WO2022119459A1 - Device and method of analyte detection - Google Patents

Abstract

The present invention provides a portable and low-cost colorimetric reader for in vitro diagnostics and bioanalytical applications. Further provided are methods of using the device to detect an analyte in a biological sample. The colorimetric reader device of the invention may be used for the readout of membrane-based and membrane-free spot-based microarrays, as well as the readout of conventional ELISAs and LFIAs. 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; one or more electronics modules 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.

Description

DEVICE AND METHOD OF ANALYTE DETECTION

BACKGROUND OF THE INVENTION

FIELD OF THE INVENTION

[0001] 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.

BACKGROUND INFORMATION

[0002] The field of medicine relies heavily on investigative testing performed on various compositions of biological material. Testing biological samples, such as bodily fluids containing cells or other analytes, often involves collecting a biological sample and exposing the sample to a diagnostic testing assay.

[0003] 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. Such tests have been designed, manufactured, produced, and marketed for use in such fields as home, industrial, veterinary, environmental, food, bioanalytical and occupational testing, among others.

[0004] 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. 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.

[0005] Existing conventional colorimetric readers for multiplex immunoassays have several limitations. For example, conventional readers are bulky, expensive, complex, and take more time for readout. As such, there is a need for simple, portable, and low-cost colorimetric readers for in vitro diagnostics and bioanalytical applications, such as multiplexed immunoassays in a microarray format. SUMMARY OF THE INVENTION

[0006] In one embodiment, 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. In various aspects, 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.

[0007] In another embodiment, 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.

[0008] In yet another embodiment, 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.

BRIEF DESCRIPTION OF THE FIGURES

[0009] Figure 1 is a top view of a reader device showing the layout of device components in accordance with aspects of the invention. [00010] 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.

[00011] Figure 3 is a right side view of the device depicted in Figure 1 showing the back panel and assay substrate, e.g., PictArray™ slide, aligned and facing the camera, attached to the inner side of the door in accordance with aspects of the invention.

[00012] Figures 4A-4E illustrate the design layout of the invention's reader device, e.g., Pictlmager96+™, 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; and, Figure 4E shows the intended detection area of the 96 well substrate for a single camera. The labelled parts in Figure 4A correspond to: 1) top board: PCB panel with mounted camera surrounded by LEDs; 2) diffuser, typically opal acrylic sheet; 3) lines illustrating the field of view of cameras; 4) 96- well microtiter plate for multiplex immunoassay (MIA) or conventional ELISA; 5) bottom diffuser; and 6) bottom board: PCB panel with LED arrays.

[00013] Figures 5A-5E illustrate the design layout of the reader device of the invention, e.g., Pictlmager+™, 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; and, Figure 5E shows the intended detection area of the 16 well assay substrate for a single camera. The labelled parts in Figure 5 A correspond to: 1) top board: PCB panel with mounted camera surrounded by LEDs; 2) diffuser, typically opal acrylic sheet; 3) lines illustrating the field of view of cameras; 4) 16- well PictArray™ assay substrate; 5) bottom diffuser; and 6) bottom board: PCB panel with LED arrays.

[00014] Figure 6 shows images captured by the reader device of the invention, e.g., Pictlmager+™ 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 PictArray™ assay substrate.

[00015] 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.

[00016] Figure 8 shows the LED panel designs of the reader devices of the invention, i.e., Pictlmager+™ and Pictlmager96+™, in accordance with aspects of the invention.

[00017] 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+™ and Pictlmager96+™, in accordance with aspects of the invention.

[00018] Figure 10 shows aspects of the LED layout for a reader device of the invention, i.e., Pictlmager+™, in accordance with aspects of the invention.

[00019] Figure 11 shows the LED panel design for a reader device of the invention, i.e., Pictlmager+™, in accordance with aspects of the invention.

[00020] Figure 12 shows the LED panel design for a reader device of the invention, i.e., Pictlmager96+™, in accordance with aspects of the invention.

[00021] Figure 13 is a block diagram showing the hardware functions for a reader device of the invention, i.e., Pictlmager+™, in accordance with aspects of the invention.

[00022] Figure 14 is a block diagram showing the hardware functions for a reader device of the invention, i.e., Pictlmager96+™, in accordance with aspects of the invention.

[00023] 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.

DETAILED DESCRIPTION OF THE INVENTION

[00024] 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). In various aspects, 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.

