The present invention relates to an analytical test device.
Biological testing for the presence and/or concentration of an analyte may be conducted for a variety of reasons including, amongst other applications, preliminary diagnosis, screening samples for presence of controlled substances and management of long term health conditions.
Lateral flow devices (also known as “lateral flow immunoassays”) are one variety of biological testing. Lateral flow devices may be used to test a liquid sample, such as saliva, blood or urine, for the presence of an analyte. Examples of lateral flow devices include home pregnancy tests, home ovulation tests, tests for other hormones, tests for specific pathogens and tests for specific drugs. For example, EP 0 291194 A1 describes a lateral flow device for performing a pregnancy test.
In a typical lateral flow testing strip, a liquid sample is introduced at one end of a porous strip which is then drawn along the strip by capillary action (or “wicking”). A portion of the lateral flow strip is pre-treated with labelling particles which are activated with a reagent which binds to the analyte to form a complex, if the analyte is present in the sample. The bound complexes and also unreacted labelling particles continue to propagate along the strip before reaching a testing region which is pre-treated with an immobilised binding reagent which binds bound complexes of analyte and labelling particles and does not bind unreacted labelling particles. The labelling particles have a distinctive colour, or other detectable optical or non-optical property, and the development of a concentration of labelling particles in the test regions provides an observable indication that the analyte has been detected. Lateral flow test strips may be based on, for example, colorimetric labelling using gold or latex nanoparticles, fluorescent marker molecules or magnetic labelling particles.
Another variety of biological testing involves assays conducted in liquids held in a container such as a vial, a PCR well/plate, a cuvette or a microfluidic cell. Liquid assays may be measured based on colorimetry or fluorescence. An advantage of some liquid based assays is that they may allow tests to be conducted using very small (e.g. picolitre) volumes.
Sometimes, merely determining the presence or absence of an analyte is desired, i.e. a qualitative test. In other applications, an accurate concentration of the analyte may be desired, i.e. a quantitative test. For example, WO 2008/101732 A1 describes an optical measuring instrument and measuring device. The optical measuring instrument includes at least one source for providing at least one electromagnetic beam to irradiate a sample and to interact with the specimen within the sample, at least one sensor for detecting an output of the interaction between the specimen and the electromagnetic beam, an integrally formed mechanical bench for the optical and electronic components and a sample holder for holding the sample. The at least one source, the at least one sensor, and the mechanical bench are integrated in one monolithic optoelectronic module and the sample holder can be connected to this module.
Quantitative detectors for biological testing methods may require optical components such as beamsplitters, lenses, monochromators, filters and so forth. Such components may be complex, expensive and/or bulky, and may have properties which vary considerably with the wavelength of light. Optical components such as beamsplitters, lenses, monochromators, filters and so forth are typically too bulky for integration into a single use, self-contained lateral flow immunoassay test or a self-contained microfluidic assay test.
Biological samples which may contain an analyte of interest may be coloured, for example blood or urine. Conventionally, coloured samples have been treated by filtering out the coloured dye (eg filtering full red blood to obtain clear serum) or by introducing a washing/flushing step.
According to a first aspect of the invention there is provided an analytical test device including two or more sets of emitters, each set of emitters comprising one or more light emitters configured to emit light within a range around a corresponding wavelength. Each set of light emitters is configured to be independently illuminable. The test device also includes one or more photodetectors arranged such that light from each set of emitters reaches the photodetectors via an optical path comprising a sample receiving portion. The emitters and photodetectors are configured such that, at the sample receiving portion of the optical path, a normalised spatial intensity profile generated by each set of emitters is substantially equal to a normalised spatial intensity profile generated by each other set of emitters. The test device also includes a liquid transport path comprising a first end, a second end and a liquid sample receiving region. The liquid transport path is configured to transport a liquid sample received in the liquid sample receiving region towards the second end and through the sample receiving portion of the optical path.
Absorbance measurements obtained using the two or more sets of emitters may be de-convoluted (de-mixed) to quantify the concentration of one or more analytes whilst also compensating for optical scattering due to defects or other inhomogeneities of a sample.
Thus, the analytical test device can provide improved signal to noise ratio for simultaneous measurements of one or more analytes.
Thus, the analytical test device may include a simplified optical path which does not require optical components such as filters or monochromators to perform dual-wavelength measurements. Thus, the analytical test device may be less bulky and simpler to manufacture.
The test device may also include a controller configured to sequentially illuminate each set of emitters and to obtain a corresponding measured absorbance value using the photodetectors, such that only one set of emitters is illuminated at any time. The controller may also be configured to generate an absorbance vector using the measured absorbance values. The controller may also be configured to determine a concentration vector by multiplying the absorbance vector with a de-convolution matrix (also referred to as a de-mixing matrix).
Each set of emitters emits light within a range around a wavelength which is different to each of the other sets of emitters.
The two or more sets of emitters may include a set of first light emitters configured to emit within a range around a first wavelength, and a set of second light emitters configured to emit within a range around a second wavelength. The two or more sets of emitters may also include a set of third light emitters configured to emit within a range around a third wavelength. The two or more sets of emitters may include a set of fourth light emitters configured to emit within a range around a fourth wavelength.
The controller may be configured to subtract a signal obtained at a reference wavelength, for example the second wavelength, from a signal obtained at a measurement wavelength, for example the first wavelength, in order to compensate for optical scattering due to defects or other inhomogeneities in a medium or on a substrate holding a sample.
Thus, using first and second separate, alternately illuminable emitters which provide substantially equal normalised spatial intensity profiles, absorbance measurements may be corrected using measurements at a reference wavelength. In this way, the analytical test device can provide improved signal to noise ratio.
The wavelength corresponding to each set of emitters may correspond to a peak emission wavelength of the emitters. Each set of emitters may emit light within a range having a full-width at half maximum of no more than 10 nm, no more than 25 nm, no more than 50 nm, no more than 100 nm or no more than 200 nm.
The optical path may include no monochromators. The optical path may include no beamsplitters between the sample receiving portion and the photodetectors. The optical path may include no fibre couplers and/or fibre splitters between the sample receiving portion and the photodetectors.
Normalised spatial intensity profiles may be substantially equal at an entrance to, an exit from, or on any plane perpendicular to the optical path and within the sample receiving portion of the optical path. Normalised spatial intensity profiles may be substantially equal throughout the sample receiving portion of the optical path.
Normalised spatial intensity profiles may be considered to be substantially equal on a plane perpendicular to the path if the normalised intensity values for the first and second wavelengths are within 5%, within 10%, within 15% or within 20% of one other at each point on that plane. Normalised spatial intensity profiles may be considered to be substantially equal on a plane perpendicular to the path if the normalised intensity values for the first and second wavelengths differ, at each point on that plane, by less than two times, less than three times or less than five times the standard error of normalised intensities at the first wavelength or the second wavelength, whichever has the larger standard error.
The wavelengths corresponding to each set of light emitters may be selected in dependence upon the absorbance spectrum of one or more target analytes. The wavelength corresponding to each set of light emitters may be selected such that a target analyte has relatively higher absorbance at said wavelength than at a wavelength corresponding to each other set of light emitters. A target analyte may be any suitable labelling molecule or particles such as, for example, gold nanoparticles.
The first and second wavelengths may be selected in dependence upon the absorbance spectrum of a target analyte. The first and second wavelengths may be selected such that a target analyte has relatively higher absorbance at the first wavelength than at the second wavelength. The ratio of target analyte absorbance at the first and second wavelengths may be at least two, up to an including five, up to an including ten or more than ten. A target analyte may be any suitable labelling molecule or particles such as, for example, gold nanoparticles.
The wavelengths corresponding to each set of emitters may lie in the range between 300 nm and 1500 nm inclusive. The wavelengths corresponding to each set of emitters may lie in the range between 400 nm and 800 nm inclusive.
Each set of light emitters may include inorganic light emitting diodes. Each set of light emitters may include organic light emitting diodes. Organic light emitting diodes may be solution processed. The analytical test device may include a plurality of sets of emitters arranged to form an array. The array may include more emitters in a first direction than in a second, perpendicular direction.
