Assay with digital readout

Abstract

A device and a method for performing an assay is provided. The assay device, which may be used for determining the concentration of an analyte in a sample, includes a plurality of microchambers and a Field-effect transistor (FET) arranged at the bottom of each of the plurality of microchambers. Capture probe molecules for the analyte can be arranged within the plurality of microchambers such that each microchamber contains at most one capture probe molecule. The FET can be arranged in said microchamber to give a readable output signal based on binding of the analyte, or competitor to the analyte, with the capture probe molecule.

Claims

1. A method for performing an assay for determining a concentration of an analyte in a sample, the method comprising the steps of a) providing an assay device comprising: a plurality of microchambers; a Field-effect transistor (FET) arranged at the bottom of each microchamber of the plurality of microchambers; and capture probe molecules for the analyte arranged within the plurality of microchambers such that each microchamber in the plurality of microchambers contains at most one capture probe molecule, wherein the FET is arranged in the microchamber to give a readable output signal based on binding of said analyte, or a competitor to the analyte, with the capture probe molecule; b) supplying the sample to the assay device; c) analysing the output signals from the FETs to determine a number of true positives and/or true negatives, wherein the number of true positives is a number of microchambers in which the capture probe molecule is present and in which a binding event between the capture probe molecule and the analyte has occurred and wherein the number of true negatives is a number of microchambers in which the capture probe molecule is present and in which a binding event between the capture probe molecule and the analyte has not occurred; and d) determining the concentration of the analyte in the sample based on the determination of step c).

2. The method according to claim 1, wherein step d) comprises determining the concentration of the analyte in the sample by partitioning statistics using the number of true positives and/or true negatives.

3. The method according to claim 2, wherein step d) comprises determining the concentration of the analyte in the sample without using a calibration curve.

4. The method according to claim 1, wherein the output signals analysed in step c) are triggered by a detector reagent bound to the analyte in the sample.

5. The method according to claim 4, wherein step b) comprises a step b1) of labelling the analyte with the detector reagent and a step b2) of supplying the labelled analyte in the sample to the assay device.

6. The method according to claim 4, wherein step b) comprises supplying the detector reagent to the assay device after supplying the sample to the assay device.

7. The method according to claim 4, wherein step d) comprises determining the concentration of the analyte using the number of true positives only.

8. The method according to claim 1, wherein the output signals are triggered by a detector reagent bound to a competitor for the analyte present in the sample.

9. The method according to claim 8, wherein step d) comprises determining the concentration of the analyte using the ratio of true positives to true negatives for a given concentration of the competitor for the analyte.

10. The method according to claim 4, further comprising a step of supplying a substrate to the detector reagent, wherein the detector reagent and the substrate have the capacity to give a pH change in the microchamber upon conversion of the substrate by the detector reagent, thereby triggering the output signal in the FET.

11. The method according to claim 1, further comprising a step of determining the absence or presence of a capture probe molecule in each microchamber by analysing the output signals from the FETs.

12. The method according to claim 1, wherein the plurality of microchambers comprises at least 100, at least 1000, at least 10,000, or at least 100,000 microchambers.

13. The method according to claim 1, wherein the plurality of microchambers comprises at least 1000, at least 10,000, or at least 100,000 of the capture probe molecules.

14. The method according to claim 1, wherein the capture probe molecule is adapted to give a pH change in the microchamber upon conversion of a detection substrate, and wherein the pH change is detectable by said FET.

15. The method according to claim 1, wherein the capture probe molecule is attached to a detection probe that is adapted to give a pH change in the microchamber upon conversion of a detection substrate, and wherein the pH change is detectable by said FET.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) The above, as well as additional objects and features of the present disclosed concept, will be better understood through the following illustrative and non-limiting detailed description, with reference to the appended drawings. In the drawings like reference numerals will be used for like elements unless stated otherwise.

