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US20170307514A1 - Binding assay analysis - Google Patents

Binding assay analysis Download PDF

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Publication number
US20170307514A1
US20170307514A1 US15/517,228 US201515517228A US2017307514A1 US 20170307514 A1 US20170307514 A1 US 20170307514A1 US 201515517228 A US201515517228 A US 201515517228A US 2017307514 A1 US2017307514 A1 US 2017307514A1
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Prior art keywords
assay
sample
area
concentration
entities
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Inventor
João MANUEL DE OLIVEIRA GARCIA DA FONSECA
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Biosurfit SA
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Biosurfit SA
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Priority claimed from GB201417640A external-priority patent/GB201417640D0/en
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Assigned to BIOSURFIT, S.A. reassignment BIOSURFIT, S.A. ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: MANUEL DE OLIVEIRA GARCIA DA FONSECA, João
Publication of US20170307514A1 publication Critical patent/US20170307514A1/en
Priority to US15/928,148 priority Critical patent/US10330589B2/en
Assigned to THE EUROPEAN INVESTMENT BANK reassignment THE EUROPEAN INVESTMENT BANK SECURITY INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: BIOSURFIT S.A.
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/01—Arrangements or apparatus for facilitating the optical investigation
    • G01N21/03—Cuvette constructions
    • G01N21/05—Flow-through cuvettes
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
    • G01N21/25—Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
    • G01N21/25—Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands
    • G01N21/251—Colorimeters; Construction thereof
    • G01N21/253—Colorimeters; Construction thereof for batch operation, i.e. multisample apparatus
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
    • G01N21/55—Specular reflectivity
    • G01N21/552—Attenuated total reflection
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
    • G01N21/55—Specular reflectivity
    • G01N21/552—Attenuated total reflection
    • G01N21/553—Attenuated total reflection and using surface plasmons
    • G01N21/554—Attenuated total reflection and using surface plasmons detecting the surface plasmon resonance of nanostructured metals, e.g. localised surface plasmon resonance
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/645—Specially adapted constructive features of fluorimeters
    • G01N21/6452—Individual samples arranged in a regular 2D-array, e.g. multiwell plates
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00

Definitions

  • the present disclosure relates to methods and systems for determining a concentration of target entities in a sample, for example, determining a concentration of target antigens or antibodies in a biological sample.
  • Immunoassays can be used to quantitatively determine a concentration of target entities, for example antigens, present in a sample.
  • a concentration of target entities for example antigens
  • an arrangement comprising a microfluidic chamber into which a sample is introduced may be used.
  • Such a chamber comprises a plurality of probe entities, for example antibodies, immobilized on a surface of the chamber such that, as the sample is passed over the surface, antigens in the sample bind to the antibodies in the chamber.
  • the amount of antibody-antigen binding may be detected and quantified using, for example, fluorescence or surface plasmon resonance measurements (SPR) and a concentration of target entities in the sample can be determined from this amount.
  • SPR surface plasmon resonance measurements
  • the range of concentrations of target entities in a sample which can be detected by known microfluidic immunoassays is limited.
  • detection and quantification will only work in a sensitive (ideally linear) range of the relationship between target concentration and detection signal. Below this range, signal to noise ratios are too low and above this range the assay saturates. In either case, the measured signal becomes independent of sample concentration.
  • a method for determining a sample concentration of target entities in a sample, for example, determining a concentration of target antigens or antibodies in a blood sample or other biological sample.
  • the method comprises obtaining assay data comprising data points of respective local measurements indicative of a local concentration of target entities immobilised at each of a plurality of assay areas of an assay assembly from an assay using the assay assembly.
  • the assay areas are connected in series such that a sample flowing through the assay assembly flows past each assay area in sequence.
  • Each assay area comprises a plurality of probe entities immobilized at a surface of the assay area, the probe entities being arranged to bind to the target entities in the sample, such that the concentration of the target entities is depleted as the sample flows from one of the assay areas to the next.
  • the assay data is modeled with a parameterized function of the local measurements against a quantity indicative of the position of the respective assay areas in the sequence, wherein one or more of the parameters are dependent on the sample concentration.
  • a value indicative of the sample concentration is determined based on at least one of the one or more parameters.
  • modeling the data may involve adjusting the one or more parameters to fit the parameterized function to the data points, for example by reducing or minimizing a corresponding sum function capturing a discrepancy between values of the parameterized function and the data points, as is well known in the art.
  • Examples of such known techniques are non-linear regression, gradient descent and least square optimization in general
  • one of the one or more parameters is a variable parameter indicative of an offset amount which offsets the quantity indicative of the position of the assay area in the sequence such that the parameterized function is a function of the local measurements against the quantity indicative of the position of the assay area in the sequence offset by the offset amount.
  • the parameterized function is a function of (DZ i +Offset), where ‘Offset’ is the offset amount.
  • the parameterized function may be derived from a plurality of assay data sets, each obtained for a respective sample concentration, the sample concentrations covering a range of sample concentrations.
  • the parameterized function may be thought of by way of illustration to aid understanding and not limitation, as representative of a master depletion curve defining the depletion of a concentration of a target entities in a system characteristic of the assay assembly, the system comprising a plurality of notional assay areas arranged such that the concentration of target entities is depleted from one notional assay area in the sequence to the next notional assay area in the sequence.
  • the number of notional assay areas is greater than the number of assay areas in the assay assembly.
  • the fit of the master depletion curve to the assay data sets can be thought of by notionally offsetting the assay areas by the offset amount such that the assay areas of the assay assembly are mapped to a set of notional assay areas corresponding to a sample concentration.
