JPWO2018175346A5 - - Google Patents

Description

Cross-reference This application claims the benefits of US Provisional Patent Application No. 62 / 614,216 filed January 5, 2018 and US Provisional Patent Application No. 62 / 473,876 filed March 20, 2017. These provisional patent applications are incorporated herein by reference in their entirety.

Introduction Raman spectroscopy is used to characterize vibrations, rotations, and other low-frequency molecular motions of compounds, and is generally used to provide data to identify the presence of specific molecules in a sample. used. Raman scattering is the inelastic scattering of monochromatic light (eg, ultraviolet, visible, or near-infrared) in which light interacts with molecular vibrations and causes energy shifts due to scattered photons. The application of this method to the study of the three-dimensional structure of biomolecules is due to many difficulties such as the fairly complex spectrum, the poor quality of the spectrum obtained from the dilute solution, and the high quantity required. In addition, it is developing only slowly.

When the chromophore is contained in a very low concentration in the composite biomedia due to the strong enhancement (10 to ¹⁰⁶ times) observed when the irradiation used to excite the Raman spectrum is in the electron absorption band of the chromophore. Even, it is possible to analyze a particular vibration mode of the chromophore. Raman spectroscopy is often used to investigate a variety of biological processes, as many natural chromophores play an important role in many biological activities. In the presence of large amounts of non-absorbent species, extremely low concentrations of biopigments can be detected and structurally analyzed.

Summary The aspects of this disclosure include methods and systems for resonant Raman spectroscopy. The method according to a particular embodiment includes the following steps. The step of irradiating a sample with a monochromatic light source at the first and second irradiation intensities, one or more of the resonance Raman scattering and fluorescence scattering at the first and second irradiation intensities. The step of determining the rate of change of one or more of the intensity of resonance Raman scattering and fluorescence scattering according to the change of irradiation intensity from the first irradiation intensity to the second irradiation intensity, and the sample. A step of comparing one or more of the rate of change in the intensity of resonance Raman scattering and the rate of change in the intensity of fluorescence scattering to the rate of change in irradiation intensity from a monochromatic light source to determine the spectral response of. In some embodiments, the spectral response of the sample (eg, resonant Raman scattering and / or fluorescent scattering) indicates the physical time course of the sample. In another embodiment, the spectroscopic response of the sample indicates the chemical aging of the sample. In yet another embodiment, the spectroscopic response of the sample indicates the presence of actively metabolizing microorganisms in the sample. In some embodiments, the monochromatic light source is a laser. In some embodiments, the sample comprises a hydrophobic compound and an albumin protein. For example, hydrophobic compounds may be incorporated into albumin proteins. Hydrophobic compounds are, in certain cases, carotenoids such as lycopene. In certain embodiments, the sample comprises a hydrophobic compound, an albumin protein, and a reducing substance. The reducing substance is, in certain cases, glutathione or a derivative thereof. In other embodiments, the sample comprises a hydrophobic compound, an albumin protein, and a free radical scavenger. Free radical scavengers are, in certain cases, bilirubin or its derivatives.

In some cases, the method comprises the following steps: A step of irradiating a sample over a plurality of irradiation intensities for a certain period of time with a monochromatic light source, and a step of determining one or more of the rate of change in the intensity of resonance Raman scattering and the rate of change in the intensity of fluorescence scattering. In other cases, the method comprises the following steps: The step of irradiating a first sample over a period of time with a monochromatic light source to determine one or more of the rate of change in resonance Raman scattering intensity and the rate of change in fluorescence scattering intensity for the first sample; One or more of the normalized rate of change in the intensity of the resonance Raman scattering and the normalized rate of change in the intensity of the fluorescence scattering for the first sample, the normalization of the irradiation intensity of the first sample with a monochromatic light source. The step of calculating the net signal of the first sample by comparing it to the converted rate of change; the second sample is irradiated over the period of time by a monochromatic light source and the resonance Raman scattering for the second sample. The step of determining one or more of the rate of change in intensity and the rate of change in the intensity of the fluorescence scattering; and the normalized rate of change in the intensity of the resonance Raman scattering and the normalized change in intensity for the second sample. The step of calculating the net signal of the second sample by comparing one or more of the rates with the normalized rate of change of the irradiation intensity of the second sample with a monochromatic light source. In some embodiments, the method further comprises comparing the net signal of the first sample with the net signal of the second sample. Based on the calculated net signal compared, the method may include one or more of the following: 1) the step of determining that the first sample and the second sample are different; 2) the first The step of determining that the sample contains a different gas composition than the second sample; and 3) the step of determining that the first or second sample contains actively metabolizing microorganisms.

Aspects of the present disclosure also include methods for determining the presence of microorganisms in a sample. The method according to a particular embodiment includes the following steps. Combining a sample with an albumin-containing reagent containing a ligand in a sample holder; focused at the interface between the sample and the surface of the sample holder with either constant or time-varying light intensity and absorbed by the ligand. The process of irradiating a sample with a monochromatic light source; the process of collecting scattered light from the irradiated sample at multiple different times and measuring the Raman and fluorescent signals from the scattered light; the intensity of the Raman and fluorescent signals for the sample. The step of calculating the rate of change over time; the step of correcting the calculated rate of change in the intensity of the Raman and fluorescent signals to obtain the net signal; and with the net signal and one or more predetermined thresholds. The step of determining the presence of microorganisms in a sample based on the comparison of.

In some embodiments, one or more predetermined thresholds are set by performing the method on one or more control samples containing the inoculum at the lower limit of the concentration of the clinically infected sample. To.

In some cases, the step of correcting the rate of change in intensity of the Raman signal and the fluorescent signal comprises the step of characterizing the spectral output from the standard sample. In other cases, the step of correcting the rate of change in the intensity of the fluorescence signal comprises the step of characterizing the fluorescence output from the reagent.

In a particular embodiment, the step of correcting the rate of change in the intensity of the Raman signal and the fluorescent signal includes the following steps. The step of determining the rate of change of the total output from the standard reference sample; and the net signal is calculated as the rate of change in the intensity of the resonant Raman scattering minus the rate of change in the intensity of the resonant Raman scattering from the standard sample. Process.

In another embodiment, the step of correcting the rate of change in the intensity of the Raman signal and the fluorescent signal includes the following steps. The step of determining the rate of change in the intensity of the fluorescence scattering from the standard reference sample; and the net signal is calculated as the rate of change in the intensity of the fluorescence scattering from the standard sample subtracted from the rate of change in the intensity of the resonant Raman scattering. Process to do.

の化合物である。 In some embodiments, the method further comprises the step of pretreating albumin prior to incorporation of the ligand, for example by contacting albumin with a reducing substance (eg, glutathione). The reducing material is sufficient to reduce the disulfide bonds of albumin in certain cases. In another embodiment, albumin is pretreated with a disulfide crosslinker. In some cases, the disulfide crosslinker comprises a core that is cleaved by an enzyme or metabolite produced by a microorganism in the sample. In certain cases, the disulfide cross-linking material is of formula (I) :.

It is a compound of.

Aspects of the present disclosure also include the step of determining the presence of microorganisms in a sample (eg, actively metabolizing microorganisms) using resonant Raman scattering. The method according to a particular embodiment includes the following steps. The step of irradiating a sample over a period of time with a monochromatic light source and calculating the rate of change in the intensity of the resonant Raman scattering for the first sample; the normalized rate of change in the intensity of the resonant Raman scattering of the sample, the monochromatic source. The step of calculating the net signal of the sample by comparing it with the normalized rate of change of the irradiation intensity of the sample; and the step of determining the presence of microorganisms in the sample based on the net signal of the sample. In certain cases, the presence of microorganisms is determined when the net signal of the sample exceeds a predetermined threshold. In other embodiments, the step of determining the presence of a microorganism comprises the following steps: a monochromatic light source irradiates the first sample over multiple intensities for a period of time to determine the rate of change in the intensity of resonance Raman scattering for the first sample. Step of calculation; First sample by comparing the normalized rate of change in the intensity of resonance Raman scattering for the first sample with the normalized rate of change in the intensity of irradiation of the first sample with a monochromatic light source. The step of calculating the net signal of the The step of calculating the net signal of the second sample by comparing the normalized rate of change of Raman scattering intensity with the normalized rate of change of the irradiation intensity of the second sample by a monochromatic light source; and the second. The step of determining the presence of a microorganism in one or more of the first sample or the second sample by comparing the net signal of one sample with the net signal of the second sample. In some cases, if the net signal of the second sample is greater than the net signal of the first sample, it is determined that the microorganism is present in the second sample. In other cases, if the net signal of the first sample is greater than the net signal of the second sample, it is determined that the microorganism is present in the first sample.

Aspects of the present disclosure also include determining the presence of microorganisms (eg, actively metabolizing microorganisms) in the sample by fluorescence spectroscopy. The method according to a particular embodiment includes the following steps. The step of irradiating a sample with a monochromatic light source for a period of time and detecting fluorescence from the sample over that period; a normalized rate of change in the intensity of the detected fluorescence produced by the sample of the fluorescence produced by the control. The step of calculating the rate of change in fluorescence due to the presence of microorganisms by comparing with the normalized rate of change; and the microorganisms in the sample based on the calculated changes in fluorescence of the sample compared to a given threshold. The process of determining the existence of.

In the step of determining the presence of microorganisms in the sample (eg, actively metabolizing microorganisms), the method comprises irradiating the sample in the sample holder. In some embodiments, the sample holder is a container, such as a cuvette, vial, planar substrate, or microfluidic device. The sample container is, in certain cases, a glass vial. In some embodiments, the glass vial has a wall with a zwitterion coating, such as a zwitterion silane coating. In these embodiments, the sample is with the surface of the sample and the sample holder (eg, the wall of the container), such as by focusing a monochromatic light source at a position at the interface between the sample and the surface of the sample holder (eg, the wall of the container). Can be irradiated at the interface of. In some cases, monochromatic light is located 0.01 mm to 2 mm from the surface of the surface of the sample holder (eg, the wall of the container), such as about 0.2 mm from the surface of the surface of the sample holder (eg, the wall of the container). Be focused. Monochromatic light may be focused, for example, by a collimating optical system (including, for example, a collimating lens).

Aspects of the present disclosure also include methods for calculating the signal-to-noise ratio. In some embodiments, the method comprises the following steps: The step of irradiating the first sample over a period of time with a monochromatic light source and determining the rate of change in the intensity of the resonance Raman scattering for the first sample; the first sample to determine the first average rate of change. The step of calculating the average rate of change of the intensity of the resonance Raman scattering by; the process of calculating the standard deviation of the first average rate of change; The step of determining the rate of change in the intensity of the resonance Raman scattering for the sample; the step of calculating the average rate of change in the intensity of the resonance Raman scattering by the second sample to determine the second average rate of change; the second average The process of calculating the standard deviation of the rate of change; the process of subtracting the second average rate of change from the first rate of change to determine the average rate of change of the signal; the first standard to determine the standard deviation of the signal. The step of adding the deviation and the second standard deviation; and the step of dividing the average rate of change of the signal by the standard deviation of the signal to determine the signal noise ratio of the resonance Raman response.

Aspects of the present disclosure are for determining and correcting variations in optical measuring instruments (eg, thermal drift of monochromatic light sources, changes in the position of optical components, etc.) and sample variations (eg, fluorescence from non-target compounds). Further includes methods. In some embodiments, the method comprises the following steps: A step of irradiating a reference composition over a plurality of intensities with a monochromatic light source for a certain period of time to determine one or more of the rate of change in the intensity of resonance Raman scattering and the rate of change in the intensity of fluorescence scattering . The irradiation and determination step; The step of calculating the net signal of a reference composition by comparing one or more of the rates of change in scattering intensity with the rate of change in the irradiation intensity of the reference composition by a monochromatic light source. In certain cases, the method further comprises the following steps: The step of irradiating a sample over a range of intensities over a period of time with a monochromatic light source to determine one or more of the rate of change in the intensity of the resonant Raman scattering; the intensity of the resonant Raman scattering of the sample to generate a correction factor. The step of acquiring a net signal by correcting the determined rate of change of the sample using the rate of change in the intensity of the sample from a monochromatic light source; and the correction coefficient from the determined rate of change in the intensity of resonance Raman scattering for the sample. The process of reducing.

Aspects of the present disclosure further include methods for determining the antimicrobial agent susceptibility of a microorganism to an antimicrobial agent using resonant Raman scattering. In some embodiments, the method comprises the following steps: It is a step of irradiating a plurality of samples containing microorganisms and antimicrobial agents over a plurality of intensities with a monochromatic light source for a certain period of time, and determining the rate of change in the intensity of resonance Raman scattering for each irradiated sample. Irradiation and determination steps, including the same concentration of microorganisms and different concentrations of antimicrobial agent; normalized changes in the intensity of resonance Raman scattering for each sample, normalized changes in the intensity of irradiation for each sample with a monochromatic light source. The step of calculating the net signal of each sample by comparing with the rate; and the step of determining the susceptibility of the microorganism to the antimicrobial agent based on the net signal of multiple samples. In some cases, the method further comprises plotting the net signal of each sample as a function of the logarithm of the concentration of antimicrobial agent in each sample. In certain cases, the method may also include determining the minimum inhibitory concentration of the antimicrobial agent by determining the concentration that initially indicates a decrease in the plotted net signal. In other cases, the method may also include determining the maximum bactericidal concentration of the antimicrobial agent by determining the concentration that initially indicates an increase in the plotted net signal. In another aspect, the method further comprises determining the metabolic activity of the microorganism in each sample based on the net signal of each sample. In some cases, the method comprises determining the concentration of an antimicrobial agent that indicates a decrease in metabolic activity. In other cases, the method comprises determining the concentration of an antimicrobial agent that exhibits increased metabolic activity.

Aspects of the present disclosure further include methods for determining the antimicrobial agent susceptibility of a microorganism to an antimicrobial agent by fluorescence spectroscopy. In some embodiments, the method comprises the following steps: A step of irradiating a plurality of samples having microorganisms and antimicrobial agents with a monochromatic light source for a certain period of time and detecting fluorescence from each of the samples irradiated for a certain period of time, wherein each sample has the same concentration of microorganisms and different concentrations. Irradiation and detection steps with antimicrobial agents in; comparing the normalized rate of change in the detected fluorescence intensity produced by each sample with the normalized rate of change in fluorescence produced by the control. To calculate the rate of change in fluorescence in each sample; and to determine the susceptibility of the microorganism to the antimicrobial agent based on the calculated rate of change in fluorescence in multiple samples. In some embodiments, the method comprises irradiating each of the samples over multiple intensities of a monochromatic light source.

Each sample in these embodiments may contain microorganisms of the same concentration, eg, a concentration of 10 colony forming units (CFU) or higher (such as 14 CFU or higher). In a particular embodiment, the microbial sample prepares a composition having a predetermined concentration of microorganisms (eg, 100 CFU), and each sample is provided with a predetermined volume of microbial composition so that each sample has the same concentration of microorganisms. Prepared by aliquoting. In embodiments, the concentration range of the antimicrobial agent in the plurality of samples ranges from less than the minimum inhibitory concentration of the antimicrobial agent to higher than the minimum bactericidal concentration of the antimicrobial agent. The antimicrobial agent can be incubated with the microorganism in the sample for a predetermined period of time, such as 10 minutes or longer or 20 minutes or longer, prior to irradiation with a monochromatic light source.

Aspects of the present disclosure further include methods for characterizing unknown microbial phenotypes using resonant Raman scattering. In some embodiments, the method comprises the following steps: A step of irradiating a sample containing microorganisms, crosslinkers, and albumin proteins with a monochromatic light source over a period of time with a monochromatic light source to determine the rate of change in the intensity of resonant Raman scattering; normal resonance Raman scattering intensity for the sample. The step of calculating the net signal of the sample by comparing the normalized rate of change with the normalized rate of change of the irradiation intensity of the sample with a monochromatic light source; as well as the cross-linking cleavage of the albumin protein based on the net signal of the sample. A determination step in which the degree of cross-linking cleavage indicates the phenotype of the microorganism.

Aspects of the present disclosure further include methods for characterizing unknown microbial phenotypes by fluorescence spectroscopy. In some embodiments, the method comprises the following steps: A step of irradiating a sample containing microorganisms, crosslinkers, and albumin proteins with a monochromatic light source for a period of time to detect fluorescence from the sample over a period of time; a normalized change in the intensity of the detected fluorescence produced by the sample. The step of calculating the rate of change in fluorescence by comparing the rate to the normalized rate of change in fluorescence produced by the control; and the step of determining cross-linking cleavage based on the calculated rate of change in fluorescence of the sample. A determination step in which the degree of cross-linking cleavage indicates the phenotype of the microorganism. In some embodiments, the method comprises irradiating the sample over multiple intensities of a monochromatic light source.

の化合物である。 In some embodiments, the cross-linking material can be a disulfide crosslinker. In some cases, the cross-linking material is a glutamic acid derivative. In certain embodiments, the cross-linking material is of formula (I) :.

It is a compound of.

The cross-linking material may be present in the sample in an amount such that the cross-linking material: albumin protein molar ratio is 1: 10-10: 1, for example, the cross-linking material: albumin protein molar ratio is approximately 1: 2. In some embodiments, an increase in the net signal of the sample over time indicates that the microorganism produces a metabolite that cleaves one or more crosslinks in the albumin protein. Based on this, the phenotype of the microorganism can be characterized.

A system for carrying out the method of interest with a monochromatic light source and a detector for detecting resonant Raman scattering or fluorescence is also provided. In some embodiments, the monochromatic light source is a laser. For example, the laser may be an Nd: YAG laser, such as a double wave Nd: YAG laser that outputs 532 nm light. In some cases, the detector is a charge-coupled device (CCD). In some embodiments, the system further comprises a sample container. The sample container may be, for example, a glass vial. In some embodiments, the glass vial has a wall with a zwitterion coating, such as a zwitterion silane coating. In these embodiments, the system of interest is configured to illuminate the sample at the interface between the sample and the wall of the container, such as by focusing a monochromatic light source at a position at the interface between the sample and the wall of the container. In some cases, monochromatic light is focused at a location 0.01 mm to 2 mm from the surface of the vessel wall, such as about 0.2 mm from the surface of the vessel wall. These systems may also include an optical adjustment component for focusing a monochromatic light source at the interface between the sample and the vessel wall, such as a collimating lens.

The present invention will be best understood by reading the following detailed description in conjunction with the accompanying drawings. The drawings include the following figures.

FIG. 6 is a graph showing the normalized rate of change of resonance Raman intensity from lycopene plotted against the normalized rate of change of laser intensity according to a particular embodiment. It is a graph which shows the variation of the nonlinear coefficient by the amount of the dissolved gas (and supersaturated gas) present in a liquid by a specific aspect. It is a graph which shows the mean resonance Raman scattering peak height from four non-infected control samples and three 10CFU Staphylococcus aureus (S. aureus) infected samples. FIG. 6 is a graph showing mean resonant Raman scattering peak heights from 4 uninfected control samples and 3 10 CFU Staphylococcus aureus infected samples, each containing 1 μM bilirubin. It is a graph which shows the signal-to-noise ratio of a control sample and a 10CFU Staphylococcus aureus sample according to a specific embodiment. FIG. 6 is a graph showing a plot of a signal of resonant Raman scattering estimated at t = 600 seconds, plotted as a function of added bilirubin, according to a particular embodiment. FIG. 5 is a graph showing variation in the signal-to-noise ratio of compositions with different amounts of bilirubin, plotted as a function of experimental time, according to a particular embodiment. FIG. 6 is a graph showing variation in a timescale in which the signal-to-noise ratio is optimized for the amount of bilirubin present in the assay, according to a particular embodiment. 9A-9B are graphs showing changes in measured values of 4 non-infected samples and 3 infected samples as a function of bilirubin content. FIG. 9A shows the measured raw data values. 9A-9B are graphs showing changes in measured values of 4 non-infected samples and 3 infected samples as a function of bilirubin content. FIG. 9B shows the corrected data values for the drift observed at the laser output. FIG. 5 is a graph showing instrument stability determination using the normalized rate of change of resonant Raman scattering of the reference composition compared to the normalized rate of change of photodetector shutter time according to a particular embodiment. It is a figure which shows the layer of the sample chamber by a specific embodiment. It is a flow chart for minimizing the influence of heat on the laser output by a specific aspect. It is a flow chart for checking the thermal instability of a system by a specific aspect. It is a graph of the YCAL test for checking the thermal instability of the system according to a specific aspect. FIG. 5 is a flow chart of a control process for minimizing thermal fluctuations during system operation according to a specific aspect. It is a graph which shows the relationship of the magnitude of the signal from the sample which has the reducing substance of a different concentration by a specific aspect. It is a graph which shows the relationship between the magnitude of a signal and an incubation time at a predetermined measurement time according to a specific aspect. It is a graph which shows the relationship between the rate of change of Raman peak height and the laser exposure time by a specific aspect. FIG. 6 is a graph showing the relationship between the confidence interval of the NIST profile and the distance from the sample-container interface and the focal point of light irradiation, according to a particular embodiment. It is a graph which shows the relationship between the Raman intensity profile and the collimating lens position from a sample container by a specific aspect. FIG. 6 is a graph showing the relationship between the gradient of a non-infected control sample and the focal position of a monochromatic light source from the edge of the sample-vessel interface according to a particular embodiment. FIG. 22A is a graph showing the signal-to-noise ratio of the Raman spectrum obtained for a sample in a zwitterion coated glass vial according to a particular embodiment. FIG. 22B is a graph showing the signal-to-noise ratio of the Raman spectrum obtained for a sample in an uncoated glass vial according to a particular embodiment. It is a graph which shows the relationship between a signal-to-noise ratio and a focal point distance from a sample-container interface according to a specific aspect. FIG. 6 is a graph showing Lieber's method of fitting a fifth-order polynomial to a spectrum with contributions from Raman peaks and background fluorescence, according to a particular embodiment. FIG. 25A is a graph showing the relationship between the signal-to-noise ratio and the gradient according to a specific embodiment. FIG. 25B is a graph showing the relationship between the signal-to-noise ratio and the confidence interval of the gradient according to a specific embodiment. FIG. 26A is a graph showing the amplitude of aggregated Raman peaks for 4 non-infected control samples and 3 infected samples according to a particular embodiment. FIG. 26B is a graph showing the estimated rate of change of uninfected and infected samples from the area under the curve. FIG. 26C is a graph showing the estimated signal-to-noise ratios of non-infected and infected samples. FIG. 3 is a graph showing an estimated gradient aggregated for controls and infected samples according to a particular embodiment and corrected for variability observed in NIST standards. FIG. 28A is a graph showing the estimated rate of change of non-infected and infected samples estimated from the area under the curve according to a particular embodiment. FIG. 28B is a graph showing the signal-to-noise ratio estimated as the signal divided by the rms standard deviation of the rate of change of non-infected and infected samples, according to a particular embodiment. FIG. 28C is a graph showing a signal as a function of the time of this sample set in which bacteria were centrifuged and removed, according to a particular embodiment. FIG. 29A is a graph showing the rate of change of Raman peak in a clinical sample according to a particular embodiment. FIG. 29B is a graph showing the amplitude of Raman peaks in a series of trials completed with clinical samples supplemented with plasma from different patients, according to a particular embodiment. It is a graph which shows the relationship between the change rate of Raman peak in a blood culture test, and the time until it becomes positive by a specific aspect. FIG. 31A is a graph showing changes in signal intensity of samples with different bacterial concentrations in a clinical sample according to a particular embodiment. FIG. 31B is a graph showing signal intensities at 1000 seconds for clinical samples with different bacterial concentrations in a particular embodiment. FIG. 32A is a graph showing the measured gradient of Raman intensity as a function of antimicrobial agent (vancomycin) concentration, according to a particular embodiment. FIG. 32B is a graph showing the time it takes for the test sample to become cloudy. FIG. 33A is a graph showing the results of absorbance at 650 nm of a peptide crosslinker compound containing two disulfide bonds according to a particular embodiment. FIG. 33B is a graph showing the rise of the Raman signal over time as a function of microbial concentration. FIG. 5 is a graph showing an increase in Raman signal as a function of the molar ratio of the added disulfide crosslinker according to a particular embodiment. FIG. 35A is a graph showing aggregated fluorescence measurements for three non-infected control samples according to a particular embodiment. FIG. 35B is a graph showing the signal-to-noise ratio for fluorescence detection estimated as the signal divided by the rms standard deviation of the rate of change of non-infected and infected samples, according to a particular embodiment.

Selected Definitions As used herein, "Raman scattering" and other similar terms and / or phrases refer to any method in which light incident on a sample at one wavelength is scattered at another wavelength. .. Scattering is due to an incoherent process due to the absorption of incident photons by the excitation of the structure from the initial lower vibration level (ground state) to the higher vibration level, followed by relaxation down to different ground state levels. possible.

As used herein, the term "Raman band" and similar terms and / or phrases refer to spectral profiles (eg, intensity vs. frequency) that correspond to Raman scattering from specific chemical bonds within the molecule. It will be appreciated that each chemical bond appears as a Raman band at a separate frequency, and in some cases these Raman bands may overlap, making it difficult to distinguish between Raman bands. Further, it will be understood that the Raman cross-sectional area of a chemical bond is a constant that defines the intensity of the corresponding Raman peak. Furthermore, it will be appreciated that this cross-sectional area can change with wavelength and / or with resonance. Such resonance changes occur during resonance Raman enhancement.

As used herein, the "Raman spectrum" of a sample and similar terms and / or phrases refer to the sum of all Raman bands, and the relative height of the individual Raman bands in the Raman spectrum is the corresponding chemistry. It will be understood that it is proportional to the relative abundance of bonds multiplied by their Raman cross-sectional area.

As used herein, "absorption" and similar terms and / or phrases refer to any method by which incident light is absorbed by the sample of interest. Incident photons interact with the structure by any number of mechanisms, including excitation of outer shell electrons (eg, corresponding to absorption of UV or visible radiation), or excitation of molecules to higher vibration / rotational energy states. Can be.

As used herein, "resonant Raman scattering" and similar terms and / or terms are specific with the excitation of the molecule from the initial ground state to a significant excited state corresponding to a significant vibrational state. Refers to a process that is understood to be a type of Raman scattering. Thus, for the purposes of this discussion, resonance "Raman enhancement" (or "resonance Raman") and other similar terms and / or phrases are any method in which the Raman cross section of a particular band is enhanced by strong optical absorption. Point to.

As used herein, "resonant Raman non-linearity (RRNL)" refers to the rate of change in the intensity of the resonant Raman peak (plot on the Y-axis) and the rate of change in the intensity of the incident laser beam (plot on the X-axis). Refers to the gradient of the trace between.

As used herein, "net signal" is instrument-related variation (eg, drift of monochromatic light sources, drift of optical components such as collimated optics) and / or variation in sample vials (eg, not covered). Refers to the rate of change in intensity of one or more of Raman scattering or fluorescence scattering after correction of (fluorescence from a compound). In some embodiments, the "net signal" is the resonance Raman defined above in that net signal = 0 when RRNL = 1 and RRNL <1 when the net signal is less than 0. Is related to the non-linearity (RRNL) of.

As used herein, "vial" and other similar terms and / or phrases refer to, for example, a test vessel containing a test sample with any other component of the assay. It will be appreciated that vials can be constructed from any reasonably transparent material, such as glass and plastic, as described in detail below.

Detailed Description Aspects of the present disclosure include methods and systems for resonant Raman spectroscopy. The method according to a particular embodiment includes the following steps. One or more of the steps of irradiating a sample with a monochromatic light source at first and second irradiation intensities, resonance Raman scattering and fluorescence scattering at first and second irradiation intensities. The step of determining the intensity of, one or more of the intensity of resonance Raman scattering and the intensity of fluorescence scattering according to the change of irradiation intensity from the first irradiation intensity to the second irradiation intensity is calculated. The step of 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 fluorescent scattering with the rate of change in irradiation intensity from a monochromatic light source to determine the spectral response of the sample. The method also includes the following steps. A step of determining the presence or absence of microorganisms in a sample, and a step of correcting variability associated with a measuring instrument (eg, a monochromatic light source) and variability associated with the sample (eg, fluorescence from a non-target compound). Also provided are methods for determining the antimicrobial agent susceptibility of a microorganism to an antimicrobial agent and for characterizing the phenotype of an unknown microorganism in a sample. A system for implementing the method of interest is also provided.

Before describing the invention in more detail, it should be understood that the invention is not limited to the particular embodiments described, as such may vary. As the scope of the invention is limited only by the appended claims, the terms used herein are for and intended to describe only certain embodiments. Please also understand that there is no such thing.

If a range of values is provided, each intervening value between the upper and lower bounds of the range and any other described value or intervening value in the described range shall be distinctly different in context. It should be understood that up to one tenth of the lower limit unit is included in the invention unless stated. The upper and lower limits of these smaller ranges may be included independently within the smaller range and are included in the invention depending on any limits specifically excluded in the described range. If the ranges described include one or both of the limits, the scope of the invention also includes excluding any or both of those included limits.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention belongs. In practice or testing of the present invention, any method and material similar to or equivalent to that described herein can be used, but representative exemplary methods and materials are described herein.

All publications and patents cited herein are incorporated herein by reference as if the individual publications or patents were specifically and individually indicated for inclusion by reference. Incorporated herein by reference to disclose and describe the methods and / or materials cited as relevant to the publication. Citations of any publication are intended for disclosure of publications prior to the filing date and should not be construed as admitting that the invention is not entitled to precede such publications. In addition, the dates of publications provided may differ from the actual publication dates, and the actual publication dates may need to be confirmed independently.

It should be noted that, as used herein and in the appended claims, the singular form (a, an, the) includes multiple referents unless explicitly stated otherwise in the context. .. It should also be noted that the claims may be drafted to exclude any arbitrary element. Therefore, this statement is due to the use of exclusive terms such as "solely" or "only" in connection with the description of the elements of the claims, or the use of "negative" limitation. It is intended to serve as an earlier description of.

As will be apparent to those of skill in the art by reading the present disclosure, each of the individual embodiments described and exemplified herein will not deviate from the scope or intent of the invention. It has separate components and features that can be easily separated or combined with any of the features of. Any of the described methods can be carried out in the order of the described events or in any other logically possible order.

Resonance Raman spectroscopy As outlined above, aspects of the present disclosure include methods for resonance Raman spectroscopy. The method according to a particular embodiment includes the following steps. One or more of the steps of irradiating a sample with a monochromatic light source at first and second irradiation intensities, resonance Raman scattering and fluorescence scattering at first and second irradiation intensities. The step of determining the intensity of, one or more of the intensity of resonance Raman scattering and the intensity of fluorescence scattering according to the change of irradiation intensity from the first irradiation intensity to the second irradiation intensity is calculated. The step of 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 fluorescent scattering with the rate of change in irradiation intensity from a monochromatic light source to determine the spectral response of the sample.