[00025] Before the present device, system and method are described, it is to be understood that this invention is not limited to the particular device, system, method and/or experimental conditions described herein, as such devices, systems, methods, and conditions may vary. It is also to be understood that the terminology used herein is for purposes of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only in the appended claims.

[00026] As used in this specification and the appended claims, the singular forms “a”, “an”, and “the” include plural references unless the context clearly dictates otherwise. Thus, for example, references to “the device” include one or more devices, references to “the system” include one or more systems, and 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.

[00027] As discussed herein, the invention pertains to the development of compact, simple, low-cost, and robust colorimetric readers for the readout of various in vitro diagnostic formats. In some aspects, the reader is specifically designed for imaging of spot microarrays on a 16-well membrane-based assay substrate, e.g., PictArray™ platform, and a membrane- free 96-well microtiter plate assay substrate. In various aspects, these formats are used for the development of multiplex immunoassays (MIAs) for human and veterinary diagnostics. The present disclosure illustrates multiple reader designs, e.g., Pictlmager+™ and Pictlmager96+™ for the readout of membrane-based MIAs on a 16-well membrane-based assay substrate, e.g., PictArray™ platform, and a membrane-free 96-well microtiter plate assay substrate, e.g., PictArrays™ platform. The technical specification and characteristics of these colorimetric readers are specified in extensive detail herein.

[00028] It will be appreciated that 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.

[00029] Accordingly, in one embodiment, 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. With reference to Figure 1, 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. In various aspects, 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.

[00030] As shown in Figure 1, in various aspects, the optical system includes one or more cameras 80, which can be arranged as an array as detailed further herein.

[00031] In some aspects, the reader device further includes a light diffuser substrate 80 disposed adjacent to the illumination source 40.

[00032] It will be appreciated that certain technical requirements exist for colorimetric readout in various immunoassay formats. Some technical aspects of colorimetric readout in various immunoassay formats that are read by the reader device of the invention are illustrated below. [00033] Membrane-based MIAs

[00034] 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+™ enables the colorimetric readout of membrane-based MIAs performed on a 16-well PictArray™ platform, while Pictlmager96+™ is used for the colorimetric readout of membrane-based MIAs performed on 96-well MTPs.

[00035] Membrane-free MIAs

[00036] 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+™ enables the colorimetric readout of membrane-free MIAs using the bottom as well as top illumination.

[00037] ELISA and Other Colorimetric Bioanalytical Tests

[00038] As the ELISA and other colorimetric bioanalytical tests employ the same 96-well microplate platform as membrane-free MIAs, they have the same technical requirements for the reader. Pictlmager96+™ is the most appropriate low-cost device for the readout of such assays.

[00039] LFIAs

[00040] As LFIA strips and cassettes are opaque, they can only be read from above. Therefore, the reader device needs to have the same technical requirements as desired for membrane-based MIAs. The illumination and imaging must be done from the top. Pictlmager+™ may be used for colorimetric readout of a single LFIA cassette, while Pictlmager96+™ may be employed for colorimetric readout of at least three LFIA cassettes. [00041] Components of the Colorimetric Reader Device of the Invention

[00042] In various aspects, the reader devices of the invention include a slide/plate holder. For example, the colorimetric readers of the invention include a holder either for a 16-well PictArray™ slide (in case of Pictlmager+™) or a standard 96-well MTP (in case of Pictlmager96+™). Both the Pictlmager+™ and Pictlmager96+™ 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.

[00043] In some aspects, 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.

[00044] In various aspects, 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.

[00045] In various aspects, the reader devices of the invention include an illumination source. In some aspects, 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 PictArray™ or 96-well MTP platform. Pictlmager+™ includes top illumination only, while Pictlmager96+™ includes both top and bottom illumination.

[00046] It will be appreciated that the function of the panels of illumination sources is to provide uniform and controlled light intensity to the 16-well PictArray™ or 96-well MTP, which enables the camera to image the colorimetric spot microarray in the wells. As 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. In one aspect, 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

(D50). Therefore, in one aspect, Cree’s CLM3C-WKW-CWBYA453 LEDs are used as they have lower intensity (brightness) of only 4.2 Im and close colour temperature approximation to the D50 needed at 5500K for the Pictlmager+™. [00047] In various aspects, the reader devices of the invention include an optical imaging system. In some aspects, 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 PictArray™ 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 PictArray™. On the other hand, Pictlmager96+™ has six cameras covering the entire 96-well MTP with one camera located in the centre of each 4x4 wells.

[00048] In another aspect, 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.

