EP4127679A1 - Vorrichtungen, systeme und verfahren zur pathogendetektion auf der basis von ramanspektroskopie - Google Patents

Vorrichtungen, systeme und verfahren zur pathogendetektion auf der basis von ramanspektroskopie

Info

Publication number
EP4127679A1
EP4127679A1 EP21721717.3A EP21721717A EP4127679A1 EP 4127679 A1 EP4127679 A1 EP 4127679A1 EP 21721717 A EP21721717 A EP 21721717A EP 4127679 A1 EP4127679 A1 EP 4127679A1
Authority
EP
European Patent Office
Prior art keywords
sample
raman
cuvette
exemplary embodiments
spectrometer
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP21721717.3A
Other languages
English (en)
French (fr)
Inventor
Gregory William AUNER
Michelle Ann BRUSATORI
Charles James SHANLEY
Satya Kiran KOYA
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Wayne State University
Original Assignee
Wayne State University
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Wayne State University filed Critical Wayne State University
Publication of EP4127679A1 publication Critical patent/EP4127679A1/de
Withdrawn legal-status Critical Current

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Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/65—Raman scattering
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
    • C12Q1/18—Testing for antimicrobial activity of a material
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01J—MEASUREMENT OF INTENSITY, VELOCITY, SPECTRAL CONTENT, POLARISATION, PHASE OR PULSE CHARACTERISTICS OF INFRARED, VISIBLE OR ULTRAVIOLET LIGHT; COLORIMETRY; RADIATION PYROMETRY
    • G01J3/00—Spectrometry; Spectrophotometry; Monochromators; Measuring colours
    • G01J3/02—Details
    • G01J3/0205—Optical elements not provided otherwise, e.g. optical manifolds, diffusers, windows
    • G01J3/0208—Optical elements not provided otherwise, e.g. optical manifolds, diffusers, windows using focussing or collimating elements, e.g. lenses or mirrors; performing aberration correction
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01J—MEASUREMENT OF INTENSITY, VELOCITY, SPECTRAL CONTENT, POLARISATION, PHASE OR PULSE CHARACTERISTICS OF INFRARED, VISIBLE OR ULTRAVIOLET LIGHT; COLORIMETRY; RADIATION PYROMETRY
    • G01J3/00—Spectrometry; Spectrophotometry; Monochromators; Measuring colours
    • G01J3/02—Details
    • G01J3/0205—Optical elements not provided otherwise, e.g. optical manifolds, diffusers, windows
    • G01J3/021—Optical elements not provided otherwise, e.g. optical manifolds, diffusers, windows using plane or convex mirrors, parallel phase plates, or particular reflectors
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01J—MEASUREMENT OF INTENSITY, VELOCITY, SPECTRAL CONTENT, POLARISATION, PHASE OR PULSE CHARACTERISTICS OF INFRARED, VISIBLE OR ULTRAVIOLET LIGHT; COLORIMETRY; RADIATION PYROMETRY
    • G01J3/00—Spectrometry; Spectrophotometry; Monochromators; Measuring colours
    • G01J3/02—Details
    • G01J3/0205—Optical elements not provided otherwise, e.g. optical manifolds, diffusers, windows
    • G01J3/0218—Optical elements not provided otherwise, e.g. optical manifolds, diffusers, windows using optical fibers
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01J—MEASUREMENT OF INTENSITY, VELOCITY, SPECTRAL CONTENT, POLARISATION, PHASE OR PULSE CHARACTERISTICS OF INFRARED, VISIBLE OR ULTRAVIOLET LIGHT; COLORIMETRY; RADIATION PYROMETRY
    • G01J3/00—Spectrometry; Spectrophotometry; Monochromators; Measuring colours
    • G01J3/12—Generating the spectrum; Monochromators
    • G01J3/18—Generating the spectrum; Monochromators using diffraction elements, e.g. grating
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01J—MEASUREMENT OF INTENSITY, VELOCITY, SPECTRAL CONTENT, POLARISATION, PHASE OR PULSE CHARACTERISTICS OF INFRARED, VISIBLE OR ULTRAVIOLET LIGHT; COLORIMETRY; RADIATION PYROMETRY
    • G01J3/00—Spectrometry; Spectrophotometry; Monochromators; Measuring colours
    • G01J3/12—Generating the spectrum; Monochromators
    • G01J3/18—Generating the spectrum; Monochromators using diffraction elements, e.g. grating
    • G01J3/1838—Holographic gratings
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01J—MEASUREMENT OF INTENSITY, VELOCITY, SPECTRAL CONTENT, POLARISATION, PHASE OR PULSE CHARACTERISTICS OF INFRARED, VISIBLE OR ULTRAVIOLET LIGHT; COLORIMETRY; RADIATION PYROMETRY
    • G01J3/00—Spectrometry; Spectrophotometry; Monochromators; Measuring colours
    • G01J3/28—Investigating the spectrum
    • G01J3/44—Raman spectrometry; Scattering spectrometry ; Fluorescence spectrometry
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56983—Viruses
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01J—MEASUREMENT OF INTENSITY, VELOCITY, SPECTRAL CONTENT, POLARISATION, PHASE OR PULSE CHARACTERISTICS OF INFRARED, VISIBLE OR ULTRAVIOLET LIGHT; COLORIMETRY; RADIATION PYROMETRY
    • G01J3/00—Spectrometry; Spectrophotometry; Monochromators; Measuring colours
    • G01J3/12—Generating the spectrum; Monochromators
    • G01J3/18—Generating the spectrum; Monochromators using diffraction elements, e.g. grating
    • G01J2003/1842—Types of grating
    • G01J2003/1861—Transmission gratings
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/65—Raman scattering
    • G01N2021/651—Cuvettes therefore
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
    • G01N2333/08—RNA viruses
    • G01N2333/11—Orthomyxoviridae, e.g. influenza virus
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
    • G01N2333/08—RNA viruses
    • G01N2333/165—Coronaviridae, e.g. avian infectious bronchitis virus

Definitions

  • the present disclosure generally relates to the field of spectroscopy, including apparatuses, systems, and methods for performing Raman spectroscopy.
  • Disclosed embodiments relate to, among other things, apparatuses, systems, and methods for detecting biological or chemical targets based on Raman spectroscopy.
  • Raman spectroscopy is a vibrational spectroscopic technique that provides spectroscopic “fingerprints” by which organic and inorganic molecules and substances can be identified.
  • Raman scattering occurs when light interacts with a molecular vibration or rotation, and a change in polarizability takes place during molecular motion. This results in light being inelastically scattered (Raman-scattered light) at a vibrational frequency shifted up or down from that of the excitation light.
  • the frequency difference between excitation light and Raman-scattered light is the Raman shift, typically represented as cm 1 .
  • a Raman spectrum is the intensity profile of the inelastically scattered light as a function of frequency or the frequency difference.
  • a Raman spectrum may include one or more Raman bands or Raman peaks.
  • the Raman bands or Raman peaks occur at vibrational frequencies characteristic of vibrational modes of specific bond types in a molecule or substance and directly provide information of the atomic or molecular composition of a molecule or substance.
  • the unique spectroscopic fingerprint of a molecule or substance can be obtained, with the intensity directly proportional to the concentration of the molecule or substance that gives rise to the bands or peaks.
  • the present disclosure provides, among other things, apparatuses, systems, and methods for detecting biological or chemical targets based on Raman spectroscopy.
  • a spectrometer includes a plurality of optical elements.
  • the plurality of optical elements includes an entrance aperture, a collimating element, a volume phase holographic grating, a focusing element, and a detector array.
  • the entrance aperture is configured to receive a light beam.
  • the collimating element is configured to direct the light beam to the volume phase holographic grating.
  • the volume phase holographic grating is configured to disperse the light beam over a preselected spectral band of at least 50 nm.
  • the focusing element is configured to focus the dispersed light beam to the detector array.
  • the plurality of optical elements are configured to transfer the light beam from the entrance aperture to the detector array with an average transfer efficiency from 60% to 98% for first order diffraction over the preselected spectral band of at least 50 nm.
  • a spectrometer includes a plurality of optical elements.
  • the plurality of optical elements includes an entrance aperture, a collimating element, a volume phase holographic grating, a focusing element, and a detector array.
  • the entrance aperture is configured to receive a light beam.
  • the collimating element is configured to direct the light beam to the volume phase holographic grating.
  • the volume phase holographic grating is configured to disperse the light beam over a preselected spectral band of at least 50 nm.
  • the focusing element is configured to focus the dispersed light beam to the detector array.
  • the plurality of optical elements are configured to detect the dispersed light beam at the detector array with a spectral resolution from 0.1 cm 1 to 2.5 cm 1 over the preselected spectral band of at least 50 nm. In some embodiments, the plurality of optical elements are configured to detect the dispersed light beam at the detector array with a spectral resolution from 0.1 cm 1 to 2.5 cm 1 over the preselected spectral band of at least 1600 cm 1 as an alternative representation. In such instances, the spectral resolution refers to the average spectral resolution of the spectrometer over the preselected spectral band.
  • a spectrometer includes a plurality of optical elements.
  • the plurality of optical elements includes an entrance aperture, a collimating element, a volume phase holographic grating, a focusing element, and a detector array.
  • the entrance aperture is configured to receive a light beam.
  • the collimating element is configured to direct the light beam to the volume phase holographic grating.
  • the volume phase holographic grating is configured to disperse the light beam over a preselected spectral band of at least 50 nm.
  • the focusing element is configured to focus the dispersed light beam to the detector array.
  • the plurality of optical elements are configured to provide a performance ratio and a performance product in the preselected spectral band.
  • the performance ratio is a ratio between a transfer efficiency and a path length of the light beam traveled from the focusing element to the detector array in units of % transfer efficiency per cm of path length.
  • the performance ratio is from 3 %-cm 1 to 12.3 %-cm 1 .
  • the performance product is a product of a spectral resolution in cm 1 and the path length of the light beam traveled from the focusing element to the detector array in cm.
  • the performance product is from 0.8 to 100.
  • a cuvette for containing a sample includes a chamber that has at least one tapered wall, a top end, and a bottom end.
  • the tapered wall has a tilt angle relative to the bottom end configured to concentrate a portion of the sample to a central region on an interior surface of the bottom end.
  • a cuvette for containing a sample includes a chamber that has at least one tapered wall, a top end, and a bottom end.
  • the tapered wall has a tilt angle relative to the bottom end configured to homogenize a portion of the sample across a central region on an interior surface of the bottom end.
  • an interrogation apparatus for receiving an optical signal from a sample.
  • the interrogation apparatus includes a cuvette configured to contain the sample.
  • the cuvette includes a chamber that has at least one wall, a top end, and a bottom end.
  • the interrogation apparatus further includes a focusing back reflector above the bottom end of the cuvette.
  • the focusing back reflector has a focal point on or above the bottom end.
  • the focusing back reflector is configured to reflect and focus light from the bottom end to the focal point.
  • a Raman spectroscopic system includes an excitation light source to radiate a light beam into a cuvette through a bottom end of the cuvette and onto a portion of a sample contained in the cuvette.
  • the Raman spectroscopic system further includes a Raman spectrometer.
  • the spectrometer includes an entrance aperture, a collimating element, a transmission diffraction grating, a focusing element, and a detector array.
  • the entrance aperture is configured to receive a Raman signal from the portion of the sample through the bottom end of the cuvette.
  • the collimating element is configured to receive the Raman signal from the entrance aperture and direct the Raman signal to the transmission diffraction grating.
  • the transmission diffraction grating is configured to disperse the Raman signal over a preselected spectral band.
  • the focusing element is configured to focus the dispersed Raman signal to the detector array.
  • a Raman spectroscopic system includes an excitation light source to radiate a light beam into a cuvette through a bottom end of the cuvette and onto a portion of a sample contained in the cuvette.
  • the cuvette includes a chamber, a top end, and a bottom end.
  • the Raman spectroscopic system further includes a focusing back reflector above the bottom end configured to reflect and focus light from the bottom end to a focal point on or above the bottom end.
  • the Raman spectroscopic system further includes a Raman spectrometer.
  • the spectrometer includes an entrance aperture, a collimating element, a transmission diffraction grating, a focusing element, and a detector array.
  • the entrance aperture is configured to receive a Raman signal from the portion of the sample through the bottom end of the cuvette.
  • the Raman signal includes Raman signal reflected by the focusing back reflector.
  • the collimating element is configured to receive the Raman signal from the entrance aperture and direct the Raman signal to the transmission diffraction grating.
  • the transmission diffraction grating is configured to disperse the Raman signal over a preselected spectral band.
  • the focusing element is configured to focus the dispersed Raman signal to the detector array.
  • a method for detecting the presence or absence of at least one feature of a Raman signal indicative of the presence or absence of a target in a sample includes concentrating a portion of the sample to a central region on an interior surface of a bottom end of a cuvette.
  • the cuvette includes a chamber, at least one tapered wall, a top end, and the bottom end.
  • the method further includes focusing a light beam to the central region.
  • the method further includes directing a Raman signal from the central region to a Raman spectrometer.
  • the method further includes detecting the presence or absence of at least one feature of the Raman signal indicative of the presence or absence of the target in the sample.
  • a method for detecting the presence or absence of at least one feature of a Raman signal indicative of the presence or absence of a target in a sample includes focusing a light beam onto a portion of the sample on an interior surface of a bottom end of a cuvette.
  • the cuvette includes a chamber, at least one tapered wall, a top end, and the bottom end.
  • the method further includes reflecting and focusing light from the bottom end of the cuvette.
  • the light includes a portion of the light beam and a Raman signal from the portion of the sample to a focal point on or above the interior surface of the bottom end.
  • the method further includes directing a Raman signal from the portion of the sample to a Raman spectrometer.
  • the method further includes detecting the presence or absence of at least one feature of the Raman signal indicative of the presence or absence of the target in the sample.
  • a method for detecting the presence or absence of at least one feature of a Raman signal indicative of the presence or absence of a target in a sample includes focusing a light beam onto a portion of the sample on an interior surface of a bottom end of a cuvette.
  • the cuvette includes a chamber, at least one tapered wall, a top end, and the bottom end.
  • the method further includes directing a Raman signal from the portion of the sample passing through the bottom end to a Raman spectrometer.
  • the method further includes detecting the presence or absence of at least one feature of the Raman signal indicative of the presence or absence of the target in the sample.
  • a method for detecting the presence or absence of at least one feature of a Raman signal indicative of the presence or absence of a target in a sample includes focusing a light beam onto a portion of the sample. The method further includes directing a Raman signal from the portion of the sample to a spectrometer.
  • the spectrometer includes a plurality of optical elements. The plurality of optical elements includes an entrance aperture, a collimating element, a volume phase holographic grating, a focusing element, and a detector array.
  • the entrance aperture is configured to receive a light beam.
  • the collimating element is configured to direct the light beam to the volume phase holographic grating.
  • the volume phase holographic grating is configured to disperse the light beam over a preselected spectral band of at least 50 nm.
  • the focusing element is configured to focus the dispersed light beam to the detector array.
  • the plurality of optical elements are configured to transfer the light beam from the entrance aperture to the detector array with an average transfer efficiency from 60% to 98% for first order diffraction over the preselected spectral band of at least 50 nm.
  • the method further includes detecting the presence or absence of at least one feature of the Raman signal indicative of the presence or absence of the target in the sample.
  • a method for performing an analysis on a sample within a cuvette includes concentrating a portion of the sample to a central region on an interior surface of a bottom end of a cuvette.
  • the cuvette includes a chamber, at least one tapered wall, a top end, and the bottom end.
  • the method further includes focusing a light beam to the central region.
  • the method further includes directing a Raman signal from the central region to a Raman spectrometer.
  • the method further includes analyzing the Raman signal.
  • a method for performing an analysis on a sample within a cuvette includes focusing a light beam onto a portion of the sample on an interior surface of a bottom end of a cuvette.
  • the cuvette includes a chamber, at least one tapered wall, a top end, and the bottom end.
  • the method further includes reflecting and focusing light from the bottom end of the cuvette.
  • the light includes a portion of the light beam and a Raman signal from the portion of the sample to a focal point on or above the interior surface of the bottom end.
  • the method further includes directing a Raman signal from the portion of the sample to a Raman spectrometer.
  • the method further includes analyzing the Raman signal.
  • a method for performing an analysis on a sample within a cuvette includes focusing a light beam onto a portion of the sample on an interior surface of a bottom end of a cuvette.
  • the cuvette includes a chamber, at least one tapered wall, a top end, and the bottom end.
  • the method further includes directing a Raman signal from the portion of the sample passing through the bottom end to a Raman spectrometer.
  • the method further includes analyzing the Raman signal.
  • a method for performing an analysis on a sample within a cuvette includes focusing a light beam onto a portion of the sample.
  • the method further includes directing a Raman signal from the portion of the sample to a spectrometer.
  • the spectrometer includes a plurality of optical elements.
  • the plurality of optical elements includes an entrance aperture, a collimating element, a volume phase holographic grating, a focusing element, and a detector array.
  • the entrance aperture is configured to receive a light beam.
  • the collimating element is configured to direct the light beam to the volume phase holographic grating.
  • the volume phase holographic grating is configured to disperse the light beam over a preselected spectral band of at least 50 nm.
  • the focusing element is configured to focus the dispersed light beam to the detector array.
  • the plurality of optical elements are configured to transfer the light beam from the entrance aperture to the detector array with an average transfer efficiency from 60% to 98% for first order diffraction over the preselected spectral band of at least 50 nm.
  • the method further includes analyzing the Raman signal.
  • FIG. 1 is a schematic representation of an exemplary Raman spectrometer.
  • FIG. 2 is a schematic representation of another exemplary Raman spectrometer.
  • FIG. 3 is a schematic representation of an exemplary Raman spectroscopic system, according to some embodiments of the present disclosure.
  • FIG. 4 is a graphical representation of the diffraction efficiency over a spectral band of an exemplary transmission grating, according to some embodiments of the present disclosure.
  • FIG. 5 is a graphical representation of theoretically predicted spectra output of an optical signal by an exemplary Raman spectrometer, according to some embodiments of the present disclosure.
  • FIG. 6 is a graphical representation of theoretically predicted spectra output of an optical signal by an exemplary Raman spectrometer, according to some embodiments of the present disclosure.
  • FIG. 7 is a schematic representation of an exemplary interrogation apparatus, according to some embodiments of the present disclosure.
  • FIG. 8 is a graphical illustration of an example of focusing an excitation light beam to, or receiving an optical signal from, a focal point, according to some embodiments of the present disclosure.
  • FIG. 9A is a top view of an exemplary chamber of an exemplary cuvette that can contain a sample, according to some embodiments of the present disclosure.
  • FIG. 9B is a side view of the exemplary chamber of FIG. 9A.
  • FIG. 9C is a bottom view of the exemplary chamber of FIG. 9A.
  • FIG. 10A is a top view of an exemplary chamber of an exemplary cuvette that can contain a sample, according to some embodiments of the present disclosure.
  • FIG. 10B is a side view of the exemplary chamber of FIG. 10A.
  • FIG. IOC is a bottom view of the exemplary chamber of FIG. 10A.
  • FIG. 11 A is a top view of another exemplary chamber of an exemplary cuvette that can contain a sample, according to some embodiments of the present disclosure.
  • FIG. 1 IB is a side view of the exemplary chamber of FIG. 11 A.
  • FIG. 11C is a bottom view of the exemplary chamber of FIG. 11 A.
  • FIG. 12 illustrates an exemplary interior surface of a tapered wall of an exemplary cuvette that can contain a sample, according to some embodiments of the present disclosure.
  • FIG. 13 illustrates an exemplary interior surface of a tapered wall of an exemplary cuvette that can contain a sample, according to some embodiments of the present disclosure.
  • FIG. 14 is a schematic representation of another exemplary cuvette for containing a sample, according to embodiments of the present disclosure.
  • FIG. 15 is a schematic representation of another exemplary interrogation apparatus, according to some embodiments of the present disclosure.
  • FIG. 16 illustrates an exemplary preparation of a functionalized surface of an exemplary cuvette that can contain a sample, according to some embodiments of the present disclosure.
  • FIG. 17A is an exploded perspective view of an exemplary cuvette, according to some embodiments of the present disclosure.
  • FIG. 17B is a top view of the exemplary cuvette of FIG. 17 A, according to some embodiments of the present disclosure.
  • FIG. 17C is a cross-sectional view of the exemplary cuvette of FIG. 17A, according to some embodiments of the present disclosure.
  • FIG. 17D is a cross-sectional view of the exemplary cuvette of FIG. 17A, according to some embodiments of the present disclosure.
  • FIG. 17E is a cross-sectional view of another exemplary cuvette having an exemplary filter, according to some embodiments of the present disclosure.
  • FIG. 17F is a magnified image of the exemplary filter of FIG. 17E, according to some embodiments of the present disclosure.
  • FIG. 17G is a perspective view of an exemplary filter for trapping a target in a sample, according to some embodiments of the present disclosure.
  • FIG. 18A is a perspective view of an exemplary sample collection cartridge, according to some embodiments of the present disclosure.
  • FIG. 18B is an exploded perspective view of the exemplary sample collection cartridge of FIG. 18 A.
  • FIG. 18C is a top cross-sectional view of the exemplary sample collection cartridge of FIG. 18 A.
  • FIG. 18D is a cross-sectional view of the exemplary sample collection cartridge of FIG. 18 A.
  • FIG. 18E is a perspective view of another exemplary sample collection cartridge, according to some embodiments of the present disclosure.
  • FIG. 19 is a flowchart of an exemplary method for detecting the presence or absence of a target in a sample, according to some embodiments of the present disclosure.
  • FIG. 20 illustrates an exemplary mean Raman spectrum of a cytotoxic and invasive strain of Pseudomonas aeruginosa in water.
  • FIG. 21 illustrates an exemplary decision tree for detecting Gram-negative bacteria.
  • FIG. 22 illustrates an exemplary decision tree for detecting Gram-positive bacteria.
  • FIG. 23 illustrates eighteen exemplary preselected spectral bands for detecting the presence or absence of bacteria.
  • FIG. 24 illustrates an exemplary mean Raman spectrum of Escherichia coli.
  • FIG. 25 illustrates an exemplary mean Raman spectrum of Proteus mirabilis.
  • FIG. 26 illustrates an exemplary mean Raman spectrum of Klebsiella pneumoniae.
  • FIG. 27 illustrates an exemplary mean Raman spectrum of Leptospira interrogans.
  • FIG. 28 illustrates an exemplary mean Raman spectrum of Pseudomonas aeruginosa.
  • FIG. 29 illustrates an exemplary mean Raman spectrum of Enterococcus faecalis.
  • FIG. 30 illustrates an exemplary mean Raman spectrum of Streptococcus zooepidemicus or Streptococcus canis.
  • FIG. 31 illustrates an exemplary mean Raman spectrum of Staphylococcus pseudintermedius .
  • FIG. 32 illustrates exemplary spectral bands (gray bands) for detecting the presence or absence of magnesium ammonium phosphate and an exemplary measured Raman spectrum.
  • FIG. 33 illustrates exemplary preselected spectral bands (gray bands) for detecting the presence or absence of calcium oxalate dihydrate and an exemplary measured Raman spectrum.
  • FIG. 34 illustrates an exemplary preselected spectral band (gray band) for detecting the presence or absence of calcium oxalate monohydrate and an exemplary measured Raman spectrum.
