Lateral flow device, assay device and kit and method for analyzing a fluid sample

Abstract

The invention concerns a lateral flow device, system, kit and method for analyzing a fluid sample. The device comprises a support structure, a flow channel formed in the support structure, and an injection zone in fluidic connection with the flow channel for introducing the fluid sample into the flow channel. According to the invention the flow channel comprises at least two indicator zones at least one of which is capable of producing an optically detectable response signal when interacting with the fluid sample, and the indicator zones are arranged at least partly adjacent to each other on different sections of the flow channel. The invention allows for low-cost imaging devices, such as cameras of mobile phones to be used for making challenging lateral flow diagnostics on a variety of fields of technology.

Claims

1. A lateral flow device for an assay device for analyzing a fluid sample, the lateral flow device comprising: a support structure, a flow channel formed into the support structure, wherein the flow channel comprises a first indicator zone capable of facilitating an optically detectable response signal of the fluid sample interacting with an indicator substance, an injection zone in fluidic connection with the flow channel for introducing the fluid sample into the flow channel, and a second indicator zone capable of facilitating an optically detectable response signal of the fluid sample interacting with an indicator substance and arranged: parallel to the first indicator zone, wherein the first and second indicator zones are located side-by-side in a direction transverse to the direction of fluid flow in the flow channel, or to another flow channel formed into the support structure, wherein; the first and second indicator zones are arranged at least partly adjacent to each other so as to fit into an imaging area, the indicator zones fit into a circular imaging area having a diameter of 5 mm in a plane of the support structure, the injection zone comprises an indicator substance capable of providing an optical response signal upon interaction with a matrix of the fluid sample, at least one of the indicator zones comprises test substance capable of providing an optical response signal upon interaction with an analyte of the fluid sample, the first indicator zone is a test or reference zone and the second indicator zone is a reference or test zone, respectively, and wherein at least one of the indicator zones comprises a substance capable of providing a fluorescent optical response signal upon interaction with the fluid sample.

2. The lateral flow device according to claim 1, wherein the first and second indicator zones are arranged to the same flow channel and at least partly adjacent to each other at different portions of the flow channel.

3. The lateral flow device according to claim 1, wherein the flow channel is hollow.

4. The lateral flow device according to claim 3, wherein the hollow flow channel is free of a lateral flow matrix and dimensioned to provide a capillary flow of the fluid sample.

5. The lateral flow device according to claim 2, wherein the flow channel comprises a turning portion, whereby said portions are defined by different sides of the turning portion.

6. The lateral flow device according to claim 1, wherein the flow channel comprises a branching portion, whereby said portions are defined by different branches of the flow channel.

7. The lateral flow device according to claim 1, wherein the shortest distance between the indicator zones is less than 10 times the width of the flow channel.

8. The lateral flow device according to claim 1, wherein the lateral flow device contains at least two indicator zones comprising different test substances capable of providing optical response signals upon interaction with different analytes of the fluid sample.

9. The lateral flow device according to claim 8, wherein at least one of the indicator zones comprises a substance capable of providing a fluorescent optical response signal upon interaction with the fluid sample.

10. The lateral flow device according to claim 1, wherein the support structure is transparent on at least one side, at least in the region of the indicator zones, so as to allow optical imaging thereof during flow of the fluid sample.

11. The lateral flow device according to claim 1, wherein the support structure comprises a planar plate comprising a uniform bottom section, at least a partly transparent top section comprising said injection zone, and an intermediate section between the bottom and top section, the intermediate section being patterned to define the flow channel in the lateral dimension of the plate.

12. The lateral flow device according to claim 1, wherein the location of the indicator zones is marked thereon using visually detectable markers.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1A shows a lateral top view of the present device according to one embodiment of the invention.

(2) FIG. 1B shows a cross-sectional of an exemplary structure of the present device according to one embodiment.

(3) FIG. 1C illustrates a lateral flow plate 10 with a branched flow channel 13 according to an alternative embodiment.

(4) FIG. 2A illustrates a measurement setup using a mobile phone, intermediate lens and a lateral flow device of the present kind.

(5) FIG. 2B shows as a cross section an exemplary optical setup according to one embodiment of the invention.

(6) FIG. 2C shows a docking station 34 comprising on its upper surface a first docking zone for a mobile phone 32 and a second docking zone for a lateral flow plate 30.

