WO2022119459A1 - Dispositif et procédé de détection d'analyte - Google Patents

Dispositif et procédé de détection d'analyte Download PDF

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Publication number
WO2022119459A1
WO2022119459A1 PCT/NZ2021/050215 NZ2021050215W WO2022119459A1 WO 2022119459 A1 WO2022119459 A1 WO 2022119459A1 NZ 2021050215 W NZ2021050215 W NZ 2021050215W WO 2022119459 A1 WO2022119459 A1 WO 2022119459A1
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Prior art keywords
colorimetric
reader
assay
colorimetric reader
sample
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PCT/NZ2021/050215
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English (en)
Inventor
Sandeep Kumar VASHIST
Xuzhi TAN
Howard Stanley MOORE
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Pictor Limited
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Publication of WO2022119459A1 publication Critical patent/WO2022119459A1/fr

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    • 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
    • G01N21/25—Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands
    • G01N21/251—Colorimeters; Construction thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
    • A61B5/00—Measuring for diagnostic purposes; Identification of persons
    • A61B5/145—Measuring characteristics of blood in vivo, e.g. gas concentration, pH value; Measuring characteristics of body fluids or tissues, e.g. interstitial fluid, cerebral tissue
    • A61B5/14546—Measuring characteristics of blood in vivo, e.g. gas concentration, pH value; Measuring characteristics of body fluids or tissues, e.g. interstitial fluid, cerebral tissue for measuring analytes not otherwise provided for, e.g. ions, cytochromes
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
    • G01N15/06—Investigating concentration of particle suspensions
    • 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
    • G01N21/25—Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands
    • G01N21/255—Details, e.g. use of specially adapted sources, lighting or optical systems
    • 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
    • G01N21/25—Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands
    • G01N21/256—Arrangements using two alternating lights and one detector
    • 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
    • G01N33/54386—Analytical elements
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N35/00—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor
    • G01N35/00029—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor provided with flat sample substrates, e.g. slides
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N35/00—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor
    • G01N35/00584—Control arrangements for automatic analysers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
    • A61B5/00—Measuring for diagnostic purposes; Identification of persons
    • A61B5/103—Detecting, measuring or recording devices for testing the shape, pattern, colour, size or movement of the body or parts thereof, for diagnostic purposes
    • A61B5/1032—Determining colour for diagnostic purposes
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
    • G01N15/01—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials specially adapted for biological cells, e.g. blood cells
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
    • G01N15/06—Investigating concentration of particle suspensions
    • G01N15/075—Investigating concentration of particle suspensions by optical means
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
    • G01N15/06—Investigating concentration of particle suspensions
    • G01N2015/0681—Purposely modifying particles, e.g. humidifying for growing
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
    • G01N15/06—Investigating concentration of particle suspensions
    • G01N2015/0687—Investigating concentration of particle suspensions in solutions, e.g. non volatile residue
    • 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
    • G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
    • G01N21/77—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator
    • G01N21/78—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator producing a change of colour
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2474/00—Immunochemical assays or immunoassays characterised by detection mode or means of detection
    • 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
    • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
    • G01N33/581—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with enzyme label (including co-enzymes, co-factors, enzyme inhibitors or substrates)
    • 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
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere

Definitions

  • the present invention relates generally to diagnostics, and more specifically to a colorimetric reader device and related method of use of the device to detect an analyte in a sample.
  • An assay generally involves bringing a portion of the sample into contact with an assay in order to qualitatively assess or measure the presence and/or amount of an anticipated analyte in the sample.
  • Many such test assays are known to detect for the presence of a specific antigen, antibody, biomarker, such as a protein, virus, cell, chemical, nucleic acid, hormone, or other material in a biological sample, including a biological fluid sample, such as blood, urine, saliva and the like.
  • a biological fluid sample such as blood, urine, saliva and the like.
  • Colorimetric immunoassays are considered an accepted standard for analyte detection. Such assays typically involve a primary antigen-specific antibody that binds to a target analyte, such as an antigen, from the sample, with antigen binding detected using a secondary antibody linked to a colorimetric detection system.