The first emitters may be inorganic light emitting diodes. The first emitters may be organic light emitting diodes. The second emitters may be inorganic light emitting diodes. The second emitters may be organic light emitting diodes. The analytical test device may include a plurality of first and second emitters arranged in an array. The array may include more emitters in a first direction than in a second, perpendicular direction.
The photodetectors may take the form of photodiodes, photoresistors, phototransistors, complementary metal-oxide semiconductor (CMOS) pixels, charge coupled device (CCD) pixels, photomultiplier tubes or any other suitable photodetector. The photodetectors may take the form of organic photodiodes. Organic photodiodes may be solution processed. The analytical test device may include a plurality of photodiodes arranged in an array. The array may include more photodiodes in a first direction than in a second, perpendicular direction.
The optical path may be configured such that the photodetectors receive light transmitted through the sample receiving portion of the optical path.
The optical path may be configured such that the photodetectors receive light reflected from the sample receiving portion of the optical path.
The photodetectors may form an image sensor arranged to image all or a portion of the sample receiving portion of the optical path.
The liquid transport path may take the form of a porous medium. The porous medium may include nitrocellulose or other fibrous materials capable of transporting an aqueous liquid by capillary action, whether inherently or following appropriate surface treatments. The liquid transport path may include at least one microfluidic channel. The microfluidic channel may form a part of a microfluidic device.
The optical path may include a slit arranged before the sample receiving portion and each set of emitters may be arranged to illuminate the slit.
The optical path may include a slit arranged on the optical path before the sample receiving portion. Each first emitter and each second emitter may have a cylindrically symmetric angular emission profile, and each pair of first and second emitters may be arranged such that the slit perpendicularly bisects the pair.
Thus, equal normalised spatial intensity profiles of light at the first and second wavelengths may be provided at the sample receiving portion using a particularly simple and compact arrangement of first and second emitters.
A diffuser may be included between each set of emitters and the slit. The slit may have adjustable width. The slit may have a width between 100 μm and 1 mm inclusive. The slit may have a width between 300 μm and 500 μm inclusive. The light emitters belong to each set may have Gaussian angular emission profiles. The first and second emitters may have Gaussian angular emission profiles.
The two or more sets of emitters may include a set of second emitters, and each second emitter may be substantially transparent at the wavelengths emitted by each other set of emitters, and each other emitter may emit light into the optical path through a corresponding second emitter.
Each second emitter may be substantially transparent at the first wavelength, and each first emitter may emit light onto the optical path through a corresponding second emitter. Each second emitter may be substantially transparent at the wavelengths emitted by each first emitter and each third emitter, and wherein each first emitter and each third emitter may emit light into the optical path through a corresponding second emitter.
Thus, the optical path may be a gap between a second emitter and a photodetector. In this way, optical components such as beamsplitters, lenses, filters, monochromators or the like may be omitted.
Transparency at the wavelengths emitted by each other set of emitters may correspond to a transmittance of more than 50%, more than 75%, more than 85%, more than go % or more than 95%. Transparency at the first wavelength may correspond to a transmittance of more than 50%, more than 75%, more than 85%, more than go % or more than 95%.
The two or more sets of emitters may be arranged into an array including a plurality of pixels. Each pixel may include at least one subpixel, and each subpixel may include a light emitter corresponding to each set of emitters.
A plurality of first light emitters and a plurality of second light emitters may be arranged into an array, wherein the first and second light emitters alternate in a chessboard configuration.
Thus, the optical path may be a gap between an array of light emitters and a photodetector. In this way, optical components such as beamsplitters, lenses, filters, monochromators of the like may be omitted.
Two or three sets of emitters may be interdigitated with one another to form an array. The liquid transport path may take the form of a lateral flow type strip. The liquid transport path may take the form of the whole, a part, or at least one channel of a microfluidic device.
The controller may be further configured to intersperse illumination of each set of emitters with periods when none of the sets of emitters is illuminated.
The analytical test device may also include at least one output device.
The at least one output device may take the form of one or more light emitting diodes, and the controller may be configured to illuminate each light emitting diode in response to a corresponding value of the concentration vector exceeding a predetermined threshold.
The at least one output device may take the form of a display element, and the controller may be configured to cause the display element to display one or more outputs in response to determining the concentration vector. The controller may be configured, in response to a value of the concentration vector exceeding a predetermined threshold, to cause the display element to display a corresponding symbol or symbols. The controller may be configured to cause the display element to display one or more values of the concentration vector.
The at least one output device may take the form of a wired or wireless communications interface for connection to a data processing apparatus, and the controller may be configured to output the concentration vector to the data processing apparatus via the wired or wireless communications interface.
The controller may be configured to normalise absorbance values with respect to a reference calibration absorbance value.
The controller may be configured to illuminate the first emitters and obtain a first set of measurements using the photodetectors, to illuminate the second emitters and obtain a second set of measurements using the photodetectors, and to subtract the second set of measurements from the first set of measurements.
The controller may be configured to multiply the second set of measurements by a weighting factor before subtracting the second set of measurements from the first set of measurements.
According to a second aspect of the invention there is provided a method of operating the analytical test device. The method includes applying a liquid sample to the liquid sample receiving region of the analytical test device.
According to a third aspect of the invention there is provided a method of determining a de-convolution matrix. The method includes providing an optical path which includes a sample receiving portion. The method also includes providing a number, N, of sets of emitters, each set of emitters including one or more light emitters configured to emit light within a range around a corresponding wavelength into the optical path. At the sample receiving portion, a normalised spatial intensity profile generated by a given set of emitters is substantially equal to a normalised spatial intensity profile generated by each other set of emitters. The method also includes providing a number, N, of calibration samples. Each calibration sample includes a known concentration of N different analytes. The method also includes, for each calibration sample, arranging the calibration sample wholly or partly within the sample receiving portion of the optical path. The method also includes, for each calibration sample, sequentially illuminating each set of emitters and obtaining a corresponding measured absorbance value using the photodetectors, wherein only one set of emitters is illuminated at any time. The method also includes, for each calibration sample, generating an absorbance vector using the N measured absorbance values. The method also includes, for each calibration sample, generating a concentration vector using the N known concentrations of analytes. The method also includes generating a first N by N matrix by setting the values of each column, or each row, to be equal to the values of the absorbance vector of a corresponding calibration sample. The method also includes inverting the first matrix. The method also includes generating a second N by N matrix by setting the values of each column, or each row, to be equal to the values of the concentration vector of a corresponding calibration sample. The method also includes determining a deconvolution matrix by multiplying the second matrix by inverse of the first matrix.
Absorbance and concentration values may be normalised with respect to a reference calibration absorbance value.
A deconvolution matrix determined according to the method of determining a de-convolution matrix may be used by the controller of the analytical test device.
The method of determining a de-convolution matrix may be carried out using the analytical test device.
Certain embodiments of the present invention will now be described, by way of example, with reference to the accompanying drawings in which:
If the number and complexity of optical components in a quantitative detector could be reduced, then the size and cost of the detector could be reduced. This would be of particular advantage for handheld or portable testing devices, and for single use home testing kits.
The minimum threshold for detecting an analyte may be improved if the signal to noise ratio of the measurement could be improved. Additionally, improvements in the signal to noise ratio may also allow for an analyte concentration to be determined with improved resolution.
Referring to
Each first light emitter is configured to emit light 5 within a range around a first wavelength λ1, and each second light emitter is configured to emit light 6 within a range around a second wavelength λ2. The first light emitter(s) 2 may take the form of, for example, organic or inorganic light emitting diodes. Similarly, the second light emitter(s) 3 may take the form of, for example, organic or inorganic light emitting diodes. Organic light emitting diodes may be solution processed. If the first light emitter(s) 2 take the form of organic light emitting diodes, the second light emitter(s) need not take the form of organic light emitting diodes and vice versa. The analytical test device may include a plurality of first and second light emitters 2, 3 arranged in an array. The array may include more light emitters 2, 3 in a first direction than in a second, perpendicular direction.
The one or more photodetector(s) are sensitive across a broad wavelength range which includes at least the first and second wavelengths λ1, λ2. The photodetector(s) 4 may take the form of, for example, photodiodes, photoresistors, phototransistors, complementary metal-oxide semiconductor (CMOS) pixels, charge coupled device (CCD) pixels, photomultiplier tubes or any other suitable photodetector. Photodiodes may be organic or inorganic. Organic photodiodes may be solution processed. The analytical test device 1 may include a plurality of photodetectors 4 arranged in an array. The array may include more photodetectors in a first direction y than in a second, perpendicular direction x.
