WO2022008931A1 - Améliorations apportées ou se rapportant à un appareil d'imagerie - Google Patents

Améliorations apportées ou se rapportant à un appareil d'imagerie Download PDF

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Publication number
WO2022008931A1
WO2022008931A1 PCT/GB2021/051768 GB2021051768W WO2022008931A1 WO 2022008931 A1 WO2022008931 A1 WO 2022008931A1 GB 2021051768 W GB2021051768 W GB 2021051768W WO 2022008931 A1 WO2022008931 A1 WO 2022008931A1
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WIPO (PCT)
Prior art keywords
assay
spots
substrate
spot
assay spots
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PCT/GB2021/051768
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English (en)
Inventor
Marko Dorrestijn
David R. Klug
Hajra BASIT
Stefan Leo VAN WORKUM
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Vidya Holdings Ltd
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Publication of WO2022008931A1 publication Critical patent/WO2022008931A1/fr

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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
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/645—Specially adapted constructive features of fluorimeters
    • G01N21/648—Specially adapted constructive features of fluorimeters using evanescent coupling or surface plasmon coupling for the excitation of fluorescence
    • 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
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/645—Specially adapted constructive features of fluorimeters
    • G01N21/6452—Individual samples arranged in a regular 2D-array, e.g. multiwell plates
    • 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
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/645—Specially adapted constructive features of fluorimeters
    • G01N21/6456—Spatial resolved fluorescence measurements; Imaging
    • G01N21/6458—Fluorescence microscopy
    • 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
    • G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
    • G01N21/47—Scattering, i.e. diffuse reflection
    • G01N21/49—Scattering, i.e. diffuse reflection within a body or fluid
    • G01N21/51—Scattering, i.e. diffuse reflection within a body or fluid inside a container, e.g. in an ampoule
    • 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
    • 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
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54366—Apparatus specially adapted for solid-phase testing
    • G01N33/54373—Apparatus specially adapted for solid-phase testing involving physiochemical end-point determination, e.g. wave-guides, FETS, gratings

Definitions

  • the present invention relates to improvements in or relating to an apparatus for imaging and in particular, an apparatus for imaging a plurality of assay spots deposited on a substrate using total internal reflection excitation microscope.
  • detecting a component such as a biomolecule has attracted great interest in early disease diagnosis, including cancers, inflammation and neurodegenerative diseases.
  • the detection of multiple components such as antibodies and other components of interest can facilitate early clinical or diagnostic treatment.
  • Commonly known standard methods, such as ELISA and immuno-blotting often require cumbersome multiple purification and pre-treatment processes, and, in addition, require a relatively large volume of sample. These problems often hinder the accuracy and throughput of the assays and therefore limit their applications.
  • TIR Total internal reflection
  • Total internal reflection occurs at the interface between a higher refractive-index medium and a lower refractive-index medium. Above the critical angle, defined by the refractive indices of the two media, light travelling in a higher refractive-index medium incident on a lower refractive-index medium is totally reflected. This total internal reflection generates an exponentially decaying light field, known as an evanescent wave.
  • the evanescent wave can be used to excite luminescent molecules or scattering particles in very close proximity to the boundary between the two media. These luminescent molecules will consequently emit light at a certain wavelength, which can be selectively detected to provide information on the boundary region.
  • the luminescence may arise from fluorescence or phosphorescence.
  • TIR techniques such as total internal reflection fluorescence (TIRF), specifically lens based TIR provides a highly sensitive method for detecting assay spots on a surface but TIR also has a limited field of view and therefore often requires interrogating each deposited component, such as an antibody spot, individually. Thus the apparatus set up tends to require moving parts to allow the system to move between each spot. Furthermore, multiple spots in an assay array have been previously imaged with TIR but not using a microscope objective for both launching the excitation light and collection of the emitted, reflected or scattered light, thus lacking single molecule sensitivity detection and the dynamic range. In addition, the throughput rate is limited by the number of printed spots that can fit within the reduced field of view of the imaging system.
