Abstract
An apparatus for creating and correcting a two dimensional intensity map of one or more assay spots in a detection zone is provided. The apparatus comprising, a locator for affirming the location of the detection zone; a total internal reflection excitation device comprising a light source for illuminating the one or more assay spots in the detection zone; a detector configured to receive light that is emitted, reflected or scattered from the one or more assay spots and to create a two dimensional intensity map of the one or more assay spots comprising a two dimensional array of quantitative pixel values; and a processor configured to correct the intensity map to remove noise through analysis of corresponding pixel values from an earlier intensity map; wherein the analysis includes curve fitting.
Claims
1: An apparatus for creating and correcting an image of one or more assay spots in a detection zone prior to the assay reaching equilibrium, the apparatus comprising: a substrate comprising one or more assay spots deposited thereon; a reference marker for affirming the location of the detection zone; a total internal reflection excitation device comprising a light source for illuminating the one or more assay spots in the detection zone; a detector configured to receive light that is emitted, reflected or scattered from the one or more assay spots and to create an image of the one or more assay spots comprising a two dimensional array of quantitative pixel values; and a processor configured to correct the image to remove noise by fitting a curve to the quantitative pixel values using corresponding pixel values from an earlier image, wherein the processor is configured to identify reference markers provided in or adjacent to the detection zone to align the earlier image with the new image.
2: The apparatus according to claim 1, wherein the analysis comprises subtraction of corresponding pixel values from an earlier image.
3. (canceled)
4: The apparatus according to claim 1, wherein the earlier image is detected at the time of manufacture of the detection zone and is provided to the apparatus.
5. (canceled)
6: The apparatus according to claim 1, wherein the earlier image is obtained in the apparatus.
7: The apparatus according to claim 1, wherein the earlier image is obtained at T˜0 of the assay.
8: The apparatus according to claim 1, wherein a plurality of images is taken and each image is corrected by subtraction of the directly preceding image.
9: The apparatus according to claim 1, wherein the processor is further configured to process a plurality of corrected images to calculate the change of intensity over time of the one or more assay spots.
10: The apparatus according to claim 1, wherein the detection zone is provided within a cartridge that is removable from the apparatus and wherein the reference marker is a support configured to hold the cartridge when it is inserted into the apparatus.
11: The apparatus according to claim 10, wherein the cartridge further comprises a unique identifier.
12: The apparatus according to claim 1, wherein the earlier image is detected at the time of manufacture of the cartridge and associated with the unique identifier.
13: The apparatus according to claim 1, wherein the detection zone is integral with the apparatus and the reference marker is configured to confirm the location of the detection zone.
14: The apparatus according to claim 1, wherein the total internal reflection excitation device further comprises one or more lenses or mirrors in series configured to launch light towards the cartridge and to collect the emitted, reflected or scattered light from the one or more assay spots.
15: The apparatus according to claim 1, wherein the one or more assay spots comprise a liquid sample.
16: The apparatus according to claim 15, wherein the liquid sample is a saliva sample.
Description
[0041] The present invention will now be described, by way of example only, with reference to the accompanying figures in which:
[0042] FIGS. 1A and 1B provide an apparatus according to the present invention;
[0043] FIGS. 2A to 2C provide a schematic of the background subtraction according to the present invention;
[0044] FIGS. 3A to 3D provide a schematic of a single frame subtraction;
[0045] FIGS. 4A to 4D provide a schematic of the continuous frame subtraction method according to the present invention;
[0046] FIG. 5 provides a schematic for background subtraction in assays stored in a different medium to incubation;
[0047] FIGS. 6A to 6C provide a schematic of background subtraction after realignment of the substrate;
[0048] FIGS. 7A to 7D provide a schematic of kinetic multiplexing monitoring according to the present invention;
[0049] FIGS. 8A to 8D illustrate a subtraction method on a saliva sample;
[0050] FIG. 9 shows a graph depicting assay intensity as a function of time;
[0051] FIG. 10A shows a graph depicting assay signal as a function of time;
[0052] FIG. 10B shows a graph depicting a curve fit to measured assay points;
[0053] FIG. 10C shows a graph depicting assay signal as a function of time after a background subtraction; and
[0054] FIGS. 11A to 11D illustrate various graphs of assay signal as a function of time which may undergo curve fitting analysis.