[00049] In various aspects, the reader devices of the invention include image processing functionality and electronics. In some aspects, image processing is achieved by a microprocessor in the device or via interfacing the data to an external computer. In one aspect, 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 PictArray™ or 96-well MTP.

[00050] In various aspects, 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.

[00051] In various aspects, the reader devices of the invention include functionality, such as a processor having software for image processing and/or analysis. In some aspects, 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 Excel™ 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.

[00052] In certain aspects, Pictorial® is used for image analysis. In various aspects, 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 PictArray™ 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 PictArray™ 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 PictArray™ spots will be in the range of 0 to 255. The data generated from each spot is collated with the layout of PictArray™ and tested samples to provide a final test report for the samples being analysed. [00053] In various aspects, the data processing steps are as follows.

1. Receive image(s) from camera.

2. Detect wells of interest in the images.

3. Identify alignment spot(s) in each well.

4. Create a grid with equal width and height of pre-determined size.

5. Detect spots in each cell of the grid.

6. Find the average pixel intensities of Red, Green, and Blue channels of the spots, and calculate the composite mean pixel intensity.

7. Inverse each average value and composite mean pixel intensity by subtracting from 255.

8. Report the quantitative values of determined pixel intensity of each spot into a useable file format, and then perform the desired analysis.

[00054] In some aspects, the software functionality also provides low-level control functions of the devices to adjust the right settings for various situations.

[00055] In related aspects, the software functionality provides the user interface, where operator(s) can provide input and extract relevant data from the devices. [00056] Figures 4A-4E illustrate the design layout of the reader device in one aspect of the invention.

[00057] Figures 5A-5E illustrate the design layout of the reader device in one aspect of the invention.

[00058] Table 1 provides the physical specifications of aspects of the reader devices of the invention and certain components thereof.

[00059] Table 1: Physical Specifications

[00060] LED Panel Design

[00061] In various aspects, the LEDs for both Pictlmager+™ and Pictlmager96+™ 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.

[00062] With reference to Figure 8, in some aspects, 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.

[00063] With reference to Figure 9, in some aspects, 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.

[00064] With specific reference to the reader device shown in Figures 5A-5E, e.g., Pictlmager+™, the camera is situated at the center of the LED panel, while 32 LEDs surround the camera. For the light to go from the edge of the LEDs to the edge of the 16- well slide and return to the camera to be captured uniformly, the LED panel needs to be double the length of the 16 well slide as shown in Figure 10. As such, 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.

[00065] With specific reference to the reader device shown in Figures 4A-4E, e.g., Pictlmager96+™, 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.

[00066] The top panel design considerations for Pictlmager96+™ are similar to the Pictimager+™ design, with clearances for the six camera lenses. In some aspects, the LEDs are spaced 10 mm apart horizontally and 8 mm vertically and are integrated with the camera’s multiplex board.

[00067] Circuit Block Diagrams

[00068] Hardware functionality for Pictlmager+™ is illustrated In Figure 13 in accordance with aspects of the invention. [00069] Hardware functionality for Pictlmager96+™is illustrated In Figure 13 in accordance with aspects of the invention, by the following block diagrams for both devices. [00070] Imaging Algorithms

[00071] Device operation and image capturing using Pictlmager+™is performed as follows. The procedure for the user operation of Pictlmager+™ includes the following in one aspect of the invention.

1. Connect the Pictlmager+™ with the supplied USB cable to a PC.

2. Insert the processed 16-well PictArray™ slide into the Pictlmager+™ door frame.

3. Ensure that the slide is inserted correctly with the alignment notch.

4. Close the door of the device.

5. Turn the switch ON at the back of the device.

6. Launch Pi ctori al ® to proceed .

7. Go to the Capture screen.

8. The camera shall start steaming video and available for capturing stills.

9. Press the Analyze button to receive the final still image to be used for image processing.

[00072] Device operation and image capturing using Pictlmager96+™ are performed as follows. The procedure for the user operation of Pictlmager96+™ includes the following in one aspect of the invention.

1. Connect Pictlmager96+™ to the local network for remote web app access via ethemet cable, or connect to a screen via HDMI, a keyboard, and a mouse via USB.

2. Connect the AC wall supply to Pictlmager96+™.

3. Power on Pictlmager96+™.

4. Launch Pictorial® 96 web app or Linux app.

5. Open/eject the sample tray via software or manually.

6. Place processed multiplex immunoassay, ELISA, or lateral flow immunoassay with receiving adapter onto the sample tray.