In carrying out the method according to a particular embodiment, the sample is irradiated with a monochromatic light source. As used herein, the term "monochromatic" is used in its conventional sense to refer to a light source that outputs light irradiation with a narrow bandwidth. In a plurality of embodiments, the monochromatic light source outputs light having a narrow wavelength range including, for example, 25 nm or less, 20 nm or less, 15 nm or less, 10 nm or less, 5 nm or less, and 2 nm or less. In certain embodiments, the monochromatic light source outputs light of a single wavelength (eg, light of 532 nm). Any convenient monochromatic light source can be used, such as a laser or a single wavelength light emitting diode. In certain embodiments, the light source is a broadband light source that optically communicates with an optical conditioning component that narrows the irradiation bandwidth to a single wavelength. For example, monochromatic light irradiation of a sample for resonance Raman spectroscopy by the method of interest uses one or more optical bandpass filters, diffraction grids, monochrome meters, or a broadband light source coupled to any combination thereof. And, for example, wideband halogen lamps, heavy hydrogen arcclamps, xenon arcclamps, stabilized fiber coupled wideband light sources, continuous spectrum wideband LEDs, super luminescent light emitting diodes, semiconductor light emitting diodes, wide area spectrum LED white light sources, or This can be achieved by using a multi-LED white light source or the like.

In certain embodiments, the sample is irradiated with a laser. In some cases, the laser is a continuous wave laser. In other cases, the laser is a pulsed laser. In certain cases, the laser is a diode laser such as an ultraviolet diode laser, a visible light diode laser, and a near infrared diode laser. In some cases, the monochromatic light source is a diode laser that outputs light at wavelengths from 375 nm to 1000 nm, including, for example, 405 nm to 875 nm, 450 nm to 800 nm, 500 nm to 650 nm, and 525 nm to 625 nm. In other cases, the laser is a pulsed laser, such as a solid-state laser. In certain cases, the monochromatic light source is a solid-state laser that outputs light at wavelengths from 375 nm to 1000 nm, including, for example, 405 nm to 875 nm, 450 nm to 800 nm, 500 nm to 650 nm, and 525 nm to 625 nm. Other suitable lasers are helium neon (HeNe) laser, argon laser, krypton laser, xenon ion laser, nitrogen laser, carbon dioxide laser, carbon monoxide laser, excima laser, hydrogen fluoride laser, among other laser types. , Hydrofluoride hydrogen laser, oxygen iodine laser, vapor phase iodine laser, helium cadmium laser, helium mercury laser, helium silver laser, strontium steam laser, neon copper laser, copper steam laser, gold steam laser, manganese steam laser, ruby laser , Nd: YAG laser, NdCrYAG laser, Er: YAG laser, Nd: YLF laser, Nd: YVO ₄ laser, Nd: YCa ₄ O (BO ₃ ) ₃ laser, Nd: glass laser, titanium sapphire laser, thurium YAG laser, Itterbium YAG laser, Yb ₂ O ₃ laser, Itterbium-added glass laser, Holmium YAG laser, Chromium ZnSe laser, Serium-added lithium fluoride strontium aluminum laser, Promethium 147-added phosphate glass laser, Chromium-attached chrysoberyl laser, Elbium-added And may include, but are not limited to, erbium itterbium co-added glass lasers, trivalent uranium-added calcium fluoride lasers, sumalium-added calcium fluoride lasers, GaN lasers, InGaN lasers, AlGaInP lasers, AlGaAs lasers, InGaAsP lasers. In a particular embodiment, the monochromatic light source is a double wave neodymium-added yttrium aluminum garnet that outputs 532 nm light.

In some embodiments, the sample is continuously irradiated. In other embodiments, the sample is sampled by a monochromatic light source, eg, 0.001 ms or greater, 0.005 ms or higher, 0.01 ms or higher, 0.05 ms or higher, 0.1 ms or higher, 0.5 ms or higher, 1 ms or higher, 5 Milliseconds or more, 10 milliseconds or more, 100 milliseconds or more, 1000 milliseconds or more, 5 seconds or more, 15 seconds or more, 30 seconds or more, 60 seconds or more, 100 seconds or more, 200 seconds or more, 300 seconds or more, 400 seconds or more Irradiated at discrete intervals, including 500 seconds or more, 1000 seconds or more, and 1500 seconds or more, or at some other interval. The time between each interval may be different, for example 0.005 ms or more, 0.01 ms or more, 0.05 ms or more, 0.1 ms or more, 0.5 ms or more, 1 ms or more, 5 ms or more, 10 It may be 0.001 milliseconds or more, including milliseconds or more, 100 milliseconds or more, and 1000 milliseconds or more.

In certain embodiments, the monochromatic light source is a strobe light source in which the sample is illuminated with a periodic flash of light. For example, the frequency of strobe light is, for example, 0.05kHz or more, 0.1kHz or more, 0.5kHz or more, 1kHz or more, 2.5kHz or more, 5kHz or more, 10kHz or more, 25kHz or more, 50kHz or more, and 0.01kHz or more including 100kHz or more. May be.

Samples are sampled, for example, 2 or more, 3 or more, 4 or more, 5 or more, 10 or more, 25 or more, and 50 or more to obtain sufficient resonant Raman scattering and / or fluorescent scattering. It may be irradiated at any number of intervals, such as irradiating the sample once or more including.

Depending on the type of light source and optical components (lens, mirror, collimator, etc.), the sample may be, for example, 2.5 mm or more, 5 mm or more, 10 mm or more, 15 mm or more, 25 mm or more, 50 mm or more, 100 mm or more, 500 mm or more from the sample. Can be illuminated by a monochromatic light source from any suitable distance, such as 1000 mm or greater, 2500 mm or greater, 5000 mm or greater, and 1 mm or greater from a sample containing 10000 mm or greater. Similarly, the sample is sampled by a monochromatic light source from any suitable angle, including, for example, 5 ° to 90 °, 15 ° to 85 °, 20 ° to 80 °, 25 ° to 75 °, and 30 ° to 60 °. Can be irradiated.

As outlined above, the method comprises determining the intensity of one or more of resonant Raman scattering and fluorescent scattering at one or more wavenumbers. Resonant Raman scattering depends on the chromophore, for example, 250 cm ^-1 to 1900 cm ^-1 , 300 cm ^-1 to 1800 cm ^-1 , 350 cm ^-1 to 1700 cm ^-1 , and 200 cm ^-1 to include 400 nm to 1600 cm ^-1 . It can be detected at any suitable wavenumber in the range 2000 cm ^-1 . In certain embodiments, the chromophore is lycopene and resonant Raman scattering is detected and measured in one or more of 1512 cm ^-1 , 1515 cm ^-1 , 1525 cm ^-1 , and 1535 cm ^-1 . In other embodiments, the method is also one or more in the range of 1500 cm ^-1 to 4000 cm ^-1 , including, for example, 1750 cm ^-1 to 3750 cm ^-1 , 2000 cm ^-1 to 3500 cm ^-1 , and 3000 cm ^-1 . Includes a step of detecting fluorescence by wave number.

The duration for detecting light from a sample (eg, resonant Raman scattering, fluorescence) by the method of interest (discussed in more detail below) is, for example, 30 seconds to 1750 seconds, 45 seconds to 1500 seconds, It can range from 60 seconds to 1250 seconds, 120 seconds to 1000 seconds, 200 seconds to 800 seconds, etc., and 10 seconds to 2000 seconds including 400 seconds to 600 seconds.

Light from the sample (eg, resonant Raman scattering, fluorescence), among other photodetectors, is an active pixel sensor (APS), 4-split photodiode, wedge-type detector, image sensor, charge-coupled device (CCD), Augmented Charge Coupled Devices (ICCDs), light emitting diodes, photon counters, borometers, photodetectors, photoregisters, solar cells, photodiodes, photoelectron multipliers, phototransistors, quantum dot photoconductors or photodiodes, etc., and It can be detected by any convenient detection protocol including, but not limited to, a combination of optical sensors or photodetectors. In certain embodiments, the light from the sample (eg, resonant Raman scattering, fluorescence) is detected on one or more CCDs.

In the spectroscopy methods described in the present specification (for example, non-linear resonance Raman spectroscopy, fluorescence spectroscopy), the temperature change in the target method is, for example, 4.5 ° C or lower, 4 ° C or lower, 3.5 ° C or lower, 3 ° C or lower. , 2.5 ℃ or less, 2 ℃ or less, 1.5 ℃ or less, 1 ℃ or less, 0.5 ℃ or less, 0.1 ℃ or less, 0.05 ℃ or less, 0.01 ℃ or less, 0.005 ℃ or less, 0.001 ℃ or less, 0.0001 ℃ or less, 0.00001 ℃ or less, etc. And can be done while maintaining a constant temperature such as 5 ° C or lower, including 0.000001 ° C or lower. Temperatures include, but are limited to, heat sinks, fans, exhaust pumps, vents, refrigeration, coolants, heat exchanges, Pelche or resistance heating elements, and any combination thereof, among other types of temperature control protocols. It can be controlled by any convenient protocol that is not.

In some embodiments, the sample is irradiated through one or more optical conditioning components. "Optical adjustment" means changing or adjusting the light from a monochromatic light source before shining it on the sample. For example, optical adjustment can be to change the profile of the light beam, change the focus of the light beam, change the direction of beam propagation, or collimate the light beam. In some cases, optical adjustment involves collimating light. The term "colimate" is used in the traditional sense to optically adjust the co-linearity of light propagation or to suppress the spread of light from a common axis of propagation. In some cases, collimating involves narrowing the spatial cross-sectional area of the light beam. In other cases, the optical adjustment is, for example, 5 ° or more, 10 ° or more, 15 ° or more, 20 ° or more, 25 ° or more, 30 ° or more, 45 ° or more, 60 ° or more, 75 ° or more, and 90. Includes changing the direction of the light beam, including changing the direction of light propagation by 1 ° or more. In yet other cases, the optical adjustment may reduce the dimensions of the light by, for example, 5% or more, including reductions of 10% or more, 25% or more, 50% or more, and 75% or more. , Beam spot) is a reduction protocol for reducing.

The optical adjustment component can be any convenient device or structure that gives the desired change to the light beam, such as lenses, mirrors, beam splitters, collimating optics (eg, lenses), pinholes, slits, gratings, refractors. It may include, but is not limited to, a vessel and any combination thereof. As described in more detail below, the system of interest requires one or more optical conditioning components, including, for example, two or more, three or more, four or more, and five or more optical tuning components. May be included depending on.

In certain embodiments, the photodetection system comprises a collimator placed adjacent to the sample holder. The collimator can be any convenient collimating protocol, such as one or more mirrors or curved lenses or a combination thereof. For example, a collimator is, in certain cases, a single collimator lens. In other cases, the collimator is a collimator mirror. In yet other cases, the collimator includes two lenses. In yet other cases, the collimator includes a mirror and a lens. If the collimator contains one or more lenses, the focal length of each collimator lens may be, for example, 6mm-475mm, 7mm-450mm, 8mm-425mm, 9mm-400mm, 10mm-375mm, 12.5mm-350mm, and so on. It can vary in the range of 5mm to 500mm including the focal length in the range of 15mm to 300mm. In certain embodiments, the focal length ranges from 400 mm to 500 mm, including, for example, 405 mm to 475 mm, 410 mm to 450 mm, and 410 mm to 425 mm, such as 410 mm or 420 mm.

In some embodiments, the sample is irradiated in a sample holder, eg, a sample container. The sample holder can be any suitable shaped substrate or container for irradiating the sample and detecting one or more of resonant Raman scattering and fluorescent scattering. In some embodiments, the sample holder is a flat substrate (eg, a microscope slide). In another embodiment, the sample holder is a microfluidic device with one or more microfluidic channels. In yet another embodiment, the sample holder is a container having a cross-sectional shape, and the cross-sectional shape of interest is a straight cross-sectional shape, eg, a curved cross-sectional shape such as a square, a rectangle, a trapezoid, a triangle, a hexagon, etc. It includes, but is not limited to, circles, ellipses, etc., and irregular shapes, such as a parabolic lower part coupled to a planar upper part. The size of the sample holder can vary depending on the volume of the sample being irradiated, and the length of the holder of interest can be, for example, 10 mm to 90 mm, 15 mm to 85 mm, 20 mm to 80 mm, 25 mm to 75 mm, 30 mm to 70 mm, and 35 mm. The range is 5 mm to 100 mm including ~ 65 mm, and the width of the holder of interest (or the cross section when the container is cylindrical) is, for example, 10 mm to 90 mm, 15 mm to 85 mm, 20 mm to 80 mm, 25 mm to 75 mm, 30 mm. It is 5 mm to 100 mm including ~ 70 mm and 35 mm to 65 mm. In multiple embodiments, the sample holders are, for example, 0.5 cm 3-9 cm ³ , 1 cm ^3-8 cm ³ , 1.5 cm 3-7 cm ³ , ² cm ^3-6 cm ³ , 2.5 cm ^3-5 cm ³ , etc., and ³ cm ^3-4 cm ³ . Can have a volume of 0.1 cm ³ to 10 cm ³ including.

The sample holder can be formed from any transparent material that transmits a desired wavelength range, including but not limited to optical glass, borosilicate glass, Pyrex glass, ultraviolet quartz, infrared quartz, sapphire. In certain embodiments, the sample container is a glass with a wall having a zwitterion coating, such as, for example, a zwitterion silane coating (as described in the experimental section below). The sample container may be a plastic such as, for example, polycarbonate, polyvinyl chloride (PVC), polyurethane, polyether, polyamide, polyimide, or a copolymer of these thermoplastics, including polyester, eg PETG (glycol) among other polymer plastic materials. Polyethylene of interest, which can be formed from modified polyethylene terephthalate, etc., is based on poly (ethylene terephthalate) (PET), bottle grade PET (monoethylene glycol, terephthalic acid, and other commonomers such as isophthalic acid, cyclohexene dimethanol). Poly (alkylene terephthalate) such as poly (butylene terephthalate) (PBT), and poly (hexamethylene terephthalate); poly (ethylene adipate), poly (1,4-butylene adipate), poly (hexa). Poly (alkylene adipate) such as methylene adipate; poly (alkylene svelate) such as poly (ethylene svelate); poly (alkylene sebacate) such as poly (ethylene sebacate); poly (ε-caprolactone) and poly (ε-caprolactone) β-Propiolactone); Poly (alkylene isophthalate) such as poly (ethylene isophthalate); Poly (alkylene 2,6-naphthalenedicarboxylate) such as poly (ethylene 2,6-naphthalenedicarboxylate); Poly Poly (alkylene sulfonyl-4,4'-dibenzoate) such as (ethylene sulfonyl-4,4'-dibenzoate); poly (p-phenylene alkylene dicarboxylate) such as poly (p-phenylene ethylene dicarboxylate) Poly (trans-1,4-cyclohexanediylalkylene dicarboxylate) such as poly (trans-1,4-cyclohexanediylethylene dicarboxylate); poly (1,4-cyclohexane-dimethyleneethylene dicarboxylate) etc. Poly (1,4-cyclohexane-dimethylenealkylenedicarboxylate); poly ([2.2.2]-bicyclooctane-1,4-dimethyleneethylenedicarboxylate) and other polys ([2.2.2]-bicyclo Octane-1,4-dimethylenealkylene dicarboxylate); (S)-polylactice, (R, S)-polylactice, poly (tetramethylglycolide), and poly (lactide-co-glycolide) lactic acid polymers And Copolymers; Polycarbonate of Bisphenol A, 3,3'-Dimethyl Bisphenol A, 3,3', 5,5'-Tetrachlorobisphenol A, 3,3', 5,5'-Tetramethylbisphenol A; Poly (p) -Polycarbonates such as phenylene terephthalamide); polyesters such as polyethylene terephthalate, such as Mylar ™ polyethylene terephthalate, may include, but are not limited to.

In multiple embodiments, the sample holder ranges from 100 nm to 1500 nm including, for example, 150 nm to 1400 nm, 200 nm to 1300 nm, 250 nm to 1200 nm, 300 nm to 1100 nm, 350 nm to 1000 nm, 400 nm to 900 nm, and 500 nm to 800 nm, such as 532 nm. Can pass the light of.

As outlined above, the method irradiates the sample with a monochromatic light source at the first and second irradiations and resonates Raman scattering at the first and second irradiations. Includes a step of determining strength. To detect resonant Raman scattering, the sample of interest in the method of interest comprises a compound that exhibits resonant Raman scattering upon irradiation with a monochromatic light source. In some embodiments, the compound is a chromophore whose incident irradiation frequency of a monochromatic light source is close in energy to the electronic transitions of the compound. For example, in certain cases, the method is, for example, 90 cm ^-1 or less, 80 cm ^-1 or less, 70 cm ^-1 or less, 60 cm ^-1 or less, 50 cm ^-1 or less, 40 cm ^-1 or less, 30 cm ^-1 or less, 25 cm ^-1 . Below, 20 cm ^-1 or less, 15 cm ^-1 or less, 10 cm ^-1 or less, 5 cm ^-1 or less, 4 cm ^-1 or less, 3 cm ^-1 or less, 2 cm ^-1 or less, 1 cm ^-1 or less, 0.5 cm ^-1 or less, 0.1 cm The chromophore in the sample, including the steps of irradiating with a monochromatic light source having a frequency of ^-1 or less, 0.05 cm ^-1 or less, 0.01 cm ^-1 or less, and 0.001 cm ^-1 or less from the electronic transition of the chromophore in the sample. Including the step of irradiating the sample with a monochromatic light source having a frequency of 100 cm ^-1 or less from the electronic transition of.

Depending on the wavelength of irradiation and the type of monochromatic light source (eg, laser), the chromophores in the sample of the method of interest may differ. In some embodiments, the chromophore is a hydrophobic compound that exhibits resonant Raman scattering in response to laser irradiation. In some cases, the chromophore is a carotene or carotenoid. For example, the carotenoids of interest are carotene (eg, α-carotene, β-carotene, γ-carotene, δ-carotene, ε-carotene, lycopene, etc.) and xanthophylls (eg, lutein, zeaxanthin, neoxanthin, violaxanthin, etc.). Fravoxanthin, α- and β-cryptoxanthin, etc.), but not limited to these. In certain embodiments, the chromophore is lycopene.

In some embodiments, the chromophore binds to another component in the sample, such as to increase the solubility of the chromophore in the sample. In certain cases, the chromophore is hydrophobic and is non-covalently bound to the albumin protein. Albumin proteins of interest are human serum albumin (HSA; gene ID: 213); bovine serum albumin (BSA; gene ID: 280717); mouse albumin (gene ID: 11657); rat albumin (gene ID: 24186); goat. Albumin (gene ID: 100860821); donkey albumin (gene ID: 106835108); horse albumin (gene ID: 1000034206); camel albumin (gene ID: 105080389 or 105091295), etc., or human serum albumin (HSA; gene ID: 213) Bovine serum albumin (BSA; gene ID: 280717); mouse albumin (gene ID: 11657); rat albumin (gene ID: 24186); goat albumin (gene ID: 100860821); donkey albumin (gene ID: 106835108); horse Albumin (gene ID: 1000034206); camel albumin (gene ID: 105080389 or 105091295) and at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or It includes, but is not limited to, proteins having an amino acid sequence having 100% albumin sequence identity.

In certain embodiments, the samples of the present disclosure are hydrophobic compounds that are non-covalently attached to an albumin protein, such as those described in US Patent Application Publication No. 2016/0324933, filed May 6, 2016. The disclosure of the publication of the US patent application, including (eg, chromogen), is incorporated herein by reference in its entirety.

Samples of the method of interest may also include free radical scavengers. As used herein, the term "free radical scavenger" is used in its conventional sense to refer to a compound that reacts with free radicals in a composition, forms a complex, or removes free radicals in a composition. .. Free radical scavengers of the method of interest can be antioxidants, oxygen scavengers, gas ion scavengers, hydroxyl radical scavengers and the like. In certain embodiments, the free radical scavenger is bilirubin or a derivative thereof, eg, unconjugated bilirubin that is not water soluble at pH 7.2.

In some embodiments, bilirubin or a derivative thereof non-covalently binds to a protein such as the albumin protein described above. The term "non-covalent" is used herein in the conventional sense to refer to the interaction of bilirubin or a derivative thereof with a protein, and among other types of non-covalent bonds, dipole-dipole bonds, It can include van der Waals interactions, ionic bonds, ionic-dipole bonds, and hydrogen bonds. In some cases, bilirubin or its derivatives are located at least partially within the albumin protein. In certain cases, bilirubin or its derivatives bind to the binding site of albumin protein.

Free radical scavengers are, for example, 0.005 μM to 4.9 μM, 0.01 μM to 4.8 μM, 0.05 μM to 4.7 μM, 0.1 μM to 4.6 μM, 0.5 μM to 4.5 μM, 0.75 μM to 4 μM, and 0.75 μM to 1.5 μM, It may be present in the sample in varying amounts ranging from 0.001 μM to 5 μM, including, for example, 0.75 μM to 1.25 μM. In certain cases, the free radical scavenger is bilirubin or a derivative thereof, eg 0.005 μM to 4.9 μM, 0.01 μM to 4.8 μM, 0.05 μM to 4.7 μM, 0.1 μM to 4.6 μM, 0.5 μM to 4.5 μM, 0.75 μM. It is present in the sample in an amount of 0.001 μM to 5 μM, including up to 4 μM and 0.75 μM to 1.5 μM, eg 0.75 μM to 1.25 μM.

In some embodiments, the sample comprises a reducing material. As used herein, the term "reducing substance" is used in its conventional sense to refer to a compound that loses (or "donates") electrons to another species in a redox chemical reaction. In certain embodiments, the reducing substance is glutathione or a derivative thereof.

The reducing substance can be present in the sample in varying amounts ranging from 0.001 mg / mL to 10 mg / mL, 0.005 mg / mL to 9 mg / mL, 0.01 mg / mL to 8 mg / mL, 0.05 mg / mL to 7 mg. For example, 0.1 mg / mL to 1 mg / mL, such as / mL and 0.1 mg / mL to 6 mg / mL. In certain cases, the reducing substance is glutathione or a derivative thereof and may be present in the sample in an amount of 0.001 mg / mL-10 mg / mL, 0.005 mg / mL-9 mg / mL, 0.01 mg / mL-8 mg /. mL, 0.05 mg / mL to 7 mg / mL, 0.1 mg / mL to 6 mg / mL, etc., for example, 0.1 mg / mL to 1 mg / mL.

の化合物であり、式中、
R₁およびR₂は、水素、アルキル、置換アルキル、シクロアルキル、置換シクロアルキル、アリール、置換アリール、ヘテロアルキル、置換ヘテロアルキル、ヘテロシクロアルキル、置換ヘテロシクロアルキル、ヘテロアリール、および置換ヘテロアリールから独立に選択され、
X₁およびX₂は、N、O、またはSから独立に選択され、
R₃は、水素、アルキル、置換アルキル、アミノ、ハロゲン、シアノ、アルコール、またはアルコキシである。 In some embodiments, the sample comprises a cross-linking material. In some cases, the cross-linking material can be a disulfide crosslinker (eg, a compound having a disulfide link that exchanges a disulfide bond with a cysteine group on a protein). In some cases, the cross-linking material has a core that is a glutamic acid derivative, such as a glutamic acid derivative with one or more disulfide linkages. In certain embodiments, the cross-linking material is of formula (I) :.

In the formula,
R ₁ and R ₂ are from hydrogen, alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, aryl, substituted aryl, heteroalkyl, substituted heteroalkyl, heterocycloalkyl, substituted heterocycloalkyl, heteroaryl, and substituted heteroaryl. Selected independently,
X ₁ and X ₂ are selected independently of N, O, or S,
R ₃ is hydrogen, alkyl, substituted alkyl, amino, halogen, cyano, alcohol, or alkoxy.

In some embodiments, R ₁ and R ₂ are heteroaryl, X ₁ and X ₂ are N, and R ₃ is amino.

の化合物である。 In certain embodiments, the cross-linking material is of formula (DS-1).

It is a compound of.

The cross-linking substance has a molar ratio of cross-linking substance: albumin protein, for example, 1: 9 to 9: 1, 1: 8 to 8: 1, 1: 7 to 7: 1, 1: 6 to 6: 1, 1: 5. May be present in the sample in an amount of 1:10 to 10: 1, including ~ 5: 1, 1: 4 to 4: 1, 1: 3 to 3: 1, etc., and 1: 2 to 2: 1. .. In certain embodiments, the molar ratio of cross-linking material: albumin protein in the sample of the method of interest is approximately 1: 2. According to a particular aspect, the method comprises the following steps: The step of irradiating a sample over a period of time with a monochromatic light source, and the step of determining the rate of change of one or more of the resonance Raman scattering and fluorescence scattering. In these embodiments, the sample is, for example, 10 seconds to 1500 seconds, 30 seconds to 1400 seconds, 45 seconds to 1300 seconds, 60 seconds to 1200 seconds, 120 seconds to 1000 seconds, 200 seconds to 800 seconds, and 400 seconds. It can be irradiated for any desired period, including up to 600 seconds. In some embodiments, a normalized rate of change in the intensity of the resonant Raman scattering is calculated. In another embodiment, a normalized rate of change in irradiation intensity from a monochromatic light source (eg, a laser) is also calculated.

In certain embodiments, the method irradiates the sample (eg, by focusing a monochromatic light source with a collimator as described above) at the interface between the sample and the surface of the sample holder (eg, the wall of the sample container). Including the process. "Interface" means the space where the surface of the sample holder (eg, the wall of the container) comes into contact with the sample. In some embodiments, the interface illuminated by the monochromatic light source in the method of interest can range from about 0.01 mm to 2 mm from the surface of the wall of the container (ie, where the sample contacts the container), eg 0.02 mm to 1.9. mm, 0.03mm-1.8mm, 0.04mm-1.7mm, 0.05mm-1.6mm, 0.06mm-1.5mm, 0.07mm-1.4mm, 0.08mm-1.3mm, 0.09mm-1.2mm, 0.1mm-1mm, for example , 0.2 mm from the surface of the sample holder (eg, the wall of the container). In these embodiments, the sample holder (eg, a glass vial) remains substantially stationary (eg, no vibration, agitation, etc.) and the rate of the interface liquid layer irradiated by the monochromatic light source is near zero or It is zero. For example, the liquid velocity of the interface sample layer illuminated by a monochromatic light source is, for example, 10 ^-3 cm ³ / sec or less, 10 ^-4 cm ³ / sec or less, 10 ^-5 cm ³ / sec, 10 ^-6 cm ³ /. In 10 ^-2 cm ³ / sec or less, including 10 ^-7 cm ³ / sec or less, 10 ^-8 cm ³ / sec or less, 10 ^-9 cm ³ / sec or less, and 10 ^-10 cm ³ / sec or less. possible. In certain embodiments, the sample velocity in the interface layer illuminated by a monochromatic light source in the method of interest is 0 cm ³ / sec.

The method also calculates the rate of change of one or more of the resonance Raman scattering and fluorescence scattering in response to the change in irradiation intensity from the first irradiation intensity to the second irradiation intensity, and the resonance Raman. It comprises the step of determining the spectral response of a sample by comparing the rate of change in intensity of one or more of the scattering and fluorescent scattering with the rate of change in irradiation intensity from a monochromatic light source. In some embodiments, the spectroscopic response of the sample indicates the physical time course of the sample. In another embodiment, the spectral response of the sample indicates the chemical aging of the sample. In yet another embodiment, the spectroscopic response of the sample indicates the presence of actively metabolizing microorganisms in the sample.

In a plurality of embodiments, the method comprises the following steps. The step of irradiating a sample over a period of time with a monochromatic light source, the step of determining the rate of change of one or more of the resonance Raman scattering and the fluorescence scattering, and the resonance Raman scattering to determine the net signal. And the step of comparing the rate of change of one or more of the fluorescence scattering with the rate of change of the normalized irradiation intensity by a monochromatic light source. In the step of calculating the net signal, the method may further include the following steps: Resonance The step of calculating the normalized rate of change of one or more intensity of Raman scattering and fluorescence scattering and the normalized rate of change of irradiation intensity, and resonance to determine the net signal. The step of comparing the normalized rate of change of one or more of Raman scattering and fluorescent scattering with the normalized rate of change of irradiation intensity.

In aspects of the present disclosure, the term "normalized" as used herein is used in its conventional sense to refer to adjusting values measured at different scales to a nominally common scale. For example, normalized values allow the corresponding normalized values to be compared for different datasets, eliminating the effect of the overall effect. In certain embodiments, determining the normalized rate of change involves dividing the gradient (eg, the gradient of the linear fitting of the dataset) by the absolute value observed at time = 0.

The range of normalized intensity can vary depending on the chromophore and monochromatic light source. In some embodiments, the monochromatic light source is a laser, and the normalized intensity of the laser is, for example, 15% or more, 25% or more, 50% or more, 75% or more, 90% or more, and 99% or more. Changes by 10% or more including. In these embodiments, the normalized rate of change in laser intensity is, for example, -9 × 10 ^-5 per second to 9 × 10 ^-5 per second, -8 × 10 ^-5 per second to 8 × 10 ^-5 per second, -7. × 10 ^-5 every second to 7 × 10 ^-5 every second, -6 × 10 ^-5 every second to 6 × 10 ^-5 every second, -5 × 10 ^-5 every second to 5 × 10 ^-5 every second, -4 × 10 ^-5 every second ~ 4 x 10 ^-5 per second, -3 x 10 ^-5 per second ~ 3 x 10 ^-5 per second, -2 x 10 ^-5 per second ~ 2 x 10 ^-5 per second, etc., and -1 x 10 ^-5 per second ~ 1 x It can be in the range of -10 × 10 ^-5 per second to 10 × 10 ^-5 per second, including 10 ^-5 per second. The normalized rate of change in the intensity of the resonance Raman scattering is also, for example, -9 × 10 ^-5 / s to 9 × 10 ^-5 / s, -8 × 10 ^-5 / s to 8 × 10 ^-5 / s, -7 ×. 10 ^-5 every second to 7 x 10 ^-5 every second, -6 x 10 ^-5 every second to 6 x 10 ^-5 every second, -5 x 10 ^-5 every second to 5 x 10 ^-5 every second, -4 x 10 ^-5 every second ~ 4 x 10 ^-5 per second, -3 x 10 ^-5 per second to 3 x 10 ^-5 per second, -2 x 10 ^-5 per second to 2 x 10 ^-5 per second, etc., and -1 x 10 ^-5 per second to 1 x 10 ^-5 including -5 per second -10 x 10 ^-5 per second to 10 x 10 ^-5 per second.