(2) FIGS. 1a-1c shows a representative assay device and determination in which microchamber a capture probe molecule is present. FIG. 1a illustrates a schematic assay device 1 for determining the concentration of an analyte in a sample according to the present disclosure. FIG. 1b illustrates a detection probe for an unlabelled capture probe molecule 4 can be supplied to the device so that it may bind to the capture probe molecules in the microchambers. The capture probe molecules with attached detection probes may in this way be adapted to give pH change in the microchambers. FIG. 1c illustrates that the presence or absence of such a readout signal for each microchamber may be monitored as shown by arrows 7a-7i.

(3) FIGS. 2a-2c show a schematic embodiment of a sandwich assay method using the assay device 1 of FIGS. 1a-1c. FIG. 2a shows a schematic embodiment of a sandwich assay method using the assay device 1. FIG. 2b shows a sample comprising the analyte of interest being supplied to the device 1. FIG. 2c shows that when analysing the output signals from the microchambers, and with information about in which microchambers a capture probe molecule is present, it may be possible to discriminate between different types of microchambers.

(4) FIGS. 3a-3c show a further example of a representative assay device and a sandwich method. FIG. 3a show a representative assay device and sandwich method. FIG. 3b shows that the analyte is unlabelled when supplied to the assay device, and a detector reagent is, after the analyte in the sample has been allowed to bind to the capture probe molecule, subsequently supplied to the assay device to allow binding of detector reagent to analyte. The detector reagent may for example be an antibody with affinity to the analyte. FIG. 3c show a representative assay device and sandwich method.

(5) FIGS. 4a and 4b illustrate an embodiment in which a competitor labelled with a detector reagent may be mixed with the sample prior to supplying the sample to the assay device. FIGS. 4a and 4b thus each show an example of a competitive assay in which the competitor may be supplied to the assay device concurrently with the sample.

DETAILED DESCRIPTION OF THE DISCLOSURE

(6) Detailed embodiments of the present disclosure will now be described with reference to the drawings.

(7) The above disclosed concept has mainly been described with reference to a limited number of examples. However, as is readily appreciated by a person skilled in the art, other examples than the ones disclosed above are equally possible within the scope of the inventive concept, as defined by the appended claims.

(8) The present disclosure is related to measuring concentrations in of analytes in so-called digital assays, such as immunoassays.

(9) A schematic assay device 1 for determining the concentration of an analyte in a sample according to the present disclosure is shown in FIG. 1a. The device 1 can comprise a substrate 2, such onto which a plurality of microchambers 5a-5i can be arranged. The microchambers may be arranged in an array and the plurality of microchambers may be at least 100, at least 1000, at least 10,000, at least 100,000, at least 200,000, or at least 500,000 microchambers.

(10) Arranged at the bottom of each well is a Field-effect transistor (FET), in this case a FinFET 3. Further, in the plurality of microchambers 5a-5i, either none or a single capture probe molecule 4 can be arranged. In the schematic example of FIG. 1a, microchambers 5a, 5c, 5f, 5g and 5j has a single capture molecule 4, whereas microchambers 5b, 5d, 5e, 5h and 5i has no capture probe molecule 4. The capture probe molecules 4 in this example may be attached to the outer surface of the FinFETs 3.

(11) The capture probe molecules may be arranged within the plurality of microchambers in an amount of capture probe per microchamber that allows for subsequent analysis of the binding events in the microchambers by the use of partitioning statistical analysis. As an example less than three, such as less than two such as at most 1 capture probe molecule may be present in a microchamber.

(12) Further, the number of microchambers being occupied with a capture probe molecule may be at least 10%, such as between 30-40%.

(13) A higher total number of microchambers allow for a lower number of microchambers being occupied with a capture probe molecule but still give enough reading points. The number of microchambers being occupied with a capture probe molecule may also be dependent on the actual concentration range of the analyte you want to measure.

(14) The assay device 1 further comprises a fluidic inlet (not shown), a fluidic outlet (not shown) as well as fluidics (not shown) for distributing a sample supplied to the fluidic inlet over the plurality of microchambers 5a-5i. The fluidic outlet may thus be used for discharging sample from the device 1.

(15) Furthermore, the FinFETs may be arranged to give a readable output signal based on binding of an analyte, or competitor to the analyte, with said capture probe molecule 4.

