GB2582619A

GB2582619A - Enzyme linked assay methods and apparatuses

Info

Publication number: GB2582619A
Application number: GB1904315.7A
Authority: GB
Inventors: Lee Andrew
Original assignee: Sumitomo Chemical Co Ltd
Current assignee: Sumitomo Chemical Co Ltd
Priority date: 2019-03-28
Filing date: 2019-03-28
Publication date: 2020-09-30
Also published as: GB201904315D0; WO2020193934A1

Abstract

A method of measuring the concentration of a target analyte using a kinetic enzyme linked-assay (e.g. ELISA, K-ELISA) comprises i) mixing a sample with an enzyme linked affinity reagent specific for the analyte and ii) capturing analyte-enzyme complexes using a second affinity reagent to immobilise enzyme in a flow cell iii) introducing a vast excess of substrate to the flow cell iv) detecting the enzymatic conversion of the substrate to a reporter over a time series v) identifying a first time point corresponding to the introduction of the substrate vi) identifying a second time point corresponding to the depletion of the substrate such that zero order reaction (linear) kinetics cease vii) estimating based on the reporter concentration at the first and second time points, the concentration of the target analyte. A corresponding method for processing the time series of measured values, a computer program for data analysis, a flow cell preferably configured into an apparatus system to perform a microfluidic kinetic enzyme-linked assay are all disclosed.

Description

ENZYME LINKED ASSAY METHODS AND APPARATUSES

BACKGROUND

Embodiments of the present disclosure relate to methods and apparatuses for conducting enzyme linked assays.

Enzyme linked assays, for example enzyme-linked immunosorbent assay (ELISA), employ an enzyme which converts a substrate into a reporting substance which may be detected by colorimetric, fluorescence or chemiluminescence measurements. Enzyme linked assays may be carried out for any target analyte which can be ligand immobilised and linked to an enzyme, for example DNA strands, cells, viruses, bacteria, antigens, antibodies, or other small molecules. Enzyme linked assays are often conducted in well assay plates, or similar types of containers.

Enzyme linked assays may be measured kinetically or using an end point measurement. An endpoint measurement is the most commonly used method, in which a signal is simply read off after a set amount of time has elapsed. The signal at the end point is proportional to the concentration of analyte in an analysed sample. The time picked for the end point should be before a reaction plateau and/or before a change in a measured optical property such as absorbance becomes non-linear. In some protocols, a stop solution (or quenching solution) may be added to stop the enzyme substrate reaction at the end point. Use of a stop solution may be useful when carrying out multiple assays together, as a stop solution may allow multiple assays to be stopped together, and may permit measuring those assays over a longer period of time without concerns about further changes in a measured optical property.

In contrast to end point measurements, for kinetic measurements the sample is read multiple times during a period in order to measure a rate of signal change corresponding to an optical property. The rate of signal changes may be proportional to the concentration of analyte. Kinetic measurements may be used less often in lab based ELISA techniques, as kinetic measurements may be slower because multiple samples cannot be read at the same time using a well assay plate and a plate reader.

Xuan Weng, Gautam Gaur, and Suresh Neethirajan, "Rapid Detection of Food Allergens by Microfluidics ELISA-Based Optical Sensor", Biosensors (Basel), 2016 Jun, 6(2), 24, describes a microfluidic ELISA platform combined with an optical sensor, developed for the quantitative analysis of the proteins wheat gluten and Ara h 1.

Aung Thiha and Fatimah Ibrahim, "A Colorimetric Enzyme-Linked Immunosorbent Assay (ELISA) Detection Platform for a Point-of-Care Dengue Detection System on a Lab-onCompact-Disc", Sensors (Basel), 2015 May, 15(5), 11431-11441, describes an integrated device to detect and interpret the ELISA test results on a Lab-on-Compact-Disc platform.

ShuQi Wang, Savas Tasoglu, Paul Z. Chen, Michael Chen, Ragip Akbas, Sonya Wach, Cenk Ibrahim Ozdemir, Umut Atakan Gurkan, Francoise F. Giguel, Daniel R. Kuritzkes and Utkan Demirci, "Micro-a-fluidics ELISA for Rapid CD4 Cell Count at the Point-of-Care", Nature Scientific Reports volume 4, Article number: 3796 (2014), doi:10.1038/srep03796, describes a portable micro-a-fluidic platform for performing an automated CD4 cell count using an enzyme-linked immunosorbent assay (ELISA).

WO 2016/175708 Al describes an enzyme-linked immunosorbent assay (ELISA) plate comprising at least one row of reaction chambers.

US 2009/181411 Al describes microfluidic methods and devices for heterogeneous binding and agglutination assays.

US 5,073,029 describes optoelectronic device for rapid sequential measurement of the optical density of multiple samples.

US 4,857,454 A describes a method for kinetic measurement of enzyme activity bound to a solid matrix.

US 2005/036142 Al describes apparatus and methods for detecting analytes in a sample.

EP 2,839,280 A describes Enzyme-based diagnostic testing systems for detecting and quantifying at least one of the activity level or the concentration of an enzyme or a biochemical analyte in a biological sample.

SUMMARY

In some embodiments there is provided a method of measuring a concentration of a target analyte using an enzyme linked assay. The method includes functionalising a flow cell using an enzyme linked assay applied to a sample, such that in response to the sample contains the target analyte, the flow cell will become functionalised with an immobilised concentration of enzyme molecules bound to the target analyte. The method also includes introducing a substrate to a flow cell. The substrate is convertible into a reporting substance by the enzyme molecules. The method also includes recording a time series of measured values corresponding to an optical property of the flow cell which depends on a concentration of the reporting substance. The method also includes determining a first time point corresponding to the introduction of the substrate to the flow cell. The method also includes determining a second time point corresponding to an endpoint of linear kinetics for the conversion of substrate into reporting substance. The method also includes estimating, based on the measured values obtained between the first and second time points, a concentration of the target analyte in the sample.

Determining a second time point may include, for each given time point until the second time-point has been set, calculating a coefficient of determination corresponding to a linear model of the measured values between the first time point and the given time point. Determining a second time point may include, for each given time point until the second time-point has been set, in response to the coefficient of determination is less than or equal to a linearity threshold, setting the given time point as the second time point.

Determining a second time point may include calculating a first gradient of the measured values at the first time point. Determining a second time point may include, for each given time point until the second time-point has been set, calculating a difference between a gradient of the measured values at the given time point and the first gradient. Determining a second time point may include, for each given time point until the second time-point has been set, in response to the magnitude of the difference is greater than or equal to a gradient threshold, setting the given time point as the second time point.

Determining a first time point may include receiving a signal corresponding to activation of a substrate supply pump which is configured to introduce substrate into the flow cell. The substrate supply pump may comprise a peristaltic pump. The substrate supply pump may comprise a syringe pump. The substrate supply pump may comprise a pressure driven pump. The substrate supply pump may comprise a diaphragm pump.

Determining a first time point may include determining that the optical property of the flow cell has departed from a baseline value by more than an activation threshold.

Determining the first time point and determining the second time point may be carried out concurrently with recording the time series of measured values.

Determining the first time point and determining the second time point may be carried out subsequently to recording the time series of measured values.

Determining the first time point and/or the second time point may be carried out by a local data processing apparatus. Determining the first time point and/or the second time point may be carried out by a data processing apparatus which is remote from the flow cell. There may be a delay between recording the time series of measurements and determining the first and second time points. The delay may be seconds, days, weeks, months and so forth.

The optical property may be an absorbance of the reporting substance in the flow cell. The absorbance may be measured using a light source to illuminate the flow cell and a photodetector arranged to receive light transmitted through the flow cell, or reflected from the flow cell.

The optical property may be a fluorescence of the reporting substance in the flow cell. The fluorescence may be measured using a light source to illuminate any reporting substance in the flow cell with excitation light, and a photodetector arranged to receive fluorescence light emitted from the flow cell. An optical path between the flow cell and the photodetector may include a filter which absorbs the excitation light.

The light source may be a light-emitting diode, an organic light-emitting diode, a laser diode, a laser, a filament bulb, a tungsten halogen bulb, a fluorescent bulb, and so forth. Organic light-emitting diodes may be solution processed.

The optical property may be a chemiluminescence of the reporting substance in the flow cell. The chemiluminescence may be measured using a photodetector arranged to receive chemiluminescence light emitted from the flow cell. The flow cell may be screened from ambient light. When the optical property is a chemiluminescence, there may be no light source directed at the flow cell.

The photodetector may be a photodiode, an organic photodiode, a photovoltaic, a photoresistor, a phototransistor, and so forth.

The method may be carried out for a plurality of flow cells concurrently.

Two or more flow cells of the plurality of flow cells may be functionalised using a common sample. Two of more flow cells of the plurality of flow cells may be functionalised using different samples.

The substrate may be pumped through each flow cell in a closed loop. The substrate may be pumped through each flow cell in a reciprocating motion.

Apparatus may be specifically adapted to carry out the method.

In some embodiments there is provided a method of processing a time series of measured values. The time series of measured values is obtained by functionali sing a flow cell using an enzyme linked assay applied to a sample, such that if the sample contains the target analyte, the flow cell becomes functionalised with an immobilised concentration of enzyme molecules bound to the target analyte. Obtaining the time series of measured values also includes introducing a substrate to a flow cell. The substrate is convertible into a reporting substance by the enzyme molecules. Obtaining the time series of measured values also includes recording the time series of measured values corresponding to an optical property of the flow cell which depends on a concentration of the reporting substance. The method of processing the time series of measured values includes receiving the time series of measured values. The method of processing the time series of measured values also includes determining a first time point corresponding to the introduction of the substrate to the flow cell. The method of processing the time series of measured values also includes determining a second time point corresponding to an endpoint of linear kinetics for the conversion of substrate into reporting substance. The method of processing the time series of measured values also includes estimating, based on the measured values obtained between the first and second time points, a concentration of the target analyte in the sample.

In some embodiments there is provided a computer program product stored on a non-transitory computer readable medium and configured, when executed by one or more physical electronic processers of a data processing apparatus, to cause the data processing apparatus to carry out the method.

A computer program may be configured to cause a data processing apparatus to carry out the method.

In some embodiments there is provided a fluidic device including one or more flow cells. Each flow cell includes a channel and at least two ports in fluid communication with the channel. At least one internal surface of each channel is functionalised with a capture molecule. The fluidic device also includes a photodiode corresponding to each flow cell and arranged to receive light from the corresponding flow cell. Each photodiode is attached to the corresponding flow cell or each photodiode is integrally formed with the corresponding flow cell. The capture molecules enable functionalising the flow cell using an enzyme linked assay applied to a sample, such that in response to the sample contains the target analyte, the capture molecules will immobilise complexes of enzyme molecules bound to target analyte. The enzyme molecules act to convert a substrate into a reporting substance.

The photodiode may be an organic photodiode. Organic photodiodes may be solution processed directly onto one or more surfaces defining a channel of a flow cell. The fluidic device may be a micro-fluidic device.