  • FIG. 35 illustrates exemplary mean Raman spectra of three different fecal samples for detecting the presence or absence of hookworm and roundworm.
  • FIG. 36 illustrates an exemplary mean Raman spectrum of a sample containing A/PR/8 (H1N1) serotype influenza virus.
  • FIG. 37 illustrates an exemplary structure of SARS-CoV-2.
  • FIG. 38 illustrates receiving at least a portion of a swab in an exemplary cuvette, according to some embodiments of the present disclosure.
  • FIG. 39 illustrates an exemplary functionalized magnetic bead, according to some embodiments of the present disclosure.
  • FIG. 40 illustrates the complex of a virus captured by an exemplary functionalized magnetic bead and bound to an exemplary labeled (e.g., fluorescently) anti-S antibody, according to some embodiments of the present disclosure.
  • exemplary labeled e.g., fluorescently
  • FIG. 41 illustrates an exemplary magnetic field generating device for trapping exemplary functionalized magnetic microbeads and its use with an exemplary cartridge to provide rotation and agitation of the magnetic microbeads, according to some embodiments of the present disclosure.
  • FIG. 42 illustrates three exemplary mean Raman spectrum of three samples containing different strains of influenza viruses.
  • FIG. 43 illustrates five exemplary mean Raman spectra of five samples containing four different strains of influenza viruses compared to bacteria.
  • FIG. 44 illustrates three exemplary mean Raman spectra of three dried samples containing same strain of virus inactivated respectively by three distinct methods.
  • FIG. 45 illustrates three exemplary mean Raman spectra of water, a sample containing neutrophils, a sample containing neutrophils and Escherichia coli.
  • FIG. 46 illustrates three exemplary mean Raman spectra of water, a sample containing neutrophils, a sample containing neutrophils and Leptospira interrogans.
  • FIG. 47 illustrates two exemplary mean Raman spectra of a sample containing neutrophils and Escherichia coli and a sample containing neutrophils and Leptospira interrogans .
  • FIG. 48 illustrates differentiation, based on analyzing Raman signals and discriminant function analysis (DFA), of methicillin resistant staphylococcus aureus (MRSA) in the presence and absence of an antibiotic (cefoxitin), according to some embodiments of the present disclosure.
  • DFA discriminant function analysis
  • FIG. 49 illustrates time-course mean Raman spectra of exemplary spectral regions corresponding to phospholipid in in both MRSA (black lines) and methicillin sensitive staphylococcus aureus (MSS A) (gray lines), according to some embodiments of the present disclosure.
  • FIG. 50 illustrates time-course mean Raman spectra of exemplary spectral regions corresponding to proteins inhibiting disruption of the cell membrane in MRSA, according to some embodiments of the present disclosure.
  • FIG. 51 illustrates time-course mean Raman spectra of exemplary regions corresponding to lipid and protein of the cell membrane in MSS A, according to some embodiments of the present disclosure.
  • FIG. 52 illustrates an exemplary testing flow diagram for detecting antibiotic resistance of bacteria, according to some embodiments of the present disclosure.
  • FIG. 53 A illustrates an exemplary Raman spectroscopic system, according to some embodiments of the present disclosure.
  • FIG. 53B illustrates a multi-sample carousel of the exemplary Raman spectroscopic system of FIG. 53 A configured to receive a plurality of cuvettes.
  • FIG. 53C illustrates centrifuging a plurality of samples placed in the cuvettes by the exemplary Raman spectroscopic system of FIG. 53 A.
  • FIG. 53D illustrates an exemplary sample preparation process performed by the exemplary Raman spectroscopic system of FIG. 53 A.
  • FIG. 53E illustrates interrogating a sample by the exemplary Raman spectroscopic system of FIG. 53 A.
  • Some embodiments of the present disclosure may be implemented using a microscope, a spectrograph or spectrometer, or apparatuses or systems built according to certain embodiments of the present disclosure.
  • target refers to any substance, chemical, organism, and material, including biological material.
  • Biological material refers to any biological matter, such as molecules, cells, tissue, molecular structures, toxins, metabolites, biomarkers, and pathogens, including bacteria, parasites, and viruses.
  • sample refers to a sample to be interrogated for one or more targets. The target may be unidentified before the interrogation, and the sample may or may not contain the target to be detected. The interrogated sample may be a portion of a sample obtained from the source, and that portion would contain the target if the target is present in the sample. When a sample is described as being concentrated, homogenized, and/or interrogated, such concentration, homogenization, and/or interrogation may include only a portion of the sample obtained from the source.
  • a Raman signal refers to Raman-scattered light.
  • Raman spectrum refers to a representation of the intensity of Raman-scattered light as a function of its frequency.
  • the frequency of Raman-scattered light is typically converted to the Raman shift, which is the frequency difference between excitation light and Raman-scattered light, according to the following formula: where Dn is the Raman shift represented as cm 1 , lo is the excitation wavelength represented as nm, and l is the wavelength of the Raman-scattered light represented as nm.
  • the Raman shift refers to a stokes shift, an anti-stokes shift, or both.
  • preselected spectral band refers to a spectral band or spectral region in an optical signal that may contain a feature characteristic of or indicative of one or more targets to be detected.
  • a feature of a Raman signal refers to one or more of a shape, height, slope, area, and location of one or more Raman bands and/or Raman peaks of the Raman spectrum.
  • a feature may include only one of a shape, height, slope, area, or location.
  • a feature may include any combination of two of a shape, height, slope, area, or location.
  • a feature may include any combination of three of a shape, height, slope, area, or location.
  • a feature may include any combination of a shape, height, slope, area, or location.
  • a feature may include all five (a shape, height, slope, area, and location).
  • one feature is indicative of the presence or absence of a target.
  • more than one feature is indicative of the presence or absence of a target.
  • One or more features may correspond to the molecular structure, composition, and inter-molecular interactions of a target in the sample to be interrogated.
  • a feature is indicative of one or more changes to the molecular structure, composition, and/or inter-molecular interaction of a target in the sample to be interrogated.
  • one or more features may correspond to one or more changes of a target in the sample to be interrogated.
  • one or more features may correspond to one or more genetic and/or phenotypic changes of a target in the sample to be interrogated.
  • more than one preselected spectral band is detected or used for detecting the presence or absence of the target or one or more changes of the target in a sample.
  • the preselected spectral band for a certain target may be experimentally or theoretically determined before being used for detecting the presence or absence of the target in a sample.
  • a preselected spectral band can be determined based on one or more predefined Raman bands or Raman peaks corresponding to the vibration, rotation, interaction, and/or inter-connection of one or more functional groups of atoms in a pure sample of the target.
  • one or more functional groups include, but not limited to, chemical or molecular groups in nucleic acids, proteins, lipids, phospholipids, or carbohydrates.
  • an “optical signal” refers to electromagnetic radiation from a sample to be interrogated.
  • the electromagnetic radiation can be elastically or inelastically scattered light emitted from the sample to be interrogated, such as fluorescence emission or Raman-scattered light.
  • path length refers to the physical linear distance traveled by a light beam from a focusing element of the spectrometer to a detector of the spectrometer. Unless indicated otherwise, path length as used herein is denominated in units of cm. The path length of a spectrometer can affect, among other things, the overall physical dimension of a spectrometer.
  • the “light throughput” of a spectrometer refers to a transfer efficiency of a spectrometer for transferring a light beam entering the entrance aperture to the detector of the spectrometer.
  • the transfer efficiency is the percent of the light that enters the entrance aperture that reaches the detector.
  • Such transfer efficiency of the spectrometer may vary among different wavelengths of the light beam, and may also vary for different diffraction orders.
  • an average transfer efficiency is used to describe the light throughput of a spectrometer.
  • the average transfer efficiency refers to the average transfer efficiency of the spectrometer for different wavelengths within a spectral band of the light beam for a certain diffraction order. Given a certain detector, increasing the light throughput or transfer efficiency of a spectrometer improves the sensitivity of the spectrometer or reduces the integration time for detecting low intensity optical signals, such as Raman- scattered light.
  • the “performance ratio” of a spectrometer refers to a ratio between (1) the transfer efficiency as a percent and (2) the path length of the spectrometer measured in cm.
  • the performance ratio is directly proportional to the performance of the spectrometer. Given a certain path length or spectral resolution of a spectrometer, increasing the transfer efficiency of the spectrometer improves the overall performance of the spectrometer and increases the performance ratio. The performance ratio may vary for different wavelengths. Alternatively, given a transfer efficiency of the spectrometer, reducing the path length of the light beam in the spectrometer improves the performance of the spectrometer and increases the performance ratio.
  • the performance ratio of a spectrometer refers to the average performance ratio of the spectrometer over a spectral band, which is the ratio between (1) the average transfer efficiency of the spectrometer over a spectral band as a percent and (2) the path length of the spectrometer measured in cm.
  • the “performance product” of a spectrometer refers to a product of a spectral resolution measured in cm 1 and the path length of the spectrometer measured in cm. All else equal, a smaller performance product corresponds to better spectral resolution and/or a shorter path length, either or both of which may be preferred according to certain embodiments.
  • the performance product may vary for different wavelengths.
  • the performance product of a spectrometer refers to the average performance product of the spectrometer over a spectral band, which is the product of an average spectral resolution over a spectral band measured in cm 1 and the path length of the spectrometer measured in cm.
  • Raman spectroscopic systems Due to the inherent low intensity of Raman-scattered light and complexities of interrogating biological material, such as urine, saliva, blood, contaminated water, and fecal matter, it is desirable for Raman spectroscopic systems to have both high light throughput and high spectral resolution to increase the signal to noise (S/N) ratio of the measured Raman spectra to obtain information from the measured Raman spectra. It is also desirable for Raman spectroscopic systems to have smaller physical dimensions such that the systems can be setup on a standard laboratory countertop or for deployment at point of need in the field. However, the desirable benefits of high light throughput, high spectral resolution, and small physical dimension are difficult to achieve in typical Raman spectrometers.
  • a typical Raman spectrometer has a long path length to increase the spectral resolution, which in turn increases the physical dimension of the spectrometer. Furthermore, there exists a fundamental tradeoff between spectral resolution and light throughput in typical Raman spectrometers. These difficulties are further described below with reference to FIGS.
  • FIG. 1 is a schematic representation of a system, including a typical Raman spectrometer that is used to obtain Raman spectra from a sample.
  • excitation light from a laser 10 or monochromatic source is reflected off a long pass edge filter 22 (or notch filter) and is directed through lens 20, which in turn focuses the excitation light onto a sample.
  • Raman-scattered light from the sample is received by lens 20 and is directed to the edge filter 22 that blocks the excitation light and passes only the Raman- scattered light through.
  • Lens 24 then focuses the Raman-scattered light onto the entrance slit 32 of a spectrometer 30.
  • FIG. 2 is a schematic representation of another system, including a typical Raman spectrometer, where optical fibers 12 are used to direct the excitation light and Raman-scattered light to the spectrometer 30.
  • the spectral resolution of a spectrometer can be affected by various factors, including 1) the size of the entrance aperture or slit, 2) optical characteristics of the collimating and focusing mirrors (e.g., focal lengths and focal spot sizes), 3) the dispersive element (e.g., a grating), 4) the excitation wavelength of the laser, and 5) the detector (e.g., the pixel size of a CCD).
  • the size of the entrance aperture or slit may affect the minimum image size that the collimating and focusing mirrors can form in the detector plane.
  • the type of dispersive element may affect the total wavelength range and/or the spectral resolution of the spectrometer.
  • the type of detector may affect the maximum number and size of discreet points in which the spectrum can be digitized.
  • the light throughput of a spectrometer can also be affected by various factors, including 1) the etendue of the spectrometer, 2) the diffraction efficiency of the dispersive element over the spectral region of interest, 3) the quantum efficiency of the detector, and 4) light losses in optical components (e.g., through adsorption or reflection), such as filters, lenses, and mirrors.
  • the etendue is the ability of the spectrometer to accept light and is a function of the entrance aperture area (S) times the solid angle (W) of the accepted light beam.
  • the long path length can increase the physical dimension of the spectrometer such that the spectrometer is too large to be setup on a standard laboratory shelf or for deployment at point of need in the field.
  • the long path length of the spectrometer can lead to low light throughput of the spectrometer, which in turn degrades the sensitivity of the system for detecting targets.
  • Spectrometers with low light throughput may also need to use longer integration time for collecting an optical signal, during which biological or other dynamic changes of the sample being interrogated may confound the Raman spectra and/or reduce the quality of the Raman spectra for accurate analysis and detection.
  • the reflecting grating may need to be turned to scan over the spectral range. Such scanning further increases the amount of time for interrogating a sample.
  • the present disclosure provides spectrometers and spectroscopic systems having both high spectral resolution and high light throughput.
  • the present disclosure provides spectrometers and spectroscopic systems having small physical dimensions suitable to be setup on a standard laboratory countertop or for deployment at a point of need in the field.
  • the present disclosure provides spectrometers and spectroscopic systems allowing for short interrogation time with high sensitivity.
  • such spectrometers and spectroscopic systems can be used for various spectroscopic applications, including for Raman spectroscopic analysis in biomedical applications.
  • Raman spectrometers and Raman spectroscopic systems may allow for rapid and sensitive acquisition of high quality Raman spectra and real-time detection of biological or chemical targets.
  • FIG. 3 is a schematic representation of an exemplary Raman spectroscopic system 100, according to some embodiments of the present disclosure.
  • Raman spectroscopic system 100 includes an illumination system 101, a detection system 201, and an interrogation apparatus 300, each having a plurality of components.
  • the illumination system 101 of Raman spectroscopic system 100 includes an excitation light source 110 that emits an excitation light beam 200.
  • excitation light source 110 may be a laser or a monochromatic light source.
  • the illumination system 101 includes a beam expander 112 that expands excitation light beam 200 to a larger excitation light beam 210.
  • beam expander 112 further collimates excitation light beam 200 such that the divergence of the expanded excitation light beam 210 is smaller than excitation light beam 200.
  • the illumination system 101 includes a line bandpass filter 113 that has a passband to transmit wavelengths of excitation light source 110 and suppresses other wavelengths.
  • Line bandpass filter 113 can be used to block ambient light outside of the passband of the filter.
  • the illumination system 101 includes one or more mirrors or beamsplitters for directing excitation light beam 200 or expanded excitation light beam 210 towards the interrogation apparatus 300 of Raman spectroscopic system 100. For example, as shown in FIG. 3, mirror 114 and beamsplitter 116 reflect and direct excitation light beam 210 towards the interrogation apparatus 300.
  • the detection system 201 of Raman spectroscopic system 100 may receive an optical signal 220 from interrogation apparatus 300, such as Raman-scattered light, and provide one or more outputs based on the received optical signal 220.
  • the one or more outputs of the detection system 201 may include a Raman spectrum of the optical signal, an analysis of the Raman spectrum, a result of the analysis, and an alert based on the result of the analysis.
  • optical signal 220 may be received by the detection system 201 over a period of interrogation time.
  • the detection system 201 of the Raman spectroscopic system 100 includes a spectrometer 120, a notch filter 117, and an aperture focusing lens 118.
  • Notch filter 117 blocks wavelengths of excitation light source 110 while transmitting other wavelengths, including wavelengths of optical signal 220 from interrogation apparatus 300.
  • Aperture focusing lens 118 focuses optical signal 220 onto an entrance aperture 122 of spectrometer 120.
  • Entrance aperture 122 may be a slit or a pinhole for receiving a light beam, such as optical signal 220.
  • entrance aperture 122 may receive a fiber or fiber bundle that carry a light beam.
  • aperture focusing lens 118 focuses optical signal 220 from interrogation apparatus 300 to a point at entrance aperture 122.
  • aperture focusing lens 118 is designed to be a multi-element diffraction limited lens.
  • aperture focusing lens 118 may have a diameter equal to or greater than the diameter of optical signal 220 such that a substantial amount or all of optical signal 220 are collected and focused to entrance aperture 122
  • spectrometer 120 includes a collimating element 124, a transmission grating 126, a focusing element 128, and a detector 130.
  • Collimating element 124 receives a light beam that has passed through entrance aperture 122, collimates the light beam, and directs the light beam towards transmission grating 126.
  • Transmission grating 126 disperses the light beam and directs the dispersed light beam to focusing element 128.
  • Focusing element 128 focuses the dispersed light beam to detector 130.
  • the optical components of spectrometer 120 are designed and configured individually and as a whole to provide both high spectral resolution and high light throughput in one or more preselected spectral bands.
  • the path length is 3 ⁇ 4 to 1 meter, which can result in low light throughput and may require a spectrometer size that is not optimal for certain applications. Shortening path length, however, has been associated with reducing the spectral resolution.
  • the optical components of spectrometer 120 are designed and configured individually and as a whole such that the spectrometer 120 has a reduced path length compared to typical spectrometers, but does not have reduced spectral resolution.
  • the advantages of high light throughput, high spectral resolution, and reduced path length are at least partially achieved from the design and use of a lens-grating-lens configuration. Any one or more of the following design considerations may be used according to some embodiments.
  • an exemplary embodiment of spectrometer 120 uses a lens-grating-lens configuration.
  • This lens-grating-lens configuration reduces the path length of spectrometer 120 compared to typical spectrometers as shown in FIGS. 1 and 2, which in turn increases the light throughput of the spectrometer.
  • the path length of the spectrometer 120 i.e., from the focusing element 128 to the detector 120, is from 8 cm to 20 cm. In some exemplary embodiments, the path length is 12.5 cm.
  • the spectrometer 120 has a path length less than one third of that of the typical spectrometers as shown in FIGS. 1 and 2.
  • Reducing the path length of spectrometer 120 can reduce the overall physical dimension of spectrometer 120, making it optimal for standard laboratory uses or to be deployed in the field.
  • Another benefit of the lens-grating-lens configuration shown in FIG. 3 is that it provides more flexibility in replacing one type of detector 130 with another type of detector, each of which may be more suitable for different applications.
  • a consideration for designing spectrometer 120 is selecting a collimating element 124.
  • the collimating element 124 that receives light from the entrance aperture is configured to receive a most of the light entering the entrance aperture. The more light that the collimating element 124 receives from the entrance aperture, the greater the light throughput. In certain embodiments, larger lenses may be used as the collimating element 124 achieve this result.
  • the f-number of collimating element 124 in FIG. 3 is selected such that collimating element 124 receives all or substantially all of the diverging optical signal 220 that enters entrance aperture 122, thereby increasing the light throughput of spectrometer 120.
  • the collimating element 124 receives from 80% to 98% of optical signal 220 that enters entrance aperture 122.
  • collimating element 124 is a multi-element lens that has two or more elements.
  • collimating element 124 is a multi-element diffraction limited lens.
  • a collimating element 124 having a small f-number can be used for receiving the light beam that entered entrance aperture 122.
  • collimating element 124 has an f-number from F/4 to F/1.2, where F is the focal length of collimating element 124.
  • collimating element 124 has an f-number of F/4 or F/2.
  • Certain exemplary optical elements that can used as collimating element 124 include (i) fixed focus multi-element lenses that allows for correction of chromatic aberrations and (ii) reflective optics that collimate and direct light towards the grating.
  • f-number refers to the ratio of the focal length to the diameter of the entrance pupil of the collimating element or the focusing element.
  • a consideration for designing spectrometer 120 is selecting a transmission grating 126.
  • transmission grating 126 having both high spectral resolution and high diffraction efficiency in one or more preselected spectral bands is used in the lens-grating-lens configuration.
  • Transmission grating 126 is a transmissive diffraction grating designed based on specific specifications to achieve high diffraction efficiency and high spectral resolution in an operational wavelength range. Those specifications may include angle of incidence, line density, and bandwidth or operational wavelength range.
  • Such transmissive diffraction gratings used in the lens-grating- lens configuration are designed to allow for shorter path lengths without significantly sacrificing the desired resolution.
  • the diffraction efficiency of transmission grating 126 may be higher for first order diffraction than other orders of diffraction at a designed angle of incidence.
  • the spectral resolution of transmission grating 126 may be higher for first order diffraction than other orders of diffraction at a designed angle of incidence. Therefore, description of the diffraction efficiency and spectral resolution of transmission grating 126 herein refers to the diffraction efficiency and spectral resolution of transmission grating 126 for first order diffraction at a designed angle of incidence for one or more wavelengths of vertically polarized light, horizontally polarized light, and/or the combination of vertically polarized light and horizontally polarized light.
  • description of the diffraction efficiency and spectral resolution of transmission grating 126 herein refers to the diffraction efficiency and spectral resolution of transmission grating 126 for first order diffraction at a designed angle of incidence for one or more wavelengths of the combination of vertically polarized light and horizontally polarized light.
  • the diffraction efficiency of the transmission grating refers to the average diffraction efficiency of the transmission grating over the preselected spectral band.
  • the spectral resolution of the transmission grating refers to the average spectral resolution of the transmission grating over the preselected spectral band.
  • transmission grating 126 may depend on various design factors, including wavelength, line density, polarization, angle of incidence, and diffraction order.
  • transmission grating 126 is designed to disperse an incident light beam with high spectral resolution and high diffraction efficiency over all wavelengths in an operational wavelength range for vertically and/or horizontally polarized light.
  • a center wavelength or a design wavelength refers to the wavelength where transmission grating 124 has the highest diffraction efficiency for first order diffraction when the designed angle of incidence into the Bragg planes is equal to the angle of diffraction out of the Bragg planes.
  • transmission grating 126 is designed to disperse an incident light beam with, as compared to the operational wavelength as a whole, highest spectral resolution and/or the highest or peak diffraction efficiency at a first wavelength for vertically polarized light, a second wavelength for horizontally polarized light, and/or a center wavelength for the combination of vertically and horizontally polarized light.
  • the first wavelength is the same as the second wavelength.
  • the first wavelength is the same as the center wavelength.
  • the second wavelength is the same as the center wavelength.
  • the first wavelength, the second wavelength, and the center wavelength are the same.
  • the center wavelength of transmission grating 126 refers to the Bragg wavelength at which the angle of incidence into the Bragg planes of transmission grating 126 is equal to the angle of diffraction out of the Bragg planes.
  • the initial step for designing the transmission grating 126 is determining the operational wavelength range and center wavelength where transmission grating 126 disperses an incident light beam with high diffraction efficiency.
  • the operational wavelength range may include one or more preselected spectral bands.
  • the center wavelength is a wavelength within the one or more preselected spectral bands, such as a wavelength at or around the middle of the one or more preselected spectral bands.
  • the center wavelength is a selected wavelength within one or more preselected spectral bands of interest, and may or may not be at the middle of the one or more preselected spectral bands.
  • the angle of incidence can be determined according to the grating equation below.
  • AOI angle of incidence
  • AOD angle of diffraction
  • l the wavelength.
  • a center wavelength can be tuned by adjusting the incidence angle, a.
  • transmission grating 126 is designed to have an angle of incidence greater than 0° for wavelengths in the operational wavelength range for first order diffraction. In some exemplary embodiments, transmission grating 126 may have an angle of incidence from 10° to 60° for all the wavelengths in the operational wavelength range. In some exemplary embodiments, transmission grating 126 may have an angle of incidence from 10° to 60° for a center wavelength in the operational wavelength range.
  • transmission grating 126 is designed to have a higher angle of incidence to reduce the overall dimension of spectrometer 120, making spectrometer 120 suitable to be set up on standard laboratory countertop or to be used in the field.