  • TIRF total internal reflection fluorescence
  • an apparatus for imaging a plurality of assay spots comprising, a plurality of assay spots deposited onto a substrate, each assay spot comprising one or more capture components and/or one or more detection reagents; and a total internal reflection excitation microscope comprising a light source for illuminating the assay spots; a lens system configured to launch light towards the substrate and to collect the emitted, reflected or scattered light from the assay spots, wherein the lens system comprises an objective lens; and a detector configured to detect the collected emitted, reflected or scattered light from the lens system, wherein the detector is further configured to image multiple assay spots simultaneously within a field of view.
  • an apparatus for imaging a plurality of assay spots comprising, a plurality of assay spots deposited onto a substrate, each assay spot comprising one or more capture components and/or one or more detection reagents; and a total internal reflection excitation microscope comprising a light source for illuminating the assay spots; a lens system configured to launch light towards the substrate and to collect the emitted, reflected or scattered light from the assay spots, wherein the lens system comprises an objective lens; and wherein the objective lens has a numerical aperture of 1.3 or above an index matching fluid provided between the lens system and the assay spots deposited on the substrate; and a detector configured to detect the collected emitted, reflected or scattered light from the lens system, wherein the detector is further configured to image multiple assay spots simultaneously within a field of view.
  • an apparatus for imaging a plurality of assay spots comprising, a plurality of assay spots deposited onto a substrate, each assay spot comprising one or more capture components and/or one or more detection reagents; and a total internal reflection excitation microscope comprising a light source for illuminating the assay spots; a lens system is configured to launch light towards the substrate and to collect the emitted, reflected or scattered light from the assay spots; and a detector configured to detect the collected emitted, reflected or scattered light from the lens system, wherein the detector is further configured to image multiple assay spots simultaneously within a field of view.
  • one or more lenses can be arranged in series to form a lens system.
  • the lens may be an objective lens.
  • the lens system can comprise a plurality of lenses arranged in series.
  • the apparatus of the present invention includes an objective lens which is advantageous because it allows the apparatus to achieve detection with single molecule sensitivity and provide a large dynamic range of the order of 106. Moreover, the apparatus of the present invention can also be used to provide a high throughput assay system based on objective or a lens system launched total internal reflection (TIR). This approach enables a user to image multiple spots simultaneously using TIR delivered through a microscope objective lens or a lens system in a high throughput manner.
  • TIR total internal reflection
  • TIR Total internal Reflection Fluorescence
  • Other examples of TIR techniques include, but is not limited to, Raman scattering, Rayleigh scattering, Mie scattering and photon upconversion.
  • At least one of the lenses in series is an objective lens.
  • the objective lens based TIR which can sometimes be referred to as through-lens illumination, avoids many of the limitations of utilising a prism to introduce light above the critical angle for TIR to be achieved.
  • the angle is achieved by focusing the beam off-axis to the periphery of a high numerical aperture lens system such as the objective lens.
  • numerical aperture >1.33 is essential to obtain critical angle.
  • the objective lens may be configured to collect the emitted, reflected or scattered illuminated light from the assay spots and focus the collected emitted, reflected or scattered light onto a detector.
  • the objective lens may also be configured to provide magnification of the sample deposited on the surface of the substrate. Imaging with a high magnification objective lens offers single molecule resolution in addition to the advantage of reducing stray reflections and ease of TIR angle alignment compared with prism or light guide/waveguide TIR.
  • the apparatus includes a detector which is provided to detect the emitted light from the capture components using fluorescence. Additionally or alternatively, the detector can be used to detect scattered light such as Raman scattering, Rayleigh scattering, Mie scattering and/or upconversion.
  • total internal reflection excitation microscopy may include, but is not limited to, Total internal reflection fluorescence microscopy (TIRFM), Rayleigh scattering microscopy, Raman microscopy.
  • TRFM Total internal reflection fluorescence microscopy
  • Rayleigh scattering microscopy Raman microscopy.
  • spots of arrays can also be detected by upconversion.
  • the detector can be an optical detector such as a camera.
  • the detector can be a low read- noise camera or detector and along with the objective lens, can offer single molecule sensitivity detection.
  • the term “high throughput” may refer to a single TIRF reader that is able to image more than 10, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 45000, 5000, 6000, 7000, 8000, 9000 or more than 10,000 spots per day, potentially leading to >10,000 assays per day.
