The invention relates generally to assay methods and systems for use thereof to carry out bioassays such as specific binding assays, and more particularly multiplexed bioassays for protein and nucleic acid analysis. Immunoassay and molecular assays often require a large dynamic range for quantitative or semi-quantitative assays. The invention generally relates to adjusting and expanding the dynamic ranges for all analytes in the multiplex assay panel to match the intrinsic electronic dynamic range of the detector.
A growing demand for specific binding assays including immunoassays and molecular assays has put pressure on the diagnostic industry to reduce costs and optimize sample usage by designing multiplex assays to test for several analytes simultaneously. Instead of performing one test per sample by the conventional method, many tests can be conducted which generate many data points per sample. Factor in the time saved by running the equivalent of 20, 100, or more different assays in a single assay workflow, and the benefits of multiplexing are clear. For example, the usage of 20 microplates, one plate per test, to run 20 tests, can be reduced to 1 microplate producing significant savings in manufacturing costs. An increase in testing volume and a pressure of rapid turnaround results further enhance the value of multiplex assays. The ability to measure multiple targets in a single assay allows researchers and laboratory personnel to detect hundreds of analytes and generate thousands of test data points in a single run. While being judicious with precious sample volumes, it is known that it is difficult to get sufficient blood samples from many pediatric patients. From screening biomarkers for further evaluation to validating results for drug development, multiplex assays provide many important benefits, helping to save time, save labor, save material resources, conserve limited sample volume, reduce test cost, and optimize productivity.
The presence or absence of analyte in a sample is determined by contacting the sample with agents which will specifically bind to the desired analyte but not with other analytes which have been practiced for decades and are well known in the art. The presence or absence of a binding reaction can be determined by use of light signals to detect the presence or absence of the specific binding pair. There are multiple multiplex technologies, such as color coded microbead, digitally coded magnetic bead, microarray, and multiple color fluorophores. In the multiplex assay, the coded micro particles are used for analyte identification. The number of analytes measured is determined by the number of different bead colors, digital barcodes, or micro spots in the microarray. Multiplex assays within a given application area or class of technology can be further stratified based on how many analytes can be measured per assay, where “multiplex” refers to those with the highest number of analyte measurements per assay. The “low-plex” (>2 and <5) or “mid-plex” refers to procedures that process fewer (>5 and <50), though there are no formal guidelines for calling a procedure multi-, mid-, or low-plex based on number of analytes measured. In the biological sciences, a multiplex assay can use magnetic beads to simultaneously measure multiple analytes in a single experiment. A multiplex immunoassay is one form of a specific binding assay and is a derivative of an ELISA using beads for binding the capture antibody. In the past few years, multiplex assays have started to migrate from research applications into clinical settings.
Many factors can affect the dynamic range of bioassays, such as the power of light source, sample volume, matrix volume or matrix surface area, particle or bead number, electronic gain factors, optical elements, and quality and nature of optical detectors. Among them, optical detectors play a very significant role in term of dynamic range. Most of the quantitative biological tests are performed using an optical sensor or detector, such as CCD, CMOS, photo multiplied tube or the like. The sensor chip is the key element of bioassay detection, but the intrinsic dynamic range of the sensor is limited by its electronic capacity.
For example, Charge Coupled Device (CCD) is a highly sensitive photon detector. The sensor chip is divided up into many light-sensitive small areas (known as pixels) which can be used to build up an image of the scene of interest. A photon of light which falls within the area defined by one of the pixels will be converted into one (or more) electrons and the number of electrons collected will be directly proportional to the intensity of the scene at each pixel. When the sensor is clocked out (i.e., the integration duration is run), the number of electrons in each pixel is measured and the number of photons can be reconstructed. For example, the dynamic range of a CCD is typically defined as the full-well capacity divided by the camera noise and relates to the ability of a camera to record simultaneously very low light signals alongside bright signals. The ratio is often expressed in decibels which is calculated as DR (full well capacity/read noise) or in the equivalent number of A/D units required to digitize the signal. Because all optical sensors can reach photon saturation at full-well capacity, the sensor ability of the sensor to expand the upper end of the dynamic range is limited. At the same time electronic noise limits the lower end of the dynamic range. Because the electronic dynamic range of a sensor is fixed. The dynamic range (DR) of every analyte in the sample need to fall into or be able to match the DR of the sensor in order for its presence and/or quantity to be accurately assessed.