  • the parameterized function may be a logistic function. In some cases an n th order polynomial function or a spline may be used, or any other suitable functional form may be used.
  • the parameterized function may comprise a look up table to represent the master depletion curve, for example, using interpolation to construct data points between data entries in the look up table.
  • the offset amount is determined by minimizing a difference between the respective local measurement and a corresponding value of the parameterized function for each assay area of the assay assembly.
  • the offset amount may be determined using a least squares approximation or any other suitable approximation.
  • the offset amount, ‘Offset’ is determined by minimizing the following function:
  • DP(DZ i ) is the local measurement at the respective assay area, i, and f is the corresponding value of the parameterized function.
  • DZ i is the quantity indicative of the position of the assay area in the sequence.
  • Minimization of any suitable cost function may be carried out to obtain the best fit of the parameterized function to the assay data. For example, Chi-squared minimization techniques may be used. Other minimization approaches may be to apply a weighting to the local measurements. For example, a lower weighting might be given to measurements close to a noise threshold. For example, a higher weighting may be given to the assay areas earlier in the sequence.
  • the one or more parameters may comprise one or more fixed parameters characteristic of a given assay assembly.
  • one fixed parameter may be indicative of a maximum amplitude of the local measurement which can be detected from the assay area sometimes referred to as DP max below.
  • another fixed parameter may be indicative of an amount by which or a rate at which the concentration of target entities is depleted as the sample flows from one assay area to the next, sometimes referred to as Shape below.
  • these parameters are referred to as fixed in the sense that they are characteristic of an assay assembly (or a batch or assy assemblies manufactured under substantially indentical conditions) and substantially do not vary as a function of the composition of the sample to by assayed.
  • each assay assembly is only used once in some embodiments and hence experiments are carried out using respective assay assemblies from the same manufacturing batch to characterise the batch.
  • the determined fixed parameters may be verified as being representative of the batch by validation experiments using other assemblies of the batch with samples of known sample concentration or target entities.
  • the values of DP max ), and Shape may be determined and fixed for the assay assembly.
  • the one or more fixed parameters may be determined using experimental data, for example, by minimizing a difference between the respective local measurement and a corresponding value of the parameterized function for each assay area, i, in the assay assembly for each of a plurality of experiments, j, wherein each experiment is carried out using a sample having a given concentration of target entities and the concentrations span a range of concentrations.
  • the offset amount may be determined using a least squares approximation or any other suitable approximation.
  • the one or more fixed parameters or set of constants, ⁇ may be determined by minimizing the following function:
  • a value of ‘Offset’ can also be determined for each of the plurality of experiments, j.
  • Concentration j may be defined as a calibration function giving a concentration value for a corresponding value of Offset.
  • a calibration function can be fitted to data points of ⁇ Concentration j , Offset j ⁇ .
  • the calibration function may be used to determine the sample concentration of target entities based on at least one of the one or more the variable parameters, in particular Offset in the example above.
  • the parameterized function may be a logistic function.
  • the parameterized function is proportional to:
  • DP max is indicative of a maximum amplitude of the local measurement which can be detected from anassay area
  • Shape is indicative of a rate at which the concentration of target entities is depleted as the sample flows from one assay area to the next
  • Offset is a variable parameter determined by data fitting
  • DZ i is indicative of the position of the respective assay area, i, in the sequence.
  • DZ i may take a positive half integer value for each assay area i.e. 0.5, 1.5, 2.5, etc. This is because an amount of depletion occurs in the first assay area prior to the locus where the first measurement is taken.
  • DZ i may take an integer value, or any other suitable value. In some embodiments a mixture of integer and half integer values may be used.
  • the quantity indicative of position in the sequence is indicative of an amount of probe entities (able to interact with target entities) present upstream of the assay area—the locus where the corresponding measurement is taken.
  • the change in the quantity from one assay area to the next may be non-constant and may depend on the amount of probe entities or the capacity to bind target entities between the two assay areas concerned.
  • the parameterized function may be fit to the obtained assay data by adjusting the value of Offset. This, by way of illustration, can be thought of as mapping the assay data to the master depletion curve.
  • the value indicative of the concentration of target entities in the sample may be determined using a value of Offset with a calibration function.
  • the assay assembly is characterized by determining DP max and Shape. More complex models, for example the 4PL and 5PL functions mentioned below may be more accurate in describing the system however, in such complex models, additional fitting parameters are used.
  • the value indicative of the sample concentration may be determined, for example calculated, using a calibration function.
  • the calibration function may be a logistic function, an exponential function, or any other suitable function.
  • the calibration function comprises a first function for use at sample concentrations of a target entity above a given value, and a second function for use at sample concentrations of a target entity below the given value.
  • the first function is a function of Offset
  • Determining the value indicative of the sample concentration of the target entities may comprise calculating the sample concentration itself or calculating any transformation of the sample concentration. Likewise, determining the value indicative of sample concentration may include modeling the local measurements directly or any transformation thereof.
  • Determining the sample concentration may be an iterative process. For example, a first step may be applied initially followed by a second step that may provide a more refined result. Specifically, in some embodiments, the first function is used in the first step to determine a value indicative of concentration. If the value is below a threshold, the second step re-calculates the value using the second calibration function. In some embodiments, the order is reversed and the first function is used in the second step if the value from the first step (from the second function) is above a threshold.
  • the calibration function is a 4 parameter logistic (4PL) nonlinear regression model as shown in equation (2) below.
  • the calibration function is a 5 parameter logistic (5PL) nonlinear regression model as shown in equation (3) below.
  • the 4PL and 5PL functions can also be used as the parameterized function.