The method according to a particular embodiment comprises the step of characterizing the sample composition with a net signal. For example, a net signal can be used to detect changes in the sample composition or to determine if the two compositions have equivalent or different configurations. In certain embodiments, the net signal is used to characterize the amount of gas present in the sample. In other embodiments, the net signal can be used to determine if an actively metabolizing microorganism is present in the sample.

In one aspect, the method comprises the following steps. The step of irradiating a first sample over a period of time with a monochromatic light source (eg, a laser) for a period of time to determine the first rate of change at one or more of the Raman and fluorescent scattering; and monochromatic light sources. A step of irradiating a second sample over a plurality of intensities over a period of time and determining the rate of change at one or more of the Raman and Fluorescent Scattering for the second sample. The net signal of the first sample is the normalized rate of change of the intensity of one or more of the resonant Raman and fluorescence scattering, and the normalized rate of change of the intensity of the first sample irradiated by a monochromatic light source. Calculated by comparing with. The net signal of the second sample is the normalized rate of change in the intensity of one or more of the resonant Raman and fluorescence scattering, and the normalized rate of change in the intensity of the second sample's irradiation with a monochromatic light source. It is determined by comparing with.

In some embodiments, the net signal of the first sample and the net signal of the second sample are the same. In other embodiments, the net signal of the first sample and the net signal of the second sample are different. In some cases, this difference indicates that the amount of gas (eg, carbon dioxide, oxygen, methane, nitrogen, etc.) present in the first and second samples is different. In other cases, this difference indicates that the types of gas present in the first sample and the second sample are different. In yet other cases, the difference between the first net signal and the second net signal indicates that the amount of solubilized gas (ie, the gas dissolved in the sample solution) is different.

Methods of Determining the Presence of Microorganisms in a Sample by Resonant Raman Scattering Aspects of the present disclosure also include determining the presence of microorganisms in a sample (eg, actively metabolizing microorganisms) by resonant Raman scattering. The method according to a particular embodiment includes the following steps. The step of irradiating a sample over a period of time with a monochromatic light source and calculating the rate of change in the intensity of the resonant Raman scattering for the first sample; the normalized rate of change in the intensity of the resonant Raman scattering of the sample, the monochromatic source. The step of calculating the net signal of the sample by comparing it with the normalized rate of change of the irradiation intensity of the sample; and the step of determining the presence of microorganisms in the sample based on the net signal of the sample.

The method according to a particular embodiment includes the following steps. Combining a sample with an albumin-containing reagent containing a ligand in a sample holder; focused at the interface between the sample and the surface of the sample holder with either constant or time-varying light intensity and absorbed by the ligand. The process of irradiating a sample with a monochromatic light source; the process of collecting scattered light from the irradiated sample at multiple different times and measuring the Raman and fluorescent signals from the scattered light; the intensity of the Raman and fluorescent signals for the sample. The step of calculating the rate of change over time; the step of correcting the calculated rate of change in the intensity of the Raman and fluorescent signals to obtain the net signal; and with the net signal and one or more predetermined thresholds. The step of determining the presence of microorganisms in a sample based on the comparison of.

In some cases, the step of correcting the rate of change in intensity of the Raman signal and the fluorescent signal comprises the step of characterizing the spectral output from the standard sample. In other cases, the step of correcting the rate of change in intensity of the Raman signal comprises the step of characterizing the fluorescence output from the reagent.

In a particular embodiment, the step of correcting the rate of change in the intensity of the Raman signal and the fluorescent signal includes the following steps. The step of determining the rate of change in total output from the standard reference sample; and the net signal is calculated as the rate of change in the intensity of the resonant Raman scattering minus the rate of change in the intensity of the resonant Raman scattering from the standard sample. Process.

In another embodiment, the step of correcting the rate of change in the intensity of the Raman signal and the fluorescent signal includes the following steps. The step of determining the rate of change in the intensity of the fluorescent scattering from the standard reference sample; and the net signal calculated as the rate of change in the intensity of the resonant Raman scattering minus the rate of change in the intensity of the fluorescent scattering from the standard sample. Process to do.

In certain embodiments, the presence of microorganisms is determined when the net signal of the sample exceeds a predetermined threshold. Depending on the chromophore (eg, lycopene non-covalently bound to albumin) and microorganisms in the sample, the thresholds are, for example, -0.5 to -1.0 x 10 ^-5 / sec, -1 to -2 x 10 ^-5 / sec. Etc., which can vary in the range of -0.5 to -4 × 10 ^-5 / sec and include a predetermined threshold when the net signal is 1. In some embodiments, one or more predetermined thresholds are set by performing the above method on one or more control samples containing the inoculum at the lower limit of the concentration of the clinically infected sample. Will be done. In some embodiments, the net signal of the sample is a threshold value that comprises, for example, 5% or more, 10% or more, 15% or more, 25% or more, 50% or more, 75% or more, and 90% or more. If the threshold is exceeded by 1% or more, it is determined that microorganisms are present in the sample. In certain cases, if the net signal of the sample exceeds the threshold by more than 2 times, including, for example, 3 times or more, 5 times or more, and 10 times or more, it is determined that the sample has microorganisms.

In other embodiments, determining the presence of a microorganism comprises comparing the net signals of two different samples. In some embodiments, one of the samples is a microbe-free reference sample. In these embodiments, the method may include the following steps. The step of irradiating the first sample over a period of time with a monochromatic light source and calculating the rate of change in the intensity of the resonant Raman scattering for the first sample; the normalized intensity of the resonant Raman scattering for the first sample. The step of calculating the net signal of the first sample by comparing the rate of change with the normalized rate of change of the irradiation intensity of the first sample by a monochromatic light source; the second sample over multiple intensities by a monochromatic light source. The step of determining the rate of change in the intensity of the resonance Raman scattering with respect to the second sample by irradiating the sample for the fixed period; The step of calculating the net signal of the second sample by comparing it to the normalized rate of change of the irradiation intensity of the sample; and by comparing the net signal of the first sample with the net signal of the second sample. , The step of determining the presence of a microorganism in one or more of the first sample or the second sample.

In some cases, if the net signal of the second sample is greater than the net signal of the first sample, it is determined that the microorganism is present in the second sample. In other cases, if the net signal of the first sample is greater than the net signal of the second sample, it is determined that the microorganism is present in the first sample. If the first sample is a reference sample and the net signal of the second sample is greater than the net signal of the reference sample, it is determined that the microorganism is present in the second sample.

Methods of Determining the Presence of Microorganisms in a Sample by Fluorescence The aspects of the present disclosure also include the step of determining the presence of microorganisms in a sample (eg, actively metabolizing microorganisms) by fluorescence. The method according to a particular embodiment includes the following steps. The step of irradiating a sample with a monochromatic light source for a period of time and detecting fluorescence from the sample over that period of time; a normalized rate of change in the intensity of the detected fluorescence produced by the sample of the fluorescence produced by the control. The step of calculating the rate of change in fluorescence due to the presence of microorganisms by comparing with the normalized rate of change; and the microorganisms in the sample based on the calculated changes in fluorescence of the sample compared to a given threshold. The process of determining the existence of.

In some embodiments, the sample is irradiated with a monochromatic light source over multiple intensities. Fluorescence produced by the sample and control is detected at 2500 cm ^-1 to 3500 cm ^-1 , such as 3000 cm ^-1 . In some embodiments, the control and sample comprise a reagent composition comprising a hydrophobic compound that is non-covalently bound to an albumin protein. The control sample is, in some cases, a composition comprising a reagent composition and free of microorganisms. For example, the control is a composition having the same components as the sample in the absence of microorganisms. In some embodiments, the control comprises only the components of the reagent composition. In another embodiment, the control comprises components of the reagent composition and microorganism-free plasma.

Methods for Calculating Signal-to-noise Ratios Aspects of the present disclosure also include methods for calculating signal-to-noise ratios in nonlinear Resonance Raman spectroscopy. In some embodiments, the signal-to-noise ratio is calculated by the following steps. The step of irradiating the first sample over a period of time with a monochromatic light source and determining the rate of change in the intensity of the resonance Raman scattering for the first sample; the first sample to determine the first average rate of change. The step of calculating the mean rate of change of the intensity of the resonance Raman scattering by; the step of calculating the standard deviation of the first mean rate of change; The step of determining the rate of change in the intensity of the resonance Raman scattering for the sample; the step of calculating the average rate of change in the intensity of the resonance Raman scattering by the second sample to determine the second average rate of change; the second average The process of calculating the standard deviation of the rate of change; the process of subtracting the second average rate of change from the first rate of change to determine the average rate of change of the signal; the first standard to determine the standard deviation of the signal. The step of adding the deviation and the second standard deviation; and the step of dividing the average rate of change of the signal by the standard deviation of the signal to determine the signal noise ratio of the resonance Raman response.

In some embodiments, the first sample comprises a hydrophobic compound non-covalently bound to an albumin protein and the second sample comprises a hydrophobic compound non-covalently bound to an albumin protein and a microorganism. For example, the first sample can contain lycopene non-covalently bound to albumin protein and the second sample can contain lycopene and microorganisms non-covalently bound to albumin protein.

In certain embodiments, the method comprises the following steps: The step of increasing the signal-to-noise ratio by contacting the first sample and the second sample with a free radical scavenger such as bilirubin or a derivative thereof. In certain embodiments, the free radical scavenger (eg, bilirubin or a derivative thereof) is, for example, 10% or more, 15% or more, 25% or more, 50% or more, 75% or more, 90% or more, 95% or more, 1.5 times. Increase the signal-to-noise ratio by 5% or more, including 2 times or more, 3 times or more, 5 times or more, and 10 times or more.

Free radical scavengers in these embodiments are, for example, 0.005 μM to 4.9 μM, 0.01 μM to 4.8 μM, 0.05 μM to 4.7 μM, 0.1 μM to 4.6 μM, 0.5 μM to 4.5 μM, 0.75 μM to 4 μM, and 0.75. It can be present in varying amounts in the range 0.001 μM to 5 μM, including μM to 1.5 μM, eg 0.75 μM to 1.25 μM. In certain cases, bilirubin or derivatives thereof can be used, for example, 0.005 μM to 4.9 μM, 0.01 μM to 4.8 μM, 0.05 μM to 4.7 μM, 0.1 μM to 4.6 μM, 0.5 μM to 4.5 μM, 0.75 μM to 4 μM, and the like. Contact with the first and second samples in an amount of 0.001 μM to 5 μM, including 0.75 μM to 1.5 μM, eg 0.75 μM to 1.25 μM.

Depending on the amount of free radical scavenger (eg, birylbin or a derivative thereof), the period of irradiation of the first and second samples is, for example, 10 seconds to 2000 seconds, 30 seconds to 1750 seconds, 45 seconds to 1500 seconds, It changes from 60 seconds to 1250 seconds, 120 seconds to 1000 seconds, 200 seconds to 800 seconds, etc., and 400 seconds to 600 seconds. In some embodiments, the free radical scavenger (eg, bilirubin or a derivative thereof) is present in the first and second samples in an amount ranging from 0.75 μM to about 1.25 μM, the first sample and the second. Sample 2 is irradiated for a period of 600 to 900 seconds. In one example, a free radical scavenger (eg, bilirubin or a derivative thereof) is present in the first and second samples in an amount of about 1 μM, with the first and second samples 600-900 seconds. Irradiated for the period of. In another example, a free radical scavenger (eg, bilirubin or a derivative thereof) is present in the first and second samples in an amount of about 1.5 μM, with the first and second samples being 900 seconds. Irradiated for a period of ~ 1200 seconds.

Methods for Compensating for Thermal Drift The aspects of the present disclosure also include methods for compensating for thermal drift of monochromatic light sources in non-linear resonance Raman spectroscopy. In some embodiments, the monochromatic light source is a laser and the method of interest compensates for the thermal drift of the laser in nonlinear resonance Raman spectroscopy.

In certain embodiments, the method comprises first determining whether or not the monochromatic light source exhibits thermal drift. In these embodiments, the method comprises the following steps: A step of irradiating a reference composition over a plurality of intensities with a monochromatic light source for a certain period of time to determine the rate of change in the intensity of resonance Raman scattering . Irradiation and determination steps, including reference compounds that do not show a change in the intensity of the reference composition; A step of determining the net signal of the reference composition by comparing it with the rate of change of the irradiation intensity of the reference composition.

In these embodiments, the reference composition comprises a reference compound, such as NIST calibration standard SRM 2242, which does not show a change in the intensity of resonant Raman scattering in response to changes in irradiation intensity.

The method also comprises compensating for thermal drift of a monochromatic light source. To compensate for laser drift, the method may include the following steps: A step of irradiating a sample over a period of time with a monochromatic light source to determine the rate of change in the intensity of resonant Raman scattering; a step of determining the rate of change in output from the reference composition to generate a correction factor; The step of reducing the correction factor from the determined rate of change of the intensity of the resonant Raman scattering for the sample in order to correct for the thermal drift of the monochromatic light source.

Methods for Characterizing Microbial Sensitivity to Antimicrobial Agents Using Resonant Raman Scattering Aspects of the present disclosure also include methods for determining the susceptibility of microorganisms to antimicrobial agents using resonant Raman scattering. In some embodiments, the method comprises determining the minimum inhibitory concentration (MIC) of the antimicrobial agent (ie, the minimum concentration of the antimicrobial agent that inhibits the growth of the microorganism). In another aspect, the method comprises determining the minimum bactericidal concentration (MBC) of the antimicrobial agent (ie, the minimum concentration of antimicrobial agent required to kill the microorganism).

In a plurality of embodiments, the method comprises the following steps. It is a step of irradiating a plurality of samples containing microorganisms and antimicrobial agents over a plurality of intensities with a monochromatic light source for a certain period of time, and determining the rate of change in the intensity of resonance Raman scattering for each irradiated sample. Irradiation and determination steps, including the same concentration of microorganisms and different concentrations of antimicrobial agent; normalized changes in the intensity of resonance Raman scattering for each sample, normalized changes in the intensity of irradiation for each sample with a monochromatic light source. The step of determining the net signal of each sample by comparing with the rate; and the step of determining the susceptibility of the microorganism to the antimicrobial agent based on the net signal of multiple samples.

In some embodiments, the method further comprises comparing the net signal for each sample. For example, the step of comparing net signals for each sample may include plotting the net signal of each sample as a function of the logarithm of the concentration of antimicrobial agent in each sample. In these embodiments, the method may further comprise the following steps: A step of determining one or more of the concentration of an antimicrobial agent indicating a decrease in net signal and the concentration of an antimicrobial agent indicating an increase in net signal. In some embodiments, the concentration of antimicrobial agent that initially exhibits a decrease in the net signal on the plot is determined to be the minimum inhibitory concentration (MIC) of the antimicrobial agent against the microorganism. In another embodiment, the concentration of antimicrobial agent indicating an increase in the net signal on the plot is determined to be the minimum bactericidal concentration (MBC) of the antimicrobial agent against the microorganism. In some embodiments, the method comprises determining the metabolic activity of the microorganism in each sample based on the net signal of each sample. For example, the method may include the following steps. A step of determining the concentration of an antimicrobial agent indicating a decrease in metabolic activity, or a step of determining the concentration of an antimicrobial agent indicating an increase in metabolic activity. In these embodiments, the minimum inhibitory concentration of the antimicrobial agent can be determined to be the concentration of the antimicrobial agent that exhibits a decrease in the metabolic activity of the microorganism. The minimum bactericidal concentration of the antimicrobial agent can be determined to be the concentration of the antimicrobial agent that indicates an increase in the metabolic activity of the microorganism.

Depending on the type of microorganism, the antimicrobial agents of interest may include, but are not limited to, antibacterial agents, antifungal agents, antiviral agents, antiparasitic agents, and antimicrobial repellents. Suitable antibiotics are fluoroquinolones such as cyprofloxacin, norfloxacin, ofloxacin, enoxacin, penicillin, freroxacin, enlofloxacin, malvofloxacin, salafloxacin, orbifloxacin, danofloxacin, streptomycin, netylmycin, canamycin. , Neomycin, Tobramycin, Amicacin, Sisomycin, Ribostamycin, Dibecacin, Flamycetin, Gentamycin and other aminoglycosides, Penicillin, Ampicillin, Amoxycillin, Nafcillin, Oxacillin, and Ticarcillin and other penicillin and aminopenicillin, Ceftriaxons, Cephalosporins, Cephalosporins such as cephalosporins and cephalosporins such as cefthioflu, β-lactams such as clavillin which can be used in combination with penicillin or aminopenicillin, macrolides such as clarislomycin and erythromycin, and dactinomycin. , Clindamycin, Naridicic acid, Chloramphenicol, Riphanpin, Clofadimin, Spectinomycin, Polymixin B, Collistin, Minocillin, Bancomycin, Hyglomycin B or C, Fushidic acid, Trimethoprim, and Cefotaxim Including, but not limited to these. The concentration of the antimicrobial agent in each sample vial is, for example, 0.005 μg / mL to 900 μg / mL, 0.01 μg / mL to 800 μg / mL, 0.05 μg / mL to 700 μg / mL, 0.1 μg / mL to 500 μg / mL, It may vary depending on the type of antimicrobial agent, such as 0.5 μg / mL to 250 μg / mL, and 0.001 μg / mL to 1000 μg / mL containing 1 μg / mL to 100 μg / mL. In some embodiments, the range of antimicrobial agent amounts across the sample ranges from concentrations below the minimum inhibitory concentration of the antimicrobial agent to concentrations above the minimum bactericidal concentration of the antimicrobial agent. For example, the amount of antimicrobial agent may include increasing the amount in the sample vial under test. For example, the sample vial is 0.125 μg / mL-8 μg / mL, such as 0.125 μg / mL, 0.25 μg / mL, 0.5 μg / mL, 1 μg / mL, 2 μg / mL, 4 μg / mL, and 8 μg / mL concentrations. May contain an antimicrobial agent in a concentration in the range of.

In the method for characterizing the susceptibility of microorganisms to antimicrobial agents according to the present disclosure, the amount of microorganisms in each sample may be the same and may be 10 colony forming units (CFU) or greater, eg, 11 CFU or greater. Includes 12CFU and above, 13CFU and above, 14CFU and above, 15CFU and above, 20CFU and above, 25CFU and above, and 50CFU and above. In certain embodiments, the method may include the following steps: A step of preparing a microorganism-containing composition having a predetermined amount of microorganisms (for example, 100 CFU), and a step of aliquoting an equal amount of the microorganism-containing composition to each of the samples.

Microorganisms and antimicrobial agents, in certain embodiments, may be used, for example, 0.5 minutes or longer, 1 minute or longer, 2 minutes or longer, 5 minutes or longer, 10 minutes or longer, 15 minutes or longer, 20 minutes before irradiating the sample with a monochromatic light source. Incubate for a predetermined period of time, including more than 30 minutes, more than 45 minutes, and more, and more than 60 minutes. For example, antimicrobial agents and microorganisms may be used, for example, 1 minute to 55 minutes, 2 minutes to 50 minutes, 3 minutes to 45 minutes, 4 minutes to 40 minutes, 5 minutes to 35 minutes, etc. before being irradiated by a monochromatic light source. , And can be incubated in the sample for a predetermined period of 0.5-60 minutes, including 10-30 minutes, eg 20 minutes.

Methods for Characterizing Microbial Sensitivity to Antimicrobial Agents by Fluorescence Spectroscopy The aspects of the present disclosure also include methods for determining the susceptibility of microorganisms to antimicrobial agents by fluorescence spectroscopy. In some embodiments, the method comprises determining the minimum inhibitory concentration (MIC) of the antimicrobial agent (ie, the minimum concentration of the antimicrobial agent that inhibits the growth of the microorganism). In another aspect, the method comprises determining the minimum bactericidal concentration (MBC) of the antimicrobial agent (ie, the minimum concentration of antimicrobial agent required to kill the microorganism).

In a plurality of embodiments, the method comprises the following steps. A step of irradiating a plurality of samples having a microorganism and an antimicrobial agent with a monochromatic light source for a certain period of time and detecting fluorescence from each of the samples irradiated for a certain period of time, wherein each sample has the same concentration. Irradiation and detection steps with microorganisms and different concentrations of antimicrobial agent; normalized rate of change in the intensity of the detected fluorescence produced by each sample, normalized rate of change in fluorescence produced by the control. The step of calculating the rate of change in fluorescence in each sample by comparison with; and the step of determining the susceptibility of the microorganism to the antimicrobial agent based on the calculated rate of change in fluorescence of multiple samples. In some embodiments, each sample is irradiated with a monochromatic light source over multiple intensities.

In some embodiments, the method further comprises comparing the calculated rate of change of fluorescence for each sample. In some cases, the step of comparison involves plotting the calculated rate of change in fluorescence of each sample as a function of the logarithm of the concentration of antimicrobial agent in each sample. In another aspect, the method further comprises determining the concentration of antimicrobial agent that exhibits a decrease or increase in the rate of change in fluorescence. In yet another embodiment, the method further comprises determining the metabolic activity of the microorganism in each sample based on the calculated rate of change in fluorescence of each sample. For example, in other cases, the method comprises determining the concentration of an antimicrobial agent that exhibits an increase or decrease in metabolic activity.

Fluorescence produced by the sample and control is detected at 2500 cm ^-1 to 3500 cm ^-1 , such as 3000 cm ^-1 . In some embodiments, the control and sample comprise a reagent composition comprising a hydrophobic compound that is non-covalently bound to an albumin protein. The control sample is, in some cases, a composition comprising a reagent composition and free of microorganisms. For example, the control is a composition having the same components as the sample in the absence of microorganisms. In some embodiments, the control comprises only the components of the reagent composition. In another embodiment, the control comprises components of the reagent composition and microorganism-free plasma.

Methods for Determining the Expression of Unknown Microorganisms Using Resonant Raman Scattering Aspects of the present disclosure also include methods for determining the phenotype of unknown microorganisms using resonant Raman scattering. In some embodiments, the method comprises determining whether an unknown microorganism produces a reactive metabolite (eg, a free radical-containing metabolite). In certain embodiments, the metabolite is a reactive species capable of reacting with or cleaving the crosslinks.

In a plurality of embodiments, the method comprises the following steps. A step of irradiating a sample containing microorganisms, cross-linking substances, and albumin proteins with a monochromatic light source over a period of time with a monochromatic light source to determine the rate of change in the intensity of resonant Raman scattering; normalizing the intensity of resonant Raman scattering for the sample. The step of calculating the net signal of the sample by comparing the normalized rate of change with the normalized rate of change of the irradiation intensity of the sample by a monochromatic light source; and the step of determining the cross-linking cleavage based on the net signal of the sample. A determination step in which the degree of cross-linking cleavage indicates the phenotype of the microorganism. In certain embodiments, an increase in the net signal of the sample over time indicates that the microorganism produces a metabolite that cleaves one or more crosslinks in the albumin protein.

の化合物であり、式中、
R₁およびR₂は、水素、アルキル、置換アルキル、シクロアルキル、置換シクロアルキル、アリール、置換アリール、ヘテロアルキル、置換ヘテロアルキル、ヘテロシクロアルキル、置換ヘテロシクロアルキル、ヘテロアリール、および置換ヘテロアリールから独立に選択され、
X₁およびX₂は、N、O、またはSから独立に選択され、
R₃は、水素、アルキル、置換アルキル、アミノ、ハロゲン、シアノ、アルコール、またはアルコキシである。 For example, if the microorganism of interest produces a peptidase enzyme (eg, a peptidase that cleaves a glutamate peptide bond) in a particular embodiment, any suitable reactive cross-linking material is used, depending on the type of microorganism being tested. obtain. In some embodiments, the cross-linking material is a disulfide crosslinker (eg, a compound having a disulfide link that exchanges disulfide bonds with proteins). In certain cases, the cross-linking material is also a glutamic acid derivative, such as a glutamic acid derivative having one or more disulfide linkages. In certain embodiments, the cross-linking material is of formula (I) :.

In the formula,
R ₁ and R ₂ are from hydrogen, alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, aryl, substituted aryl, heteroalkyl, substituted heteroalkyl, heterocycloalkyl, substituted heterocycloalkyl, heteroaryl, and substituted heteroaryl. Selected independently,
X ₁ and X ₂ are selected independently of N, O, or S,
R ₃ is hydrogen, alkyl, substituted alkyl, amino, halogen, cyano, alcohol, or alkoxy.

In some embodiments, R ₁ and R ₂ are heteroaryl, X ₁ and X ₂ are N, and R ₃ is amino.

の化合物である。 In certain embodiments, the cross-linking material is of formula (DS-1).

It is a compound of.

The cross-linking substance has a molar ratio of cross-linking substance: albumin protein, for example, 1: 9 to 9: 1, 1: 8 to 8: 1, 1: 7 to 7: 1, 1: 6 to 6: 1, 1: 5. May be present in the sample in an amount of 1:10 to 10: 1, including ~ 5: 1, 1: 4 to 4: 1, 1: 3 to 3: 1, etc., and 1: 2 to 2: 1. .. In certain embodiments, the molar ratio of cross-linking material: albumin protein in the sample of the method of interest is approximately 1: 2.

Methods for Determining the Expression of Unknown Microorganisms by Fluorescence Spectroscopy The aspects of the present disclosure also include methods for determining the phenotype of unknown microorganisms using fluorescence spectroscopy. In some embodiments, the method comprises determining whether an unknown microorganism produces a reactive metabolite (eg, a free radical-containing metabolite). In certain embodiments, the metabolite is a reactive species capable of reacting with or cleaving the crosslinks.

In a plurality of embodiments, the method comprises the following steps. A step of irradiating a sample containing a microorganism, a cross-linking substance, and an albumin protein with a monochromatic light source for a certain period of time to detect fluorescence from the sample over a certain period of time; a normalized change in the intensity of the detected fluorescence generated by the sample. The step of calculating the rate of change in fluorescence by comparing the rate to the normalized rate of change in fluorescence produced by the control; and the step of determining cross-linking cleavage based on the calculated rate of change in fluorescence of the sample. A determination step in which the degree of cross-linking cleavage indicates the phenotype of the microorganism. In some embodiments, the sample is irradiated with a monochromatic light source over multiple intensities.

Fluorescence produced by the sample and control is detected at 2500 cm ^-1 to 3500 cm ^-1 , such as 3000 cm ^-1 . In some embodiments, the control and sample comprise a reagent composition comprising a hydrophobic compound that is non-covalently bound to an albumin protein. The control sample is, in some cases, a composition comprising a reagent composition and free of microorganisms. For example, the control is a composition having the same components as the sample in the absence of microorganisms. In some embodiments, the control comprises only the components of the reagent composition. In another embodiment, the control comprises components of the reagent composition and microorganism-free plasma.

Systems for Nonlinear Resonance Raman spectroscopy Aspects of the present disclosure also include systems for nonlinear Resonance Raman spectroscopy. The system according to a particular embodiment includes a monochromatic light source, a detector for detecting resonance Raman scattering, and a processor, the processor including a memory operably attached to the processor, and the memory stores instructions. , The instruction is to irradiate the sample with a monochromatic light source at the first and second irradiation intensities when executed by the processor; resonance Raman scattering at the first and second irradiation intensities. To determine the rate of change in the intensity of resonance Raman scattering in response to the change in irradiation intensity from the first irradiation intensity to the second irradiation intensity; and to determine the resonance Raman response of the sample. In addition, the processor is made to compare the rate of change of the intensity of the resonance Raman scattering with the rate of change of the irradiation intensity by the monochromatic light source. The system of interest is also a system that includes (a) a monochromatic light source, (b) an optical tuning component, (c) an optical detector, and (d) a processor, which can be operated by the processor. Containing concatenated memory, the memory stores instructions, which, when executed by the processor, send a monochromatic light source to the sample in the sample holder with a first and second fluorescence intensity for a given period of time. Irradiation; Measuring scattered light from the sample using an optical detector; Determining the intensity of resonance Raman and fluorescence scattering at the first and second irradiation intensities; Intensity of resonance Raman scattering. And to calculate the rate of change in the intensity of the fluorescence scattering; and to have the processor correct for the intensity of the resonance Raman scattering and the rate of change in the intensity of the fluorescence scattering in order to obtain the net signal.

As outlined above, the system includes one or more monochromatic light sources. In a plurality of embodiments, the monochromatic light source of interest outputs light having a narrow wavelength range, including, for example, 25 nm or less, 20 nm or less, 15 nm or less, 10 nm or less, 5 nm or less, and 2 nm or less. In certain embodiments, the monochromatic light source outputs light of a single wavelength. In some cases, the monochromatic light source is a single wavelength laser. In other cases, the monochromatic light source is a monochromatic LED.

In certain embodiments, the light source is a broadband light source that optically communicates with an optical conditioning component that narrows the irradiation bandwidth to a single wavelength. For example, monochromatic light irradiation of a sample for resonance Raman spectroscopy by the method of interest uses a broadband light source coupled to one or more optical bandpass filters, diffraction grids, monochrome meters, or any combination thereof. And, for example, wideband halogen lamps, heavy hydrogen arcclamps, xenon arcclamps, stabilized fiber coupled wideband light sources, continuous spectrum wideband LEDs, super luminescent light emitting diodes, semiconductor light emitting diodes, wide area spectrum LED white light sources, or This can be achieved by using a multi-LED white light source or the like.

In certain embodiments, the system comprises a laser. In some cases, the laser is a continuous wave laser. In other cases, the laser is a pulsed laser. In certain cases, the laser is a diode laser such as an ultraviolet diode laser, a visible light diode laser, and a near infrared diode laser. In some cases, the monochromatic light source is a diode laser that outputs light at wavelengths from 375 nm to 1000 nm, including, for example, 405 nm to 875 nm, 450 nm to 800 nm, 500 nm to 650 nm, and 525 nm to 625 nm. In other cases, the laser is a pulsed laser, such as a solid-state laser. In certain cases, the monochromatic light source is a solid-state laser that outputs light at wavelengths from 375 nm to 1000 nm, including, for example, 405 nm to 875 nm, 450 nm to 800 nm, 500 nm to 650 nm, and 525 nm to 625 nm. Other suitable lasers are helium neon (HeNe) laser, argon laser, krypton laser, xenon ion laser, nitrogen laser, carbon dioxide laser, carbon monoxide laser, excima laser, hydrogen fluoride laser, among other laser types. , Hydrofluoride hydrogen laser, oxygen iodine laser, vapor phase iodine laser, helium cadmium laser, helium mercury laser, helium silver laser, strontium steam laser, neon copper laser, copper steam laser, gold steam laser, manganese steam laser, ruby laser , Nd: YAG laser, NdCrYAG laser, Er: YAG laser, Nd: YLF laser, Nd: YVO ₄ laser, Nd: YCa ₄ O (BO ₃ ) ₃ laser, Nd: glass laser, titanium sapphire laser, thurium YAG laser, Itterbium YAG laser, Yb ₂ O ₃ laser, Itterbium-added glass laser, Holmium YAG laser, Chromium ZnSe laser, Serium-added lithium fluoride strontium aluminum laser, Promethium 147-added phosphate glass laser, Chromium-attached chrysoberyl laser, Elbium-added And may include, but are not limited to, erbium itterbium co-added glass lasers, trivalent uranium-added calcium fluoride lasers, sumalium-added calcium fluoride lasers, GaN lasers, InGaN lasers, AlGaInP lasers, AlGaAs lasers, InGaAsP lasers. In a particular embodiment, the monochromatic light source is a double wave neodymium-added yttrium aluminum garnet that outputs 532 nm light.