Bioassays, both immunoassay and molecular specific binding assays often require a large dynamic range for quantifying analyte with a wide range of analytical concentrations. The need becomes even more challenging for multiplex assays, in which the multiple analytes may have very different range of concentrations in a sample. For single analyte target, sample dilutions are commonly method to adjust the analytical concentration for detection purpose, but it becomes impossible to adjust this accurately for multiple analytes having significantly different concentration and/or signal ranges. Every individual target in the multiplex assays needs to operate within its own proper dynamic range for quantitative assays, but the range of concentration can vary significantly.
Bioassays often require a large dynamic range for quantifying analyte with a wide range of concentrations. For bioassays, the dynamic range of an assay is described as the lowest to the highest concentration of an analyte that can be reliably detected by the assay. This is referred to as the lower and upper limits of detection (LLOD and ULOD, respectively). With a commonly available bioassay systems, the useful dynamic range is limited to approximately 2 to 4 decades (2 log-4 log).
For detection of a single target, dilutions are commonly used to adjust the analyte concentration in the sample to be detected by the detector. The concentration of the analyte may be very different from sample to sample under different clinical conditions. It is important to have a large dynamic range to cover concentration ranges of clinical significance. Although sample dilutions have become common practice, it becomes very difficult to carry out appropriate dilutions for multiple analytes in one sample. The concentration range of interest for an individual analyte can vary widely, for example, in the immunoassays: Analyte 1: from 0.1 pg/ml to 1,000 pg/ml, Analyte 2: from 1.0 pg/ml to 10,000 pg/ml, 3: from 10 pg/ml to 100,000 pg/ml etc. In the nucleic acid, DNA, RNA molecular assays: the analyte concentrations can be ranged from a single copy to 1,000 s of copies. The quantitation can be even more difficult for post PCR reactions, it can range from 0.001 fmole/μl to 1,000 fmole/μl with a wide dynamic range. If the sample for one highly concentrated analyte is diluted it may become impossible to detect other analytes with low concentrations. Considering the number of analytes in the multiplex assays, it becomes a daunting task to have proper dilutions, wherein all analytes of all samples fall into the dynamic range of the single sensor.
A method and system are provided with adjustable and expandable dynamic ranges for two or more of the analytes in multiplex bioassays using a plurality of optical integration durations. Depending on the analyte, a short exposure time is used to avoid sensor saturation in order to extend the upper limit of the concentration. To extend the lower limit of the concentration, a long exposure time is used to generate more photons and overcome the read noise. In this manner, the dynamic range for detection of each analyte in multiplex assays can be optimized and customized.
The present invention is directed to enhancing the dynamic range of bioassays for quantitative measurements. More importantly, it solves the problems of limited dynamic ranges for multiplex assays, in which each target analyte has a wide range of concentration range. Because all optical sensors are limited by full-well electron capacity for the upper limit of the dynamic range and electronic noise for the lower end of the dynamic range the extent of the dynamic range of the sensor is a fixed number. However, the amount of photons detected by the sensor depends on the time duration of the photon flux generated by the analyte. In the case of fluorescence, the total measurable fluorescence intensity is the multiplied product of “analyte concentration” and “time duration of the light intensity”.
The present invention is directed to obtain a plurality of signals from a series of integration durations for every measurement. A short integration duration is used to generate a smaller number of photons to avoid the sensor saturation for high concentration; and a long integration duration is used to generate a greater number of photons to overcome the detector noise for low concentration. A series of integration durations is thus utilized for each sample measurement.