The first and second light emitters 2, 3 are each coupled to an optical path 7 along which the light 5, 6 travels to reach the photodetector(s) 4. The optical path 7 includes a sample receiving portion 8. The analytical test device 1 is arranged to receive a sample 9. When a sample 9 is received into the analytical test device 1, the sample, or at least a portion of the sample 9, intersects the sample receiving portion 8 of the optical path 7.
The sample receiving portion 8 of the optical path 7 may be configured to receive a sample 9 in the form of a lateral flow test strip 18 (
The first light emitter(s) 2 and the second light emitter(s) 3 are alternately illuminable. Illumination of the first and second light emitters 2, 3 may be interspersed with periods when neither of the first and second light emitters 2, 3 is illuminated. A period between turning off the first light emitter(s) 2 and illuminating the second light emitter(s) 3 can be used for detecting fluorescence excited by the light 5 from the first light emitter(s) 2. Similarly, fluorescence excited by light 6 from the second light emitter(s) 3 may be detected during a period after turning off the second light emitter(s) and before turning on the first light emitter(s) 2.
The analytical test device 1 also includes a controller 27. The controller 27 is configured to sequentially illuminate the first and second emitters 2, 3 and to obtain corresponding measured absorbance values using the photodetectors 4. Only one set of emitters 2, 3 is illuminated at any time. The controller 27 is also configured to generate an absorbance vector using the measured absorbance values, and to determine a concentration vector by multiplying the absorbance vector with a de-convolution matrix as described hereinafter. The controller may optionally be configured to intersperse illumination of each set of emitters with periods when none of the sets of emitters is illuminated. The controller 27 may be configured to normalise absorbance so values with respect to a reference calibration absorbance value.
In the particular case of first and second emitters 2, 3, the controller 27 may be configured to illuminate the first emitters 2 and obtain a first set of measurements using the photodetectors 4, to illuminate the second emitters 3 and obtain a second set of measurements using the photodetectors 4, and to subtract the second set of measurements from the first set of measurements, a s further described hereinafter. The controller may be configured to multiply the second set of measurements by a weighting factor before subtracting the second set of measurements from the first set of measurements. Further details of the methods, processes and calculations carried out by the controller 27 are described hereinafter.
The analytical test device 1 also includes at least one output device 28. For example, the output device 28 may take the form of one or more light emitting diodes which are arranged for viewing by a user of the analytical test device 1. The controller 27 may be configured to illuminate each light emitting diode in response to a concentration of a specific analyte vector exceeding a predetermined threshold.
In a further example, the output device 28 may take the form of a display element. The controller may be configured to cause the display element to display one or more outputs in response to determining the concentrations of one or more analytes. The controller may be configured, in response to a determined concentration of an analyte exceeding a predetermined threshold, to cause the display element to display a corresponding symbol or symbols. The controller may be configured to cause the display element to display the determined concentrations of one or more analytes.
In another example, the at least one output device 28 may take the form of a wired or wireless communications interface for connection to a data processing apparatus (not shown). The data processing apparatus may take the form of, for example, a mobile telephone, tablet computer, laptop, desktop or a server. The controller may be configured to output the measured concentrations of one or more analytes to the data processing apparatus (not shown) via the wired or wireless communications interface.
Referring also to
For example, referring in particular to
Referring in particular to
The beam profiler 15 measures light 5, 6 intensities in a measurement plane 17 which is rotated by an angle θ about the second direction y with respect to the notional surface 14. Positions on the notional surface 14, for example the bounds xA, xB, yA, yB of the notional surface 14 of the sample receiving portion 8, are projected onto positions xA′, xB′, yA′, yB′ on the measurement plane 17 according to xA′=xA/sin θ, xB′=xB/sin θ, yA′=yA and yB′=yB. Preferably, a light sensitive area of the beam profiler 15 detection surface 16 is large enough to encompass the projected bounds xA′, xB′, yA′, yB′ of the notional surface 14.
Referring in particular to
The first and second beam profiles 10, 11 are preferably substantially equal on the measurement plane 17, i.e. on entering the sample receiving portion 8. Preferably, the normalised spatial intensity profiles 10, 11 are substantially equal throughout the sample receiving portion 8 of the optical path 7. However, uniformity throughout the sample receiving portion 8 is not necessary since, in use, the scattering from the sample 9 will be more significant than effects of diverging beam profiles 10, 11.
A number of difference metrics may be used to quantify the extent of differences between the first and second beam profiles 10, 11. For example, a maximum beam profile difference Δmax may be defined according to:
Similarly, an average beam profile difference Δavg may be defined according to:
If the output of the beam profiler 15 is an array of intensities corresponding to an array of positions x′, y′, the integral defined in Equation 2 may be readily converted to a sum in order to determine the average beam profile difference Δavg.
Alternatively, a root-mean-square (RMS) difference ΔRMS may be defined according to:
If the output of the beam profiler 15 is an array of intensities corresponding to an array of positions x′, y′, the integral defined in Equation 3 may be converted to a sum to determine the average beam profile difference Δavg. Difference metrics are not limited to the maximum, average and/or RMS beam profile differences Δmax, Δmean, ΔRMS) and alternative difference metrics may be defined to quantify the extent of differences between the first and second beam profiles 10, 11.
The first and second emitters 2, 3 and the optical path 7 are arranged such that the first and second beam profiles 10, 11 are substantially equal on the measurement plane 17. The following description shall refer to an example in which light 5 from the first emitter 2 is used to quantify the sample 9, whilst light 6 from the second emitter 3 is used as a reference (as explained hereinafter). However, the same principles are applicable if light 6 from the second emitter 3 is used to quantify the sample 9, whilst light 5 from the first emitter 2 is used as a reference.
The beam profiles 10, 11 may be considered to be substantially equal when the maximum difference Δmax, average difference Δavg or RMS difference ΔRMS are less than or equal to an absolute threshold determined by prior experiments. Preferably, whether or not the beam profiles 10, 11 may be considered to be substantially equal can be evaluated by comparing the maximum difference Δmax average difference Δavg or RMS difference ΔRMS to a relative threshold determined from the beam profiles 10, 11 themselves.
For example, a first threshold may be based on a fraction of the maximum normalised intensity of light 5 from the first emitter 2, i.e. I1max=max(I1(x′,y′)). The first and second beam profiles 10, 11 may be considered to be substantially equal if the maximum Δmax, average Δavg or RMS difference ΔRMS is less than or equal to 0.05×I1max (≤5%), less than or equal to 0.1×I1max (≤10%), less than or equal to 0.2×I1max (≤20%) or less than or equal to 0.5×I1max (≤50%).
In an ideal case, the first and second beam profiles are equal to each other at each point, i.e. for all x′, y′ measured by the beam profiler 15. In practice, an alternative determination of whether the first and second beam profiles are sufficiently similar to be regarded as substantially equal may be performed using the inequality:
In which 0≤f≤0.5 is a fraction. For example, a value of f=0.1 corresponds to testing whether the difference between first and second beam profiles 10, 11 is less than or equal to 10% of the first beam profile 10. In one example, the first and second beam profiles 10, 11 may be considered to be substantially equal if the inequality of Equation (4) is satisfied for all xA′≤x′≤xB′ and all yA′≤y′≤yB′. Alternatively, the first and second beam profiles 10, 11 may be considered to be substantially equal if the inequality of Equation (4) is satisfied for a threshold percentage of the area measured by the beam profiler 15, for example, if the inequality of Equation (4) is satisfied for greater than or equal to 90%, greater than or equal to 75% or greater than or equal to 50% of the measured area.
The beam profiler 15 may be any suitable form of beam profiler such as, for example, a camera based beam profiler, a translating slit beam profiler, a translating step beam profiler and so forth. The relative sensitivity of the beam profiler 15 to different wavelengths need not be the same at the first and second wavelengths λ1, λ2, since any difference should be compensated for through the use of relative spatial intensities. Filters are not required in order to determine the beam profiles 10, 11, since the first and second emitters 2, 3 are independently illuminable.