  • a target analyte such as an antigen
  • the most widely used format is enzyme-linked immunosorbent assays (ELISA), having well-established protocols for the measurement of an analyte in a biological sample.
  • the invention provides a portable and low-cost colorimetric reader for in vitro diagnostics and bioanalytical applications, namely, detecting an analyte in a biological sample.
  • the reader includes: a platform configured to hold an assay substrate, the assay substrate being configured to perform an assay of an analyte; an illumination source configured to illuminate the assay substrate; an optical imaging system operable to capture an image of all or a portion of the assay substrate; an electronics module(s) having the functionality to control the intensity of light emitted from the illumination source; and an image processor having the functionality to receive and process the images generated by the optical imaging system.
  • the processing includes detecting a fiduciary marker(s) present on the assay substrate, automated gridding and orientation of the assay substrate to analyze light intensity associated with the analyte's binding to a reagent of the assay substrate, and detection of the analyte.
  • the invention provides a method of analyzing a sample.
  • the method includes: providing a colorimetric reader of the invention; loading a sample onto the assay substrate; generating an image of the assay substrate via the optical imaging system; and processing the optical image via the image processor, wherein the processing comprises detection of a fiduciary marker(s) present on the assay substrate, automated gridding and orientation of the assay substrate to analyze light intensity associated with the binding of an analyte in the sample to a reagent of the assay substrate, and detection of the analyte, thereby analyzing the sample.
  • the invention provides a method of detecting a disease or disorder in a human or non-human subject.
  • the method includes: obtaining a sample from a subject; analyzing the sample using the method of analyzing a sample of the invention; and detecting a disease or disorder in the subject based on the analysis, thereby detecting a disease or disorder in the subject.
  • Figure 1 is a top view of a reader device showing the layout of device components in accordance with aspects of the invention.
  • Figure 2 is a isometric view of the device depicted in Figure 1 showing the door's outer surface when it is closed in accordance with aspects of the invention.
  • Figure 3 is a right side view of the device depicted in Figure 1 showing the back panel and assay substrate, e.g., PictArrayTM slide, aligned and facing the camera, attached to the inner side of the door in accordance with aspects of the invention.
  • back panel and assay substrate e.g., PictArrayTM slide
  • Figures 4A-4E illustrate the design layout of the invention's reader device, e.g., Pictlmager96+TM, for use with a 96 well microtiter assay substrate (excluding enclosure, the receiving member, and control electronics) in accordance with aspects of the invention.
  • Figure 4A is a 3D view
  • Figure 4B is a 3D sectional view with a single camera in the system
  • Figure 4C is a cross-sectional view including an illustration of the viewing window
  • Figure 4D shows the top half of the system setup
  • Figure 4E shows the intended detection area of the 96 well substrate for a single camera.
  • top board PCB panel with mounted camera surrounded by LEDs
  • diffuser typically opal acrylic sheet
  • MIA multiplex immunoassay
  • bottom diffuser PCB panel with LED arrays.
  • Figures 5A-5E illustrate the design layout of the reader device of the invention, e.g., Pictlmager+TM, for use with a 16 well microtiter assay substrate (excluding enclosure, the receiving member, and control electronics) in accordance with aspects of the invention.
  • Figure 5A is a 3D view
  • Figure 5B is a 3D sectional view with a single camera in the system
  • Figure 5C is a front view including an illustration of the viewing window
  • Figure 5D shows the top half of the system setup
  • Figure 5E shows the intended detection area of the 16 well assay substrate for a single camera.
  • top board PCB panel with mounted camera surrounded by LEDs
  • diffuser typically opal acrylic sheet
  • 16- well PictArrayTM assay substrate PCB panel with LED arrays.
  • Figure 6 shows images captured by the reader device of the invention, e.g., Pictlmager+TM for the biotinylated goat anti-mouse IgG antibody titration slide in accordance with aspects of the invention. The images were used to determine the average composite pixel intensity of colorimetric spots corresponding to a particular concentration across all wells in a PictArrayTM assay substrate.
  • the reader device of the invention e.g., Pictlmager+TM for the biotinylated goat anti-mouse IgG antibody titration slide in accordance with aspects of the invention.