  • transmission grating 126 has a peak diffraction efficiency of 60% or more, including all percentages from 60% to 100%, at a center wavelength of a preselected spectral band for the combination of vertically and horizontally polarized light. In some exemplary embodiments, transmission grating 126 has a peak diffraction efficiency of 60% or more, including all percentages from 60% to 100%, at a first wavelength of a preselected spectral band for vertically polarized light. In some exemplary embodiments, transmission grating 126 has a peak diffraction efficiency of 60% or more, including all percentages from 60% to 100%, at a second wavelength of a preselected spectral band for horizontally polarized light.
  • the first wavelength is the same as the second wavelength. In some exemplary embodiments, the first wavelength is the same as the center wavelength. In some exemplary embodiments, the second wavelength is the same as the center wavelength. In some exemplary embodiments, the first wavelength, the second wavelength, and the center wavelength are the same.
  • transmission grating 126 has a diffraction efficiency higher than 60% for most or all the wavelengths in a preselected spectral band. In some exemplary embodiments, transmission grating 126 is designed to provide a high and substantially flat dispersion of all wavelengths of a preselected spectral band, allowing for fast concurrent identification of a number of spectral features.
  • “flat” or “flatness” refers to the generally smooth and even performance of an optical component in a preselected spectral band, such as the diffraction efficiency of transmission grating 126 as shown in FIG. 4. In some embodiments, “substantially flat” indicates that the performance of the optical components in the preselected spectral band vary less than 30%, 20%, or 10%. [0130] In some exemplary embodiments, the preselected spectral band is determined based on the wavelengths of the excitation light source suitable for detecting a target and/or the Raman peaks associated with the molecular structures or compositions in the target.
  • the preselected spectral band for a certain target may be experimentally or theoretically determined before being used for detecting the presence or absence of the target in a sample.
  • the preselected spectral band can be from 537 nm to 596 nm or a sub-spectral band at any points from 537 nm to 596 nm, or alternatively represented as a preselected spectral band from 200 cm 1 to 2000 cm 1 or a sub- spectral band at any points from 200 cm 1 to 2000 cm 1 .
  • the preselected spectral band can be from 408 nm to 441 nm or a sub-spectral band at any values from 408 nm to 441 nm, or alternatively represented as a preselected spectral band from 180 cm 1 to 2016 cm 1 or a sub-spectral band at any points from 200 cm 1 to 2000 cm 1 .
  • the preselected spectral band can be from 791 nm to 1048 nm or a sub- spectral band at any values from 791 nm to 1048 nm, or alternatively represented as a preselected spectral band from 100 cm 1 to 3200 cm 1 or a sub-spectral band at any points from 100 cm 1 to 3200 cm 1 .
  • the preselected spectral band can be from 1075 nm to 1613 nm or a sub- spectral band at any values from 1075 nm to 1613 nm, or alternatively represented as a preselected spectral band from 100 cm 1 to 3200 cm 1 or a sub-spectral band at any points from 100 cm 1 to 3200 cm 1 .
  • the excitation wavelength can be selected based on the characteristic Raman bands or Raman peaks of the target to be detected.
  • transmission grating 126 may include more than one sub-transmission grating, such as two or more volume phase holographic gratings, that disperse an incident light beam over more than one preselected spectral bands.
  • transmission grating 126 can be designed to disperse light in a number of preselected spectral bands, such as a plurality of spectral bands selected at any points from 100 cm 1 to 3200 cm 1 .
  • transmission grating 126 is designed to have high spectral resolution to improve the spectral resolution of spectrometer 120 to resolve spectral features of an incident light beam, such as optical signal 220.
  • the next step for designing the transmission grating 126 is determining the line density k based on the desired spectral resolution in the operational wavelength range and/or the desired spectral resolution at the center wavelength. Increasing the line density increases the spectral resolution of the transmission grating 126, which in turn increases the spectral resolution of spectrometer 120 to resolve spectral features of an incident light beam, such as optical signal 220. However, increasing the line density may reduce the bandwidth of the operational wavelength range of the transmission grating 126.
  • a maximum line density is used that does not change the operational wavelength range.
  • the line density is from 380 lines/mm to 6000 lines/mm. In some exemplary embodiments, the line density is from 2500 lines/mm to 6000 lines/mm.
  • high spectral resolution refers to a spectral resolution sufficient for resolving one or more characteristic features of a target in the preselected spectral band, such as Raman bands or Raman peaks.
  • transmission grating 126 disperses an incident light beam with a spectral resolution of less than 5 cm 1 in one or more preselected spectral bands, such as a spectral resolution from 1.5 cm 1 to 2.5 cm 1 .
  • the desired spectral resolution of spectrometer 120 and transmission grating 126 may be designed based on the spectral features of the target to be detected.
  • the spectral resolution of transmission grating 126 may be designed to be from 1.5 cm 1 to 2.5 cm 1 in a preselected spectral band from 537 nm to about 596 nm or from 200 cm 1 to 2000 cm 1 . In some exemplary embodiments, when the target is bacteria and excitation wavelengths from 400 nm to 532 nm are used, the spectral resolution of transmission grating 126 may be designed to be from 1.5 cm 1 to 2.5 cm 1 in a sub-spectral band at any points from 537 nm to 596 nm or a sub-spectral band at any values from 200 cm 1 to 2000 cm 1 .
  • the spectral resolution of transmission grating 126 may be designed to be from 2 cm 1 to 5 cm 1 in a preselected spectral band from about 791 nm to 1048 nm, and/or from 1075 nm to 1613 nm.
  • spectrometer 120 when the target is bacteria, spectrometer 120 provides a spectral resolution of 2.5 cm 1 or less, such as a spectral resolution from 1.5 cm 1 to 2.2 cm 1 in a preselected spectral band from 200 cm 1 to 2000 cm 1 or a sub-spectral band at any values from 200 cm 1 to 2000 cm 1 .
  • transmission grating 126 is a volume phase holographic grating that provides both high spectral resolution and high diffraction efficiency for both vertically and horizontally polarized light in a preselected spectral band.
  • the volume phase holographic (VPH) grating is formed in a layer of transmissive material, such as dichromated gelatin, and is sealed between two layers of optically transparent glass or fused silica.
  • Various design factors affect the diffraction efficiency, polarization sensitivity, and bandwidth of the volume phase holographic grating. These factors include the Bragg angle, the average refractive index, the refractive index differential, and the thickness of the transmissive material.
  • the volume phase holographic grating is designed to have a thickness from 0.5 mm to 10 mm.
  • the layer of the transmissive material of the volume phase holographic grating is designed to have a thickness from 0.1 pm to 0.1 mm.
  • the refractive index is modulated, forming a periodic structure in the transmissive material, which can be referred to as fringes.
  • the refractive index of the volume phase holographic grating is from 1 to 2.42.
  • the periodic structure or fringes can be interferometrically produced with a predetermined line density or spatial frequency.
  • FIG. 4 is a graphical representation of the diffraction efficiency of an exemplary volume phase holographic grating (VPH Grating) over a preselected spectral band.
  • the spatial frequency of the exemplary volume phase holographic grating is about 26501/mm and the angle of incidence for first order diffraction for a center wavelength of 568 nm is about 48.8°.
  • the exemplary volume phase holographic grating has a diffraction efficiency higher than 80% at a center wavelength of 568 nm for first order diffraction at the angle of incidence of 48.8°.
  • the spectrometer 120 including the exemplary volume phase holographic grating also provides a spectral resolution of at least about 2 cm 1 in the preselected spectral band.
  • the exemplary volume phase holographic grating offers both a high and flat diffraction efficiency curve for both vertically polarized light (E s ) and horizontally polarized light (E p ) over a broad spectral band that spans over 50 nm, where E p represents the average of the diffraction efficiency of both E s and E p.
  • E s vertically polarized light
  • E p horizontally polarized light
  • Such high and flat diffraction efficiency improves the light throughput or sensitivity of spectrometer 120 in the preselected spectral band.
  • This high and flat diffraction efficiency of the exemplary volume phase holographic grating is further confirmed by theoretical predictions of spectra output of spectrometer 120 as shown in FIGS. 5 and 6.
  • FIG. 5 is a graphical representation of theoretically predicted spectra output of an optical signal by spectrometer 120 without the exemplary volume phase holographic grating.
  • FIG. 6 is a graphical representation of theoretically predicted spectra output of the same optical signal by spectrometer 120 with the exemplary volume phase holographic grating. As illustrated in FIGS.
  • the addition of the exemplary volume phase holographic grating reduced the power of predicted spectra output by less than 40% across the preselected spectral band, showing that the diffraction efficiency of the exemplary volume phase holographic grating is higher than 60%, where the diffraction efficiency of the exemplary volume phase holographic grating can be calculated as the ratio of the power of predicted spectra output of the spectrometer with the exemplary volume phase holographic grating to the power of predicted spectra output of the spectrometer without the exemplary volume phase holographic grating.
  • the diffraction efficiency of the exemplary volume phase holographic grating is 80%.
  • dispersive elements may be used in place of the transmission grating 126, such as a dispersive prism, a staircase reflective element as described in US 2015/0029504 Al, or a step-style reflective element as described in US 2015/0085284 Al.
  • a consideration for designing spectrometer 120 is selecting a suitable detector 130, which provides a spectral profile of intensity over wavelengths or wavenumbers of the dispersed optical signal 230, such as a Raman spectrum.
  • the detector 130 may be a detector array having an array of pixels, such as a CCD, a CMOS, a GaAs detector array, or an array of micro photo multiplier tubes.
  • the detector 130 may be selected to obtain the desired sensitivity and/or spectral resolution.
  • detector 130 having high quantum efficiency is used to improve the sensitivity of spectrometer 120. Examples of such detectors include a cryogenically cooled CCD and a deep cooled back thinned CCD.
  • detector 130 may be selected based on the design parameters of other components of spectrometer 120 to obtain the desired spectral resolution and sensitivity, such as the size of entrance aperture 122, the magnification of entrance aperture 122 by collimating element 124 and focusing element 128, and the dispersion of optical signal 230 by transmission grating 126.
  • a detector 130 having smaller pixel sizes may be used to increase the spectral resolution of spectrometer 120.
  • the size of detector 130 is selected such that the image of entrance aperture 122 extends across the image area of detector 130 along a dimension.
  • detector 130 has 2048 by 70 pixels, the size of each pixel being 14 pm x 14 pm, resulting in an image area of 28.7 mm by 0.98 mm. In some exemplary embodiments, detector 130 has 2048 pixels by 248 pixels, the size of each pixel being from 8 pm x 8 pm to 16 pm x 16 pm.
  • detector 130 may be selected such that the operational wavelength range of the dispersed spectrum detected by the detector 130 spreads across the image area of the detector 130.
  • Reciprocal linear dispersion defines the extent to which a spectral interval is physically spread out across a focal field in a spectrometer, e.g., the image area of the detector 130, and is represented as nm/mm.
  • the operational wavelength range of the transmission rating 126 includes a preselected spectral band from 540.17 nm to 592.63 nm and the detector 130 has an image area of 28.7 mm by 0.98 mm
  • the reciprocal linear dispersion of spectrometer 120 can be about 1.83 nm/mm.
  • the reciprocal linear dispersion of spectrometer 120 and the size of the pixels of detector 130 can affect the spectral resolution of spectrometer 120.
  • the spectral resolution of the spectrometer 120 can be determined to be 2 x 14 pm x 1.84 nm/mm, which is about 0.052 nm or 1.63 cm 1 .
  • other factors of the components of spectrometer 120 can also affect the spectral resolution, such as the size of entrance aperture 122.
  • a consideration for designing spectrometer 120 is selecting the size of entrance aperture 122, such as an entrance slit, which can affect the spectral resolution of the spectrometer 120.
  • the spectral resolution of a spectrometer is equal to the bandpass (BP) of the spectrometer at Nyquist sampling.
  • BP is defined as the FWHM spectral response of a spectrometer to incident monochromatic light.
  • the total or net bandpass is a result of the natural line width of the spectrum of the incident monochromatic light source used to measure the FWHM, the limiting instrumental line profile that includes system aberrations and diffraction effects, and the influence of the entrance slit.
  • FWHM can be approximated by the following generalized bandpass equation: where BPnet is the net bandpass, BPnat is the natural spectral bandwidth of the emitting source, BPsiit is the bandpass determined by the reciprocal linear dispersion and the width of the image of the entrance slit on detector 130 formed by collimating element 124 and focusing element 128, and BPres is the limiting resolution of the instrument (the ultimate bandpass with a line emission source).
  • the bandpass is dominated by the bandpass determined by the slit width, BPsiit, which can be calculated according to
  • W s ii t (mm) 10 6 (nm/mm) cos (ct) ⁇ where RLD is the reciprocal nk(lines/mm)L a (mm ) linear dispersion and W sUt image is the width of the image of the entrance slit on detector 130 generated by collimating element 124 and focusing element 128. RLD defines the extent to which a spectral interval is physically spread out across a focal field in a spectrometer.
  • the width of the image of the slit, W sUt ima g e is the product of the magnification of the image of entrance aperture 122 and the physical width of the entrance slit.
  • the magnification of the image of entrance slit is a function of the ratio of the focal length of the focusing element 124 to the focal length of the collimating element 124.
  • the product of the image of the entrance slit width and the reciprocal linear dispersion, W sUt image RLD, is a function of the physical width of the entrance slit (W sUt ) the diffraction order (n), the angle of incidence (a), the line density of the grating (k) represented as line/mm, and the focal length of the collimating element 124 ( L a ).
  • BPsiit for the first order diffraction is calculated to be 0.046 nm.
  • the FWHM or the spectral resolution of spectrometer 120 can be approximated as 0.046 nm or alternatively represented as 1.43 cm 1 at a wavelength of 568 nm when the excitation wavelength is 532.02 nm. Because the spectral resolution can be improved with the narrowing of the width of the image of the entrance slit on detector 130, in some exemplary embodiments, a narrower image of the entrance slit on the detector 130 is desired.
  • a consideration for designing spectrometer 120 is selecting the magnification of spectrometer 120.
  • the width of entrance aperture 122 can also affect the light throughput of spectrometer 120 by limiting the etendue of the spectrometer. As the width of the entrance aperture 122 increases, more light is received through the entrance aperture 122. More light is desirable in certain embodiments.
  • a narrower image of entrance aperture 122 on the detector 130 is desired to obtain a better spectral resolution. Therefore, the size of entrance aperture 122 can be increased to increase the light throughput of spectrometer 120 or decreased to increase the spectral resolution of spectrometer 120.
  • the size of entrance aperture 122 is based on the designed spectral resolution and bandwidth of transmission grating 126, the magnification of entrance aperture 122 in spectrometer 120, and the pixel size of detector 130.
  • the magnification of spectrometer 120 is selected to be 1 or close to 1 such that the width of the image of the entrance aperture 122 equals the physical width of the entrance aperture 122.
  • the focal length of focusing element 128 is designed to be the same as the focal length of collimating element 124.
  • entrance aperture 122 may be a pinhole having a diameter from 10 pm to 25 pm.
  • entrance aperture 122 may be a slit having a width from 5 pm to 25 pm.
  • focusing element 128 is identical to collimating element 124. In some exemplary embodiments, the focal length of focusing element 128 is equal to the path length of spectrometer 120. In some exemplary embodiments, focusing element 128 and collimating element 124 have the same f-number. In other exemplary embodiments, focusing element 128 and collimating element 124 have different f-numbers. In some exemplary embodiments, focusing element 128 has an f-number from F/4 to F/1.2, where F is the focal length of focusing element 128. In some exemplary embodiments, focusing element 128 has an f-number of F/4 or F/2.
  • focusing element 128 is a multi-element diffraction limited lens having a substantially flat response across a preselected spectral band.
  • response of focusing element 128 refers to the optical transfer function of focusing element 128.
  • configuring the optical components of spectrometer 120 individually and as a whole to provide high spectral resolution, high light throughput, and short path length in one or more preselected spectral bands involve adjusting different factors based on the practical limitations, applications, and/or test results. For example, if the width of the entrance slit selected for a desired spectral resolution is very narrow such that it only allows a very limited amount of light to enter the spectrometer 120, it can affect sensitivity of spectrometer 120 in detecting low intensity optical signals, such as Raman signals.
  • the width of the entrance slit may be adjusted to be wider and the other factors may be adjusted to achieve the desired spectral resolution, such as selecting a wider detector 130 and/or choosing a grating with a higher line density.
  • the spectral resolution of spectrometer 120 is less than 5 cm 1 in one or more preselected spectral bands, such as a spectral resolution from 1.5 cm 1 to 2.5 cm 1 , a spectral resolution from 1.69 cm 1 to 2.08 cm 1 , or a spectral resolution from 1.85 cm 1 to 2.16 cm 1 .
  • One or more parameters can be used to characterize the performance of spectrometer 120.
  • the desired performance of spectrometer 120 may vary for different applications. In biomedical applications, it typically is desired to have a compact spectroscopic system or a compact spectrometer with high spectral resolution, high light throughput, and short interrogation time.
  • such compact spectroscopic system or spectrometer may be set up on a standard laboratory shelf or used in resource-limited settings.
  • the performance ratio is used to evaluate the performance of spectrometer 120 in the preselected spectral band.
  • the lens-grating-lens configuration of spectrometer 120 and the combined use of collimating element 124, transmission grating 126, and focusing element 128 increase both the spectral resolution and transfer efficiency of spectrometer.
  • the designs and configurations of these components in some exemplary embodiments improve the performance of spectrometer 120 and increase the performance ratio of spectrometer 120.
  • the performance ratio of spectrometer 120 is from 3 %-cm 1 to 12.3 %-cm 1 . For example, when the focal length of focusing element 128 is 135 mm and the transfer efficiency of spectrometer 120 is 60%, the performance ratio of the spectrometer 120 is approximately 4.4 %-cm 1 .
  • the performance product is used to evaluate the performance of spectrometer 120 in the preselected spectral band.
  • the lens-grating4ens configuration of spectrometer 120 and the combined use of collimating element 124, transmission grating 126, and focusing element 128 improves performance of spectrometer 120 and reduces the performance product of spectrometer 120.
  • the performance product of spectrometer 120 is from 0.8 to 100.
  • the performance product is from 12 to 50. For example, when the focal length of focusing element 128 is 135 mm and the spectral resolution of spectrometer 120 is 2 cm 1 , the performance product of the spectrometer 120 is approximately 27.
  • the length of interrogation time for obtaining an optical signal is used to evaluate the performance of spectrometer 120 in the preselected spectral band. Due to the inherent low intensity of Raman signals, in typical Raman spectrometers, long periods of interrogation time are needed to increase the signal to noise (S/N) ratio of the measured Raman spectra to obtain information from the measured Raman spectra.
  • the interrogation time of typical Raman spectrometers can be from 20 minutes to 60 minutes. However, the long periods of interrogation time can in turn degrade the optical signal because the sample, such as a sample containing biological materials, may change during a period of interrogation time.
  • the high light throughput or high efficiency of spectrometer 120 allows obtaining Raman spectra having a high sensitivity, such as a high signal to noise ratio, in a short period of interrogation time.
  • the period of interrogation time of spectrometer is from 30 seconds to 10 minutes. Such short period of interrogation time mitigate the changes in the sample that confounds the determination of the presence or absence of a target.
  • successive short periods of interrogation time allows monitoring or tracking the changes within the sample over time, such as growth phase changes of a bacteria colony.
  • Raman spectroscopic system 100 includes an interrogation apparatus 300.
  • Interrogation apparatus 300 is designed to collect or concentrate the sample to be interrogated at a focal point of the illumination and signal receiving optics to reduce the technical complexity for focusing on the sample, thereby improving the accuracy and reliability of the collected optical signal. Additionally, in some exemplary embodiments, interrogation apparatus 300 is designed to increase the amount of signal that can be received from the same sample to reduce the interrogation time and improve the sensitivity of Raman spectroscopic system 100. In some exemplary embodiments, interrogation apparatus 300 may further allow Raman spectroscopic system 100 to meet the requirements of the Clinical Laboratory Improvement Amendments of 1988.
  • FIG. 7 is a schematic representation of an exemplary interrogation apparatus 300, according to some embodiments of the present disclosure.
  • interrogation apparatus 300 includes a cuvette 310 that can contain a sample to be interrogated.
  • interrogation apparatus 300 includes one or more optical elements for focusing an incoming excitation beam to a focal point in cuvette 310.
  • interrogation apparatus 300 may include a lens 320 that focuses excitation light beam 210 to a focal point 350 on or above a bottom end of cuvette 310.
  • Lens 320 further receives and collimates optical signal 220 from focal point 350 as described below with reference to FIG. 8.
  • at least one optical element 324 is used to direct optical signal 220 to the detection system.
  • optical element 324 can be a mirror that reflects and directs the collimated optical signal 220 to the detection system.
  • a focal point refers to the focus of an optical element, such as a lens or a concave mirror.
  • the focus of an optical beam can be a focal spot when the optical beam is focused by an optical element, such as a spherical lens, that focuses incoming light into a spot.
  • focal point 350 overlaps with the focal spot of the optical beam, such as excitation light beam 210.
  • FIG. 8 is a graphical illustration of an example of focusing an excitation light beam to, or receiving an optical signal from, a focal point of an exemplary interrogation apparatus 300, according to some embodiments of the present disclosure. As shown in FIG.
  • a focal spot (shown as dotted circle in FIG. 8) of the excitation light beam overlaps with focal point 350.
  • receiving an optical signal from focal point 350 refers to receiving the optical signal from a measurement volume 222 at and surrounding focal point 350.
  • the focus of an optical beam can be a focal line when the optical beam is focused by an optical element, such as a cylindrical lens, that focuses incoming light into a line.
  • focal point 350 overlaps with the focal line (shown as broken line in FIG. 8) of the optical beam.
  • cuvette 310 of interrogation apparatus 300 reduces the technical complexity for performing focusing on the sample and increases the amount of optical signal 220 that can be received from the same sample.
  • typical cuvettes used in spectroscopic measurements are small tube-like containers having straight walls. When interrogating a sample, the excitation light beam is focused through a straight wall onto the sample in a solution form or diluted in a solution.
  • cuvette 310 of interrogation apparatus 300 is designed to concentrate or collect the sample to be interrogated to focal point 350 of cuvette 310. As described herein, concentrating or collecting the sample to be interrogated to focal point 350 refers to concentrating or collecting the sample at and/or around focal point 350.
  • concentrating or collecting the sample to be interrogated to the focal point 350 allows the system to automatically focus on the sample to be interrogated when cuvette 310 is placed at a known place with a fixed focal point in the system, which does not require a highly trained investigator or technician to perform.
  • cuvette 310 is designed to concentrate the sample to be interrogated to the focal point 350 such that greater amount of optical signal 220 can be received during the same period of interrogation time, improving the sensitivity of the system.
  • concentrating or collecting the sample to be interrogated to focal point 350 also may allow a shorter period of interrogation time to be used and/or increases the quality of the obtained optical signal 220 for further spectral analysis.
  • excitation light beam 210 focused to focal point 350 can attract certain targets, such as pathogens, to focal point 350, such as Escherichia coli , which can further concentrate such targets to focal point 350.
  • cuvette 310 includes a chamber having a top end 314, a bottom end 312, and at least one tapered wall 340.
  • Bottom end 312 is narrower than top end 314.
  • Tapered wall 340 has a tilt angle relative to a centerline perpendicular to the bottom end 312.