  • x DZ i +01:15 et, for example.
  • each local measurement is indicative of variation in a refractive index at the surface of the respective assay area due to target-probe binding.
  • the local measurement may correspond to a change in Surface Plasmon Resonance (SPR) behavior at the detection area.
  • SPR Surface Plasmon Resonance
  • Such a change may be detected by a change in the peak of SPR absorption, for example, a diffusion angle value at which the peak occurs.
  • Other SPR detection paradigms for example based on wavelength or phase may of course be used in some embodiments.
  • changes in the local concentration of target entities from one assay area to the next of 0.5 nM may be detected.
  • any other suitable means for quantitatively detecting an amount of target-probe binding at the surface of the respective assay area may be used, for example, fluorescence or absorption detection (for example UV absorption) and/or detection of a label (fluorescent or otherwise) bound to the target entities may be used.
  • Variation in the refractive index at the surface of the respective assay area may be amplified using an amplifier solution, in some embodiments.
  • the amplifier solution is arranged to interact with target entities bound to the surface of the assay area such that the variation in the refractive index at the respective assay area is amplified when the amplifier has interacted with the bound target entities.
  • the amplifier solution may comprise entities which are arranged to bind to the target entities which are in turn bound to the surface of the assay area, for example gold nanoparticles that are functionalized to bind to the target entities to give target specific amplification, other suitable nanoparticles, secondary antibodies, and beads may be used.
  • the amplifier solution may amplify the variation in the refractive index at the surface of the respective assay area by 2-20 times, for example 5-10 times, for example 10 times.
  • the target and/or probe entities may be molecules or other suitable entities, for example proteins, DNA, peptides, enzymes, viruses, bacteria, cells, etc.
  • the sample may be a blood sample or any other liquid biological (or other) sample.
  • each local measurement comprises a difference between a baseline signal detected prior to interaction of the sample with the assay area and a post-amplification signal detected after interaction of the amplifier solution with target entities bound to the respective assay area.
  • the post-amplification signal may be detected after the respective assay area has been washed with a buffer solution.
  • each local measurement comprises a difference between a pre-amplification signal and post-amplification signal.
  • the pre-amplification signal is detected after interaction of the sample with the respective assay area and before interaction of the amplifier solution with target entities bound to the respective assay area.
  • the post-amplification signal is detected after interaction of the amplifier solution with target entities bound to the respective assay area.
  • the pre-amplification signal may comprise a contribution from a bulk sample refractive index of the sample.
  • the post-amplification signal may be obtained after unbound amplifier and the sample have been substantially washed away by buffer solution in a wash step subsequent to the application of amplifier.
  • the parameterized function of the local measurements may comprise an adjustment term to account for the bulk sample refractive index of the sample affecting the pre-amplification signal but not the post-amplification signal.
  • the adjustment term may be fit to the data points as part of the one or more variable parameters, for example it may be fit simultaneously together with Offset in some embodiments.
  • the adjustment term may be determined based on a difference between a baseline signal detected prior to interaction of the sample with the assay area and the pre-amplification signal. In such embodiments there is no need to fit this term but rather the adjustment term can simply be subtracted from the local measurement (the difference between the pre and post amplification signals).
  • Embodiments that account for bulk sample refractive index contribution to the pre-amplification signal advantageously dispense with the need for a separate wash step prior to amplification if bulk sample refractive index changes are to be accounted for.
  • the concentration of amplifier solution is such that the assay assembly (i.e. all assay areas) is saturated with amplifier.
  • the concentration of amplifier solution is such that the assay assembly is not saturated with amplifier.
  • the local measurements are dependent on the concentration of the amplifier solution as well as the concentration of target in the sample.
  • the parameterized function may contain an additional parameter to account for the depletion of amplifier or an additional term.
  • the ‘Shape’ parameter may be a vector varying with both the sample concentration and the amplifier concentration.
  • Shape may be a function of the quantity indicative of position/upstream binding capacity to capture the varying concentration of amplifier.
  • the effect of the concentration of amplifier can be thought of as being akin to the effect of the density of probe entities present in the assay assembly. Accordingly, for example, the value of DZ i may be adjusted to account for the amplifier concentration in a similar way to how DZ i is adjusted to take into account the relative binding capacity of the assay assembly as will be described in detail below.
  • the assay areas have the same binding capacities for the target entities.
  • the assay areas have different binding capacities for the target entities.
  • DZ i is indicative of an amount of probe entities upstream of the position of the assay assembly, i.
  • the assay areas may be connected by microfluidic circuitry.
  • the circuitry between the assay area as a binding capacity for target entities.
  • Each assay area may be located in a respective chamber connected to adjacent chambers housing respective assay area(s) in the sequence by a conduit between pairs of chambers.
  • Each assay area may occupy a portion of a chamber, wherein the local measurements are made at each respective portion.
  • the assay area may occupy the whole chamber.
  • a plurality of assay areas is provided in a single chamber, for example as part of a contiguous functionalized surface, the assay areas being solely defined by the locus where measurements are taken.
  • Each local measurement may be indicative of a rate at which the amplifier solution interacts with the respective assay area, for example measured as a rate of change of the measurement signal at a defined point.
  • Each local measurement may comprise a measurement indicative of the time taken from introduction of the amplifier solution into the respective assay area to detection of a threshold signal amplitude, for example a maximum signal amplitude.
  • obtaining assay data may comprise carrying out the local measurements.
  • assay data may be obtained from a third party.