  • Assay spots can be deposited in any form onto the surface of the substrate which includes, but is not limited to, immobolising or printing the assay spots onto surface of the substrate.
  • An example of printing as disclosed in the present invention involves stamping the assay spots onto the surface of the substrate.
  • the assay spots are in liquid form.
  • the assay spots are in a dry form.
  • the assay spots, which may be in liquid form can be deposited onto the surface of the substrate and dry out.
  • the assay spots deposited on the substrate can be washed.
  • the assay spots may be deposited onto the surface of the substrate by contact printing methods i.e. directly contacting the substrate. In some embodiments, the assay spots may be deposited onto the surface of the substrate by non-contact printing methods. In some embodiments, the assay spots may be deposited onto the surface of the substrate by liquid printing such as inkjet printing.
  • the assay spots may be printed onto the surface of the substrate by a lithographic printing process. This is particularly advantageous because it can improve the resolution of the assay spots during detection.
  • Each assay spot can be made into any suitable shape that is capable of containing at least one capture component and/or one or more detection reagents.
  • the assay spot may be a circular shape or it may be a non-circular shape. In other examples, the assay spot may have a rectangular, a triangular, a square or a crescent-shaped geometry. In some embodiments, the assay spot may be a partial spot. In some embodiments, the assay spot may have an irregular shape.
  • a portion of an assay spot containing at least one capture component and/or one or more detection reagents may be activated by light to provide an activated area.
  • One or more components and/or reagents can be flowed across the surface of the substrate. Component and/or reagents can then bind onto the immbolised capture component and/or detection reagent within the activated area.
  • the equivalent diameter of each assay spot is between 20 to 100 pm but it may exceed 20, 30, 40, 50, 60, 70, 80 or 90 pm. In some embodiments, the diameter of each assay spot is less than 100, 90, 80, 70, 60, 50, 40, 30 or 20 pm. In some embodiments, the diameter of each assay spot is 50 pm. If the assay spots are printed onto the substrate with a diameter smaller than 10 pm, the sensitivity of detecting the assays spots is reduced. In addition, assay spots smaller than 10 pm provide a reduced dynamic range. The smaller the spot the lower the sensitivity and dynamic range. Therefore, the diameter of the spot can be any suitable size.
  • the diameter of the spot is selected to be a particular size such that a specific sensitivity and/or dynamic range can be achieved whilst also being able to fit multiple spots into the field of view.
  • a portion of an assay spot, with a large diameter may be within field of view.
  • the spots can overlap or merge together. In this case, it may be possible to select an area of interest such as an unmerged portion of the assay spots and detect the unmerged portion of assay spots within the field of view.
  • the assay spots can be printed onto the surface of the substrate. The behaviour of the spot once applied to the substrate will depend on the fluid properties of the spot and the wetting properties of the substrate onto which is it printed. The surface tension will define the spot boundary. If the spot is very small, the signal produced will be correspondingly low and it may provide a reduced certainty of reading.
  • the spot will remain constrained in a comparatively small area on the substrate. Conversely, if the substrate material has a positive wetting coefficient and/or the viscosity of the spot is low, then the spot will wet across the substrate and may merge with neighboring spots. By spreading the same volume of liquid across a larger area, the dynamic range will be reduced. It is still possible to obtain useful information from partially merged spots provided the centre of the spot is at a known position and therefore the central, unmerged portion of each spot can be interrogated.
  • Printing microarrays onto the surface of a substrate using non-contact microarrayers such as inkjet, piezo or acoustic dispensing can produce assay spots of approximately >20 pm diameter with high spatial accuracy, resolution and reproducibility i.e. variability of the location of the assay spot center of approximately 5 pm. This will allow in the imaging of 5 printed assay spots in the field of view.
  • the assay spots must be printed onto the substrate at a sufficient size i.e. 50 pm in diameter in order to prevent or minimize the risk of assay spots on the surface of the substrate merging together.
  • a surface treatment may be applied onto the surface of the substrate to stop assay spots merging.
  • An example of a surface treatment may include an anti-wetting treatment.