7. Close the tray via software or manually.

8. Go to the Capture screen.

9. 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.

11. Switch to the second set of cameras.

12. Repeat steps 9, 10.

13. Switch to the third set of cameras.

14. Repeat steps 9, 10.

15. Display all six images taken by the six cameras on screen and proceed to image processing.

16. Return to step 5 to start a new test or shut down via the Shutdown button.

[00073] Device operation and image capturing using Pictlmager™4 Cam is performed as follows. The procedure for the user operation of Pictlmager™4 Cam includes the following in one aspect of the invention.

1. Connect Pictlmager™ 4 Cam to the local network for remote web app access via ethemet cable, or connect to a screen via HDMI, a keyboard, and a mouse via USB.

2. Connect the AC wall supply to Pictlmager™ 4 Cam.

3. Power on Pictlmager™ 4 Cam.

4. Launch Pictorial© 4 Cam web app.

5. Open/eject the sample tray via software or manual control.

6. Place processed multiplex immunoassay, ELISA, or lateral flow immunoassay with a receiving adapter onto the sample tray.

7. Close the tray via software control.

8. Go to the Capture screen.

9. 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.

10. The still images are then taken automatically after a fixed time and stored for both cameras at full 8 MP resolution

11. Switch to the second set of cameras.

12. Repeat steps 9, 10.

13. Images of 2x 8-well strips are now captured. 14. Move the tray by a distance of 2 wells (18mm for 96-well microtiter plates) with automatic software operation. The cameras are now aligned with the next two strips of 8 wells.

15. Repeat steps 9-14 five more times to capture the entire 96 well microtiter plate.

16. Display all 24 images taken in reduced resolution, and proceed to image processing.

17. Return to step 5 to start a new test or shut down the device.

[00074] Image Processing

[00075] Figure 15 illustrates the data processing steps for Pictlmager+™, Pictlmager96+™’ and Pictlmager™4 Cam in various aspects of the invention.

[00076] Detection Applications

[00077] MIA - 16-well PictArray™ (Membrane Based)

[00078] In some aspects, Pictlmager+™ may be used for the colorimetric readout of assays on a conventional membrane-based spot microarray platform including 16 wells, e.g., PictArray™. It is a compact, lightweight, rapid, and robust colorimetric reader, with only a single camera, for membrane-based PictArrays™.

[00079] MIA - 96-well PictArray™ (Membrane Free and Membrane Based)

[00080] In some aspects, Pictlmager96+™ 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+™ 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 CL2™ by Scienion AG, Germany, although it will be far more compact, lightweight, and rapid.

[00081] Conventional Colorimetric ELISA in 96-well MTP (Membrane-free and Membrane-b ased)

[00082] In some aspects, Pictlmager96+™ may be used for the colorimetric readout of conventional colorimetric ELISAs in 96-well MTP. Pictlmager96+™ based colorimetric readout is similar to that of absorbance readout in a conventional ELISA. However, Pictlmager96+™ 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. However, Pictlmager96+™ is at least 10-fold lower in cost, compact, faster, and more robust than a commercial microplate reader.

[00083] LFIAs

[00084] In some aspects, as both Pictlmager+™ and Pictlmager96+™ has top illumination, they may be used for the readout of colorimetric LFIAs. Pictlmager+™ can read one LFIA cartridge, while Pictlmager96+™ can read up to 4 LFIA cartridges.

[00085] Automated Clinical Analyzer Based Multiplex Immunoassays

[00086] In some aspects, Pictlmager96+™ 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.

[00087] Automated Centrifugal Microfluidics-Based Immunoassay

[00088] In some aspects, Pictlmager96+™ may be used for the colorimetric readout of centrifugal microfluidics-based automated immunoassays that are performed on membrane- free and transparent LabDisk™ platforms.

[00089] Manual Lab-On-A-Chip Based Rapid Immunoassay

[00090] In some aspects, Pictlmager96+™ 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.

[00091] Manual Paper-Based Immunoassays

[00092] In some aspects, both Pictlmager+™ and Pictlmager96+™ 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

[00094] In some aspects, both Pictlmager+™ and Pictlmager96+™ 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+™ and Pictlmager96+™ may be used. But if the color determination needs to be done on a transparent substrate or platform, Pictlmager96+™ may be used.

[00095] Methods of Use

[00096] In another embodiment, 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.

[00097] In yet another embodiment, 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.