  • bottom end 312 of cuvette 310 includes an optical window 313.
  • optical window 313 is made of an optically transparent material that has low Raman emission at the excitation wavelengths.
  • optical window 313 may be made of any of the following exemplary materials: fused silica, glass, sapphire, and quartz.
  • Optical window 313 has an interior surface 313a and an exterior surface 313b. As shown in FIG. 7, in some exemplary embodiments, focal point 350 is on or above interior surface 313a. In some embodiments, the focal point is above interior surface 313a by a distance from 100 pm to 5 mm. In some embodiments, interior surface 313a includes a functionalized surface to attract and/or retain a sample to be interrogated. In some exemplary embodiments, the functionalized surface is a layer of material covering the interior surface 313a or a central region of the interior surface 313a. In some exemplary embodiments, the functionalized surface is formed by modifying the interior surface 313a. In some exemplary embodiments, focal point 350 is designed to be on or above the functionalized surface.
  • Certain exemplary functionalized surfaces may have physical, chemical, or biological characteristics suitable to attract or retain the sample to be interrogated.
  • the functionalized surface can be hydrophobic to attract the sample to be interrogated, such as proteins, through Van der Waals attraction.
  • the functionalized surface can be hydrophilic to attract the target through dipole-dipole interactions.
  • the functionalized surface can include biomolecules or chemicals that bind to the target.
  • the functionalized surface may include antibodies that bind to target pathogens, cells, or biomolecules.
  • the functionalized surface may include protein G.
  • the functionalized surface may include functionalized bacteria specific phages.
  • the functionalized surface is prepared with Oseltamivir, a synthetic derivative prodrug of ethyl ester with antiviral activity, or Zanamivir for attracting and retaining viruses, such as influenza viruses.
  • Oseltamivir a synthetic derivative prodrug of ethyl ester with antiviral activity
  • Zanamivir for attracting and retaining viruses, such as influenza viruses.
  • the chemical structure of Oseltamivir is illustrated below.
  • tapered wall 340 of cuvette 310 allows the sample to be interrogated, subject to the effect of gravity, to first settle onto the tapered wall 340 and move down along the tapered wall 340 towards the bottom end 312 of cuvette 310.
  • such guided settlement of the sample to be interrogated allows the sample to be interrogated to settle in a higher concentration at a narrow region at the bottom end 312, such as a central region on the interior surface 313a of optical window 313.
  • central region refers to an area at and surrounding a center region of the interior surface 313a that overlaps or is close to focal point 350.
  • the tilt angle of the tapered wall 340 may be influenced by the sample to be interrogated. In some exemplary embodiments, the tilt angle is from 9 degrees to 19 degrees. In some exemplary embodiments, a settling period allows the cuvette 310 to concentrate the sample to be interrogated to focal point 350 before receiving an optical signal 220. In some exemplary embodiments, the settling period may depend on the type of the sample to be interrogated, the density of the sample to be interrogated, and the tilt angle of tapered wall 340.
  • a settling period from 5 seconds to 5 minutes may be used before receiving the signal.
  • a settling period from 10 milliseconds to 5 minutes may be used before receiving the signal.
  • FIG. 9A is a top view of an exemplary chamber of cuvette 310.
  • FIG. 9B is a side view and
  • FIG. 9C is a bottom view of the exemplary chamber of FIG. 9A.
  • FIG. 10A is a top view of another exemplary chamber of cuvette 310.
  • FIG. 10B is a side view and
  • FIG. IOC is a bottom view of the exemplary chamber of FIG. 10A.
  • FIG. 11 A is a top view of another exemplary chamber of cuvette 310.
  • FIGS. 11A is a top view of an exemplary chamber of cuvette 310.
  • FIG. 9B is a side view
  • FIG. 9C is a bottom view of the exemplary chamber of FIG. 9A.
  • FIG. 10A is a top view of another exemplary chamber of cuvette 310.
  • FIG. 10B is a side
  • the chamber of cuvette 310 has a shape of a truncated cone.
  • the chamber of cuvette 310 has a shape of a truncated pyramid having a plurality of tapered walls 340, such as three, four, six, eight, and ten tapered walls 340.
  • the chamber of cuvette 310 has a shape of a truncated pyramid having four tapered walls 340.
  • FIGS. 10A-10C As another non-limiting example, as shown in FIGS.
  • the chamber of cuvette 310 has a shape of a truncated pyramid having six tapered walls 340.
  • the chamber of cuvette 310 has a shape of a truncated pyramid having a number of tapered walls 340 from 3 to 10.
  • the shape of the chamber of cuvette 310 and number of tapered walls 340 can be configured based on the sample to be interrogated. In some exemplary embodiments, it is beneficial to detect optical signal 220 from a homogenized layer of the sample to be interrogated where the sample to be interrogated is evenly or substantially evenly distributed. In some exemplary embodiments, this is achieved with a cuvette 310 having a chamber with four tapered walls 340. After being received in cuvette 310 with four tapered walls 340, the sample to be interrogated can settle towards bottom end 312 and form a homogenized layer across interior surface 313a of optical window 313 or across a central region of interior surface 313a.
  • the homogenized layer is a monolayer.
  • the sample to be interrogated can settle towards interior surface 313a and concentrate to a central region of interior surface 313a, forming an accumulated mass of the sample to be interrogated.
  • the sample to be interrogated can settle towards interior surface 313a and concentrate to a central region of interior surface 313a, forming an accumulated mass of the sample to be interrogated at the central region and a homogenized layer around the central region.
  • the shape of the chamber of cuvette 310 and number of tapered walls 340 can be configured based on the geometry of the excitation beam 210.
  • the shape of the chamber of cuvette 310 and number of tapered walls 340 can be configured such that a homogenized layer of the sample to be interrogated is formed across interior surface 313a of optical window 313 to which the excitation beam 210 is focused. This configuration can result in a larger exposure area of the sample to be interrogated.
  • the configuration may be used, for certain motile or heterogeneous samples where it may be desirable to interrogate a larger area across the interior surface 313a.
  • a motile sample may include targets that are mobile, such as Escherichia coli.
  • a heterogenous sample may have different concentrations of the targets distributed in the sample, such as a bacterial biofilm or a non-uniform chemical.
  • the shape of the chamber of cuvette 310 and number of tapered walls 340 can be configured such that the sample to be interrogated is concentrated to a central region of interior surface 313a.
  • such a configuration can be used to increase the amount of optical signal 220 received from the sample to be interrogated when the concentration of the sample to be interrogated is low.
  • Tapered walls 340 of cuvette 310 may be made of various suitable materials.
  • the tapered walls 340 of cuvette 310 may be made of glass, Teflon, fluorocarbon-based materials, or a polymer, such as polystyrene (PS) or polymethyl methacrylate (PMMA).
  • the interior surface of tapered wall 340 is modified to have a suitable physical, chemical, or biological characteristic to facilitate concentrating the sample to be interrogated to a focal point 350.
  • FIG. 12 illustrates an exemplary interior surface 342 of a tapered wall 340, according to some embodiments of the present disclosure.
  • interior surface 342 of a tapered wall 340 includes a smooth hydrophobic coating, such as a hydrophobic fluorine terminated polymer coating.
  • exemplary hydrophobic coatings include glass, sapphire, fused silica, lacquers, hydrophobic self- assembled monolayers, fluorocarbons, acrylics, vinyls, olefins, carbonates, and amides.
  • the interior hydrophobic coating may inhibit the sample to be interrogated from adhering to tapered wall 340 and facilitates movement of the sample to be interrogated to the bottom end 312.
  • FIG. 13 illustrates another exemplary interior surface 342 of tapered wall 340, according to some embodiments of the present disclosure.
  • interior surface 342 of tapered wall 340 includes a micro textured hydrophilic coating that is wetted when immersed in liquid.
  • the micro textured hydrophilic coating is a hydrophilic-terminated polymer coating, such as poly(N-isoproplacrylamide) (PNIPAM), polyacrylamide (PAM), polyethylenimine, poly (acrylic acid), poly(vinl alcohol), copolymers, and polyethers, a metal-terminated polymer coating, or a self-assembled monolayer.
  • PNIPAM poly(N-isoproplacrylamide)
  • PAM polyacrylamide
  • polyethylenimine poly (acrylic acid), poly(vinl alcohol), copolymers
  • polyethers such as poly(N-isoproplacrylamide) (PNIPAM), polyacrylamide (PAM), polyethylenimine, poly (acrylic acid), poly(vinl
  • Such hydrophilic coating can have a slip stream effect that impedes the sample to be interrogated from adhering to the tapered wall 340 and thus facilitates movement of the sample to be interrogated to the bottom end 312.
  • the tilt angle can be increased up to 80 degrees.
  • an interior surface or an exterior surface of the tapered wall 340 is coated with a metallic material that reflects optical signal 220 off the tapered wall 340 back into cuvette 310 and/or keeps ambient light from entering cuvette 310.
  • the metallic coating of the tapered wall 340 reduces Raman emission of the material of the tapered wall 340.
  • interrogation apparatus 300 includes a focusing back reflector 330.
  • focusing back reflector 330 is a concave mirror, such as a spherical or parabolic mirror. Focusing back reflector 330 is placed above bottom end 312 of cuvette 310 at a distance such that focal point 350 overlaps the focus of focusing back reflector 330.
  • a portion of the excitation light beam 210 that is not absorbed by the sample to be interrogated at focal point 350 can be reflected and focused back to focal point 350 by focusing back reflector 330.
  • the portion of optical signal 220 that is emitted towards focusing back reflector 330 can also be reflected and focused back to focal point 350.
  • Both of the reflected portions of the excitation light beam 210 and optical signal 220 can be absorbed by the sample to be interrogated at focal point 350 to generate more optical signal 220, creating a resonance effect.
  • such resonance effect of optical signal 220 enhances optical signal 220 by increasing the overall amount of optical signal 220 that can be received from the same sample during the same period of interrogation time. This in turn improves the sensitivity of Raman spectroscopic system 100.
  • optical signal 220 is a Raman signal emitted by a sample to be interrogated concentrated to focal point 350
  • excitation light beam 210 not absorbed by the sample to be interrogated is reflected and focused back to focal point 350 to cause more Raman emission from the sample to be interrogated, resulting in an enhanced optical signal 220 to be directed to spectrometer 120.
  • the portion of the Raman signal emitted towards focusing back reflector 330, having the same energy as required to cause Raman emission is reflected and focused back to focal point 350 to cause more Raman emission of the sample to be interrogated, resulting in a further enhanced optical signal 220.
  • such enhancement of optical signal 220 can increase the amount of optical signal 220 from two to twenty orders of magnitude.
  • focusing back reflector 330 is part of a cover of cuvette 310. As shown in FIG. 14, after cuvette 310 is filled with a sample or a solution containing the sample, focusing back reflector 330 covers and seals cuvette 310. In some exemplary embodiments, focusing back reflector 330 is formed by metalizing an interior side of a curved cover of cuvette 310.
  • a concave mirror 322 is used to focus excitation light beam 210 to focal point 350 and receive optical signal 220 from focal point 350.
  • concave mirror 322 collimates and directs optical signal 220 to the detection system.
  • concave mirror 322 can be a spherical mirror or a parabolic mirror.
  • the interior surface 313a of the bottom end 312 of the cuvette 310 includes a plurality of nanostructures to enhance optical signal 220 emitted from a sample being interrogated through surface-enhanced Raman scattering (SERS).
  • the nanostructures are metal nanoparticles or metal nanodots, such as gold, silver, or platinum nanoparticles.
  • the nanostructures are arranged in an array.
  • the nanostructures may increase optical signal 220 by several orders of magnitude, allowing Raman spectroscopic system 100 to detect low levels of targets, such as low concentrations of pathogens or biomarkers.
  • the sample being interrogated may also be retained to the interior surface 313a by being attracted to or binding to the nanostructures.
  • FIG. 16 illustrates exemplary preparation of nanoparticles on the surface of an exemplary cuvette 310 that can contain a sample, according to some embodiments of the present disclosure.
  • a photoresist layer 311 is used to pattern open columns on interior surface 313a of optical window 313 at a high aspect ratio.
  • the high aspect ratio of the open columns creates a lensing effect that create regular repeatable patterns.
  • gold, silver, or platinum nanoparticles are sputter deposited onto interior surface 313a of optical window 313 through the openings or channels of photoresist layer 311 during a short deposition time.
  • photoresist layer 311 is removed, leaving an array of nanoparticles 315 that can enhance optical signal 220 through SERS.
  • the length or diameter of the nanoparticles is less than 100 nm.
  • a fill factor of the nanoparticles on interior surface 313a is between 50% and 90%.
  • a sample to be interrogated is a solution or mixed in a solution before being interrogated.
  • air bubbles in the chamber of cuvette 310 can act like lenses, cause scattering and light loss, and impair the performance of the excitation and collection optics.
  • cuvette 310 may include a cover that reduces or eliminates air bubbles in the chamber of cuvette 310.
  • the cover is an upper optical window 316 that pushes out air bubbles and seals the solution between upper optical window 316 and bottom end 312.
  • cuvette 310 may include a lid that seals cuvette 310 while allowing air bubbles to be pushed out of the cuvette 310.
  • FIG. 17A is an exploded perspective view of an exemplary cuvette 310 having a lid 360, according to some embodiments of the present disclosure.
  • FIG. 17B is top view of the exemplary cuvette 310 of FIG. 17A.
  • FIGS. 17C and 17D are cross-sectional views of the exemplary cuvette of FIG.
  • lid 360 includes a seal 362 that meets ridge 364 on top end 314 of cuvette 310 when lid 360 is closed onto top end 314. When lid 360 is closed, seal 362 and ridge 364 can form a liquid tight seal.
  • lid 360 of cuvette 310 has a recessed portion 366.
  • upper optical window 316 forms the bottom end of recessed portion 366. Upper optical window 316 may be attached to recessed portion 366 using an adhesive layer 332 or other suitable attachment method. As shown in FIGS.
  • lid 360 when lid 360 is closed onto top end 314, recessed portion 366 displaces some solution in a top part of the chamber of cuvette 310 to a space 363 formed between recessed portion 366 and tapered wall 340 of cuvette 310. Such displacement allows solution to be filled and surround upper optical window 316 to reduce air bubbles in the chamber of cuvette 310.
  • lid 360 includes at least one air hole 368. Air hole 368 is covered with a hydrophobic membrane 369 that allows air to escape but retains liquid therein. Therefore, when cuvette 310 is filled with a solution as shown in FIG.
  • top end 314 of cuvette 310 includes one or more clamps 318 for locking and securing lid 360.
  • optical window 313 is attached to bottom end 312 using an adhesive layer 317 or other suitable attachment method.
  • cuvette 310 includes a bar code 365 or other suitable type of labeling for identifying the sample to be interrogated.
  • the target in the sample to be interrogated may be highly motile, such as a motile bacterium, where it may be desirable to trap the target to the focal point 350 on or above the interior surface 313a of the cuvette 310 for interrogation.
  • a filter is used to trap the motile bacterium.
  • at least one filter 319 that traps the target in a sample to be interrogated is included toward the bottom of the cuvette 310.
  • Leptospirosis is an infection caused by corkscrew shaped gram negative like bacteria of the genus Leptospira. In humans, it can cause a wide range of symptoms, some of which may be mistaken for other diseases. Some infected persons, however, may have no symptoms at all. Without treatment, Leptospirosis can lead to kidney damage, meningitis (inflammation of the membrane around the brain and spinal cord), liver failure, respiratory distress, and even death. Leptospira are spiral-shaped bacteria that are 6- 20 pm long and 0.1 pm in diameter with a wavelength of about 0.5 pm. One or both ends of the spirochete are usually hooked. Leptospira are represented in urine due to kidney infections.
  • the filter 319 that traps the motile bacteria in a sample is placed toward the bottom end 312 of the cuvette 310 to concentrate the motile bacteria at the bottom end.
  • the filter 319 concentrates the trapped motile bacteria to the focal point 350 on or above the interior surface 313a of the cuvette 310.
  • the filter 319 is a membrane filter. In some exemplary embodiments, the filter 319 is a polycarbonate membrane filter. In some exemplary embodiments, the filter 319 is a NucleporeTM membrane filter. In some exemplary embodiments, the filter 319 is a cellulose filter. FIG. 17F is a magnified image of the exemplary filter 319 of FIG. 17E, according to some embodiments of the present disclosure. In some exemplary embodiments, the filter 319 has a diameter equal to or smaller than the diameter of the bottom end 312 of cuvette 310. In some exemplary embodiments, the filter 319 has a diameter of 8 mm.
  • filter 319 is configured to pass the pathogens, such as viruses, in the sample toward the bottom end 312 of the cuvette 310 to concentrate the pathogens at the bottom end of the cuvette.
  • the pathogens in the sample may pass through the filter and accumulate at the bottom end of the cuvette during and/or post centrifugation.
  • the filter 319 has a plurality of holes or pores.
  • the size of the holes of the filter 319 can be selected based on the target to be trapped.
  • the diameter of the holes or the pore size of filter 319 is from 0.1 pm to 10.0 pm.
  • the diameter of the holes or the pore size of filter 319 is from 0.2 pm to 1.0 pm.
  • filter 319 is made of a metallic material.
  • a surface of the filter 319 is a metallic material or is coated with a metallic material.
  • the metallic material is or comprises one or more metal or metal alloy, such as platinum (Pt), palladium (Pd), silver (Ag), copper (Cu), Tantalum (Ta), or stainless steel.
  • the filter 319 has a platinum coating having a thickness from 250 Angstroms to 1000 Angstroms.
  • the platinum coating of the filter 319 may inhibit background fluorescence signal emitted from the base material of the filter 319, such as the polycarbonate substrate.
  • the platinum coating of the filter 319 may allow surface-enhanced Raman scattering effects to be induced at the edges of the holes where the radii of the platinum-coated holes are at a nanostructure level.
  • filter 319 is used as follows: (1) aspirate 5 ml of the sample (such as urine, bacteria in water, bacteria in urine) into a 10 ml syringe; (2) place the filter 319 on the tip of the syringe and gently press the plunger of the syringe to filter the sample through the filter 319 until the barrel of the syringe is empty; (3) remove the filter 319 and aspirate 1 ml of water into the syringe; (4) place the same filter 319 back on to the tip of the syringe and gently press the plunger until the barrel is empty (this step may be repeated as needed to remove residue sample solution on the filter 319).
  • the filter 319 includes the target on its surface if the target is present in the sample.
  • the filter 319 is placed face down in the cuvette 310 such that the target is trapped or concentrated to the bottom end 312 of the cuvette 310 or the focal point 350 of the cuvette 310.
  • FIG. 17G illustrates an exemplary filter 319 with trapped exemplary bacteria on its surface to be placed face down at the bottom end of the cuvette 310, according to some embodiments of the present disclosure.
  • FIGS. 18A-18E cuvette 310 is part of a sample collection cartridge 400 that can receive a sample directly from a swab.
  • FIG. 18A is a perspective view of an exemplary sample collection cartridge 400, according to some embodiments of the present disclosure.
  • FIG. 18B is an exploded perspective view of the exemplary sample collection cartridge 400 of FIG. 18 A.
  • FIG. 18C is a top cross-sectional view of the exemplary sample collection cartridge 400 of FIG. 18 A.
  • FIG. 18D is a cross- sectional view of the exemplary sample collection cartridge 400 along the cross-section A-A as shown in FIG. 18C.
  • FIG. 18E is a perspective view of another exemplary sample collection cartridge 400.
  • an exemplary sample collection cartridge 400 includes cuvette 310, a reservoir 410 that can contain a washing solution, and a chamber 420 that can receive a swab. Reservoir 410, chamber 420, and cuvette 310 are connected via channel 430.
  • the washing solution is water.
  • reservoir 410 can be actuated such that the wash solution in reservoir 410 can be pushed out from reservoir 410.
  • Cuvette 310 then concentrates and/or homogenizes at least a portion of the sample that has been carried into cuvette 310 to bottom end 312.
  • FIG. 18E is a perspective view of another exemplary sample collection cartridge 400, according to some embodiments of the present disclosure.
  • the view in FIG. 18E shows the interior of cartridge.
  • sample collection cartridge 400 includes cuvette 310, a reservoir 410 that can contain a washing solution, a chamber 420 that can receive a swab 422, and a channel 430 connecting reservoir 410, the absorbent material of swab 422 in chamber 420, and cuvette 310.
  • chamber 420 When chamber 420 receives swab 422 that has absorbed a sample, the wash solution in reservoir 410 can be pushed out from reservoir 410 and pass by the absorbent material of swab 422 such that at least a portion of the sample is washed off from the absorbent material of swab 422 and carried into cuvette 310 by the wash solution.
  • Cuvette 310 then concentrates and/or homogenizes at least a portion of the sample that has been carried into cuvette 310 to bottom end 312.
  • sample collection cartridge 400 allows a sample obtained directly from a source using a swab to be ready for interrogation without any manual processing. This greatly simplifies and reduces the time required for sample collection and gathering and reduces the total time for determining the presence or absence of a target in the sample.
  • FIG. 19 is a flowchart of an exemplary method 500 for determining the presence or absence of a target in a sample. Method 500 uses all or a selection of features of Raman spectroscopic system 100 described above in reference to FIGS. 3-18E.
  • method 500 includes steps 510-550.
  • a sample is received in a cuvette and at least a portion of the sample to be interrogated is concentrated and/or homogenized to a central region on interior surface 313a of bottom end 312 of cuvette 310.
  • an excitation light beam is focused to the central region.
  • the excitation light beam is a coherent light beam emitted by a laser.
  • an optical signal is received from the sample to be interrogated and directed to a spectrometer.
  • the optical signal is a Raman signal.
  • the optical signal is dispersed over a preselected spectral band.
  • a spectrum of the optical signal obtained by the spectrometer is analyzed to detect the presence or absence of a target in the sample.
  • Method 500 may further include additional steps. Each of steps 510-550 of method 500 may further include additional steps or be replaced by one or more steps.
  • method 500 includes reflecting and focusing light from a bottom end of the cuvette to a focal point on or above an interior surface of the bottom end using a focusing back reflector.
  • light from the bottom end of the cuvette includes a portion of the excitation light beam that has not been absorbed by the sample to be interrogated and the optical signal from the sample to be interrogated emitted towards the focusing back reflector.
  • step 510 a sample is received in the cuvette and at least a portion of the sample to be interrogated is concentrated and/or homogenized to a central region on the interior surface of the bottom end of the cuvette.
  • step 510 further includes attracting or immobilizing the sample to be interrogated on the interior surface of the cuvette.
  • step 530 further includes directing the optical signal from the sample to be interrogated passing through the bottom end of the cuvette to the spectrometer.
  • step 530 includes dispersing the optical signal with a spectral resolution from 0.1 cm 1 to 5 cm 1 over the preselected spectral band.
  • step 530 further includes dispersing the optical signal with an average transfer efficiency from 60% to 98% for first order diffraction over the preselected spectral band.
  • step 550 further includes analyzing one or more preselected spectral bands of the optical signal.
  • the preselected spectral band for assessing or detecting a certain target is experimentally or theoretically determined based on one or more predefined Raman bands or Raman peaks corresponding to the vibration, rotation, interaction, and/or inter-connection of one or more functional groups of atoms or molecules in a pure sample of the target.