  • the images were used to determine the average composite pixel intensity of colorimetric spots corresponding to a particular concentration across all wells in a PictArrayTM assay substrate.
  • Figure 7 shows the graph of composite pixel intensity and biotinylated goat antimouse IgG antibody concentration as shown in Figure 6 in accordance with aspects of the invention.
  • Figure 8 shows the LED panel designs of the reader devices of the invention, i.e., Pictlmager+TM and Pictlmager96+TM, in accordance with aspects of the invention.
  • Figure 9 shows the typical surface-mount LED light radiation pattern and LED spacing for a reader device of the invention, i.e., for both Pictlmager+TM and Pictlmager96+TM, in accordance with aspects of the invention.
  • Figure 10 shows aspects of the LED layout for a reader device of the invention, i.e., Pictlmager+TM, in accordance with aspects of the invention.
  • Figure 11 shows the LED panel design for a reader device of the invention, i.e., Pictlmager+TM, in accordance with aspects of the invention.
  • Figure 12 shows the LED panel design for a reader device of the invention, i.e., Pictlmager96+TM, in accordance with aspects of the invention.
  • FIG. 13 is a block diagram showing the hardware functions for a reader device of the invention, i.e., Pictlmager+TM, in accordance with aspects of the invention.
  • FIG 14 is a block diagram showing the hardware functions for a reader device of the invention, i.e., Pictlmager96+TM, in accordance with aspects of the invention.
  • Figure 15 is a block diagram showing the data processing steps of image processing and/or analysis for a reader device of the invention in accordance with aspects of the invention.
  • the present invention relates to the field of portable and low-cost colorimetric readout devices, which are primarily used for the readout of membrane-based and membrane-free spot-based microarrays but may also be used for the readout of conventional ELISAs and lateral flow immunoassays (LFIAs).
  • the present invention provides a colorimetric reader device for detecting an analyte in a biological sample that obviates the limitations of the existing commercial readers for multiplex immunoassays, which are bulky, expensive, complex, and have long readout times.
  • the device of the invention has many-fold lower manufacturing cost as it is highly simplified and doesn’t require any costly mechanical assembly parts and movement of cameras, light sources or multi-well microtiter plates (MTPs).
  • the device of the invention is many-fold compact and lightweight in comparison to other commercial readers. Additionally, as there is no mechanical movement and imaging is done instantly in one image capture, the readout is also many-fold faster than the existing commercial readers.
  • the invention provides a very robust colorimetric reader device, which is easy to maintain and repair and doesn’t require costly and complex engineering steps. Further, the same device may be used for the colorimetric analysis of conventional ELISAs and LFIAs.
  • references to “the device” include one or more devices
  • references to “the system” include one or more systems
  • references to “the method” include one or more methods, and/or steps of the type described herein which will become apparent to those persons skilled in the art upon reading this disclosure and so forth.
  • the invention pertains to the development of compact, simple, low-cost, and robust colorimetric readers for the readout of various in vitro diagnostic formats.
  • the reader is specifically designed for imaging of spot microarrays on a 16-well membrane-based assay substrate, e.g., PictArrayTM platform, and a membrane- free 96-well microtiter plate assay substrate.
  • these formats are used for the development of multiplex immunoassays (MIAs) for human and veterinary diagnostics.
  • MIAs multiplex immunoassays
  • the present disclosure illustrates multiple reader designs, e.g., Pictlmager+TM and Pictlmager96+TM for the readout of membrane-based MIAs on a 16-well membrane-based assay substrate, e.g., PictArrayTM platform, and a membrane-free 96-well microtiter plate assay substrate, e.g., PictArraysTM platform.
  • a 16-well membrane-based assay substrate e.g., PictArrayTM platform
  • a membrane-free 96-well microtiter plate assay substrate e.g., PictArraysTM platform.
  • the technical specification and characteristics of these colorimetric readers are specified in extensive detail herein.
  • the reader devices described herein may be used for the colorimetric readout of conventional ELISAs, as well as other bioanalytical tests (such as those of protein quantification) with the same sensitivity and performance as conventional bulky and costly microplate readers, such as those manufactured by Tecan and Thermo Fischer Scientific. Additionally, the reader devices described herein may be used for the colorimetric readout of single and multiple conventional LFIA strips, respectively.