In an embodiment, a method of conducting an assay for multiple analytes in a sample, wherein the presence or absence of each analyte in said sample is indicated by a light signal, is provided. The method may include the steps of: determining the presence of an analyte by: providing a light source to the sample wherein a light signal is emitted associated with the presence of individual analytes; capturing, by one or more photodetectors, a plurality of images, each including the plurality of analytes, at a plurality of durations; for a first analyte of the plurality of analytes, determining, by one or more processors, a first range of integration durations associated with a light signal associated with the presence of the first analyte; identifying, by the one or more processors, a first image, of the plurality of images, captured at a duration within the first range of integration durations associated with the light signal associated with the presence of the first analyte; and analyzing, by the one or more processors, the first image to determine the presence of the first analyte; and for a second analyte of the plurality of analytes, determining, by one or more processors, a second range of integration durations associated with the presence of the second analyte; identifying, by the one or more processors, a second image, of the plurality of images, captured at a duration within the second range of integration durations associated with the light signal associated with the presence of the second analyte; and analyzing, by the one or more processors, the second image to determine the presence of the second analyte.
In another embodiment, a method of conducting a specific binding assay for multiple analytes in a sample wherein the presence or absence of each analyte in said sample is indicated by a light signal, is provided. The method may include providing a specific binding pair member for each analyte, contacting the sample with the specific binding pair members under conditions in which analytes will stably bind with their specific binding pair member but will not bind to other specific binding pair members and in which stably bound specific binding pairs are capable of being detected by emitting a light signal determining the presence of stably bound specific binding pair members by: providing a light source to the plurality of specific binding pair members and analytes over a period of time wherein stably bound specific binding pairs will emit a light signal; capturing, by one or more photodetectors, a plurality of images, each including the plurality of analytes, at a plurality of durations; for a first analyte of the plurality of analytes, determining, by one or more processors, a first range of integration durations associated with a light signal associated with the presence of the first analyte; identifying, by the one or more processors, a first image, of the plurality of images, captured at a time within the first range of integration durations associated with the light signal associated with the presence of the first analyte; and analyzing, by the one or more processors, the first image to determine the presence of the first analyte; and for a second analyte of the plurality of analytes, determining, by one or more processors, a second range of integration durations associated with the presence of the second analyte; identifying, by the one or more processors, a second image, of the plurality of images, captured at a time within the second range of integration durations associated with the light signal associated with the presence of the second analyte; and analyzing, by the one or more processors, the second image to determine the presence of the second analyte.
In yet another embodiment, a method for detecting a signal associated with the quantitative measurement of an analyte in a multiplex bioassay, wherein measuring the concentration of an analyte in a sample is indicated by a light signal strength, is provided. The method may include identifying each analyte, of a plurality of analytes on solid substrates in a multiplex bioassay, by optical imaging; capturing a plurality of signals, N, at each of a series of integration durations, IT-N, for each analyte in said multiplex assay, wherein each image contains light signals for each analyte in the multiplex assay; for each respective analyte, selecting a respective light signal, of the plurality of light signals, captured at a respective integration duration, of the series of integration durations, that is within a linear range associated with the respective analyte; normalizing the other light signals, of the plurality of light signals by compensating for their integration durations based on a ratio of their integration durations to the respective integration duration that is within the linear range associated with the respective analyte; and combining the normalized light signals in order to expand the linear range associated with the respective analyte of the plurality of analytes.
In still another embodiment, a system with an analyte-dependent dynamic range for measuring a concentration of each analyte, of a plurality of analytes in a multiplex bioassay, may be provided, comprising: a light source configured to provide a series of pulses having different time durations to excite multiple analytes on solid substrates in said sample; an imaging detector configured to capture a plurality of light image signals (N), having a respective plurality of optical integration durations (ID-N) corresponding to the pulses of said light source, wherein each of said light image contains light signals for each analyte in said multiplex bioassay; one or more processors; and one or more memories storing instructions that, when executed by the one or more processors, cause the one or more processors to, for each analyte of the plurality of analytes: for each respective analyte, select a respective light signal, of the plurality of light signals, captured at a respective integration duration, of the series of integration durations, that is within a linear range associated with the respective analyte; normalize the other light signals, of the plurality of light signals by compensating for their integration durations based on a ratio of their integration durations to the respective integration duration that is within the linear range associated with the respective analyte; and combine the normalized light signals in order to expand the linear range associated with the respective analyte of the plurality of analytes.