The fluidic device may include a number of flow cells.

The fluidic device may also include a light-emitting diode corresponding to each flow cell. Each light-emitting diode may be arranged to illuminate the corresponding flow cell. Each light-emitting diode may be attached to the corresponding flow cell or each light-emitting diode may be integrally formed with the corresponding flow cell.

The light-emitting diode may be an organic light-emitting diode. Organic light-emitting diodes may be solution processed directly onto one or more surfaces defining a channel of a flow cell.

The fluidic device may also include a recording module connected to each photodiode and configured to, for each flow cell record a time series of measured values corresponding to an optical property of said flow cell. The optical property may depend on a concentration of the reporting substance in said flow cell. The fluidic device may also include a time point determination module configured to, for each flow cell, determine a first time point corresponding to the introduction of the substrate to said flow cell. The time point determination module may also be configured to, for each flow cell, determine a second time point corresponding to an endpoint of linear kinetics for the conversion of the substrate into the reporting substance. The fluidic device may also include an estimation module configured to, for each flow cell, estimate a concentration of a target analyte in a sample used to functionalise that flow cell using an enzyme linked assay, based on the measured values obtained between the first and second time points.

The fluidic device may also include one or more substrate reservoirs. The fluidic device may also include one or more substrate supply pumps connected between the one or more substrate reservoirs and the one or more flow cells. Each substrate supply pump may be configured to introduce the substrate into the channels of one or more flow cells.

Each substrate supply pump may introduce substrate into a single flow cell. Each substrate supply pump may introduce substrate into two or more flow cells concurrently. Each substrate supply pump may be connected to a corresponding substrate reservoir. Two or more substrate supply pumps may be connected to a common substrate reservoir. The substrate supply pump may comprise a peristaltic pump. The substrate supply pump may comprise a syringe pump.

Each substrate supply pump may be further configured to pump substrate through each flow cell in a closed loop. Each substrate supply pump may be further configured to pump substrate through each flow cell in a reciprocating motion.

The time point determination module may determine the first time point based on a time of activating the one or more substrate supply pumps.

In some embodiments there may be provided a system for measuring a concentration of a target analyte using an enzyme linked assay. The system may include the fluidic device and an apparatus. The apparatus may include a recording module connectable to one or more photodiodes of the fluidic device. The recording module may be configured to, for each flow cell, record a time series of measured values corresponding to an optical property of a flow cell. The optical property may depend on a concentration of the reporting substance in the flow cell. The apparatus may also include a time point determination module configured to, for each flow cell determine a first time point corresponding to the introduction of the substrate to the flow cell. The time point determination module may be further configured to, for each flow cell, determine a second time point corresponding to an endpoint of linear kinetics for the conversion of the substrate into the reporting substance. The apparatus may also include an estimation module configured to, for each flow cell, estimate a concentration of a target analyte in a sample used to functionalise that flow cell using an enzyme linked assay, based on the measured values obtained between the first and second time points.

The system may also include one or more substrate reservoirs. The system may also include one or more substrate supply pumps connectable between the one or more substrate reservoirs and the one or more flow cells of the fluidic device. Each substrate supply pump may be configured to introduce the substrate into the channels of one or more flow cells.

Each substrate supply pump may introduce substrate into a single flow cell. Each substrate supply pump may introduce substrate into two or more flow cells simultaneously. Each substrate supply pump may be connected to a corresponding substrate reservoir. Two or more substrate supply pumps may be connected to a common substrate reservoir. The substrate supply pump may comprise a peristaltic pump. The substrate supply pump may comprise a syringe pump.

In some embodiments there is provided apparatus for measuring a concentration of a target analyte using an enzyme linked assay. The apparatus is configured to receive a fluidic device which includes one or more flow cells. Each flow cell includes a channel and at least two ports in fluid communication with the channel. At least one internal surface of each channel is functionalised with a capture molecule which enables functionalising the flow cell using an enzyme linked assay applied to a sample, such that in response to the sample contains the target analyte, the capture molecules will immobilise complexes of enzyme molecules bound to target analyte. The enzyme molecules act to convert a substrate into a reporting substance. The apparatus includes a photodetector corresponding to each flow cell and arranged such that when the fluidic device is received by the apparatus, each photodetector will receive light from the corresponding flow cell. The apparatus also includes a recording module connected to each photodetector and configured to, for each flow cell record a time series of measured values corresponding to an optical property of said flow cell. The optical property depends on a concentration of the reporting substance in the flow cell. The apparatus also includes a time point determination module configured to, for each flow cell, determine a first time point corresponding to the introduction of the substrate to said flow cell. The time point determination module is further configured to, for each flow cell, determine a second time point corresponding to an endpoint of linear kinetics for the conversion of the substrate into the reporting substance. The apparatus also includes an estimation module configured to, for each flow cell, estimate a concentration of a target analyte in a sample used to functionalise said flow cell using an enzyme linked assay, based on the measured values obtained between the first and second time points.

Each photodetector may be a photodiode. Each photodiode may be an organic photodiode. Organic photodiodes may be solution processed. The fluidic device may be a micro-fluidic device.

The apparatus may be configured to receive a fluidic device comprising a plurality of flow cells.

The apparatus may also include a light source corresponding to each flow cell and arranged such that when the fluidic device is received by the apparatus, each light source will illuminate the corresponding flow cell.

The apparatus may also include one or more substrate reservoirs. The apparatus may also include one or more substrate supply pumps connectable between the one or more substrate reservoirs and the one or more flow cells of the fluidic device. Each substrate supply pump may be configured to introduce the substrate into the channels of one or more flow cells.

The apparatus may be local to the flow cell(s). The apparatus may be remote from the flow cell(s). The recording module may be connectable to the one or more photodiodes via a wired or wireless network.

The time point determination module may be configured to determine each second time point by, for each given time point until the second time-point is determined, calculating a coefficient of determination corresponding to a linear model of the measure values between the first time point and the given time point, and in response to the coefficient of determination is less than or equal to a linearity threshold, setting the given time point as the second time point.

The time point determination module may be configured to determine each second time point by calculating a first gradient of the measured values at the first time point. The time point determination module may be further configured to determine each second time point by, for each given time point until the second time-point is determined, calculating a difference between the gradient of the measured values at the given time point and the first gradient, and in response to the magnitude of the difference is greater than or equal to a gradient threshold, setting the given time point as the second time point.

The time point determination module may be configured to determine each first time point based on determining that the optical property of the corresponding flow cell has departed from a baseline value by more than an activation threshold.

The time point determination module may be configured to determine the first time point and the second time point concurrently with the recording module recording the time series of measured values.

The time point determination module may be configured to determine the first time point and the second time point subsequently to recording the time series of measured values.

There may be a delay between recording the time series of measurements and determining the first and second time points. The delay may be seconds, days, weeks, months and so forth.

The time determination module and/or the estimation module may be remote from the recording module. The time determination module and/or the estimation module may be connected or connectable to the recording module via one or more wired or wireless networks.

The recording module, the time determination module and/or the estimation module may be integrated as a single data processing device. The data processing device may take the form of a computing device including one or more digital electronic processors. The data processing device may take the form of a microcontroller, a field programmable gate array, and so forth.

The optical property may be an absorbance of the reporting substance in the flow cell. The absorbance may be measured using a light source to illuminate the flow cell and a photodetector arranged to receive light transmitted through the flow cell or reflected from the flow cell.

The optical property may be a fluorescence of the reporting substance in the flow cell. The fluorescence may be measured using a light source to illuminate any reporting substance in the flow cell with excitation light, and a photodetector arranged to receive fluorescence light emitted from the flow cell. The photodetector may be arranged at an angle to the light source to avoid or minimise detection of the excitation light. An optical path between the flow cell and the photodetector may include a filter which absorbs the excitation light.

The photodetector may be a photodiode, an organic photodiode, a photovoltaic, and so forth.

The optical property may be a chemiluminescence of the reporting substance in the flow cell. The chemiluminescence may be measured using a photodetector arranged to receive chemiluminescence light emitted from the flow cell. The flow cell may be screened from ambient light.

DESCRIPTION OF THE DRAWINGS

The disclosed technology and accompanying figures describe some implementations of the disclosed technology.

Figure 1 is a schematic illustration of a first fluidic device for conducting one or more enzyme linked assays; Figure 2 is a schematic illustration of a first example of a flow cell for a fluidic device; Figures 3 to 5 schematically illustrate using an enzyme linked assay method to functionalise a flow cell; Figure 6 is a schematic process flow diagram of a first method of performing a kinetic enzyme linked assay; Figure 7 is a schematic process flow diagram of a first approach to determining a time point corresponding to an endpoint of linear kinetics for the conversion of a substrate into a reporting substance; Figure 8 is a schematic process flow diagram of a second approach to determining a time point corresponding to an endpoint of linear kinetics for the conversion of a substrate into a reporting substance; Figure 9 is a schematic process flow diagram of a second method of performing a kinetic enzyme linked assay, Figure 10 is a schematic illustration of a second example of a flow cell; Figure 11 is a schematic illustration of an example of a flow cell for fluorescence measurements; Figure 12 is a schematic illustration of an example of a flow cell for chemiluminescence measurements.

Figure 13 is a schematic illustration of a system including a second fluidic device and an external computing device; Figure 14 is a schematic illustration of a second system including a third fluidic device and a reader device; Figure 15 is a schematic illustration of a third system including a fourth fluidic device and a second reader device; Figure 16 is a schematic illustration of a first arrangement for delivery of substrate solution into channels of a flow cell; Figure 17 is a schematic illustration of a second arrangement for delivery of substrate solution into channels of a flow cell; Figure 18 is a schematic illustration of a third arrangement for delivery of substrate solution into channels of two or more flow cells; Figure 19 is a schematic illustration of a fourth arrangement for delivery of substrate solution into channels of two or more flow cells; Figure 20 is a schematic illustration of an exemplary enzyme linked assay used to obtain illustrative experimental data' Figure 21 presents experimentally measured transmission values for the exemplary enzyme linked assay, as a function of assay time; Figure 22 presents values of a coefficient of determination r2 for a linear model fitted to the experimentally measured transmission values shown in Figure 21, as a function of assay time; Figure 23 presents kinetic assay measurements obtained using methods according to some embodiments of the present disclosure; and Figure 24 presents end point assay measurements for comparison with Figure 23.

The drawings are not drawn to scale and have various viewpoints and perspectives. The drawings are some implementations and examples. Additionally, some components and/or operations may be separated into different blocks or combined into a single block for the purposes of discussion of some of the embodiments of the disclosed technology. Moreover, while the technology is amenable to various modifications and alternative forms, specific embodiments have been shown by way of example in the drawings and are described in detail below. The intention, however, is not to limit the technology to the particular implementations described. On the contrary, the technology is intended to cover all modifications, equivalents, and alternatives falling within the scope of the technology as defined by the appended claims.