  • Table 1 lists exemplary Raman bands or Raman peaks corresponding to the vibration or rotation of exemplary functional groups of pure samples at the excitation wavelength of 532 nm. For example, if a target contains a number of functional groups, a preselected spectral band including a combination of features corresponding to the functional groups contained in the target can be selected. In certain exemplary embodiments, a method involves detecting the presence or absence of one or more features in the preselected spectral band that are indicative of the presence or absence of the target in a sample being interrogated.
  • Raman spectra are complex in nature and often contain broad Raman bands or Raman peaks due to an ensemble effect with contributions arising from all the molecules present in the sample, such as contributions arising from molecular interactions and/or bonding with neighboring molecules or atoms, the preselected spectral bands can shift from the experimentally or theoretically determined spectral bands.
  • analyzing optical signal 220 refers to analyzing the spectrum of optical signal 220 in the preselected spectral band.
  • analyzing optical signal 220 includes analyzing and/or detecting one or more features formed by one or more Raman bands or Raman peaks of the spectrum of optical signal 220.
  • analyzing one or more features of the spectrum of optical signal 220 includes comparing and determining the differences between the spectrum in one or more preselected spectral bands of optical signal 220 to a reference spectrum in the same preselected spectral bands.
  • differences of the slope, the shape, the height, the shift of location, and/or the area, of one or more Raman bands or Raman peaks between the spectrum of optical signal 220 and the reference spectrum can be identified.
  • the Raman bands or Raman peaks are measured as stokes shifts, or anti-stokes shifts, and/or a combination of stokes and anti-stokes shifts that may be used, for example, to verify the accuracy of the Raman shift.
  • a selection of these identified differences can be used as a unique fingerprint for identifying the presence of a target or for distinguishing one target from another substance, material, or molecule.
  • the reference spectrum is the Raman spectrum of an optical signal 220 received from a control sample, such as water or buffer solution containing known molecules, materials, or substances, or a biological sample whose Raman bands or Raman peaks have been previously determined, such as a biological sample containing a particular protein.
  • a control sample such as water or buffer solution containing known molecules, materials, or substances
  • a biological sample whose Raman bands or Raman peaks have been previously determined, such as a biological sample containing a particular protein.
  • analyzing the spectrum of optical signal 220 in the preselected spectral band allows the quantitation of the amount of the target in the sample to be interrogated, such as determining the concentration of the target.
  • the amount of the target can be determined based on the ratio of the height of a Raman band or Raman peak to the height of a reference Raman band or Raman peak.
  • FIG. 20 illustrates an exemplary mean Raman spectrum of a cytotoxic and invasive strain of Pseudomonas aeruginosa in water. As shown in FIG.
  • a Raman peak corresponding to a C-C bond in the molecular composition of Pseudomonas aeruginosa and a Raman peak corresponding to water are identified.
  • the ratio between the height of the Raman peak of the C-C bond and the height of the Raman peak of water can be used to determine the concentration of Pseudomonas aeruginosa in the sample based on a calibration curve previously determined.
  • the amount of the target can be determined based on the ratio of the area covered by a Raman band or Raman peak to the area covered by a reference Raman band or Raman peak.
  • the area under the Raman band or Raman peak may be determined from a Gaussian fit to the Raman band or Raman peak.
  • Raman spectroscopic system 100 is used to assess or detect the presence or absence of a bacterium or to determine the strain of a bacterium. Bacteria are typically systematically classified so that different strains can be differentiated, and similar strains can be grouped in one or more broader categories. Such differentiation and grouping may be beneficial for determining treatment for bacterial infection or for studying microbial colonies.
  • FIGS. 21 and 22 illustrate exemplary decision trees for detecting Gram-negative bacteria and Gram-positive bacteria. As illustrated by FIGS. 21 and 22, different strains of bacteria can share many similarities, making it difficult to distinguish among them.
  • each species of bacterium can have a distinct cell envelope composition, mole fraction of amino acids, virulence factors, and capsule constitutes, each species of bacterium can have a unique Raman spectral fingerprint due to the stretching and bending of molecular bonds in proteins, nucleic acids, lipids, and saccharides.
  • Gram-negative bacteria and Gram-positive bacteria can be distinguished based on various distinct molecular components of the outer cell wall.
  • the outer cell wall of Gram-positive bacteria is comprised of several layers of peptidoglycan.
  • Gram-positive cells bacteria contain teichoic acids absent in Gram-negative bacteria.
  • teichoic acids There are two types of teichoic acids in Gram-positive cells bacteria: lipoteichoic acid, which is physically connected to the plasma membrane and traverses the peptidoglycan layer, and wall teichoic acid, which is covalently bound to peptidoglycan.
  • Teichoic acids play a role in providing rigidity to the cell wall as well as in the regulation of cell growth.
  • Gram-negative bacteria have an outer membrane comprised of a complex of lipopolysaccharide (LPS), protein, and phospholipid.
  • LPS is made up of a hydrophobic lipid (lipid A), which is responsible for the toxic properties of the molecule, a hydrophilic core polysaccharide chain, and a hydrophilic O-antigenic polysaccharide side chain.
  • lipid A hydrophobic lipid
  • Other antigens that are associated with strains of Gram-negative bacteria include the K or capsule antigen and the flagella H antigen.
  • Raman spectroscopic system 100 is used to identify and analyze one or more preselected spectral bands of the optical signal received from a sample to assess or detect the presence or absence of a bacterium of a certain class, order, family, genus, species, and/or strain for the particular application.
  • FIG. 23 illustrates eighteen exemplary preselected Raman spectral bands for detecting the presence or absence of bacteria. These preselected Raman spectral bands were determined based on experimentally collected Raman spectra of the bacteria shown in FIGS. 21 and 22. One or more of these preselected Raman spectral bands include distinguishing features associated with the unique molecular components of the outer cell wall of a bacterium.
  • the eighteen exemplary preselected Raman spectral bands shown in FIG. 23 include 610-630 cm 1 , 630-650 cm 1 , 715-735 cm 1 , 950- 979 cm 1 , 990-1010 cm 1 , 1115-1135 cm 1 , 1155-1165 cm 1 , 1160-1180 cm 1 , 1200-1220 cm 1 , 1240-1260 cm 1 , 1290-1310 cm 1 , 1315-1325 cm 1 , 1330-1350 cm 1 , 1410-1430 cm 1 , 1440- 1460 cm 1 , 1570-1590 cm 1 , 1600-1620 cm 1 , and 1650-1670 cm 1 .
  • one or more of the eighteen Raman spectral bands are analyzed.
  • the preselected Raman spectral bands may include 735-874 cm 1 and/or 1013-1116 cm 1 (not shown).
  • the preselected spectral band of 610-630 cm 1 includes a Raman peak at 621 cm 1 , corresponding to vibrations associated with amino acids.
  • the preselected spectral band of 630-650 cm 1 includes a Raman peak at 643 cm 1 , corresponding to vibrations associated with proteins and amino acids.
  • the preselected spectral band of 715-735 cm 1 includes a Raman peak at 725 cm 1 , corresponding to vibrations associated with proteins and amino acids.
  • the preselected spectral band of 950-979 cm 1 includes a Raman peak at 960 cm 1 , corresponding to vibrations associated with proteins and amino acids.
  • the preselected spectral band of 990-1010 cm 1 includes a Raman peak at 1003 cm 1 , corresponding to vibrations associated with proteins.
  • the preselected spectral band of 1115-1135 cm 1 includes a Raman peak at 1126 cm 1 , corresponding to vibrations associated with lipids and carbohydrates.
  • the preselected spectral band of 1155-1165 cm 1 includes a Raman peak at 1158 cm 1 , corresponding to vibrations associated with proteins.
  • the preselected spectral band of 1160-1180 cm 1 includes a Raman peak at 1173 cm 1 , corresponding to vibrations associated with amino acids.
  • the preselected spectral band of 1200-1220 cm 1 includes a Raman peak at 1209 cm 1 , corresponding to vibrations associated with amino acids.
  • the preselected spectral band of 1240-1260 cm 1 includes a Raman peak at 1249 cm 1 , corresponding to vibrations associated with proteins (Amide III) and nucleic acids.
  • the preselected spectral band of 1290-1310 cm 1 includes a Raman peak at 1296 cm 1 , corresponding to vibrations associated with lipids.
  • the preselected spectral band of 1315- 1325 cm 1 includes a Raman peak at 1320 cm 1 , corresponding to vibrations associated with proteins and nucleic acids.
  • the preselected spectral band of 1330-1350 cm 1 includes a Raman peak at 1338 cm 1 , corresponding to vibrations associated with proteins, lipids, amino acids, and nucleic acids.
  • the preselected spectral band of 1410-1430 cm 1 includes a Raman peak at 1420 cm 1 , corresponding to vibrations associated with lipids and nucleic acids.
  • the preselected spectral band of 1440-1460 cm 1 includes a Raman peak at 1448 cm 1 , corresponding to vibrations associated with proteins and lipids.
  • the preselected spectral band of 1570-1590 cm 1 includes a Raman peak at 1578 cm 1 , corresponding to vibrations associated with amino acids.
  • the preselected spectral band of 1600-1620 cm 1 includes a Raman peak at 1606 cm 1 , corresponding to vibrations associated with amino acids.
  • the preselected spectral band of 1650-1670 cm 1 includes a Raman peak at 1657 cm 1 , corresponding to vibrations associated with proteins (Amide I) and lipids.
  • the preselected spectral band of 735-874 cm 1 includes Raman peaks corresponding to vibrations associated with nucleic acids and amino acids.
  • the preselected spectral band of 1013-1116 cm 1 includes Raman peaks corresponding to vibrations associated with protein, carbohydrates, and lipids.
  • a combination of at least two of the preselected Raman spectral bands shown in FIG. 23 are selected for spectral analysis for detecting the presence or absence of a bacterium. In some exemplary embodiments, all of these preselected Raman spectral bands are analyzed to determine the species or strain of a bacterium or to distinguish the species or strain of a bacterium from another species or strain.
  • one preselected spectral band is analyzed to determine the species of a bacterium.
  • the preselected spectral band may include a plurality of features. For example, a preselected spectral band from 600 cm 1 to 1200 cm 1 was used to detect different Gram-negative and Gram-positive species of bacteria in water.
  • Specimens of each of the bacteria species Escherichia coli , Proteus mirahilis , Klebsiella pneumoniae , Pseudomonas aeruginosa, Enterococcus faecalis, Streptococcus zooepidemicus or Streptococcus canis and Staphylococcus pseudintermedius were prepared separately from bacteria plated on tryptic soy agar plates. A single isolated colony was added to 5 ml of tryptic soy broth in a 14 ml culture tube. The culture tube was placed on a shaker in a 37°C incubator and incubated overnight for 18 hours. The overnight culture was centrifuged at room temperature for 5 minutes at 3500 rpms. The supernatant was removed and the bacteria pellet was re-suspended in 5 ml of filtered (sterilized) tap water.
  • the supernatant was removed and the bacteria pellet was re-suspended in 5 ml of filtered (sterilized) tap water. The bacteria were centrifuged and the washing process was repeated once. After the final wash, filtered tap water was added to the bacteria pellet until the OD, measured at a wavelength of 600 nm, of the solution was adjusted to the desired value of 1.
  • the preselected spectral band from 600 cm 1 to 1200 cm 1 was selected to include five Raman bands, including 600-800 cm 1 , 800-1200 cm -1 , 1200-1400 cm -1 , 1400- 1500 cm -1 , and 1500-1760 cm -1 .
  • the Raman band of 600-800 cm 1 corresponds to vibrations associated with nucleotide conformation.
  • the Raman band of 800-1200 cm -1 corresponds to contributions from nucleic acids, lipids, proteins, and C-0 stretching of carbohydrates.
  • the Raman band of 1200-1400 cm -1 corresponds to contributions from proteins, polysaccharides, lipids, and nucleic acids.
  • the Raman band of 1400-1500 cm -1 corresponds to C-H, CFE, and CEE vibrations.
  • the Raman band of 1500-1760 cm -1 corresponds to the Amine I band with contributions of water, proteins, nucleic acids, and lipids.
  • FIGS. 24-31 illustrate exemplary mean Raman spectra of different species of bacteria in water obtained by Raman spectroscopic system 100 in one exemplary embodiment.
  • system 100 included the excitation light source 110 and the spectrometer 120.
  • the spectrometer 120 included the transmission grating 126 (manufactured by Wasatch Photonics), the collimating element 124 (Zeiss Interlock 2/135), the focusing element 128, the entrance aperture 122, and the detector 130 (deep cooled back thinned CCD).
  • the excitation light source 110 was a laser having a wavelength of 532.02 nm and a power of 100 mW.
  • the transmission grating 126 had a line density of 2650 lines/mm, an operational wavelength range of 540.17 nm to 592.63 nm, a diffraction efficiency of approximately 60% to 80% with an average diffraction efficiency of approximately 65% in the operational wavelength range as more specifically shown in FIG. 4, and an angle of incidence of 48.816° at a center wavelength of 568 nm for first order diffraction.
  • the collimating element 124 and the focusing element 128 both had a focal length of 135 mm and an f-number of F/2.
  • the detector 130 was a deep cooled back thinned CCD (Horiba SyncerityTM deep cooled CCD Camera, Model 354308) having an array of 2048 x 70 pixels, a pixel size of 14 pm x 14 pm, and an image area of 28.7 mm by 0.98 mm.
  • CCD Horiba SyncerityTM deep cooled CCD Camera
  • the spectrometer 120 had a preselected spectral band of 540.17 nm to 592.63 nm, or alternatively represented as a preselected spectral band of 278.5 cm 1 to 1905.1 cm 1 based on the 532.02 nm excitation wavelength, a transfer efficiency of approximately 60% to 80% with an average transfer efficiency of approximately 65% over the preselected spectral band, a spectral resolution of 1.43 cm 1 for a center wavelength of 568 nm, a spectral resolution of 1.31 cm 1 to 1.58 cm 1 for different wavelengths of the preselected spectral band with an average spectral resolution of 1.43 cm 1 of all the wavelengths of the preselected spectral band over the 2048 pixels of the CCD array, a path length from the focusing element 128 to the detector 130 of 13.5 cm, and a width of the entrance aperture 122 of 25 pm.
  • the performance ratio of the spectrometer 120 was 4.8 %-cm 1 , which is the ratio between the 65% average transfer efficiency and the 13.5 cm path length.
  • the performance product of the spectrometer 120 was 19.31, which is a product of the 1.43 cm 1 average spectral resolution and the 13.5 cm path length of the spectrometer 120.
  • a volume of 5 ml of each sample to be interrogated was received in a cuvette 310 of Raman spectroscopic system 100 as shown in FIGS. 17A-17D (manufactured by WI Inc.).
  • the tapered wall 340 of cuvette 310 was made of Pro-fax PD702 polypropylene homopolymer and had a tilt angle of 16.2 degrees with a tolerance of 0.5 degree.
  • mean Raman spectra are obtained and analyzed to assess or detect the presence or absence of a bacterium.
  • a mean Raman spectrum refers to the average of a plurality of Raman spectra measured by a spectrometer. As shown in FIGS. 24-31, the mean Raman spectra of each species of bacteria has unique Raman features resulting from the superposition of the five Raman bands in the preselected spectral band from 600 cm 1 to 1200 cm 1 .
  • Raman spectroscopic system 100 is used to assess or detect the presence or absence of urinary crystals.
  • the existence of urinary crystals and types of urinary crystal can provide useful information for the underlying diseases of humans or animals.
  • proper identification of urinary crystals is important in determining a suitable medical treatment.
  • struvite crystals are comprised of magnesium ammonium phosphate, which is likely to appear when the urine becomes too alkaline. Struvite is a natural constituent of urine. It remains dissolved (and not precipitate) in urine for as long as the urine remains slightly acidic and not too concentrated.
  • the exemplary system 100 including the spectrometer 120 and cuvette 310, described in paragraph [0196] was used.
  • Raw Raman spectra were preprocessed through subtraction of background noise and normalization and mean Raman spectra were obtained and analyzed to assess or detect the presence or absence of a urinary crystal.
  • FIG. 32 illustrates exemplary predetermined spectral bands (gray bands) for detecting the presence or absence of magnesium ammonium phosphate and an exemplary measured Raman spectrum.
  • exemplary predetermined spectral bands for detecting the presence or absence of magnesium ammonium phosphate and an exemplary measured Raman spectrum.
  • FIG. 32 to detect the presence of magnesium ammonium phosphate, two spectral bands 544-572 cm 1 and 910-982 cm 1 , containing the Raman peaks of POb vibration at 563 cm 1 and 944 cm 1 , respectively, were selected and analyzed.
  • FIG. 33 illustrates exemplary predetermined spectral bands (gray bands) for detecting the presence or absence of calcium oxalate dihydrate and an exemplary measured Raman spectrum. As shown in FIG.
  • FIG. 34 illustrates an exemplary predetermined spectral band (gray band) for detecting the presence or absence of calcium oxalate monohydrate and an exemplary measured Raman spectrum.
  • one spectral band 1437-1481 cm 1 containing the Raman peak of COO- vibration at 1463 cm 1 was selected and analyzed. The assessment and analysis of these preselected spectral bands of the Raman spectra obtained using Raman spectroscopic system 100 allowed for the detection of the presence of calcium oxalate dihydrate and magnesium ammonium phosphate in water.
  • Raman spectroscopic system 100 is used to assess or detect the presence or absence of parasites in fecal samples , such as hookworms and roundworms. Hookworms are intestinal, blood-feeding, parasitic worms that cause types of infection known as helminthiases.
  • fecal samples were prepared: a fecal sample containing hookworm eggs, a fecal sample containing roundworm eggs, and a control sample without hookworm or roundworm.
  • the exemplary system 100 including the spectrometer 120 and cuvette 310, described in paragraph [0196] was used.
  • Each fecal sample was prepared according to the following procedure: (1) obtain and homogenize a fecal sample within a bag or a vial; (2) weigh 1 gram of the fecal sample in a wax coated cup; (3) add 15 mL of water to the cup and homogenize the fecal sample using a spatula or tongue depressor; (4) homogenize the fecal sample in the cup thoroughly to obtain a solution of the fecal sample; (5) drain the solution of the fecal sample through gauze into a new cup; (6) transfer the filtered solution into a 15 mL test tube; (7) add water to the filtered solution to make the total volume to 15 mL (or enough to balance); (8) centrifuge for 10 minutes at 1500 rpm; (9) discard the supernatant carefully not to disturb the upper layer of sediment; (10) add Sheather’s sugar solution in two steps to obtain a homogeneous solution (homogenize with a vortex or a wooden stick); (11) add Sheat
  • the samples can be prepared according to the following procedure: (1) place a clean cuvette on the a scale (e.g., an Acculab VI-200 scale), and zero the scale; (2) add unprocessed fecal sample to the cuvette using a spatula or wood stick until the appropriate mass of fecal sample has been reached; (3) pipet 5 ml of filtered tap water into the cuvette; (4) mix the fecal sample with a clean spatula or wood stick until a homogeneous slurry has been achieved; and (5) seal the cuvette for measurement.
  • a scale e.g., an Acculab VI-200 scale
  • FIG. 35 illustrates exemplary mean Raman spectra of the three different fecal samples (the fecal sample containing hookworm eggs, the fecal sample containing roundworm eggs, and the control sample without hookworm or roundworm) prepared by the 19-step procedure described above obtained using Raman spectroscopic system 100.
  • one or more Raman bands or Raman peaks contain distinguishing features that enable distinguishing the fecal sample containing hookworm from the fecal sample containing roundworm eggs; and enable distinguishing the fecal samples containing parasites from the control sample without the parasites.
  • the relative heights, slopes, locations, areas, and/or shapes of the various Raman bands or Raman peaks differ with each sample and can be used for distinguishing these three samples.
  • Exemplary Raman bands or Raman peaks include a Raman peak at 607 cm 1 , corresponding to vibrations associated with glycerol; a Raman peak at 608 cm 1 , corresponding to vibrations associated with cholesterol; a Raman band of 1540-1680 cm 1 , corresponding to vibrations associated with Amide carbonyl group and aromatic hydrogens; a Raman peak at 1602 cm 1 , corresponding to vibrations associated with phenylalanine, 5(C-C), and/or phenylalanine (protein assignment); a Raman peak at 1603 cm 1 , corresponding to vibrations associated with C-C in-plane bending mode of phenylalanine and tyrosine, and/or vibrations associated with ring C-C stretch of phenyl (1); a Raman peak at 1605 cm 1 ,
  • Raman spectroscopic system 100 is used to identify and analyze one or more preselected spectral bands of the optical signal received from a sample to assess or detect the presence or absence of a virus of a certain class, order, family, genus, species, and/or for the particular application. Influenza virus continues to be responsible for widespread respiratory disease, deaths, and significant economic loss despite worldwide vaccination and eradication programs.
  • Raman spectroscopic system 100 can be used to detect the presence or absence of influenza virus by identifying and analyzing characteristic Raman bands or Raman peaks associated with functional groups of the virus and/or the chemical structures or groups of an antiviral agent bound to the virus.
  • the antiviral inhibitor when bound to influenza virus, provides at least one unique Raman feature associated with the chemical interaction between the antiviral inhibitor and the influenza virus, which can be detected using Raman spectroscopic system 100.
  • Influenza virus can be classified into three influenza types, Influenza A, Influenza B, and Influenza C, based on the antigenic difference between their internal matrix and nucleocapsid proteins.
  • the strains of influenza A and B viruses include A/PR/8 (H1N1), A/FW/50 (H1N1, A/USSR/77 (H1N1), A/WSN/33 (H1N1), A/Udorn/72 (H3N2), A/Udom/72 1A spherical variant (H3N2), A/Udorn/72 10A filamentous variant (H3N2), A/Memphis/96 (H3N2), A/Arizona/94 (H3N2, A/Chick/California/2000 (H6N2), B/Beijing/96, and H5N1.
  • the Raman spectrum includes characteristic Raman bands or Raman peaks (as indicated by arrows in the figure) associated with the pleated sheet structure amide I group, distinct carbon-carbon, nucleic acids, and other amide groups. At least one feature of at least one of these Raman bands or Raman peaks is indicative of the presence or absence of A/PR/8 (HIM) serotype influenza virus.
  • Raman spectroscopic system 100 can be used to assess or detect the presence or absence of various types or strains of viruses besides influenza virus, such as human parainfluenza virus types 1, 2 and 3, respiratory syncytial virus (RSV), Adenovirus, or vesicular stomatitis virus (VSV).
  • RSV respiratory syncytial virus
  • VSV vesicular stomatitis virus
  • Raman spectroscopic system 100 can be used to assess or detect the presence or absence of pseudo type viruses, such as liposomes or virosomes injected with viral proteins or viral nucleic acids.
  • Raman spectroscopic system 100 can be used to assess or detect the presence or absence of coronavirus, such as severe acute respiratory syndrome coronavirus (SARS-CoV), Middle East respiratory syndrome coronavirus (MERS-CoV), and severe acute respiratory syndrome coronavirus 2 (SARS- CoV-2).
  • coronavirus such as severe acute respiratory syndrome coronavirus (SARS-CoV), Middle East respiratory syndrome coronavirus (MERS-CoV), and severe acute respiratory syndrome coronavirus 2 (SARS- CoV-2).
  • SARS-CoV severe acute respiratory syndrome coronavirus
  • MERS-CoV Middle East respiratory syndrome coronavirus
  • SARS- CoV-2 severe acute respiratory syndrome coronavirus 2
  • Raman spectroscopic system 100 allows for real- time detection and/or identification of coronavirus at the point-of-care.