In some embodiments, the system comprises an optical tuning component optically coupled to a laser. "Optical adjustment" means that the laser beam can be changed as desired before it reaches the sample. For example, the system may include one or more lenses, collimators, pinholes, mirrors, beam choppers, slits, diffraction gratings, filters, photorefractors, and any combination thereof. In certain embodiments, the optical tuning component is a wavelength separator. As used herein, the term "wavelength separator" is used in its conventional sense to refer to an optical protocol for separating multicolored light into its component wavelengths. According to certain embodiments, wavelength separation may include selectively passing or blocking a particular wavelength or wavelength range of multicolored light. The wavelength separation protocol of interest, which can be part of the connector of interest or can be combined with the connector of interest, is colored glass, bandpass filters, interference filters, dichroic mirrors, diffraction gratings, among other wavelength separation protocols. Includes, but is not limited to, monochromators and combinations thereof.

In some cases, a monochromatic light source for illuminating the sample is coupled to a collimator. The term "colimate" is used in the traditional sense to optically adjust the co-linearity of light propagation or to suppress the spread of light from a common axis of propagation. In some cases, the collimator is configured to narrow the spatial cross-sectional area of the light beam from a monochromatic light source. In other cases, the optical adjustment component may be, for example, 5 ° or higher, 10 ° or higher, 15 ° or higher, 20 ° or higher, 25 ° or higher, 30 ° or higher, 45 ° or higher, 60 ° or higher, 75 ° or higher, and the like. It is configured to change the direction of the light beam from the monochromatic light source, such as changing the propagation of the light beam from the monochromatic light source by 1 ° or more, including changing the propagation direction of the light by 90 ° or more. In yet other cases, the optical adjustment may reduce the dimensions of the light by, for example, 5% or more, including reductions of 10% or more, 25% or more, 50% or more, and 75% or more. , Beam spot) is a reduction protocol for reducing.

In certain embodiments, the system is configured to irradiate the sample (eg, by focusing a monochromatic light source with a collimator as described above) at the interface between the sample and the wall of the sample container. "Interface" means the space where the surface of the wall of the container contacts the sample in the container. In some embodiments, the system of interest is such that the monochromatic light source is approximately 0.01 mm to 2 mm, eg 0.02 mm to 1.9 mm, 0.03 mm to 1.8 mm from the surface of the container wall (ie, where the sample contacts the container). , 0.04mm-1.7mm, 0.05mm-1.6mm, 0.06mm-1.5mm, 0.07mm-1.4mm, 0.08mm-1.3mm, 0.09mm-1.2mm, 0.1mm-1mm, for example 0.2 from the surface of the container wall It can be configured to illuminate the interface at points such as mm. In these embodiments, the system of interest substantially populates a sample container (eg, a glass vial as described below) such that the velocity of the interfacial liquid layer illuminated by a monochromatic light source is near zero or zero. It is configured to remain stationary (eg, no vibration, no agitation, etc.). For example, the liquid velocity of the interface sample layer illuminated by a monochromatic light source is, for example, 10 ^-3 cm ³ / sec or less, 10 ^-4 cm ³ / sec or less, 10 ^-5 cm ³ / sec, 10 ^-6 cm ³ /. Includes 10 ^-7 cm ³ / sec or less, 10 ^-8 cm ³ / sec or less, 10 ^-9 cm ³ / sec or less, and 10 ^-10 cm ³ / sec or less, 10 ^-2 cm ³ / sec or less. Can be. In certain embodiments, the system of interest is configured such that the speed of the sample in the interface layer is 0 cm ³ / sec when illuminated by a monochromatic light source in the method of interest.

As mentioned above, the method comprises irradiating the sample with monochromatic light and determining the intensity of resonant Raman scattering and fluorescence. The system for implementing the method of interest includes one or more detectors for detecting fluorescence and resonant Raman scattering. Among other photodetectors, active pixel sensor (APS), 4-split photodiode, wedge type detector, image sensor, charge-coupled device (CCD), augmented charge-coupled device (ICCD), light-emitting diode, photon counter, boro These include meters, photodetectors, photoregisters, solar cells, photodiodes, photoelectron multipliers, phototransistors, quantum dot photoconductors or photodiodes, and combinations thereof, including photosensors or photodetectors. Any convenient photodetection protocol, but not limited, may be used. In certain embodiments, the system comprises one or more CCDs.

If the system of interest includes two or more photodetectors, each photodetector may be the same or a combination of different types of photodetectors. For example, if the system of interest includes two photodetectors, in some embodiments, the first photodetector is a CCD-type device and the second photodetector is a CMOS-type device. In another embodiment, both the first and second photodetectors are CCD-type devices. In yet another embodiment, both the first and second photodetectors are CMOS devices. In yet another embodiment, the first photodetector is a CCD or CMOS device and the second photodetector is a photomultiplier tube. In yet another embodiment, the first photodetector and the second photodetector are photomultiplier tubes.

The detector may be optically coupled to one or more optical tuning components. For example, the system may include one or more lenses, collimators, pinholes, mirrors, beam choppers, slits, diffraction gratings, filters, photorefractors, and any combination thereof. In some embodiments, the detector is coupled to a wavelength separator such as colored glass, bandpass filters, interference filters, dichroic mirrors, diffraction gratings, monochromators, and combinations thereof. In certain embodiments, the resonant Raman scattering from the sample is collected by an optical fiber (eg, an optical fiber relay bundle) and the resonant Raman scattering is transmitted to the detector surface through the optical fiber. Any fiber optic optical relay system can be used to transfer scattered light to the active surface of the detector.

In certain embodiments, the photodetection system comprises a collimator placed adjacent to the sample container. The collimator can be any convenient collimator, such as one or more mirrors or curved lenses or a combination thereof. For example, a collimator is, in certain cases, a single collimator lens. In other cases, the collimator is a collimator mirror. In yet other cases, the collimator includes two lenses. In yet other cases, the collimator includes a mirror and a lens. If the collimator contains one or more lenses, the focal length of each collimator lens may be, for example, 6mm-475mm, 7mm-450mm, 8mm-425mm, 9mm-400mm, 10mm-375mm, 12.5mm-350mm, and so on. It can vary in the range of 5mm to 500mm including the focal length in the range of 15mm to 300mm. In certain embodiments, the focal length ranges from 400 mm to 500 mm, including, for example, 405 mm to 475 mm, 410 mm to 450 mm, and 410 mm to 425 mm, such as 410 mm or 420 mm.

In some embodiments, spectroscopy (eg, nonlinear resonance Raman spectroscopy, fluorescence spectroscopy, etc.) is performed at a substantially constant temperature. Therefore, in the target system, the temperature change of the system is, for example, 4.5 ° C or less, 4 ° C or less, 3.5 ° C or less, 3 ° C or less, 2.5 ° C or less, 2 ° C or less, 1.5 ° C or less, 1 ° C or less, 0.5 ° C or less. , 0.1 ℃ or less, 0.05 ℃ or less, 0.01 ℃ or less, 0.005 ℃ or less, 0.001 ℃ or less, 0.0001 ℃ or less, 0.00001 ℃ or less, and 0.000001 ℃ or less, 5 ℃ or less, etc. It is configured to do. In some embodiments, the temperature of the system can be controlled by a temperature control subsystem that measures the system temperature and, if necessary, controls ambient conditions to maintain the desired system temperature. The temperature subsystem may include, but is not limited to, heat sinks, fans, exhaust pumps, vents, refrigeration, coolants, heat exchanges, Pelche elements or resistance heating elements, among other types of temperature control protocols. Temperature control protocol may be included. As described in more detail below, in some embodiments, the system includes instructions that include an algorithm that measures the spectral drift of the laser at a given time and calculates the thermal drift of the laser in response to changes in temperature. Includes processors with memory. The system according to these aspects also includes an algorithm for calculating the correction factor of resonance Raman spectroscopy for the measured thermal drift of the laser.

The system of interest may also include a sample holder for illuminating the sample with a monochromatic light source. The sample holder can be any suitable shaped substrate or container for irradiating the sample and detecting one or more of resonant Raman scattering and fluorescent scattering. In some embodiments, the sample holder is a flat substrate (eg, a microscope slide). In another embodiment, the sample holder is a microfluidic device with one or more microfluidic channels. In yet another embodiment, the sample holder is a container having a cross-sectional shape, and the cross-sectional shape of interest is a straight cross-sectional shape, eg, a curved cross-sectional shape such as a square, a rectangle, a trapezoid, a triangle, a hexagon, etc. It includes, but is not limited to, circles, ellipses, etc., and irregular shapes, such as a parabolic lower part coupled to a planar upper part. The size of the sample holder can vary depending on the volume of the sample being irradiated, and the length of the holder of interest can be, for example, 10 mm to 90 mm, 15 mm to 85 mm, 20 mm to 80 mm, 25 mm to 75 mm, 30 mm to 70 mm, and 35 mm. The range is 5 mm to 100 mm including ~ 65 mm, and the width of the holder of interest (or the cross section when the container is cylindrical) is, for example, 10 mm to 90 mm, 15 mm to 85 mm, 20 mm to 80 mm, 25 mm to 75 mm, 30 mm. It is 5 mm to 100 mm including ~ 70 mm and 35 mm to 65 mm. In multiple embodiments, the sample holders are, for example, 0.5 cm 3-9 cm ³ , 1 cm ^3-8 cm ³ , 1.5 cm 3-7 cm ³ , ² cm ^3-6 cm ³ , 2.5 cm ^3-5 cm ³ , etc., and ³ cm ^3-4 cm ³ . Can have a volume of 0.1 cm ³ to 10 cm ³ including.

The sample holder can be formed from any transparent material that transmits a desired wavelength range, including but not limited to optical glass, borosilicate glass, Pyrex glass, ultraviolet quartz, infrared quartz, sapphire. In certain embodiments, the sample container is a glass with a wall having a zwitterion coating, such as, for example, a zwitterion silane coating (as described in the experimental section below). The sample container may be a plastic such as, for example, polycarbonate, polyvinyl chloride (PVC), polyurethane, polyether, polyamide, polyimide, or a copolymer of these thermoplastics, including polyester, eg PETG (glycol) among other polymer plastic materials. Polyethylene of interest, which can be formed from modified polyethylene terephthalate, etc., is based on poly (ethylene terephthalate) (PET), bottle grade PET (monoethylene glycol, terephthalic acid, and other commonomers such as isophthalic acid, cyclohexene dimethanol). Poly (alkylene terephthalate) such as poly (butylene terephthalate) (PBT), and poly (hexamethylene terephthalate); poly (ethylene adipate), poly (1,4-butylene adipate), poly (hexa). Poly (alkylene adipate) such as methylene adipate; poly (alkylene svelate) such as poly (ethylene svelate); poly (alkylene sebacate) such as poly (ethylene sebacate); poly (ε-caprolactone) and poly (ε-caprolactone) β-Propiolactone); Poly (alkylene isophthalate) such as poly (ethylene isophthalate); Poly (alkylene 2,6-naphthalenedicarboxylate) such as poly (ethylene 2,6-naphthalenedicarboxylate); Poly Poly (alkylene sulfonyl-4,4'-dibenzoate) such as (ethylene sulfonyl-4,4'-dibenzoate); poly (p-phenylene alkylene dicarboxylate) such as poly (p-phenylene ethylene dicarboxylate) Poly (trans-1,4-cyclohexanediylalkylene dicarboxylate) such as poly (trans-1,4-cyclohexanediylethylene dicarboxylate); poly (1,4-cyclohexane-dimethyleneethylene dicarboxylate) etc. Poly (1,4-cyclohexane-dimethylenealkylenedicarboxylate); poly ([2.2.2]-bicyclooctane-1,4-dimethyleneethylenedicarboxylate) and other polys ([2.2.2]-bicyclo Octane-1,4-dimethylenealkylene dicarboxylate); (S)-polylactice, (R, S)-polylactice, poly (tetramethylglycolide), and poly (lactide-co-glycolide) lactic acid polymers And Copolymers; Polycarbonate of Bisphenol A, 3,3'-Dimethyl Bisphenol A, 3,3', 5,5'-Tetrachlorobisphenol A, 3,3', 5,5'-Tetramethylbisphenol A; Poly (p) -Polycarbonates such as phenylene terephthalamide); polyesters such as polyethylene terephthalate, such as Mylar ™ polyethylene terephthalate, may include, but are not limited to.

In multiple embodiments, the sample holder ranges from 100 nm to 1500 nm including, for example, 150 nm to 1400 nm, 200 nm to 1300 nm, 250 nm to 1200 nm, 300 nm to 1100 nm, 350 nm to 1000 nm, 400 nm to 900 nm, and 500 nm to 800 nm, such as 532 nm. Can pass the light of. As outlined above, the system includes one or more processors with memory containing instructions stored to perform the above method. In some embodiments, the memory stores and contains instructions, which, when executed by the processor, illuminate the sample with a monochromatic light source at a first and second irradiation intensity; Determining the intensity of resonance Raman scattering at 1 irradiation intensity and 2nd irradiation intensity; the rate of change of resonance Raman scattering intensity according to the change of irradiation intensity from the 1st irradiation intensity to the 2nd irradiation intensity. To calculate; as well as having the processor compare the rate of change in the intensity of the resonant Raman scattering with the rate of change in the intensity of the irradiation with a monochromatic light source to determine the change in the sample's response to the resonance Raman response.

In another embodiment, the memory contains instructions that, when executed by the processor, illuminate the sample over a period of time with a monochromatic light source and cause the processor to determine the rate of change in the intensity of the resonant Raman scattering. .. In certain cases, the memory, when run by a processor, illuminates the first sample over a period of time with a monochromatic light source to determine the rate of change in the intensity of resonant Raman scattering for the first sample. The net signal of the first sample by comparing the normalized rate of change in the intensity of the resonance Raman scattering with respect to the first sample to the normalized rate of change in the intensity of irradiation of the first sample with a monochromatic light source. To determine the rate of change in the intensity of the resonant Raman scattering for the second sample by irradiating the second sample over the period of time with a monochromatic light source; and the resonant Raman scattering for the second sample. Have the processor perform the calculation of the net signal of the second sample by comparing the normalized rate of change of the intensity of the second sample with the normalized rate of change of the irradiation intensity of the second sample by a monochromatic light source. Includes instructions. In certain embodiments, the memory also performs on the processor to determine, when executed by the processor, that the first sample contains a different gas composition than the second sample, based on the compared net signals. Includes instructions to make. In another embodiment, the memory determines that the first sample or the second sample contains actively metabolizing microorganisms based on the compared net signals when executed by the processor. Includes instructions that cause the processor to execute.

Multiple aspects also include a system for determining the presence or absence of microorganisms in a sample. A system according to a particular embodiment includes a processor that includes a memory operably attached to the processor, the memory containing instructions, which, when executed by the processor, are over multiple intensities by a monochromatic light source. Irradiating the sample for a period of time and calculating the rate of change in the intensity of the resonance Raman scattering for the first sample; Have the processor calculate the net signal of the sample by comparing it to the converted rate of change; and determine the presence or absence of microorganisms in the sample based on the net signal of the sample. In some cases, the memory further includes instructions that, when executed by the processor, cause the processor to determine that microorganisms are present if the net signal of the sample exceeds a predetermined threshold. The threshold can vary in the range -0.5 to -4 x 10 ^-5 / sec, for example -0.5 to -1 x 10 ^-5 / sec, -1 to -2 x 10 ^-5 / sec, and the net signal Includes a given threshold for -1 × 10 ^-5 / sec. In some embodiments, one or more predetermined thresholds are set by performing the above method on one or more control samples containing the inoculum at the lower limit of the concentration of the clinically infected sample. Will be done. In some embodiments, when the memory is executed by the processor, the net signal of the sample is, for example, 5% or more, 10% or more, 15% or more, 25% or more, 50% or more, 75% or more, and the like. And, for example, if the threshold exceeds 1% or more of the threshold value including 90% or more, including 2 times or more, 3 times or more, 5 times or more, and 10 times or more, it is determined that the microorganism is present in the sample. Includes instructions that let the processor do that.

In certain embodiments, the system comprises a processor that includes a memory operably attached to the processor, the memory containing instructions, and the instructions are net two different samples when executed by the processor. Have the processor perform the determination of the presence of microorganisms by comparing the signals. In some embodiments, one of the samples is a microbe-free reference sample. In these embodiments, the memory comprises an instruction, which, when executed by the processor, irradiates the first sample over a period of time with a monochromatic light source for a period of time and the intensity of resonance Raman scattering with respect to the first sample. Calculating the rate of change; by comparing the normalized rate of change in the intensity of the resonance Raman scattering with respect to the first sample to the normalized rate of change in the intensity of irradiation of the first sample with a monochromatic light source. Calculating the net signal of one sample; irradiating the second sample over multiple intensities over a period of time with a monochromatic light source to determine the rate of change in the intensity of resonant Raman scattering for the second sample; second Computing the net signal of the second sample by comparing the normalized rate of change of the intensity of the resonance Raman scattering with respect to the sample to the normalized rate of change of the intensity of irradiation of the second sample with a monochromatic light source. Also, the processor is to determine the presence or absence of microorganisms in one or more of the first sample or the second sample by comparing the net signal of the first sample with the net signal of the second sample. Let it be carried out. In some cases, the memory determines that microorganisms are present in the second sample if the net signal of the second sample is greater than the net signal of the first sample when run by the processor. , Includes instructions to be executed by the processor. In other cases, the memory determines that the microorganism is present in the first sample if the net signal of the first sample is greater than the net signal of the second sample when run by the processor. Contains instructions to be executed by the processor. If the first sample is a reference sample, the memory determines that microorganisms are present in the second sample if the net signal of the second sample is greater than the net signal of the reference sample when run by the processor. Includes instructions to let the processor do what it does.

Multiple aspects also include a system for calculating the signal-to-noise ratio in nonlinear resonant Raman spectroscopy. A system according to a particular embodiment includes a processor that includes memory operably attached to the processor, the memory containing instructions, which, when executed by the processor, are over multiple intensities by a monochromatic light source. Irradiating the first sample for a period of time to determine the rate of change in the intensity of the resonance Raman scattering for the first sample; to determine the first average rate of change, the intensity of the resonance Raman scattering by the first sample. Calculating the mean rate of change; calculating the standard deviation of the first mean rate of change; irradiating the second sample over a range of intensities over a period of time with a monochromatic light source for the resonance Raman scattering for the second sample. To determine the rate of change in intensity; to calculate the average rate of change in the intensity of the resonance Raman scattering by the second sample to determine the second average rate of change; to calculate the standard deviation of the second average rate of change. To do; to subtract the second mean change rate from the first mean change rate to find the average rate of change of the signal; to find the standard deviation of the signal, the first standard deviation and the second standard deviation And to have the processor divide the average rate of change of the signal by the standard deviation of the signal to determine the signal noise ratio of the resonant Raman response.

Aspects of the present disclosure also include a subsystem for compensating for thermal drift of a monochromatic source in non-linear resonance Raman spectroscopy. If the system of interest includes a laser, the subsystem of interest is configured to compensate for the thermal drift of the laser. A system according to a particular embodiment includes a processor that includes memory operably attached to the processor, which stores instructions, which, when executed by the processor, are over multiple intensities by a monochromatic light source. By irradiating the reference composition for a certain period of time and determining the rate of change in the intensity of the resonance Raman scattering, the reference composition does not show a change in the intensity of the resonance Raman scattering in response to the change in the irradiation intensity by the monochromatic light source. Irradiation and determination, including the reference compound; and to determine if the monochromatic light source exhibits thermal drift, the rate of change in the intensity of the resonant Raman scattering with respect to the reference composition, the irradiation intensity of the reference composition with the monochromatic light source. Have the processor perform the calculation of the net signal of the reference composition by comparing with the rate of change of.

If the system determines that a monochromatic light source (eg, a laser) exhibits thermal drift, the system may be configured to compensate for thermal drift in non-linear resonance Raman spectroscopy. In these embodiments, the system of interest can include a memory with instructions, which, when executed by a processor, irradiate the sample with a monochromatic light source over a range of intensities for a period of time to cause resonance Raman scattering. To determine the rate of change in intensity; to multiply the net signal of the reference composition by the rate of change in the intensity of the sample from the monochromatic source to generate a correction factor; and to correct for thermal drift of the monochromatic source. The processor is made to reduce the correction factor from the determined rate of change of the intensity of the resonance Raman scattering with respect to the sample.

Multiple aspects also include a system for characterizing the antimicrobial agent susceptibility of microorganisms to antimicrobial agents using resonant Raman scattering. A system according to a particular embodiment comprises a processor that includes a memory operably attached to the processor, the memory containing instructions, which, when executed by the processor, are over multiple intensities by a monochromatic light source. , Microorganisms and antimicrobial agents, respectively, are irradiated for a period of time to determine the rate of change in the intensity of resonance Raman scattering for each irradiated sample, where each sample has the same concentration of microorganisms and different concentrations. Irradiation and determination, including anti-microbial agents of the , Computing the net signal for each sample; and having the processor determine the susceptibility of the microorganism to the antimicrobial agent based on the calculated net signal for multiple samples. In some embodiments, the memory contains instructions that, when executed by the processor, cause the processor to perform a comparison of net signals for each sample. In some cases, the memory stores instructions in memory that, when executed by the processor, cause the processor to plot the net signal of each sample as a function of the log of the concentration of antimicrobial agent in each sample. And further included. In some cases, the memory contains an instruction in the memory that causes the processor to determine the concentration of antimicrobial agent indicating a decrease in the net signal when executed by the processor. For example, the memory may contain instructions in the memory that, when executed by the processor, cause the processor to determine the concentration of antimicrobial agent indicating an increase in the net signal.

In another embodiment, the memory contains an instruction in the memory that, when executed by the processor, causes the processor to determine the metabolic activity of the microorganism in each sample based on the net signal of each sample. .. For example, the memory causes the processor to determine the concentration of an antimicrobial agent that indicates a decrease in metabolic activity when executed by the processor, or determines the concentration of an antimicrobial agent that indicates an increase in metabolic activity. Can be stored and contained in memory for instructions that cause the processor to execute.

Multiple aspects also include a system for characterizing the antimicrobial agent susceptibility of microorganisms to antimicrobial agents using fluorescence spectroscopy. A system according to a particular embodiment comprises a processor that includes a memory operably attached to the processor, the memory containing instructions, which, when executed by the processor, are microbes and anti-microbes by a monochromatic light source. By irradiating a plurality of samples containing microorganisms each containing a microbial agent for a certain period of time and detecting fluorescence from each of the samples irradiated for a certain period of time, each sample has the same concentration of microorganisms and different concentrations of anti-microorganisms. Irradiation and detection, including agent; by comparing the normalized rate of change in the detected fluorescence intensity produced by each sample with the normalized rate of change in fluorescence produced by the control. Calculating the rate of change in fluorescence in each sample; as well as having the processor determine the susceptibility of the microorganism to the antimicrobial agent based on the calculated rate of change in fluorescence in multiple samples. In some embodiments, the system is configured to illuminate each sample over multiple intensities with a monochromatic light source. In some embodiments, the memory further comprises an instruction to cause the processor to compare the calculated rate of change of fluorescence for each sample when performed by the processor. In these cases, the comparison may include plotting the calculated rate of change in fluorescence of each sample as a function of the logarithm of the concentration of antimicrobial agent in each sample. In another embodiment, the memory further comprises instructions that cause the processor to determine the concentration of antimicrobial agent that, when executed by the processor, indicates a decrease or increase in the rate of change in fluorescence. In yet another embodiment, the memory further comprises an instruction to cause the processor to determine the concentration of an antimicrobial agent that, when executed by the processor, indicates a decrease or increase in metabolic activity.

Multiple aspects also include a system for determining the phenotype of unknown microorganisms in a sample using resonant Raman scattering. A system according to a particular embodiment includes a processor that includes a memory operably attached to the processor, the memory containing instructions, which, when executed by the processor, are over multiple intensities by a monochromatic light source. Irradiating a sample containing microorganisms, cross-linking material, and albumin protein with a monochromatic light source for a period of time to determine the rate of change in the intensity of resonance Raman scattering; the normalized rate of change in the intensity of resonance Raman scattering for the sample. Computing the net signal of the sample by comparing it to the normalized rate of change of the irradiation intensity of the sample by a monochromatic light source; and determining the cross-linking cleavage based on the calculated net signal of the sample. Have the processor perform the determination that the degree of cross-linking cleavage indicates the phenotype of the microorganism.

Multiple aspects include a system for determining the phenotype of an unknown microorganism in a sample using fluorescence spectroscopy. A system according to a particular embodiment includes a processor that includes a memory operably attached to the processor, the memory containing instructions, which, when executed by the processor, microorganisms, crosslinks, and albumin. Irradiating a sample containing a protein with a monochromatic light source for a period of time and detecting fluorescence from the sample over that period; a normalized rate of change in the intensity of the detected fluorescence produced by the sample is generated by the control. To calculate the rate of change in fluorescence by comparing it to the normalized rate of change in fluorescence; as well as to determine cross-linking cleavage based on the calculated rate of change in fluorescence of the sample. Have the processor perform the determination that the degree indicates the phenotype of the microorganism. In some embodiments, the system is configured to illuminate the sample over multiple intensities with a monochromatic light source.

の化合物であり、式中、
R₁およびR₂は、水素、アルキル、置換アルキル、シクロアルキル、置換シクロアルキル、アリール、置換アリール、ヘテロアルキル、置換ヘテロアルキル、ヘテロシクロアルキル、置換ヘテロシクロアルキル、ヘテロアリール、および置換ヘテロアリールから独立に選択され、
X₁およびX₂は、N、O、またはSから独立に選択され、
R₃は、水素、アルキル、置換アルキル、アミノ、ハロゲン、シアノ、アルコール、またはアルコキシである。 In some embodiments, the cross-linking material is a disulfide crosslinker (eg, a compound having a disulfide link that exchanges disulfide bonds with proteins). In certain cases, the cross-linking material is also a glutamic acid derivative, such as a glutamic acid derivative having one or more disulfide linkages. In certain embodiments, the cross-linking material is of formula (I) :.

In the formula,
R ₁ and R ₂ are from hydrogen, alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, aryl, substituted aryl, heteroalkyl, substituted heteroalkyl, heterocycloalkyl, substituted heterocycloalkyl, heteroaryl, and substituted heteroaryl. Selected independently,
X ₁ and X ₂ are selected independently of N, O, or S,
R ₃ is hydrogen, alkyl, substituted alkyl, amino, halogen, cyano, alcohol, or alkoxy.

In some embodiments, R ₁ and R ₂ are heteroaryl, X ₁ and X ₂ are N, and R ₃ is amino.

の化合物である。 In certain embodiments, the cross-linking material is of formula (DS-1).

It is a compound of.

The cross-linking substance has a molar ratio of cross-linking substance: albumin protein, for example, 1: 9 to 9: 1, 1: 8 to 8: 1, 1: 7 to 7: 1, 1: 6 to 6: 1, 1: 5. May be present in the sample in an amount of 1:10 to 10: 1, including ~ 5: 1, 1: 4 to 4: 1, 1: 3 to 3: 1, etc., and 1: 2 to 2: 1. .. In certain embodiments, the molar ratio of cross-linking material: albumin protein in the sample of the method of interest is approximately 1: 2. As mentioned above, the system of interest includes a processor including memory operably coupled to the processor with instructions for performing the methods described herein. The system may include a non-temporary computer-readable storage medium for storing the above instructions. Computer-readable storage media may be used on one or more computers for full or partial automation of the system for performing the methods described herein. In certain embodiments, instructions by the methods described herein can be encoded in a computer-readable medium in the form of "programming", and the term "computer-readable medium" as used herein is used. Refers to any non-temporary storage medium involved in providing instructions and data to a computer for execution and processing. Examples of suitable non-volatile storage media are floppy disks, hard disks, optical disks, optical magnetic disks, CD-ROMs, CD-Rs, magnetics, whether such devices are inside or outside a computer. Includes tapes, non-volatile memory cards, ROMs, DVD-ROMs, Blue-ray discs, solid state discs, and network attached storage (NAS). Files containing information can be "stored" on computer-readable media. "Storing" is the recording of information so that the computer can access and retrieve it at a later date.

The computer implementation methods described herein can be performed using programming that can be described in one or more of any number of computer programming languages. Such languages include, for example, Java (Sun Microsystems, Inc., Santa Clara, CA), Visual Basic (Microsoft Corp., Redmond, WA), C ++ (AT & T Corp., Bedminster, NJ), and many any. Including other languages.

Computer-readable storage media may be used in one or more computer systems with displays and operator input devices. The operator input device can be, for example, a keyboard, a mouse, or the like. The processing module includes a processor that can access the memory in which the instructions for performing the steps of the method of interest are stored. Processing modules can include operating systems, graphical user interface (GUI) controllers, system memory, memory storage devices, input / output controllers, cache memory, data backup units, and many other devices. The processor may be a commercially available processor or one of the other processors available or will be available. The processor runs the operating system, the operating system interfaces with firmware and hardware in a well-known manner, and the processor is known in Java, Perl, C ++, other high-level or low-level languages, and in the art. Allows you to coordinate and execute the functions of various computer programs that can be written in different programming languages, such as their combinations. The operating system typically works with the processor to coordinate and perform the functions of other components of the computer. The operating system also provides scheduling, input / output control, file and data management, memory management, and communication control and related services, all in accordance with known techniques.

Technical Features Specific technical features of the target method are shown below.

で表すことができ、
式中、I_oおよびν_oはレーザーの強度および周波数であり、I_Rは周波数ν_mnで観測されるラマンバンドの強度である。また、分極率は、レーザー周波数の差および基底状態と励起状態との間のエネルギー差によって定義され得る。

Rate of change of resonance Raman peak In resonance Raman spectroscopy, the observed Raman band intensity is given by Equation 1:

Can be represented by
In the equation, I _o and ν _o are the intensity and frequency of the laser, and _{IR is the intensity of the Raman band observed at the frequency ν mn .} Polarizability can also be defined by the difference in laser frequency and the energy difference between the ground state and the excited state.

Using these basic terms, it can be seen that the rate of change in Raman band intensity corresponds to the rate of change in laser intensity and the rate of change in the polarizability term.