DETAILED DESCRIPTION

Unless the context clearly requires otherwise, throughout the description and the claims, the words "comprise," "comprising," and the like are to be construed in an inclusive sense, as opposed to an exclusive or exhaustive sense; that is to say, in the sense of "including, but not limited to." As used herein, the terms "connected," "coupled," or any variant thereof means any connection or coupling, either direct or indirect, between two or more elements; the coupling or connection between the elements can be physical, logical, electromagnetic, or a combination thereof Additionally, the words "herein," "above," "below," and words of similar import, when used in this application, refer to this application as a whole and not to any particular portions of this application. Where the context permits, words in the Detailed Description using the singular or plural number may also include the plural or singular number respectively. The word "or," in reference to a list of two or more items, covers all of the following interpretations of the word: any of the items in the list, all of the items in the list, and any combination of the items in the list. As used herein, by a material "over" a layer is meant that the material is in direct contact with the layer or is spaced apart therefrom by one or more intervening layers. As used herein, by a material "on" a layer is meant that the material is in direct contact with that layer. A layer "between" two other layers as described herein may be in direct contact with each of the two layers it is between or may be spaced apart from one or both of the two other layers by one or more intervening layers.

The teachings of the technology provided herein can be applied to other systems, not necessarily the system described below. The elements and acts of the various examples described below can be combined to provide further implementations of the technology. Some alternative implementations of the technology may include not only additional elements to those implementations noted below, but also may include fewer elements.

These and other changes can be made to the technology in light of the following detailed description. While the description describes certain examples of the technology, and describes the best mode contemplated, no matter how detailed the description appears, the technology can be practiced in many ways. Details of the system may vary considerably in its specific implementation, while still being encompassed by the technology disclosed herein. As noted above, particular terminology used when describing certain features or aspects of the technology should not be taken to imply that the terminology is being redefined herein to be restricted to any specific characteristics, features, or aspects of the technology with which that terminology is associated. In general, the terms used in the following claims should not be construed to limit the technology to the specific examples disclosed in the specification, unless the Detailed Description section explicitly defines such terms.

Accordingly, the actual scope of the technology encompasses not only the disclosed examples, but also all equivalent ways of practicing or implementing the technology under the claims.

To reduce the number of claims, certain aspects of the technology are presented below in certain claim forms, but the applicant contemplates the various aspects of the technology in any number of claim forms. For example, while some aspect of the technology may be recited as a computer-readable medium claim, other aspects may likewise be embodied as a computer-readable medium claim, or in other forms, such as being embodied in a meansplus-function claim.

In the following description, for the purposes of explanation, numerous specific details are set forth in order to provide a thorough understanding of implementations of the disclosed technology. It will be apparent, however, to one skilled in the art that embodiments of the disclosed technology may be practiced without some of these specific details.

The techniques introduced here can be embodied as special-purpose hardware (e.g., circuitry), as programmable circuitry appropriately programmed with software and/or firmware, or as a combination of special-purpose and programmable circuitry. Hence, embodiments may include a machine-readable medium having stored thereon instructions which may be used to program a computer (or other electronic devices) to perform a process. The machine-readable medium may include, but is not limited to, floppy diskettes, optical disks, compact disc read-only memories (CD-ROMs), magneto-optical disks, ROMs, random access memories (RAMs), erasable programmable read-only memories (EPROMs), electrically erasable programmable read-only memones (EEPROMs), magnetic or optical cards, flash memory, or other type of media / machine-readable medium suitable for storing electronic instructions. The machine-readable medium includes non-transitory medium, where non-transitory excludes propagation signals. For example, a processor can be connected to a non-transitory computer-readable medium that stores instructions for executing instructions by the processor.

Kinetic measurements of enzyme linked assays may offer higher accuracy when compared to endpoint assays. Another advantage associated with kinetic measurements of an enzyme linked assay may be an enhanced dynamic range. With an endpoint assay, the possible dynamic range may be limited by the time at which the assay is stopped or measured. High concentrations may need to be stopped quickly before saturation, whereas low concentrations may need more time for the signal to develop sufficiently to allow accurate and repeatable measurement. Despite the potential benefits, end point measurements have typically been employed more frequently. This may have been a result of difficulties in obtaining kinetic measurements for multiple assays in parallel, compared to conducting end point assays using well assay plates. Another issue is that there is often a time delay between adding a substrate and being able to arrange the assay within a measurements device or reader, and initial data may be missed. Consequently, many prior art point of care devices utilise an end point assay.

This disclosure concerns fluidic devices in which the measurement electronics, for example a pair of a light-emitting diode and a photodiode, are integrated with flow cells for conducting enzyme linked assays. Many such flow cells may be fabricated in a single fluidic device, and the integration of measurement electronics with each flow cell may enable effective parallelisation of kinetically measured enzyme linked assays. In addition, the inventors have developed methods which enable the time of introducing the substrate to be determined, as well as determining the duration of an initial period of linear kinetics. By limiting the measurements to the initial linear (or substantially linear) period, the accuracy and repeatability of an enzyme linked assay may be further improved, because it may be empirically observed that divergence between assays typically occurs in a subsequent phase of nonlinear kinetics.

According to methods and apparatus described in the present disclosure, fluidic flow cells may be permanently coupled to a detector (and a light source for some assays), or may be received into a device in which the flow cells are aligned with a detector (and a light source in some assays). This may enable a baseline signal to be established prior to in-situ addition of a substrate, so that the very start of the conversion reaction may be identified and used. In some examples according to the present disclosure, the integration of detectors and/or light sources with fluidic devices may be performed by solution processing organic photodiodes and/or organic light emitting diodes directly onto a structure defining a flow cell. In this way, fluidic devices having a number of flow cells, each with integrated organic photodiodes and organic light-emitting diodes may be produced relatively simply and cheaply. This may be advantageous for disposable or single use point of care applications.

Figure 1 is a schematic illustration of a first fluidic device 1 for conducting one or more enzyme linked assays according to some embodiments of the present disclosure.

The first fluidic device 1 includes one or more (at least one) flow cell 2, a photodiode 3 corresponding to each flow cell 2, a recording module 4, a time point determination module 5 and an estimation module 6.

Figure 2 is a schematic illustration of a first example of a flow cell 2 according to some embodiments of the present disclosure.

Each flow cell 2 includes a channel 7 and at least two ports 8, including first and second ports 8a, 8b in fluid communication with the channel 7. At least one internal surface 9 of each channel 7 is functionalised with a capture molecule 10. The structure and function of the first example flow cell 2 shown in Figure 2 shall be discussed in further detail hereinafter. In some example, the first fluidic device 1 may be a micro-fluidic device so that the channel 7 is a micro-fluidic channel.

Each photodiode 3 corresponds to a flow cell 2, and arranged to receive light 11 from that flow cell 2. In the first fluidic device 1, each photodiode 2 is attached to, or integrally formed with, the corresponding flow cell 2. Each photodiode 3 may take the form of an organic photodiode. Advantageously, organic photodiodes 4 may be solution processed directly onto one or more surfaces defining a channel 7 a flow cell 2.

Figures 3 to 5 schematically illustrate using an enzyme linked assay method to funct onalise a flow cell 2.

The capture molecules 10 enable functionalising the flow cell 2 using an enzyme linked assay applied to a sample 12. The capture molecules 10 may take the form of for example, an antibody, an antigen, a DNA strand, a RNA strand, a protein, a molecularly imprinted polymer (MIP), DNA aptamers, RNA aptamers, and so forth.

Referring in particular to Figure 3, the sample 12 is mixed with a first reagent 13 which includes a quantity of enzyme complexes 14. Each enzyme complex 14 may include, for example, an enzyme 15 bound to a binding group 16. The binding group 16 is specific for a particular target analyte 17 and may take the form of, for example, an antibody, antigen, DNA strand, RNA stand, protein and so forth. The mixture of the sample 12 and the first reagent 13 is left for a period so that any concentration of target analyte 17 in the sample 12 has sufficient time to react with binding groups 16 of the enzyme complexes 14 to form target-enzyme complexes 18. The time needed depends on the particular enzyme linked assay being conducted, and may be seconds, minutes or up to several hours. The quantity of enzyme complexes 14 should be greater than any expected quantity of target analyte 17, in order to avoid saturation of the assay. This is usually not an issue as enzyme linked assays are primarily intended for detection of relatively low concentrations of target analyte 17, as compared to other assay methods.

Referring in particular to Figure 4, the reacted mixture 19 of sample 12 and first reagent 13 is pumped into the channel 7 of a flow cell 2 in order to functionalise the flow cell 2. For example, the reacted mixture 19 may be pumped in through the first port 8a whilst air 20 escapes through the second port 20. In some example, the reacted mixture 19 may be pumped in through the first port 8a until the channel 7 is filled and the reacted mixture 19 flows out of the second port 8b. The reacted mixture 19 is left in the channel 7 for at least longer enough for target-enzyme complexes 18 to bind to capture molecules 10 to form immobilised target-enzyme complexes 21. The quantity of capture molecules 10 should be larger than the quantity of target-enzyme complexes 18 in order to avoid saturation of the assay. The reacted mixture 19 may be left in the channel 7 for a period of seconds, minutes or hours.

The reacted mixture 19 is then flushed out of the channel 7, for example using distilled water, deionised water, a buffer solution, and so forth. The flow cell 2 is now functionalised, and the results of the assay may be read by introducing a substrate solution 22 into the channel 7. The substrate solution 22 contains a substrate 23 which is converted into a reporting substance 24 by the enzyme 15 of the immobilised target-enzyme complexes 21. The reporting substance 24 has one or more detectable optical properties such as, for example, absorbance of visible, ultraviolet and/or infrared light, fluorescence and/or chemiluminescence. In a chemiluminescent assay, the reporting substance 24 takes the form of a high energy intermediate molecule which decays accompanied by emission of visible, ultraviolet and/or infrared light.

Since the rate of conversion of substrate 23 into reporting substance 24 depends on the concentration of immobilised target-enzyme complexes 21, a rate of change of an optical property arising from the reporting substance 24 may be linked to the concentration of target analyte 17 in the sample 12 used to flinctionalise the flow cell 2. For example, calibration experiments may be conducted using known concentrations of the target analyte 17.

In examples where the optical property is an absorbance or fluorescence, the first fluidic device 1 also includes a light emitting diode 25 corresponding to each flow cell 2. Each light emitting diode 25 is arranged to illuminate the corresponding flow cell with light 26. Each light-emitting diode 25 is attached to, or integrally formed with, the corresponding flow cell 2. Each light-emitting diode 25 may be an organic light-emitting diode. Advantageously, organic light-emitting diodes 25 may be solution processed directly onto one or more surfaces defining a channel 7 of a flow cell 2. In some examples, a single organic light emitting diode 25 may have a large area which is arranged to illuminate two or more flow cells 2.