  • Raman spectroscopic system 100 may detect and/or identify the presence or absence of one or more types of coronavirus in 3 to 5 minutes.
  • Coronaviruses are a family of RNA viruses that typically cause mild respiratory disease in humans.
  • SARS-CoV severe acute respiratory disease coronavirus
  • novel coronavirus (SARS-CoV-2) was identified in Wuhan, China.
  • SARS-CoV-2 may be asymptomatic or develop flu-like symptoms, including fever, cough, fatigue, and shortness of breath. In some of those infected, the disease may progress to pneumonia, acute respiratory distress syndrome, multi-organ failure, and death.
  • methods and devices according to the present invention can be implemented in conjunction with the following additional methods and/or devices for rapid, sensitive, and high-throughput assessment or detection of the presence or absence of SARS-CoV-2 in a sample.
  • FIG. 37 illustrates an exemplary structure of SARS-CoV-2.
  • SARS-CoV-2 has five proteins: Spike (S), Envelop (E), Membrane (M), Hemagglutinin-esterase dimer (HE), and Nucleocapsid (N).
  • S Spike
  • E Envelop
  • M Membrane
  • HE Hemagglutinin-esterase dimer
  • N Nucleocapsid
  • S and M proteins are more abundant compared to others making them the ideal candidates as antibody targets for detection.
  • anti-S and anti-E antibodies may be used to capture SARS-CoV-2 particles in a sample for detection.
  • a sample swab is directly placed in a cuvette for interrogation using Raman spectroscopic system 100.
  • the cuvette may be similar to the cuvette 310 as described above with reference to FIGS. 7-17G.
  • a portion of the swab, such as the tip of the swab may be separated and placed into the cuvette.
  • the tip of a swab may be submerged in an elution solution in the cuvette for a period of time, after which the swab is removed.
  • the solution in the cuvette 310 is then interrogated using Raman spectroscopic system 100.
  • an exemplary sample collection cartridge is used for preparing a sample for interrogation of a sample that may contain, for example, SARS-CoV-2.
  • the cartridge may be similar to the exemplary sample collection cartridge 400 as described above with reference to FIGS. 18A-18D.
  • a sample swab may be inserted into chamber 420 for receiving a swab.
  • a blister 410 contains an elution solution.
  • the elution solution can be released from the blister manually or automatically, thereby releasing one or more viruses (or other sample) from the swab into a container.
  • the container is a funnel.
  • the container is a cuvette, such as cuvette 310. The elution solution containing the released viruses is then collected in the container and directly interrogated using Raman spectroscopic system 100.
  • capture-activated fluorescent antibodies are used to detect, for example, SARS-CoV-2.
  • a capture- activated fluorescent antibody may target, for example, a selected protein on the surface of SARS-CoV-2 and undergoes a conformational change or distortion upon binding. Such conformational change or distortion causes a fluorophore tagged to a capture-activated fluorescent antibody to emit fluorescence signal upon excitation. The fluorescent signal is then detected using Raman spectroscopic system 100.
  • a solution containing capture-activated fluorescent antibodies are placed in a cuvette.
  • a sample swab is submerged in the solution for a period of time for SARS-CoV-2 to bind to the capture-activated fluorescent antibodies.
  • the sample in the cuvette may be agitated to expedite binding and then interrogated using Raman spectroscopic system 100.
  • a cartridge such as cartridge 400, is used.
  • the swab sample is received in the swab chamber of the cartridge.
  • the elution solution in the blister of the cartridge includes the capture-activated fluorescent antibodies.
  • the elution solution is released from the blister manually or automatically, thereby washing and releasing one or more viruses in the swab sample into a container.
  • the capture-activated fluorescent antibodies may bind with the released viruses in the container.
  • the container is a cuvette, such as cuvette 310.
  • the container may be agitated to expedite binding.
  • the elution solution containing the capture-activated fluorescent antibodies and the released viruses is then interrogated using Raman spectroscopic system 100
  • functionalized magnetic beads are used to capture and detect an analyte, such as SARS-CoV-2.
  • an analyte such as SARS-CoV-2.
  • the surfaces of these magnetic beads are coated with protein A, protein G, or a combination of protein A and protein G.
  • the protein A and/or protein G of the magnetic beads are then bound to the Fc region of anti-S or anti-E antibodies, whose Fab regions can further bind to SARS-CoV-2.
  • An exemplary functionalized magnetic bead 600 is shown in FIG. 39.
  • a magnetic field generating device 800 is used.
  • functionalized magnetic microbeads modified with selective antibodies are added to a container, such as a funnel or cuvette 310, and trapped in the container by the magnetic field 812 generated by a magnetic ring structure 810 of the magnetic field generating device 800.
  • the magnetic ring structure 810 may include alternating magnets 814 and magnet return 816.
  • the magnetic ring structure 810 may be subjected to low RPM rotation to generate the magnetic field.
  • the magnetic ring structure 810 may be rotated at a frequency ranging from 1 Hz to 60 Hz.
  • a blister 410 of the cartridge 400 contains an elution solution.
  • the elution solution can be released from the blister manually or automatically, thereby washing and releasing one or more viruses in the swab into a container, such as a funnel.
  • the functionalized magnetic beads remain trapped by the magnetic field in the container and capture and trap the released viruses.
  • the magnetic field generated by the magnetic ring structure 810 is configured to agitate or mix the sample in the container to accelerate binding of the functionalized magnetic beads to the released viruses.
  • a wash cycle (or a purification step) is performed where a wash solution is flushed through the container to remove waste out the bottom of the container while the functionalized magnetic beads remain trapped in the container. After the wash, the cartridge 400 is then removed from magnetic ring structure 810.
  • the container is a funnel.
  • the container is cuvette 310. The sample remaining in the cuvette is interrogated using Raman spectroscopic system 100. In some other exemplary embodiments, the sample in the container is expelled and collected into a cuvette 310 for interrogation using Raman spectroscopic system 100.
  • protein A, protein, G, anti-S antibody, and/or anti-E antibody can have characteristic Raman bands or Raman peaks distinguishing from that of the lipid outer layer of the virus.
  • a direct detection of protein A, protein, G, anti-S antibody, and/or anti-E antibody bound to SARS-CoV-2 using Raman spectroscopic system 100 allows for detecting trapped or purified SARS-CoV-2, and may provide a sensitive test for SARS- CoV-2.
  • fluorophore tagged antibodies 700 are used for detecting the viruses captured and trapped by the functionalized magnetic beads.
  • a solution containing fluorophore tagged anti-S antibodies are added to the sample in the container of cartridge 400.
  • a portion of the fluorophore tagged anti-S antibodies may bind to the viruses that have been captured by the functionalized magnetic beads.
  • FIG. 40 shows a virus captured by a functionalized magnetic bead 600 and bound to an anti-S antibody (AF-antiS) 700.
  • Another wash cycle is performed where a wash solution is flushed through the container to remove unbound fluorophore tagged anti-S antibodies.
  • the container is a cuvette.
  • the sample remaining in the cuvette is interrogated using Raman spectroscopic system 100.
  • the sample in the container is expelled and collected into a cuvette for interrogation using Raman spectroscopic system 100.
  • fluorophores are excited using a 532 nm excitation laser and the emitted fluorescence signal is detected by Raman spectroscopic system 100.
  • the use of fluorescence detection may provide for sensitive and pre-symptom ultra-low level of detection.
  • the fluorescence signal is detected to test or detect the presence or absence of viruses, such as coronavirus.
  • both the Raman signal and fluorescence signal are detected for sensitive testing or detecting the presence or absence of viruses.
  • diffracted and/or scattered light is emitted from the functionalized magnetic beads and/or viral particles irradiated by the excitation light source 110 of Raman spectroscopic system 100.
  • a diffracted light signal, a scattered light signal, or a combination of diffracted and/or scattered light signals is detected by Raman spectroscopic system 100.
  • both the Raman signal and the diffracted and/or scattered light signals are detected for sensitive testing or detecting the presence or absence of viruses.
  • the Raman signal, the diffracted and/or scattered light signals, and the fluorescence signal are detected for sensitive testing or detecting the presence or absence of viruses.
  • contrived clinical nasal swabs were prepared, some which are virus-positive nasal swabs containing SARS-CoV-2, and some of which are virus negative nasal swabs.
  • Each contrived virus-positive nasal swab was prepared according to the following procedure: (1) obtaining a viral stock aliquot or a diluted virus samples from storage; if the viral stock aliquot is frozen, allow the virus stock to thaw slowly at 4°C; (2) dilute the viral stock or diluted virus sample in microtubes to achieve intended concentration with phosphate buffered saline (PBS); (3) transfer 100 uL of desired concentration of the virus sample to the bottom of a cuvette already containing scrape secretion of a virus negative nasal swab; (4) pipette the sample in the cuvette to mix nasal secretion gently but thoroughly with the virus dilution in PBS.
  • PBS phosphate buffered saline
  • Each contrived virus-negative nasal swab was prepared without the above steps 1-2 and uses 100 uL of PBS solution in step 3. Samples from both the virus-positive nasal swabs and virus-negative nasal swabs were eluted and collected into a cuvette for interrogation using Raman spectroscopic system 100.
  • Table 3 below shows the results of the detection of SARS-CoV-2 in the contrived clinical nasal swabs using both Raman spectroscopic system 100 and polymerase chain reaction (PCR) test.
  • the PCR was performed as the “gold standard” for detecting SARS-CoV-2 after each sample in the cuvette was interrogated using Raman spectroscopic system 100.
  • both Raman signals and diffracted and/or scattered light signals were detected and analyzed for detecting the presence or absence of SARS-CoV-2 using Raman spectroscopic system 100.
  • Raman spectroscopic system 100 has a positive percent agreement (PPA) of 97.1% and a negative percent agreement (NPA) of 97.8% with the PCR test.
  • Table 3 Exemplary results of the detection of SARS-CoV-2 using Raman spectroscopic system 100.
  • Renishaw inVia Reflex Raman Microscope is used to demonstrate the identification and analysis of one or more preselected spectral bands of the optical signal received from a sample to distinguish different strains of viruses.
  • FIG. 42 illustrates three exemplary mean Raman spectra of three samples containing different strains of influenza viruses, including a sample containing A/PR/8 (H1N1), a sample containing A/WSN/33 (H1N1), and a sample containing A/Udom/72 (H3N2). These samples were prepared from purified influenza viruses in phosphate buffer solution. An excitation wavelength of 514.5 nm was used. As shown in FIG.
  • a number of Raman bands or Raman peaks associated with molecular functional groups in the viruses are distinct for these three different samples, and can be used for distinguishing the different strains of viruses in these samples.
  • the relative heights, slopes, areas, shapes, and/or locations of the various Raman bands or Raman peaks differ with each virus strain, which can be used for distinguishing the various strains of viruses from one another.
  • Exemplary Raman bands or Raman peaks for distinguishing virus strains are summarized in Table 4 below.
  • Renishaw inVia Reflex Raman Microscope is used to demonstrate the identification and analysis of one or more preselected spectral bands of the optical signal received from a sample to distinguish viruses from bacteria.
  • FIG. 43 illustrates five exemplary mean Raman spectra of five samples containing four different strains of influenza viruses or bacteria, including a sample containing A/PR/8 (H1N1), a sample containing WSN (H1N1), a sample containing Udorn (H3N2), a sample containing PR8, and a sample containing Methicillin-resistant Staphylococcus aureus (MRSA 2R).
  • the samples containing viruses were prepared from purified viruses in phosphate buffer solution.
  • the sample containing bacteria was prepared according to the method described above in paragraphs [0193] and [0194] An excitation wavelength of 514.5 nm was used.
  • a number of Raman bands or Raman peaks associated with molecular functional groups in the viruses and bacteria are distinct for these five different samples, and can be used for distinguishing the different strains of viruses and for distinguishing each virus strain from the bacteria in these samples.
  • the relative heights, slopes, areas, shapes, and/or locations of the various Raman bands or Raman peaks differ with each virus strain and differ between the virus strains from the bacteria, which can be used for distinguishing the various strains of viruses from one another and for distinguishing the virus strains from the bacteria.
  • Exemplary Raman bands or Raman peaks for distinguishing virus strains and for distinguishing the virus strains from the bacteria are summarized in Table 5 below.
  • Renishaw inVia Reflex Raman Microscope is used to demonstrate the identification and analysis of one or more preselected spectral bands of the optical signal received from a sample to distinguish viruses inactivated by different inactivation methods.
  • FIG. 44 illustrates three exemplary mean Raman spectra of three dried samples containing A/PR/8 (H1N1) inactivated respectively by three distinct methods: UV, heat, and chemical deactivation. An excitation wavelength of 785 nm was used. As shown in FIG. 44, a number of Raman bands or Raman peaks associated with molecular functional groups in A/PR/8 are different for these three different samples.
  • the relative heights, slopes, locations, areas, and/or shapes of the various Raman bands or Raman peaks differ with each sample and can be used for distinguishing these three samples. Exemplary differences are included in the shift of the Raman peak from 1040 to 1080 cm 1 and in the shift of the Raman peak located near 1340 cm 1 . These results indicate that very minor changes, even in the same strain of virus, can be identified by some exemplary embodiments of the present disclosure. Such capability of some exemplary embodiments of the present disclosure may further allow the detection and identification of pseudo type viruses or engineered viruses. Furthermore, such capability of some exemplary embodiments of the present disclosure may be valuable in assessing the virulence and effect of treatment for influenza in the clinical setting.
  • Raman spectroscopic system 100 is used to identify and analyze one or more preselected spectral bands of the optical signal received from a sample to detect target cells, such as white blood cells.
  • target cells such as white blood cells.
  • white blood cells There are five different types of white blood cells: neutrophils (45%-73% normal), monocytes (2%-8% normal), lymphocytes (20%-40% normal), eosinophils (0-4% normal), and basophils (0-1% normal).
  • the concentration of white blood cells in the blood typically ranges from 3.4 x 10 3 -10 x 10 3 cells/mm 3 .
  • An increase or decrease from the normal ranges of white blood cell concentration in the blood may be due to infection, disease, and drugs.
  • an abnormal increase of neutrophils in the blood can be due to a bacterial infection
  • an abnormal increase of eosinophils in the blood can be due to a parasitic infection and a hypersensitivity reaction (drug/allergy)
  • an abnormal increase of basophils in the blood can be due to chronic inflammation and leukemia
  • an abnormal increase of lymphocytes in the blood can be due to mononucleosis, tuberculosis, syphilis, and viral infection
  • an abnormal decrease of lymphocytes in the blood can be due to due to HIV infection, radiation, and steroids
  • an abnormal increase of monocytes in the blood can occur during recovery from bacterial infection, leukemia, or a disseminated tuberculosis infection.
  • an abnormal amount of white blood cells in urine may be due to infection, inflammation, disease, allergies, or drugs.
  • a concentration of white blood cells in urine greater than 10 cells/mm 3 or 10,000 cells/ml can be indicative of a bacteria urinary tract infection (UTI).
  • a concentration of neutrophils in urine higher than a threshold can be due to UTI and/or pyelonephritis
  • a concentration of eosinophils in urine higher than a threshold can be due to acute interstitial nephritis caused by an allergic reaction, typically to drugs
  • a concentration of lymphocytes in urine higher than a threshold can be due to an inflammation, usually a chronic condition like bladder stones or bladder cancer, or viral diseases
  • a concentration of monocytes in urine higher than a threshold can be due to viral infection.
  • An abnormal increase of neutrophils in the blood can be due to viral, bacterial, or fungal infections or stress.
  • An abnormal increase of eosinophils in the blood can be due to parasitic infection and allergic reactions.
  • An abnormal increase of basophils in the blood may indicate bone marrow problems, and when found with an increase of eosinophils may indicate allergies.
  • An abnormal increase of lymphocytes in the blood can be due to autoimmune diseases, such as colitis.
  • An abnormal increase of monocytes in the blood may indicate Leukemia or other types of cancer.
  • Raman spectroscopic system 100 is used to detect the presence of neutrophils or the presence of neutrophils and bacteria in the same sample.
  • Neutrophils are the most common white blood cells observed in urine and are the inflammatory cells seen in cystitis.
  • a fresh human peripheral blood neutrophil sample containing 120 million cells in 15 ml of media from Human Cells Biosciences were used. The fresh human peripheral blood neutrophil sample was washed at 1200 rpm for 5 minutes twice to remove the media and diluted to 1 million/mL in PBS buffer at pH 7.2.
  • a sample containing neutrophils was prepared by spiking water with the diluted fresh human peripheral blood neutrophil sample such that the concentration of neutrophils in the sample was 100,000 cells/ml.
  • the exemplary system 100 including the spectrometer 120 and cuvette 310, described in paragraph [0196] was used. A volume of 5 ml of the sample containing neutrophils was then placed in the cuvette of Raman spectroscopic system 100 for measurement.
  • FIG. 45 illustrates three exemplary mean Raman spectra of water, a sample containing neutrophils, and a sample containing neutrophils and Escherichia coli.
  • FIG. 46 illustrates three exemplary mean Raman spectra of water, a sample containing neutrophils, and a sample containing neutrophils and Leptospira interrogans.
  • FIG. 47 illustrates two exemplary mean Raman spectra of a sample containing neutrophils and Escherichia coli and a sample containing neutrophils and Leptospira interrogans.
  • a number of Raman bands or Raman peaks associated with molecular functional groups in the neutrophils and bacteria are distinct for these three different samples, and can be used for distinguishing the three different samples and detecting the presence of only neutrophils and the presence of both neutrophils and bacteria in these samples.
  • the relative heights, shapes, areas, slopes, and/or location of the various Raman bands or Raman peaks differ with each sample and can be used for distinguishing these samples.
  • a number of Raman bands or Raman peaks associated with molecular functional groups in bacteria are distinct for Escherichia coli and Leptospira interrogans , and can be used to determine what type of bacteria is in the sample. Such capability is useful for determining the type of bacterial infection and the suitable treatment when the presence of neutrophils has been detected.
  • any one or more exemplary Raman bands or Raman peaks that can be used in exemplary embodiments of detecting the presence or absence of neutrophils and/or bacteria include a Raman peak at 416.42 cm 1 , corresponding to vibrations associated with fatty acid; a Raman peak at 431.60 cm 1 , corresponding to vibrations associated with carboxylic acid; a Raman peak at 1105.05 cm 1 , corresponding to vibrations associated with carbohydrate or lipid; a Raman peak at 437.28 cm 1 , corresponding to vibrations associated with saccharide; a Raman peak at 408.82 cm 1 , corresponding to vibrations associated with saccharide; a Raman peak at 435.38 cm 1 , corresponding to vibrations associated with carboxylic acid; a Raman peak at 435.38 cm 1 , corresponding to vibrations associated with carboxylic acid; a Raman peak at 427.81 cm 1 , corresponding to vibrations associated with saccharide; a Raman peak at 433.49 cm 1 ,
  • the excitation light source 110 of Raman spectroscopic system 100 could be a laser having a wavelength of 532.02 nm.
  • the transmission grating 126 of Raman spectroscopic system 100 could have a line density of 2650 lines/mm, an operational wavelength range of 540.173 nm to 592.629 nm, a diffraction efficiency of approximately 60% to 80% with an average diffraction efficiency of approximately 65% in the operational wavelength range, and an angle of incidence of 48.816 degrees at a center wavelength of 568 nm for first order diffraction.
  • the collimating element 124 and the focusing element 128 could have a focal length of 135 mm and an f-number in the range of F/1.2 to F/4.
  • the detector 130 could be a deep cooled back thinned CCD having an array of 2048 x 70 pixels, a pixel size of 14 pm x 14 pm, and an image area of 28.7 mm by 0.98 mm.
  • spectrometer 120 of Raman spectroscopic system 100 could have a preselected spectral band of 540.173 nm to 592.629 nm, or alternatively represented as a preselected spectral band of 283.69 cm 1 to 1922.33 cm 1 based on the excitation wavelength of 532.02 nm, an average transfer efficiency of 65% over the preselected spectral band, a spectral resolution of 1.43 cm 1 at the center wavelength of 568 nm, a spectral resolution from 1.31 cm 1 to 1.58 cm 1 for different wavelengths of the preselected spectral band with an average spectral resolution of 1.43 cm 1 of all the wavelengths of the preselected spectral band over the 2048 pixels of the CCD array, a path length from the focusing element 128 to the detector 130 of 13.5 cm, and a width of the entrance aperture 122 of 25 pm.
  • the performance ratio of the spectrometer 120 could be 4.8 %-cm 1 , which is the ratio between the 65% average transfer efficiency and the 13.5 cm path length of the spectrometer 120.
  • the performance product of the spectrometer 120 could be 26, which is the product of the 2 cm 1 average spectral resolution and the 13.5 cm path length of the spectrometer 120.
  • the excitation light source 110 of Raman spectroscopic system 100 could be a laser having a wavelength of 488 nm.
  • the transmission grating 126 of Raman spectroscopic system 100 could have a line density of 2650 lines/mm, an operational wavelength range of 540.173 nm to 592.629 nm, a diffraction efficiency of approximately 60% to 80% with an average diffraction efficiency of approximately 65% in the operational wavelength range, and an angle of incidence of 48.816 degrees at a center wavelength of 568 nm for first order diffraction.
  • the collimating element 124 and the focusing element 128 could have a focal length of 135 mm and an f-number in the range from F/1.2 to F/4.
  • the detector 130 could be a deep cooled back thinned CCD having an array of 2048 x 70 pixels, a pixel size of 14 pm x 14 pm, and an image area of 28.7 mm by 0.98 mm.
  • spectrometer 120 of Raman spectroscopic system 100 could have a preselected spectral band of 540.173 nm to 592.629 nm, or alternatively represented as a preselected spectral band of 1979.2 cm 1 to 3617.8 cm 1 based on the excitation wavelength of 488 nm, an average transfer efficiency of 65% over the preselected spectral band, a spectral resolution of 1.43 cm 1 at the center wavelength of 568 nm, a spectral resolution from 1.31 cm 1 to 1.58 cm 1 for different wavelengths of the preselected spectral band with an average spectral resolution of 1.43 cm 1 of all the wavelengths of the preselected spectral band over the 2048 pixels of the CCD array, a path length from the focusing element 128 to the detector 130 of 13.5 cm, and a width of the entrance aperture 122 of 25 pm.
  • the performance ratio of the spectrometer 120 could be 4.8 %-cm 1 , which is the ratio between the 65% average transfer efficiency and the 13.5 cm path length of the spectrometer 120.
  • the performance product of the spectrometer 120 could be 19.31, which is the product of the 1.43 cm 1 average spectral resolution and the 13.5 cm path length of the spectrometer 120.
  • Raman spectroscopic system 100 could have the 488 nm laser and the 532.02 nm laser as two separate excitation light sources to allow the spectrometer 120 to acquire spectra in both the preselected spectral band of 1979.2 cm 1 to 3617.8 cm 1 and the preselected spectral band of 283.69 cm 1 to 1922.33 cm 1 .
  • the sample is excited sequentially at the excitation wavelength of 488 nm and the excitation wavelength of 532.02 nm separately (or vice versa).
  • the spectra acquired in each of the preselected spectral bands are combined into a single spectrum spanning 283.69 cm 1 to 1922.33 cm 1 and 1979.2 cm 1 to 3617.8 cm 1 .