  • a system for determining a sample concentration of target entities in a sample comprises a processor arranged to obtain assay data comprising data points of respective local measurements indicative of a local concentration of target entities immobilized at each of a plurality of assay areas of an assay assembly from an assay using the assay assembly, wherein the assay areas are connected in series such that a sample flowing through the assay assembly flows past each assay area in sequence, and wherein each assay area comprises a plurality of probe entities immobilized at a surface of the assay area, the probe entities being arranged to bind to the target entities, such that the concentration of the target entities is depleted as the sample flows from one of the assay areas to the next.
  • the processor is also arranged to model the assay data with a parameterized function of the local measurements against a quantity indicative of the position of the respective assay areas in the sequence, wherein one or more of the parameters are dependent on the sample concentration.
  • the processor is further arranged to determine a value indicative of the sample concentration based on at least one of the one or more parameters.
  • a method for determining a sample concentration of target entities in a sample comprises introducing a sample into an assay assembly from an assay using the assay assembly, the assay assembly comprising a plurality of assay areas wherein the assay areas are connected in series such that a sample flowing through the assay assembly flows past each assay area in sequence, and wherein each assay area comprises a plurality of probe entities immobilized at a surface of the assay area, the probe entities being arranged to bind to the target entities, such that the concentration of the target entities is depleted as the sample flows from one of the assay areas to the next.
  • the sample is caused to flow through the assay assembly and local measurements are carried out at each assay area to obtain assay data comprising data points of respective local measurements indicative of a local concentration of the target entities immobilized at each of the plurality of assay areas of the assay assembly.
  • the assay data is modeled with a parameterized function of the local measurements against a quantity indicative of the position of the assay area in the sequence, wherein one or more of the parameters are dependent on the sample concentration.
  • a value indicative of the sample concentration is determined based on at least one of the one or more parameters.
  • a system for determining a sample concentration of target entities in a sample.
  • the system comprises an assay assembly comprising a plurality of assay areas connected in series such that a sample flowing through the assay assembly flows past each assay area in sequence, and wherein each assay area comprises a plurality of probe entities immobilized at a surface of the assay area, the probe entities being arranged to bind to the target entities, such that the concentration of the target entities is depleted as the sample flows from one of the assay areas to the next.
  • the system further comprises at least one detector arranged to carry out local measurements at each assay area to obtain assay data comprising data points of respective local measurements indicative of a local concentration of the target entities immobilized at each of the plurality of assay areas.
  • the system further comprises a processor arranged to model the assay data with a parameterized function of the local measurements against a quantity indicative of the position of the assay area in the sequence, wherein one or more of the parameters are dependent on the sample concentration.
  • the processor is also arranged to determine a value indicative of the sample concentration based on at least one of the one or more parameters.
  • a single detector is provided for carrying out local measurements at the plurality of assay areas, for example by moving one of the detector and the assay areas relative to the other.
  • a detector may be provided for each assay area.
  • a method for determining a sample concentration of target entities in a sample comprising obtaining assay data comprising a local measurement indicative of a local concentration of the target entity immobilised at an assay area of an assay assembly from an assay using the assay assembly, wherein the assay area comprises a plurality of probe entities immobilized at a surface of the assay area, the probe entities being arranged to bind to target entities.
  • the local measurement is based on signals indicative of a variation in a refractive index at the surface of the assay area, such variation being amplified following interaction of an amplifier solution with target entities bound to the surface of the assay area.
  • the local measurement comprises a difference between a pre-amplification signal and post-amplification signal, wherein the pre-amplification signal is detected after interaction of the sample with the assay area and before interaction of the amplifier solution with the target entities bound to the assay area, and the post-amplification signal is detected after interaction of the amplifier solution with the target entities bound to the assay area and after the assay area has been washed with a buffer solution.
  • the method further comprises adjusting the local measurement using an adjustment term such that a bulk sample refractive index of the sample is taken into account and using the adjusted local measurement to determine a value indicative of the sample concentration.
  • the adjustment term is determined based on a difference between a baseline signal detected prior to interaction of the sample with the assay area and the pre-amplification signal.
  • an assay assembly for determining a sample concentration of target entities in a sample.
  • the assay assembly comprises a plurality of assay areas serially connected such that a sample flowing through the assay assembly flows through each assay area in sequence.
  • Each assay assembly comprises an inlet and an outlet and for each pair of assay areas in the plurality of assay areas, the outlet of a first assay area is coupled to the inlet of a second assay area by a coupling portion, such that a sample flowing through the assay assembly flows from first assay area to the second assay area via the coupling portion for each pair of assay areas in the plurality of assay areas.
  • each assay area comprises a plurality of probe entities immobilized at a surface of the assay area, the probe entities being arranged to bind to the target entities, such that the concentration of the target entities is detectably depleted as the sample flows from one of the assay areas to the next.
  • the flow of the sample may be a laminar flow such that diffusion or other mixing effects are substantially negligible.
  • only target entities in a portion of the sample adjacent the chamber surface will be available for binding with the probe entities.
  • the same portion of sample will be adjacent the surface of each chamber and only target entities present in that same portion of the sample are available for binding.
  • the concentration of target entities in the portion of the sample adjacent each surface is depleted as the sample flows from one chamber to the next. While the depletion may be only a small fraction of the amount of target in the bulk of the sample, due to diffusion limited laminar flow the depletion of target entities represents a significant detectable change in concentration.
  • microfluidic device comprising an assay assembly described above is provided.