In some examples, the first fluidic device 1 may also include one or more output devices 33. The one or more output devices 33 may provide a user with an indication of one or more estimated concentrations 32. For example, the one or more output devices 33 may include one or more light-emitting diodes or liquid crystal display elements. Alternatively, the one or more output devices 33 may take the form of a communications interface which permits exporting one or more estimated concentrations 32 to a separate device via a wired or wireless network. In some example, the one or more output devices 33 may provide par-processed output (not shown) to a user of the fluidic device 1. In some examples, the estimation module 6 and/or output device(s) may compare an estimated concentration against an assay threshold value, and then output the result of the comparison in a positive or negative format. For example, present/not present, pregnant/not pregnant, high risk/low risk, and so forth.

In some examples, the first fluidic device 1 includes a number of flow cells 2, for example at least two and preferably more. This may allow a number of different samples 12 to be analysed in parallel. Additionally, or alternatively, using a number of flow cells 2 may allow multiple enzyme linked assays to be conducted in parallel on the same sample 12.

In some examples, the recording module 4, the time determination module 5 and/or the estimation module 6 may be integrated as a single data processing device such as, for example, a computing device including one or more digital electronic processors, a microcontroller, a field programmable gate array, an application specific integrated circuit, and so forth.

First method of performing a kinetic assay Figure 6 is a schematic process flow diagram of a first method of performing a kinetic enzyme linked assay, according to some embodiments of the present disclosure.

The first method shall be described in relation to the first fluidic device 1. However, the first method is not limited to being carried out using the first fluidic device 1, and may be carried out using any suitable flow cell(s) and any suitable hardware for recording an optical property related to the concentration of a reporting substance 24 as a function of time. The first method shall be described in relation to a single flow cell 2. However, the first method may be carried out for two or more flow cells in parallel.

The flow cell 2 is functionalised with a concentration of immobilised target-enzyme complexes 21 (step St). For example, as described hereinbefore with reference to Figures 2 to 5.

The substrate solution 22 including the substrate 23 is introduced into the channel 7 of the flow cell 2 (step S2). Either before the substrate solution 22 is introduced, or substantially simultaneously with introduction of the substrate solution 22, recording of a time series 30 of measured values is commenced (step S3). The time series 30 of measured values correspond to an optical property of the flow cell 2 which depends on a concentration of the reporting substance 24. The time series 30 may span a predetermined duration which is expected to encompass the period of linear kinetics for the assay. Whilst the predetermined duration has not elapsed (step S4), the recording of the time series 30 continues (step S3).

The optical property may be an absorbance, fluorescence or chemiluminescence of the reporting substance 24. In general, an absorbance may be measured using a light source 27 (Figure 15) arranged to illuminate the flow cell 2, and a photodetector 28 (Figure 15) arranged to receive light which has passed through the flow cell 2 in a transmission (Figure 2) or reflection (Figure 10) geometry. In general, a fluorescence may be measured using a light source 27 to excite the reporting substance 24 with incident light 26 at a first wavelength, and using a photodetector 28 to detect light 11 re-emitted at a second wavelength. The photodetector 28 may be screened from detecting the incident light 26 geometrically and/or using filters. A light source 27 may take the form of a light-emitting diode, an organic light-emitting diode, a laser diode, a laser, a filament bulb, a tungsten halogen bulb, a fluorescent bulb, and so forth. A photodetector 28 may be a photodiode, an organic photodiode, a photovoltaic device, and so forth.

For example, in the first fluidic device 1 shown in Figure 1, the recording module 4 receives a photocurrent 29 from the photodiode 3. The recording module 4 is configured to a time series of record measured values of the photocurrent 29. The recording module 4 may also be configured to perform processing such as, for example, amplifying and or filtering the photocurrent 29. When the optical property is absorbance or fluorescence, the recording module 4 may be further configured to control illumination of the corresponding light emitting diode 25. The recording module 4 may also be configured to convert the photocurrent 29 into a derived measurement, for example a transmittance or absorbance of the flow cell 2. The time series 30 of measured values output to the time point determination module 5 may correspond to absorbance, florescence or chemiluminescence, depending on the specific assay being conducted.

Once the predetermined duration has elapsed (step S4), the time series 30 is analysed in order to determine a first time point 0 corresponding to the time of introduction of the substrate solution 22 and substrate 23 to the flow cell 2 (step S5).

The first time point ti may be determined in any one of a number of suitable ways. In some examples, the first time point tl may correspond to activation of a substrate supply pump 31 (Figure 16) which supplies the substrate solution 22 to the flow cell 2, for example a signal may be received which corresponds to activation of the substrate supply pump 31. In other examples, the first time point 0 may be determined as a time at which the monitored optical property of the flow cell (absorbance, fluorescence, chemiluminescence) departs from a baseline value by more than an activation threshold. The activation threshold may be determined from calibration experiments using known concentrations of target analyte 17 and a known first time point ti of introducing the substrate solution 22.

For example, in the first fluidic device I the time point determination module 5 provides the function of determining the first time point tr.

The time series 30 following the first time point II is further analysed to determine a second time point 12 which corresponds to an endpoint of linear kinetics for the conversion of the substrate 23 into the reporting substance 24 (step S6). The first time point ti may be determined in any one of a number of suitable ways.

For example, Figure 7 is a schematic process flow diagram of a first approach to determining the second time point 12.

A time parameter T is set equal to r = tr (step S9). The time parameter r is incremented by an amount ót corresponding to a sampling interval of the time series 30 (step S10) This corresponds to incrementing time parameter r by one sample. A measured value from the time series 30 may be denoted kit). A linear model is fitted to the time series 30 of measured values 1/(i) for the interval tr < t <r (step S11). The coefficient of determination r2 is calculated for the fitted model and the values TAO for it < t < r (step S12). If the value of the coefficient of determination r2 remains larger than a linearity threshold Chn (step S13), then the time parameter T is incremented (step S10) and the linear model re-fitted for measured values V(t) with /7 <t <r 61.

However, if the value of the coefficient of determination r2 is less than or equal to the linearity threshold Cr" (step S13), then the second time point 12 is set equal to the present value of the time parameter T, i.e. 12 = T (step S14). If the final sample in the time series 30 is reached and the second time point t2 is not yet set, then the second time point t2 may be set to a final value of the time parameter mast, i.e. t = Tlart.

Figure 8 is a schematic process flow diagram of a second approach to determining the second time point t2.

Similarly to the first approach, a time parameter T is set equal to r= ti (step S15). A gradient of the time series 30 of measured values V(t) is calculated corresponding to the first time point ti (step S16). For example, the gradient at the first time point may be denoted as: dV G(ti) = dt t=ti (1) The gradient G(t) may be calculated using any suitable numerical approximation, for example a forward difference, backwards difference or central difference numerical gradient, and so forth.

The time parameter r is incremented by an amount 61 corresponding to a sampling interval of the time series 30 (step S17). This corresponds to incrementing time parameter r by one sample. The gradient G(r) corresponding to the value of the time parameter is calculated (step S18). A difference AG is calculated as the magnitude of the difference between the gradient G(tt) at the first time point and the gradient G(r) at the current value of the time parameter r, i.e. AG = G(r) -G(I t)I (step S19). As the kinetics of reporting substance 24 generation depart from linearity, the gradient difference AG from start to end of the interval between the first time point 17 and the time parameter T will increase. If the gradient difference AG remains less than a gradient threshold Gm, (step S20), then the time parameter r is incremented (step S17), and the gradient calculations are repeated (steps S18 and S19).

However, if the gradient difference AG exceeds the gradient threshold Gem (step S20), then the second time point t2 is set equal to the current time parameter r, t2 = T. If the final sample in the time series 30 is reached and the second time point 12 is not yet set, then the second time point 12 may be set to a final value of the time parameter Vail, i.e. t = Tact.

In the example of the first fluidic device 1 shown in Figure 1, the time point determination module 5 also provides the function of determining the second time point t2.

Referring again to Figure 6, following determination of the second time point t2 (step S6), the first and second time points ft, t2 are used along with the time series 30 to estimate a concentration 32 of the target analyte 17 which was present in the sample 12 (step S7). The estimation of the concentration 32 of the target analyte 17 is based on the gradient of the measured values V(1) between the first and second time points 11 <l< b. For example, the concentration 32 may be estimated based on the gradient of a linear model fitted to the values It(t) between the first and second time points ti < t < 12. In another example, the estimated concentration 32 may by based on an average value of the gradient G(t) between the first and second time points II < t <12. The estimate of concentration 32 may be based on an empirical relationship determined using data from calibration experiments conducted using known concentrations of target analyte 17. Alternatively, the estimate of concentration 32 may be based on interpolating (or extrapolating) between (or beyond) the data from calibration experiments.

In the example of the first fluidic device I shown in Figure I, the estimation module 6 provides the function of estimating one or more estimated concentrations 32 based on the time series 30, the first time point Ii and the second time point t2.

If further samples 12 are to be measured subsequently (step S8), the first method is repeated.

By basing estimated concentrations 32 only on the initial period of linear kinetics, the reliability and repeatability of an enzyme linked assay may be improved. Application of the first method requires integration of the flow cell(s) 2 and monitoring of the relevant optical property of the flow cell 2, because it is important that the first time point It is captured in the time series 30. This may be difficult to achieve in a conventional assay well plate, as typically a substrate solution 22 must be added to all of the wells before the well plate is transferred to a measurement/reading device. Using the first fluidic device 1, which incorporates channels 7 having ports 8 for direct injection of substrate solution 2 integrated with photodiodes 3 and, when necessary, light-emitting diodes 25, enables starting to record the time series 30 before or substantially at the same time as introducing the substrate solution 22. In this way, the initial, linear kinetic behaviour may be reliably recorded.

Using the initial phase of linear kinetics may also improve the dynamic range, because even if the assay subsequently saturates, the initial linear period will allow estimating a concentration. By contrast, for an end point assay a set stop point might mean that faster assays may be saturated before slower assays have reached the stop point.

Although described in relation to a single flow cell 2, the first method is equally applicable to any number of flow cells 2 being analysed in parallel (concurrently). Two or more flow cells 2 may be functionalised using different samples 12. Additionally or alternatively, two or more flow cells 2 may be used to perform different assays on the same sample 12.

Second method of performing a kinetic assay Figure 9 is a schematic process flow diagram of a second method of performing a kinetic enzyme linked assay, according to some embodiments of the present disclosure.

The first method (Figure 6) relates to examples in which the time series 30 is recorded, then subsequently analysed to determine the first and second time points tr, t2. However, in a second method according to the present specification the first and second time points ft, t2 may be determined concurrently with recording the time series 30. The second method is substantially similar to the first method, and unless self-evidently incompatible, any feature described in relation to the first method should be considered equally applicable to the second method.

The flow cell 2 is functionalised with a concentration of immobilised target-enzyme complexes 21 (step S22) in the same way as the first method (step St).

The substrate solution 22 including the substrate 23 is introduced into the channel 7 of the flow cell 2 (step S23) in the same way as the first method (step S2). In the same way as the first method (step S3), either before the substrate solution 22 is introduced, or substantially simultaneously with introduction of the substrate solution 22, recording of a time series 30 of measured values is commenced (step S24). The time series 30 of measured values 1/(i) again corresponds to an optical property of the flow cell 2 which depends on a concentration of the reporting substance 24.