For an understanding of the scope and nature of the invention, as well as the preferred mode of use, reference should be made to the following detailed description read in conjunction with the accompanying drawings. In the following drawings, which are examples, like reference numerals designate like or similar parts throughout the drawings.
In order to conduct quantitative and semi-quantitative biological assays it is important to have a large dynamic range to cover all analytes and samples for all patients under all health conditions. Recently, multiplex assays, which offer multiple tests have become very popular and have offer tremendous advantages over the conventional one test per sample. However, for multiplex assays, it also creates challenges for quantitative assays because the dynamic range needed to cover all the analytes. Considering the number of analytes in the multiplex assays, it becomes a daunting task. Conventional wisdom which is to dilute a sample to a certain concentration, and let all analytes fall into the single set of dynamic range of the single sensor becomes impractical to carry out because of the wide range of analyte concentrations and resulting signals ranging over many logs. Accordingly, a method and system which can adjust and expand the dynamic ranges depend on each of the analyte is needed.
The present invention is directed to a method for adjustable and expandable dynamic ranges for multiplex bioassays is comprising a) detecting a plurality of signals, N, from a series of optical integration durations, IT-N, for each analyte in said multiplex assays; b) normalizing each of multiple signals to a corresponding integration duration or normal integration duration; and c) selecting and combining some of the plurality of the normalized signals operating in linearity to adjust and expand the dynamic range
The present invention is directed to a method for adjustable and expandable dynamic ranges for multiplex bioassays wherein the plurality of signals, N=2-5 signals.
The present invention is directed to a method for adjustable and expandable dynamic ranges for multiplex bioassays of wherein the series of optical integration durations range from 0.1 millisecond to 10 second but can also range from 1 millisecond to 1 second, to from 10 milliseconds to 0.1 seconds. The multiple signals are obtained from optical sensors, comprising CCD, CMOS camera, photo diode, and photomultiplier tubes. The bioassay comprises specific binding assays including but not limited to immunoassays or molecular assays. The plurality of signals can be of any light spectrum including and outside of the human visual spectrum and according to preferred aspects of the invention include fluorescent or chemiluminescent signals.
The present invention is directed to a system for adjustable and expandable dynamic ranges for multiplex bioassays comprising a) a light source generating a plurality of excitation lights with different time duration; b) a sensor detecting a plurality of signals with a series of integration durations; c) a processor normalizing the plurality of signals from the sensor according to a corresponding integration duration; and selecting and combining a plurality of the normalized signals operating in linearity to adjust and expand the dynamic range for each analyte in the multiplex assays.
The present invention is directed to a system for adjustable and expandable dynamic ranges for multiplex bioassays wherein the plurality of signals, N=2-5 signals.
The present invention is directed to a system for adjustable and expandable dynamic ranges for multiplex bioassays of wherein the series of optical integration durations range from 0.1 millisecond to 10 second as well as other narrower and broader ranges. The multiple signals are obtained from optical sensors, comprising CCD, CMOS camera, photo diode, and photomultiplier tubes. The bioassay comprising immunoassays or molecular assays. The plurality of signals is fluorescence or chemiluminescence signal.
The present invention is directed to a system for adjustable and expandable dynamic ranges for multiplex bioassays, the multiple optical integration durations are in synchronizing with the excitation of the light source.
The concentration range of interest for individual analyte can vary widely. For example, for an immunoassay, it can be ranged from 0.01 pg/ml to 100,000 pg/ml; and for nucleic acid assays, from 0.001 to 1,000 fmole/μl.