  • the surface of the substrate can be wetted as long as the assay spots don’t merge together or a sufficient region within each spot hasn’t substantially merged or mixed together.
  • small assays spots can spread along the surface of the substrate.
  • four assay spots can be imaged in a field of view where the spots wet the surface.
  • the substrate can be made out of plastic or glass. Providing a plastic substrate can be particularly cost effective. The main barriers for providing a plastic substrate may be due to difficulties in chemical modification of the plastic substrate, thickness of the plastic substrate and/or providing a suitable refractive index matching material.
  • the substrate may be a flat surface. In some embodiments, the boundary of the assay spot on the substrate may be defined by the surface tension i.e. the contact between the surface of the assay spot and the surface of the substrate.
  • dynamic range refers to the ratio between the smallest signal and the largest signal detected by the detector.
  • the substrate may comprise one or more wells.
  • the assay spots can be deposited onto the surface of the well. Providing wells in the substrate can increase the surface area of the substrate which in turn, increases the area on which the assay spots can be printed on.
  • each well comprises 2 to 128 assay spots within the field of view. In some embodiments, each well comprises more than 2, 20, 40, 60, 80, 100 or 120 assay spots. In some embodiments, each well comprises less than 128, 120, 100, 80, 60, 40, or 20 assay spots. In some embodiments, each well comprises 2 to 10 assay spots within the field of view.
  • the apparatus as disclosed in the present invention can be used to image 2 to 10 assays spots for example, 2 to 5, 3 to 5, or 4 to 5 assay spots within a field of view of an objective lens or a lens system based TIR setup. This would greatly increase the throughput of the measurements without compromising on the sensitivity of detection.
  • each well may contain between 1 to 100 assay spots.
  • the well may have a diameter of between 1 to 10 mm or it may be within a range of 1 to 10 mm, 1 to 9 mm, 1 to 8 mm,1 to 7 mm, 1 to 6mm, 1 to 5mm, 1 to 4 mm, 1 to 3 mm or 1 to 2 mm.
  • the capture component and/or detection reagent in each assay spot can be different from one another. Imaging multiple spots within a field of view can be highly desirable for multiplexed assays where each spot can be a different capture antibody, protein, RNA, DNA or small molecules, thereby reducing the amount of sample required for the assay.
  • the capture component and/or detection reagent in each assay spot on the substrate can be the same.
  • the total internal reflection excitation microscope can be a total internal reflection fluorescence microscope (TIRFM).
  • TRFM Total Internal Reflection Fluorescence microscopy
  • the substrate may be mounted onto a multi-axis controlled stage or any other devices or apparatuses that are capable of providing motion.
  • the substrate can be mounted on a multi-axis controlled stage, where the multi-axis motion controlled stage can be configured to provide a motion and move the substrate into any desired position.
  • the multi-axis motion stage can be controlled manually by a user or automatically by a controller such as a software controller.
  • the multi-axis controlled stage also allows the sample to be moved across the objective lens rapidly.
  • the multi-axis controlled stage is an X, Y axis or an X,Y, Z controlled stage.
  • the objective lens has a numerical aperture of 1 , 1.1 , 1.2, 1.3, 1.31 , 1.32, 1.33, 1.34, 1.35, 1.36, 1.37, 1.38, 1.39, 1.4 or above. In some embodiments, the objective lens has a numerical aperture of 1.33 or above. In some embodiments, the objective lens can be an oil immersion objective lens.
  • Oil immersion lens provides large numerical apertures >1.4 for collecting coherent light.
  • a large numerical aperture is >1.33.
  • Objective lenses also offer high magnification of the sample to provide a high resolution image of the sample.
  • objective lens can be used to direct the laser beam at suitable angle to enable TIR phenomenon, where the angle of incidence is greater than the critical angle, and that the luminescent or scattered light from the sample can also be detected using the objective lens.
  • TIR microscopy is TIRF microscopy.
  • the objective lens may be a water immersion lens.
  • the apparatus may further comprise an index matching fluid and/or an index matching gel may be provided between the lens and the assay spots deposited on the surface of the substrate.
  • the lens system may comprise one or more lenses arranged in series. At least one lens arranged in series is an objective lens.