  • the invention provides a portable and low-cost colorimetric reader device for in vitro diagnostics and bioanalytical applications, namely, detecting an analyte in a biological sample.
  • the reader 10 includes: a platform 20 configured to hold an assay substrate 30, the assay substrate 30 being configured to perform an assay of an analyte; an illumination source 40 configured to illuminate the assay substrate 30; an optical imaging system 50 operable to capture an image of all or a portion of the assay substrate 30; an electronics module(s) 60 having the functionality to control the intensity of light emitted from the illumination source 40; and an image processor having the functionality to receive and process the images generated by the optical imaging system 50.
  • the processing includes detection of a fiduciary marker(s) present on the assay substrate, automated gridding, and orientation of the assay substrate to analyze light intensity associated with analyte’s binding to a reagent of the assay substrate, and detection of the analyte.
  • the optical system includes one or more cameras 80, which can be arranged as an array as detailed further herein.
  • the reader device further includes a light diffuser substrate 80 disposed adjacent to the illumination source 40.
  • Membrane-based MIAs require illumination from the top as the membranes have low transparency, making it difficult for the light to penetrate and thus illuminate the colorimetric spot microarray. Further, the camera must be positioned at the top to image the colorimetric spots.
  • Pictimager+TM enables the colorimetric readout of membrane-based MIAs performed on a 16-well PictArrayTM platform, while Pictlmager96+TM is used for the colorimetric readout of membrane-based MIAs performed on 96-well MTPs.
  • Membrane-free MIAs are performed using conventional 96-well MTPs, having a very high transparency index, which enables light to be transmitted at very high efficiency. Therefore, it can be illuminated from the bottom or the top. However, the imaging is always done from the camera that is mounted on the top. Pictlmager96+TM enables the colorimetric readout of membrane-free MIAs using the bottom as well as top illumination.
  • the reader devices of the invention include a slide/plate holder.
  • the colorimetric readers of the invention include a holder either for a 16-well PictArrayTM slide (in case of Pictlmager+TM) or a standard 96-well MTP (in case of Pictlmager96+TM). Both the Pictlmager+TM and Pictlmager96+TM include mechanical alignment mechanisms that attach to the base holder to create holders for one and up to 4 LFIA cassettes, respectively. The mechanical attachments can easily attach/detach to the base holders.
  • the holder may be motorized to incorporate defined insertion and ejection of the 96-well MTP. This is to improve the handling of the plates after it is placed on the holder. Accurate motion and positioning of the tray can be defined in the software-controlled motor function, with end-stop sensors implemented for mechanical collision protection as a last resort. The motor function can also detect any possible jamming due to obstacles when the tray is inserted. When ELISA tests are being carried out, the motorized tray will eliminate any spillage from human error during inserting or ejecting the holder tray.
  • the reader devices of the invention include housing.
  • the housing provides an optically opaque black body environment that prevents ambient light from interfering with internal illumination and image capture.
  • the reader devices of the invention include an illumination source.
  • the illumination source includes one or more panels of illumination sources (e.g., an array of light-emitting diodes (LEDs) or organic LED (OLED) panel) located either above and/or below the 16-well PictArrayTM or 96-well MTP platform.
  • Pictlmager+TM includes top illumination only, while Pictlmager96+TM includes both top and bottom illumination.
  • the function of the panels of illumination sources is to provide uniform and controlled light intensity to the 16-well PictArrayTM or 96-well MTP, which enables the camera to image the colorimetric spot microarray in the wells.
  • the luminous intensity of LEDs is directly proportional to the applied current through each LED, the inter-device variation of the light intensity is controlled and held constant via controlling the current.
  • the sRGB specification which is currently standardized as IEC 61966-2-1 : 1999/ AMDl :2003, assumes a dimly lit encoding (creation) environment with an ambient correlated colour temperature (CCT) of 5000K
  • the reader devices of the invention include an optical imaging system.
  • the optical imaging system includes one or more camera modules, which capture the images of colorimetric spot microarrays in several wells of the 16-well PictArrayTM or 96-well MTP. It is located at the top of the device with the camera(s) facing downwards.