(16) In this example, the capture probe molecules 4 may be adapted to give a pH change in the microchamber 5a-5i upon conversion of a detection substrate, wherein said pH change can be detectable by said FET. This is illustrated in FIGS. 1b and 1c. In FIG. 1b, a detection probe for an unlabelled capture probe molecule 4 can be supplied to the device so that it may bind to the capture probe molecules in the microchambers. The capture probe molecules with attached detection probes may in this way be adapted to give pH change in the microchambers. Thus, after washing, a substrate for the detection probe may be supplied to the device 1. An oil seal 6 may be added to the device to allow for sealing the individual microchambers. However, other types of seals may also be used for sealing the individual microchambers. Upon conversion of the substrate by the detection probes, protons may be released. This release leads to a pH change within the microchamber, which in turn yield a readable output signal from the FinFET. As an alternative, hydroxyl ions or other ions may be released by conversion of the substrate, and this change in ion concentration may yield a readable output signal. Another alternative, an enzyme that consumes a proton or a hydroxyl ion upon conversion of a substrate can be used, since such an enzyme can also yield a pH change that could be measurable.

(17) The presence or absence of such a readout signal for each microchamber may be monitored, as illustrated by arrows 7a-7i in FIG. 1c.

(18) Consequently, different methodologies may be used to determine if a capture probe molecule, such as a receptor, is present in a microchamber, for example: i) having an enzyme attached to the capture probe molecule covalently and measure its activity (using pH changes as described above); ii) determining the presence of capture probes molecules (e.g. IgG) by incubating them with an enzymatically labelled anti-receptor probe (e.g. HRP-labelled IgG), determine presence/absence of the capture probe by measuring pH changes (as described above); iii) determining the presence of capture probes molecules by incubating the microchambers with magnetic, metallic, or dielectric beads with affinity for the capture probe molecule The presence or absence of such a bead may be detected by conventional means. As an alternative, a magnetic, metallic, or dielectric bead may be coupled to the capture probe molecule before it is immobilized in the microchambers.

(19) FIGS. 2a-2c show a schematic embodiment of a sandwich assay using the assay device 1 of FIGS. 1a-1c. In FIG. 2b, the sample comprising the analyte of interest may be supplied to the device 1. In this example, the analyte has previously been labelled with a detector reagent, such as an enzyme. This leads to binding of the analyte in microchambers. After washing, a substrate for the detector reagent may be added. Addition of oil may also create an oil seal so that the reaction between capture probe and substrate may continue undisturbed. The substrate turnover by the detector reagent leads to the release of protons and thus a pH change in the microchambers. When analysing the output signals from the microchambers, and with information about in which microchambers a capture probe molecule is present, it may be possible to discriminate between microchambers of the following types, as also illustrated by FIG. 2c:

(20) TABLE-US-00001 true microchambers in which a capture probe molecule is positives present and in which a binding event between capture probe molecule and analyte has occurred true microchambers in which a capture probe molecule is negatives present and in which a binding event between capture probe molecule and analyte has not occurred false microchambers in which a capture probe molecule is positives absent but still indicate that substrate turnover reaction has occurred in the microchamber when detecting the analyte irrelevant microchambers in which a capture probe molecule is absent and indicate that substrate turnover reaction has occurred in the microchamber

(21) In some embodiments, the false positives can be considered as irrelevant.

(22) For a sandwich assay, it may be enough to count the number of true positives to be able to determine the analyte concentration. The number of true positives may this be regarded as “1” microchambers in the digital assay, whereas the number of true negatives may be regarded as “0” microchambers in the digital assay.