If the first time point tt has not been determined (step S25), then it is determined whether the currently sampled value V(r) is the first time point (step S26), and the next value V(r-(50 is recorded (step S24). If the currently sampled value V(r) is the first time point (step S26), then the first time point tt = I-is recorded. The conditions for determining the first time point ft (step S26) are the same as for the first method (step S5).

If the first time point tt has been determined and recorded (step S25), then it is determined whether the currently sampled value V(T) is the second time point (step S27). In other words, it is checked whether the linear kinetic region is still occurring. The conditions for determining the first time point ti (step S27) are the same as for the first method (step S6).

For example, referring again to Figure 7, the second time point (2 may be determined using a minor modification of the coefficient of determination (-2 approach. In particular, the process may flow from determining that the first time point ti has been found (step S25) directly to fitting the linear model (step S11) followed by calculation of the coefficient of determination r2 (step S12). If the coefficient of determination ri) > Ch, (step S13), then the process continues without setting the second time point (2 (to step S28). If the coefficient of determination r2 < Om (step 513), then second time point (2 is set equal to the current time (2 = T (step S14).

Alternatively, referring again to Figure 8, the second time point t2 may be determined using a minor modification of the gradient difference AG approach. In particular, the gradient G(/,) at the first time point ti may be calculated at the same time that the first time point (i is determined (step S26) For the next measured value F(T), the process may flow from determining that the first time point ti has been found (step S25) directly to calculating the current gradient G(r) (step S18) and the gradient difference AG (step S19). If the gradient difference AG < Gem (step 520), then the process continues without setting the second time point t2 (to step 528). If the gradient difference AG> Gtin (step S20), then second time point (2 is set equal to the current time (2 = r (step S21) If the gradient difference AG approach is used on the most recent sample V(v), only backward difference numerical gradients may be used. However, the gradient difference AG approach may be employed using a delay of one or more samples in order to allow use of forward and/or central difference numerical gradients, for example considering at time or later.

Returning to consideration of Figure 9, it is checked whether the second time point 12 has been found (step S28), and if not the next measured value T(T--fit) is recorded (step S24). If the second time point (2 has been found (step S28), then the concentration(s) 32 are estimated (step S29) in the same way as for the first method (S7).

If further samples 12 are to be measured subsequently (step S30), the second method is repeated.

The second method may reduce the time taken to read out the assay, since the time series 30 is only recorded for long enough to span the initial phase of linear kinetics.

Example of a flow cell for measurements of absorbance in transmission Referring back to Figure 2, an example of a flow cell 2 shown is configured for measurements of absorbance in a transmission geometry.

The photodiode 3 is directly supported on a first surface 34 of a first transparent substrate 35. In some examples, the photodiode 3 may be an organic photodiode 3 formed directly on the first transparent substrate 35 using solution processing methods. Solution processing of organic photodiodes 3, for example printed organic photodiodes 3, may permit relatively low cost direct integration with the flow cell 2. This may be an advantage because each flow cell 2 is effectively single use. In this context, attachment of separate organic or inorganic photodiodes 3 may be more complex and/or expensive. The first transparent substrate 35 may be formed of, for example, poly(methyl methacrylate) (PMIN4A), or similar transparent plastics. Examples of materials which may be used to form the first transparent substrate 35 include, but are not limited to, polydimethylsiloxane(PDMS), thermoset polyester, polystyrene, polycarbonate, poly-ethylene glycol diacrylate, polyurethane, cyclic-olefin copolymer (COC). In some examples, the first transparent substrate 35 may be formed from glass.

A second, opposite surface 36 of the first transparent substrate 35 supports a functional substrate 37, for example the first transparent substrate 35 may be bonded to the functional substrate 37. The capture molecules 10 are bound to the functional substrate 37. In some examples, functional substrate 37 may be treated with the capture molecules 10 prior to assembling the flow cell(s) 2. In some examples, the flow cell(s) 2 may be assembled, and the functional substrates 37 in contact with each channel 7 may be functionalised with capture molecules 10 in-situ using the ports 8a, 8b. The functional substrate 37 may be formed of; for example, glass or plastics materials having surfaces compatible with the capture molecules 10. The functional substrate 37 may be formed from any material suitable for forming the first transparent substrate 35.

Depending on the choice of materials for the first transparent substrate 35 and the nature of the capture molecules, it may be possible to omit the functional substrate 37. For example, if the capture molecules 10 may be bonded directly to the material of the first transparent substrate 35.

The shape of each channel 7 is defined by a gasket 38 supported on the functional substrate 37 (or first transparent substrate 35), which defines a perimeter of the channel 7 of the flow cell 2. The gasket 38 may be formed of silicone rubber, or other similar materials suitable for forming a liquid-tight seal when compressed or bonded between a pair of plates.

A second transparent substrate 39 is placed over the gasket 38 to seal the channels 7. The second substrate 39 may have a first surface 40 contacting the gasket 38 and defining one surface of the channel 7, and a second opposite surface 41. Fluid communication into/out of the channel 7 is provided by first and second ports 8a, 8b which penetrate through the second transparent substrate 39. The ports 8a, 8b may be through holes which are always open. Alternatively, in some examples each port 8a, 8b may take the form of a rubber plug or bung having a central aperture which may be accessed using, for example, a needle, but which is otherwise held closed by pressure. In other examples, the ports 8a, 8b may be permanently coupled to tubes and/or pumps for supplying and/or removing fluids. Examples of fluids which may be supplied and/or removed from a channel 7 may include a sample solution for functionalising the channel 7, one or more washing, flushing or buffer solutions, the substrate solution 22, and so forth.

The light-emitting diode 25 is directly supported on the second surface 41 of the second transparent substrate 39. In some examples, the light-emitting diode 25 may be an organic light-emitting diode 25 formed directly on the second transparent substrate 39 using solution processing methods. Solution processing of organic light-emitting diodes 25, for example printed organic light-emitting diodes 25, may permit relatively low cost direct integration with the flow cell 2. This may be an advantage because each flow cell 2 is effectively single use. In this context, attachment of separate organic or inorganic light-emitting diodes 25 may be more complex and/or expensive. The second transparent substrate 39 may be formed of, for example, poly(methyl methacrylate) (PMIVIA), or any material suitable for forming the first transparent substrate 35.For measurements of absorbance of the channel 7 in transmission, the light 11 received at the photodiode 3 represents the fraction of the incident light 26 from the light-emitting diode 25 which is not absorbed or scatted by the reporting substance 24 (or other intervening features).

In some examples, the shape of each channel 7 need not be defined by a gasket 38. Instead, the channel 7 may be formed by machining, for example micromilling, the first transparent substrate 35 and/or the functional substrate 37. In other examples, the channels 7 may be formed during the production the first transparent substrate 35 and/or the functional substrate 37, for example by 3D printing or injection moulding. In examples which do not include a gasket 38, the channels 7 may be sealed by forming a liquid-tight seal between the second transparent substrate 37 and the first transparent substrate 35 and/or the functional substrate 37.

Example of a flow cell for measurements of absorbance in reflection The flow cell 2 configuration is not limited to measurements of absorbance in transmission.

Figure 10 is a schematic illustration of a second example of a flow cell 42 (second flow cell 42) according to some embodiments of the present disclosure.

The second flow cell 2 is the same as the first flow cell 2, except that the first transparent substrate 35 is replaced with a reflector 43, and the photodiode 3 is supported, or formed, on the second surface 41 of the second transparent substrate 39. One or more photodiodes 3 may form an alternating pattern with one or more light-emitting diodes 25. Incident light 26 is reflected from within the volume of the channel 7 or from reflector 43, and the reflected light 11 is incident on the photodiode 3. In some examples, the light-emitting diode(s) 25 may form a ring around a central photodiode 3, or the reverse.

Example of a flow cell for measurements of fluorescence Flow cell 2, 42 configurations are not limited to measurements of absorbance, and fluorescence may alternatively be measured.

Figure 11 is a schematic illustration of an example of a fluorescence flow cell 44 according to some embodiments of the present disclosure.

The fluorescence flow cell 44 is similar to the second flow cell 42, except that the first transparent substrate 35 replaces the reflector 43, and a layer of filters 45, 46 is disposed between the second surface 41 of the second transparent substrate 39 and the photodiode(s) 3 and light-emitting diode(s) 25.

Incident light 26 from the light-emitting diodes 24 passes through a first wavelength filter 45 and into the channel 7. The first wavelength filter 45 may provide that the incident light 26 has a relatively narrow spectral bandwidth compared to the raw output of the light-emitting diode(s) 25. This may improve the selectivity of the photocurrent signal 29 from the photodiode(s) 3. Incident light 26 may pass through the flow cell 44, may be scattered within the channel 7, or may be reflected from an internal surface of the flow cell 44. However, a second wavelength filter 46 attenuates light in the spectral bandwidth corresponding to the incident light, reducing the corresponding signal detected by the photodiode 3. When reporting substance 24 is present, the incident light 26 may excite the reporting substance to fluoresce and re-emit light 11 having a longer wavelength (the wavelength difference may be referred to as Stokes shift). The second wavelength filter 46 does not significantly attenuate the re-emitted light 11. In this way, fluorescence of a reporting substance 24 may be used as the optical property.

Example of a flow cell for measurements of chemiluminescence Flow cell 2, 42, 44 configurations are not limited to active measurements requiring incident light 26, for example absorbance or fluorescence.

Figure 12 is a schematic illustration of an example of a chemiluminescence flow cell 47 according to some embodiments of the present disclosure The chemiluminescence flow cell 47 is the same as the first example of a flow cell 2, except that the light-emitting diode 25 is omitted. Chemiluminescent reporting substance 24 generated in the channel 7 emits light 11 which is detected by the photodiode 3.

No matter what type or configuration of flow cell 2, 42, 44, 47 is used, the fluidic device 1 may be screened from ambient light.

Second fluidic device and system Figure 13 is a schematic illustration of a system 48 including a second fluidic device 49 and an external computing device 50, according to some embodiments of the present disclosure.

The second fluidic device 49 is the same as the first fluidic device 1, except that it does not include the time point determination module 5 or the estimation module 6. Instead, the functions of the time point determination module 5 and the estimation module 6 are provided by an external computing device SO connected to the second fluidic device 49. In some examples, the external computing device 50 is connected to the second fluidic device 49 via a wired or wireless network 51. The external computing device 50 may be remote from the second fluidic device 49, for example the external computing device SO may take the form of a server.