  • the Raman spectroscopic system 100 can have other combinations of multiple excitation light sources having different excitation wavelengths.
  • the multiple excitation light sources can be used to sequentially excite a sample to acquire spectra in multiple preselected spectral bands, which can be combined into a single spectrum.
  • the excitation light source 110 of Raman spectroscopic system 100 could be a laser having a wavelength of 514.5 nm.
  • the transmission grating 126 of Raman spectroscopic system 100 could have a line density of 2747 lines/mm, an operational wavelength range of 521.2 nm to 571.8 nm, a diffraction efficiency of approximately 60% to 80% with an average diffraction efficiency of approximately 65% in the operational wavelength range, and an angle of incidence of 48.823 degrees at a center wavelength of 548 nm for first order diffraction.
  • the collimating element 124 and the focusing element 128 could have a focal length of 135 mm and an f-number in the range of F/1.2 to F/4.
  • the detector 130 could be a deep cooled back thinned CCD having an array of 2048 x 70 pixels, a pixel size of 14 pm x 14 pm, and an image area of 28.7 mm by 0.98 mm.
  • spectrometer 120 of Raman spectroscopic system 100 could have a preselected spectral band of 521.2 nm to 571.8 nm, or alternatively represented as a preselected spectral band of 248.32 cm 1 to 1946.4 cm 1 based on the excitation wavelength of 514.5 nm, an average transfer efficiency of 65% over the preselected spectral band, a spectral resolution of 1.48 cm 1 at the center wavelength of 548 nm, a spectral resolution from 1.36 cm 1 to 1.63 cm 1 for different wavelengths of the preselected spectral band with an average spectral resolution of 1.48 cm 1 of all the wavelengths of the preselected spectral band over the 2048 pixels of the CCD array, a path length from the focusing element 128 to the detector 130 of 13.5 cm, and a width of the entrance aperture 122 of 25 pm.
  • the performance ratio of the spectrometer 120 could be 4.8 %-cm 1 , which is the ratio between the 65% average transfer efficiency and the 13.5 path length of the spectrometer 120.
  • the performance product of the spectrometer 120 could be 19.98, which is the product of the 1.48 cm 1 average spectral resolution and the 13.5 cm path length of the spectrometer 120.
  • the excitation light source 110 of Raman spectroscopic system 100 could be a laser having a wavelength of 473 nm.
  • the transmission grating 126 of Raman spectroscopic system 100 could have a line density of 2747 lines/mm, an operational wavelength range of 521.2 nm to 571.8 nm, a diffraction efficiency of approximately 60% to 80% with an average diffraction efficiency of approximately 65% in the operational wavelength range, and an angle of incidence of 48.823 degrees at a center wavelength of 548 nm for first order diffraction.
  • the collimating element 124 and the focusing element 128 could have a focal length of 135 mm and an f-number in the range of F/1.2 to F/4.
  • the detector 130 could be a deep cooled back thinned CCD having an array of 2048 x 70 pixels, a pixel size of 14 pm x 14 pm, and an image area of 28.7 mm by 0.98 mm.
  • spectrometer 120 of Raman spectroscopic system 100 could have a preselected spectral band of 521.2 nm to 571.8 nm, or alternatively represented as a preselected spectral band of 1953.6 cm 1 to 3651.7 cm 1 based on the excitation wavelength of 473 nm, an average transfer efficiency of 65% over the preselected spectral band, a spectral resolution of 1.48 cm 1 at the center wavelength of 548 nm, a spectral resolution from 1.36 cm 1 to 1.63 cm 1 for different wavelengths of the preselected spectral band with an average spectral resolution of 1.48 cm 1 of all the wavelengths of the preselected spectral band over the 2048 pixels of the CCD array, a path length from the focusing element 128 to the detector 130 of 13.5 cm, and a width of the entrance aperture 122 of 25 pm.
  • the performance ratio of the spectrometer 120 could be 4.8 %-cm 1 , which is the ratio between the 65% average transfer efficiency and the 13.5 path length of the spectrometer 120.
  • the performance product of the spectrometer 120 could be 19.98, which is the product of the 1.48 cm 1 average spectral resolution and the 13.5 cm path length of the spectrometer 120.
  • Raman spectroscopic system 100 could have the 514.5 nm laser and the 473 nm laser as two separate excitation light sources 110 to allow the spectrometer 120 to acquire spectra in both the preselected spectral band of 248.32 cm 1 to 1946.4 cm 1 and the preselected spectral band of 1953.6 cm _1 to 3651.7 cm 1 .
  • the excitation light source 110 of Raman spectroscopic system 100 could be a laser having a wavelength of 638 nm.
  • the transmission grating 126 of Raman spectroscopic system 100 could have a line density of 2178 lines/mm, an operational wavelength range of 657.1 nm to 720.97 nm, a diffraction efficiency of approximately 60% to 80% with an average diffraction efficiency of approximately 65% in the operational wavelength range, and an angle of incidence of 48.807 degrees at a center wavelength of 691 nm for first order diffraction.
  • the collimating element 124 and the focusing element 128 could have a focal length of 135 mm and an f-number in the range of F/1.2 to F/4.
  • the detector 130 could be a deep cooled back thinned CCD having an array of 2048 x 70 pixels, a pixel size of 14 pm x 14 pm, and an image area of 28.7 mm by 0.98 mm.
  • spectrometer 120 of Raman spectroscopic system 100 could have a preselected spectral band of 657.1 nm to 720.97 nm, or alternatively represented as a preselected spectral band of 456.4 cm 1 to 1803.8 cm 1 based on the excitation wavelength of 638 nm, an average transfer efficiency of 65% over the preselected spectral band, a spectral resolution of 1.17 cm 1 at the center wavelength of 691 nm, a spectral resolution from 1.08 cm 1 to 1.30 cm 1 for different wavelengths of the preselected spectral band with an average spectral resolution of 1.18 cm 1 of all the wavelengths of the preselected spectral band over the 2048 pixels of the CCD array, a path length from the focusing element 128 to the detector 130 of 13.5 cm, and a width of the entrance aperture 122 of 25 pm.
  • the performance ratio of the spectrometer 120 could be 4.8 %-cm 1 , which is the ratio between the 65% average transfer efficiency and the 13.5 cm path length of the spectrometer 120.
  • the performance product of the spectrometer 120 could be 15.93, which is the product of the 2 cm 1 average spectral resolution and the 13.5 cm path length of the spectrometer 120.
  • the excitation light source 110 of Raman spectroscopic system 100 could be a laser having a wavelength of 589 nm.
  • the transmission grating 126 of Raman spectroscopic system 100 could have a line density of 2178 lines/mm, an operational wavelength range of 657.1 nm to 720.97 nm, a diffraction efficiency of approximately 60% to 80% with an average diffraction efficiency of approximately 65% in the operational wavelength range, and an angle of incidence of 48.807 degrees at a center wavelength of 691 nm for first order diffraction.
  • the collimating element 124 and the focusing element 128 could have a focal length of 135 mm and an f-number in the range of F/1.2 to F/4.
  • the detector 130 could be a deep cooled back thinned CCD having an array of 2048 x 70 pixels, a pixel size of 14 pm x 14 pm, and an image area of 28.7 mm by 0.98 mm.
  • spectrometer 120 of Raman spectroscopic system 100 could have a preselected spectral band of 657.1 nm to 720.97 nm, or alternatively represented as a preselected spectral band of 1760.4 cm 1 to 3107.8 cm 1 based on the excitation wavelength of 638 nm, an average transfer efficiency of 65% over the preselected spectral band, a spectral resolution of 1.17 cm 1 at the center wavelength of 691 nm, a spectral resolution from 1.08 cm 1 to 1.30 cm 1 for different wavelengths of the preselected spectral band with an average spectral resolution of 1.18 cm 1 of all the wavelengths of the preselected spectral band over the 2048 pixels of the CCD array, a path length from the focusing element 128 to the detector 130 of 13.5 cm, and a width of the entrance aperture 122 of 25 pm.
  • the performance ratio of the spectrometer 120 could be 4.8 %-cm 1 , which is the ratio between the 65% average transfer efficiency and the 13.5 cm path length of the spectrometer 120.
  • the performance product of the spectrometer 120 could be 15.93, which is the product of the 1.18 cm 1 average spectral resolution and the 13.5 cm path length of the spectrometer 120.
  • Raman spectroscopic system 100 could have the 638 nm laser and the 589 nm laser as two separate excitation light sources 110 to allow the spectrometer 120 to acquire spectra in both the preselected spectral band of 456.4 cm 1 to 1803.8 cm 1 and the preselected spectral band of 1760.4 cm 1 to 3107.8 cm 1 .
  • the excitation light source 110 of Raman spectroscopic system 100 could be a laser having a wavelength of 514.5 nm.
  • the transmission grating 126 of Raman spectroscopic system 100 could have a line density of 2704 lines/mm, an operational wavelength range of 520.23 nm to 572.67 nm, a diffraction efficiency of approximately 60% to 80% with an average diffraction efficiency of approximately 65% in the operational wavelength range, and an angle of incidence of 47.8 degrees at a center wavelength of 548 nm for first order diffraction.
  • the collimating element 124 and the focusing element 128 could have a focal length of 135 mm and an f-number in the range of F/1.2 to F/4.
  • the detector 130 could be a deep cooled back thinned CCD having an array of 2048 x 70 pixels, a pixel size of 14 pm x 14 pm, and an image area of 28.7 mm by 0.98 mm.
  • spectrometer 120 of Raman spectroscopic system 100 could have a preselected spectral band of 520.23 nm to 572.67 nm, or alternatively represented as a preselected spectral band of 214.4 cm 1 to 1974.4 cm 1 based on the excitation wavelength of 514.5 nm, an average transfer efficiency of 65% over the preselected spectral band, a spectral resolution of 1.53 cm 1 at the center wavelength of 548 nm, a spectral resolution from 1.40 cm 1 to 1.70 cm 1 for different wavelengths of the preselected spectral band with an average spectral resolution of 1.54 cm 1 of all the wavelengths of the preselected spectral band over the 2048 pixels of the CCD array, a path length from the focusing element 128 to the detector 130 of 13.5 cm, and a width of the entrance aperture 122 of 25 pm.
  • the performance ratio of the spectrometer 120 could be 4.8 %-cm 1 , which is the ratio between the 65% average transfer efficiency and the 13.5 cm path length of the spectrometer 120.
  • the performance product of the spectrometer 120 could be 20.79, which is the product of the 1.54 cm 1 average spectral resolution and the 13.5 cm path length of the spectrometer 120.
  • the excitation light source 110 of Raman spectroscopic system 100 could be a laser having a wavelength of 488 nm.
  • the transmission grating 126 of Raman spectroscopic system 100 could have a line density of 2786 lines/mm, an operational wavelength range of 490.3 nm to 542.8 nm, a diffraction efficiency of approximately 60% to 80% with an average diffraction efficiency of approximately 65% in the operational wavelength range, and an angle of incidence of 46.185 degrees at a center wavelength of 518 nm for first order diffraction.
  • the collimating element 124 and the focusing element 128 could have a focal length of 135 mm and an f-number in the range of F/1.2 to F/4.
  • the detector 130 could be a deep cooled back thinned CCD having an array of 2048 x 70 pixels, a pixel size of 14 pm x 14 pm, and an image area of 28.7 mm by 0.98 mm.
  • spectrometer 120 of Raman spectroscopic system 100 could have a preselected spectral band of 490.3 nm to 542.8 nm, or alternatively represented as a preselected spectral band of 96.5 cm 1 to 2067.8 cm 1 based on the excitation wavelength of 488 nm, an average transfer efficiency of 65% over the preselected spectral band, a spectral resolution of 1.715 cm 1 at the center wavelength of 518 nm, a spectral resolution from 1.56 cm 1 to 1.91 cm 1 for different wavelengths of the preselected spectral band with an average spectral resolution of 1.72 cm 1 of all the wavelengths of the preselected spectral band over the 2048 pixels of the CCD array, a path length from the focusing element 1
  • the performance ratio of the spectrometer 120 could be 4.8 %-cm 1 , which is the ratio between the 65% average transfer efficiency and the 13.5 cm path length of the spectrometer 120.
  • the performance product of the spectrometer 120 could be 23.22, which is the product of the 1.72 cm 1 average spectral resolution and the 13.5 cm path length of the spectrometer 120.
  • the excitation light source 110 of Raman spectroscopic system 100 could be a laser having a wavelength of 638 nm.
  • the transmission grating 126 of Raman spectroscopic system 100 could have a line density of 2343 lines/mm, an operational wavelength range of 665.8 nm to 718.3 nm, a diffraction efficiency of approximately 60% to 80% with an average diffraction efficiency of approximately 65% in the operational wavelength range, and an angle of incidence of 54.39 degrees at a center wavelength of 694 nm for first order diffraction.
  • the collimating element 124 and the focusing element 128 could have a focal length of 135 mm and an f-number in the range of F/1.2 to F/4.
  • the detector 130 could be a deep cooled back thinned CCD having an array of 2048 x 70 pixels, a pixel size of 14 pm x 14 pm, and an image area of 28.7 mm by 0.98 mm.
  • spectrometer 120 of Raman spectroscopic system 100 could have a preselected spectral band of 665.8 nm to 718.3 nm, or alternatively represented as a preselected spectral band of 654.8 cm 1 to 1751.8 cm 1 based on the excitation wavelength of 638 nm, an average transfer efficiency of 65% over the preselected spectral band, a spectral resolution of 0.96 cm 1 at the center wavelength of 694 nm, a spectral resolution from 0.89 cm 1 to 1.03 cm 1 for different wavelengths of the preselected spectral band with an average spectral resolution of 0.96 cm 1 of all the wavelengths of the preselected spectral band over the 2048 pixels of the CCD array, a path length from the focusing element 128 to the detector 130 of 13.5 cm, and a width of the entrance aperture 122 of 25 pm.
  • the performance ratio of the spectrometer 120 could be 4.8 %-cm 1 , which is the ratio between the 65% average transfer efficiency and the 13.5 cm path length of the spectrometer 120.
  • the performance product of the spectrometer 120 could be 12.96, which is the product of the 0.96 cm 1 average spectral resolution and the 13.5 cm path length of the spectrometer 120.
  • the excitation light source 110 of Raman spectroscopic system 100 could be a laser having a wavelength of 514.5 nm.
  • the transmission grating 126 of Raman spectroscopic system 100 could have a line density of 2789 lines/mm, an operational wavelength range of 522.05 nm to 570.87 nm, a diffraction efficiency of approximately 60% to 80% with an average diffraction efficiency of approximately 65% in the operational wavelength range, and an angle of incidence of 49.83 degrees at a center wavelength of 548 nm for first order diffraction.
  • the collimating element 124 and the focusing element 128 could have a focal length of 135 mm and an f-number in the range of F/1.2 to F/4.
  • the detector 130 could be a deep cooled back thinned CCD having an array of 2048 x 70 pixels, a pixel size of 14 pm x 14 pm, and an image area of 28.7 mm by 0.98 mm.
  • spectrometer 120 of Raman spectroscopic system 100 could have a preselected spectral band of 522.05 nm to 570.87 nm, or alternatively represented as a preselected spectral band of 280.97 cm 1 to 1919.18 cm 1 based on the excitation wavelength of 514.5 nm, an average transfer efficiency of 65% over the preselected spectral band, a spectral resolution of 1.43 cm 1 at the center wavelength of 548 nm, a spectral resolution from 1.31 cm 1 to 1.57 cm 1 for different wavelengths of the preselected spectral band with an average spectral resolution of 1.43 cm 1 of all the wavelengths of the preselected spectral band over the 2048 pixels of the CCD array, a path length from the focusing element 128 to the detector 130 of 13.5 cm, and a width of the entrance aperture 122 of 25 pm.
  • the performance ratio of the spectrometer 120 could be 4.8 %-cm 1 , which is the ratio between the 65% average transfer efficiency and the 13.5 cm path length of the spectrometer 120.
  • the performance product of the spectrometer 120 could be 19.31, which is the product of the 1.43 cm 1 average spectral resolution and the 13.5 cm path length of the spectrometer 120.
  • the excitation light source 110 of Raman spectroscopic system 100 could be a laser having a wavelength of 488 nm.
  • the transmission grating 126 of Raman spectroscopic system 100 could have a line density of 3018 lines/mm, an operational wavelength range of 494.73 nm to 538.36 nm, a diffraction efficiency of approximately 60% to 80% with an average diffraction efficiency of approximately 65% in the operational wavelength range, and an angle of incidence of 51.41 degrees at a center wavelength of 518.1 nm for first order diffraction.
  • the collimating element 124 and the focusing element 128 could have a focal length of 135 mm and an f-number in the range of F/1.2 to F/4.
  • the detector 130 could be a deep cooled back thinned CCD having an array of 2048 x 70 pixels, a pixel size of 14 pm x 14 pm, and an image area of 28.7 mm by 0.98 mm.
  • spectrometer 120 of Raman spectroscopic system 100 could have a preselected spectral band of 494.73 nm to 538.36 nm, or alternatively represented as a preselected spectral band of 283.05 cm 1 to 1919.97 cm 1 based on the excitation wavelength of 488 nm, an average transfer efficiency of 65% over the preselected spectral band, a spectral resolution of 1.43 cm 1 at the center wavelength of 518 nm, a spectral resolution from 1.31 cm 1 to 1.57 cm 1 for different wavelengths of the preselected spectral band with an average spectral resolution of 1.43 cm 1 of all the wavelengths of the preselected spectral band over the 2048 pixels of the CCD array, a path length from the focusing element 128 to the detector 130 of 13.5 cm, and a width of the entrance aperture 122 of 25 pm.
  • the performance ratio of the spectrometer 120 could be 4.8 %-cm 1 , which is the ratio between the 65% average transfer efficiency and the 13.5 cm path length of the spectrometer 120.
  • the performance product of the spectrometer 120 could be 19.31, which is the product of the 1.43 cm 1 average spectral resolution and the 13.5 cm path length of the spectrometer 120.
  • the excitation light source 110 of Raman spectroscopic system 100 could be a laser having a wavelength of 638 nm.
  • the transmission grating 126 of Raman spectroscopic system 100 could have a line density of 1983 lines/mm, an operational wavelength range of 649.75 nm to 727.35 nm, a diffraction efficiency of approximately 60% to 80% with an average diffraction efficiency of approximately 65% in the operational wavelength range, and an angle of incidence of 43.21 degrees at a center wavelength of 690.5 nm for first order diffraction.
  • the collimating element 124 and the focusing element 128 could have a focal length of 135 mm and an f-number in the range of F/1.2 to F/4.
  • the detector 130 could be a deep cooled back thinned CCD having an array of 2048 x 70 pixels, a pixel size of 14 pm x 14 pm, and an image area of 28.7 mm by 0.98 mm.
  • spectrometer 120 of Raman spectroscopic system 100 could have a preselected spectral band of 649.75 nm to 727.35 nm, or alternatively represented as a preselected spectral band of 283.56 cm 1 to 1925.48 cm 1 based on the excitation wavelength of 638 nm, an average transfer efficiency of 65% over the preselected spectral band, a spectral resolution of 1.43 cm 1 at the center wavelength of 690.5 nm, a spectral resolution from 1.29 cm 1 to 1.61 cm 1 for different wavelengths of the preselected spectral band with an average spectral resolution of 1.43 cm 1 of all the wavelengths of the preselected spectral band over the 2048 pixels of the CCD array, a path length from the focusing element 128 to the detector 130 of 13.5 cm, and a width of the entrance aperture 122 of 25 pm.
  • the performance ratio of the spectrometer 120 could be 4.8 %-cm 1 , which is the ratio between the 65% average transfer efficiency and the 13.5 cm path length of the spectrometer 120.
  • the performance product of the spectrometer 120 could be 19.31, which is the product of the 1.43 cm 1 average spectral resolution and the 13.5 cm path length of the spectrometer 120.
  • the excitation light source 110 of Raman spectroscopic system 100 could be a laser having a wavelength of 785 nm.
  • the transmission grating 126 of Raman spectroscopic system 100 could have a line density of 1388 lines/mm, an operational wavelength range of 797.5.57 nm to 931.2 nm, a diffraction efficiency of approximately 60% to 80% with an average diffraction efficiency of approximately 65% in the operational wavelength range, and an angle of incidence of 36.94 degrees at a center wavelength of 866 nm for first order diffraction.
  • the collimating element 124 and the focusing element 128 could have a focal length of 135 mm and an f-number in the range of F/1.2 to F/4.
  • the detector 130 could be a deep cooled back thinned CCD having an array of 2048 x 70 pixels, a pixel size of 14 pm x 14 pm, and an image area of 28.7 mm by 0.98 mm.
  • spectrometer 120 of Raman spectroscopic system 100 could have a preselected spectral band of 797.5.57 nm to 931.2 nm, or alternatively represented as a preselected spectral band of 282.02 cm 1 to 1920.22 cm 1 based on the excitation wavelength of 785 nm, an average transfer efficiency of 65% over the preselected spectral band, a spectral resolution of 1.43 cm 1 at the center wavelength of 866 nm, a spectral resolution from 1.25 cm 1 to 1.65 cm 1 for different wavelengths of the preselected spectral band with an average spectral resolution of 1.43 cm 1 of all the wavelengths of the preselected spectral band over the 2048 pixels of the CCD array, a path length from the focusing element 128 to the detector 130 of 13.5 cm, and a width of the entrance aperture 122 of 25 pm.
  • the performance ratio of the spectrometer 120 could be 4.8 %-cm 1 , which is the ratio between the 65% average transfer efficiency and the 13.5 cm path length of the spectrometer 120.
  • the performance product of the spectrometer 120 could be 19.31, which is the product of the 1.43 cm 1 average spectral resolution and the 13.5 cm path length of the spectrometer 120.
  • the excitation light source 110 of Raman spectroscopic system 100 could be a laser having a wavelength of 1064 nm.
  • the transmission grating 126 of Raman spectroscopic system 100 could have a line density of 775 lines/mm, an operational wavelength range of 1097.03 nm to 1337.03 nm, a diffraction efficiency of approximately 60% to 80% with an average diffraction efficiency of approximately 65% in the operational wavelength range, and an angle of incidence of 28.22 degrees at a center wavelength of 1220.5 nm for first order diffraction.
  • the collimating element 124 and the focusing element 128 could have a focal length of 135 mm and an f-number in the range of F/1.2 to F/4.
  • the detector 130 could be a deep cooled back thinned CCD having an array of 2048 x 70 pixels, a pixel size of 14 pm x 14 pm, and an image area of 28.7 mm by 0.98 mm.
  • spectrometer 120 of Raman spectroscopic system 100 could have a preselected spectral band of 1097.03 nm to 1337.03 nm, or alternatively represented as a preselected spectral band of 283 cm 1 to 1919.25 cm 1 based on the excitation wavelength of 1064 nm, an average transfer efficiency of 65% over the preselected spectral band, a spectral resolution of 1.43 cm 1 at the center wavelength of 1220.5 nm, a spectral resolution from 1.18 cm 1 to 1.75 cm 1 for different wavelengths of the preselected spectral band with an average spectral resolution of 1.43 cm 1 of all the wavelengths of the preselected spectral band over the 2048 pixels of the CCD array, a path length from the focusing element 128 to the detector 130 of 13.5 cm, and a width of the entrance aperture 122 of 25 pm.