[0055] The present invention as disclosed herein provides an apparatus and method for creating and correcting a two dimensional intensity map of one or more assay spots in a detection zone. Referring to FIG. 1A, there is provided an apparatus 100 for illustrating prism TIR geometry. The apparatus 100 comprises a substrate 102 such as an optical element 102 as shown in FIG. 1A. The optical element 102 comprises a light pathway 104 comprising an input surface 106, a reflective surface 108 and an output surface 110. The input surface 106 enables light 104, such as an incident light beam, to enter into the optical element 102. The input surface 106 and/or the output surface 110 can be a refractive or a diffractive or a transmissive surface. In some instances, the incident light beam is refracted at the input surface 106 upon entry into the optical element 102. The light 104 is directed towards the reflective surface 108 where one or more assay spots 109 comprising one or more capture components and/or one or more detection reagents are deposited onto the reflective surface 108 of the optical element 102. The assay spots are located in one or more detection zone on the surface of the optical element 102. In some instances, the incident light beam can be scattered or emitted upon entry into the optical element 102. The optical element 102 may be a prism as shown in FIG. 1A.
[0056] The apparatus 100 further comprises a total internal reflection excitation device 112 comprising a light source 114 for illuminating the one or more assay spots in the detection zone on the surface of the optical element 102. As shown in FIG. 1A, there is also provided a lens 118 such as an objective lens to collect the emitted, reflected or scattered light 116 at a specific wavelength from the assay spots deposited on the substrate 102. The lens 118 may be used to focus the emitted, reflected or scattered light 116 onto a detector 120. The detector 120 is configured to receive light that is emitted, reflected or scattered 116 from the one or more assay spots and to create a two dimensional intensity map of the one or more assay spots comprising a two dimensional array of quantitative pixel values. A processor 122, which can be directly or indirectly coupled with the detector 120, is configured to correct the intensity map to remove noise through analysis of corresponding pixel values from an earlier intensity map.
[0057] The apparatus 100 as disclosed herein involves acquiring more than one image of an immunoassay at various time points during an incubation period. By subtracting out pixel intensities between the frames, a ‘background removal’ method is attained. The residual between the subtracted frames is therefore the rate of change of the assay signal between the time points. This is in contrast with the traditional method of analysis whereby the signal of the assay is measured as a single acquisition and the intensity is calibrated through known standards. Kinetic measurements can be especially valuable in situations where the concentration of the component of interest or the label concentration varies with time or with respect to the absolute position of the field of view.
[0058] Referring to FIG. 1B, there is provided an apparatus 100 for illustrating objective TIR geometry. The apparatus 100 comprises an autofocus feedback system using a lens system 130. The lens system 130 may comprise one or more lenses arranged in series which can be configured to direct an incident light to a substrate 132 and to collect the emitted light, reflected or scattered light from the assay spots deposited on the substrate 132. The lens system 130 may comprise one or more objective lens. The apparatus 100 as shown in FIG. 1B comprises a substrate 132 where one or more assay spots are deposited onto the surface of the substrate 132. The assay spots are deposited, preferably printed onto the surface of the substrate 132. Each assay spot comprises at least one capture component and/or at least one detection reagent. A light source 134, for example a laser beam such as an exication laser beam, provides an incicident light 136 that can be directed at the substrate 132 to excite the molecules on the assay spots.
[0059] The apparatus 100 also comprises a refractive index matching liquid 137, which is provided between the lens system 130 and the substrate 132. Objective based TIR imaging requires refractive index matching liquid such as an index matching oil in between the assay spots deposited on the substrate and the lens system to achieve TIR. Additionally or alternatively, an index matching gel may be provided.
[0060] As shown in FIG. 1B, an exictation dichroic 138 is provided and configured to reflect the incident laser beam 136 into the lens system 130 and to reflect out the reflected, emitted or scattered light 140, as shown in FIG. 1B.
[0061] A detector 142 is then configured to receive the collected emitted, reflected or scattered light 140 from the lens system 130 to create a two dimensional intensity map of the one or more assay spots comprising a two dimensional array of quantitative pixel values. A processor 144, which can be directly or indirectly coupled with the detector 142, is configured to correct the intensity map to remove noise through analysis of corresponding pixel values from an earlier intensity map.
[0062] Referring to FIGS. 2A to 2C, there is provided an array 10 of assays spots 12 comprising one or more capture component and/or one or more detection reagents. The assay spots 12 that develop over time, as indicated by arrow 14, have a steady increase in intensity, whereas background contamination 16 occurs on different timescales or even remains unchanged over time. By subtracting out a prior frame, as indicated by arrow 15, such as subtracting frame 2 18 from frame 1 17, the subsequent images are cleaned from the background, as shown in frame 3 19 in FIG. 2C. By utilising this method, the detector which may be a camera, is able to detect the change in signal between different time points of the assay.