The system 48 may be used to implement either the first method (Figure 6) or the second method (Figure 9). Since each flow cell 2, 42, 44, 47 of the second fluidic device 49 is effectively single use (cannot be used for a second assay), it may be advantageous to reduce the amount of processing which is performed by the second fluidic device 49, which may reduce the cost and complexity of the consumable second fluidic device 49. However, be retaining the integrated photodiode(s) 3, and for some assay types the integrated light-emitting diode(s) 25, the second fluid device 49 remains suitable for obtaining a time series 30 of measured values 17(t) which includes the initial period of linear kinetics. In this way, the system 48 is compatible with the first and second methods according to the present specification, and may enable improved reliability and repeatability for quantifying an enzyme linked assay.

There may be a delay between recording the time series 30 of measured values ITO and determining the first and second time points, for example a delay may be seconds, days, weeks, months and so forth.

Third fluidic device and system Figure 14 is a schematic illustration of a second system 52 including a third fluidic device 53 and a reader device 54, according to some embodiments of the present disclosure.

The third fluidic device 53 is the same as the first fluidic device I, except that it does not include the time point determination module 5, the estimation module 6 or the recording module 4. Instead, the functions of the recording module 4, the time point determination module 5 and the estimation module 6 are provided by a separate reader device 54 connected to the third fluidic device 53. In some examples, the reader device 54 is connected to the third fluidic device 53 via a wired or wireless network 51.

The second system 52 may be used to implement either the first method (Figure 6) or the second method (Figure 9). Since each flow cell 2, 42, 44, 47 of the third fluidic device 53 is effectively single use (cannot be used for a second assay), it may be advantageous to remove the need for any processing to be performed by the third fluidic device 53, which may reduce the cost and complexity of the consumable third fluidic device 53. However, by retaining the integrated photodiode(s) 3, and for some assay types the integrated light-emitting diode(s) 25, the third fluidic device 53 remains suitable for obtaining a time series 30 of measured values V(r) which includes the initial period of linear kinetics. In this way, the second system 54 is compatible with the first and second methods according to the present specification, and may enable improved reliability and repeatability for quantifying an enzyme linked assay.

Additionally, the reader device 54 may provide power and/or control signals 55 to light-emitting diodes of the third fluidic device 53. For example, the third fluidic device 53 may include a battery (not shown) to power one or more light-emitting diodes 25, and the reader device 54 may provide power and/or control signals 55 to switch the one or more light-emitting diodes 25 on or off. Alternatively, the reader device 54 may be connected to the third fluidic device 53 in order to provide power and/or control signals 55 which directly power one or more light-emitting diodes 25 in order to illuminate corresponding flow cells 2, 42, 44, 47.

The recording module 4, the time determination module 5 and/or the estimation module 6 may be provided as separate components. Alternatively, the recording module 4, the time determination module 5 and/or the estimation module 6 may be provided by a microcontroller, one or more digital electronic processors, a field programmable gate array, and so forth.

Fourth fluidic device and system Figure 15 is a schematic illustration of a third system 56 including a fourth fluidic device 57 and a second reader device 58, according to some embodiments of the present disclosure.

The fourth fluidic device 58 is the same as the third fluidic device 53, except that it does not include any photodiodes 3 or light-emitting diodes 25. The second reader device 58 is the same as the reader device 54, except that it additionally includes one or more photodetectors 28, and optionally includes one or more light sources 27, depending on the type of assay to be conducted using the fourth fluidic device 57.

The second reader device 58 is configured to receive a fourth fluidic device 57 so that each photodetector 28 may receive light 11 from a corresponding flow cell 2, 42, 44, 47. Depending on the type of assay, a fourth fluidic device 57 received by the second reader device 58 may additionally be arranged so that each flow cell 22 receives incident light 26 emitted by a corresponding light source 27 of the second reader device 58.

The recording module 4 receives signals 59 directly from the one or more photodetectors 28, and measures the time series 30 of measured values VW. The signals 59 may take the form of a photocurrent 29, depending on the type of photodetector 28 which is used. The one or more photodetectors 28 may be photodiodes, organic photodiodes, phototransistors, photoresistors, photovoltaics, and so forth. Organic photodiodes may be solution processed.

When the assay to be performed requires incident light 26, the second reader device 58 may also include light sources 27. When light sources 27 are included, the recording module 4 provides power and/or control signals 55 for controlling when to illuminate each light source 27. Each light source 27 may take the form of a light-emitting diode, an organic light-emitting diode, a laser diode, a laser, a filament bulb, a tungsten halogen bulb, a fluorescent bulb, and so forth. Organic light-emitting diodes may be solution processed.

The third system 56 may be used to implement either the first method (Figure 6) or the second method (Figure 9). Since each flow cell 2, 42, 44, 47 of the fourth fluidic device 57 is effectively single use (cannot be used for a second assay), it may be advantageous to remove the need for any electronic components to be included in the fourth fluidic device 57, which may reduce the cost and complexity of the consumable fourth fluidic device 57. The fourth fluidic device 57 retains one or more integrated flow cells 2 having channels 7 and ports 8 enabling in-situ injection of substrate solution 22. In this way, the fourth fluidic device 58 may be used for obtaining a time series 30 of measured values V(t) which includes the initial period of linear kinetics. In this way, the third system 56 is compatible with the first and second methods according to the present specification, and may enable improved reliability and repeatability for quantifying an enzyme linked assay.

First arrangement for substrate solution delivery Figure 16 is a schematic illustration of a first arrangement 60 for delivery of substrate solution 22 into channels 7 of a flow cell 2, according to some embodiments of the present disclosure.

The first arrangement 60 includes a reservoir 61 containing substrate solution 22 and a substrate supply pump 33. The substrate supply pump 31 is connected to the reservoir 61, and connectable to one port 8, 8a of a flow cell 2, 42, 44, 47 of a fluidic device 1, 49, 53, 57. The substrate supply pump 31 may be used to pump substrate solution 22 into and/or through a connected flow cell 2, 42, 44, 47. In some examples, the first arrangement 60 may also include an overflow sump 62 connected to a second port 8, 8b of a connected flow cell 2, 42, 44, 47, to collect substrate solution 22 which has been pumped through the channel 7 of the flow cell 2, 42, 44, 47.

The substrate supply pump 31 may take the form of, for example, a rotary pump, a peristaltic pump, a syringe pump, a membrane pump, a diaphragm pump, a pressure driven pump, or any other type of pump suitable for pumping appropriate volumes of substrate solution 22 to fill the channel 7 of a flow cell 2, 42, 44, 47 of a fluidic device (or microfluidic device) 1, 49, 53, 57. The substrate supply pump 31 may be activated and/or actuated manually or automatically.

The first arrangement 60 may be wholly separate from a fluidic device 1, 49, 53, 57, and one of more of the first arrangement 60 may be connected to one or more corresponding flow cells 2, 42, 44, 47 of a fluidic device 1, 49, 53, 57 in order to introduce the substrate solution 22 as part of the first or second methods (Figures 6, 9) Alternatively, one or more of the first arrangement 60 may be directly integrated into a fluidic device 1, 49, 53, 57. When activation and/or actuation of the substrate supply pump 31 is automatic, the second, third and/or fourth fluidic devices 49, 53, 57 may receive pump power and/or control signals (not shown) from a corresponding external computing device 50, first reader device 54 or second reader device 58.

In other examples, one of more of the first arrangement 60 may be integrated into a reader device 54, 58, and arranged to be connectable to corresponding flow cells 2, 42, 44, 47 of one or more third or fourth fluidic devices 53, 57.

In still other examples, the elements of the first arrangement 60 may be divided between a third or fourth fluidic device 53, 57 and the corresponding reader device 54, 58. For example, a third or fourth fluidic device 53, 57 may include one or more reservoirs 61 and the reader device 31 may include one of more corresponding substrate supply pumps 31 which may be connected between the reservoirs 61 ad flow cells 2, 42, 44, 47.

In one particular example, a fluidic device 1, 49, 53, 57 may include one or more first arrangements 60 including a substrate supply pump 31 in the form of a peristaltic pump, but may not include a motor or similar means of driving the substrate supply pump 31. The substrate supply pump 31 may be configured to receive motive power from a reader device 54, 58 or other external device (not shown) by means of, for example, an axle received into the substrate supply pump 31 or a gear which may be meshed with a drive chain of the substrate supply pump 3.

In some examples, the first arrangement 60 may simply take the form of a syringe filled with substrate solution 22, which a user may actuate when a connected flow cell 2, 42, 44, 47 is arranged for monitoring by a corresponding photodiode 3 or photodetector 28.

Second arrangement for substrate solution delivery Figure 17 is a schematic illustration of a second arrangement 63 for delivery of substrate solution 22 into channels 7 of a flow cell 2, according to some embodiments of the present disclosure.

The second arrangement 63 is similar to the first arrangement 60, except that the second arrangement 63 further includes a valve 64 and a recirculation path 65. The substrate supply pump 31 connects to a first port 8, 8a of the channel 7 of the flow cell 2, 42, 44, 47, whilst the recirculation path 65 connects a second port 8, 8b of the channel 7 back to the valve 64. With the valve 64 connecting the substrate supply pump 31 to the reservoir 61, the channel 7 of the flow cell 2, 42, 44, 47 may be filled with substrate solution 22. Subsequently, the valve 64 may be switched to connect the recirculation path 65 to the substrate supply pump 31. In this way, a finite volume of substrate solution 22 may be recirculated through the flow cell 2 whilst the time series 30 is measured. The kinetics of converting substrate 23 to reporting substance 23 may deviate from linearity due to diffusion limited supply of unreacted substrate 23. By continuously circulating the substrate solution 22 using the second arrangement 63 (or an equivalent pumping circuit), it may be possible to maintain the assay within a region of linear kinetics for a longer period of time. Having a longer time between the first and second time points ti, t2, may reduce the relative influence noise on the estimate gradient. In this way, the second arrangement 63 may enable greater accuracy and reliability in determining a concentration of target analyte 17 in a sample 12.

In the same way as the first arrangement 60, the second arrangement 63 may be separate from a fluidic device 1, 49, 53, 57, may be integrated within a fluidic device 1, 49, 53, 57, or may be integrated with an external computing device 50 or reader device 54, 58. In the same way as the first arrangement 60, the reservoir 61, valve 64 and/or pump 31 of the second arrangement 63 may be distributed between a fluidic device 49, 53, 57 and an external computing device 50 or reader device 54, 58 in any permutation.

Third arrangement for substrate solution delivery Figure 18 is a schematic illustration of a third arrangement 66 for delivery of substrate solution 22 into channels 7 of two or more flow cells 2, according to some embodiments of the present disclosure.

The third arrangement 66 is similar to the first arrangement 60, except that a single substrate supply pump 31 is connected to two or more flow cells 2, 42, 44, 47. In this way, a single reservoir 61 and substrate supply pump 31 may be used to supply substrate solution 22 to two or more flow cells 2, 42, 44, 47 in parallel. This may reduce the complexity of providing substrate solution 22 to two or more flow cells 2, 42, 44, 47, as compared to providing a first arrangement corresponding to each flow cell 2, 42, 44, 47.

