for determining the antimicrobial susceptibility of a microorganism to an antimicrobial agent as well as methods for characterizing a phenotype of an unknown microorganism in a sample. Methods further include determining and correcting for variations in optical measurement instrumentation and variations in the sample.
The disclosure describes reagent and sample-holder features to enhance measurement reliability, including embodiments where the sample includes a hydrophobic compound non-covalently associated with an albumin protein, embodiments where the reagent includes a free radical scavenger such as bilirubin, and embodiments where the sample holder comprises walls having a zwitterionic coating. The methods further include irradiating at an interface between the sample and a surface of the sample holder and calculating a net signal after corrections to determine the presence of actively metabolizing microorganisms, antimicrobial susceptibility, or crosslink cleavage indicative of phenotype.
Claims Coverage
One independent claim was identified. Five main inventive features were extracted from the independent claim.
Albumin with incorporated carotenoid ligand
combining in a sample holder a liquid sample and a reagent that contains albumin having an incorporated ligand which is a carotenoid
Interface-focused monochromatic irradiation
irradiating the sample with a monochromatic light source that is absorbed by the carotenoid, wherein the irradiation is achieved with a focusing lens that is focused at a position located at the interface between the surface of the sample holder that is in contact with the sample, with either an invariant light intensity or one that varies over time and that is focused at an interface between the sample and a surface of the sample holder
Time-resolved Raman and fluorescence measurement
collecting scattered light from the irradiated sample and measuring a Raman signal and a fluorescence signal from the scattered light at a plurality of different times
Net signal correction and threshold-based determination
calculating a rate of change in intensity of the Raman signal and fluorescence signal for the sample over time; correcting the calculated rates of change in the intensities of the Raman signal and the fluorescence signal to obtain a net signal; and determining the presence of a microorganism in the sample based on a comparison of the net signal versus one or more preset thresholds
Carotenoid resonant Raman sensitivity to dissolved gases and free radicals
wherein the carotenoid absorbs the monochromatic light from the light source and has a resonant Raman output that varies in the presence of dissolved gases and free radicals in the sample
The independent claim centers on a reagent comprising albumin with a carotenoid ligand, interface-focused monochromatic irradiation, time-resolved acquisition of Raman and fluorescence signals, correction to obtain a net signal compared to preset thresholds, and the carotenoid's resonant Raman sensitivity to dissolved gases and free radicals.
Stated Advantages
Determining the presence or absence of a microorganism in a sample after calculating and correcting spectroscopic rates of change.
Correcting for variations associated with measurement instrumentation and variations associated with the sample.
Determining antimicrobial susceptibility of a microorganism to an antimicrobial agent, including methods to estimate minimum inhibitory concentration and minimum bactericidal concentration.
Characterizing a phenotype of an unknown microorganism by detecting crosslink cleavage in an albumin protein.
Enabling analysis of specific vibrational modes of chromophores at very low concentration by use of Resonant Raman enhancement.
Documented Applications
Determining the presence of a microorganism in a sample by Resonant Raman scattering or by fluorescence spectroscopy.
Determining antimicrobial susceptibility of a microorganism to an antimicrobial agent, including plotting net signal versus antimicrobial concentration to estimate MIC and MBC.
Characterizing a phenotype of an unknown microorganism by assessing crosslink cleavage of an albumin protein using Resonant Raman or fluorescence.
Correcting for thermal drift and other variations in optical measurement instrumentation using a reference composition and calculating a correction factor.
Calculating signal-to-noise ratio for Resonant Raman response by comparing averaged rates of change and standard deviations between control and test samples.
Using systems comprising a monochromatic light source, optical adjustment components, a photodetector, and a processor to irradiate samples, measure scattered light, calculate rates of change, and obtain net signals.
Irradiating samples at an interface between the sample and a surface of a sample holder, including embodiments using glass vials with zwitterionic coatings.
Applying reagent formulations containing a hydrophobic compound non-covalently associated with albumin and optional components such as a reducing agent, free radical scavenger, or crosslinking agent to enable the described spectroscopy-based diagnostics.
Diagnosing clinical samples, including blood-plasma derived samples, for infection status based on corrected spectroscopic net signals.
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