A signal obtained using the second light emitter(s) may be subtracted from a signal obtained using the first emitter(s) in order to compensate for optical scattering due to defects or other inhomogeneities in a medium, or substrate which forms part of the sample 9. The subtraction is carried out by the controller 27.
Referring also to
Lateral flow test strips 18 (also known as “lateral flow immunoassays”) are a variety of biological testing kit. Lateral flow test strips 18 may be used to test a liquid sample, such as saliva, blood or urine, for the presence of an analyte. Examples of lateral flow devices include home pregnancy tests, home ovulation tests, tests for other hormones, tests for specific pathogens and tests for specific drugs.
In a typical lateral flow test strip 18, a liquid sample is introduced at one end of a porous strip 19, and the liquid sample is then drawn along the lateral flow test strip 18 by capillary action (or “wicking”). A portion of the lateral flow strip 18 is pre-treated with labelling particles 21 (
Referring also to
The fibres 22 scatter and/or absorb light across a broad range of wavelengths in an approximately similar way. For example, the proportion of light 5 from the first light emitter(s) 2 which is scattered by fibres 22 is approximately the same as the proportion of light 6 from the second light emitter(s) 3. However, the fibrous porous strip 19 is not uniform, and the density of fibres 22 may vary from point to point along the porous strip 19. As explained further hereinafter, such background variations of absorbance, which are due to the inhomogeneity of the porous strip 19, may limit the sensitivity of a measurement, i.e. the minimum detectable concentration of labelling particles 21.
Referring also to
The first and second wavelengths λ1, λ2 may lie in the range between 300 nm and 1500 nm inclusive. The first and second wavelengths λ1, λ2 may lie in the range between 400 nm and 800 nm inclusive.
Referring in particular to
Referring also to
The absorbance A1(x) at the first wavelength X, and the absorbance A2(x) at the second wavelength λ2 have substantially equal contributions from scattering and/or absorption by the fibres 22 of the porous strip 19. The background level of absorbance varies with position x along the porous strip 19 due to the inhomogeneity of fibre 22 density. Absorbance signals resulting from the labelling particles 21 cannot be reliably detected unless they are at least larger than the background variance which results from inhomogeneity of the porous strip 19. This restricts the lower limit of labelling particle concentration which can be detected using a lateral flow test strip 18. The same background variance also limits the resolution of a quantitative measurement of labelling particle 21 concentration/optical density.
However, since the fibres 22 scatter light at the first and second wavelengths λ1, λ2 in approximately the same way, the absorbance values A2(x) values at the second wavelength λ2 may be subtracted from the absorbance values A1(x) at the first wavelength λ1 to reduce or remove the effect of the variations in background absorbance which result from the inhomogeneous distribution of fibres 22 in the porous strip.
Although in practice some amount of background variance in absorbance will remain when the difference A1(x)-A2(x) is obtained, the relative size of the signal which is specific to the labelling particles 21 may be increased, in some cases substantially, with respect to background variations. In this way, the lower limit of labelling particle 21 concentrations/optical densities which may be detected may be reduced. Similarly, the resolution of a quantitative measurement of labelling particle 21 concentration/optical density may be increased.
Although the normalised spatial intensity profiles, i.e. first and second beam profiles 10, 11 generated by the first and second light emitter(s) are preferably substantially equal for the correction to be effective (as described hereinbefore), the absolute spatial intensity profiles (not shown) need not be equal.
When the absolute intensities of light 5, 6 from the first and second light emitters 2, 3 are not equal, the intensity ratio α of the first and second light emitters 2, 3 may be measured in the absence of a sample 9 and used to perform a weighted correction, i.e. A1(x)-αA2(x). Alternatively, the weighting factor α may account for differing sensitivity of the photodetector(s) 4 at the first and second wavelengths λ1, λ2.
Through alternately illuminating the first and second emitters 2, 3, the analytical test device 1 may include a relatively simple optical path 7 which does not require optical components such as beamsplitters, filters or monochromators to perform dual-wavelength measurements. Thus, the analytical test device 1 may be less bulky, simpler and less expensive to manufacture. Additionally, many optical components such as beamsplitters have wavelength dependent properties, which may restrict the choice of wavelengths λ1, λ2. By reducing the number of optical components in the optical path 7, or in some examples removing the need for intermediate optical components altogether, the wavelengths λ1, λ2 for a dual-wavelength measurement may be less constrained.
Referring also to
A sample 9 is placed so that a region of interest on the sample 9 coincides with the sample receiving portion 8 of the optical path 7 (step S1). When the analytical test device 1 is integrated in a self-contained assay including a lateral flow strip or a microfluidic device, this stage may be omitted. The first light emitter(s) 2 are turned on for a period of duration δt1, and the photodetector(s) 4 measure the light 5 transmitted through (or reflected from) the sample receiving portion 8 of the path (step S2). Optionally, the first light emitter(s) 2 may be switched off for a period of duration δt0, so that the photodetector(s) 4 may also measure fluorescence excited by the light 5 from the first light emitter(s) 2 (step S3).
The second light emitter(s) 3 are turned on for a period of duration δt2, and the photodetector(s) 4 measure the light 6 transmitted through (or reflected from) the sample receiving portion 8 of the path (step S4). Optionally, the second light emitter(s) 3 may be switched off for a period of δt0, so that the photodetector(s) 4 may also measure fluorescence excited by the light 6 from the second light emitter(s) 2 (step S5).
The absorbance values A2(x) determined using the second light emitter(s) 3 are subtracted to correct the absorbance values A1(x) determined using the first light emitter(s) 2 according to A1(x)-αA2(x), in which a is a weighting factor to account for differences in the absolute intensity of illumination between the first and second wavelengths λ1, λ2 and/or differing sensitivity of the photodetector(s) 4 at the first and second wavelengths λ1, λ2 (step S6).
Alternatively, for measurements in transmission, a simple calculation may be performed by dividing the transmission of the light 5 from the first emitter 2 by the transmission of the light 6 from the second emitter 3.
If further samples 9 are to be measured, then the next sample 9 may be placed (step S7). Alternatively, if there are additional regions of interest on the same sample 9, for example if the sample 9 is a lateral flow test strip 18 having more than one test region 20, the sample 9 may be repositioned with the next region of interest within the sample receiving portion 8.
The periods δt1 and δt2 may lie in a range between, for example, 10 ms and 500 ms inclusive.
The analytical test device 1 may be configured to use a range of emitter 2, 3 and photodetector 4 geometries.
Referring also to
For example, if a sample 9 in the form of a lateral flow test strip 18 is used, the lateral flow test strip 18 may be arranged with a testing region 20 positioned between the light emitter(s) 2, 3 and photodiode(s) 4. The sample receiving portion 8 of the path 7 corresponds to the thickness of the lateral flow test strip 18 which intersects the optical path 7.
Additional optical components may be included in the optical path 7. For example, the light from the light emitters 2, 3 into the optical path 7 and/or the light from the optical path 7 to the photodiode(s) 4 may be restricted by slits or other apertures. Optionally, a diffuser, one or more lenses and/or other optical components may also be included in the optical path 7.
Referring also to
Additional optical components may be included in the optical path 7. For example, the light from the light emitters 2, 3 into the optical path 7 and/or the light from the optical path 7 to the photodiode(s) 4 may be restricted by slits or other apertures. Optionally, a diffuser, one or more lenses and/or other optical components may also be included in the optical path 7.
Referring also to
An image sensor 24 may be used to image transmitted or reflected light. Additional optical components may be included in the optical path 7. For example, the light from the light emitters 2, 3 into the optical path 7 and/or the light from the optical path 7 to the photodiode(s) 4 may be restricted by slits or other apertures. Optionally, a diffuser, or more lenses and/or other optical components may also be included in the optical path 7.