In the same way as the first and second arrangements 60, 63, the third arrangement 66 may be separate from a fluidic device 1, 49, 53, 57, may be integrated within a fluidic device 1, 49, 53, 57, or may be integrated with an external computing device 50 or reader device 54, 58. In the same way as the first and second arrangements 60, 63, the reservoir 61 and pump 31 of the third arrangement 63 may be distributed between a fluidic device 49, 53, 57 and an external computing device 50 or reader device 54, 58 in any permutation.

Fourth arrangement for substrate solution delivery Figure 19 is a schematic illustration of a fourth arrangement 67 for delivery of substrate solution 22 into channels 7 of two or more flow cells 2, according to some embodiments of the present disclosure.

The fourth arrangement 67 is similar to the first arrangement 60, except that a single substrate reservoir 61 is connected to two or more flow cells 2, 42, 44, 47 via two or more corresponding substrate supply pumps 31. If more than two flow cells 2, 42, 44, 47 are present, then the fourth arrangement 67 includes a separate substrate supply pump 31 for each flow cell. By used a separate substrate supply pump 31 for each flow cell 2, 42, 44, 47, the supply of substrate solution 22 to each flow cell 2, 42, 44, 47 may be independently controlled. The fourth arrangement 67 may reduce the complexity of providing substrate solution 22 to two or more flow cells 2, 42, 44, 47, as compared to providing a first arrangement corresponding to each flow cell 2, 42, 44, 47. The fourth arrangement 67 may have a reduced possibility of contamination arising from back-diffusion between flow cells 2, 42, 44, 47.

In the same way as the first, second and third arrangements 60, 63, 66, the fourth arrangement 67 may be separate from a fluidic device 1, 49, 53, 57, may be integrated within a fluidic device 1, 49, 53, 57, or may be integrated with an external computing device 50 or reader device 54, 58. In the same way as the first, second and third arrangements 60, 63, 66, the reservoir 61 and pumps 31 of the fourth arrangement 67 may be distributed between a fluidic device 49, 53, 57 and an external computing device 50 or reader device 54, 58 in any permutation.

In a modification of the fourth arrangement 67, a valve 64 may be provided in series with each substrate supply pump 31 in the same way as the second arrangement 63. In this way, substrate solution 22 may be separately recirculated through each of two or more flow cells 2, 42, 44, 47, without the need for multiple reservoirs 61.

In a modification of first, second or fourth arrangements 60, 66, 67, each substrate supply pump 31 may operate to cause substrate solution to undergo reciprocating motion through the channel 7 or one of more connected flow cells 2, 42, 44, 47. For example, in the first arrangement 60 a quantity of substrate solution 22 may be moved backwards and forwards between the pump 31 and an overflow sump 62 via the channel 7 of the flow cell 2, 42, 44, 47. Each pump 31 of the fourth arrangement 67 may be configured in the same way. In the third arrangement 66, lengths of tubing or channels connecting each flow cell 2, 42, 44, 47 to a common outlet of the substrate supply pump 31 would need to be sufficiently long to prevent mixing between the flow cells 2, 42, 44, 47.

Illustrative experimental data The preceding discussion may be better understood with reference to illustrative experimental data obtained using an example of a third fluidic device 53. The fluidic devices, 1, 49, 53, 57, and external computing device 50, reader devices 54, 58 and flow cells 2, 42, 44, 47 described herein are not limited to the specific conditions and samples which were used to obtain the illustrative experimental data presented hereinafter.

Enzyme linked assays were performed using an example of the third fluidic device 53 including organic photodiodes 3 and organic light emitting diodes 25 arranged on opposite sides of the channels 7 of flow cells 2. The optical property of the flow cells 2 which was measured was absorbance, measured in a transmission geometry. The mixing of reagents and pumping of substrate solution 22 was separate from the third fluidic device used for the illustrative experiments.

The absorption based assays which were conducted used have used,3',5,5'-tetramethylbenzidine (TMB) as a substrate 23, which is a commonly used chromogenic substrate, acted on by the enzyme 15 horseradish peroxidase. As described hereinafter, the accuracy and reliability of the assay results may be improved using kinetic data obtained according to the methods of the present specification, as compared to a conventional method based on "endpoint" values obtained at a fixed time after starting an assay.

Figure 20 is a schematic illustration of an exemplary enzyme linked assay 68 used to obtain illustrative experimental data.

The exemplary assay 68 is for detecting short single strand DNA oligomers 69, which for the illustrative experimental data were 40 base pairs in length. The short single strand DNA oligomers (or target strand) 69 provide the target analyte 17. Two further DNA strands were used in the exemplary assay 68, a first labelled strand 70 having a digoxigenin (DIG) molecule 71 covalently attached to one end, and a second labelled strand 72 having a biotin molecule 73 attached to one end. The first labelled strand 70 is complementary to a first half 74 of the target strand 69, and the second labelled strand 72 is complementary to a second half 75 of the target strand 69.

The target strand 69 used had the sequence: AAG ATA GCT GGA GAA CTA AGA GTG AAC CTA CTG AAA GAC T (SEQ ID NO:1).

The first labelled strand 70 had the sequence: TTA GTT CTC CAG CTA TCT T/3'Dig (SEQ ID NO: 2/3'Digoxigenin).

The second labelled strand had the sequence 5'Biotin/AGT CTT TCA GTA GGT TCA C (5'Biotin/SEQ ID NO:3).

A functional substrate 37 in the form of a glass microscope slide was prepared with a capture molecule 10 in the form of streptavidin 77 covalently attached to a surface which defines one side of the channel 7 of a flow cell 2 constructed substantially as shown in Figure 2. The first and second transparent substrates 35, 39 were formed of PMMA, and the gasket 38 was formed from silicone rubber. The ports 8 were lengths of stainless steel tubing inserted through holes in the PMMA. The organic photodiode 3 and organic light-emitting diode 25 were attached to the first and second transparent substrates 35, 39 on opposite sides of the flow cell 2 channel 7 A sample 12 containing a known concentration of target strands 69 was mixed in a suitable buffer with the first and second labelled strands 70, 72, and left to hybridise for one hour. In these experiments, a 5xSSC buffer was used, which is a saline-sodium citrate buffer having a standard concentration of 0.15M sodium chloride and 0.015M sodium citrate. Therefore, 5xSSC buffer includes 0.75M sodium chloride and 0.075M sodium citrate. The first and second labelled strands 70, 72 hybridise with the target strands 69 in the buffer to form a hybrids 76 having the DIG molecule 71 attached to one end and the biotin molecule 73 attached to the other end.

The reacted solution containing the hybrids 76 was injected into the channel 7 of a flow cell 2 and left for 30 minutes. During this period, the biotin groups 73 of a majority of any hybrids 76 present bind to the exposed streptavidin 77 capture molecules 10 supported on the slide 37 to form a concentration of immobilised hybrids 78. The channel 7 of the flow cell 2 was then washed by injecting with 5xSSC wash buffer.

A solution including anti-DIG Fab fragments 79 conjugated to an enzyme 15 in the form of poly horseradish peroxidase 80 was pumped into the channel 7 of the flow cell 2 and left for 30 minutes. Antibody fragments are formed when an antibody is split or fragmented. The parts of the antibody that bind to antigens are referred to as the Fab fragments. In these experiments, the Fab fragments were conjugates to alkaline phosphatase. During this time a majority of the anti-dig Fab fragments 79 will bind onto the exposed DIG groups 71 of any immobilised hybrids 78 attached to the surface of the functional substrate 37 in the form of the glass slide, forming an immobilised target-enzyme complex 21 in the form of an immobilised horseradish peroxidase complex 81. The channel 7 of the flow cell 2 was then washed by injecting with TBS (tris-buffered saline) buffer containing 0.01% tween (TBST buffer). TBST is a mixture of tris-buffered saline and tween 20 (RTM) (tween 20 (RTM) is a trade name for polysorbate 20). In these experiments, the buffer composition was 50mM Tris (tris(hydroxymethyl)aminomethane), 150mM NaCI and 0.01% (v/v) tween 20.

A measurement program was started, which included driving the organic light-emitting diode 25 with a constant current of 500p.A for approximately 500ms, measuring the photocurrent 29 from the organic photodiode 3 when biased at 0 V, and then turning the organic light-emitting diode 25off again. Measurements were made at 30 second intervals to accumulate the time series 30. After starting the measurement program, the substrate solution 22 including a substrate 23 in the form of TMB ultra obtained from Thermofisher (RTM) was injected into the channel 7 of the flow cell 2. In this way, the substrate 23 is injected with the channel 7 already arranged between the organic light-emitting diode 25 and organic photodiode 3 pair. This enables capturing the initial period of linear (or substantially linear) kinetics. The time series 30 was recorded spanning a total period of 20 minutes, then analysed to determine the first and second time points 11, /2.

The measured values V(t) making up the time series 30 were converted from the raw format (photocurrent 29 in amps) into a transmission Tusing the formula: V (t) T = V (to) (2) In which V(to) is the initial photocurrent 29 value at an initial time t = to. The initial photocurrent 29 current was taken as the value measured before the substrate 23 was injected.

Figure 21 presents experimentally measured transmission values as a function of assay time. The data shown in Figure 21 correspond to a target strand 69 having a concentration of 1 pM.

It may be observed that the transmission T of the flow cell 2 drops over time as more of the substrate 23 in the form of TMB is converted into the corresponding reporting substance 24, increasing the absorbance of the channel 7. It may also be observed that the kinetics appear to be initially linear, before transitioning to non-linear behaviour. Without wishing to be bound by theory, the onset of the non-linear behaviour may correspond to local depletion of substrate 23 in the form of TM13 in the vicinity of the immobilised horseradish peroxidase complexes 81, and diffusion limited transport of the TMI3 to equalise the concentration gradient. In this example, the first time point 1/ was known, and corresponded to t = 0 minutes.

Figure 22 presents the coefficient of determination r2 for fitting a linear model to the experimentally measured transmission values shown in Figure 21.

A linear regression line was calculated at each time t spanning the period from the first time point 17 to that time t. For each time point t (and therefore each model), a coefficient of determination r2 was also calculated, and these values are presented in Figure 22. Applying for the purposes of this example a threshold of r2 = 0.999, it may be observed that the initial linear region lasted until a second time point 12 between 4 and 5 minutes. The gradient of the linear region was calculated, and used as a metric related to the concentration of the target strand 69 in a sample 12. As described hereinbefore, an alternative method (Figure 8) based on gradient of the transmission T values could also be used to determine an initial linear region.

Figure 23 presents gradients of the initial linear region as a function of the concentration of target strand 69, obtained using methods according to some embodiments of the present disclosure.

Figure 24 presents transmission values T obtained at a fixed end-point of 15 minutes as a function of the concentration of target strand 69.

A total of twelve measurements were carried out using the methods of the present disclosure and a conventional end-point method. The measurements were spread across four different compositions. Measurements were repeated three times for each concentration, and the error bars in Figures 23 and 24 represent standard deviations.