  • Pictlmager+ employs only one camera, located in the centre of the device at the top, to image all the 16 wells in the PictArrayTM.
  • Pictlmager96+TM has six cameras covering the entire 96-well MTP with one camera located in the centre of each 4x4 wells.
  • the device includes a 4-camera setup, where 4 cameras are mounted in a column. Each camera is located in the center of 2x2 wells, which enables the 4 cameras to image the 2x8-well strips within the 96-well MTP.
  • the motorized tray then moves the MTP to 5 different locations of 2x8 well strips to image the entire 96-well MTP.
  • a possible variant of the 4-camera setup includes a motorized camera array instead to achieve the same outcome.
  • the reader devices of the invention include image processing functionality and electronics.
  • image processing is achieved by a microprocessor in the device or via interfacing the data to an external computer.
  • the imaging system transmits one or more images from the optical image sensor to the image processing unit, where it is digitally processed.
  • Software and processing functionality then takes the image and extracts the optical intensity of each colorimetric spot in the microarrays in different wells of 16-well PictArrayTM or 96-well MTP.
  • additional electronics functionality is provided to perform one or more of the following: controlling the light intensity of LED panels; powering and physically communicating with the camera(s); and/or provide motor functions to control the inversion and ejection of the plate holder tray.
  • the reader devices of the invention include functionality, such as a processor having software for image processing and/or analysis.
  • the colour intensity of each spot in the microarray is determined by the use of a gridding technique in the software or application, which places grids on all the microarrays using a fiduciary marker(s) to align the grid at the appropriate position.
  • the intensity values are obtained in a database or spreadsheet (e.g., Microsoft ExcelTM spreadsheet file), which is used for the analysis of results.
  • the software or application then generates quantitative index values for samples contained in each spot and performs qualitative clinical determinations.
  • Pictorial® is used for image analysis.
  • the software functionality analyses the captured images to detect the microarray spots in each well and generate the pixel intensity results.
  • the software first identifies the PictArrayTM wells and then detects the positive control spots within each well that appears after the MIA.
  • the positive control spots act as alignment anchors, which are used by the software to place a microarray grid for all spots within each well.
  • the software algorithm then splits the colorimetric image of the PictArrayTM into the Red, Green and Blue (RGB) channels, and extracts the composite pixel intensity for each spot based on the weighted contribution of each channel.
  • the composite pixel intensity of the colorimetric PictArrayTM spots will be in the range of 0 to 255.
  • the data generated from each spot is collated with the layout of PictArrayTM and tested samples to provide a final test report for the samples being analysed.
  • the data processing steps are as follows.
  • the software functionality also provides low-level control functions of the devices to adjust the right settings for various situations.
  • the software functionality provides the user interface, where operator(s) can provide input and extract relevant data from the devices.
  • Figures 4A-4E illustrate the design layout of the reader device in one aspect of the invention.
  • Figures 5A-5E illustrate the design layout of the reader device in one aspect of the invention.
  • Table 1 provides the physical specifications of aspects of the reader devices of the invention and certain components thereof.
  • the LEDs for both Pictlmager+TM and Pictlmager96+TM include LED strings of 4 in series, with combined voltage of 12V (around 3 V per LED). This configuration is the industrial standard on DC 12V systems.
  • the LED panel supply is provided through a current-controlled DC/DC converter to about 12V across the LED strings in parallel.
  • the luminous intensity of LEDs is directly proportional to that of the applied current through each LED. So, for a string of LEDs, the current must be identical, and thus the light intensity must also be the same. It is highly likely that the LEDs used on a panel will be from the same manufacturing batch, which means that the characteristics of each of them will be highly consistent.
  • the resistor’s inclusion for each of the LEDs string provides an additional crude/simplest intensity balancing of the LEDs. Therefore, these features ensure the uniform intensity of each LED on the whole panel.
  • the nominal viewing angle of LEDs is 120 degrees and they retain >85% intensity within 30 degrees from the center. So for LEDs 10 mm apart, the light reaching the diffuser 8+ mm away will be sufficiently mixed. The diffuser would provide another layer of mixing so that the light exiting the diffuser would be uniform across the whole diffuser.