  • the index matching fluid may be provided between an objective lens, and the substrate.
  • Objective based TIR imaging such as TIRF imaging requires refractive index matching oil in between the sample deposited on the substrate and the objective lens to achieve TIR. To be able to scan an entire multiwell chip containing multiple assay spots in each well, the thickness of index matching oil between the lens and the substrate needs to be maintained.
  • the index matching fluid can be a liquid.
  • the index matching liquid can be oil. Oil is an ideal index matching fluid because the refractive index can be the same or similar as the substrate that is made out of glass.
  • an index matching solid or an index matching gel can be provided.
  • the apparatus further comprises an automated feeding mechanism configured to maintain the required thickness of refractive index matching fluid or index matching gel to allow high throughput imaging.
  • An example of an automated feeding mechanism may be an auto-oiler.
  • An auto-oiler can be utilise to automatically supply and replenish an index matching fluid e.g. index matching oil to the objective lens which removes the requirement for manual replacement of the index matching fluid. Hence, this mininises or reduces any interruptions to the apparatus set up during high throughput imaging.
  • the light source may be a laser beam.
  • the light source may be an LED or a Super Luminescent Diode (SLD) or a diode beam.
  • the illuminating light may be spatially and temporally coherent by using a laser beam as a light source.
  • the laser beam can be a collimated beam because of its high coherence and low divergence.
  • a collimated beam of light is a beam which has a low beam divergence.
  • the illumination of the assay spots can utilise a Gaussian beam profile.
  • Using a laser with a Gaussian profile for imaging the array spots can be advantageous because the illumination intensity is greatest at the centre of the Gaussian.
  • Using a Gaussian profile for imaging can be particularly effective when imaging a single spot within a field of view.
  • the illumination of the assay spots may utilise a square beam profile.
  • the apparatus may be modified to include a top-hat beam using or a square core fiber and a despeckler to convert the Gaussian beam profile into a homogenous square profile.
  • the size of the beam can be adjusted such that it either matches the field of view or exceeds it slightly to ensure even illumination of all the deposited assay spots in the field of view. This can ensure the high sensitivity of detection for all the assay spots across the field of view.
  • the apparatus set up may include a square core fiber or a Fourier transform filter to convert the Gaussian beam profile into a homogenous square profile.
  • a square profile the noise around the edges compared to the centre of illumination can be reduced.
  • the illumination of the assay spots around the edges or corners of the field of view can be significantly enhanced.
  • the field of view may be between 100 x 100 pm; 130 x 130 pm or 410 x 410 pm. It may be larger.
  • the image field of view is 136 pm x 136 miti
  • the imaging field of view is 205 x 205 m
  • the imaging field of view is 410 x 410 Mm.
  • the field of view may be between 100 Mm x 400 Mm to 100 Mm x 400 Mm. The upper bound is not limiting as the drops could be very large.
  • Figure 1A provides an illustration of five assay spots deposited on a substrate according to the present invention
  • Figure 1 B provides an illustration of four assay spots deposited on a substrate according to the present invention
  • Figure 1 C provides an illustration of one central spot and a portion of four assay spots within a well imaged in a field of view;
  • Figure 2 provides an illustration of a multiplexed assay where the four printed spots comprises different capture components and/or different detection reagents in a well on a substrate;
  • Figure 3 provides a schematic showing a total internal reflection excitation microscope.
  • an image 10 showing a plurality array spots 12 i.e. four or five array spots 12 on the surface of a substrate 14 within a field of view of a TIR microscope using an objective lens.
  • the substrate may comprise one or more wells where the assay spots can be deposited within the wells.
  • Around four to five assay spots can be imaged using a TIR excitation microscope such as a TIRF microscope within a field of view, as illustrated in Figures 1A and 1 B.
  • the assay spots can be deposited onto the substrate using any means.
  • the assay spots can be printed directly onto the substrate using a non-contact microarrayer.
  • Printing microarrays using non-contact microarrayers can produce spots of 20 pm diameter with high spatial accuracy, resolution and reproducibility i.e. distance between spots of ⁇ 5 pm. This enables the printing of four assay spots 12 in a field of view where the assay spots 12 wet the surface as illustrated in Figure 1 B or the printing of up to 5 assay spots 12 within the field of view, as illustrated in Figure 1A.