に変化がない場合、ラマンバンドの正規化された変化率は、レーザー強度の正規化された変化率に等しくなる。分極率の項がレーザー強度とともに変化する場合（例えば、励起状態がレーザー強度の変化により変化している場合）、または分極率の項が単独で変化する場合（例えば、励起状態が進行中の化学/物理反応により変化する場合）、2つの項は等しくならない。このシナリオでは、分極率の項に影響を与え得る変化を特徴づけるためにラマンピーク強度の変化率が使用され得る。 Polarizability term

If there is no change in, the normalized rate of change of the Raman band is equal to the normalized rate of change of the laser intensity. If the polarizability term changes with laser intensity (eg, if the excited state changes due to a change in laser intensity), or if the polarizability term changes alone (eg, chemistry in which the excited state is in progress). / If it changes due to a physical reaction), the two terms are not equal. In this scenario, the rate of change in Raman peak intensity can be used to characterize changes that can affect the polarizability term.

Aspects of the subject matter of the invention described herein, including embodiments, can be useful alone or in combination with one or more other aspects or aspects. As will be apparent to those of skill in the art by reading this disclosure, each of the individually numbered aspects may be used or combined with any of the above or below individually numbered aspects. It is intended to provide support for all such combinations of aspects and is not limited to the combinations of aspects explicitly presented below.

1. A method for determining the resonance Raman response of a sample, which includes the following steps:
The step of irradiating a sample with a monochromatic light source at the first and second irradiation intensities;
Step of determining the intensity of resonant Raman scattering at the first irradiation intensity and the second irradiation intensity;
The step of calculating the rate of change in the intensity of the resonance Raman scattering in response to the change in irradiation intensity from the first irradiation intensity to the second irradiation intensity; and to determine the change in the sample's resonance Raman response to the resonance Raman scattering. The process of comparing the rate of change in intensity with the rate of change in irradiation intensity by a monochromatic light source.

2. The method according to 1, wherein the resonant Raman response of the sample indicates the physical time course of the sample.

3. The method according to 1, wherein the resonant Raman response of the sample indicates the chemical aging of the sample.

4. The method according to 1, wherein the resonant Raman response of the sample indicates the presence of actively metabolizing microorganisms in the sample.

5. The method according to any one of 1 to 4, wherein the monochromatic light source is a laser.

6. The method according to any one of 1-5, wherein the sample comprises a hydrophobic compound and an albumin protein.

7. The method according to 6, wherein the hydrophobic compound is a carotenoid.

8. The method according to 7, wherein the hydrophobic compound is lycopene.

9. The method according to any of 6-8, wherein the hydrophobic compound non-covalently binds to the albumin protein.

10. The method according to any of 1-9, wherein the sample contains a free radical scavenger.

11. The method according to 10, wherein the free radical scavenger non-covalently binds to the albumin protein.

12. The method according to any of 10-11, wherein the free radical scavenger comprises bilirubin or a derivative thereof.

13. The method according to 12, wherein bilirubin or a derivative thereof is present in the sample at a concentration of 0.5 μM to 2 μM.

14. The method according to 12, wherein bilirubin or a derivative thereof is present in the sample at a concentration of 0.25 μM to 1.75 μM.

15. The method according to any one of 1 to 9, wherein the sample contains a reducing substance.

16. The method according to 15, wherein the reducing substance is glutathione or a derivative thereof.

17. The method according to any of 15-16, wherein the reducing substance is present in the sample at a concentration of 0.1 mg / mL to 1 mg / mL.

18. The method according to any one of 1 to 17, comprising a step of irradiating a sample with a monochromatic light source over a plurality of irradiation intensities for a certain period of time and determining the rate of change in the intensity of resonance Raman scattering.

19. The method according to any one of 1 to 17, including the following steps:
A step of irradiating a first sample over a plurality of intensities with a monochromatic light source for a certain period of time to determine the rate of change in the intensity of resonant Raman scattering for the first sample;
By comparing the normalized rate of change in the intensity of the resonant Raman scattering with respect to the first sample to the normalized rate of change in the intensity of irradiation of the first sample with a monochromatic light source, the net signal of the first sample can be obtained. Calculation process;
A step of irradiating a second sample over a plurality of intensities over a period of time with a monochromatic light source to determine the rate of change in the intensity of the resonant Raman scattering for the second sample; and normalizing the intensity of the resonant Raman scattering for the second sample. The step of calculating the net signal of the second sample by comparing the resulting rate of change with the normalized rate of change of the irradiation intensity of the second sample by a monochromatic light source.

20. The method according to 18, further comprising comparing the calculated net signal of the first sample with the calculated net signal of the second sample.

21. The method according to 20, further comprising determining that the first sample and the second sample are different based on the calculated net signal compared.

22. The method according to 20, further comprising determining that the first sample contains a different gas composition than the second sample, based on the calculated net signal compared.

23. The method according to 20, further comprising determining that the first sample or the second sample contains actively metabolizing microorganisms based on the calculated net signal compared.

24. The method according to any of 1 to 23, wherein the sample is irradiated in the sample container.

25. The method according to 24, wherein the sample container is a glass vial.

26. The method according to 25, wherein the glass vial comprises a wall with a zwitterionic coating.

27. The method of 26, wherein the glass vial comprises a wall with a zwitterionic silane coating.

28. The method according to any of 25-27, wherein the sample is irradiated at the interface between the sample and the wall of the container.

29. The method of 28, wherein the monochromatic light source is focused at the interface between the sample and the wall of the container.

30. The method of 29, wherein the monochromatic light source is focused on the sample at a position 0.01 mm to 2 mm from the surface of the container wall.

31. The method of 30 in which a monochromatic light source is focused on a sample at a position approximately 0.2 mm from the surface of the container wall.

32. The method according to any of 29-31, wherein the monochromatic light source is focused using a collimating lens.

33. The method according to any of 1-32, wherein the sample further comprises a cross-linking substance.

34. The method according to 33, wherein the cross-linking material is a disulfide crosslinker.

35. The method according to any of 33-34, wherein the cross-linking substance is a glutamic acid derivative.

の化合物を含む、35に記載の方法。 36. The cross-linking material is formula (I) :.

35. The method according to 35.

37. The method according to any of 33-36, wherein the cross-linking material is present in the sample in an amount such that the cross-linking material: albumin protein molar ratio is 1:10 to 10: 1.

38. The method according to 37, wherein the cross-linking material is present in the sample in an amount such that the molar ratio of cross-linking material: albumin protein is approximately 1: 2.

Methods for detecting microorganisms in samples
1. A method for determining the presence of microorganisms in a sample, which includes the following steps:
The process of irradiating a sample over a period of time with a monochromatic light source and calculating the rate of change in the intensity of resonant Raman scattering for the sample;
The step of calculating the net signal of the sample by comparing the normalized rate of change of the intensity of the resonant Raman scattering of the sample with the normalized rate of change of the intensity of the sample irradiated by a monochromatic light source; and the calculation of the sample. A step of determining the presence of microorganisms in a sample based on the net signal.

2. The method according to 1, wherein the presence of microorganisms is determined when the calculated net signal of the sample exceeds a predetermined threshold.

3. The method according to 2, wherein the predetermined threshold is the net signal of 1.

4. The method according to any one of 1 to 3, wherein the monochromatic light source is a laser.

5. The method according to any one of 1-4, wherein the sample comprises a hydrophobic compound that does not covalently bind to an albumin protein.

6. The method according to any one of 1-5, further comprising a free radical scavenger.

7. The method according to 6, wherein the free radical scavenger comprises bilirubin or a derivative thereof.

8. The method according to 7, wherein bilirubin or a derivative thereof is present at a concentration of 0.5 μM to 2 μM.

9. The method according to 7, wherein bilirubin is present at a concentration of 0.25 μM to 1.75 μM.

10. The method according to any one of 1 to 9, wherein the sample contains a reducing substance.

11. The method according to 10, wherein the reducing substance is glutathione or a derivative thereof.

12. The method according to any of 10-11, wherein the reducing substance is present in the sample at a concentration of 0.1 mg / mL to 1 mg / mL.

13. The method according to any of 1 to 12, wherein the sample is irradiated in the sample container.

14. The method according to 13, wherein the sample container is a glass vial.

15. The method according to 14, wherein the glass vial comprises a wall with a zwitterionic coating.

16. The method according to 15, wherein the glass vial comprises a wall with a zwitterionic silane coating.

17. The method according to any of 14-16, wherein the sample is irradiated at the interface between the sample and the wall of the container.

18. The method according to 17, wherein the monochromatic light source is focused at the interface between the sample and the wall of the container.

19. The method according to 18, wherein the monochromatic light source is focused on the sample at a position 0.01 mm to 2 mm from the surface of the wall of the container.

20. The method according to 19, wherein the monochromatic light source is focused on the sample at a position approximately 0.2 mm from the surface of the container wall.

21. The method of any of 18-20, wherein the monochromatic light source is focused using a collimating lens.

22. A method for determining the presence of microorganisms in a sample, which includes the following steps:
The process of irradiating a first sample over a period of time with a monochromatic light source and calculating the rate of change in the intensity of resonant Raman scattering for the first sample;
By comparing the normalized rate of change in the intensity of the resonant Raman scattering with respect to the first sample to the normalized rate of change in the intensity of irradiation of the first sample with a monochromatic light source, the net signal of the first sample can be obtained. Calculation process;
A step of irradiating a second sample over a plurality of intensities with a monochromatic light source for the period of time and determining the rate of change in the intensity of resonant Raman scattering for the second sample;
The net signal of the second sample is obtained by comparing the normalized rate of change in the intensity of the resonant Raman scattering with respect to the second sample to the normalized rate of change in the intensity of irradiation of the second sample with a monochromatic light source. The step of calculating; and the step of determining the presence of microorganisms in one or more of the first sample or the second sample by comparing the net signal of the first sample with the net signal of the second sample. ..

23. The method according to any of 22, wherein the microorganism is determined to be present in the second sample if the calculated net signal of the second sample is greater than the calculated net signal of the first sample.

24. The method according to any of 22, wherein the microorganism is determined to be present in the first sample if the calculated net signal of the first sample is greater than the calculated net signal of the second sample.

25. The method according to any of 22-24, wherein the monochromatic light source is a laser.

26. The method according to any of 22-25, wherein the sample comprises a hydrophobic compound that does not covalently bind to an albumin protein.

27. The method according to 26, further comprising a free radical scavenger.

28. The method according to 27, wherein the free radical scavenger comprises bilirubin or a derivative thereof.

29. The method of 28, wherein bilirubin or a derivative thereof is present at a concentration of 0.5 μM to 2 μM.

30. The method of 28, wherein bilirubin is present at a concentration of 0.25 μM to 1.75 μM.

31. The method according to any of 22-30, wherein the sample contains a reducing substance.

32. The method according to 31, wherein the reducing substance is glutathione or a derivative thereof.

33. The method according to any of 31-32, wherein the reducing substance is present in the sample at a concentration of 0.1 mg / mL to 1 mg / mL.

34. The method according to any of 22-33, wherein the sample is irradiated in a sample container.

35. The method according to 34, wherein the sample container is a glass vial.

36. The method according to 35, wherein the glass vial comprises a wall with a zwitterionic coating.

37. The method of 36, wherein the glass vial comprises a wall with a zwitterionic silane coating.

38. The method according to any of 34-37, wherein the sample is irradiated at the interface between the sample and the wall of the container.

39. The method of claim 38, wherein the monochromatic light source is focused at the interface between the sample and the wall of the container.

40. The method of 39, wherein the monochromatic light source is focused on the sample at a position 0.01 mm to 2 mm from the surface of the container wall.

41. The method of 39, wherein the monochromatic light source is focused on the sample at a position approximately 0.2 mm from the surface of the container wall.

42. The method of any of 39-41, wherein the monochromatic light source is focused using a collimating lens.

Methods for determining the presence of microorganisms in a sample
1. A method for determining the presence of microorganisms in a sample, which includes the following steps:
(A) A step of combining a sample and a reagent containing albumin containing a ligand in a sample holder;
(B) The step of irradiating the sample with a monochromatic light source that is focused at the interface between the sample and the surface of the sample holder with either constant or time-varying light intensity and is absorbed by the ligand;
(C) A step of collecting scattered light from an irradiated sample at a plurality of different times and measuring a Raman signal and a fluorescence signal from the scattered light;
(D) The step of calculating the rate of change of the intensity of the Raman signal and the fluorescence signal with respect to the sample over time;
(E) The step of correcting the calculated rate of change of the intensity of the Raman and fluorescent signals to obtain the net signal; and (f) based on the comparison of the net signal with one or more predetermined thresholds. , The step of determining the presence of microorganisms in the sample.

2. One or more predetermined thresholds of 2.1f by performing the method of claim 1 for one or more control samples comprising an inoculum in an amount at the lower limit of the concentration of the clinically infected sample. The method described in 1, which is set.

3. The method according to 1, wherein the ligand is lycopene.

4. The method according to 1, wherein the monochromatic light source is a laser.

5. The method according to 4, wherein the laser irradiates the sample at 532 nm.

6. The method according to 1, wherein the step of correcting the rate of change in intensity of the Raman signal and the fluorescent signal comprises the step of characterizing the spectral output from the standard sample.

7. The method described in 6, where the standard is NIST SRM 2242.

8. The method of claim 1, wherein the step of correcting the rate of change in intensity of the Raman signal comprises the step of characterizing the fluorescence output from the reagent.

9. The method according to 1, further comprising the step of pretreating albumin of the reagent with a reducing substance prior to incorporating the ligand.

10. The method according to 9, wherein the step of pretreating albumin with a reducing substance is sufficient to reduce the disulfide bond.

11. The method according to 9, wherein the reducing substance is glutathione or bilirubin.

12. The method according to 10, further comprising contacting the pretreated albumin with a disulfide crosslinker.

13. The method according to 12, wherein the disulfide cross-linking material comprises a core that is cleaved by an enzyme or metabolite produced by a microorganism in the sample.

の化合物を含む、10に記載の方法。 14. The cross-linking material is formula (I) :.

10. The method of 10.

15. The method according to 1, where the sample holder is a glass vial.

16. The method according to 15, wherein the surface of the glass vial comprises a coating that alters the absorption of albumin on the glass surface.

17. The method according to 16, wherein the coating is a zwitterionic coating.

18. The method according to 1, wherein the reagent comprises an antimicrobial composition.

19. The method according to 18, further comprising the following steps:
The process of plotting the net signal against the antimicrobial concentration,
The minimum value of the plot is used to estimate the minimum bactericidal concentration of the antimicrobial composition; and the breakpoints in the plot are used to estimate the minimum inhibitory concentration of the antimicrobial composition. Plot process.

A method for compensating for laser output drift during nonlinear Raman spectroscopy
1. Method including the following steps:
A step of irradiating a reference composition over a plurality of intensities with a monochromatic light source for a certain period of time to determine the rate of change in the intensity of resonance Raman scattering . Irradiation and determination steps, including reference compounds that do not show a change in the intensity of the reference composition; The step of calculating the net signal of the reference composition by comparing it with the rate of change of the irradiation intensity of the reference composition.

2. The method according to 1, further comprising the following steps:
A step of irradiating a sample with a monochromatic light source over a certain range of intensities for a certain period of time to determine the rate of change in the intensity of resonant Raman scattering;
The step of multiplying the net signal of the reference composition by the rate of change of the irradiation intensity of the sample by the monochromatic light source to generate the correction factor; and the intensity of the resonant Raman scattering with respect to the sample to compensate for the thermal drift of the monochromatic light source. The process of reducing the correction factor from the determined rate of change.

3. The method according to any one of 1 and 2, wherein the monochromatic light source is a laser.

4. The method according to 3, wherein the laser is a solid-state laser that irradiates at 532 nm.

5. The method according to any one of 1-4, wherein the sample comprises a hydrophobic compound that does not covalently bind to an albumin protein.

6. The method according to 5, further comprising a free radical scavenger.

7. The method according to 6, wherein the free radical scavenger comprises bilirubin or a derivative thereof.

8. The method according to 7, wherein bilirubin or a derivative thereof is present at a concentration of 0.5 μM to 2 μM.

9. The method according to 7, wherein bilirubin is present at a concentration of 0.25 μM to 1.75 μM.

10. The method according to any one of 1 to 9, wherein the fixed period is 200 seconds to 1500 seconds.

11. The method according to 10, wherein the fixed period is 600 seconds.

12. The method according to any of 1 to 11, wherein the sample contains a reducing substance.

13. The method according to 12, wherein the reducing substance is glutathione or a derivative thereof.

14. The method according to any of 12 to 13, wherein the reducing substance is present in the sample at a concentration of 0.1 mg / mL to 1 mg / mL.

Methods for characterizing antimicrobial susceptibility
1. A method for determining the antimicrobial agent susceptibility of a microorganism to an antimicrobial agent, which comprises the following steps:
A step of irradiating a plurality of samples containing microorganisms and antimicrobial agents with a monochromatic light source over a plurality of intensities for a certain period of time, and determining the rate of change in the intensity of resonance Raman scattering for each irradiated sample. Irradiation and determination steps, where each sample contains the same concentration of microorganisms and different concentrations of antimicrobial agent;
The step of calculating the net signal of each sample by comparing the normalized rate of change in the intensity of the resonant Raman scattering for each sample with the normalized rate of change in the intensity of each sample from a monochromatic light source; The step of determining the susceptibility of a microorganism to an antimicrobial agent based on the calculated net signal of the sample.

2. The method according to 1, further comprising a step of comparing the calculated net signals for each sample.

3. The method according to 2, wherein the step of comparison comprises plotting the calculated net signal of each sample as a function of the logarithm of the concentration of the antimicrobial agent in each sample.

4. The method according to any one of 2 to 3, further comprising a step of determining the concentration of an antimicrobial agent indicating a decrease in net signal.

5. The method according to 4, further comprising the step of determining the concentration of the antimicrobial agent indicating an increase in the net signal.

6. The method according to 1, further comprising determining the metabolic activity of the microorganisms in each sample based on the calculated net signal of each sample.

7. The method according to 6, further comprising the step of determining the concentration of an antimicrobial agent indicating a decrease in metabolic activity.

8. The method according to 6, further comprising the step of determining the concentration of an antimicrobial agent showing an increase in metabolic activity.

9. The method according to any of 1-8, wherein each sample contains microorganisms at a concentration of 10 colony forming units (CFU) or higher.

10. The method according to 9, wherein each sample contains microorganisms at a concentration of 14 CFU or higher.

11. The method according to 9, comprising the step of aliquoting each sample from a microbial composition having a concentration of microorganisms above 100 colony forming units (CFU).

12. Described in one of 1 to 11, wherein the concentration range of the antimicrobial agent in multiple samples ranges from a concentration below the minimum inhibitory concentration of the antimicrobial agent to a concentration higher than the minimum bactericidal concentration of the antimicrobial agent. the method of.

13. The method according to any of 1-12, further comprising the step of incubating the antimicrobial agent with the microorganism for a predetermined period of time prior to irradiation of the sample.

14. The method according to 13, wherein the antimicrobial agent is incubated with the microorganism for at least 10 minutes.

15. The method according to 13, wherein the antimicrobial agent is incubated with the microorganism for at least 20 minutes.

16. The method according to any of 1 to 15, wherein the sample contains a reducing substance.

17. The method according to 16, wherein the reducing substance is glutathione or a derivative thereof.

18. The method according to any of 16-17, wherein the reducing substance is present in the sample at a concentration of 0.1 mg / mL to 1 mg / mL.

19. The method according to any of 1-18, wherein the sample is irradiated in the sample container.

20. The method according to 19, wherein the sample container is a glass vial.

21. The method according to 20, wherein the glass vial comprises a wall with a zwitterionic coating.

22. The method of 21. The glass vial comprises a wall with a zwitterionic silane coating.

23. The method of any of claims 19-22, wherein the sample is irradiated at the interface between the sample and the wall of the container.

24. The method of 23, wherein the monochromatic light source is focused at the interface between the sample and the wall of the container.

25. The method of 24, wherein the monochromatic light source is focused on the sample at a position 0.01 mm to 2 mm from the surface of the container wall.

26. The method of 24, wherein the monochromatic light source is focused on the sample at a position approximately 0.2 mm from the surface of the container wall.

27. The method according to any of 24-27, wherein the monochromatic light source is focused using a collimating lens.

Methods for determining the phenotype of unknown microorganisms
1. A method of characterizing the phenotype of a microorganism, which includes the following steps:
A step of irradiating a sample containing a microorganism, a cross-linking substance, and an albumin protein with a monochromatic light source over a plurality of intensities for a certain period of time to determine the rate of change in the intensity of resonant Raman scattering;
The process of calculating the net signal of a sample by comparing the normalized rate of change in the intensity of resonance Raman scattering with respect to the sample to the normalized rate of change in the intensity of irradiation of the sample with a monochromatic light source; as well as the calculation of the sample. A step of determining cross-linking cleavage based on a net signal, wherein the degree of cross-linking cleavage indicates the phenotype of a microorganism.

2. The method according to 1, wherein the cross-linking substance is a disulfide crosslinker.

3. The method according to any one of 1 and 2, wherein the cross-linking substance is a glutamic acid derivative.

の化合物を含む、3に記載の方法。 4. The cross-linking substance is formula (I) :.

3. The method according to 3, comprising the compound of.

5. The method according to any of 1 to 4, wherein the cross-linking substance is present in the sample in an amount such that the cross-linking substance: albumin protein molar ratio is 1:10 to 10: 1.

6. The method according to 5, wherein the cross-linking material is present in the sample in such an amount that the molar ratio of cross-linking material: albumin protein is approximately 1: 2.

7. The method according to any of 1-6, wherein an increase in the calculated net signal of the sample over time indicates that the microorganism produces a metabolite that cleaves one or more crosslinks in the albumin protein.

8. The method according to any one of 1 to 7, wherein the monochromatic light source is a laser.

9. The method according to 8, wherein the laser is a solid-state laser that irradiates at 532 nm.

10. The method according to any of 1-9, wherein the sample comprises a hydrophobic compound and an albumin protein.

11. The method according to 10, wherein the hydrophobic compound is a carotenoid.

12. The method according to 11, wherein the hydrophobic compound is lycopene.

13. The method according to any of 10-12, wherein the hydrophobic compound non-covalently binds to the albumin protein.

14. The method according to any of 1 to 13, wherein the sample contains a reducing substance.

15. The method according to 14, wherein the reducing substance is glutathione or a derivative thereof.

16. The method according to any of 14-15, wherein the reducing substance is present in the sample at a concentration of 0.1 mg / mL to 1 mg / mL.

17. The method according to any of 1 to 16, wherein the sample is irradiated in the sample container.

18. The method according to 17, wherein the sample container is a glass vial.

19. The method according to 18, wherein the glass vial comprises a wall with a zwitterionic coating.

20. The method according to 19, wherein the glass vial comprises a wall with a zwitterionic silane coating.

21. The method according to any of 17-20, wherein the sample is irradiated at the interface between the sample and the wall of the container.

22. The method according to 21, wherein the monochromatic light source is focused at the interface between the sample and the wall of the container.

23. The method of 22., wherein the monochromatic light source is focused on the sample at a position 0.01 mm to 2 mm from the surface of the container wall.

24. The method of 22 in which a monochromatic light source is focused on a sample at a position approximately 0.2 mm from the surface of the container wall.

25. The method according to any of 22-24, wherein the monochromatic light source is focused using a collimating lens.

Methods for Detecting Microorganisms in Samples Using Fluorescence
1. A method for determining the presence of microorganisms in a sample, which includes the following steps:
A step of irradiating a sample with a monochromatic light source for a certain period of time and detecting fluorescence from the sample over the certain period of time;
By comparing the normalized rate of change in the detected fluorescence intensity produced by the sample with the normalized rate of change in fluorescence produced by the control, the rate of change in fluorescence due to the presence of microorganisms can be determined. The step of calculating; and the step of determining the presence of microorganisms in the sample based on the calculated change in the fluorescence of the sample compared to a given threshold.

2. The method according to 1, comprising the step of irradiating the sample over multiple intensities using a monochromatic light source.

3. The method according to any of 1-2, wherein the fluorescence produced by the sample and control is detected at 2500 cm ^-1 to 3500 cm ^-1 .

4. The method according to 3, wherein the fluorescence produced by the sample and control is detected at 3000 cm ^-1 .

5. The method according to any one of 1 to 4, wherein the control is a composition free of microorganisms.

6. The method according to any one of 1 to 5, wherein the microorganism is a pathogenic microorganism.

7. The method according to any one of 1 to 6, wherein the monochromatic light source is a laser.

8. The method according to any one of 1-7, wherein the sample comprises a hydrophobic compound that does not covalently bind to an albumin protein.

9. The method according to any one of 1-8, further comprising a free radical scavenger.

10. The method according to 9, wherein the free radical scavenger comprises bilirubin or a derivative thereof.

11. The method according to 10, wherein bilirubin or a derivative thereof is present at a concentration of 0.5 μM to 2 μM.

12. The method according to 11, wherein bilirubin is present at a concentration of 0.25 μM to 1.75 μM.

13. The method according to any of 1 to 12, wherein the sample contains a reducing substance.

14. The method according to 13, wherein the reducing substance is glutathione or a derivative thereof.

15. The method according to any of 13-14, wherein the reducing substance is present in the sample at a concentration of 0.1 mg / mL to 1 mg / mL.

16. The method according to any of 1 to 15, wherein the sample is irradiated in the sample container.

17. The method according to 16, wherein the sample container is a glass vial.

18. The method according to 17, wherein the glass vial comprises a wall with a zwitterionic coating.

19. The method according to 18, wherein the glass vial comprises a wall with a zwitterionic silane coating.

20. The method according to any of 17-19, wherein the sample is irradiated at the interface between the sample and the wall of the container.

21. The method according to 20, wherein the monochromatic light source is focused at the interface between the sample and the wall of the container.

22. The method according to 20, wherein the monochromatic light source is focused on the sample at a position 0.01 mm to 2 mm from the surface of the container wall.

23. The method of 22 in which a monochromatic light source is focused on a sample at a position approximately 0.2 mm from the surface of the container wall.

24. The method according to any of 21-23, wherein the monochromatic light source is focused using a collimating lens.

25. The method according to any of 1-24, wherein the control is a composition having the same components as the sample in the absence of microorganisms.

Methods for characterizing antimicrobial susceptibility
1. A method for determining the antimicrobial agent susceptibility of a microorganism to an antimicrobial agent, which comprises the following steps:
A step of irradiating a plurality of samples containing microorganisms and antimicrobial agents with a monochromatic light source for a certain period of time to detect fluorescence from each of the samples irradiated for a certain period of time, wherein each sample has the same concentration of microorganisms and different concentrations. Irradiation and detection steps, including antimicrobial agents;
Calculate the rate of change in fluorescence in each sample by comparing the normalized rate of change in the detected fluorescence intensity produced by each sample to the normalized rate of change in fluorescence produced by the control. Steps; as well as determining the susceptibility of microorganisms to antimicrobial agents based on the calculated rate of change in fluorescence of multiple samples.

2. The method according to 1, further comprising the step of comparing the calculated rate of change of fluorescence for each sample.

3. The method according to 2, wherein the step of comparison comprises plotting the calculated rate of change of fluorescence of each sample as a function of the logarithm of the concentration of antimicrobial agent in each sample.

4. The method according to any one of 2 to 3, further comprising a step of determining the concentration of an antimicrobial agent indicating a decrease in the rate of change in fluorescence.

5. The method according to 4, further comprising the step of determining the concentration of an antimicrobial agent indicating an increase in the rate of change in fluorescence.

6. The method according to 1, further comprising determining the metabolic activity of the microorganisms in each sample based on the calculated rate of change of fluorescence of each sample.

7. The method according to 6, further comprising the step of determining the concentration of an antimicrobial agent indicating a decrease in metabolic activity.

8. The method according to 6, further comprising the step of determining the concentration of an antimicrobial agent showing an increase in metabolic activity.

9. The method according to any of 1-8, wherein each sample contains microorganisms at a concentration of 10 colony forming units (CFU) or higher.

10. The method according to 9, wherein each sample contains microorganisms at a concentration of 14 CFU or higher.

11. The method according to 9, comprising the step of aliquoting each sample from a microbial composition having a concentration of microorganisms above 100 colony forming units (CFU).

12. Described in one of 1 to 11, wherein the concentration range of the antimicrobial agent in multiple samples ranges from a concentration below the minimum inhibitory concentration of the antimicrobial agent to a concentration higher than the minimum bactericidal concentration of the antimicrobial agent. the method of.

13. The method according to any of 1-12, further comprising the step of incubating the antimicrobial agent with the microorganism for a predetermined period of time prior to irradiation of the sample.

14. The method according to 13, wherein the antimicrobial agent is incubated with the microorganism for at least 10 minutes.

15. The method according to 13, wherein the antimicrobial agent is incubated with the microorganism for at least 20 minutes.

16. The method according to any of 1 to 15, wherein the sample contains a reducing substance.

17. The method according to 16, wherein the reducing substance is glutathione or a derivative thereof.

18. The method according to any of 16-17, wherein the reducing substance is present in the sample at a concentration of 0.1 mg / mL to 1 mg / mL.

19. The method according to any of 1-18, wherein the sample is irradiated in the sample container.

20. The method according to 19, wherein the sample container is a glass vial.

21. The method according to 20, wherein the glass vial comprises a wall with a zwitterionic coating.

22. The method of 21. The glass vial comprises a wall with a zwitterionic silane coating.

23. The method according to any of 19-22, wherein the sample is irradiated at the interface between the sample and the wall of the container.

24. The method of 23, wherein the monochromatic light source is focused at the interface between the sample and the wall of the container.

25. The method of 24, wherein the monochromatic light source is focused on the sample at a position 0.01 mm to 2 mm from the surface of the container wall.

26. The method of 24, wherein the monochromatic light source is focused on the sample at a position approximately 0.2 mm from the surface of the container wall.

27. The method according to any of 24-27, wherein the monochromatic light source is focused using a collimating lens.

28. The method according to any of 1-27, wherein the fluorescence produced by the sample and control is detected at 2500 cm ^-1 to 3500 cm ^-1 .

29. The method according to 28, wherein the fluorescence produced by the sample and control is detected at 3000 cm ^-1 .

30. The method according to any of 1-29, wherein the control is a microbial-free composition.

31. The method according to any of 1-30, wherein the control is a composition having the same components as the sample in the absence of microorganisms.

32. The method according to any of 1-31, comprising the step of irradiating each sample over multiple intensities using a monochromatic light source.

Methods for determining the phenotype of unknown microorganisms
1. A method of characterizing the phenotype of a microorganism, which includes the following steps:
A step of irradiating a sample containing a microorganism, a cross-linking substance, and an albumin protein with a monochromatic light source for a certain period of time to detect fluorescence from the sample over the certain period of time;
The step of calculating the rate of change in fluorescence by comparing the normalized rate of change in the detected fluorescence intensity generated by the sample with the normalized rate of change in the fluorescence produced by the control; as well as the sample. A step of determining cross-linking cleavage based on the calculated rate of change of fluorescence of, wherein the degree of cross-linking cleavage indicates the phenotype of the microorganism.