  • the camera is situated at the center of the LED panel, while 32 LEDs surround the camera.
  • the LED panel needs to be double the length of the 16 well slide as shown in Figure 10.
  • the LED panel’s final design has the LEDs 10 mm apart and 8 mm from the diffuser.
  • Figure 11 illustrates the LED design in this aspect of the invention.
  • the bottom panel of the 96 well for backlight has LEDs that are 10 mm apart horizontally, 8 mm apart vertically, and 14 mm away from the diffuser. It only needs to be the 96 well plate’s size to uniformly backlit the backlight diffuser and the 96-well plate.
  • Figure 12 illustrates the LED design in this aspect of the invention.
  • the top panel design considerations for Pictlmager96+TM are similar to the Pictimager+TM design, with clearances for the six camera lenses.
  • the LEDs are spaced 10 mm apart horizontally and 8 mm vertically and are integrated with the camera’s multiplex board.
  • the camera shall start steaming video and available for capturing stills.
  • the first two cameras shall start streaming on the screen at 8 MP setting and focused on imaging 4x4 wells each possibly with reduced displayed resolution. 10.
  • the still images are then taken automatically after a fixed time and stored for both cameras at full 8 MP resolution.
  • the first two cameras shall start streaming on the screen at 8 MP setting and focused on imaging of 2x2 wells each possibly with reduced displayed resolution.
  • the still images are then taken automatically after a fixed time and stored for both cameras at full 8 MP resolution
  • step 5 Return to step 5 to start a new test or shut down the device.
  • Figure 15 illustrates the data processing steps for Pictlmager+TM, Pictlmager96+TM’ and PictlmagerTM4 Cam in various aspects of the invention.
  • MIA - 16-well PictArrayTM (Membrane Based)
  • Pictlmager+TM may be used for the colorimetric readout of assays on a conventional membrane-based spot microarray platform including 16 wells, e.g., PictArrayTM. It is a compact, lightweight, rapid, and robust colorimetric reader, with only a single camera, for membrane-based PictArraysTM.
  • MIA - 96-well PictArrayTM (Membrane Free and Membrane Based)
  • Pictlmager96+TM may be used for the colorimetric readout of assays on Pictor’s new membrane-free spot microarray platform comprising of 96 wells. It is a portable (small benchtop model) and lightweight colorimetric readout device, enabling rapid readout of spot microarrays printed in each well of an entire 96-well MTP. As Pictlmager96+TM has both top and bottom illumination, which may be selected by the user depending on whether they will be reading membrane-based or membrane-free spot microarrays, it can be used for the readout of both spot microarray formats in the 96-well MTP. It is expected that this device would be similar in performance to sciREADER CL2TM by Scienion AG, Germany, although it will be far more compact, lightweight, and rapid.
  • Pictlmager96+TM may be used for the colorimetric readout of conventional colorimetric ELISAs in 96-well MTP.
  • Pictlmager96+TM based colorimetric readout is similar to that of absorbance readout in a conventional ELISA.
  • Pictlmager96+TM signal is equivalent to that of an absorbance signal obtained from a commercial microplate reader such as from Tecan, which will be demonstrated by normalizing both the signals and plotting them for several ELISAs.
  • Pictlmager96+TM is at least 10-fold lower in cost, compact, faster, and more robust than a commercial microplate reader.
  • Pictlmager+TM and Pictlmager96+TM may be used for the readout of colorimetric LFIAs.
  • Pictlmager+TM can read one LFIA cartridge, while Pictlmager96+TM can read up to 4 LFIA cartridges.
  • Pictlmager96+TM may be used for the readout of membrane-free multiplex immunoassays in 96-well MTP. It could be an integral part of the automated clinical analyzer.
  • Pictlmager96+TM may be used for the colorimetric readout of centrifugal microfluidics-based automated immunoassays that are performed on membrane- free and transparent LabDiskTM platforms.
  • Pictlmager96+TM may be used for the colorimetric readout of lab- on-a-chip (LOC) based rapid immunoassays that are performed in a fully-integrated LOC using paramagnetic beads or solid substrates.