[0063] Referring to FIGS. 3A to 3D, there is a provided a single frame subtraction schematic. The recorded images 22, 24, 26 (FIGS. 3B to 3D) each have the initial recording 20 (see FIG. 3A) subtracted from them, yielding a background-free assay which increases with time. For assays that can be monitored continuously, the frame 20 when the incubation starts, or a short time after the start of the assay 27 as shown in FIG. 3A, can be used as the background level 28 and be removed from all subsequent frames 22, 24, 26 (FIGS. 3B to 2D). In some embodiments, it would be desirable and preferable for the subtracted frame as shown in FIG. 3D to be close to the start of the assay, because substantially all of the signal at that time will be background signal at T=0. This mode of assay tracking relies on the biggest difference between the images being the binding of the detection component of the assay for example, secondary antibodies to the capture component such as primary antibodies. The subtraction method as illustrated in FIGS. 3A to 3D is particularly useful when the profile of the laser illumination or the excess concentration of the label does not change significantly over time, which may occur from for example a moving liquid-air boundary due to evaporation or a changing concentration gradient of the detection reagents.
[0064] Referring to FIGS. 4A to 4D, there is provided an illustration of the continuous frame subtraction methodology. As illustrated in FIGS. 4A to 4D, frames 32, 34, 36, 38 are subtracted from each other after a pre-determined time. The background signal 31 that has developed during the assay 30 appears in only a single analysed frame.
[0065] The T=0 subtraction highlights the difference of the recorded image between the initial assay and all subsequent images. In some instances, the signal can in principle be subtracted from any time point. The signal subtraction can be particularly effective when the signal of the background 31 contamination is introduced prior to the assembly of the assay 30. If on the other hand the assay accumulates additional contamination during the incubation period, then T=0 subtraction will carry the additional background over. Given that the signal intensity of the assays follows approximately a linear relation at intermediate incubations, it is possible to continually subtract consecutive frames to afford a steady assay difference signal. Additional background during the incubation then contaminates only a single image before being removed again. Using this method, it can be critical to select a sufficient time between the subtracted frames to ensure that the assay intensity has changed substantially. During this time, the acquisition can either be inactive e.g. if the label is damaged by the acquisition, as is the case for fluorophores or the in-between frames can have further processing to produce a representative snapshot of the assay with the selected time window.
[0066] Referring to FIG. 5, a schematic is provided to show two methods for achieving a T<0 background subtraction in assays stored in a different medium to incubation. As shown in FIG. 5, the assay is filled and correlated for difference in intensity between the different media. The background signal is then subtracted.
[0067] Some detection reagents such as fluorophores can undergo photo-bleaching upon light exposure, which means that continuous acquisition would reduce the assay intensity and is therefore not feasible. Although photo-bleaching of a large number of molecules can be taken into the account, it may be beneficial to avoid unnecessary exposure of the assay. Provided that the background signal is introduced to the assay during manufacturing process and not from the assay itself, it may then be possible to record this before the start of the incubation. It may be a challenge in that the optical detection is sensitive to the medium in which the acquisition occurs. The assays are performed in a solution containing the sample and the detection reagents, whereas prior to the start of the incubation, the detection reagents are usually kept dry. The background of the dry state before the incubation cannot therefore be simply subtracted from the subsequent images when the incubation starts to remove the contaminant signal. This can be alleviated in two ways; the dry and the filled state is correlated and accounted for or the assay is pre-loaded with a liquid with similar optical properties to the sample/detection reagent mixture.
[0068] Referring to FIGS. 6A to 6C, there is shown a T<<0 subtraction schematic. After the initial background measurement as shown in FIG. 6A, the consumable 50 comprising one or more assay spots 54 needs to be re-aligned with respect to the reader (not shown in the accompanying drawings) as shown in FIG. 6B. In some instances where the consumable 50 is not perfectly aligned, the two reference spots 52 can help correlate both the positional and rotational mismatch to perform the background subtraction, as shown in FIG. 6C. The consumable 50 can be rotated at any angle such that it is correctly aligned with the reader.
[0069] To achieve a good quality T<0 background subtraction, it is desirable that the assay consumable 50 does not degrade or accumulate additional background. The subtraction can be done at any point prior to the assay. In some instances, the major source of background signal can be introduced during the manufacture of the consumable 50, then a T<<0 background subtraction can be performed as part of the quality control of the manufacture in batches, then stored until needed, thereby improving the quality of the assay and negating the need for a blank measurement when performing the assay. As an example, this can be required when performing a rapid test.
[0070] The following method as illustrated in FIGS. 6A to 6C requires an alignment process whereas the other methods as disclosed herein have relied on the sample being in the same location with respect to the reader. A T<<0 background subtraction requires the sample to be taken off the reader and the same location found when the assay is read. In some cases, it may be possible to align the sample such that there is a negligible positional difference between the initial background reading and the latter assay measurements. In some examples, the reference alignment marks or spots 52 can be used to provide the positional information of both stages of the reading. One of the most straightforward ways to provide the positional references is through two spots spaced as far from each other as possible, while still within the field of view of a single frame. There are numerous other reference markers that can be used in this way. Examples of markers may include, but is not limited to, writing a line on more than two spots.