2. The method according to 1, wherein the cross-linking substance is a disulfide crosslinker.

3. The method according to any one of 1 and 2, wherein the cross-linking substance is a glutamic acid derivative.

の化合物を含む、3に記載の方法。 4. The cross-linking substance is formula (I) :.

3. The method according to 3, comprising the compound of.

5. The method according to any of 1 to 4, wherein the cross-linking substance is present in the sample in an amount such that the cross-linking substance: albumin protein molar ratio is 1:10 to 10: 1.

6. The method according to 5, wherein the cross-linking material is present in the sample in such an amount that the molar ratio of cross-linking material: albumin protein is approximately 1: 2.

7. The method according to any one of 1-6, indicating that an increase in the calculated rate of change of fluorescence of a sample produces a metabolite that cleaves one or more crosslinks in an albumin protein.

8. The method according to any one of 1 to 7, wherein the monochromatic light source is a laser.

9. The method according to 8, wherein the laser is a solid-state laser that irradiates at 532 nm.

10. The method according to any of 1-9, wherein the sample comprises a hydrophobic compound and an albumin protein.

11. The method according to 10, wherein the hydrophobic compound is a carotenoid.

12. The method according to 11, wherein the hydrophobic compound is lycopene.

13. The method according to any of 10-12, wherein the hydrophobic compound non-covalently binds to the albumin protein.

14. The method according to any of 1 to 13, wherein the sample contains a reducing substance.

15. The method according to 14, wherein the reducing substance is glutathione or a derivative thereof.

16. The method according to any of 14-15, wherein the reducing substance is present in the sample at a concentration of 0.1 mg / mL to 1 mg / mL.

17. The method according to any of 1 to 16, wherein the sample is irradiated in the sample container.

18. The method according to 17, wherein the sample container is a glass vial.

19. The method according to 18, wherein the glass vial comprises a wall with a zwitterionic coating.

20. The method according to 19, wherein the glass vial comprises a wall with a zwitterionic silane coating.

21. The method according to any of 17-20, wherein the sample is irradiated at the interface between the sample and the wall of the container.

22. The method according to 21, wherein the monochromatic light source is focused at the interface between the sample and the wall of the container.

23. The method of claim 22, wherein the monochromatic light source is focused on the sample at a position 0.01 mm to 2 mm from the surface of the wall of the container.

24. The method of 22 in which a monochromatic light source is focused on a sample at a position approximately 0.2 mm from the surface of the container wall.

25. The method according to any of 22-24, wherein the monochromatic light source is focused using a collimating lens.

26. The method according to any of 1-25, wherein the fluorescence produced by the sample and control is detected at 2500 cm ^-1 to 3500 cm ^-1 .

27. The method according to 26, wherein the fluorescence produced by the sample and control is detected at 3000 cm ^-1 .

28. The method according to any of 1-27, wherein the control is a microbial-free composition.

29. The method according to any of 1-28, wherein the control is a composition having the same components as the sample in the absence of microorganisms.

30. The method according to any of 1 to 29, comprising the step of irradiating each sample over multiple intensities using a monochromatic light source.

System for Resonance Raman spectroscopy
1. Monochromatic light source and
With the detector,
With the processor
In a system containing, the processor contains memory operably attached to the processor, the memory stores instructions, and the instructions are executed by the processor.
Irradiating the sample with a monochromatic light source at the first and second intensity;
Determining the intensity of resonant Raman scattering at the first and second irradiation intensities;
To calculate the rate of change in the intensity of the resonant Raman scattering in response to the change in intensity from the first irradiation intensity to the second irradiation intensity; and to determine the change in the sample's resonant Raman response to the resonant Raman scattering. A system that causes the processor to compare the rate of change in intensity with the rate of change in irradiation intensity from a monochromatic light source.

2. The system according to 1, where the monochromatic light source is a laser.

3. The system described in 2, where the laser is an Nd: YAG laser.

4. The system described in 3, where the laser is a double wave Nd: YAG laser.

5. The system according to any one of 1-4, wherein the detector is a charge-coupled device (CCD) detector.

6. Further includes instructions that, when executed by the processor, irradiate the sample with a monochromatic light source over multiple intensities for a period of time and force the processor to determine the rate of change in the intensity of the resonant Raman scattering. The system described in any of 5 to 5.

7. If the memory is executed by the processor
Irradiating the first sample over a period of time with a monochromatic light source to determine the rate of change in the intensity of the resonant Raman scattering for the first sample;
By comparing the normalized rate of change in the intensity of the resonant Raman scattering with respect to the first sample to the normalized rate of change in the intensity of irradiation of the first sample with a monochromatic light source, the net signal of the first sample can be obtained. To calculate;
Irradiating the second sample over multiple intensities over a period of time with a monochromatic light source to determine the rate of change in the intensity of the resonant Raman scattering for the second sample; and normalizing the intensity of the resonant Raman scattering for the second sample. Further included is an instruction to force the processor to calculate the net signal of the second sample by comparing the resulting rate of change with the normalized rate of change of the intensity of the second sample from a monochromatic light source. The system described in any of 1 to 6.

8. Have the processor determine that the first sample contains a different gas composition than the second sample, based on the calculated net signal compared, when the memory is run by the processor. The system described in 7, which further contains instructions.

9. The memory determines that the first or second sample contains actively metabolizing microorganisms based on the calculated net signals compared when executed by the processor. 8 The system according to 8, further including instructions for causing the processor to perform.

System for detecting microorganisms in samples
1. Monochromatic light source and
With the detector,
With the processor
In a system that includes, the processor contains memory operably attached to the processor, the memory stores instructions, and the instructions are executed by the processor.
Irradiating a sample over a period of time with a monochromatic light source over one or more intensities and calculating the rate of change in the intensity of resonant Raman scattering for the sample;
Compute the net signal of the sample by comparing the normalized rate of change of the intensity of the resonance Raman scattering of the sample with the normalized rate of change of the intensity of the sample irradiated by a monochromatic source; and the calculated sample. A system that causes a processor to determine the presence or absence of microorganisms in a sample based on the net signal.

2. The instruction described in 1, further comprising an instruction to cause the processor to determine that a microorganism is present if the calculated net signal of the sample exceeds a predetermined threshold when the memory is executed by the processor. system.

2. The system according to 2, wherein the predetermined threshold is the net signal of 1.

3. The system described in any of 1-2, where the monochromatic light source is a laser.

4. The system according to 3, wherein the laser is a solid-state laser that irradiates at 532 nm.

5. The system according to any of 1 to 4, wherein the sample contains a hydrophobic compound that does not covalently bind to an albumin protein.

6. The system according to any of 1-5, wherein the sample contains a reducing substance.

7. The system according to 6, wherein the reducing substance is glutathione or a derivative thereof.

8. The system according to any of 6-7, wherein the reducing substance is present in the sample at a concentration of 0.1 mg / mL to 1 mg / mL.

9. The system according to any of 1-8, further comprising a free radical scavenger.

10. The system according to 9, wherein the free radical scavenger contains bilirubin or a derivative thereof.

11. The system according to 10, wherein bilirubin or a derivative thereof is present at a concentration of 0.5 μM to 2 μM.

12. The system according to 11, wherein bilirubin is present at a concentration of 0.25 μM to 1.75 μM.

13. The system according to any of 1-12, wherein the detector is a charge-coupled device (CCD) detector.

14. The system according to any of 1 to 13, further including a sample container.

15. The system according to 14, wherein the sample container is a glass vial.

16. The system according to 15, wherein the glass vial contains a wall with a zwitterionic coating.

17. The system according to 16, wherein the glass vial contains a wall with a zwitterionic silane coating.

18. The system according to any of 14-17, which is configured to irradiate the sample at the interface between the sample and the wall of the container.

19. The system according to 18, wherein the monochromatic light source is focused at the interface between the sample and the wall of the container.

20. The system according to 19, wherein the monochromatic light source is focused on the sample at a position 0.01 mm to 2 mm from the surface of the container wall.

21. The system according to 20, wherein the monochromatic light source is focused on the sample at a position approximately 0.2 mm from the surface of the container wall.

22. The system according to any of 19-21, wherein the monochromatic light source is focused using a collimating lens.

23. Monochromatic light source and
With the detector,
With the processor
In a system containing, the processor contains memory operably attached to the processor, the memory stores instructions, and the instructions are executed by the processor.
Irradiating the first sample over a period of time with a monochromatic light source and calculating the rate of change in the intensity of the resonant Raman scattering for the first sample;
By comparing the normalized rate of change in the intensity of the resonant Raman scattering with respect to the first sample to the normalized rate of change in the intensity of irradiation of the first sample with a monochromatic light source, the net signal of the first sample can be obtained. To calculate;
Irradiating the second sample over a plurality of intensities over a period of time with a monochromatic light source to determine the rate of change in the intensity of resonant Raman scattering for the second sample;
The net signal of the second sample is obtained by comparing the normalized rate of change in the intensity of the resonant Raman scattering with respect to the second sample to the normalized rate of change in the intensity of irradiation of the second sample with a monochromatic light source. To calculate; and to determine the presence or absence of microorganisms in one or more of the first sample or the second sample by comparing the net signal of the first sample with the net signal of the second sample. A system that causes the processor to perform.

24. If the memory is run by the processor and the calculated net signal of the second sample is greater than the calculated net signal of the first sample, then the microorganism is determined to be present in the second sample. 23. The system according to 23, further including instructions for causing the processor to do so.

25. If the memory is run by the processor and the calculated net signal of the first sample is greater than the calculated net signal of the second sample, then the microorganism is determined to be present in the first sample. 24. The system described in 24, further including instructions that cause the processor to do so.

26. The system according to any of 23-25, wherein the monochromatic light source is a laser.

27. The system according to 26, wherein the laser is a solid-state laser that irradiates at 532 nm.

28. The system according to any of 23-27, wherein the sample comprises a hydrophobic compound that is non-covalently bound to an albumin protein.

29. The system according to any of 23-28, wherein the sample contains a reducing substance.

30. The system according to 29, wherein the reducing substance is glutathione or a derivative thereof.

31. The system according to any of 29-30, wherein the reducing substance is present in the sample at a concentration of 0.1 mg / mL to 1 mg / mL.

32. The system according to any of 23-31, further comprising a free radical scavenger.

33. The system according to 32, wherein the free radical scavenger comprises bilirubin or a derivative thereof.

34. The system according to 33, wherein bilirubin or a derivative thereof is present at a concentration of 0.5 μM to 2 μM.

35. The system according to 33, wherein bilirubin is present at a concentration of 0.25 μM to 1.75 μM.

36. The system according to any of 70-79, wherein the detector is a charge-coupled device (CCD) detector.

37. The system according to any of 23-36, further comprising a sample container.

38. The system according to 37, wherein the sample container is a glass vial.

39. The system according to 38, wherein the glass vial contains a wall with a zwitterionic coating.

40. The system according to 39, wherein the glass vial contains a wall with a zwitterionic silane coating.

41. The system according to any of 37-40, which is configured to irradiate the sample at the interface between the sample and the wall of the container.

42. The system according to 41, wherein the monochromatic light source is focused at the interface between the sample and the wall of the container.

43. The system according to 42, wherein the monochromatic light source is focused on the sample at a position 0.01 mm to 2 mm from the surface of the container wall.

44. The system according to 42, wherein the monochromatic light source is focused on the sample at a position approximately 0.2 mm from the surface of the container wall.

45. The system according to any of 42-44, wherein the monochromatic light source is focused using a collimating lens.

46. (a) Monochromatic light source and
(B) Optical adjustment components and
(C) Photodetector and
(D) With the processor, which contains the memory that stores the instructions and is operably coupled to the processor.
In a system that contains an instruction, when executed by a processor,
Irradiating the sample in the sample holder with a monochromatic light source at the first irradiation intensity and the second irradiation intensity for a predetermined period;
Measuring scattered light from a sample using a photodetector;
To determine the intensity of resonant Raman scattering and fluorescence scattering at the first and second irradiation intensities;
To calculate the rate of change in the intensity of the resonant Raman scattering and the intensity of the fluorescent scattering; and to have the processor correct the rate of change in the intensity of the resonant Raman scattering and the intensity of the fluorescent scattering to obtain a net signal. system.

47. The system according to 46, which includes collimating optics mounted on a linear table in which the optical adjustment component is configured to produce an adjustable focus.

48. The system described in 46, where the optical adjustment component includes a mechanical shutter.

49. The system according to 46, further comprising a turntable containing multiple sample chambers.

50. The system described in 49, where the turntable contains eight sample chambers.

51. Collimated optics and
It also includes a subsystem for characterizing incoherently scattered light, including an optical tuning component configured to spread the incoherently scattered light onto a linearly arranged photodetector, 50. The system described in.

52. The processor further includes a memory containing the instructions, which, when executed by the processor, causes the processor to move the collimating optics to focus light on each of the sample chambers, 51. Described system.

53. The system according to 46, wherein the sample contains albumin incorporating a ligand.

54. If the memory is executed by the processor
Determining the rate of change in the intensity of fluorescence scattering from a standard reference sample;
The system according to 1, further comprising an instruction to cause the processor to calculate the net signal as the rate of change in the intensity of the resonant Raman scattering minus the rate of change in the intensity of the fluorescent scattering from the standard sample.

55. If the memory is executed by the processor
Determining the rate of change of total output from the standard reference sample;
53 or 54, further comprising an instruction to force the processor to calculate the net signal as the rate of change in the intensity of the resonant Raman scattering minus the rate of change in the intensity of the resonant Raman scattering from the standard sample. system.

56. When the memory is executed by the processor, it compares the net signal with a given threshold and, based on this comparison, determines that the first sample contains actively metabolizing microorganisms. 53 or 54. The system according to 53 or 54, further including instructions to be performed by the processor.

57. The system according to 46, wherein the sample contains a reducing substance.

58. The system according to 57, wherein the sample contains a disulfide crosslinker.

59. The system according to 58, wherein the disulfide cross-linking material comprises a core that is cleaved by an enzyme or metabolite produced by a microorganism in the sample.

の化合物を含む、59に記載の方法。 60. The cross-linking material is formula (I):

59. The method according to 59, comprising the compound of.

61. The system according to 46, wherein the sample holder is a glass vial.

62. The system according to 46, wherein the sample holder contains a coating that alters the absorption of albumin.

63. The system of claim 62, wherein the coating comprises a zwitterionic coating.

64. The system according to 46, wherein the processor is configured to expose the sample to a monochromatic light source to minimize the net signal.

65. The system according to 46, which is configured to irradiate the sample at the interface between the sample and the surface of the sample holder.

66. The system according to 65, which is configured to maximize the net signal.

A system for compensating for laser output drift during nonlinear Raman spectroscopy
1. Monochromatic light source and
With the detector,
With the processor
In a system containing, the processor contains memory operably attached to the processor, the memory stores instructions, and the instructions are executed by the processor.
The reference composition is irradiated with a monochromatic light source over a plurality of intensities for a certain period of time, and the rate of change in the intensity of resonance Raman scattering is determined . Irradiation and determination, including reference compounds that do not show a change in the intensity of the monochromatic light source; and the rate of change in the intensity of resonance Raman scattering for the reference composition to determine if the monochromatic light source exhibits thermal drift. A system that causes a processor to calculate the net signal of a reference composition by comparing it to the rate of change of the irradiation intensity of the reference composition.

2. If the memory is executed by the processor
Irradiating a sample with a monochromatic light source over a range of intensities for a period of time to determine the rate of change in the intensity of resonant Raman scattering;
To generate a correction factor, multiply the net signal of the reference composition by the rate of change of the irradiation intensity of the sample by the monochromatic light source; and to compensate for the thermal drift of the monochromatic light source, the intensity of the resonant Raman scattering for the sample. The system according to 1, further comprising an instruction to cause the processor to reduce the correction factor from the determined rate of change.

3. The system described in any of 1-2, where the monochromatic light source is a laser.

4. The system according to 3, wherein the laser is a solid-state laser that irradiates at 532 nm.

5. The system according to any of 1 to 4, wherein the sample contains a hydrophobic compound that does not covalently bind to an albumin protein.

6. The system according to any of 1-5, wherein the sample contains a reducing substance.

7. The system according to 6, wherein the reducing substance is glutathione or a derivative thereof.

8. The system according to any of 6-7, wherein the reducing substance is present in the sample at a concentration of 0.1 mg / mL to 1 mg / mL.

9. The system according to any of 1-8, further comprising a free radical scavenger.

10. The system according to 9, wherein the free radical scavenger contains bilirubin or a derivative thereof.

11. The system according to 10, wherein bilirubin or a derivative thereof is present at a concentration of 0.5 μM to 2 μM.

12. The system according to 10, wherein bilirubin is present at a concentration of 0.25 μM to 1.75 μM.

13. The system according to any of 1 to 12, wherein the fixed period is 200 to 1500 seconds.

14. The system according to 13, wherein the fixed period is 600 seconds.

15. The system according to any of 1-14, wherein the detector is a charge-coupled device (CCD) detector.

System for characterizing antimicrobial susceptibility
1. Monochromatic light source and
With the detector,
With the processor
In a system containing, the processor contains memory operably attached to the processor, the memory stores instructions, and the instructions are executed by the processor.
A monochromatic light source irradiates a plurality of samples containing microorganisms and antimicrobial agents over a plurality of intensities for a certain period of time, and determines the rate of change in the intensity of resonance Raman scattering for each irradiated sample. Irradiation and determination, including the same concentration of microorganisms and different concentrations of antimicrobial agent;
Compute the net signal for each sample by comparing the normalized rate of change in the intensity of the resonance Raman scattering for each sample with the normalized rate of change for the intensity of each sample irradiated by a monochromatic source; A system that causes a processor to determine the susceptibility of a microorganism to an antimicrobial agent based on the calculated net signal of a sample of.

2. The system according to 1, wherein when the memory is executed by the processor, the instructions are stored in the memory to cause the processor to compare the net signals calculated for each sample.

3. When the memory is executed by the processor, the instruction to make the processor execute to plot the calculated net signal of each sample as a function of the logarithm of the concentration of the antimicrobial agent of each sample is stored in the memory. Including, the system described in 2.

4. Described in one of 2-3, which contains an instruction in memory that causes the processor to determine the concentration of antimicrobial agent that indicates a decrease in the net signal when the memory is executed by the processor. System.

5. The system according to 4, wherein the memory contains an instruction that causes the processor to determine the concentration of antimicrobial agent indicating an increase in the net signal when the memory is executed by the processor.

6. The memory contains instructions that, when executed by the processor, cause the processor to determine the metabolic activity of the microorganisms in each sample based on the calculated net signal of each sample. , The system described in 1.

7. The system according to 6.

8. The system according to 6, wherein the memory contains an instruction that causes the processor to determine the concentration of an antimicrobial agent indicating an increase in metabolic activity when executed by the processor.

9. The system according to any of 1-8, wherein each sample contains microorganisms at a concentration of 10 colony forming units (CFU) or higher.

10. The system according to 9, wherein each sample contains microorganisms at a concentration of 14 CFU or higher.

11. Described in one of 1 to 10, wherein the concentration range of the antimicrobial agent in multiple samples ranges from a concentration below the minimum inhibitory concentration of the antimicrobial agent to a concentration higher than the minimum bactericidal concentration of the antimicrobial agent. System.

12. The system according to any of 1 to 11, wherein the sample contains a reducing substance.

13. The system according to 12, wherein the reducing substance is glutathione or a derivative thereof.

14. The system according to any of 12-13, wherein the reducing substance is present in the sample at a concentration of 0.1 mg / mL to 1 mg / mL.

15. The system according to any of 1-14, further comprising a sample container.

16. The system according to 15, where the sample container is a glass vial.

17. The system according to 16, wherein the glass vial contains a wall with a zwitterionic coating.

18. The system according to 17, wherein the glass vial contains a wall with a zwitterionic silane coating.

19. The system according to any of 15-18, which is configured to irradiate the sample at the interface between the sample and the wall of the container.

20. The system according to 19, wherein the monochromatic light source is focused at the interface between the sample and the wall of the container.

21. The system according to 20, wherein the monochromatic light source is focused on the sample at a position 0.01 mm to 2 mm from the surface of the container wall.

22. The system according to 20, wherein the monochromatic light source is focused on the sample at a position approximately 0.2 mm from the surface of the container wall.

23. The system according to any of 20-22, wherein the monochromatic light source is focused using a collimating lens.

24. The system according to any of 1 to 23, wherein the detector is a charge-coupled device (CCD) detector.

A system for determining the phenotype of unknown microorganisms
1. Monochromatic light source and
With the detector,
With the processor
In a system containing, the processor contains memory operably attached to the processor, the memory stores instructions, and the instructions are executed by the processor.
To determine the rate of change in the intensity of resonant Raman scattering by irradiating a sample containing microorganisms, cross-linking materials, and albumin proteins with a monochromatic light source over a period of time with a monochromatic light source;
Compute the net signal of the sample by comparing the normalized rate of change in the intensity of the resonance Raman scattering with respect to the sample to the normalized rate of change in the intensity of the sample irradiated by a monochromatic light source; as well as the calculation of the sample. A system in which a processor is made to determine that the degree of cross-linking cleavage indicates the phenotype of a microorganism based on the net signal.

2. The system according to 1, wherein the cross-linking material is a disulfide crosslinker.

3. The system according to any one of 1-2, wherein the cross-linking substance is a glutamic acid derivative.

の化合物を含む、3に記載のシステム。 4. The cross-linking substance is formula (I) :.

The system according to 3, which comprises a compound of.

5. The system according to any of 1-4, wherein the cross-linking material is present in the sample in an amount such that the cross-linking material: albumin protein molar ratio is 1:10 to 10: 1.

6. The system according to 5, wherein the cross-linking material is present in the sample in such an amount that the cross-linking material: albumin protein molar ratio is approximately 1: 2.

7. The system according to any one of 1-6, wherein an increase in the calculated net signal of the sample over time indicates that the microorganism produces a metabolite that cleaves one or more crosslinks in the albumin protein.

8. The system described in any of 1-7, wherein the monochromatic light source is a laser.

9. The system according to 8, wherein the laser is a solid-state laser that irradiates at 532 nm.

10. The system according to any of 1-9, wherein the sample contains a hydrophobic compound and an albumin protein.

11. The system according to 10, wherein the hydrophobic compound is a carotenoid.

12. The system according to 11, wherein the hydrophobic compound is lycopene.

13. The system according to any of 10-12, wherein the hydrophobic compound non-covalently binds to the albumin protein.

14. The system according to any of 1 to 13, wherein the sample contains a reducing substance.

15. The system according to 14, wherein the reducing substance is glutathione or a derivative thereof.

16. The system according to any of 14-15, wherein the reducing substance is present in the sample at a concentration of 0.1 mg / mL to 1 mg / mL.

17. The system according to any of 1-16, further comprising a sample container.

18. The system according to 17, wherein the sample container is a glass vial.

19. The system according to 18, wherein the glass vial contains a wall with a zwitterionic coating.

20. The system according to 19, wherein the glass vial contains a wall with a zwitterionic silane coating.

21. The system according to any of 17-20, which is configured to irradiate the sample at the interface between the sample and the wall of the container.

22. The system according to 21, wherein the monochromatic light source is focused at the interface between the sample and the wall of the container.

23. The system according to 22 in which a monochromatic light source is focused on a sample at a position 0.01 mm to 2 mm from the surface of the container wall.

24. The system according to 22 in which a monochromatic light source is focused on the sample at a position approximately 0.2 mm from the surface of the container wall.

25. The system according to any of 22-24, wherein the monochromatic light source is focused using a collimating lens.

26. The system according to any of 1-25, wherein the detector is a charge-coupled device (CCD) detector.

A system for detecting microorganisms in a sample via fluorescence
1. Monochromatic light source and
With the detector,
With the processor
In a system containing, the processor contains memory operably attached to the processor, the memory stores instructions, and the instructions are executed by the processor.
Irradiating the sample with a monochromatic light source for a certain period of time and detecting the fluorescence from the sample over the certain period of time;
Calculate the rate of change in fluorescence due to the presence of microorganisms by comparing the normalized rate of change in the intensity of the detected fluorescence generated by the sample to the normalized rate of change in fluorescence from the control. That; and a system that causes the processor to determine the presence or absence of microorganisms in the sample based on the calculated rate of change of fluorescence of the sample.

2. The instruction according to 1, further comprising an instruction to cause the processor to determine that a microorganism is present when the calculated rate of change of fluorescence exceeds a predetermined threshold when the memory is executed by the processor. system.

3. The system described in any of 1-2, where the monochromatic light source is a laser.

4. The system according to 3, wherein the laser is a solid-state laser that irradiates at 532 nm.

5. The system according to any of 1 to 4, wherein the sample contains a hydrophobic compound that does not covalently bind to an albumin protein.

6. The system according to any of 1-5, wherein the sample contains a reducing substance.

7. The system according to 6, wherein the reducing substance is glutathione or a derivative thereof.

8. The system according to any of 6-7, wherein the reducing substance is present in the sample at a concentration of 0.1 mg / mL to 1 mg / mL.

9. The system according to any of 1-8, wherein the detector is a charge-coupled device (CCD) detector.

10. The system according to any of 1-9, further including a sample container.

11. The system according to 10, wherein the sample container is a glass vial.

12. The system according to 11, wherein the glass vial contains a wall with a zwitterionic coating.

13. The system according to 12, wherein the glass vial contains a wall with a zwitterionic silane coating.

14. The system according to any of 11-13, which is configured to irradiate the sample at the interface between the sample and the wall of the container.

15. The system according to 14, wherein the monochromatic light source is focused at the interface between the sample and the wall of the container.

16. The system according to 15, wherein the monochromatic light source is focused on the sample at a position 0.01 mm to 2 mm from the surface of the container wall.

17. The system according to 16, wherein a monochromatic light source is focused on the sample at a position approximately 0.2 mm from the surface of the container wall.

18. The system according to any of 15-17, wherein the monochromatic light source is focused using a collimating lens.

19. The system according to any of 1-18, wherein the fluorescence produced by the sample and control is detected at 2500 cm ^-1 to 3500 cm ^-1 .

20. The system according to 19, wherein the fluorescence produced by the sample and control is detected at 3000 cm ^-1 .

21. The system according to any of 1-20, wherein the control is a microbial-free composition.

22. The system according to any of 1-21, wherein the microorganism is a pathogenic microorganism.

23. The system according to any of 1-22, wherein the control is a composition having the same components as the sample in the absence of microorganisms.

24. The system according to any of 1-23, which is configured to irradiate a sample over multiple intensities using a monochromatic light source.

System for characterizing antimicrobial susceptibility
1. Monochromatic light source and
With the detector,
With the processor
In a system containing, the processor contains memory operably attached to the processor, the memory stores instructions, and the instructions are executed by the processor.
Multiple samples containing microorganisms, each containing a microorganism and an antimicrobial agent, are irradiated with a monochromatic light source for a certain period of time, and fluorescence from each of the samples irradiated for the certain period is detected, and each sample has the same concentration. Irradiation and detection, including microorganisms and different concentrations of antimicrobial agents;
Calculate the rate of change in fluorescence in each sample by comparing the normalized rate of change in the detected fluorescence intensity produced by each sample to the normalized rate of change in fluorescence produced by the control. That; as well as a system that causes the processor to determine the susceptibility of a microorganism to an antimicrobial agent based on the calculated rate of change of fluorescence of multiple samples.

2. The system according to 1, further comprising an instruction to cause the processor to compare the calculated rate of change of fluorescence for each sample when the memory is executed by the processor.

3. The system according to 2, wherein the step of comparison comprises plotting the calculated rate of change of fluorescence of each sample as a function of the logarithm of the concentration of antimicrobial agent in each sample.

4. The system according to 1, further comprising an instruction to cause the processor to determine the concentration of antimicrobial agent indicating a decrease in the rate of change in fluorescence when the memory is executed by the processor.

5. The system according to 1, further comprising an instruction to cause the processor to determine the concentration of antimicrobial agent indicating an increase in the rate of change in fluorescence when the memory is executed by the processor.

6. To include an instruction that causes the processor to determine the metabolic activity of the microorganisms in each sample based on the calculated rate of change of fluorescence of each sample when the memory is executed by the processor. Described system.

7. The system according to 1, further comprising an instruction to cause the processor to determine the concentration of an antimicrobial agent indicating a decrease in metabolic activity when the memory is executed by the processor.

8. The system according to 1, further comprising an instruction to cause the processor to determine the concentration of an antimicrobial agent indicating an increase in metabolic activity when the memory is executed by the processor.

9. The system according to any of 1-8, wherein each sample contains microorganisms at a concentration of 10 colony forming units (CFU) or higher.

10. The system according to 9, wherein each sample contains microorganisms at a concentration of 14 CFU or higher.

11. The system according to 9, wherein each sample is aliquoted from a microbial composition having a concentration of 100 colony forming units (CFU) or higher.

12. Described in one of 1 to 11, wherein the concentration range of the antimicrobial agent in multiple samples ranges from a concentration below the minimum inhibitory concentration of the antimicrobial agent to a concentration higher than the minimum bactericidal concentration of the antimicrobial agent. System.

13. The system according to any of 1-12, wherein the antimicrobial agent is incubated with the microorganism for a predetermined period of time before irradiating the sample.

14. The system according to 13, wherein the antimicrobial agent is incubated with the microorganism for at least 10 minutes.

15. The system according to 13, wherein the antimicrobial agent is incubated with the microorganism for at least 20 minutes.

16. The system according to any of 1 to 15, wherein the sample contains a reducing substance.

17. The system according to 16, wherein the reducing substance is glutathione or a derivative thereof.

18. The system according to any of 16-17, wherein the reducing substance is present in the sample at a concentration of 0.1 mg / mL to 1 mg / mL.

19. The system according to any of 1-18, wherein the sample is irradiated in the sample container.

20. The system according to 19, wherein the sample container is a glass vial.

21. The system according to 20, wherein the glass vial contains a wall with a zwitterion coating.

22. The system according to 21, wherein the glass vial contains a wall with a zwitterionic silane coating.

23. The system according to any of 19-22, which is configured to irradiate the sample at the interface between the sample and the wall of the container.

24. The system according to 23, wherein the monochromatic light source is focused at the interface between the sample and the wall of the container.

25. The system according to 24, wherein the monochromatic light source is focused on the sample at a position 0.01 mm to 2 mm from the surface of the container wall.

26. The system according to 24, wherein the monochromatic light source is focused on the sample at a position approximately 0.2 mm from the surface of the container wall.