  • LOC lab- on-a-chip
  • both Pictlmager+TM and Pictlmager96+TM may be used for the colorimetric readout of immunoassays performed by researchers on a wide range of paperbased formats. These assays employ the top illumination in our readers for the readout. [00093] Other Emerging Immunoassay Formats and Bioanalytical Applications
  • both Pictlmager+TM and Pictlmager96+TM may be used for the readout of any colorimetric immunoassay, biochemical assay or other analytical application that involves the determination of color. If the color determination has to be done on opaque substrate, both Pictlmager+TM and Pictlmager96+TM may be used. But if the color determination needs to be done on a transparent substrate or platform, Pictlmager96+TM may be used.
  • the invention provides a method of analyzing a sample.
  • the method includes: providing a colorimetric reader of the invention; loading a sample onto the assay substrate; generating an image of the assay substrate via the optical imaging system; and processing the optical image via the image processor, wherein the processing comprises detection of a fiduciary marker(s) present on the assay substrate, automated gridding and orientation of the assay substrate to analyze light intensity associated with binding of an analyte in the sample to a reagent of the assay substrate, and detection of the analyte, thereby analyzing the sample.
  • the invention provides a method of detecting a disease or disorder in a human or non-human subject.
  • the method includes: obtaining a sample from a subject; analyzing the sample using the method of analyzing a sample of the invention; and detecting a disease or disorder in the subject based on the analysis, thereby detecting a disease or disorder in the subject.
  • Biotinylated Antibody Titration Slides were prepared on BA83 and Biodyne B slides by spotting various concentrations of goat anti-mouse IgG biotin-labeled in different wells of the 16-well PictArrayTM platform using a microarray printer. The procedure is specified in more detail below.
  • Goat anti-mouse IgG biotin-labeled (1 mg/ml, 69E05) was diluted to 20 pg/ml by adding 2 pl of biotinylated antibody into an Eppendorf tube containing 50 pl of 2x Print buffer (55E61) and 48 pl of deionized water (DIW). Further, 600 pl of IX Print Buffer was prepared by adding 300 pl of 2X Print Buffer to 300 pl of DIW.
  • a 2-fold dilution series was carried out using 20 pg/ml of biotinylated antibody as the highest concentration.
  • the dilution was done by taking 50 pl of biotinylated antibody (20 pg/ml) as the starting material and adding it to 50 pl of lx print buffer. This led to the biotinylated antibody concentrations of 10, 5, 2.5, 1.25, 0.625, 0.3125, 0.1563, 0.078, and 0.039 pg/ml.
  • the source plate was used to print 21 PictArrayTM slides on Percy Array er (1 lx
  • Biodyne including slide 1
  • lOx BA83 The printing was carried out overnight, followed by the visual inspection of printed slides by putting them on a bottom illuminated light panel. Subsequently, the biotinylated antibody printed slides were blocked with dispensing 75 pl of IX blocker (72E45) to each PictArrayTM well and incubating it for 60 min at room temperature. Finally, the IX blocker was discarded by inverting the PictArrayTM in a chemical waste container /sink and tapping it onto a paper towel.
  • 20X TMB Substrate solution (Scytek, Cat#ACK500, Lot#41298) was diluted to IX by mixing 1 mL of 20X TMB Substrate solution with 19 mL of Substrate buffer (Scytek, Cat#ACK500, Lot#41298). Thereafter, 50 pl of IX TMB substrate solution was added to each well of the PictArrayTM and incubated at room temperature for 5 min. This was followed by washing the PictArrayTM slide three times with IX PBST washing buffer. The colorimetric PictArrayTM slides were then incubated at 37°C for 20 min to dry the wells.
  • the colorimetric spots corresponding to various biotinylated antibody concentrations printed in the PictArrayTM after binding to SA-HRP and TMB substrate solution, were then read by Pictlmager+TM that employs a 5-MP camera module.
  • the Pictlmager+TM is a low-cost multiplex array reader for membrane-based 16-well PictArraysTM, which is powered through a standard computer USB port.
  • the biotinylated antibody titration slide was inserted into the receiving holder of Pictlmager+TM and presented directly to the camera module at the top of the device in its field of view.
  • the PictArrayTM is illuminated via light emitted from a panel of LEDs surrounding the camera, which is diffused via a diffuser before it falls onto the biotinylated antibody titration slide.