[0071] As shown in FIG. 7, there is provided a schematic of kinetic monitoring of multiplexed assays. For example, one or more assay spots 70 may comprise one or more different capture component and/or one or more different detection reagent 72, 74, 76, 77 from another assay spot 70 in the same frame 78. Assays of different rate constant in the field of view can be tracked by recording multiple frames at intermediate time points for the assay. Alternatively, a single measurement when the assays have equilibrated may show a similar intensity.
[0072] When more than one assay 72, 74, 76, 77 is in the field of view of the camera at the same time, it may be beneficial to acquire the signals of different assays 72, 74, 76, 77 at different times. For example a very fast binding and/or high biomarker concentration assay may require measurements within minutes after the start of incubation, while a slow binding assay and/or low biomarker concentration assay may require measurement after a few hours. Acquiring multiple recordings of such assays simultaneously as shown in FIG. 7 means that any type of assays can be multiplexed together without the loss of information from the assays.
[0073] Referring to FIGS. 8A to 8D, there is provided an illustration of the T=0 subtraction. FIG. 8A shows T≈0 seconds after filling of the assay mixture. FIG. 8B shows the kinetic activity 2 seconds after frame A shown in FIG. 8A. FIG. 8C illustrates the residual intensity of the subtraction of frame A from frame B. FIG. 8D illustrates the residual intensity after T=0 subtraction after 2 minutes of incubation.
[0074] The intensity of the assay can be tracked either individually if the assay is multiplexed or as a whole. For example, FIG. 9 demonstrates a single spot as a function of time. The ability to track the assay intensity as a function of time rather than acquiring a single static image allows the direct determination of the biomarker concentration rather than relying on an equivalent assay conditions using calibration standards. Thus, tracking the assay intensity as a function of time provides a more robust method and opens the possibility of de-skilled use outside laboratory conditions.
[0075] Referring to FIGS. 10A, 10B and 10C, there are provided examples of curve fitting analysis removing noise from an intensity map. FIG. 10A depicts an example of a graph showing signal as a function of time. The graph has contributions from an assay signal 146, and also a background signal 148. The background signal 148 may remain constant throughout the assay, or may change at a different rate to the assay signal 146. As shown in FIG. 10B, actual signal measurements can be plotted as points 152 on a graph. The contribution of the background to these measured points 152 is unknown. By analysis of corresponding pixel values from an earlier intensity map, a curve 150 can be fit to the measured points 152. By fitting a curve 150 to the measured points 152, it is possible for the contribution of the background signal 148 to be subtracted, and for noise to be removed from the assay signal 146. Referring to FIG. 10C, after the background subtraction, the graph of signal as a function of time is free from background signal 148, which facilitates an accurate determination of the assay signal.
[0076] Referring to FIGS. 11A to 11D, there are provided examples of graphs of signal as a function of time. The graphs illustrate the different kinetics an assay signal 146 may have. Curve fitting analysis may be used to remove the background signal 148 from any of the various assay signals 146 shown. FIG. 11A shows an example of a logarithmic increase signal. As illustrated in FIG. 11A, the signal is observed when all assay components come into contact at t=0 and one or more of the components depletes within the timescale of the measurement.
[0077] FIG. 11B shows an example of a linear signal increase. As illustrated in FIG. 11B, the signal is observed when all assay components come into contact at t=0 and none of the components deplete substantially within the measurement time. In some instances, the signals may plateau when the assay has reached equilibrium.
[0078] FIG. 11C shows an example of a linear signal decrease. As illustrated in FIG. 11C, the signal is observed when all assay components come into contact at t=0, none of the components deplete within the measurement time and the signal decreases in the presence of the analyte of interest. The graph as shown in FIG. 11C i.e. a decreased signal vs time scenario, may be a particularly useful application for assays with a high off rate.
[0079] FIG. 11D shows an example of a logarithmic increase with a lag. As illustrated in FIG. 11D, the signal is observed when there is a lag between all assay components coming into contact and one or more of the components depletes within the timescale of the measurement.
[0080] Various further aspects and embodiments of the present invention will be apparent to those skilled in the art in view of the present disclosure.
[0081] “and/or” where used herein is to be taken as specific disclosure of each of the two specified features or components with or without the other. For example “A and/or B” is to be taken as specific disclosure of each of (i) A, (ii) B and (iii) A and B, just as if each is set out individually herein.
[0082] Unless context dictates otherwise, the descriptions and definitions of the features set out above are not limited to any particular aspect or embodiment of the invention and apply equally to all aspects and embodiments which are described.
[0083] It will further be appreciated by those skilled in the art that although the invention has been described by way of example with reference to several embodiments. It is not limited to the disclosed embodiments and that alternative embodiments could be constructed without departing from the scope of the invention as defined in the appended claims.