Referring also to
The liquid transport path 41 may take the form of a porous medium, for example the porous strip 19 of a lateral flow test strip 18. The porous strip 19 may include nitrocellulose or other fibrous materials capable of transporting an aqueous liquid by capillary action. The porous strip 19 may be inherently capable of drawing liquid along the liquid transport path 41 by capillary action. Depending on the fibres used, surface treatments may be performed to permit, or enhance, the transport of liquid along the liquid transport path 41. When the liquid transport path 41 takes the form of a porous strip 19, dry and wet portions of the porous strip are separated by a flow front 45 which propagates along the liquid transport path 41. Even once the flow front 45 has reached the second end, 44, liquid may continue to flow along the liquid transport path 41 if the second end 44 is in contact with a reservoir or wicking pad 66 (
The liquid transport path 41 intersects the sample receiving portion 8 of the optical path 7 and the optical absorbance of the porous strip 19 in the sample receiving portion may be monitored as a function of time. Such measurements may sometimes be referred to as “dynamic” or “kinetic” measurements. For example, if a lateral flow test strip 18 is arranged with a test region 20 within the sample receiving portion 8, then the development of the concentration of labelling particles 21 may be tracked as a function of time by measuring the absorbance of the test region 20 at the first and second wavelengths λ1, λ2 as a function of time. If a lateral flow test strip 18 includes additional regions of interest, for example control regions 26 or further test regions 20, then the analytical test device 1 may be provided with additional pairs of emitters 2, 3 and photodetector(s) 4.
The liquid transport path 41 need not be a porous strip 19 of a lateral flow test strip 18. Alternatively, the liquid transport path 42 may take the form of one or more channels of a microfluidic device.
In this way, dynamic information about the development of an assay may be obtained. Dynamic information may be useful, for example, to check that an assay has behaved as expected or within acceptable bounds for a result to be considered reliable. The intervals δt1, δt2 and, if used, δt0, should be relatively short compared to the timescales on which an assay is developed.
There are several different ways to introduce light 5, 6 from the first and second emitters 2, 3 onto the optical path 7 so that the corresponding normalised spatial intensity profiles 10, 11 are substantially equal in the sample receiving portion 8 of the optical path 7.
For example, referring also to
Each pair of first and second emitters 2, 3 may be arranged such that the slit 46 perpendicularly bisects the pair of emitters 2, 3, when the arrangement is viewed along a direction perpendicular to the slit members 47 defining the slit 46. For example, if the slit members 47 define the slit in an x-y plane with reference to a set of Cartesian axes, then the slit 46 should perpendicularly bisect each pair of emitters 2, 3 when viewed along the z axis.
Optionally, a diffuser 48 may be arranged at a point between the slit 46 and the emitters 2, 3. One or more lenses (not shown) may also be included to collect and/or focus light 5, 6 from the light emitters 2, 3.
Referring also to
The slit 46 should be relatively narrow to provide fine spatial resolution and to ensure that the normalised intensity profiles 10, 11 are substantially equal across the width t of the slit 41. The slit may have a width between 100 μm and 1 mm inclusive. Preferably, the slit has a width between 300 μm and 500 μm inclusive.
Coupling light 5, 6 from the first and second emitters 2, 3 into the optical path 7 through a slit 46 may be used for measurements in transmission or reflection.
Referring also to
Where samples 9 include multiple regions of interest, the sample 9 may be moved in front of the light emitting diode array 60 to scan the sample 9. Alternatively, the light emitting diode array 60 and corresponding photodetector 4 may be moved to scan the sample 9. Alternatively, a light emitting diode array 60 and one or more photodetectors 4 may be arranged corresponding to each region of interest of the sample 9 so that each region may be measured concurrently.
A light emitting diode array 60 may be used for measurements in reflection or transmission.
Referring also to
For example, when a sample is in the form of a lateral flow test strip 18 which extends longitudinally in a first direction x, transversely in a second direction y and has a thickness in a third direction z, a light emitting diode array 60 may extend for substantially the width of the lateral flow test strip 18 in the transverse y direction and for a relatively shorter distance in the longitudinal x direction. If the lateral flow test strip 18 is mounted in a sample mounting stage 29 including a window for transmission measurements, then the light emitting diode array 60 may extend for substantially the width of the lateral flow test strip 18. Alternatively, the lateral flow test strip 18 may be mounted fixedly with respect to the analytical test device 1, and a pair of an LED array 60 and a photodetector 4 may be provided corresponding to each test region 20 and/or control region 26
Referring also to
Optionally, a diffuser 48 may be arranged between the light emitting diode array 60 and the sample receiving portion 8 of the optical path 7. One or more lenses (not shown) may also be included to collect and/or focus light 5, 6 from the light emitting diode array 60.
Referring also to
The second light emitter 3 may be manufactured using materials which are transparent, or substantially transparent at the first wavelength λ1. For example, the absorbance 61 of the second light emitter 3 at the first wavelength λ1 may be relatively low. Absorbance may be considered to be relatively low if it is less than 50%, less than 25%, less than 15%, less than 10% or less than 5% (i.e. transmittance of more than 50%, more than 75%, more than 85%, more than 90% or more than 95%). In this way, the light emitting diode providing the second light emitter 3 may be deposited on top of the light emitting diode providing the first light emitter 2, and the first emitter 2 may emit light 5 onto the optical path 7 through the second light emitter 2.
This arrangement may be particularly compact for transmission measurements, but may also be used for reflectance measurements.
Referring also to
This arrangement may be particularly compact for transmission measurements, but may also be used for reflectance measurements.
Referring also to
The lateral flow testing device 62 includes a porous strip 19 divided into a sample receiving portion 63, a conjugate portion 64, a test portion 65 and a wick portion 66. The porous strip 19 is received into a base 67. A lid 68 is attached to the base 67 to secure the porous strip 19 and cover parts of the porous strip 19 which do not require exposure. The lid 68 includes a sample receiving window 69 which exposes part of the sample receiving portion 63 to define the liquid sample receiving region 42. The lid and base 67, 68 are made from a polymer such as, for example, polycarbonate, polystyrene, polypropylene or similar materials.
The base 57 includes a recess 70 into which a pair of light emitting diode arrays 60 are received. Each light emitting diode array 60 may be configured as described hereinbefore. The lid 68 includes a recess 71 into which a pair of photodetectors 4 are received. The photodetectors 4 may take the form of photodiodes. One pair of a light emitting diode array 60 and a photodiode 4 are arranged on opposite sides of a testing region 20 of the porous strip 19. The second pair of a light emitting diode array 60 and a photodiode are arranged on opposite sides of a control region 26 of the porous strip 19. Slit members 47 separate the light emitting diode arrays 60 from the porous strip 19 to define narrow slits 46 with widths in the range between 300 μm to 500 μm inclusive. The slit members 47 define slits 46 which extend transversely across the width of the porous strip 19. For example, if the porous strip 19 extends in a first direction x and has a thickness in a third direction z, then the slits 46 extend in a second direction y. Further slit members 47 define slits 46 which separate the photodiodes 4 from the porous strip 19. The slits 46 may be covered by a thin layer of transparent material to prevent moisture entering into the recesses 70, 71. Material may be considered to be transparent to a particular wavelength λ if it transmits more than 75%, more than 85%, more than 90% or more than 95% of the light at that wavelength λ. A diffuser 48 may optionally be included between each light emitting diode array 60 and the corresponding slit 46.
A liquid sample 72 is introduced to the sample receiving portion 63 through the sample receiving window 69 using, for example, a dropper 73 or similar implement. The liquid sample 72 is transported along the liquid transport path 41 towards the second end 44 by a capillary, or wicking, action of the porosity of the porous strip 63, 64, 65, 66. The sample receiving portion 63 of the porous strip 18 is typically made from fibrous cellulose filter material.
The conjugate portion 64 has been pre-treated with at least one particulate labelled binding reagent for binding an analyte which is being tested for, to form a labelled-particle-analyte complex (not shown). A particulate labelled binding reagent is typically, for example, a nanometre- or micrometre-sized label particle 21 which has been sensitised to specifically bind to the analyte. The particles provide a detectable response, which is usually a visible optical response such as a particular colour, but may take other forms. For example, particles may be used which are visible in infrared, which fluoresce under ultraviolet light, or which are magnetic. Typically, the conjugate portion 64 will be treated with one type of particulate labelled binding reagent to test for the presence of one type of analyte in the liquid sample 72. However, lateral flow devices 62 may be produced which test for two or more analytes using two or more particulate labelled binding reagents concurrently. The conjugate portion 64 is typically made from fibrous glass, cellulose or surface modified polyester materials.