For quantitative assays, a calibration curve: signal vs. concentration trendline, operating in a linearity is preferred to provide better accuracy for data interpretation.
While the read noise or electronic noise limits the lower end of the dynamic range. High sensitivity is often needed for analytes having very low concentrations. This is referred to as the lower limits of detection (LLOD). The LLOD is often limited by the sensor read noise. To be considered as good signal, the signal/noise ratio should be >3. However, the optical signal depends on the number of photons being generated and being detected. If we increase the duration of the excitation time and the corresponding integration duration, the sensor can detect more photons, thus improving sensitivity.
By combining a plurality of integration durations, N, normalizing the signal to a corresponding integration duration (or normally use standard integration duration), selecting some sets, N=2-5, of the normalized signals which operating in linearity, in terms of signal versus concentration, thus the dynamic range of each analyte in the multiplex assays can be adjusted and expanded.
The system 800 may include a computing device 802, as well as a light source 804 and an image detector 806 (e.g., which may include one or more of CCD, CMOS cameras, photo diodes, photomultiplier tubes, etc.). The light source 804 may be configured to provide light to analytes on solid substrates in a sample 805, and the image detector 806 may be configured to capture images of the analytes on the solid substrates in the sample 805.
For instance, a specific binding pair member may be provided for each analyte in the sample 805. For instance, the specific binding assay may be an antigen-antibody assay. As another example, the specific binding assay may be a nucleic acid hybridization assay. The nucleic acid hybridization assay may be a DNA hybridization assay, or an RNA hybridization assay, in various examples. The sample 805 may be contacted with specific binding pair members under conditions in which analytes will stably bind with their specific binding pair member but will not bind to other specific binding pair members and in which stably bound specific binding pairs are capable of being detected by emitting a light signal. For instance, the binding pair members may be bound to a solid substrate. The solid substrate may comprise a plurality of digitally coded beads, barcoded magnetic beads, color coded microbeads, or microarray settings on a planar surface. For instance, the beads may be labeled, e.g., by a bar code. In some examples, the beads may be magnetic. Additionally, in some examples, two or more specific binding pair members may be bound to different physical locations on the solid substrate.
For instance, in the example of barcoded solid substrates, each barcoded solid substrate may be immobilized with a specific biomolecular probe which can react with a specific analyte, but not other analytes, in the multiplex assay in the sample. The resulting analyte may be labelled with fluorophore for fluorescence detection.
The computing device 802, light source 804 and image detector 806 may be configured to communicate with one another via a wired or wireless network 810. To facilitate such communications, the computing device 802, light source 804, and image detector 806 may each respectively comprise a wireless transceiver to receive and transmit wireless communications.
In some embodiments, the computing device 802 may comprise one or more servers, which may comprise multiple, redundant, or replicated servers as part of a server farm. In still further aspects, such server(s) may be implemented as cloud-based servers, such as a cloud-based computing platform. For example, such server(s) may be any one or more cloud-based platform(s) such as MICROSOFT AZURE, AMAZON AWS, or the like. Such server(s) may include one or more processor(s) 812 (e.g., CPUs) as well as one or more computer memories 814.
Memories 814 may include one or more forms of volatile and/or non-volatile, fixed and/or removable memory, such as read-only memory (ROM), electronic programmable read-only memory (EPROM), random access memory (RAM), erasable electronic programmable read-only memory (EEPROM), and/or other hard drives, flash memory, MicroSD cards, and others. Memorie(s) 122 may store an operating system (OS) (e.g., Microsoft Windows, Linux, UNIX, etc.) capable of facilitating the functionalities, apps, methods, or other software as discussed herein. Memorie(s) 814 may also store an analyte identification application 816.
Additionally, or alternatively, the memorie(s) 816 may store data from various sources. This data may also be stored in a database 810, which may be accessible or otherwise communicatively coupled to the computing system 802.