  • FIG. 1 is a schematic illustration of a device comprising an assay assembly
  • FIG. a is a schematic illustration of a cross-sectional view of target-probe binding in an assay area of the assay assembly of FIG. 1 ;
  • FIG. 2 b is schematic illustration of a cross-sectional view of amplifier-target binding an assay area of the assay assembly of FIG. 1 ;
  • FIG. 3 is a schematic illustration of a system for determining the concentration of a target entity in a sample
  • FIG. 4 is a graphical representation of a depletion master curve
  • FIG. 5 is a graphical representation of the depletion curve of FIG. 4 is using experimental data
  • FIG. 6 is a flow chart showing a method for determining the concentration of a target entity in a sample
  • FIG. 7 is a sensorgram illustrating the variation in a response amplitude with time and illustrating local measurements ⁇ 31 , ⁇ 32 and ⁇ 21 ;
  • FIG. 8 is a sensorgram illustrating the variation in a response amplitude with time and illustrating a local measurement G amp ;
  • FIG. 9 is a sensorgram illustrating the variation in a response amplitude with time and illustrating a local measurement ⁇ t .
  • a centrifugal or “lab on a disc” microfluidic device 2 is arranged for rotation about an axis 4 .
  • the microfluidic device 2 is a microfluidic polycarbonate disc having an outer diameter of 120 mm, a thickness of ⁇ 1.2 mm, and a hole in the centre of the disc measuring 15 mm in diameter.
  • the disc comprises two 0.6 mm discs bound together by a thin-film polymer.
  • the microfluidic features shown in FIG. 1 and described below are defined in one of the discs and the thin film.
  • the other of the two discs comprises SPR areas provided with a gold coated diffraction grating as described below.
  • the disc 2 comprises an assay assembly 6 which has a plurality chambers 8 arranged in series such that each pair of chambers in the plurality is linked by a conduit 10 .
  • the chambers are aligned with SPR areas.
  • a sample for example blood or other liquid, is introduced into the assay assembly 6 via an inlet conduit 14 , which forms an inlet for the first chamber 8 in the series, and the sample leaves the assay assembly via an outlet conduit 12 , which forms an outlet for the final chamber 8 in the series.
  • Each chamber 8 measures 0.02 mm in depth and is placed at a distance of 50 mm from the centre of the disc.
  • Each chamber is typically approximately 50 nl in volume and the assay assembly and device are arranged such that approximately 100 ⁇ l of liquid can flow through the assay assembly in approximately 5-6 minutes.
  • the chamber 8 comprises an inlet 16 through which a sample may enter the chamber 8 from a connecting conduit 10 (or the inlet conduit 14 in the case of the first chamber in the series) and an outlet 18 through which the sample may leave the chamber 8 via a connecting conduit 10 (or the outlet conduit 12 in the case of the final chamber in the series).
  • a connecting conduit 10 or the inlet conduit 14 in the case of the first chamber in the series
  • an outlet 18 through which the sample may leave the chamber 8 via a connecting conduit 10 (or the outlet conduit 12 in the case of the final chamber in the series).
  • the chamber 8 has a surface 20 comprising a grating of sinusoidal shape (not shown) measuring 100 nm in height and having a period of 1600 nm.
  • the surface 20 is gold coated and has a monolayer of probe entities 22 immobilized on top of the gold surface.
  • Each probe entity 22 has the ability to specifically bind to a specific corresponding target entity 24 which may be present in a sample passed through the chamber 8 , such that when a sample containing target entities 24 flows through the chamber 8 , specific target entities 24 in the sample bind to the probe entities 22 at the chamber surface 20 .
  • the flow of the sample is a laminar flow at sufficient rate such that diffusion or other mixing effects are substantially negligible throughout the assay assembly. Accordingly, only target entities 24 in a portion of the sample adjacent the chamber surface 20 will be available for binding with the probe entities 22 .
  • the same portion of sample will be adjacent the surface 20 of each chamber 8 , hence only target entities 24 present in that same portion of the sample are available for binding. In this way, the concentration of target entities 24 in the portion of the sample adjacent each surface 20 is depleted as the sample flows from one chamber 8 to the next.
  • a concentration change in target entities bound to the probe entities from one chamber to the next is detectable using SPR technology.
  • a detectable change in concentration in the liquid layer adjacent the surface 20 may be 0.5 nM from one point in a chamber 8 to a corresponding point in the next chamber 8 .
  • the system 26 comprises a microfluidic device 2 comprising an assay assembly 6 as described above.
  • a light source 28 is provided and aligned such that emitted light is incident on a detection zone 30 of the surface 20 a chamber 8 .
  • the light source 28 is a polarized monochromatic light source, for example a diode laser.
  • a detector 32 When in use, an amount of light incident on the detection zone 30 is reflected from the surface 20 and the reflected light is detected by a detector 32 .
  • the detector 32 is arranged to measure the light intensity of the reflected light beam as a function of angle over time.
  • the system further comprises a drive for rotating the device 2 to drive liquid flow in the device 2 , under the control of a controller, such that various liquids including a sample are introduced into the device 2 and flow through the assay assembly 6 in a defined sequence.
  • the drive is not illustrated in FIG. 3 for the sake of clarity but further details of how liquid flows may be controlled can be found in WO 2011/122972 and WO2012/131556, incorporated herein by reference.
  • changes in the refractive index at the surface 20 of the detection region 30 due to the presence of bound target entities or a bound target-amplifier complex cause changes in the resonant behavior of the surface 20 , specifically changes in surface plasmon resonance behaviour. This can be detected by detecting a change in the angle at which a light intensity minimum occurs in the reflected light as a function of time.
  • the binding of target entities 24 to probe entities 22 at the surface 20 of the chamber 8 causes a change in the refractive index at the surface 20 .
  • the amount of target-probe binding at the surface 20 of the chamber 8 can be quantitatively determined by detection of changes in the refractive index at the chamber surface 20 , for example by detecting change in the angle at which surface plasmon resonance occurs.