  • immunoassays can be achieved by microarray contact printing of assay spots 12 comprising one or more capture components and/or one or more detection reagents on the substrate 14, which typically produces spot sizes of the order of hundreds of microns with positional accuracy of around 50 pm. This limits the measurement to one spot per field of view, thereby reducing the throughput of imaging.
  • Each assay spots can comprise a capture component such as an antibody and/or one or more detection reagents.
  • the capture component deposited onto the surface of the substrate can be used to bind onto a target component of interest such as a biomarker.
  • biomarkers include, but are not limited to, immunoglobulins, CRP, NGAL, Leptin, Adiponectin, PIGF, DNAs and/or microRNAs.
  • the capture component may be an antibody.
  • the capture component could be a nucleic acid such as DNA, RNA, mRNA or microRNA, or chemically modified nucleic acid; it could be a protein, or a modified protein; or a peptide; or a polymer; it could be a hormone; or a tethered small molecule configured to capture a protein.
  • the capture component may be a non-specific capture component such as saliva or polylysine.
  • the capture component may be deposited as a uniform layer across the entire surface of each assay spots on the substrate.
  • the detection reagent which can be a secondary antibody, and can be bound with a label can be disposed in various configurations.
  • the label may be a fluorophore, a nanoparticle or a quantum dot.
  • the label can be attached to the detection reagent.
  • the detection reagent can bind to the target component to form a detection reagent-target component complex.
  • the detection reagent-target component complex can then bind to the capture component to form a sandwich complex.
  • the detection reagent can either have inherent light emitting or scattering properties or the detection reagent may comprise a label with light emitting or scattering properties.
  • the detection reagents may be, but is not limited to, one or more of the following: a peptide, a protein, a protein assembly, an oligonucleotide, a polynucleotide, a modified oligonucleotide, a modified polynucleotide, an aptamer, a morpholino, a small molecule, a cell, a cell membrane, a viral particle, a glycan, a conjugated solid particle, a conjugated solid bead or a cofactor.
  • the label may be, but is not limited to, one or more of the following: a luminescence molecule; a fluorescent molecule; a phosphorescence molecule; a chemiluminescent molecule; a molecule that exhibits Rayleigh scattering or Raman scattering; a photon upconversion; an enzyme and its substrate that produces a colorimetric signal; a metallic or inorganic particles e.g. nanoparticles, a polycyclic aromatic hydrocarbon, a metalized complex, a quantum dot or an ion.
  • the ion may be an atomistic ion or a salt of an organic molecule.
  • the label can be attached to the detection reagent.
  • the detection reagent may comprise an antibody.
  • the detection antibody can be fluorescently labelled.
  • each assay spot There may be a single capture component provided within each assay spot. Alternatively, a combination of multiple capture components may be provided.
  • the capture components deposited in one assay spot can be the same or it can be different from one another.
  • the each assay spots within the field of view may comprise the same capture component(s) and/or detection reagent(s) or it may comprise different capture component(s) and/or detection reagent(s). It is advantageous to combine multiple capture components since this allows a more powerful diagnostic ability.
  • the multiple capture components deposited in each assay spot may be spatially addressable.
  • the apparatus set up as disclosed in the present invention is used to detect a plurality of array spots using Total internal reflection (TIR) excitation microscopy.
  • TIR excitation microscopy used for detection of assay spots can include, but is not limited to, Total internal reflection fluorescence (TIRF) microscopes, Raman spectroscopy, Rayleigh microscopy.
  • TIRF Total internal reflection fluorescence
  • Raman spectroscopy Raman spectroscopy
  • Rayleigh microscopy averaging Raman spectroscopy
  • the apparatus may also be used to detect photon up conversion, which relates to the absorption of two or more photons that leads to the emission of light at shorter wavelength than the excitation wavelength.