27. The system according to any of 24-27, wherein the monochromatic light source is focused using a collimating lens.

28. The system according to any of 1-27, which is configured to detect the fluorescence produced by the sample and control at 2500 cm ^-1 to 3500 cm ^-1 .

29. The system according to 28, which is configured to detect fluorescence produced by samples and controls at 3000 cm ^-1 .

30. The system of any of claims 1-29, wherein the control is a microbial-free composition.

31. The system according to any of 1-30, wherein the control is a composition having the same components as the sample in the absence of microorganisms.

32. The system according to any of 1-31, which is configured to illuminate each sample with a monochromatic light source over multiple intensities.

A system for determining the phenotype of unknown microorganisms
1. Monochromatic light source and
With the detector,
With the processor
In a system containing, the processor contains memory operably attached to the processor, the memory stores instructions, and the instructions are executed by the processor.
Irradiating a sample containing microorganisms, cross-linking substances, and albumin protein with a monochromatic light source for a certain period of time to detect fluorescence from the sample over a certain period of time;
Calculating the rate of change in fluorescence by comparing the normalized rate of change in the intensity of the detected fluorescence generated by the sample to the normalized rate of change in the fluorescence produced by the control; as well as the sample. A system in which a processor is made to determine that the degree of cross-linking cleavage indicates the phenotype of a microorganism, which is to determine the cross-linking cleavage based on the calculated rate of change of fluorescence.

2. The system according to 1, wherein the cross-linking material is a disulfide crosslinker.

3. The system according to any one of 1-2, wherein the cross-linking substance is a glutamic acid derivative.

の化合物を含む、3に記載のシステム。 4. The cross-linking substance is formula (I)

The system according to 3, which comprises a compound of.

5. The system according to any of 1-4, wherein the cross-linking material is present in the sample in an amount such that the cross-linking material: albumin protein molar ratio is 1:10 to 10: 1.

6. The system according to 5, wherein the cross-linking material is present in the sample in such an amount that the cross-linking material: albumin protein molar ratio is approximately 1: 2.

7. The system according to any of 1-6, wherein the increased rate of change in the fluorescence of the sample causes the processor to indicate that the microorganism produces a metabolite that cleaves one or more crosslinks in the albumin protein.

8. The system described in any of 1-7, wherein the monochromatic light source is a laser.

9. The system according to 8, wherein the laser is a solid-state laser that irradiates at 532 nm.

10. The system according to any of 1-9, wherein the sample contains a hydrophobic compound and an albumin protein.

11. The system according to 10, wherein the hydrophobic compound is a carotenoid.

12. The system according to 11, wherein the hydrophobic compound is lycopene.

13. The system according to any of 10-12, wherein the hydrophobic compound non-covalently binds to the albumin protein.

14. The system according to any of 1 to 13, wherein the sample contains a reducing substance.

15. The system according to 14, wherein the reducing substance is glutathione or a derivative thereof.

16. The system according to any of 14-15, wherein the reducing substance is present in the sample at a concentration of 0.1 mg / mL to 1 mg / mL.

17. The system according to any of 1-16, which is configured to irradiate the sample in the sample container.

18. The system according to 17, wherein the sample container is a glass vial.

19. The system according to 18, wherein the glass vial contains a wall with a zwitterionic coating.

20. The system according to 19, wherein the glass vial contains a wall with a zwitterionic silane coating.

21. The system according to any of 17-20, which is configured to irradiate the sample at the interface between the sample and the wall of the container.

22. The system according to 21, wherein the monochromatic light source is focused at the interface between the sample and the wall of the container.

23. The system according to 22 in which a monochromatic light source is focused on a sample at a position 0.01 mm to 2 mm from the surface of the container wall.

24. The system according to 22 in which a monochromatic light source is focused on the sample at a position approximately 0.2 mm from the surface of the container wall.

25. The system according to any of 22-24, wherein the monochromatic light source is focused using a collimating lens.

26. The system according to any of 1-25, configured to detect fluorescence produced by samples and controls at 2500 cm ^-1 to 3500 cm ^-1 .

27. The system according to 26, configured to detect fluorescence produced by samples and controls at 3000 cm ^-1 .

28. The system according to any of 1-27, wherein the control is a microbial-free composition.

29. The system according to any of 1-28, wherein the control is a composition having the same components as the sample in the absence of microorganisms.

30. The system according to any of 1-29, which is configured to illuminate the sample with a monochromatic light source over multiple intensities.

As can be understood from the disclosures presented experimentally, this disclosure has a wide variety of uses. The following examples are presented to those skilled in the art to provide full disclosure and description of the fabrication and use of the invention, are not intended to limit the scope of the invention, and the following experiments are all or It is also not intended to be described as the only experiment performed. One of ordinary skill in the art should readily recognize various non-essential parameters that can be changed or modified to produce essentially similar results. Efforts are being made to ensure accuracy with respect to the numbers used (eg, quantity, temperature, etc.), but some experimental errors and deviations should be considered.

One embodiment of the method of study comprises a Raman spectrometer constructed around a laser whose wavelength is close to the absorption bandgap affected by changes in the excited state of the molecule under study. For systems containing lycopene incorporated into albumin, a laser with a wavelength of 532 nm is used because this wavelength is at the tail of the lycopene absorption triplet and is subject to changes in excited state. The system also includes components that modulate the laser intensity in small amounts over time.

A 532 nm laser beam is generated by exciting the resonator at 1064 nm using an 808 nm optical pump and Q-switching to 532 nm in an external resonator. The output of the final Q-switched resonator is very sensitive to the resonator orientation, and the resonator orientation is very sensitive to temperature. The temperature then changes due to the accumulation (and dissipation) of heat from within the resonator. Therefore, changes in the current applied to the excitation laser do not always appear as changes in the 532 nm laser light output from the external resonator.

Since it has been found that small changes in the temperature of the external resonator can affect the output from the laser, a mechanism that affects this can be used to modulate the laser output. In one embodiment, varying the CCD shutter time (ie, the time used to acquire data on the CCD) has been found to affect the laser output. In this example, the frequency with which the data is output from the CCD affects the heat buildup in the CCD, which in turn affects the temperature of the external laser cavity that is thermally close to the CCD.

The system of interest was configured to measure the time-dependent changes in resonant Raman intensity as the laser intensity was modulated. The rate of change in normalized resonance Raman intensity is compared to the rate of change in normalized laser intensity. To minimize measurement error, this comparison can be made with a normalized rate of change in laser intensity. To eliminate systematic differences between the control parameters used to modulate the laser intensity and the resulting laser modulation, use a reference composition that provides a characteristic Raman spectrum whose intensity does not change with laser output. Characterized the laser output. In one example, we used NIST SRM 2242, which gives a wide Raman spectrum with 532 nm light. An example of nonlinear resonance Raman spectroscopy according to the present disclosure is shown in FIG.

における第2項（分極率の項）がゼロに等しくないことを示す。これは、レーザー自体によって引き起こされる変化、またはサンプルの物理的または化学的変化によって引き起こされる変化のいずれかを示すことができる。 The trace gradient in Figure 1 is the calculated net signal. If the gradient is not equal to 1, this means

It is shown that the second term (polarizability term) in is not equal to zero. This can indicate either the changes caused by the laser itself, or the changes caused by physical or chemical changes in the sample.

Example 1:
FIG. 2 shows an example of nonlinear resonance Raman spectroscopy for characterizing small changes in composition. The sample contains lycopene that is non-covalently bound to an albumin protein (as described in US Patent Application Publication No. 2016/0324933, the entire disclosure of which is incorporated herein by reference), which is a microorganism. A nutrient buffer and a buffer that maintains the pH at 7.2 were added to support the metabolism of albumin.

In these examples, nonlinear resonance Raman spectroscopy measures the resonance Raman spectrum of lycopene, where the magnitude of the Raman peak is a sensitive function to small changes in the composition. Since lycopene is incorporated into albumin, the non-linearity of the resonance Raman response (ie, the calculated nonlinear resonance Raman coefficient) characterizes the environment provided by albumin and also as albumin solubilizes water-insoluble molecules. Due to its design, the non-linearity of resonance Raman corresponds to the amount of insoluble molecules present in solution.

In this example, the amount of gas present in the sample is characterized by the non-linearity of the resonant Raman response. Some common gases (such as oxygen, nitrogen, and carbon dioxide) can be dissolved in water, and the solubility decreases with increasing temperature. Thus, heating one or more portions of the test sample to a high temperature (such as 55 ° C.) for a sufficiently long time causes the dissolved gas to become supersaturated and nucleate by a time-dependent process. The liquid is then cooled to 37 ° C. and the liquid becomes unsaturated with respect to the dissolved gas. When this unsaturated liquid is mixed with an albumin / lycopene reagent that is nominally stored at 4 ° C (thus saturate at 4 ° C and supersaturated at 37 ° C), the mixture can become unsaturated with respect to the dissolved gas. .. In this scenario, some of the dissolved gas distributed to albumin is transferred to the solution, reducing the amount of gas taken up by albumin. Thus, by changing the amount of unsaturated liquid (and the degree of unsaturated), the environment around lycopene changes and is characterized by changes in the non-linearity of the resonant Raman.

FIG. 2 shows the nonlinearity of the resonant Raman nonlinearity coefficient for various solutions, including both albumin / lycopene reagents. The first example shows a comparison of reagents stored at 5 ° C for several weeks and tested at 37 ° C (and thus supersaturated with gas at 37 ° C). The non-linear coefficient of the reagent itself is 3.3. Addition of bacteria (concentration 10 CFU / mL) to the solution raises the nonlinear coefficient to 3.7.

Example 2:
Bilirubin is a potent antioxidant present in serum and exhibits very weak light absorption at 532 nm. In this example, bilirubin is used to control the signal in nonlinear resonant Raman spectroscopy to improve the signal-to-noise ratio when characterizing the test sample.

In the samples described, bilirubin is added to non-covalently bind to albumin protein. Figures 3 and 4 show the effect of bilirubin on the mean resonant Raman peak intensity. FIG. 3 shows the average Raman peak height from 4 non-infected control samples and 3 10 CFU S. aureus infected samples. In all cases, the reagent is incorporated into albumin, contains diluted lycopene in a 4 mL buffer medium, and contains 0.5-fold nutrient broth to allow bacterial metabolism. FIG. 4 shows average Raman peak heights from 4 non-infected control samples supplemented with 1 μM bilirubin and 3 10 CFU S. aureus infected samples. Compared to FIG. 3, the decline in the infected sample has moved to a longer timescale (450 s vs. 200 s) and the non-infected control sample shows no further decline.

FIG. 5 shows the determination of the signal-to-noise ratio of the above sample. In this example, the "signal" is the average intensity of the resonance Raman peaks of the non-infected control sample and the 10 CFU Staphylococcus aureus sample, calculated using a linear fit from t = 0 to the time shown on the X-axis. The difference in rate of change, "noise" is the sum of the standard deviations of the mean rate of change of the control sample and the 10 CFU sample. The signal-to-noise ratio is plotted as a function of time (on the X-axis) and the amount of bilirubin added (in μM, shown in the legend). The more bilirubin added, the slower the signal construction and the less likely it is to return to negative values.

FIG. 6 shows the signal from the above sample estimated at time = 600 seconds, plotted as a function of bilirubin addition (in μM, shown in the legend). On this timescale, 1 μM bilirubin is determined to be the optimal amount of bilirubin. When the bilirubin level is high, the signal does not have enough time to accumulate, and when the bilirubin level is low, the signal has already accumulated to the maximum value and is attenuated to a low level.

Example 3:
Without being bound by a particular theory or mechanism of action, bilirubin in the method of interest (a) enhances the signal in nonlinear resonance Raman spectroscopy by removing free radicals generated by laser light. This reduces the artifacts that can interfere with the signal and reduces the standard deviation of the target sample. Thus, the standard deviation of the mean rate of change of lycopene peaks in uninfected samples decreases as bilirubin is added to the assay and (b) removes the free radicals produced by the metabolic activity of the microorganisms present in the sample. This can slow down the changes caused by the microorganism, especially if the changes to the sample are too fast to be observed cleanly in the absence of bilirubin.

FIG. 7 shows a plot of the signal-to-noise ratio of samples containing non-covalently bound lycopene and different amounts of bilirubin as a function of data acquisition time. The signal-to-noise ratio is maximized along various points along the line shown in the figure and reaches its maximum at different points along the line. Fluctuations in signal-to-noise ratio signal-to-noise ratio SNR with respect to experimental time (“Signal” is the difference in the mean rate of change of lycopene peaks between uninfected control samples and 10CFU Staphylococcus aureus-infected samples, and “noise” is the two mean values. Is the sum of the standard deviations of). In all cases, the gradient is calculated from t = 0 to the time shown on the X-axis, so the increase in SNR reflects the decrease in standard deviation (this is due to the decrease in the confidence interval of the estimated gradient). .. The signal-to-noise ratio is plotted for time (X-axis) and for various amounts of bilirubin present in the assay (in the legend μM). The time-dependent Raman profile of the infected sample shows a decrease, and in experiments conducted beyond the timescale of this decrease, the signal magnitude decreases, so that the signal-to-noise ratio decreases as the signal begins to decrease.

FIG. 8 plots the time when the signal-to-noise ratio is maximized with respect to the added bilirubin concentration. As the bilirubin concentration increases, the optimal time point increases and the reliability of the diagnostic test increases. The absorption spectrum of bilirubin shifts slightly to blue due to absorption at 532 nm. This does not have a significant effect on the Raman signal, but means that it may have a secondary effect through a Raman enhancing or attenuating effect. The intensity of the resonant Raman peak is proportional to the square of the scattering cross section. The scattering cross section is related to the square of the transition dipole moment and therefore usually follows the absorption spectrum. However, if there is another electronic state nearby, the Raman intensity is proportional to the square of the sum of the cross sections. If the cross sections have opposite signs, destructive interference can occur, resulting in attenuation of resonance. If both bilirubin and lycopene are present in albumin, there may be destructive / constructive interference between excited states, which changes as bilirubin decomposes by removing free radicals. do. Other free radical scavengers, including hydrophilic ones such as glutathione and ascorbic acid and hydrophobic ones such as butylated hydroxytoluene incorporated into albumin, should not have a similar effect on the diagnostic signal. I know.

Example 4:
Uncontrolled fluctuations in laser power (or drift in laser power) can bias the observed rate of change. To correct for this, a reference standard (a standard in which the normalized spectrum is not expected to change with laser output) was incorporated into the instrument and monitored at the same time as the sample under test. This method can be used to characterize the actual drift of the laser output, which can be multiplied by a previously calibrated nonlinear coefficient to subtract the observed gradient bias error. An embodiment of this correction is shown in FIGS. 9a and 9b. Figures 9a and 9b show variations in measurements of 4 uninfected and 3 infected samples as a function of bilirubin content. FIG. 9a shows the values at the time of measurement, and FIG. 9b shows the values after correction for the observed drift of the laser output.

The example sample system includes eight sample chambers, one chamber reserved for calibration standards such as the NIST Raman Calibration Standard SRM 2242. The instrument monitors the spectrum from this calibration standard using the same optical components (spectrometer, fiber, etc.) and algorithms used to monitor the Raman spectrum from the test sample. From the measurement spectrum of the standard sample, the rate of change in Raman intensity is calculated as a function of time. This is then multiplied by the previously estimated RRNL coefficient for the reagent liquid. This is the offset coefficient. The offset coefficient is subtracted from the rate of change of the Raman peak.

Additional control steps are also implemented to ensure that the laser output performance is close to the desired specifications. For 532nm lasers, the laser output can drift due to slight changes in heat dissipation and accumulation. These changes can affect performance. These limitations can be relaxed with a control scheme that limits the drift of the laser performance and can be flagged when the laser is drifting outside the specified range.

Figure 10 shows the normalized rate of change of the Raman spectrum from the NIST standard as a function of the normalized rate of change of the CCD shutter time (which affects the laser output by heat accumulation in the external laser cavity). There is. This control can be used to change the laser output, but it can also be used to see if the device is working with respect to heat storage issues. If the observed rate of change of the Raman spectrum from the NIST standard is outside the 90% confidence band shown in Figure 10, it means that the thermal management problem has not been fully mitigated.

Example 5:
The system of the embodiment is shown in FIG. The layout shown in Figure 11 shows eight chambers, each maintained at 35 ° C to 37 ° C. Chamber 1 contains a reference composition such as NIST standard SRM 2242. The system collects the spectrum of each chamber and then rotates the circular stage to the next chamber. Collect one cycle of Raman spectra for all eight chambers in 15-25 seconds, then repeat the process for the next cycle until data is acquired for approximately 600 seconds. Alternatively, if a sampling rate faster than 15 seconds is required, the system can collect data in one chamber for the entire required period and then move on to the next chamber.

Figure 12 shows various subsystems designed to minimize thermal fluctuations in the laser cavity. These can (a) use a fan to blow in the outside air, circulate it in the equipment and then exhaust it, and (b) 35 the sample chamber without unduely affecting changes in the equipment chamber outside the sample chamber. Use of a combination of resistance heating element and Pelche heating element maintained at ~ 37 ° C (Pelche element transfers heat from equipment chamber to sample chamber, resistance element applies heat to both sample chamber and equipment chamber), and ( c) Includes the use of a delay of appropriate length (about 1 hour) after activating the laser prior to operation.

Additional controls are implemented to check the stability of the equipment to further minimize the effects of heat. Figure 13 shows one setup algorithm that checks the stability of the device. The spectrometer CCD was calibrated for 1 hour using NIST standard SRM 2242. Simply put, this calibration involves collecting the spectrum of the NIST standard SRM 2242 and dividing the measured output by the expected output of this standard. This calibration was performed for 1 hour. The ratio of the measured CCD output to the expected output is YCAL. Following this, all output from the CCD is divided by YCAL to provide output that is independent of device variation. YCAL was checked by repeating measurements over 10 minutes on the same NIST SRM 2242 sample. If the ratio between the measured spectrum of NIST SRM 2242 (after calibration with YCAL) and the expected spectrum shows ripple, it means that the instrument has drifted on a time scale of 1 hour and 10 minutes. This is shown in FIG. In some cases, the device stabilizes about 1-2 hours after the laser is activated.

To further minimize thermal management problems during equipment operation, one example involves the use of the control methods and algorithms shown in FIG. Light is introduced into the sample chamber through a normally closed mechanical shutter. When this shutter opens, the thermal management in the optical train changes. A short time (eg, about 300 ms) elapses until the change stabilizes. Instead of getting one spectrum per second, eight individual spectra are taken over 120 ms each (total data acquisition time is about 1 second). If the variability within these eight spectra is checked and the individual time periods are properly adjusted, the eight spectra show small systematic changes between them.

Example 6:
The details of the non-linear resonance Raman spectroscopy experiment according to the specific aspect of the method of interest are provided below.

Sample Preparation A sample of nonlinear resonance Raman spectroscopy was prepared as described in US Patent Application Publication No. 2016/0324933. Simply put, lycopene, which is non-covalently bound to albumin in the sample, was extracted from tomato. The tomato paste was mixed with hexane, and the portion of the paste that was dissolved in hexane was tilted and dried to a powder. The composition was then refluxed with ethyl acetate and dissolved in acetone. The acetone solution was mixed with an aqueous solution of albumin and the acetone was gradually removed by a rotary evaporator. The process was monitored to ensure that all acetone was removed, thereby ensuring that the organic extract from tomatoes was transferred to albumin. Additional specifications include: (1) Incubate the buffer solution at a temperature of about 60 ° C for at least 24 hours (a temperature above 50 ° C for at least 24 hours is appropriate) to ensure that the reagents are free of supersaturated gases. Prolonged incubation at high temperatures ensures that the dissolved gas exits the system through the process of nucleated bubble formation, as the high temperature reduces the solubility of the gas. The buffer is cooled to 37 ° C and used for the final treatment of the reagents. At 37 ° C, the buffer is unsaturated with respect to the dissolved gas. This procedure ensures that the final reagent is free of excess gas. (2) Add active glutathione GSH to the reagent (as a reducing substance) and incubate for 18 hours or more and up to 5 days before use. Sample testing shows an increase in signal magnitude over time for incubation and added active glutathione. FIG. 16 shows the signal magnitude (defined as a normalized reduction in Ramantrace) from a sample supplemented with 10 CFU of Staphylococcus aureus in a 4.5 mL test sample. The signal rises with increasing glutathione levels. These tests were performed within 2 hours of the addition of GSH. FIG. 17 shows the signal (difference in rate of change between mean control sample and 10 CFU / mL plasma / S. aureus sample) at a measurement time of 1700 seconds. X-axis "incubation time" refers to the time at which GSH was added to the reagent vial (legend concentration) and before the addition of infected plasma for testing.

Preparation of Zwitterion-Coated Glass Vials Glass vials are coated with a special zwitterion layer. 200 mL of acetone was added to 30.03 grams (0.246 mol) of 1,3-propanesulton using a magnetic stir bar under nitrogen gas. After vigorous stirring for 3 minutes, 50.0 grams (0.241 mmol) of 3- (N, N-dimethylaminopropyl) trimethoxysilane was added using a syringe. The resulting mixture was vigorously stirred under N ₂ gas overnight. After adding another 100 mL of acetone, the mixture was shaken well, the organic solvent was removed by rotary evaporation and the white product was quickly further dried in a high vacuum pump (the product of interest is highly hygroscopic). The white powder product was quickly transferred to another plastic bottle and weighed to give 78.5 grams (98.9%) of the zwitterionic silane reagent.

250 clear glass vials (Pacific Vial catalog number VC151965, diameter = 18.75 mm, 3 drums) and corresponding caps were rinsed once with deionized water and autoclaved for 30 minutes. After cooling to room temperature, the glass vials were dried in an oven at 80 ° C. and the autoclaved glass vials were transferred to a 6 quart aluminum pressure cooker. To this was added 5 L of autoclaved deionized water and 5 g of zwitterionic silane reagent (above). The pressure cooker was covered and shaken by hand to ensure that each vial was immersed in the silane solution. The lid was opened and a visual inspection was performed to confirm that each vial was covered with the solution. The pressure cooker was covered and shaken for 45 minutes. Then, another 5 g of the zwitterion silane reagent was added, and the process of shaking / inspection / shaking for 45 minutes by hand was repeated. This procedure was repeated two more times so that the total amount of zwitterionic silane reagent was 20 gm. The pressure cooker was then placed in an oven at 80 ° C. for 2 hours (up to 4 hours). Then, the pressure cooker was cooled to room temperature, opened, and the aqueous solution was tilted and discharged. The glass vials were washed twice with autoclaved deionized water and once with ethanol / autoclaved deionized water (3: 1 v / v) and dried in an oven at 80 ° C. After cooling to room temperature, the glass vials were transferred to a plastic bag and sealed for storage.

Laser Power Without being bound by a particular theory or mechanism of action, laser power and exposure time can affect resonance Raman spectroscopy by the method of interest through any of several mechanisms below.

At very long laser exposure times, the Raman peak height of the control sample may begin to decrease, but the pixel sum remains largely unchanged. The energy of the laser shifts the contributions that make up the entire Raman peak, which reduces the observed peak height.

At very short laser exposure times, the reading noise of the CCD can overwhelm the small effect being monitored. Since the Raman effect is non-linear, large changes in laser power (eg, fluctuations in laser power) can dominated the observed changes in Raman amplitude by changes in laser power (and thus determine the effects of the presence of bacteria). That can be impossible).

In some embodiments, suitable laser exposure conditions for carrying out the method of interest include conditions that result in a maximum CCD count of approximately 10,000-40,000. In one example, the maximum saturation of the CCD is 65536 counts (corresponding to 16 bits), so in this mode of operation the CCD will operate well below saturation, but well above the region where read noise is an issue. .. In certain cases, the mode of operation results in a total laser exposure of approximately 1 second (divided into 4 CCD exposures of 0.25 seconds each, with each 0.25 second exposure yielding a maximum CCD count of approximately 30,000). With longer laser exposures, the observed peak height decreases with time, and with shorter laser exposures, it increases with time. One way to mitigate this effect is to use the area under the curve (which remains relatively unchanged, as shown in Figure 18), but working with 1 second of laser exposure further exacerbates this problem. Can be reduced. In particular, FIG. 18 shows the rate of change of the Raman peak height and the "under-curve area" of the Raman peak height calculated as the sum of all the pixels representing the Raman peak (the SOP of the legend). When the laser exposure time exceeds 1 second in each exposure cycle, the Raman peak height drops significantly. In contrast, the area under the curve remains relatively unchanged.

Detection and signal measurement system The measurement system includes a spectroscope (equipped with a low-temperature cooled CCD detector capable of low-dark current operation) and a laser light source capable of performing resonance Raman spectroscopy (incident wavelength of 532 nm and output of 100 mW), up to 8 Includes a sample chamber with a rotating table that can be switched between two samples, the NIST SRM 2242 standard used for instrument calibration, and a computer with software suitable for control and analysis.

One exemplary method involves the following steps: (1) irradiating the sample with a monochromatic light source (eg, 532 nm), (2) measuring the scattered light intensity profile from 535 to 600 nm (and wavelength wave number). (3) Calibrating the CCD output with the calibration constants derived from the NIST standard SRM 2242, (4) Separating Raman contributions from the fluorescent background using appropriate software algorithms, ( 5) Repeat steps 1 to 4 to characterize the rate of change of the Raman peak as a function of time to obtain the desired signal noise ratio, (6) perform all necessary corrections for system bias at the rate of change. Step, (7) The step of using the corrected rate of change to diagnose the sample based on a predetermined threshold.

Detection and signal measurement system: Collimated lens alignment In some embodiments, the system of interest comprises a collimating lens that focuses light from a monochromatic light source onto a sample and collects light scattering from a sample vial. If the lens is too close to the glass vial, some of the scattered light can be lost outside the lens collection area. In addition, the magnitude of this loss can vary with small changes in laser beam position (or mode hopping). Therefore, if the position of the lens is less than ideal, this effect may cause a large change in the measured Raman intensity. By systematically changing the position of the collimating lens as shown in FIG. 19, it is determined that the position where the focal point is about 0.4 mm inside the liquid layer in the glass vial is the appropriate position. Figure 19 shows the change in confidence intervals in the NIST profile as a function of the distance between the edge of the glass-liquid layer and the focal point of the collimating lens.

In order to characterize the position of the collimating lens, the position of the lens was changed and the scattered light was measured. Starting from the glass wall, as the focus moves into the liquid layer, the Raman intensity first increases (because the focus covers more of the liquid) and then decreases (because the scattered light is attenuated by absorption in the liquid). .. Figure 20 shows the change in Raman intensity profile as a function of collimated lens position for sample chambers 2-8 (sample chamber 1 is reserved for NIST standard). The strength is maximized at the position 5.65 mm, which corresponds to the edge of the glass-liquid interface. The nature of the interface depends on the surface layer of the glass vial. FIG. 21 shows the use of the zwitterionic layer (described above) to further enhance the robustness of the measurement for small changes in focal position. FIG. 21 shows the gradient of the uninfected control sample as a function of the focal position from the edge of the glass liquid layer. In the case of an uncovered sample, the gradient changes greatly depending on the position of the focal point. For glass vials coated with a zwitterion layer, the gradient appears to be independent of the focal position, indicating that the interface layer extends deeper into the liquid.

(1) In the case of uncoated glass vials, a large signal appears in a short time. However, as shown in FIGS. 22A and 22B, this signal drops rapidly to zero and can even be inverted over longer periods of time. (2) For zwitterion-coated samples, the signal-to-noise ratio continues to rise over time. Thus, in the case of uncoated glass vials, a reasonable signal can be obtained, but it can depend on the position of the focal point and the time of signal recording. In the case of coated glass vials, it can be seen that the robustness of the measured values is significantly enhanced. FIG. 22A shows the signal-to-noise ratio of a twin ion-coated glass vial, and FIG. 22B shows the signal-to-noise ratio of an uncoated glass vial measured at a focal point 0.4 mm from the glass surface. For zwitterion-coated vials, the SNR increases steadily over time. For uncoated vials, there is an important signal at about 900 seconds, but this signal drops rapidly to zero and even inverts over longer periods of time.

Detection and signal measurement system: There is no laser-induced non-linear Raman resonance signal that can be measured if the amount of sample irradiated by the flow monochromatic light source at the interface of the sample container varies for all spectra obtained for that sample vial. .. To ensure that the same amount of sample is measured, the following steps are performed: (a) A glass vial and a glass vial and almost completely stationary so that the optical system can examine the same amount for each measurement. Keep the sample in it; (b) ensure that the optical system examines the volume close to the glass surface. There is an interface layer at the glass-liquid interface, where the relative velocity of the liquid layer is close to zero. When examining this volume, this sampled volume remains largely unchanged across multiple measurements. However, the result is extremely sensitive to both the nature of the interface layer and the precise positioning of the collimating lens. If the focus of the collimating lens is not deep enough, errors can occur due to laser mode hopping. If the focus is too deep, the sampled volume can change due to motion, which reduces the magnitude of the signal (or in some cases completely inverts). Based on these considerations, the proper position of the collimating lens in a particular embodiment is about 0.2 mm from the edge of the glass-liquid layer. FIG. 23 shows the variation in the signal-to-noise ratio, estimated as the difference in the measured rate of change of Raman peak in 4 control samples and 3 samples with 10 CFU / mL S. aureus-containing plasma, divided by the rms standard deviation. There is. As the collimating lens moves deeper into the liquid layer, the signal becomes smaller and virtually disappears. In addition, the signal is also short-lived and can be measured in 900 seconds, but extremely small in 2500 seconds. This behavior is typical of uncoated glass vials. The properties of the interface layer can be manipulated by applying a coating to the glass surface. As described above, when the zwitterion layer is applied to the glass surface, the interface layer extends deeper into the sample volume mass.

Example of instrumental sequence of operation The control software commands the spectroscope to "open" the mechanical shutter (ie, expose the test sample to laser light). After a delay of 0.05 seconds (for the shutter to move), the CCD is instructed to start collecting data for 1.6 seconds. The CCD data is then transferred to a read electronic circuit and acquired by a laptop computer.

The instrument commands the sample stage to rotate so that the NIST calibration standard (in chamber 1) is exposed to the optical path for 1.6 seconds. The rotating stage is then instructed to move to the next chamber, exposing the sample in that chamber. This process is repeated until the instrument obtains one spectrum for all seven chambers (if loaded) and the NIST calibration standard. This set of eight measurements is called a cycle and usually takes 26 seconds.

The instrument runs the test for 33 minutes (75 test cycles, each cycle taking 26 seconds). The first 5 cycles are "dark cycles" and the mechanical shutters are not opened. These five dark cycles provide an opportunity for equipment (laser / CCD, etc.) to stabilize. After these 5 cycles, the mechanical shutter opens when instructed during the cycle.