(23) As an example, the examples shown in FIGS. 1a-1c and 2a-2c may give an experimental design as follows if a capture antibody is used as the capture probe molecule: 1. Dilute sample in a dilution buffer that contains an enzyme-labelled detector reagent (or a labelled competitor for a competitive immunoassay). Ensure sufficient time for enzyme-labelled detector reagent to fully cover the analyte in question, which may be dependent on both reagent and concentration of the reagent; 2. In parallel, or prior to adding the diluted sample to the assay device and the plurality of microchambers: determine in which microchambers a capture antibody is present; 3. Remove reagents used to determine in which microchambers the capture antibody is present. This step may not be necessary if a magnetic bead is used for detection of the capture probe; 4. Apply diluted sample to the assay device and the plurality of microchambers (incubation time may be dependent on specific assay); 5. Wash away the sample and apply substrate for enzymatic reaction that generates a pH change the signal (pH change, see below); 6. Seal of the top by applying oil, which may prevent that product of enzymatic reaction diffuses out of the microchambers; 7. Determine in which well a pH change occurs (evidence of enzymatic reaction) by analysing readout from the FET in the microchamber.

(24) As known from Basu, B: Micro-and Nanotechnologies for Quantitative Biology and Medicine (2107), vol 22(4), 369-386, a digital assay in which, e.g. 200,000. events are counted, a detection limit of below 0.5 femtomolar may be reached, whereas if e.g. 5000 partitions are counted, a detection limit of about 50 femtomolar may be obtained.

(25) FIGS. 3a-3c show a further embodiment of the method of the present disclosure. The difference between this setup and the embodiment of FIGS. 1a-1c and FIGS. 2a-2c is that the analyte is unlabelled when supplied to the assay device (FIG. 3b), and a detector reagent is, after the analyte in the sample has been allowed to bind to the capture probe molecule, subsequently supplied to the assay device to allow binding of detector reagent to analyte. The detector reagent may for example be an antibody with affinity to the analyte.

(26) When analysing the output signals from the microchambers, and with information about in which microchambers a capture probe molecule is present, it may be possible to discriminate between microchambers as discussed in relation to by FIG. 2c above.

(27) FIGS. 4a and 4b illustrate an embodiment in which a competitor labelled with a detector reagent may be mixed with the sample prior to supplying the sample to the assay device. FIGS. 4a and 4b thus show an example of a competitive assay in which the competitor may be supplied to the assay device concurrently with the sample. The concentration of the labelled competitor may be higher than the expected concentration of the analyte. However, the concentration of the labelled competitor may also be lower than the concentration of the analyte. The analyte and the competitor can compete for the binding with the capture probe molecule in the microchambers. Thus, the total concentration of analyte and labelled competitor can result in complete occupation of the capture probes under the reaction conditions, and thus result in competition for free capture probes between the analyte and labelled competitor when the reaction approaches endpoint.

(28) After supply of a substrate for the detector reagent, an analysis of the readout signals from the FETs may thus give information on in which microchambers a competitor has bound. This may in turn be used for determining the concentration of the unlabelled analyte in the sample.

(29) For competitive assay, the ratio of “1” microchambers to “0” microchambers (i.e. the ratio of true positives to true negatives), given known concentration of added competitor, may be used to deduct the concentration of unlabelled analyte in sample. This may be the case if the competitor and the analyte bind ratiometrically to the capture probe molecule. However, not only the ratio of true positives to true negatives may be used, but as an alternative only the number of true positives or true negatives, as well as the total number of microchamber with a capture probe can be used to deduct the concentration of unlabelled analyte in sample.

(30) When analysing the output signals from the microchambers, and with information about in which microchambers a capture probe molecule is present, it may be possible to discriminate between microchambers as discussed in relation to by FIG. 2c above.

(31) Signal Generation and Conversion:

(32) As discussed above, a detectable readout signal in the microchamber may be generated by an enzymatic label. This enzymatic label may e.g. be on the capture probe molecule, on the sample analyte, on an antibody with affinity for the analyte, on a competitor for the analyte or on an antibody against such a competitor. Such an enzyme may convert a substrate that is specifically added to the assay device and upon conversion of the substrate ions such as protons (or in other cases hydroxyl ions) are released/consumed. A proton (/hydroxyl) release/consumption may result in a change of the pH in the microchamber where the enzyme is present and it is this pH change that may be measured by the FETs.