  • the total internal reflection excitation microscope comprises a light source such as a laser beam for illuminating the assay spots printed onto the surface of the substrate. As the light from the laser beam reaches the surface of the substrate 14, the light can be configured to excite the capture components within the assay spots 12. This may cause the capture components to emit light at a specific wavelength. The light may undergo a single reflection or it may undergo multiple reflections at the surface of the substrate. The emissions from the capture components may be luminescence for example, fluorescence or phosphorescence.
  • the light may be reflected or scattered at the surface of the substrate. Additionally or alternatively other optical assays may be deployed including chemiluminescence, photon upconversion, Raman scattering, Rayleigh scattering or Mie scattering and absorption including chromogenic mechanisms.
  • One or more lenses may be provided in series which can be configured to launch light towards the substrate and to collect the emitted, reflected or scattered light from the assay spots deposited on the substrate. At least one of the lenses in series can be an objective lens.
  • the objective lens is configured to collect the emitted, reflected or scattered light from the assay spots and a detector configured to detect the collected emitted, reflected or scattered light from the objective lens.
  • the detector is further configured to image multiple assay spots simultaneously within a field of view.
  • TIR microscopes and in particular TIRFM can be used to greatly increase the throughput of the measurements without compromising on the sensitivity of detection.
  • the image field of view is 136 pm x 136 pm.
  • the objective lens and the detection camera of the microscope can be suitably set up to provide a field of view of 205 x 205 pm and 410 x 401 pm.
  • FIG. 1C there is shown a plurality of assay spots 12 on a substrate 14 within a field of view.
  • the apparatus set up as disclosed in the present invention can also be applied for imaging of an entire central assay spot along with a portion of 4 assay spots around the edges of the field of view of 136 x 136 pm, when the spot diameters are greater than 70 pm, as illustrated in Figure 1C.
  • the apparatus of the present invention may use lasers with a Gaussian profile, where the illumination intensity is greatest at the centre of the Gaussian, dropping off considerably moving away from the centre towards the tails of the Gaussian.
  • the unevenness of illumination is flattened off by normalising the images to the laser profile.
  • Gaussian profile is most effective when imaging a single assay spot within a field of view. In the case of multiple assay spots, the spots around the edges can suffer from poor illumination. This can result in an increase of noise around the edges leading to loss of sensitivity.
  • the apparatus of the present invention further comprises a square core fiber or a Fourier transform filter to convert the Gaussian beam profile into a homogenous square profile. The size of the beam can be adjusted such that it either matches the field of view or exceeds it slightly to ensure even illumination of all the printed spots on the substrate in the field of view. This ensures the high sensitivity of detection for all the assay spots across the field of view.
  • Objective based TIR imaging requires an refractive index matching fluid such as index matching oil in between the sample and the objective lens to achieve TIR.
  • an instrument such as an Autooiler can be used to automatically feed the index matching fluid e.g. index matching oil to the one or more lenses or to an objective lens which removes the requirement for manual replacement of the index matching fluid and hence, this mininises or reduces any interruptions to the apparatus set up during high throughput imaging.
  • each assay spot comprises a different capture component and/or a different detection reagent in a well. It is highly desirable to image multiplexed assays within a field of view where each spot can comprise a different capture component such as a different antibody and/or a different detection reagent as shown in Figure 2, as it reduces the amount of biofluid sample required for the assay. High throughput imaging of multiple spots within a field of view will increase the throughput by a factor 8 to 10. This also makes it highly desirable in the context of saving reagents and saving the costs of consumables.
  • assay spots printed on the substrate may have different concentration and this in turns, means that the dynamic range can be increased.
  • the total internal excitation microscope 50 comprises a substrate 52 where a sample is deposited onto the surface of the substrate 52.
  • the assay spots are deposited, prefeably printed onto the surface of the substrate 52.
  • Each assay spot comprises a capture component and/or a detection reagent.
  • a light source 56 for example a laser beam such as an excitation laser beam, provides light that can be directed at the substrate to excite the molecules on the assay spots.
  • the lens system 54 may comprise one or more lenses arranged in series which can be configured to direct an incident light to the substrate 52 and to collect the emitted light at a specific wavelength from the assay spots deposited on the substrate 52. Additionally or alternatively, the assay spots may also scatter or reflects light at a specific wavelength. At least one of the lenses arranged in series is an objective lens.