  • alternative approaches for determining surface plasmon effects may be used. Examples of SPR measurement techniques are given in:
  • the depletion of the local concentration of target entity 24 as the sample flows from one chamber 8 to the next can be thought of as establishing a portion of a master depletion curve 35 , which is illustrated in FIG. 4 , with the starting concentration of the sample defining the portion.
  • the master depletion curve 35 characterizes the variation in a local measurement indicative of the concentration of target entity 24 at the surface 20 of each of a respective chamber 8 against a quantity indicative of the amount of probe entities upstream of the position of the chamber 8 in the sequence.
  • the master depletion curve 35 may be understood conceptually by considering a hypothetical system having an unlimited number of notional chambers (and hence detection zones) into which a sample having a very high concentration of target entities is introduced.
  • the initial chambers in the sequence are saturated with target entities, hence a measurement signal saturates at a maximum amplitude of the local measurement, DP max .
  • DP max maximum amplitude of the local measurement
  • the master depletion curve 35 may be understood by considering experimental or hypothetical data from a plurality of experiments carried out at different starting target entity concentrations of the sample using assay assemblies having fixed depletion characteristics. Depletion characteristics are determined by factors including fluidic characteristics, for example the flow rate of sample through the assay assembly 6 , the height and width of the chambers 8 , and the length of the detection circuit; characteristics of the recognition layer, for example, the density of probe entities 22 , the avidity and affinity of the probe entities 22 for the target entities 24 ; and characteristics of the target entity 24 , for example the diffusion coefficient; amongst others.
  • An assay assembly used to carry out the experiments typically has 5-10 chambers, accordingly the depletion data obtained will be representative of only a section of the master depletion curve, as explained above.
  • the master depletion curve may further be understood as a curve combining data from real or notional experiments carried out at a range of known starting concentrations.
  • the data obtained may be thought of as being ‘stitched’ together to form the master depletion curve. If the experimental data sets ‘overlap’ where certain chambers in separate experiments have the same or similar local target concentrations, the data sets may be notionally ‘shifted’ along the x axis until a smooth line is obtained.
  • the master depletion curve may be thought of as a combined depletion curve from four experiments, (i)-(iv). Each experiment is carried out using an assay assembly comprising five chambers and so a data set comprising five data points is obtained from each experiment.
  • the starting concentration of target entity used in experiment (i) is higher than that used in (ii), which is in turn higher than that used in (iii), which is higher than that used in (iv).
  • the data sets can be ‘stitched’ together to form the master depletion curve.
  • the master depletion curve is represented by a parameterized function.
  • the function is a logistic function.
  • the parameterized function models the amplitude of local measurements, DP, made at a respective chamber against a quantity, DZ i indicative of the position in the sequence of the chamber 8 and hence detection zone or area, more specifically, the amount of probe entities upstream of the position of the chamber 8 , i, in the sequence.
  • the position and amount quantities are essentially the same, save for some scaling.
  • the relationship may be more complicated as illustrated below.
  • Example values of DZ i are shown in Tables 1, 2, 3 and 4 below where ‘#DZ’ is the position of the chamber (hence detection zone) in the sequence, ‘DZ capacity’ is the relative capacity of the chamber to bind to target entities, and ‘DZ i ’ is the value indicative of the amount of probe entities upstream of the position of the chamber, i, in the sequence (which is of course also indication of the position in the sequence).
  • DZ i is used in the parameterised function.
  • detection is made in the centre of the each chamber.
  • Table 1 shows the case where the chambers each have the same relative capacity for binding target entities (for example the same amount of probe entities above to bind target entities).
  • the values of DZ i used are 0.5, 1.5, 2.5, 3.5 and 4.5 (the 0.5 offset being representative of binding occurring in each chamber upstream of the detection area).
  • Table 2 shows the case where the relative capacity of the chamber doubles from one chamber to the next. This difference in the relative capacity of the chambers is accounted for in the value of DZ i used.
  • Table 3 shows the case where the 1 st , 4 th and 5 th chambers have a relative capacity of 1 and the 2 nd and 3 rd chambers have a relative capacity of 2. Again, this difference in relative capacity is accounted for by adjustment of DZ i .
  • the chambers may be connected by microfluidic circuitry.
  • chambers are connected by microfluidic circuitry, the circuitry between the chambers having a binding capacity for target entities.
  • Table 4 above shows the case where the assay areas have a relative binding capacity of 1 and the circuitry between the chambers have a relative capacity of 0.5. Detection is not carried out in the circuitry. In this case, DZ i is adjusted according to Table 4 to account for the relative capacity of the system.
  • the parameterized function comprises constants which relate to the assay assembly and its depletion characteristics, DP max and Shape, and which are fixed for a given assay assembly and assay.
  • the function also comprises a parameter dependent on the concentration of target entities 24 in the sample, Offset, which is indicative of the starting concentration of a sample.
  • the parameter, Offset is determined by fitting the parameterised function to the data points for each experiment carried out.
  • Offset determines the location of the data points for the actual chambers/detection areas on the master depletion curve.
  • Offset can be understood as a value indicative of the extent to which the master depletion curve is shifted along the x axis.
  • the local measurement obtained at the first chamber in the sequence is a correspondingly low measurement, DP 1 .
  • the amount by which the master depletion curve must be shifted, and in which direction, in order to fit the assay data obtained will depend on the target concentration of the sample. Accordingly, the value of Offset is determined by fitting the parameterised function, and hence the master depletion curve, to the respective local measurements obtained at each chamber in the assay assembly.