[00098] The following example is provided to further illustrate the advantages and features of the present invention, but it is not intended to limit the scope of the invention. While this example is typical of those that might be used, other procedures, methodologies, or techniques known to those skilled in the art may alternatively be used.

EXAMPLE 1

Performance Evaluation of a Colorimetric Reader Device of the Invention [00099] The performance evaluation of reader device depicted in Figures 5A-5E, e.g., Pictlmager+™, was done via the readout of biotin titration slides, which were prepared on membrane-based 16-well PictArray ™ platforms. The procedure for the preparation of biotin titration slides, readout by Pictlmager+™, and analysis of results is provided below. [000100] Preparation of Biotinylated Antibody Titration Slides [000101] 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 PictArray™ platform using a microarray printer. The procedure is specified in more detail below.

[000102] 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.

[000103] 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.

[000104] Subsequently, 25 pl of the prepared diluted biotinylated antibody solutions were added to a 384-well source plate, as shown in Table 2 below.

[000105] Table 2

[000106] The source plate was used to print 21 PictArray™ 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 PictArray™ well and incubating it for 60 min at room temperature. Finally, the IX blocker was discarded by inverting the PictArray™ in a chemical waste container /sink and tapping it onto a paper towel.

[000107] Preparation of Colorimetric Biotinylated Antibody Titration Slides

[000108] 20X streptavidin HRP-labelled (SA-HRP) (72E39) was diluted to IX or 0.25 pg/ml by adding 1 mL of 20X SA-HRP to 19 mL of diluent.

[000109] Subsequently, 50 pl of the IX SA-HRP was added to all wells of the PictArray™ platform and incubated for 30 min at 37°C. This was followed by washing the SA-HRP bound biotinylated antibody-coated wells three times with IX PBST washing buffer.

[000110] Finally, 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 PictArray™ and incubated at room temperature for 5 min. This was followed by washing the PictArray™ slide three times with IX PBST washing buffer. The colorimetric PictArray™ slides were then incubated at 37°C for 20 min to dry the wells.

[000111] Colorimetric Readout By Pictlmager+™

[000112] The colorimetric spots, corresponding to various biotinylated antibody concentrations printed in the PictArray™ after binding to SA-HRP and TMB substrate solution, were then read by Pictlmager+™ that employs a 5-MP camera module. The Pictlmager+™ is a low-cost multiplex array reader for membrane-based 16-well PictArrays™, which is powered through a standard computer USB port. The biotinylated antibody titration slide was inserted into the receiving holder of Pictlmager+™ and presented directly to the camera module at the top of the device in its field of view. The PictArray™ 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. [000113] The colorimetric readout measurements of biotinylated antibody titration slide were compared with those made using commercially available colorimetric microarray reader, e.g., sciREADER CL2™ from Scienion AG, Germany.

[000114] Results Obtained By Pictlmager+™

[000115] The images captured by the Pictlmager+™ for the biotinylated antibody titration slide are shown in Figure 6 and were used to determine the average composite pixel intensity of colorimetric spots corresponding to a particular concentration across all wells in PictArray™.

[000116] The results of the composite pixel intensities obtained for various biotinylated antibody concentrations used in the titration slide are plotted in Figure 7. A four-parameter logistic-based standard curve analysis was performed in Microsoft Excel. The results show a four-parameter logistic regression relationship between the biotinylated antibody concentrations printed on the PictArray™ slide and the measured composite pixel intensity of the colorimetric spots with R² of 0.998.

[000117] Although the invention has been described with reference to the above examples, it will be understood that modifications and variations are encompassed within the spirit and scope of the invention. Accordingly, the invention is limited only by the following claims.

Claims

What is claimed is:

1. A colorimetric reader comprising: a) a platform configured to hold an assay substrate, the assay substrate being configured to perform an assay of an analyte; b) an illumination source configured to illuminate the assay substrate; c) an optical imaging system operable to capture an image of all or a portion of the assay substrate; d) an electronics module(s) having the functionality to control the intensity of light emitted from the illumination source; and e) an image processor having the functionality to receive and process the images generated by the optical imaging system, 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 the analyte to a reagent of the assay substrate, and detection of the analyte.

2. The colorimetric reader of claim 1, wherein the platform, the illumination source, and the optical imaging system are disposed within an optically opaque housing.

3. The colorimetric reader of claim 2, wherein the housing further comprises the electronics module(s) and optionally the image processor.

4. The colorimetric reader of claim 1, wherein the optical imaging system comprises one or more cameras disposed adjacent to the assay substrate.