  • the control circuit interfaces the camera module to a computer, which contains the image processing software and also controls the intensity of the light source.
  • the colorimetric readout measurements of biotinylated antibody titration slide were compared with those made using commercially available colorimetric microarray reader, e.g., sciREADER CL2TM from Scienion AG, Germany.

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Abstract

La présente invention concerne un lecteur colorimétrique portable et peu coûteux destiné à des applications diagnostiques et bioanalytiques in vitro. L'invention concerne en outre des procédés d'utilisation du dispositif pour détecter un analyte dans un échantillon biologique. Le dispositif de lecture colorimétrique de l'invention peut être utilisé pour la lecture de microréseaux à base de membrane et à base de point sans membrane, ainsi que pour la lecture de tests ELISA et LFIA classiques. Le lecteur comprend : une plateforme conçue pour contenir un substrat de test, le substrat de test étant conçu pour effectuer un test d'un analyte ; une source d'éclairement conçue pour éclairer le substrat de test ; un système d'imagerie optique pouvant être utilisé pour capturer une image de tout ou partie du substrat de test ; un ou plusieurs modules électroniques dont la fonctionnalité permet de commander l'intensité de la lumière émise par la source d'éclairement ; et un processeur d'images dont la fonctionnalité permet de recevoir et de traiter les images générées par le système d'imagerie optique.
PCT/NZ2021/050215 2020-12-03 2021-12-03 Dispositif et procédé de détection d'analyte WO2022119459A1 (fr)

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Citations (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5850472A (en) * 1995-09-22 1998-12-15 Color And Appearance Technology, Inc. Colorimetric imaging system for measuring color and appearance
EP1609874A2 (fr) * 2004-05-21 2005-12-28 Bioanalisi Centro Sud S.n.c. di Perseu Sinibaldo e C. Système pour la recherche et identification d' agents pathogéniques.
US20060222567A1 (en) * 2005-04-01 2006-10-05 Hafellner Body fluid testing component for simultaneous analyte detection
US20140072189A1 (en) * 2012-09-05 2014-03-13 Sidhant Jena Portable medical diagnostic systems and methods using a mobile device
US20180067109A1 (en) * 2006-11-28 2018-03-08 Pictor Limited Assay membrane and method of use thereof
US20180149600A1 (en) * 2016-11-30 2018-05-31 Idexx Laboratories, Inc. Lateral flow assay reader based on human perception and method relating thereto
WO2018148471A2 (fr) * 2017-02-08 2018-08-16 Essenlix Corporation Optique, dispositif et système d'essai
WO2020082029A1 (fr) * 2018-10-18 2020-04-23 The Regents Of The University Of California Dispositif et système de test sérodiagnostic pour la maladie de lyme à un stade précoce à l'aide d'un dosage immunologique multiplexé

Patent Citations (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5850472A (en) * 1995-09-22 1998-12-15 Color And Appearance Technology, Inc. Colorimetric imaging system for measuring color and appearance
EP1609874A2 (fr) * 2004-05-21 2005-12-28 Bioanalisi Centro Sud S.n.c. di Perseu Sinibaldo e C. Système pour la recherche et identification d' agents pathogéniques.
US20060222567A1 (en) * 2005-04-01 2006-10-05 Hafellner Body fluid testing component for simultaneous analyte detection
US20180067109A1 (en) * 2006-11-28 2018-03-08 Pictor Limited Assay membrane and method of use thereof
US20140072189A1 (en) * 2012-09-05 2014-03-13 Sidhant Jena Portable medical diagnostic systems and methods using a mobile device
US20180149600A1 (en) * 2016-11-30 2018-05-31 Idexx Laboratories, Inc. Lateral flow assay reader based on human perception and method relating thereto
WO2018148471A2 (fr) * 2017-02-08 2018-08-16 Essenlix Corporation Optique, dispositif et système d'essai
WO2020082029A1 (fr) * 2018-10-18 2020-04-23 The Regents Of The University Of California Dispositif et système de test sérodiagnostic pour la maladie de lyme à un stade précoce à l'aide d'un dosage immunologique multiplexé

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