Executing the analyte identification application 816 may include controlling the light source 804 to provide light to the sample 805. In various examples, the light source 804 may provide visible light or ultraviolet light to the sample 805. Furthermore, in various examples, the light source 804 may continuously provide light to the sample 805 over a period of time, or may provide pulses of light of various durations to the sample 805. Light signals (e.g., fluorescence signals, chemiluminescence signals, etc.) may be emitted by the sample 805 when particular analytes are present in the sample 805. Executing the analyte identification application 816 may further include controlling the image detector 806 to capture images of the sample 805 at various integration durations. For instance, the image detector 806 may be controlled to capture images at various times over a time period during which the light source 804 continuously provides light to the sample 805, or may be controlled to capture images associated with various pulses of light provided by the light source 804 to the sample 805. For instance, the image detector 806 may capture images at integration durations ranging from 0.01 milliseconds to 10 seconds.
Executing the analyte identification application 816 may further include determining respective ranges of integration durations associated with light signals that indicate the presence of respective analytes in the sample 805. For instance, the analyte identification application 816 may calculate these ranges for each analyte based on a signal-concentration response curve for that analyte. These signal-concentration responses curves, or the ranges themselves, may be stored in the database 810 in some examples and accessed by the analyte identification application 816. For each respective analyte, the analyte identification application 816 may identify and/or select a respective one of the images captured by the image detector 806 that was captured at an integration duration within the range of integration durations associated with that analyte. The analyte identification application 816 may analyze the identified and/or selected image for a particular analyte in order to determine the presence or absence of that analyte (e.g., based on whether the image includes a light signal emitted by the analyte). This process may be repeated for two or more of the analytes, or for all of the analytes, that may be present in the sample 805.
Additionally, in some examples, analyte identification application 816 may further normalize the other light signals by compensating for their integration durations based on a ratio of their integration durations to the respective integration duration that is within the linear range associated with the respective analyte. The analyte identification application 816 may combine these normalized light signals in order to expand the linear range associated with the respective analyte of the plurality of analytes.
In addition, memories 814 may also store additional machine readable instructions, including any of one or more application(s), one or more software component(s), and/or one or more application programming interfaces (APIs), which may be implemented to facilitate or perform the features, functions, or other disclosure described herein, such as any methods, processes, elements or limitations, as illustrated, depicted, or described for the various flowcharts, illustrations, diagrams, figures, and/or other disclosure herein. For instance, in some examples, the computer-readable instructions stored on the memory 814 may include instructions for carrying out any of the steps of any of the methods 900, 1000, or 1100 (which are described in greater detail below with respect to
At block 904, a plurality of images may be captured by a light detector (e.g., the light detector 806). Each image may include the plurality of analytes in the sample, and each image may be captured at a different integration duration. That is, over a period of time during which the light source continuously provides light to the sample, the image detector may capture a plurality of images at different times during that period of time. Alternatively, the light source may provide pulses of light to the sample, with each pulse having a different duration, and the image detector may capture respective images of the sample associated with each pulse of light.
For a first analyte of the plurality of analytes, the method 900 may proceed from block 904 to blocks 906-910, and for a second (different) analyte of the plurality of analytes, the method 900 may proceed from block 904 to blocks 912-916. In some examples, the method 900 may proceed in a similar manner with a third analyte, fourth analyte, etc., of the plurality of analytes.
At block 906, for the first analyte of the plurality of analytes of the sample, the method 900 may include determining a first range of integration durations of the light signal associated with the presence of the first analyte. For instance, the first range may be calculated based on a signal-concentration response curve for the first analyte. At block 908, a first image that is captured at an integration duration within the first range of integration durations may be identified, and at block 910, the first image may be analyzed to determine the presence (or absence) of the first analyte.
At block 912, for the second analyte of the plurality of analytes of the sample, the method 900 may include determining a second range of integration durations of the light signal associated with the presence of the second analyte. For instance, the second range may be calculated based on a signal-concentration response curve for the second analyte. At block 914, a second image that is captured at an integration duration within the second range of integration durations may be identified, and at block 916, the second image may be analyzed to determine the presence (or absence) of the first analyte.