  • the function of the excitation dichroic 58 is to reflect the incident laser beam in to the objective lens or lens system 54 and to reflect out the total internally reflected beam.
  • the autofocus set up 60 comprisesan infrared (IR) or Near-lnfra Red (NIR) laser that is reflected into the objective lens or lens system using the dichroic and its totally internally reflected beam is reflected on to the autofocus detection camera 64 using the same dichroic.
  • the emissions camera 62 receives the collected emitted or scattered light from the lens system or the objective lens 54.
  • the autofocus camera 64 tracks the position of the totally internally reflected IR or NIR beam position and serves to keep the sample in focus.
  • the criteria may include the following steps as outline below; providing a multi-axis motion controlled stage to allow the substrate containing the assay spots to be moved across the objective lens rapidly; automatic focus adjustment to allow single molecules to be resolved and light levels collected to be consistent; providing a suitable substrate is provided where the assay spots containing one or more capture components and/or one or more detection reagents, the assay spots can be deposited onto the recepactle that enables high throughput reading; providing an automated feeding refractive index matching fluid between an objective lens and the assay spots on the substrate; providing a large dynamic range and single molecule sensitivity detection; taking account of the gaussian intensity distribution of the illuminting laser beam; printing assay spots of
  • An apparatus and method may also be provided for detecting the presence and/or the amount of a target component in a sample of a biological fluid, the apparatus comprising a plurality of assay spots printed a substrate, each assay spot comprising one or more capture components and/or one or more detection reagents, a total internal reflection fluorescence microscopy comprising a light source for illuminating the assay spots; and an objective lens configured to image multiple assay spots simultaneously, wherein the total internal reflection fluorescence microscopy is further configured to detect the presence and/or the amount of emitted light to provide an indication of the presence and/or the amount of the target component within the sample.

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  • Health & Medical Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Physics & Mathematics (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Analytical Chemistry (AREA)
  • Biochemistry (AREA)
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  • General Physics & Mathematics (AREA)
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  • Investigating, Analyzing Materials By Fluorescence Or Luminescence (AREA)

Abstract

L'invention concerne un appareil destiné à imager une pluralité de points de test. L'appareil comprend une pluralité de points de test déposés sur un substrat, chaque point de test comprenant un ou plusieurs composants de capture et/ou un ou plusieurs réactifs de détection ; et un microscope à excitation par réflexion totale interne comprenant une source de lumière pour éclairer les points de test ; un système de lentille étant conçu pour projeter de la lumière vers le substrat et pour collecter la lumière émise, réfléchie ou diffusée à partir des points de test, le système de lentille comprenant une lentille d'objectif ; et un détecteur conçu pour détecter la lumière émise, réfléchie ou diffusée collectée par le système de lentille, le détecteur étant en outre conçu pour imager de multiples points de test simultanément dans un champ de vision.
PCT/GB2021/051768 2020-07-10 2021-07-09 Améliorations apportées ou se rapportant à un appareil d'imagerie WO2022008931A1 (fr)

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GBGB2010691.0A GB202010691D0 (en) 2020-07-10 2020-07-10 Improvements in or relating to an apparatus for imaging

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2006011346A1 (fr) * 2004-07-30 2006-02-02 National University Corporation NARA Institute of Science and Technology Lecteur de microreseaux
WO2006138257A2 (fr) * 2005-06-15 2006-12-28 Callida Genomics, Inc. Reseaux de molecules simples pour analyse genetique et chimique
WO2010054000A1 (fr) * 2008-11-04 2010-05-14 Richard Joseph Procédés, cellules de circulation et systèmes pour une analyse de cellule unique

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2006011346A1 (fr) * 2004-07-30 2006-02-02 National University Corporation NARA Institute of Science and Technology Lecteur de microreseaux
WO2006138257A2 (fr) * 2005-06-15 2006-12-28 Callida Genomics, Inc. Reseaux de molecules simples pour analyse genetique et chimique
WO2010054000A1 (fr) * 2008-11-04 2010-05-14 Richard Joseph Procédés, cellules de circulation et systèmes pour une analyse de cellule unique

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