As the flow front 45 moves into the test portion 65, labelled-particle-analyte complexes and unbound label particles are carried along towards the second end 44. The test portion 65 includes one or more testing regions 20 and control regions 26 which are monitored by a corresponding light emitting diode array 60 and photodiode 4 pair. A testing region 20 is pre-treated with an immobilised binding reagent which specifically binds the label particle-target complex and which does not bind the unreacted label particles. As the labelled-particle-analyte complexes are bound in the testing region 20, the concentration of the label particles 21 in the testing region 20 increases. The concentration increase may be monitored by measuring the absorbance of the testing region 20 using the corresponding light emitting diode array 60 and photodiode 4. The absorbance of the testing region 20 may be measured once a set duration has expired since the liquid sample 72 was added. Alternatively, the absorbance of the testing region 20 may be measured continuously or at regular intervals as the lateral flow strip is developed.
To provide distinction between a negative test and a test which has simply not functioned correctly, a control region 26 is often provided between the testing region 20 and the second end 44. The control region 26 is pre-treated with a second immobilised binding reagent which specifically binds unbound label particles and which does not bind the labelled-particle-analyte complexes. In this way, if the lateral flow testing device 62 has functioned correctly and the liquid sample 72 has passed through the conjugate portion 64 and test portion 65, the control region 26 will exhibit an increase in absorbance. The absorbance of the control region 26 may be measured by the second pair of a light emitting diode array 60 and a photodiode 4 in the same way as for the testing region 20. The test portion 65 is typically made from fibrous nitrocellulose, polyvinylidene fluoride, polyethersulfone (PES) or charge modified nylon materials. All of these materials are fibrous, and as such the sensitivity of the absorbance measurements may be improved by subtracting the measurements obtained using the second wavelength λ2 to correct for inhomogeneity of the porous strip 19 material.
The wick portion 66 provided proximate to the second end 44 soaks up liquid sample 72 which has passed through the test portion 65 and helps to maintain through-flow of the liquid sample 72. The wick portion 66 is typically made from fibrous cellulose filter material.
Although not shown in
The preceding discussion may be better understood with reference to illustrative experimental data. The analytical testing device 1 described herein is not limited to the specific conditions and samples used to obtain illustrative experimental data.
Referring to
Referring also to
The measurements were obtained by moving the blank nitrocellulose porous strip 19 through a gap between the photodiode 4 and the light emitting diodes 2, 3 and recording the output signal of the photodiode 4 as a function of the distance. The blank nitrocellulose porous strip 19 was moved using a stepper motor.
It may be observed that the inhomogeneities in the transmittance of the blank nitrocellulose strip 19 are reproducible over a wide wavelength range, since the measurements at the green and near-infrared wavelengths are substantially similar. Subtracting measurements made at a second wavelength may substantially correct for the background inhomogeneity of the porous strip 19. For example, for absorbance measurements A1(x) obtained with the green light emitting diode alone, the range of ΔOD was more than 00008, whereas the difference A1(x)-A2(x) (solid line) has a range of ΔOD of ≈0.001. This represents a substantial decrease in the background signal, and consequently lower optical densities of labelling particles 21 may be resolvable.
The gold nanoparticles used for the test lines 75, which are commonly used as labelling particles 21 in lateral flow test strips 18, are known to absorb strongly in the green but only relatively weakly in the infrared. Therefore, one example of an analytical test device as described herein may compare the difference in signals obtained using green and near-infrared organic light emitting diodes. The same approach may also be used with an imaging camera approach.
Referring also to
Referring also to
Referring also to
Referring also to
It may be observed that measurements obtained using the example analytical test device 1 have substantially reduced background noise compared to a single wavelength organic light emitting diode/organic photodiode pair. Although the test region 20 and control region 26 are well resolved in this illustrative data, the reduced background noise may allow the analytical test device 1 to detect lower concentrations than the single wavelength (green only) device.
Referring also to
Data corresponding to three experimental measurements are shown (triangles, solid line is a fitting line). The leftmost, or least equal point corresponds to ΔOD measured with no correction using the second emitter, i.e. the NIR wavelengths. The rightmost, or most equal point corresponds to ΔOD measured using an integrating sphere (not shown). The third (middle) experimental point corresponds to ΔOD measured using a simple (side-by-side) pair of inorganic LEDs emitting green and NIR light respectively. The values of ΔOD measured using a pair of light emitting diodes is three times higher than ΔOD measured using the integrating sphere (not shown), which may be attributable to a degree of difference between the first and second beam profiles 10, 11. However, the measurements using the pair of light emitting diodes are also ˜4.5 times lower than ΔOD measured with only the green wavelengths.
Data corresponding to the results of modelling of the ΔOD achievable for different beam profiles of first (green) and second (NIR) emitters 2, 3 are also shown (open circles, dashed line is a fitting line). Modelling was performed by convolving experimentally measured ΔOD data corresponding to a blank porous strip 19 with different beam profiles A, B, C and D shown schematically in
The measured data corresponding to the integrating sphere (not shown) is larger than the modelled value of zero. This may be attributable to the beam profiles not being perfectly identical, or may possibly be attributable to deviations from the simple nitrocellulose thickness variation model which is employed for correction by subtracting the absorbance values measured using the second colour. The value of ΔOD ˜5e-4 for the integrating sphere (not shown) measurements is nonetheless substantially reduced in comparison to the single wavelength value ΔOD >0.06.
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 analytical test devices 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.
Although applications have been predominantly described in relation to absorbance measurements with LFDs, fluorescence measurements may also be made using the same methods described hereinbefore and a measurement process which is similar to the hereinbefore described process of obtaining and correcting absorbance measurements (see
For example, as described hereinbefore, the first light emitter(s) 2 may be switched off for a period of duration δt0, so that the photodetector(s) 4 may measure fluorescence excited by the light 5 from the first light emitter(s) 2 (step S3). In a similar way, the so second light emitter(s) 3 may be switched off for a period of δt0, so that the photodetector(s) 4 may measure fluorescence excited by the light 6 from the second light emitter(s) 2 (step S5). This approach can be used to excite a first fluorescent marker using light 5 of the first wavelength λ1 and to excite a second fluorescent marker using light 6 of the second wavelength λ2.
A fraction of light 5 at the first wavelength A, will be scattered by the fibres 22, and thus not available to excite fluorescence. Similarly, a fraction of light 6 at the second wavelength λ2 will be scattered by the fibres 22, and thus not available to excite fluorescence. However, as described hereinbefore, the fibres 22 scatter light at the first and second wavelengths λ1, λ2 in approximately the same way. Thus, the impact of the inhomogeneity of the porous strip 19 on fluorescence measurements excited at the first and second wavelengths λ1, λ2 can be substantially the same. This can improve the accuracy of assays which are based on the relative concentrations of two (or more) fluorescent markers.
Alternatively, the first emitter 2 may be used to measure fluorescence excited by the first wavelength λ1 and the second emitter 3 may be used to perform a correction.
Referring again to
Although examples have been described in relation to lateral flow test strips 18, the present methods and apparatus can also be used with other types of sample 9 with minimal modifications.
For example, an analytical test device r may include an optical path 7 which has a sample receiving portion 8 adapted to receive a microfluidic channel or channels (not shown) perpendicularly to the optical path 7. The microfluidic channel(s) (not shown) can be in the form of one or more lengths of tubing or one or more channels machined into polymeric material. The microfluidic channel(s) (not shown) may be dimensioned to enable capillary transport of a liquid sample. Measurements at the second wavelength λ2 can be used to compensate for scattering or absorption from defects of or contamination on the walls of the microfluidic channel(s) (not shown).
Expansion to More than One Analyte
For some tests, it may be desirable to detect and quantify the concentrations of two or more analytes in the same sample concurrently. Additionally or alternatively, many samples which may contain one or more analytes of interest may be coloured, for example blood. Other samples may display a range of colours depending on a concentration of, for example, urine or other biologically derived substances or byproducts.
The methods and apparatus described hereinbefore can be adapted to detect two or more analytes in a single sample, whether the sample is coloured or substantially clear.
In general, concentrations of N−1 different analytes may be determined whilst correcting for inhomogeneity of a porous strip 19, or other such source of background scattering, by sequentially illuminating the sample receiving portion 8 using N different wavelengths. Each of the N wavelengths may be provided by a corresponding set of one or more light emitters. The controller 27 may illuminate each of the N sets of one or more light emitters according to a sequence, such that only one set of emitters is emitting light at any given time. Some of the N−1 analytes may not be of direct interest, for example, some of the N−1 analytes may be substances or compositions which provide the colouration of a sample. However, accounting for analytes providing coloration of a sample can allow more accurate detection and quantification of analytes of interest contained in the sample.