5. The colorimetric reader of claim 4, wherein the one or more cameras are mounted on a printed circuit board.

6. The colorimetric reader of claim 4, wherein the one or more cameras form an array.

7. The colorimetric reader of claim 4, wherein each of the cameras consists of a camera sensor, optical lenses, and a lens fixture.

8. The colorimetric reader of claim 1, wherein the illumination source is disposed adjacent to an upper surface of the assay substrate.

9. The colorimetric reader of claim 8, further comprising a second illumination source disposed adjacent to a bottom surface of the assay substrate.

10. The colorimetric reader of claim 1, further comprising light diffuser substrates disposed adjacent to the illumination sources.

11. The colorimetric reader of claim 1, wherein the assay substrate is a 96-well microtiter plate, and the platform is configured as a mechanical tray.

12. The colorimetric reader of claim 11, wherein the mechanical tray holds the 96-well microtiter plate and configured such that the plate is inserted and taken out of the colorimetric reader manually.

13. The colorimetric reader of claim 11, wherein the mechanical tray holds the 96-well microtiter plate and configured such that the plate is inserted and taken out of the colorimetric reader via automated movement facilitated by a motor.

14. The colorimetric reader of claim 1, wherein the assay is a colorimetric assay.

15. The colorimetric reader of claim 1, wherein the assay is an immunoassay.

16. The colorimetric reader of claim 1, wherein the assay is a multiplex immunoassay.

17. The colorimetric reader of claim 1, wherein the assay is a membrane-free multiplex immunoassay, a membrane-based multiplex immunoassay, or a lateral flow immunoassay.

18. The colorimetric reader of claim 1, wherein the image processor further includes functionality to quantitate an amount of the analyte.

19. The colorimetric reader of claim 1, wherein the assay substrate is configured as a microtiter plate having one or more sample wells disposed within a perimeter of the substrate, wherein each of the one or more sample wells are configured to hold a reaction mixture including a sample and a reagent.

20. The colorimetric reader of claim 19, wherein the microtiter plate comprises a plurality of sample wells, each plurality of sample wells having a sample and a reagent.

21. The colorimetric reader of claim 20, wherein each of the plurality of sample wells includes a microarray, the microarray having individual spots of reagents for detecting a plurality of analytes present in the sample.

22. The colorimetric reader of claim 21, wherein the image processor includes functionality to process the image to determine colorimetric light intensity associated with each spot in a sample well.

23. The colorimetric reader of claim 22, wherein the image processor further includes functionality to quantitate an amount of an analyte associated with each spot.

24. The colorimetric reader of claim 1, wherein the illumination source comprises a light-emitting diode (LED) or organic LED (OLED).

25. The colorimetric reader of claim 24, wherein the illumination source comprises a plurality of LEDs or OLEDs configured as an array.

26. The illumination source of claim 24, wherein the plurality of LEDs or OLEDs are mounted on a printed circuit board.

27. The colorimetric reader of claim 1, wherein the reagent is an immobilized capture molecule selected from the group consisting of nucleic acid, protein, peptide, hormone, steroid, lipid, and carbohydrate, a chemical compound, an antigen or antibody, a cell or portion thereof, or cellular nuclei, or portion thereof.

28. The colorimetric reader of claim 1, wherein the analyte is a biomolecule selected from the group consisting of a nucleic acid, protein, peptide, hormone, steroid, lipid, and carbohydrate, a chemical compound, a virus or portion thereof, or an antigen or antibody.

29. A method of analyzing a sample comprising: a) providing a colorimetric reader of any of claims 1-28; b) loading a sample onto the assay substrate; c) generating an image of the assay substrate via the optical imaging system; and d) 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.

30. The method of claim 29, further comprising quantitating an amount of the analyte.

31. The method of claim 29, wherein the light intensity associated with the analyte's binding is generated via colorimetry.

32. A method of detecting a disease or disorder in a human or non-human subject comprising: a) obtaining a sample from a subject; b) analyzing the sample using the method of any of claims 29-31; and c) detecting a disease or disorder in the subject based on the analysis of b), thereby detecting a disease or disorder in the subject.

33. The method of claim 32, wherein the sample is a biological fluid selected from the group consisting of whole blood, serum, plasma, urine, feces, bile, breast milk, breast fluid, gastric acid, mucus, pus, rheum, saliva, semen, sputum, sweat, tears, vaginal secretion, vomit, umbilical cord blood, spinal fluid, tissue, and endocervical fluid.