  • the performance ratio of the spectrometer 120 could be 4.8 %-cm 1 , which is the ratio between the 65% average transfer efficiency and the 13.5 cm path length of the spectrometer 120.
  • the performance product of the spectrometer 120 could be 19.31, which is the product of the 1.43 cm 1 average spectral resolution and the 13.5 cm path length of the spectrometer 120.
  • the excitation light source 110 of Raman spectroscopic system 100 could be a laser.
  • the excitation beam 200 emitted by the laser could be collimated by a lens, such as the beam expander 112, and then focused by a cylindrical lens onto the bottom end of the cuvette 310 in the form of a line.
  • the focused line of the excitation beam on the bottom end of the cuvette 310 could be imaged and projected vertically to the entrance slit 122 of the spectrometer 120 along its vertical direction by the cylindrical lens and aperture focusing lens 118, which could be imaged to the detector 130 by the collimating element 124 and the focusing element 128.
  • the detector 130 could be an e2v CCD having an array of 2048 x 264 pixels, a pixel size of 15 pm x 15 pm, and an image area of 30.7 mm by 4 mm.
  • the image of the focused line on the detector could have a height up to 4 mm and a width of 2 pixels or more to achieve Nyquist sampling of the spectrum of the optical signal.
  • spectrometer 120 of Raman spectroscopic system 100 could have any of the combinations of excitation light sources and optical elements of the exemplary embodiments described above in paragraphs [0228]- [0242]
  • the focused line of the excitation beam can be projected onto the bottom end of the cuvette 310.
  • the focused line of the excitation beam can be scanned over the bottom end of the cuvette 310 line by line or rotationally to interrogate one or more selected areas across the interior surface 313a as desired.
  • the focused line of the excitation beam can be scanned over the bottom end of the cuvette 310 to interrogate a rectangular area line by line, or can be scanned over an area of an X-shaped or star-shaped pattern rotationally, or can be scanned over a circular area rotationally.
  • the Raman bands or Raman peaks that are analyzed for detecting the presence or absence of a target in a sample can be selected based on additional understanding of the molecular structure and composition of the target and therefore, are not limited to the examples provided in this disclosure.
  • methods and devices according to the present invention can be implemented in conjunction with the methods and/or devices for testing antimicrobial resistance and/or antimicrobial susceptibility of pathogens, including as further described below.
  • antimicrobial refers to antibiotic, antibacterial, antiviral, antifungal, and/or antiparasitic.
  • testing is used in general to refer to any one method of detection, identification, and quantification of antimicrobial resistance and/or susceptibility of pathogens or a combination thereof.
  • AMR antimicrobial resistance
  • Exemplary embodiments of the present disclosure allow for distinguishing phenotypic changes in a pathogen due to the addition of antimicrobial, antiviral, or anti fungal treatment. Some exemplary embodiments relate to, among other things, methods for detecting biological targets indicative of antimicrobial resistance of bacteria, fungus, and viruses and/or antimicrobial effectiveness of antimicrobial agents based on the phenotypic response of treatment utilizing Raman spectroscopy. In some embodiments, exemplary embodiments of the present disclosure allow for quantitating a level of resistance or susceptibility of the pathogen to the at least one antimicrobial agent.
  • the phenotypic response may be detected over time and the phenotypic response rate may indicate the level of resistance or susceptibility of the pathogen to the at least one antimicrobial agent. For example, faster phenotypic response may indicate less resistance and more susceptibility of the pathogen to the at least one antimicrobial agent.
  • specimen handling, optical characterization, and Raman spectral analysis are fully integrated for direct interrogation of agnostic biological specimens using Raman spectroscopic system 100.
  • an agnostic biological specimen may include one or more pathogens that are previously unknown or may include one or more pathogens for which there is no or limited tests available for detection.
  • an agnostic biological specimen may contain a pathogen having a new mutation that has not been previously sequenced, and/or no antigen test or PCT primer is available for detecting the pathogen.
  • Raman spectroscopic system 100 can be used to detect phenotypic changes in the pathogen contained in the agnostic biological specimen from the treatment of an antimicrobial agent to determine the antimicrobial resistance and/or susceptibility of the pathogens, and/or the antimicrobial effectiveness of the antimicrobial agent.
  • Raman spectroscopy probes molecular vibrations/rotations associated with chemical bonds in a sample to obtain information on molecular structure, composition and inter-molecular interactions. Since each sample has a unique composition and/or structure, the spectroscopic profile arising from Raman active functional groups of nucleic acids, proteins, lipids, and carbohydrates allows for the evaluation, characterization, and discrimination of agnostic biological specimens. Unlike many diagnostic assays, Raman Spectroscopy requires little or no prior knowledge of the pathogen. The technique is unbiased rather than pathogen-specific. The principles of Raman spectroscopy can be leveraged to provide an alternative to conventional diagnostic methods without the need for complex and destructive sample handling or predetermined primers and probes and can readily detect non-cultured organisms or analyze non-cultured specimens.
  • phenotypic changes in pathogens are identified by changes in Raman spectral regions following exposure to clinically relevant doses of antibacterial, anti-fungal, or antiviral pharmacologic agents (collectively, antimicrobial agents).
  • the spectral changes may be representative of the phenotypic changes in DNA, RNA, protein, lipid and phospholipid metabolism observed following exposure to the agents.
  • the sample is interrogated for an initial baseline spectral analysis using Raman spectroscopic system 100.
  • the subsequent changes in the Raman spectra during and/or after an interrogation time period are obtained and analyzed using Raman spectroscopic system 100.
  • a pathogen contained in a sample is cultured in one or more nutrients or culture media to induce at least one replication and treated with at least one antimicrobial agent to induce one or more phenotypic changes over the at least one replication.
  • highly mutable bacteria such as gonorrhea bacteria
  • the cultured bacteria are treated with at least one antimicrobial agent during the replication to induce one or more phenotypic changes that can be detected using Raman spectroscopic system 100.
  • the interrogation time period may differ for different pathogens with various replication rates. In some exemplary embodiments, the interrogation time period is in the range of 1 to 120 minutes.
  • spectral changes may occur within an interrogation time period of 30 minutes, which represents typical time for one replication of an exemplary pathogen.
  • the interrogation time period can be longer than 120 minutes.
  • a plurality of iterations of interrogations are performed over an interrogation time period.
  • each iteration is performed over an integration or aggregated time period from 1 ms to 10 minutes.
  • Phenotypic response or changes following exposure to antimicrobial pharmacologic agents may include, but are not necessarily limited to, genetic changes, membrane reinforcement changes, antimicrobial binding changes, protein rearrangement, lipid rearrangement, lipid or protein signaling, cell or colony signaling (Quorum sensing), biofilm formation or alteration or enzymatic reaction in response to the antipathogen exposure.
  • the phenotypic response is driven by one or more genetic responses or changes following the exposure to an antimicrobial agent.
  • MRSA Methicillin-resistant Staphylococcus aureus
  • the mecA gene is activated following the exposure to penicillin, allowing the bacteria to produce penicillin resistant proteins to go to the membrane or for expression of enzymes.
  • the nucleic acid of the pathogen may change due to interaction of the antimicrobial agent with the nucleic acid after membrane disruption.
  • Phenotypic changes that are indicative of antimicrobial susceptibility or sensitivity (i.e., antimicrobial effectiveness of the agent) to a particular antipathogen agent may include, but are not necessarily limited to, membrane dissociation, membrane thinning, membrane disruption, membrane barrier, cell destruction, or inhibition of cell wall synthesis inhibition of protein synthesis, other alterations of cell membranes, inhibition of nucleic acid synthesis, and/or antimetabolite activity.
  • upregulation of phospholipids in MRSA after treatment with an antibiotic can be detected using Raman spectroscopic system 100.
  • dissociation of membrane, lipids, and/or proteins in bacteria after being treated with antibiotics can be detected using Raman spectroscopic system 100.
  • enzyme production and/or signaling (such as quorum sensing /signaling) to the rest of the colony of the bacteria to perform the same change or form a biofilm is detected using Raman spectroscopic system 100.
  • Antimicrobial resistance is ultimately propagated or transferred genetically.
  • the first response to antimicrobial treatment is a phospholipid change followed by protein and genetic changes.
  • Directly measuring the biological effectiveness of an antimicrobial agent in the sample eliminates the negative impact of mutations on testing accuracy.
  • Phenotypic differentiation between antimicrobial-sensitive and antimicrobial-resistant strains following exposure to antimicrobial agents due to alterations in DNA, RNA, protein, lipid or phospholipid metabolism is associated with reproducible Raman spectral changes in corresponding spectral regions.
  • Table 6 shows representative Raman spectral regions demonstrating intensity changes following exposure of methicillin resistant staphylococcus aureus (MRSA) to cefoxitin.
  • MRSA methicillin resistant staphylococcus aureus
  • discriminant function analysis is used for classifying pathogens into different groups.
  • Discriminant function analysis is a statistical procedure that classifies unknown individuals and the probability of their classification into a certain group, such as an antimicrobial resistant group or an antimicrobial susceptible group. It is a multivariate technique that builds a predictive model for group membership. DFA makes the assumption that the sample is normally distributed for the trait to be classified. It is the probability that an unknown case belongs to a certain group based on relative Mahalanobis’ distances measuring the distance to the center or centroid of each group. For example, each component (DNA, RNA, protein, lipid or phospholipid, etc.) is given a discriminant score to determine how well it predicts group classification. For a “N” group classification analysis, the DFA model is composed of “N-l” functions, which are linear combinations of predictor variables whose coefficients maximize separation between predefined groups. A representative function for a DFA model is shown below:
  • D ik bok + blkXil + ⁇ ⁇ ⁇ + bnkXin
  • D ik is the value of the k th discriminant function for the 1 th case
  • n is the number of predictors
  • Xy is the value of the independent variable of the i th case of the j th predictor
  • bjk is the value of the j th unstandardized canonical coefficient of the k th function.
  • MRSA methicillin resistant staphylococcus aureus
  • DFA methicillin resistant staphylococcus aureus
  • the DFA model represented by the “DF” equation including three variables, corresponding to three components of MRSA, protein, nucleic acids, and lipid.
  • the three variables represent three features of the Raman spectrum occur at or around 1234 cm 1 , 1319 cm 1 , and 1416 cm 1 .
  • a numerical “DFA” is calculated from the “DF” equation for each MRSA sample based on these selected features.
  • there is one decision boundary represented by the horizontal dashed line in FIG. 48. Samples having DFA scores fall on one side of the boundary are assigned to the first group, while samples having DFA scores on the other side of the boundary are assigned to the second group.
  • MRSA samples having DFA scores above the boundary line are assigned to the MRSA in the presence of cefoxitin group and MRSA samples having DFA scores below the boundary line are assigned to the MRSA in the absence of cefoxitin group.
  • phenotypic spectral changes in MRSA following exposure to cefoxitin exhibited shifts in phenylalanine peaks at 1004 cm 1 and 1031 cm 1 .
  • the changes and shifts in phenylalanine support the notion that specific proteins are being up regulated and down regulated as a result of exposure, consistent with published studies. See Liu et al. Label-Free Quantitative Proteomics Analysis of Antibiotic Response in Staphylococcus aureus to Oxacillin. Journal of Proteome Research 13 (3), 1223-1233 (2014).
  • the inventors predicted the most distinguishable differences between the samples involve DNA, RNA, nucleic acid, lipid, and phospholipid content.
  • FIG. 49-51 Representative spectra illustrating the time-course changes in specific Raman spectral regions following exposure of staphylococcus aureus to cefoxitin are shown in FIG. 49-51.
  • FIG. 49 illustrates time-course mean Raman spectra of spectral regions 1161-71 cm 1 and 1518-30 cm 1 corresponding to phospholipid in both MRSA (black lines) and methicillin sensitive staphylococcus aureus (MSSA) (gray lines). Both MRSA and MSSA were exposed to cefoxitin. As indicated by the data shown in FIG.
  • FIG. 50 illustrates time-course mean Raman spectra of two spectral regions corresponding to proteins inhibiting disruption of the cell membrane in MRSA.
  • the spectral changes shown in FIG. 50 indicate cell membrane protein enhancement changes in MRSA following cefoxitin exposure, demonstrating the detection of antimicrobial resistance of MRSA.
  • FIG. 50 shows changes in peak intensity over time in the spectral region from 1520 cm 1 1529 cm 1 .
  • FIG. 51 illustrates time-course mean Raman spectra of two regions corresponding to lipid and protein of the cell membrane in MSSA.
  • the spectral changes shown in the spectral regions 680-766 cm 1 and 1224-1229 cm 1 such as the changes in the intensity and shape (or morphology) of the Raman peak over time in the spectral region from 1238 cm 1 to 1243 cm 1 , in FIG. 51 indicate lipid and protein disruption in MSSA following cefoxitin exposure, demonstrating the detection of antimicrobial susceptibility of MSSA.
  • Antifungals can be grouped into three classes based on their site of action: azoles, which inhibit the synthesis of ergosterol (the main fungal sterol); polyenes, which interact with fungal membrane sterols physicochemically; and 5-fluorocytosine, which inhibits macromolecular synthesis.
  • Raman spectroscopy phenotypic indications of effectiveness include but are not limited to, inhibiting the synthesis of fungal cell wall polysaccharides or protein synthesis.
  • Raman Spectroscopy spectral or phenotypic indications of antifungal resistance include alteration in drug target, alteration in sterol biosynthesis, reduction in the intercellular concentration of target enzyme, and overexpression of the antifungal drug target.
  • Raman spectroscopy spectral or phenotypic indications of antiviral effectiveness are indicated by, but not limited to, inhibition of viral polymerase, Neuraminidase inhibitor, polymerase interactions, protease, and integrase, indicated by envelope protein, lipid changes, or virus load replication from host cell response.
  • the antiviral effectiveness may be identified based on the upregulation, down regulation, and/or dissociation of these exemplary biological molecules, structures, or processes detected using Raman spectroscopic system 100. For example, some antiviral agents may decrease or prevent replication of the virus in the host cell.
  • Raman spectroscopic system 100 can be used to detect spectral changes due to cell replication arrest and/or decrease of the number of viruses in and/or expressed from the cells.
  • Raman spectroscopy spectral or phenotypic indication of resistance may be indicated by, but not limited to, a virus latent response (becomes dormant), protein degeneration, lipid degeneration, structural changes, replication inhibition in host cells (e.g., replication inhibition in kidney cells).
  • the replication of the virus in the sample is induced by adding host cells to the sample in the cuvette.
  • FIG. 52 An exemplary approach for detecting antibiotic resistance of bacteria is illustrated in FIG. 52.
  • FIG. 52 illustrates a testing flow diagram for an exemplary approach for detecting antibiotic resistance of bacteria.
  • Raman spectroscopic system 100 is used in the exemplary approach as shown in FIG. 52.
  • Raman spectroscopic system 100 is used for antimicrobial susceptibility testing.
  • Raman spectroscopic system 100 performs interrogation of a sample, analyzes acquired Raman spectra, and presents or delivers the testing results in less than 60 minutes, such as in from 30 to 60 minutes.
  • the actual time required for Raman spectroscopic system 100 to perform interrogation of a sample, analyze acquired Raman spectra, and present or deliver the testing results may depend on the sample (e.g., concentration, volume, type, or the like), the type of pathogen in the sample, and/or the antimicrobial agent.
  • the interrogation, analysis, and result delivery may be performed in less than 120 minutes, 2-5 hours, 5-8 hours, 8-12 hours, 12-18 hours, 18-24 hours, 1-2 days, or 2-5 days.
  • Raman spectroscopic system 100 allows for testing of pathogen susceptibility of a plurality of different antimicrobial agents in one testing session.
  • a testing session may include a plurality of steps for performing sample preparation, interrogation, data analysis, and/or result generation.
  • a testing session may be completed in a short period of time, such as for 30 to 60 minutes.
  • at least some of the steps in a testing session are automated.
  • all of the steps in a testing session are automated.
  • Raman spectroscopic system 100 has a plurality of cuvettes for receiving a plurality of samples.
  • the samples may contain one or more pathogens to be tested.
  • the pathogens may include one or more types of viruses, bacteria, and fungus.
  • An exemplary Raman spectroscopic system 100 is shown in FIGS. 53A- 53E.
  • Raman spectroscopic system 100 may include a multi-sample carousel 140 that has a structure for holding the plurality of cuvettes 310.
  • the multi-sample carousel 140 may be used by Raman spectroscopic system 100 to perform sample processing and/or interrogation of a plurality of samples.
  • the multi-sample carousel 140 may include an opening 142 for receiving one cuvette at a time. The opening may be closed with a lid after all the cuvettes are put in place.
  • Raman spectroscopic system 100 includes a cartridge 160 for storing one or more materials for processing the samples and/or receiving waste from the process.
  • Cartridge 160 may be retrieved from or placed in Raman spectroscopic system 100 through an access 162.
  • Cartridge 160 may include one or more chambers.
  • cartridge 160 includes a solution chamber 166 configured to hold a solution, such as water or a buffer solution.
  • cartridge 160 includes a waste chamber 164 for receiving waste from one or more sample processing steps. For example, supernatant separated from centrifuging the samples and/or waste from one or more washing steps may be transported to and stored in waste chamber 164.
  • cartridge 160 includes a reagent chamber 168 for storing and/or discharging a reagent into the cuvette.
  • reagent chamber 168 may store a solution containing an antimicrobial agent.
  • Raman spectroscopic system 100 may include a dispensing mechanism that dispenses a predetermined volume of the antimicrobial agent into the cuvette for determining the antimicrobial resistance and/or susceptibility of a pathogen in a sample in a cuvette.
  • the multi-sample carousel 140 can be automatically rotated to centrifuge the samples in the plurality of cuvettes at the same time to at least partially separate a supernatant from each sample.
  • the separated supernatant in a sample is at least partially removed from the sample into waste chamber 164.
  • water or buffer solution in solution chamber 166 is added to the sample in the cuvette to perform a washing process to remove waste from the sample or to elute or dilute targets in the sample. As described herein, centrifuging and/or washing may be repeated for one or more times as desired.
  • the samples in the plurality of cuvettes may be interrogated according to a predetermined interrogation procedure by the Raman spectroscopic system 100.
  • the excitation beam 200 emitted by the laser could be collimated and focused onto the bottom end of the cuvette 310 in the form of a line.
  • the interrogation procedure may be predetermined by a user of Raman spectroscopic system 100 based on the type of samples and/or the antimicrobial agents to be tested.
  • the interrogation procedure can be automatically generated based on input from a user entered via a user interface of Raman spectroscopic system 100.
  • the interrogation procedure is manually set by the user using the user interface of Raman spectroscopic system 100.
  • the interrogation procedure may be stored in a computer-readable medium and executed by a processor of Raman spectroscopic system 100.
  • the user interface may also include a display that shows the user the samples currently being interrogated, the antimicrobial agents currently being evaluated, and/or the progress of the interrogation procedure.
  • An exemplary workflow using Raman spectroscopic system 100 for testing pathogen susceptibility include the following steps. First, a sample, such as a urine specimen, is collected into a test tube. The sample in the test tube is centrifuged at 4500 g for 5 minutes. In some embodiments, the sample is centrifuged using a centrifugal force from 4500 g to 20,000, such as from 4500 g to 14,000 g for most bacteria and from 4500 g to 20,000 g or higher for viruses. The supernatant in the test tube is at least partially removed and replaced with water preloaded with an antimicrobial agent. The test tube is agitated or mixed and the sample is then poured into a cuvette. .
  • the sample is centrifuged outside the Raman spectroscopic system 100 before or after being collected into the cuvette.
  • the sample is centrifuged within the Raman spectroscopic system 100 while the sample is in the cuvette .
  • the cuvette is placed in the Raman spectroscopic system 100, e.g., a cuvette holder or a space in a multi-sample carousel.
  • the sample in the cuvette is then continuously or intermittently interrogated for a first period of time for initial identification of one or more pathogens in the sample.
  • the sample is interrogated continuously or intermittently for a second period of time for evaluating the resistance and/or susceptibility of the pathogens to the antimicrobial agent.
  • the sample is interrogated for 12 iterations at 10 seconds per iteration. Then, the sample is interrogated several times over a 30 min to 120 min period to measure Raman spectra for determining antimicrobial resistance and/or susceptibility. Finally, the measured Raman spectra is analyzed to generate the antimicrobial resistance and/or susceptibility test result.
  • Another exemplary workflow using Raman spectroscopic system 100 for testing pathogen susceptibility include the following steps. First, a sample, such as a urine specimen, is collected into a test tube. A syringe is used to draw the sample from the test tube. A filter is placed at the tip of the syringe. The filter may be a membrane filter, such as a standard 25 mm cellulose filter. The filter captures the pathogens in the sample while the solution in the syringe is expressed out of the syringe into a waste container. Then, water preloaded with an antimicrobial agent is drawn into the syringe, flushing the pathogens off the filter. The pathogens are then mixed with the antimicrobial agent. The content in the syringe is then expressed into a test tube, which is then agitated or mixed and the content in the test tube is poured into a cuvette. The cuvette is interrogated as described in paragraph [0270]
  • the samples may include different patient samples.
  • the samples include the same patient’s samples for testing of antimicrobial resistance and/or susceptibility of different antimicrobial agents.
  • the samples may be placed in a multi-cuvette centrifuge to be concurrently prepared.
  • Raman spectroscopic system 100 includes a sample preparation module that allows for automatic preparation of multiple samples according to the exemplary workflows. The prepared samples can be transferred manually or automatically into a corresponding number of cuvettes. The cuvettes can then be manually or automatically placed in the multi sample carousel for interrogation.
  • the samples can be dosed with different amounts, such as different concentrations of the same volume or different volumes of the same concentration, of the same antimicrobial agent to determine a potency of the antimicrobial agent for the pathogen.
  • a potency may be a minimum inhibitory concentration, a 50% inhibitor concentration, or an optimal inhibitory concentration, which can be assessed based on detecting and analyzing the phenotypic response of the pathogen using Raman spectroscopic system 100 .
  • a clinical dose, in units of weight or concentrations (e.g., picograms per ml), of the antimicrobial agent for the pathogen may be determined based on the potency.
  • the clinical dose may be determined additionally based on pharmacokinetic properties of the antimicrobial agent and/or patient characteristics, such as the body mass of the patient.
  • the samples can be dosed with a fixed amount, such as the same volume of a fixed concentration, of different antimicrobial agents or different combinations of antimicrobial agents and be monitored over time to determine the potency of each of the antimicrobial agents or each of the combinations for the pathogen.
  • the minimum inhibitory concentration, 50% inhibitor concentration, optimal inhibitory concentration of an antimicrobial agent or a combination of antimicrobial agents for the pathogen may be assessed based on detecting and analyzing the phenotypic response of the pathogen over time using Raman spectroscopic system 100.
  • one or more parameters of a growth curve of the pathogen such as the number of pathogens, length of each phase, rapidness of growth or death, overall amount of time, can be measured based on phenotypic response detected during Raman spectroscopic system 100.
  • the number of pathogens killed over time i.e., the kill rate
  • the kill rate can be measured based on phenotypic response detected during Raman spectroscopic system 100 and used to determine a minimum inhibitory concentration, a 50% inhibitor concentration, or an optimal inhibitory concentration.

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EP21721717.3A 2020-04-03 2021-04-02 Vorrichtungen, systeme und verfahren zur pathogendetektion auf der basis von ramanspektroskopie Withdrawn EP4127679A1 (de)

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