Referring also to
Referring in particular to
Referring in particular to
The transmission geometry shown in
A sample may in general include N−1 analytes. The method of extracting concentrations for the N−1 analytes includes sequentially illumination using light emitted from N sets of one or more light emitters. Each set of light emitters emits light centred around a different wavelength. The number N−1 of analytes is one less than the number N of sets of emitters to allow correction for scattering from the background inhomogeneities of a porous strip 19, microfluidic channel(s), or any similar source of background scattering. Some of the analytes may be substances or compositions which give rise to the coloration of a sample. Quantifying substances or compositions which give rise to sample colouration may not be of direct interest, however, it can allow more sensitive detection and/or more accurate quantification of one or more analytes of interest contained within a coloured sample such as urine, blood, wine, cooking oils and so forth.
For light of the nth wavelength λn out of N−1 wavelengths, the absorbance through the sample receiving portion 8 is denoted A(λn). In general, the absorbance A(λn) corresponds to a range of wavelengths which spans the nth wavelength λn. For example, A(λn) may be calculated based on the integral of intensity across a wavelength range.
The total absorbance A(λn) may be viewed as the sum:
in which s(λn) is the absorbance at the nth wavelength λn due to scattering from background inhomogeneity of the porous strip 19 or other source of background scattering, ci is the concentration of the ith analyte out of N−1 analytes and εi(λn) is a coefficient relating the concentration ci to the absorbance of the ith analyte out of N−1 analytes at the nth wavelength λn. The concentrations ci are expressed in units of absorbance (optical density) corresponding to a reference wavelength, for example, the 1st wavelength λ1. Thus, the coefficients εi(λn) are each a ratio of the absorbance of the ith analyte between the 1st and nth wavelengths λ1, λn.
Measurement of absorbance may be direct, for example, in a transmission geometry by obtaining measurements with and without a sample 9 present within the sample receiving portion 8.
Alternatively, when the sample 9 is a lateral flow test strip 18, the absorbance values A(λn) may be obtained from an image or scan covering a testing region 20 and surrounding regions of untreated porous strip 19. Alternatively, the absorbance values A(λn) may be obtained by reference to a measurement of transmission/reflection obtained before a liquid sample is introduced to the lateral flow test strip.
Referring also to
Referring in particular to
Referring in particular to
Referring in particular to
Referring in particular to
Referring in particular to
As a first step in extracting green absorbance values, a slowly varying background profile 115, plotted against the primary Y-axis (range 0 to 4500), is fitted to the simulated green OPD signal 112b, plotted against the primary Y-axis (range 0 to 4500). The background profile 115 represents an approximation to the average intensity, T0, transmitted by the nitrocellulose fibres 22 of the porous strip 19. The simulated green OPD signal 112b represents the transmitted intensity, T, through the porous strip 19 and the gold nanoparticles. A normalised green transmission profile 116 is calculated as T/T0, plotted against the secondary Y-axis (range 0 to 1.2). It may be observed that the normalised green transmission profile 116 retains fluctuations resulting from the point-to-point fluctuations in the nitrocellulose fibre 22 concentration profile 110.
Referring in particular to
Referring in particular to
Referring in particular to
Two distinct correlations may be observed in
A second correlation is most easily seen in
The method of obtaining absorbance values described with reference to the simulated OPD signals 112, 113, 114 is expected to be equally applicable to measured data, whether obtained in transmission or reflection geometries.
Other methods of obtaining absorbance values may be used. Absorbance values measured according to any suitable method may be analysed in accordance with equations (6) to (13), (10b) to (13b) and/or equation (10c) as set out hereinafter.
In the general case, for light of the nth wavelength λn out of N−1 wavelengths, the absorbance (however measured) through the sample receiving portion 8 is denoted A(λn). If the absorbance A(λn) is measured at each wavelength λn, then an absorbance column vector may be defined as:
For example, the absorbance values A(λn) may be absorbance fingerprint values obtained as described hereinbefore with reference to
Similarly, a concentration column vector may be defined as:
in which the concentration cs corresponding to the background absorbance s(λn) is a dummy concentration which is set to the background absorbance at the reference wavelength, for example s(λ1) at the 1st wavelength λ1. The use of the dummy concentration in equivalent units to the analyte concentrations ci maintains appropriate scaling of measured absorbance values throughout the calculations described hereinafter. In practice, as explained hereinafter, calibration of the method typically includes obtaining measurements of the background scattering without any analytes, so obtaining a suitable value for the dummy concentration cs is not problematic. The absorbance vector A may be expressed in terms of the coefficients εi(λn), background absorbance s(λn) and concentration vector c using a matrix equation:
in which M is a square matrix having coefficients Mij=εj(λi) for 1≤j≤N−1 and MiN=s(λi). By inverting the matrix M, unknown concentrations ci of analytes may be determined from the measured absorbance values A(λn) at each wavelength λn:
c=M
−1
A (9)
In order to apply Equation (9), it is necessary to know the coefficients Mij of the matrix M, so that the inverse M−1 may be calculated. When evaluating Equation (9), a value calculated corresponding to the background scattering “concentration” would ideally be equal to the dummy concentration cs. In practical circumstances, the value calculated corresponding to the background scattering “concentration” may deviate from the dummy concentration cs. The size of the deviation may provide an indication of variations between different porous strips 19, microfluidic channels, and so forth. A large deviation may provide an indication of possible problems with a particular sample or with the calibration of the matrix M coefficients Mij.
The coefficients Mij of the matrix M may be determined in advance from experimental measurements using samples with known concentrations ci of each analyte. A measured set of absorbance values A1(λn) with a first calibration sample represented by the reference absorbance vector A1 and the corresponding concentrations ci−1 by the calibration concentration vector c1. In general, for N wavelengths λ1, . . . , λN, a number N of calibration samples and measurements are required. A fingerprint matrix F is defined using the set of reference absorbance vectors A1, . . . , AN by setting the coefficients of each reference absorbance vector A1, . . . , AN as the coefficients for a corresponding column of the fingerprint matrix F:
The entries of the fingerprint matrix F may constitute absorbance fingerprint values estimated as described in relation to
and the fingerprint matrix F and calibration matrix C are related according to:
F=MC (12)
The coefficients Mij of the matrix M can then be calculated as M=FC−1, and the coefficients of the inverse matrix M−1 can be calculated as M−1=CF−1. Thus, a set of unknown concentrations ci represented by a concentration vector c may be recovered using CF−1 as a deconvolution matrix (also referred to as a de-mixing matrix) for the measured absorbance values A(λn) represented by an absorbance vector A according to:
c=CF
−1
A (13)
In this way, a set of unknown concentrations c1, . . . , cN-1 of N−1 analytes may be reconstructed from measurements of the absorbance values A(λ1), . . . , A(λN) at N wavelengths, λ1, . . . , λN emitted from the corresponding N sets of light emitters. The absorbance values A(λ1), . . . , A(λN) may be in the form of absorbance fingerprint values obtained as described in relation to
The actual physical concentration or number density of each analyte, for example in units of number.cm−3, can be estimated from the reconstructed concentrations c1, . . . , cN-1 (i.e. absorbance values at the reference wavelength) using the Beer-Lambert law with the path length through the sample receiving portion 8 and an attenuation coefficient for the ith analyte at the reference wavelength (for example the 1st wavelength λ1). If the attenuation coefficient for the ith analyte is not known at the reference wavelength, then the coefficients Mij=εj(λi) (calculated by inverting the deconvolution (de-mixing) matrix to obtain M=FC−1) may be used to convert the concentration (absorbance) ci at the reference wavelength to an absorbance at a wavelength for which the attenuation coefficient is known.