(7) FIG. 3A shows a photograph of a lateral flow chip manufactured.

(8) FIG. 3B shows a microscopic fluorescence image of indicator zones of the chip according to FIG. 3A, obtained using a camera of a mobile phone, microscopic adapter lens and bandpass filter.

(9) FIGS. 4A and 4B show measured fluorescence THC assay response signals using a commercial fluorometer and a mobile phone-aided lateral flow system according to the present invention, respectively.

(10) FIGS. 4C and 4D show fluorescent partial images of the THC immunoassay chip according to FIG. 4A with THC concentrations of 0 and 250 ng/ml, respectively.

(11) FIG. 5 shows a graph of absorption and emission spectra of a fluorophore and filter spectrum of a bandpass filter used in the Example.

DETAILED DESCRIPTION OF EMBODIMENTS

(12) Lateral Flow Device

(13) Exemplary embodiments of the lateral flow device according to the invention are illustrated in FIGS. 1A-1C and described below. For simplicity, the illustrated embodiments contain only two indicator zones, i.e. a reference zone and an actual test zone, although configurations with more indicator zones are possible too.

(14) FIG. 1A illustrates a lateral flow plate 10 with a meandering flow channel according to an embodiment. The plate 10 comprises a support structure 11 defining a flow channel 13. At one end of the flow channel 13, there is an injection zone 12 for providing the sample to the flow channel 13. At the other end, there is a waste reservoir 18 (suction pad), to which the portion of the sample, which has passed the whole channel 13, is collected. The channel 13 itself comprises a first portion 14A, a second portion 14B and a U-shaped turning portion 15 interconnecting the first and second portions 14A, 14B. The first and second portions 14A, 14B are arranged parallel to each other. The first portion 14A comprises a test zone 16A and the second portion 14B a reference zone 16B (or vice versa). The test and reference zones 16A, 16B are indicator zones in the flow channel 13 and may herein be referred as the first indicator zone 16A and the second indicator zone 16B. The test and reference zones 16A, 16B are aligned with each other side by side so that they fit into an imaging area 19, which is illustrated herein with a dashed line. Naturally, the imaging area 19 may have a different shape, such as quadrilateral, oval or trilateral, for example. The sample flow direction at the test and reference zones 16A, 16B are opposite.

(15) FIG. 3A shows a real photograph of a lateral flow chip with a channel of the type described above. FIG. 3B shows the imaging area of the chip of FIG. 3A with a measurable difference in the optical response signals of the indicator zones 56A and 56B.

(16) FIG. 1B shows an exemplary structure of the support structure of the device. The structure comprises a bottom section 11A, a top section 11C (lid) and an intermediate section 11B between them. The intermediate section 11B defines the lateral shape of the flow channel 14. Chemical substance(s) of the test or reference zone 16 is/are arranged in the flow channel 13 so that the fluid becomes into contact therewith. The different sections 11A, 11B, 11C can be distinguishable layers stacked on top of each other but two or all three of the sections can also be manufactured from uniform material.

(17) The width of the flow channel may be for example 0.01-2 mm and thickness for example 0.01-1 mm.

(18) The structure can be made from plastic, glass, silicon, or a mixture thereof, to mention some examples. A plastic channel pattern can be formed for example by laser cutting, die cutting, hot pressing, extrusion molding, engraving or printing, followed by stacking of the intermediate, bottom and/or top layers, if needed. These techniques are well suitable for industrial-scale manufacturing.

(19) According to one embodiment, the structure comprises a layer structure made at least partly from plastic, such as PMMA. There may be provided an even plastic layer as the bottom section 11A, a through-cut channel pattern containing intermediate section 11B and a plastic layer with an injection hole as the top section 11C stacked. Alternatively, the bottom and intermediate sections may be provided as a unitary plastic piece with engraved channel patterns and a plastic layer as the lid. Stacking can be achieved by gluing, taping or hot-pressing, for example. Naturally, functionally similar structures can be made from other materials too.