At block 1002, a specific binding pair member may be provided for each analyte. For instance, the specific binding assay may be an antigen-antibody assay. As another example, the specific binding assay may be a nucleic acid hybridization assay. The nucleic acid hybridization assay may be a DNA hybridization assay, or an RNA hybridization assay, in various examples.
At block 1004, the sample may be contacted with specific binding pair members under conditions in which analytes will stably bind with their specific binding pair member but will not bind to other specific binding pair members and in which stably bound specific binding pairs are capable of being detected by emitting a light signal. For instance, the binding pair members may be bound to a solid substrate. The solid substrate may comprise a plurality of beads. For instance, the beads may be labeled, e.g., by a bar code. In some examples, the beads may be magnetic. Additionally, in some examples, two or more specific binding pair members may be bound to different physical locations on the solid substrate.
At block 1006, the method may include providing light (e.g., by a light source such as the light source 804) to the plurality of specific binding pair members and analytes over a period of time wherein stably bound specific binding pairs will emit a light signal.
At block 1008, a plurality of images, each including the plurality of analytes, may be captured at a plurality of durations by one or more photodetectors (e.g., the image detector 806).
For a first analyte of the plurality of analytes, the method 1000 may proceed from block 1008 to blocks 1010-1014, and for a second (different) analyte of the plurality of analytes, the method 1000 may proceed from block 1008 to blocks 1016-1020. In some examples, the method 1000 may proceed in a similar manner with a third analyte, fourth analyte, etc., of the plurality of analytes.
At block 1010, for the first analyte of the plurality of analytes of the sample, the method 1000 may include determining a first range of integration durations of the light signal associated with the presence of the first analyte. For instance, the first range may be calculated based on a signal-concentration response curve for the first analyte. At block 1012, a first image that is captured at an integration duration within the first range of integration durations may be identified, and at block 1014, the first image may be analyzed to determine the presence (or absence) of the first analyte.
At block 1016, for the second analyte of the plurality of analytes of the sample, the method 1000 may include determining a second range of integration durations of the light signal associated with the presence of the second analyte. For instance, the second range may be calculated based on a signal-concentration response curve for the second analyte. At block 1018, a second image that is captured at an integration duration within the second range of integration durations may be identified, and at block 1020, the second image may be analyzed to determine the presence (or absence) of the first analyte.
At block 1102, each analyte, of a plurality of analytes (e.g., 2-4096 analytes) on solid substrates in a multiplex bioassay (e.g., sample 805) may be identified by optical imaging (e.g., using the methods discussed above with respect to
At block 1104, a plurality of signals (N) (e.g., 2-5 signals) may be captured at each of a series of integration durations (IT-N) (e.g., 0.01 milliseconds to 10 seconds) for each analyte in said multiplex assay. Each image may contain light signals (e.g., fluorescence signals, chemiluminescence signals, etc.) for each analyte in the multiplex assay. These signals may be obtained from optical sensors (e.g., the image detector 806), such as, e.g., CCD, CMOS cameras, photo diodes, photomultiplier tubes, etc.
Blocks 1106-1110 may be performed for each respective analyte of the plurality of analytes. At block 1106, a respective light signal, of a plurality of light signals, that is captured at a respective integration duration, of a series of integration durations, that is within a linear range associated with the respective analyte, may be selected.
At block 1108, the other light signals, of the plurality of light signals, may be normalized by compensating for their integration durations based on a ratio of their integration durations to the respective integration duration that is within the linear range associated with the respective analyte.
At block 1110, the normalized light signals may be combined in order to expand the linear range associated with the respective analyte of the plurality of analytes.
Numerous modifications and variations in the practice of the invention are expected to occur to those skilled in the art upon consideration of the foregoing description on the presently preferred embodiments thereof. Consequently, the only limitations which should be placed upon the scope of the present invention are those that appear in the appended claims.