Equivalently, since AT=cTMT, an alternative fingerprint matrix G may be defined by setting the coefficients of each reference absorbance vector A1, . . . , AN as the coefficients for a corresponding row of the alternative fingerprint matrix G:
and the corresponding calibration concentration vectors c1, . . . , cN may be set as the rows of an alternative calibration matrix D:
and the alternative fingerprint matrix G and alternative calibration matrix D are related according to:
G=DM
T (12b)
Thus, a set of unknown concentrations ci represented by a concentration vector c may equivalently be recovered using G−1D as a deconvolution matrix for the measured absorbances A(λn) represented by an absorbance vector AT according to:
c=A
T
G
−1
D (13b)
It is preferable for each analyte to have an absorbance peak which corresponds to one of the illumination wavelengths λ1, . . . , λN. It is preferred for the absorbance peaks corresponding to the N−1 types of analyte to avoid substantially overlapping. If the absorbance spectra of analytes are too similar, this may lead to errors in determining the analyte concentrations ci. In practice, the number of analytes may be limited by the distinguishability of spectra.
In some examples, it may be convenient to normalise absorbance values with respect to a single reference calibration value, for example, A1(λ1). For example, with normalisation relative to A1(λ1), a normalised fingerprint matrix Fn may be expressed as:
Each of Equations 6 to 13 and Equations 10b to 13b may be normalised in this manner, to allow absorbance and concentration values to be expressed as fractions with respect to a reference calibration value, for example A1(λ1).
The calibration is simplified in the case that pure (or substantially pure) samples of the N−1 different analytes having known concentration ci are available for testing in reference conditions, for example, supported on a porous strip 19. One of the calibration samples should correspond to only the background scattering s(λn), e.g. the porous strip 19. In this case, determining the calibration matrix is simplified, since the determination of the concentration ci for each analyte at the reference wavelength can be simplified. For example, if the Nth calibration sample includes only the background scattering, then a calibration concentration ci0 of the ith calibration sample (1≤i≤N−1), which includes the pure (or substantially pure) ith analyte, using the 1st wavelength λ1 as the reference wavelength, may be approximated as:
c
i
0
A
i(λ1)−AN(λ1) (14)
In which Ai(λ1) is the measured absorbance of the pure or substantially pure sample of the ith analyte at the 1st wavelength. The calibration matrix C may be written as:
In which the dummy concentration cs=AN(λ1). In this special case, the calculation of the deconvolution matrix CF−1 may be simplified.
The calibration matrix C and the calculation of the deconvolution matrix CF−1 may be simplified further if the absorbance of pure (or substantially pure) samples of the different analytes may be tested under conditions in which the background scattering is very low or negligible. Under these optimum conditions, the calibration matrix is diagonal, and reference concentration values may be directly set to measured absorbance values at the reference wavelength:
In which the dummy concentration cs=AN(λ1). Each of Equations 14 to 16 may be normalised to a reference calibration absorbance value, for example A1(λ1), as explained hereinbefore.
The method of extracting optical densities for N−1 analytes using N illumination wavelengths may be applied to verify the previously applied result for a single analyte with sequential illumination at first and second wavelengths λ1, λ2, i.e. A1(x)-A2(x).
Simulations were conducted using the model described hereinbefore with reference to
The deconvolution (de-mixing) matrix CF−1 of Equation 14 may be calculated by inverting the fingerprint matrix F:
and substituting the deconvolution (de-mixing) matrix CF−1 into Equation 14 yields:
Thus, the concentration cAu of gold nanoparticles, in this example expressed in terms of absorbance in OD, is given as cAu=1.02(Agreen−ANIR), which is essentially the same result applied hereinbefore.
Application to One Analyte and Background Scattering with a Coloured Dye
Simulations were also conducted using the model described hereinbefore with reference to
Referring also to
The total, summed absorbance 123 is represented by a solid line. The estimated gold nanoparticle concentration 124 is represented by a dotted line. The estimated background scattering from the nitrocellulose strip 125 is represented by the dashed line.
In particular, the presence of the blue dye leads to errors in the estimated gold nanoparticle concentration 124. In particular, the baseline absorbance around the location of the gold nanoparticles is distorted by absorbance of the blue dye. The problem is that there are three unknowns in the concentration values, namely, the gold nanoparticle concentration cAu, the blue dye concentration cdye and the background scattering cNC from the nitrocellulose strip. Using green and NIR OLEDs, there are only two measurements. The solution is to increase the number of wavelength ranges to three.
The deconvolution (de-mixing) matrix method may be applied if all three of the simulated OPD signals 112, 113, 114 are utilised. A first simulated calibration sample, corresponding to gold nanoparticles having an optical density of OD=1, may be represented in the method by the concentration vector cAuT=(1, 0, 0) (cAu, cdye, cNC) and the corresponding absorbance vector is AAuT=(1, 0.17, 0.02) (green, red, NIR). The relevant absorbance values were obtained as absorbance fingerprint values according to a method analogous to that described hereinbefore with reference to
The deconvolution (de-mixing) matrix CF−1 of Equation 14 may be calculated by inverting the fingerprint matrix F:
and substituting the deconvolution (de-mixing) matrix CF−1 into Equation 14 yields:
Thus, the concentration cAu of gold nanoparticles, in this example expressed in terms of absorbance in OD, is given as cAu=1.025Agreen−0.028Ared−0.997ANIR).
Referring also to
It can be seen that applying the method of sequential measurements at three different wavelengths (green, red and NIR) is expected to allow for clear separation of the absorbance due to the gold nanoparticles, blue dye and the nitrocellulose strip. In particular, the estimated gold nanoparticle concentration 124 and the estimated concentration of the blue dye 126 are expected to be separable.
The hereinbefore described deconvolution (de-mixing) method may be carried out by the controller 27 of the analytical test device 1.
LED arrays 60 have been described in which, for example, the first and second light emitters 2, 3 are stacked on top of each other (see
For example, referring also to
In an alternative arrangement (not shown) of interdigitated first and second light emitters 2, 3, protrusions 127, 128 may extend from two sides of corresponding backbone segments 129, 130. Protrusions 127, 128 extending from opposite sides of a backbone segment 129, 130 may be arranged opposite one another, or not. Protrusions 127, 128 extending from one side of a backbone segment 129, 130 do not need to extend parallel to protrusions 127, 128 extending from the opposite side of the same backbone segment 129, 130.
A two-colour interdigitated LED array may be particularly compact for transmission measurements, but may also be used for reflectance measurements.
LED arrays 60 have been described which include pixels 99 having subpixels in the form of first, second and third light emitters 2, 398 (
Referring also to
A first light emitter 2 includes a number of protrusions 127 arranged parallel to one another, and interdigitated with a number of first protrusions 128a of a second light emitter 3. The protrusions 127 of the first light emitter 2 are joined to form a single light emitter 2 by a backbone segment 129. Similarly, the first protrusions 128a of the second light emitter 3 are joined to form a single light emitter 3 by a backbone segment 130. Unlike the first light emitter 3, the light second emitter 3 also includes second protrusions 128b which extend from the opposite edge of the backbone segment 130 to the first protrusions 128a, and which are interdigitated with a number of protrusions 131 of a third light emitter 98. The protrusions 131 of the third light emitter 98 are joined to form a single light emitter 98 by a backbone segment 132. The width of the protrusions 128a, 182b of the second light emitter 3, may be relatively smaller than the protrusions 127, 131 of the first and third light emitters 2, 98, in order to maintain comparable emissive areas between the first, second and third light emitters 2, 3, 98. For example, if the protrusions 127, 128, 131 extend from the respective backbone segments 129, 130, 132 in a first direction x, then the width of the protrusions 128a, 182b of the second light emitter 3 in a second direction y may be less than the corresponding width of the protrusions 127, 131 of the first and third light emitters 2, 98 in the second direction y.
The second light emitter 3 need not be placed between the first and third light emitters 2, 98. Alternatively, either the first light emitter 2 or the third light emitter 98 may be arranged to provide the central element of a three colour interdigitated LED array 60. An LED array 60 may include one or more triplets of such interdigitated first, second and third light emitters 2, 3. The numbers of protrusions 127, 128, 131 is not limited. A three-colour interdigitated LED array may be particularly compact for transmission measurements, but may also be used for reflectance measurements.
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.
Number | Date | Country | Kind |
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1616301.6 | Sep 2016 | GB | national |
1705161.6 | Mar 2017 | GB | national |
Filing Document | Filing Date | Country | Kind |
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PCT/GB2017/052859 | 9/25/2017 | WO | 00 |