(33) An example of proton-releasing reaction is the conversion of D-glucose by glucose oxidase (GOx) depicted below. This may be performed in the presence of FeSO.sub.4 to induce the decomposition of H.sub.2O.sub.2, as such shifting the reaction towards the right side, resulting in enhanced proton release.

(34) Further, an example of an enzyme that consumes hydroxyl ions is Urease.

(35) An example of an enzyme that consumes protons in the conversion of a substrate is horseradish peroxidase (HRP), e.g. when converting 3,3′,5,5′-Tetramethylbenzidine (TMB).

(36) Analysing Readout Signal from FET

(37) As discussed above, conversion of a substrate by e.g. an enzyme attached to a capture probe molecule or attached to a bound analyte, may lead to a change in ion concentration, such as a change in pH, in the microchamber. This change in concentration may for example be detected by the finFET in the microchamber, due to the finFET having an electrolyte as a gate electrode and the sensitivity to pH is the result of protonation/deprotonation of surface groups (e.g. SiOH (silanol) in case of a SiO.sub.2 surface).

(38) The readable output signal from the FET may be a voltage or a current. Other FETs, MOSFETs, without a liquid but with a solid gate may be arranged in the neighbourhood of each sensor FET to aid reading out the signal.

(39) Determination of Concentration

(40) As discussed above, the concentration of the analyte in the sample may be determined from the true positives and/or true negatives using partitioning statistics. Below follow some explicit examples on this may be achieved.

(41) Sandwich Immunoassays

(42) At low concentrations, the number of microchambers in which a binding event with the capture probe molecule has occurred, i.e. true positives, may be smaller than the total number of capture probe molecules in the microchambers.

(43) For example, 100 μl of a 1 femtomolar solution contains ˜60,000 analyte molecules, so if there are >>60,000 microchambers with a FET that contain a capture probe molecule, the Poisson distribution can be used to describe the probability of a microchamber being a true positive or a true negative (“1” microchamber or a “0” microchamber). The Poisson distribution describes the likelihood of a number of possible events occurring if the average number of events is known. If the expected average number of occurrences is, then the probability that there are exactly v occurrences is given by (Poisson)

(44) $P_{\mu }\left(v\right)=e^{-\mu }\left(\frac{{\mu }^v}{v!}\right)$

(45) In a digital assay, the key variable in this equation (μ) may be equal to the ratio of captured and labelled analyte molecules to the FETs in the microchambers. If we name P.sub.μ(0) or the fraction of “true negative” microchambers, then
μ=−ln[P.sub.μ(0)]

(46) Since the fraction of “true negative” may be equal to one minus the fraction of “true positive” microchambers, it is possible to determine μ or the from f.sub.on (the fraction of “true positive” microchambers) using
μ=−ln[1−f.sub.on]

(47) Since can be determined, the number of occurrences may be determined.

(48) Reagent-specific parameters (k.sub.on and k.sub.off of the reagents) may determine the relation between the analyte concentration and the number of true positive microchambers.

(49) In addition, non-specific binding may lead to assay (reagent, incubation time, buffer and temperature) specific background levels. Thus the background limit may be different for each assay (and assay comprises reagents, buffer conditions etc.). It may therefore be beneficial in some applications carry out the assay with samples containing known concentrations of the analyte to determine the number of true positive microchambers.

(50) Consequently, in embodiments of the present disclosure, the method may also comprise a step e1) of supplying samples containing known concentrations of the analyte to said device; and a step e2) of analysing the output signals from the FETs to determine the number of true positives and/or true negatives of the samples supplied in step e1).

(51) Further, the method may comprise using the determination from step e2) to create a calibration curve for the analyte.

(52) Competitive Immunoassays

(53) In competitive immunoassays, the conversion of the number of true positive microchambers to the concentration of analyte may be dependent on assay specific parameters, i.e. the concentration of labelled competitor.

(54) In addition, also for this case non-specific binding may occur. Thus, also when performing a competitive digital assay samples containing known concentrations of the analyte in question may be used to determine the number of true positive microchamber. The determination may then be used for making calibration curves for the analyte.