For each spectrum acquired, the software initiates a set of mathematical algorithms to analyze the data. (1) First, the "dark current" is subtracted from the CCD output, and the X-axis of the resulting spectrum is converted from the number of pixels to the frequency using a predefined linear conversion factor. (2) Next, the raw CCD output is multiplied by a calibration factor to provide a spectrum that is nominally independent of instrumental variability. (3) Third, an algorithm based on Lieber's method is implemented to estimate background fluorescence. (4) Then detect peaks in the spectral profile of 1156, 1516, 4000 cm-1 (major peaks associated with the biomarkers of the example; the last peak is ignored in plasma samples). (5) Finally, the heights of these peaks and the area under the curve are stored in a table and used in subsequent analysis steps.

The software begins calculating the slope of the line defined by these data points. It uses the least-squares linear fitting algorithm that comes standard with the National Instrument LabView package to provide data (peak height and under-curve area vs. time, both 1516 and 1156 cm-1 peaks and 4000 cm-1). It is done by fitting (independently and independently of the peak). The algorithm also calculates a 95% confidence interval (95% CI) for the gradient and y-intercept values. The gradient of the fluorescent background at 3000 cm ^-1 is subtracted from this gradient, and this normalization step removes the system bias to the data introduced by the strain on the interfacial layer caused by the large particle agglomerates.

The algorithm compares the gradient (along with 95% CI of the gradient) to the decision threshold set in the previous test. The threshold is set to a gradient> -0.5 × 10 ^-5 / sec to diagnose uninfected samples (samples with an observed gradient greater than this value are diagnosed as uninfected) and diagnose infected samples. In order to do so, the gradient is set to <-1.5 × 10 ^-5 / sec for a period of 600 seconds (samples with an observed gradient less than this value are diagnosed as infected), and for infected samples, a signal. Is observed to decay in the opposite direction of time. Samples with a gradient between these two values are diagnosed as "dichotomous". For diagnosis, mean gradients are calculated from 1156, 1516, and 4000 (if any) cm ^-1 Raman peaks.

An example of a software algorithm for separating the contribution of the Raman signal from the contribution of the fluorescent signal An algorithm for maximizing the performance of the test is implemented. Lieber's method is used to subtract background fluorescence. This method is shown in FIG. 24, which shows Lieber's method for fitting a fifth-order polynomial to a spectrum containing contributions from Raman peaks and background fluorescence. The raw measurement spectrum fits into a fifth-order polynomial curve. Data points with input spectra higher than the fitted polynomial are replaced with the fitted polynomial, and the modified spectrum is fitted to the new polynomial. In certain embodiments of the method of interest, Lieber's method is optimized by: (a) Lieber's method is carried out in the first spectrum. For background fluorescence, a fifth-order polynomial between wavenumbers 1050 cm ^-1 and 1650 cm ^-1 (in some cases a fourth-order polynomial is used); (b) the subsequent spectrum fits the background fluorescence. (That is, the above-mentioned fifth-order polynomial) is used with different amplitudes. Fit-constraining techniques ensure that the results do not include amplified mathematical artifacts.

Fluctuations in laser power and error in equipment In several embodiments, the rate of change of Raman peak as a function of time is measured. These measurements can be affected by changes in laser power and various instabilities in instrument alignment. Uniform drift of laser power can be corrected via the NIST standard, but higher-order instability cannot be corrected by the NIST standard. However, characterizing instability (potentially including laser mode hopping and drifting in any position of the alignment lens under test) helps to flag suspicious measurements.

To do this, the output from the NIST standard is characterized and the gradient and gradient confidence intervals are measured at the end of the experiment. As shown in Figure 25, when working with artificially added samples, the signal-to-noise ratio was found to vary with both gradient and gradient CI. Specifically, FIG. 25 shows the change in signal-to-noise ratio at the end of the measurement with the collimating lens in the ideal position and the coated glass vial used in all measurements. Each SNR value represents one measurement set of four non-infected control samples and three samples containing 10 CFU / mL S. aureus-infected plasma. The measurements are repeated and the SNR is plotted as a function of the confidence interval between the estimated gradient (top) of the NIST standard and the gradient itself (bottom).

Therefore, measurements where the NIST profile is "clean" (ie, SNR is expected to be greater than 1), the CI of the NIST gradient is less than 0.25, and the gradient itself is -1 to 0.1 x 10-5 / sec. The values are considered for further analysis.

Example 7: characterization of the presence of microorganisms in the sample Figure 26 shows the determination of the presence or absence of pathogenic microorganisms. This example demonstrates the use of the test with an artificially added sample prepared as follows: (a) First, a concentration of 1000 CFU / mL of Staphylococcus aureus stock solution in buffer. Prepare with. Add 0.05 mL (or expected 50 CFU) of this stock solution to 5 mL of plasma purchased from a blood bank and incubate for 2 hours. (B) Add 0.6 mL of this infected plasma to the standard reagent formulation with a volume of 3.9 mL. Three "infected" samples are made from this plasma. (C) Four non-infected control samples are prepared by adding 0.6 mL of uninfected plasma (which has also been incubated for 2 hours) to the reagent formulation. All seven test vials were tested simultaneously and the results are shown in Figures 26A-26C. By using these known samples, simple statistical measurements (such as signal-to-noise ratio) can be used to characterize the measurement.

During the course of the test, the height of the Raman peak and its rate of change are continuously monitored for each test vial. Figures 26A-26C show these measurements as a collection of controls and infected samples. In infected samples, Raman peak slowly declines over time. FIG. 26A shows the aggregated Raman peak amplitudes for four non-infected control samples and three infected samples. Figure 26B shows the rate of change in the area under the curve. The signal is defined as the difference in rate of change between the uninfected control and the infected sample. The uninfected control sample showed a rate of change consistent with the rate of change observed by the NIST standard, which is estimated to be (0.59 ± 0.16) x ^10-5 / sec at the end of the measurement. FIG. 26C shows the signal-to-noise ratio estimated as the signal divided by the rms standard deviation of the rate of change of the uninfected and infected samples.

At the final diagnosis, this value is subtracted from the estimated gradient, which reduces the aggregated control gradient (and individual vial values) just below the zero line, as shown in Figure 27. Specifically, Figure 27 summarizes the control and infection values and shows an estimated gradient corrected for the variability observed in the NIST standard. After correcting for NIST profile drift and collecting data long enough to have SNR >> 1, the sample can be analyzed to determine the presence of pathogenic microorganisms. Bacterial cells are added to plasma and infected plasma is added to standard reagents. Similar results were observed when bacterial cells were added to plasma, incubated for 2 hours, and centrifuged at high speed (12 krpm, 5 minutes) to remove the bacterial cells from the plasma (confirmed removal by culture in broth medium). , Add this infected / centrifuged plasma to the reagent vial.

Example 8: Pathogen-free infected samples In clinical scenarios, the effect of sampling volume needs to be considered. Blood pathogen levels in most patients with bloodstream infections are approximately 1-10 CFU / mL. In addition, the amount of blood collected during blood collection is generally limited to about 10 mL (often less). Therefore, for several infected patients, it is expected that 0 CFU will be present in the blood draw due to Poisson sampling statistics. Since the method of interest can detect free radicals produced by the bacterium as well as the bacterium itself, the methods described herein can still identify those samples as "infection".

To demonstrate this, a second experiment with the following protocol is described below: (a) First, a stock solution of Staphylococcus aureus in buffer is prepared at a concentration of 1000 CFU / mL. Add 0.05 mL (or expected 50 CFU) of this stock solution to 5 mL of plasma purchased from a blood bank and incubate for 2 hours. (B) Centrifuge this plasma at 12000 rpm for 5 minutes to remove bacteria from the plasma. The plate counting method is used to characterize the pathogen concentration in plasma before and after centrifugation. (C) Add 0.6 mL of this infected plasma to the standard reagent formulation of 3.9 mL. Three "infected" samples are made from this plasma. (D) Four non-infected control samples are prepared by adding 0.6 mL of uninfected plasma (which has also been incubated for 2 hours) to the reagent formulation. All seven test vials were tested simultaneously and the results are shown in Figures 28A-28C. FIG. 28A is a graph showing the estimated rate of change of non-infected and infected samples estimated from the area under the curve. The signal is defined as the difference in rate of change between the uninfected control and the infected sample. FIG. 28B shows the signal-to-noise ratio estimated as the signal divided by the rms standard deviation of the rate of change of the uninfected and infected samples. FIG. 28C is a graph showing the signal as a function of time for this sample set in which bacteria were centrifuged and removed. Shown for comparison is a sample set without a centrifugation step to remove the bacteria. As is clear from the traces, when the bacteria are centrifuged, the signal is weakened and, as opposed to time, diminishes. In contrast, if bacteria are present in the test sample, the signal attenuates with the square root of time.

Example 9: characterization of clinical samples For clinical samples (ie, samples containing plasma from human patients), some additional factors are further considered, for example, plasma distorts the flow at the interface, and /. Or it can contain agglomerates of various proteins and lipids that cause other changes that give systematic bias to the data. To normalize these biases, the gradient of the fluorescence output (eg at 3000 cm ^-1 ) is considered. The algorithm used for normalization is:
(A) First, the gradient of the Raman peak is calculated using the above method.
(B) Next, calculate the gradient of the fluorescence output at 3000 cm ^-1 . If this gradient is less than a certain predefined threshold, the measurement is rejected as too noisy. In this case, this threshold is set to -4.5 x 10 ^-5 / sec.
(C) If the gradient of the fluorescence output is less than the threshold, subtract this value from the gradient of the Raman peak. This is the "signal".
(D) Consider samples that may initially have very small amplitudes, which may be due to a variety of reasons, including the presence of large numbers of red blood cells in plasma, aggregation of large numbers of albumin molecules. The algorithm flags these samples for infection to prevent the possibility of false negatives that could harm the patient.
(E) Sample if the final gradient (after subtracting the fluorescence gradient) is less than -1 x 10 ^-5 / sec, or if the Raman peak amplitude is less than 0.22 (NIST profile = 1). Is diagnosed as infected. A sample is diagnosed as uninfected if the final gradient (measured at 1000 seconds) is greater than -0.8 × 10 ^-5 / sec and the amplitude of the Raman peak is greater than 0.22. The gradient threshold is significantly higher (about -0.25 x 10 ^-5 / sec) than the threshold suggested by bacterial-free scaling behavior, but slightly smaller than the bacterial-free scaling behavior ( ^-1 x 10-). ⁵ / sec).

One exemplary embodiment is shown in FIGS. 29A and 29B. The results of the subject's rapid test method are compared to the results of blood culture. This study was performed on waste plasma samples obtained from the rest of the CBC (Complete Blood Count) tests performed on patients with suspected bloodstream infections (and those who were also ordered for blood culture tests). Will be done. After the CBC test, the CBC test vacutainer is recovered and 0.6 mL is aliquoted from the plasma layer (some of these plasma samples had a significant amount of red blood cells due to residual red blood cells). .. Add 0.6 mL plasma layer to the reagent containing 0.3 mL glutathione (GSH) and incubated at 37 ° C for 18 hours. The test vial was placed in the test equipment and tested immediately. FIG. 29A shows the rate of change of Raman peak, and FIG. 29B shows the amplitude of Raman peak in a series of studies made with plasma from different patients. With the above algorithm, a sample is diagnosed as infected if the amplitude (FIG. 29B) or gradient (FIG. 29A) is in the "infected" band. The overall detection metrics are:
(A) 40 samples that have been diagnosed as "negative" by the method of interest described herein and are also negative in blood culture. "40 negatives"
(B) 0 samples diagnosed as positive for blood cultures diagnosed as negative by the method of interest described herein. "False negative of 0"
(C) Six samples that were positive for blood culture and were also diagnosed as positive by the methods of interest described herein. "Six true positives"
(D) Seven samples that were negative in blood culture and were diagnosed as positive by the methods of interest described herein. "7 false positives"
(E) Blood culture was negative, but the method of interest described herein can be diagnosed (because the gradient was in the middle range or the fluorescence gradient changed outside the set threshold). 13 samples that could not be done. The four samples were positive for blood cultures, but the diagnosis was not determined by the method of interest.

In these settings, for infected samples, the rate of change in Raman peak correlates with the time to positive in blood culture tests, as shown in FIG.

Comparison of signal magnitude over time for different sample types is shown in FIGS. 31A and 31B. Specifically, FIG. 31A shows the signal magnitudes of multiple test runs summarizing the time variation of signals of different sample types (the rate of change of the Raman peak in the infected sample from the rate of change in the Raman peak of the non-infected control sample). (Reduced) is shown. In all cases, the magnitude of the signal decays in the opposite direction of time. Multiple runs have been shown in the presence of 10 CFU / mL Staphylococcus aureus in the test sample. FIG. 31B shows the signal strength at 1000 seconds for different sample types. When the bacteria are centrifuged, the signal appears to be smaller. The signal appears to be significantly higher in the clinical sample.

Moreover, the signal intensity in the clinical sample appears to be significantly greater than the signal intensity in the sample with 10 CFU / mL S. aureus in the test sample, even with 2 hours of incubation. Pathogen concentrations in clinical samples are expected to be 1-10 CFU / mL, and only 0.6 mL of plasma is sampled, suggesting that many clinical samples are free of active bacteria. Given the large signal magnitude of these clinical samples, it is clear that the methods described herein respond to free radicals produced by bacteria.

Example 10: Measuring the Minimum Inhibitor Concentration of Antimicrobial Agents To Measure the Minimum Inhibitor Concentrations or To Characterize the Antimicrobial Agent Sensitivity of Unknown Pathogens to Candidate Antimicrobial Agents:
(A) Incubate the unknown pathogen in eutrophic medium for about 3 hours. This period should be sufficient to increase the concentration of the causative agent by more than about 100-fold. Therefore, 1 CFU (or more) of the original sample will be larger than 100 CFU.
(B) The pathogen is aliquoted into about 7 equal parts and each part is expected to have at least 14 CFUs, which is more than the square root Poisson sampling error of the expected number of CFUs (14 in this case). Is big.
(C) Add variable concentrations of candidate antimicrobial agents to each moiety, starting at a very low concentration of 0.125 μg / mL and increasing by a factor of 0.25, 0.5, 1, 2, 4, and 8 μg / mL (this). The range is designed to reach the desired MIC in 7 steps, doubling each). The mixture is incubated for an additional 20 minutes, which gives sufficient time for 10 CFU to interact with the candidate antimicrobial agent.
(D) Add the 7 mixtures to the test vial and test. Since the number of free radicals present in the mixed sample is a function of the antimicrobial agent concentration, the final concentration can be plotted against the antimicrobial agent concentration to derive an estimate of the MIC. This is shown in FIGS. 32A and 32B.

FIG. 32A shows the gradient measured as a function of antimicrobial agent (vancomycin) concentration. FIG. 32B shows the time it takes for the tested sample to become cloudy. Above 2 μg / mL, the sample remains clear for a very long incubation period (longer than 96 hours). Therefore, the minimum bactericidal concentration MBC is about 2 μg / mL. Above 0.25 mg / mL, the time to turbidity will be slightly longer. Therefore, the MIC is about 0.25 mg / mL. As shown in FIGS. 32A and 32B, the MIC corresponds to the concentration at which the signal drops slightly. In addition, MBC (minimum bactericidal concentration) corresponds to the concentration at which the signal rises again.

Example 11: Methods for Determining the Phenotype of an Unknown Microorganism The phenotype of an unknown microorganism in a sample can be determined by using additional cross-linking material. In the following, peptidases in the sample are identified by adding a cross-linking substance containing a cleaveable peptide bond next to the glutamate group. Scheme 1 shows the synthesis of glutamic acid derivatives using a disulfide crosslinker.

Scheme 1

Compound 1: 3.0 grams (26.4 mmol) of cysteamine hydrochloride (Sigma-Aldrich, catalog number: M6600) and 60 mL of methanol were added to a 250 mL round bottom flask containing a magnetic stir bar. The mixture was stirred with a magnetic stirrer to form a solution. 6.2 grams (28.2 mmol) of 2,2'-dithiodipyridine (Sigma-Aldrich, catalog number: 43791) was added to this solution in small portions. The resulting yellow solution was stirred overnight and concentrated using Rotavapor into a dry powder. The yellow product was further dried in a high vacuum pump for 4-5 hours to give 9.03 grams of yellow powder product (yield about 98.2%). This product mixture contained the unreacted starting material 2,2'-dithiodipyridine, the desired product compound 1, and the by-product 2-thiopyridine. The product mixture was used in the next step reaction without further purification. Compound 2: 0.5 gm (2.0 mmol) BOC-L-glutamic acid (Sigma-Aldrich, catalog number: 16345), 0.86 g (4.5 mmol) EDAC (N- (4.5 mmol) in a 250 mL round bottom flask containing a magnetic stirrer. 3-Dimethylaminopropyl) -N'-ethylcarbodiimide hydrochloride, Sigma-Aldrich, catalog number: E7760), 0.52 grams (4.5 mmol) of NHS (N-hydroxysuccinimide, Sigma-Aldrich, catalog number: 56480), and 40 mL of dichloromethane was added. The mixture was stirred with a magnetic stirrer for 2 hours to form a clear solution. To this solution is added 4 mL of TEA (trimethylamine, EMD, catalog number: 1200-5) and 100 mg of DMAP (4- (dimethylamino) pyridine, Aldrich, catalog number 10,770-0), followed by 1.4 grams of compound 1 mixture. Was added. The reaction mixture was stirred overnight, washed 3 times with 30 mL of 5% NaHCO ₃ aqueous solution and the bottom solution was dried over sodium sulfate. After removing the solvent with Rotovapor, the product residue was dissolved in 5 mL of CH ₂ Cl ₂ for column purification. Load 30 grams of silica gel with CH ₂ Cl ₂ , load the crude product solution onto the column, load the column CH ₂ Cl ₂ , 1% CH ₃ OH / CH ₂ Cl ₂ and 2% CH ₃ OH / CH ₂ Cl. Elution with ₂ , 3% CH ₃ OH / CH ₂ Cl ₂ . Fractions were identified on a normal phase TLC plate unfolding with 5% CH ₃ OH / CH ₂ Cl ₂ (Rf = 0.28). The desired fractions were pooled and concentrated to give 0.28 grams (0.48 mmol) of semi-fluid compound 3 in 22.2% yield. The ¹ H NMR spectrum of CDCl ₃ is consistent with the proposed structure of compound 3. Compound 4: 0.28 grams (0.48 mmol) of compound 3, 15 mL of methanol, and 3 mL of concentrated hydrochloric acid solution were added to a 100 mL round bottom flask containing a magnetic stir bar. The resulting mixture was stirred for 15 minutes and concentrated in small amounts by Rotavapor (mostly water). The product residue was further dried to dryness under high vacuum. After adding about 3 mL of ethanol, the product mixture was further placed under high vacuum to give 0.29 grams (0.479 mmol) of the powder product, Compound 4, in 99.8% yield. The product can be dissolved in water without further purification and the pH is adjusted to 5.3 using a 0.1 mM NaOH solution, which is albumin by disulfide exchange with the cysteine residue of the protein with activated disulfide. Used to cross proteins such as.

Crosslinkers increase background UV-Vis absorption as shown in Figure 33A, but UV-Vis absorption decreases over time with high crosslinker content (albumin network is recombined by GSH). ). FIG. 33A shows the results of the absorbance at 650 nm of a peptide crosslinker containing two disulfide bonds that exchange for reduced disulfide bonds on albumin, thereby forming a network of albumin molecules. Background absorbance from albumin (due to Rayleigh scattering) increases with crosslinker content (correspondingly, this reagent test vial containing crosslinker exhibits a signal similar to that observed with pathogenic microorganisms. ). Certain bacteria produce peptidases capable of cleaving glutamate peptide bonds (eg, Staphylococcus aureus), resulting in an increase in Raman signal over time (Fig. 33B). This rate of increase corresponds to the concentration of the pathogen. This also corresponds to the results of the Raman test, as shown in Figure 34. When the crosslinker content is low, the Raman peak decreases over time, consistent with the formation of the albumin network. Raman peaks increase when the crosslinker content is high. In certain embodiments, the crosslinked albumin network is reduced to monomeric albumin by a bacterial metabolite (peptidase in this case). For example, to test peptidases, the crosslinker: albumin ratio was set to 1: 2.

When mixed with albumin, the crosslinker disulfide bonds are exchanged for the cysteine group of albumin to form an albumin dimer. This also increases the background absorbance due to Rayleigh scattering (Fig. 33A). For reagent systems containing very large amounts of crosslinkers, crosslinked albumin appears to recombine disulfide bonds over time, slowly reducing Rayleigh scattering. Therefore, in these experiments, the crosslinker: albumin ratio was set to 1: 2. In this system, Raman peaks decrease in amplitude over time, similar to albumin cross-linking caused by free radicals. When a reagent system containing a peptide disulfide crosslinker is exposed to peptidase-producing bacteria, cleavage of the crosslinker disrupts the interfacial layer and increases Raman peak amplitude. This suggests inoculation dependence with a detection limit of more than 10 CFU with 4 mL reagent. (Fig. 33B)

Example 12: Fluorescence-mediated detection and characterization The presence or absence of pathogenic microorganisms is shown in FIGS. 35A and 35B. This example demonstrates the use of the test with an artificially added sample prepared as follows: (a) First, a concentration of 1000 CFU / mL of Staphylococcus aureus stock solution in buffer. Prepare with. Add 0.05 mL (or expected 50 CFU) of this stock solution to 5 mL of plasma purchased from a blood bank and incubate for 2 hours. (B) The plasma is then centrifuged to remove the bacteria from the plasma. Add 0.6 mL of this infected plasma to the standard reagent formulation in volume of 3.9 mL. Three "infected" samples are made from this plasma. (C) Three uninfected control samples are prepared by adding 0.6 mL of uninfected plasma (also centrifuged after 2 hours of incubation) to the reagent formulation. (D) One reagent control sample vial is made by taking the reagent vial without addition. All seven test vials were tested simultaneously and the results are shown in Figures 35A-35B. By using these known samples, simple statistical measurements (such as signal-to-noise ratio) can be used to characterize the measurement.

During the test run, fluorescence values at the appropriate frequency are continuously monitored and the rate of change is calculated for each test vial. FIG. 35A shows these measurements as a collection of control and infected samples as well as single reagent control samples. FIG. 35A shows aggregated fluorescence values for three non-infected control samples, three infected samples, and reagent samples. The fluorescence value of the uninfected control sample is reduced due to the process of fluorescence quenching. The rate of decline in infected samples is somewhat greater than this. The signal is defined as the difference in the rate of change in fluorescence between the uninfected control and the infected sample. The uninfected control sample showed a rate of change consistent with the rate of change observed in the reagent control sample, which is estimated to be (-2.2 ± 0.1) x ^10-5 / sec at the end of the measurement in this example. Will be done. FIG. 35B shows the signal-to-noise ratio estimated as the signal divided by the rms standard deviation of the rate of change of the uninfected and infected samples.

In the final diagnosis, the rate of change in fluorescence in the reagent vial is subtracted from the rate of change in fluorescence in the test vial. After correcting for the drift of the reagent target standard and collecting data for a sufficiently long period so that SNR >> 1, the sample can be analyzed to determine the presence or absence of pathogenic microorganisms.

The invention described above has been described in some detail by way of example and examples for clarity of understanding, but in the light of the teachings of the present disclosure, without departing from the spirit or scope of the appended claims. It is readily apparent to those skilled in the art that certain changes and modifications can be made to them.

Therefore, the above merely illustrates the principle of the present invention. It will be appreciated by those skilled in the art that, although not expressly described or illustrated herein, the principles of the invention can be embodied and various configurations included within the spirit and scope thereof can be devised. Moreover, all examples and conditional language described herein are primarily not limited to such specifically described examples and conditions, but the reader understands the principles of the invention. Intended to help. Moreover, all descriptions herein describing the principles, aspects, and embodiments of the invention are intended to include their structural and functional equivalents. In addition, such equivalents may include both currently known equivalents and future developed equivalents, that is, any developed element that performs the same function regardless of structure. Intended. Accordingly, the scope of the invention is not intended to be limited to the exemplary embodiments shown and described herein. Rather, the scope and gist of the present invention is embodied by the appended claims.

Claims (43)

(A) A step of combining a sample and a reagent containing albumin incorporating a ligand in a sample holder;
(B) A step of irradiating a sample with a monochromatic light source that is focused at the interface between the sample and the surface of the sample holder and absorbed by the ligand with either constant or time-varying light intensity;
(C) A step of collecting scattered light from an irradiated sample at a plurality of different times and measuring a Raman signal and a fluorescence signal from the scattered light;
(D) The step of calculating the rate of change of the intensity of the Raman signal and the fluorescence signal with respect to the sample over time;
(E) The step of correcting the calculated rate of change of the intensity of the Raman and fluorescent signals to obtain the net signal; and (f) based on the comparison of the net signal with one or more predetermined thresholds. , A method for determining the presence of microorganisms in a sample, comprising the step of determining the presence of microorganisms in the sample. The steps (a)-(e) according to claim 1, wherein the one or more predetermined thresholds are the lower limit amount of the concentration of the clinically infected sample and the one or more control samples containing the inoculum. The method according to claim 1, which is set by executing. The method of claim 1, wherein the ligand is lycopene. The method of claim 1, wherein the monochromatic light source is a laser. The method of claim 4, wherein the laser irradiates the sample at 532 nm. The method of claim 1, wherein the step of correcting the rate of change in intensity of a Raman signal and a fluorescent signal comprises characterizing a spectral output from a standard sample. The method of claim 6, wherein the standard is NIST SRM 2242. The method of claim 1, wherein the step of correcting the rate of change in intensity of the Raman signal comprises characterizing the fluorescence output from the reagent. The method of claim 1, further comprising pretreating albumin of the reagent with a reducing substance prior to mixing with the sample. 9. The method of claim 9, wherein the step of pretreating albumin with a reducing substance is sufficient to reduce disulfide bonds. The method of claim 9, wherein the reducing substance is glutathione or bilirubin. 10. The method of claim 10, further comprising contacting the pretreated albumin with a disulfide crosslinked material. 12. The method of claim 12, wherein the disulfide cross-linking material comprises a core that is cleaved by an enzyme or metabolite produced by a microorganism in the sample. The cross-linking material is the formula (I):

Contains the compounds of
The method of claim 10. The method of claim 1, wherein the sample holder is a glass vial. 15. The method of claim 15, wherein the surface of the glass vial comprises a coating that alters the absorption of albumin on the glass surface. 16. The method of claim 16, wherein the coating is a zwitterionic coating. The method of claim 1, wherein the reagent further comprises an antimicrobial composition. Including the step of plotting the net signal against the antimicrobial concentration,
The minimum values in the plot are used to estimate the minimum bactericidal concentration of the antimicrobial composition, and / or the breakpoints in the plot are used to estimate the minimum inhibitory concentration of the antimicrobial composition.
The method of claim 18. Calculating the net signal is
The net signal is calculated by determining the rate of change in the intensity of the fluorescence scattering from the standard reference sample and subtracting the rate of change in the intensity of the fluorescence scattering from the standard sample from the rate of change in the intensity of the resonant Raman scattering. Including doing,
The method according to claim 1. Calculating the net signal is
The net signal is calculated by determining the rate of change in the total output from the standard reference sample and subtracting the rate of change in the intensity of the resonant Raman scattering from the standard sample from the rate of change in the intensity of the resonant Raman scattering. Including that
The method according to claim 1. One of claims 20 or 21, further comprising the step of comparing the calculated net signal to a predetermined threshold and determining that the sample contains actively metabolizing microorganisms based on the comparison. The method described in the section. (A) Monochromatic light source and
(B) Optical adjustment components and
(C) Photodetector and
(D) Processor and
Is a system that includes
The processor includes memory operably attached to the processor, which stores instructions.
The instruction, when executed by the processor,
Irradiating the sample in the sample holder with a monochromatic light source at the interface between the sample and the surface of the sample holder, either with constant or time-varying light intensity;
Measuring scattered light from a sample at multiple different times using a photodetector;
Determining the Intensities of Resonant Raman Scattering and Fluorescent Scattering;
To calculate the rate of change of the intensity of the resonant Raman scattering and the intensity of the fluorescent scattering; and to correct the calculated rate of change of the intensity of the resonant Raman scattering and the intensity of the fluorescent scattering to obtain a net signal. To carry out
The system. 23. The system of claim 23, wherein the optical adjustment component comprises a collimating optical system mounted on a linear table configured to produce an adjustable focal point. 23. The system of claim 23, wherein the optical adjustment component further comprises a mechanical shutter. 23. The system of claim 23, further comprising a rotary table comprising a plurality of sample chambers. 26. The system of claim 26, wherein the turntable comprises eight sample chambers. Collimated optical system and
Further including a subsystem for characterizing the incoherently scattered light, including an optical tuning component configured to spread the incoherently scattered light onto a linearly arranged photodetector, claim. Item 27. The processor further contains memory for storing instructions,
The instruction causes the processor to move the collimating optics to focus light in each of the sample chambers when executed by the processor.
The system of claim 28. 23. The system of claim 23, wherein the sample further comprises albumin incorporating a ligand. The memory further contains instructions
The instruction, when executed by the processor,
Determining the rate of change in intensity of fluorescent scattering (or other light output) from reagent vials,
Let the processor calculate the net signal by subtracting the rate of change in the intensity of the fluorescent scattering from the standard sample from the rate of change in the intensity of the resonant Raman scattering.
The system of claim 23. The memory further contains instructions
The instruction, when executed by the processor,
Determining the rate of change of total output from the standard reference sample,
Let the processor calculate the net signal as the rate of change in the intensity of the resonant Raman scattering minus the rate of change in the intensity of the resonant Raman scattering from the standard sample.
The system of claim 23. The memory further contains instructions
The instruction, when executed by the processor,
The processor is made to compare the net signal with a predetermined threshold and, based on the comparison, determine that the sample contains actively metabolizing microorganisms.
The system according to any one of claims 31 or 32. 23. The system of claim 23, wherein the sample further comprises a reducing substance. 34. The system of claim 34, wherein the sample further comprises a disulfide crosslinker. 35. The system of claim 35, wherein the disulfide cross-linking material further comprises a core cleaved by an enzyme or metabolite produced by a microorganism in said sample. The crosslinked substance is of formula (I) :.

Contains the compounds of
The system of claim 35. 23. The system of claim 23, wherein the sample holder is a glass vial. 23. The system of claim 23, wherein the sample holder further comprises a coating that alters the absorption of albumin. 39. The system of claim 39, wherein the coating comprises a zwitterionic coating. 23. The system of claim 23, wherein the processor is configured to expose the sample to a monochromatic light source in order to minimize the net signal. 23. The system of claim 23, configured to irradiate the sample at the interface between the sample and the surface of the sample holder. 42. The system of claim 42, which is configured to maximize the net signal.