  • DP max is the maximum amplitude of the local measurement which can be obtained at a first chamber 8 of an assay assembly and remains constant throughout an experiment.
  • the parameter Shape is indicative of the rate at which the concentration of target entities at the chamber surface is depleted as the sample flows from one chamber to the next. This value depends on the depletion characteristics of the assay assembly and remains constant throughout an experiment for a given assay assembly and assay. exp is typically euler's number, e, however any other suitable base may be used with a corresponding adjustment in the other parameters.
  • DZ i corresponds to a value indicative of the amount of probe entities upstream of the position of the respective chamber, i, in the sequence.
  • the detection zone 30 for each chamber 8 is a portion at the centre of the chamber surface 20 .
  • DZ i may take a positive half integer value i.e. 0.5, 1.5, 2.5, 3.5 etc.
  • DZ i may take an integer value, or any other suitable value indicative of the amount of probe entities upstream of the position of the chamber in the assay assembly.
  • the constants Shape and DP max are determined by characterizing a batch of assay assemblies prior to carrying out an experiment to determine a sample concentration. Each assay assembly is only used once and hence experiments to characterise a batch of assemblies are carried out using respective assay assemblies from the same manufacturing batch to characterise the batch of microfluidic devices 2 .
  • the determined values of Shape and DP max are verified as being representative of the batch by validation experiments using other assemblies of the batch with samples of known sample concentration or target entities.
  • the determined values are then associated with the microfluidic devices 2 , for example, by shipping with the device 2 , for example as an indication on packaging, or marking the device itself 2 to indicate the values, for example using a bar code or other suitable means for carrying this information.
  • the packaging and/or disc may carry this information directly or may carry a link to a remote location where this information is held for access over a network for example the internet.
  • DP max and Shape which are collectively denoted by ⁇ , are determined using known experimental data obtained from a plurality of experiments, j, each having a known starting concentration of target entities (the concentrations spanning a range of concentrations of interest) and each carried out using an assay assembly having a plurality of assay areas, i. DP max and Shape are determined by minimizing the following sum:
  • DMZ is the local measurement at the assay area, i, and f is the corresponding value of the parameterized function having constants ⁇ .
  • Data from m experiments is used, each experiment having been carried out using an assay assembly having n assay areas.
  • the parameters of Shape and DP max are determined using any suitable optimization technique, e.g. least square, gradient descent, regression or Chi-squared minimization techniques. From the sum, (6), above, ‘Offset’ is also determined for each of the plurality of experiments, j, hence a relationship between ‘Offset’ and the starting concentration of target entities is determined.
  • Offset j This relationship between Offset j and concentration, Concentration j , for each of the plurality of experiment, j, defines data points ⁇ Concentration j , Offset j ⁇ that can be used to fit a calibration function. As will be described further below, this calibration function is used to determine the sample concentration based on the value of Offset.
  • DP(DZ i ) is the local measurement at the assay area, i, and f is the corresponding value of the parameterized function.
  • Minimization of this or any suitable cost function can be carried to obtain the best fit of the parameterized function to the assay data.
  • least-square regression minimization techniques are used and validated using a chi-squared test.
  • a value indicative of the starting target concentration of the sample can be determined using a calibration function.
  • the calibration function comprises a first function and a second function.
  • the first function, f 1 is used to determine the target concentration for samples where the target concentration is known to be high and is a function of ‘Offset’, for example f 1 may be found by fitting a suitable function to the data points, ⁇ Concentration j ,Offset j ⁇ , described above.
  • the first and second functions are represented in the form of an exponential function as shown by equations (9a) and (9b) below.
  • the parameters X 1,2 , Y 1,2 and Z 1,2 are be obtained by fitting to experimental data in a similar manner as described above.
  • determining the sample concentration is an iterative process. For example, a first step may be applied initially followed by a second step that provides a more refined result. Specifically, in some embodiments, the first function is used in the first step to determine a value indicative of concentration. If the value is below a threshold, the second step re-calculates the value using the second calibration function. In some embodiments, the order is reversed and the first function is used in the second step if the value from the first step (from the second function) is above a threshold.
  • the calibration function is a single function relating Offset to the target concentration of the sample.
  • the calibration curve of sample concentration against Offset is a logistic function e.g. a 4PL nonlinear regression model.
  • the sample concentration is a function of (Offset, a, b, c, d), where a, b, c and d are parameters of the model which may be obtained using minimization techniques, for example Chi-squared minimization techniques, and experimental data as described above.
  • a method for determining the concentration of target entities in a sample will now be described in overview with reference to FIG. 6 .
  • assay data is obtained.
  • the assay data obtained comprises a plurality of data points, each data point corresponding to a local measurement carried out at a respective chamber 8 .
  • the local measurements relate to the detection of changes in a refractive index at the surface of each of the respective chambers and are indicative of the concentration of target entity 24 at the surface 20 of the respective chamber 8 .
  • the assay data is modeled with the parameterized function at a second step 38 , as described above, the parameterized function comprising the parameter, Offset, dependent on the concentration of the target entity in the sample.
  • a value indicative of the concentration of the target entity in the sample is then determined at a third step 40 based on ‘Offset’ using the first and second calibration functions described above. It will be understood that at some point prior to step 38 , for example when loading the device 2 into the system 26 , the parameters ⁇ , are loaded into the system, for example by manual entry or by reading a tag, such as a barcode, carrying this information, as described above.