For the kinetic data presented in Figure 23 and the end-point date presented in Figure 24, the limit of detection (LOD, minimum detectable signal) was approximated and compared. The LOD was approximated using the following equation: 3.3 * s LOD = In which in is the gradient of a regression 82 line fitted to the a plot of signal against target strand 69 concentration, for example as plotted in Figures 23 or 24, and s is the standard deviation of the residuals of the regression line 82.

A first dataset was obtained using concentrations of 0.0, 0.3, 0.65 and 1.0 nano-molar, nM (nano-moles per litre) of target strand 69. The data presented in Figures 23 and 24 correspond to the first dataset. A second dataset was also obtained, using concentrations of 0.0, 1.0, 3.0, 10 and 30 nM of target strands 69. Table 1 presents the estimated LOD values for the first and second datasets using kinetic measurements according to the present disclosure, and using an end-point at 15 minutes.

First dataset Second dataset LOD (kinetic) 0.76 4.27 LOD (end-point) 0.91 7.71

Table 1

It may be observed that for both first and second datasets, the LOD using a kinetic measurement based on the initial linear behaviour appears to be significantly improved by comparison to a conventional end-point method.

MODIFICATIONS

The present invention is not limited to the disclosed embodiments. It will be appreciated that many modifications may be made to the embodiments hereinbefore described. Such modifications may involve equivalent and other features which are already known in the design, manufacture and use of enzyme linked assays, devices and/or readers, and which may be used instead of or in addition to features already described herein. Features of one embodiment may be replaced or supplemented by features of another embodiment.

Any of the fluidic devices 1, 49, 53, 57 may be a micro-fluidic device. an (3)

Although claims have been formulated in this application to particular combinations of features, it should be understood that the scope of the disclosure of the present invention also includes any novel features or any novel combination of features disclosed herein either explicitly or implicitly or any generalization thereof whether or not it relates to the same invention as presently claimed in any claim and whether or not it mitigates any or all of the same technical problems as does the present invention. The applicant hereby gives notice that new claims may be formulated to such features and/or combinations of such features during the prosecution of the present application or of any further application derived therefrom.

Claims

Claims 1. A method of measuring a concentration of a target analyte using an enzyme linked assay, comprising: functionalising a flow cell using an enzyme linked assay applied to a sample, such that in response to the sample contains the target analyte, the flow cell will become functionalised with an immobilised concentration of enzyme molecules bound to the target analyte; introducing a substrate to a flow cell, the substrate being convertible into a reporting substance by the enzyme molecules; recording a time series of measured values corresponding to an optical property of the flow cell which depends on a concentration of the reporting substance; determining a first time point corresponding to the introduction of the substrate to the flow cell; determining a second time point corresponding to an endpoint of linear kinetics for the conversion of substrate into reporting substance; estimating, based on the measured values obtained between the first and second time points, a concentration of the target analyte in the sample.
2. A method according to claim 1, wherein determining a second time point comprises, for each given time point until the second time-point has been set: calculating a coefficient of determination corresponding to a linear model of the measured values between the first time point and the given time point; in response to the coefficient of determination is less than or equal to a linearity threshold, setting the given time point as the second time point.
3. A method according to claims 1 or 2, wherein determining a second time point comprises: calculating a first gradient of the measured values at the first time point; for each given time point until the second time-point has been set: calculating a difference between a gradient of the measured values at the given time point and the first gradient; in response to the magnitude of the difference is greater than or equal to a gradient threshold, setting the given time point as the second time point.
4. A method according to any one of claims 1 to 3, wherein determining a first time point comprises receiving a signal corresponding to activation of a substrate supply pump configured to introduce substrate into the flow cell.
5. A method according to any one of claims 1 to 4, wherein determining a first time point comprises determining that the optical property of the flow cell has departed from a baseline value by more than an activation threshold.
6. A method according to any one of claims 1 to 5, wherein determining the first time point and determining the second time point is carried out concurrently with recording the time series of measured values.
7. A method according to any one of claims 1 to 5, wherein determining the first time point and determining the second time point is carried out subsequently to recording the time series of measured values.
8. A method comprising carrying out a method according to any one of claims 1 to 7 for a plurality of flow cells concurrently.
9. A method of processing a time series of measured values, the time series of measured values obtained by: functionalising a flow cell using an enzyme linked assay applied to a sample, such that if the sample contains the target analyte, the flow cell becomes functionalised with an immobilised concentration of enzyme molecules bound to the target analyte; introducing a substrate to a flow cell, the substrate being convertible into a reporting substance by the enzyme molecules; recording the time series of measured values corresponding to an optical property of the flow cell which depends on a concentration of the reporting substance; the method of processing the time series of measured values comprising: receiving the time series of measured values; determining a first time point corresponding to the introduction of the substrate to the flow cell; determining a second time point corresponding to an endpoint of linear kinetics for the conversion of substrate into reporting substance; estimating, based on the measured values obtained between the first and second time points, a concentration of the target analyte in the sample.
10. A computer program product stored on a non-transitory computer readable medium and configured, when executed by one or more physical electronic processers of a data processing apparatus, to cause the data processing apparatus to carry out the method according to claim 9.
11. A fluidic device comprising: one or more flow cells, each flow cell including a channel and at least two ports in fluid communication with the channel, wherein at least one internal surface of each channel is functionalised with a capture molecule; a photodiode corresponding to each flow cell and arranged to receive light from the corresponding flow cell, wherein each photodiode is attached to the corresponding flow cell or each photodiode is integrally formed with the corresponding flow cell; wherein the capture molecules enable functionali sing the flow cell using an enzyme linked assay applied to a sample, such that in response to the sample contains the target analyte, the capture molecules will immobilise complexes of enzyme molecules bound to target analyte, wherein the enzyme molecules act to convert a substrate into a reporting substance.
12. A fluidic device according to claim 11, comprising a plurality of flow cells.
13. A fluidic device according to claims 11 or 12, further comprising a light-emitting diode corresponding to each flow cell, each light-emitting diode arranged to illuminate the corresponding flow cell, wherein each light-emitting diode is attached to the corresponding flow cell or each light-emitting diode is integrally formed with the corresponding flow cell.
14. A fluidic device according to any one of claims 11 to 13, further comprising: a recording module connected to each photodiode and configured to, for each flow cell record a time series of measured values corresponding to an optical property of said flow cell, wherein the optical property depends on a concentration of the reporting substance in said flow cell; a time point determination module configured to, for each flow cell: determine a first time point corresponding to the introduction of the substrate to said flow cell; determine a second time point corresponding to an endpoint of linear kinetics for the conversion of the substrate into the reporting substance; an estimation module configured to, for each flow cell, estimate a concentration of a target analyte in a sample used to functionalise that flow cell using an enzyme linked assay, based on the measured values obtained between the first and second time points.
15. A fluidic device according to claim 14, further comprising: one or more substrate reservoirs; and one or more substrate supply pumps connected between the one or more substrate reservoirs and the one or more flow cells, each substrate supply pump configured to introduce the substrate into the channels of one or more flow cells.
16. A system for measuring a concentration of a target analyte using an enzyme linked assay, the system comprising: a fluidic device according to any one of claims 11 to 13; and an apparatus comprising: a recording module connectable to one or more photodiodes of the fluidic device, the recording module configured to, for each flow cell, record a time series of measured values corresponding to an optical property of a flow cell, wherein the optical property depends on a concentration of the reporting substance in the flow cell; a time point determination module configured to, for each flow cell: determine a first time point corresponding to the introduction of the substrate to the flow cell; determine a second time point corresponding to an endpoint of linear kinetics for the conversion of the substrate into the reporting substance; an estimation module configured to, for each flow cell, estimate a concentration of a target analyte in a sample used to functionalise that flow cell using an enzyme linked assay, based on the measured values obtained between the first and second time points.
17. A system according to claim 16, further comprising: one or more substrate reservoirs; and one or more substrate supply pumps connectable between the one or more substrate reservoirs and the one or more flow cells of the fluidic device, each substrate supply pump configured to introduce the substrate into the channels of one or more flow cells.
18. Apparatus for measuring a concentration of a target analyte using an enzyme linked assay, the apparatus configured to receive a fluidic device which includes one or more flow cells, each flow cell including a channel and at least two ports in fluid communication with the channel, wherein at least one internal surface of each channel is functionalised with a capture molecule which enables functionalising the flow cell using an enzyme linked assay applied to a sample, such that in response to the sample contains the target analyte, the capture molecules will immobilise complexes of enzyme molecules bound to target analyte, wherein the enzyme molecules act to convert a substrate into a reporting substance; the apparatus comprising: a photodetector corresponding to each flow cell and arranged such that when the fluidic device is received by the apparatus, each photodetector will receive light from the corresponding flow cell; a recording module connected to each photodetector and configured to, for each flow cell record a time series of measured values corresponding to an optical property of said flow cell, wherein the optical property depends on a concentration of the reporting substance in said flow cell; a time point determination module configured to, for each flow cell: determine a first time point corresponding to the introduction of the substrate to said flow cell; determine a second time point corresponding to an endpoint of linear kinetics for the conversion of the substrate into the reporting substance; an estimation module configured to, for each flow cell, estimate a concentration of a target analyte in a sample used to functionalise said flow cell using an enzyme linked assay, based on the measured values obtained between the first and second time points.
19. Apparatus according to claim 18, further comprising a light source corresponding to each flow cell and arranged such that when the fluidic device is received by the apparatus, each light source will illuminate the corresponding flow cell.
20. Apparatus according to claims 18 or 19, further comprising: one or more substrate reservoirs; and one or more substrate supply pumps connectable between the one or more substrate reservoirs and the one or more flow cells of the fluidic device, each substrate supply pump configured to introduce the substrate into the channels of one or more flow cells.
21. A fluidic device, system or apparatus according to any one of claims 14 to 20, wherein the time point determination module is configured to determine each second time point by, for each given time point until the second time-point is determined: calculating a coefficient of determination corresponding to a linear model of the measure values between the first time point and the given time point; in response to the coefficient of determination is less than or equal to a linearity threshold, setting the given time point as the second time point.
22. A fluidic device, system or apparatus according to any one of claims 14 to 20, wherein the time point determination module is configured to determine each second time point by: calculating a first gradient of the measured values at the first time point; for each given time point until the second time-point is determined: calculating a difference between the gradient of the measured values at the given time point and the first gradient; in response to the magnitude of the difference is greater than or equal to a gradient threshold, setting the given time point as the second time point.
23. A fluidic device, system or apparatus according to any one of claims 14 to 22, wherein the time point determination module is configured to determine each first time point based on determining that the optical property of the corresponding flow cell has departed from a baseline value by more than an activation threshold.
24. A fluidic device, system or apparatus according to any one of claims 14 to 23, wherein the time point determination module is configured to determine the first time point and the second time point concurrently with the recording module recording the time series of measured values.
25. A fluidic device, system or apparatus according to any one of claims 14 to 23, wherein the time point determination module is configured to determine the first time point and the second time point subsequently to recording the time series of measured values.