  • the system 26 described above and shown in FIG. 3 is used to obtain assay data. Firstly a buffer solution is made to flow through into the assay assembly 6 as a baseline, followed by a sample to be tested, an amplifier solution, and finally a wash with a second buffer solution. With reference to FIG. 7 , for each respective chamber 8 , changes in the refractive index at the chamber surface 20 can be detected by the detector 32 such that the amplitude of the detected signal, for example change in the angle at which surface plasmon resonance occurs, increases in direct proportion to the magnitude of the change in refractive index at the chamber surface 20 .
  • a baseline measurement 42 is measured for the detection region 30 .
  • the sample comprising an amount of target entities 24 is then introduced into the chamber 8 .
  • the refractive index at the surface 20 changes and consequently the amplitude of the measured signal for the detection region 30 increases 44 a .
  • a proportion of the target-probe binding is reversible hence a reduction 44 b in the amplitude of the measured signal for the detection region 30 may occur until a steady state is reached. Such reduction may not be observed in cases where the concentration of target entity 24 in the sample is very high.
  • the amplifier solution is then made to flow through the chamber 8 .
  • Active components 25 in the amplifier solution bind to the target entities 24 which are in turn bound to the probe entities 22 .
  • a sufficiently high concentration of amplifier is made to flow through the chamber 8 such that the bound target entities are saturated with amplifier.
  • the local measurements may be any of a number of suitable measurements, some of which are described in more detail in the embodiments below.
  • assay data may be obtained via any other suitable means, or may be obtained from a previously run assay, possibly run by a third party.
  • the assay data is modeled with the parameterized function and a value of ‘Offset’ is determined as described above.
  • the constants DP max and Shape characteristic of the assay assembly having been previously determined using the method described above and having been marked on the microfluidic device itself, for example using a bar code.
  • the parameterized function models the local measurements carried out at each respective chamber 8 against the quantity, DZ.
  • the quality of fit of the measured data to the parameterized function is evaluated, for example by calculation of Pearson's coefficient for the fit, using Chi-squared minimization techniques or using any other suitable means.
  • the quality of the fit is compared to a predetermined threshold such that, if the quality of fit is not sufficiently good to meet the threshold, the data is discarded.
  • a value indicative of the target concentration of the sample can be determined using the calibration function as outlined above.
  • DP max _ amp is the maximum amplitude of the local measurement at a chamber 8 following interaction of the amplifier with the chamber 8 .
  • measurements B 1 and B 3 are each made when the bulk solution in the chamber is buffer solution (i.e. the bulk solution at each measurement has the same refractive index), as DZ i becomes larger, DP will tend towards zero hence the amplitude DP min of the master curve/parameterised function is zero.
  • a potential drawback with the approach outlined in Embodiment 1 is that there can be drift in the signals being compared, for example, due to fluctuations in temperature, vibrations in the system etc.
  • the signal is dependent on the local refractive index near the detection surface.
  • Such a signal therefore comprises contributions from (i) the probe/target layer having a certain density of target entities bound thereto; (ii) the surrounding liquid; (iii) the metal present at the surface of the chamber e.g. gold.
  • the refractive index of these three contributions is dependent on the temperature and so drifts in temperature will cause drift in the signal detected. Similar drift effects result from mechanical vibrations in the system.
  • ⁇ 32 Local measurements, ⁇ 32 , are carried out at each of the respective chambers 8 in the assay assembly.
  • ⁇ 32 is measured by detecting the amplitude of a response, B 2 , following interaction of the sample with the chamber surface 20 and prior to interaction of the amplifier with target entities bound to the surface, shown as detection point 2 in FIG. 7 .
  • Measuring ⁇ 32 this has the advantage that the measurement is made over a shorter time period (because the time between B 2 and B 3 is shorter than the time between B 1 and B 3 ) and so the effect of drift is reduced.
  • ⁇ 32 comprises a contribution caused by the change in the bulk material from sample to buffer solution between B 2 and B 3 . Accordingly, ⁇ 32 can be represented by equation (11) shown below.
  • f(Offset) is the parameterized function/master curve and ⁇ bulk is the contribution due to the refractive index of the sample.
  • the change in the refractive index due to the difference in bulk solution between B 2 and B 3 , ⁇ bulk will vary from person to person and is accordingly is unknown quantity.
  • ⁇ bulk can be obtained as a further variable parameter by fitting the function (11) to the experimental data, that is adjusting Offset and ⁇ bulk at the same time.
  • ⁇ 21 can be measured, as will be described below, and used as an approximation to ⁇ bulk (ignoring the effect of unamplified target-probe binding).
  • the measurement ⁇ 21 can be thought of according to equations (12) below.
  • a change of the amplitude of the response signal detected by the detector 32 as the amplifier flows across the surface 20 of the chamber 8 , G amp is used as the local measurement. This measurement reflects the rate at which the active components in the amplifier bind with target entities 24 bound to the respective chamber 8 .
  • G amp equates to the gradient of the curve at time t 1 , indicated by point 4 on the sensorgram.
  • any other suitable, for example amplification, rate dependent measurement may also be taken.
  • a time taken from introduction of the amplifier into the respective chamber to detection of a threshold amplitude of the response signal or a feature of the signal is measured.
  • a threshold amplitude of the response signal or a feature of the signal e.g. a maximum
  • the parameterized function is therefore arranged to account for this dependency.
  • the parameterized function may contain an additional parameter to account for the depletion in the active component concentration.
  • the ‘Shape’ parameter may be a vector varying with both the sample concentration and amplifier concentration.
  • the effect of the concentration of amplifier can be thought of as being akin to the effect of the density of probe entities present in the assay assembly as a first approximation. Accordingly, for example, the value of DZ i may be adjusted to account for the amplifier concentration in a similar way to how DZ i is adjusted to take into account the relative binding capacity of the assay assembly as described in detail above.

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