(20) The flow channel 13 formed into the lateral flow plate 10 is hollow. That is to say the flow of liquid in the flow channel 13 is based on dimensioning the channel so as to excite capillary action as opposed to using a particular lateral flow matrix. Lateral flow matrixes are used in the prior art as a medium to advance flow by capillary action in a channel, which is not dimensioned to provide capillary action. The matrixes employ porous or bibulous materials, such as cellulose, paper, nylon, etc. The hollow flow channel 13, however, promotes lateral flow by appropriate dimensioning of the flow channel 13 and further promoted by a suction pad arranged to the waste reservoir 18 at an end of the flow channel 13. In other words, the flow channel 13 is flow medium free. As best seen from FIG. 1B, the flow channel 13 is being delimited by the bottom section 11A, intermediate section 11B and top section 11C without an intermediate flow matrix occupying the space there between. As a result, the lateral flow device does not need a separate enclosure for the flow channel, which is formed into the lateral flow plate 10. Accordingly, the channel is closed, which means that the top section 11C renders the flow channel 13 unexposed to the environment.

(21) FIG. 1C illustrates a lateral flow plate 10 with a branched flow channel 13 according to an alternative embodiment. Again, there is provided a support structure 11 defining a flow channel 13. The injection zone 12 is used for providing the sample to the flow channel 13, which contains a branching portion 15, comprising for example a Y-shaped branch. The branching portion 15 divides the channel into two branches 14A, 14B, which run in parallel towards a waste reservoir 18 at the other end of the flow channel 13. Both branches 14A, 14B may also have separate waste reservoirs. The first branch 14A comprises a test zone 16A and the second branch 14B a reference zone 16B (or vice versa). In this configuration too, the test and reference zones 16A, 16B are aligned with each other side by side so that they fit into a small imaging area 19, which may be circular, for example. Sample flow direction at the test and reference zones is the same.

(22) The flow channel or branches thereof are dimensioned such that the fluid to be used as sample flows by capillary action. In addition, the waste reservoirs 18, 18 are absorptive, i.e., designed to cause a suction force once the samples reach them. The suction empties the channel(s) at least partly.

(23) In the illustrated configurations, the test and reference zones 16A, 16B (16A, 16B) are entirely aligned, but they can also be only partially aligned as far as both, or parts of both, fit within the imaging area 19 (19). Moreover, the angle between channel orientations at the test and reference zones need not be 0 degrees, but the channels can be tilted with respect to each other.

(24) In order to ensure sufficient optical signal from the indicator zones, it is proposed that they extend to at least some extent in the direction of the flow channel. According to one embodiment, the length of the zones is larger than their width, in particular at least twice the width, such as at least 0.5 mm, in particular at least 1 mm. According to another embodiment, the channel comprises a plurality of small indicator zones for forming a micro array indicator zone for enabling multi-analyte detection.

(25) The turning portion 15 or the branching portion 15 can have an alternative shape too, as long as it interconnects the portions 14A, 14B or branches 14A 14B such that the test and reference zones 16A, 16B or 16A, 16B are placed sideways close to each other. The turning portion 15 can have for example a V-shape and the branching portion a T-shape.

(26) According to another embodiment (not shown) the first and second indicator portions are not provided as portions of the same flow channel but different ones. In particularly it is also possible to arrange two separate flow channels, namely a first and second flow channel, which comprise a first and second indicator portion, respectively. According to such an embodiment, the first and second indicator portions of the first and second flow channels are configured to appear adjacently in the imaging area, whereas the rest of the flow channels may be distant from each other. In such a case, both the first and second flow channel may comprise the necessary injection zones and waste reservoirs at opposing ends of the flow channels.

(27) Fluorophores are optionally contained in the system. Antibodies provided with fluorophores may be e.g. printed onto the injection zone, where they are dissolved into the sample. The sample conveys the fluorophores to the indicator zones, where a sandwich comprising capture antibody, analyte and fluorescently labeled detection antibody is formed. The imaging is based on detecting fluorescent emissions of the indicators, shown at distinct wavelengths or a broader wavelength range, using a suitable optical setup, which is described in more detail below. Typically, the emission wavelength or wavelengths are higher than the excitation wavelength.

(28) According to one embodiment, the indicator substance(s) is/are printed in the channel before stacking of the support structure, i.e., closing of the channel.

(29) According to one embodiment, the flow channel comprises, a blood filtration membrane in front of at least one of the indicator zones.

(30) Imaging Device

(31) FIG. 2A shows the principle of an exemplary imaging device utilizing an above described lateral flow plate 20. Imaging is carried out using a camera 28 of a terminal device such as a mobile phone 22, tablet, smart watch or the like. In the following examples, the terminal device is being described as a mobile phone. Between the mobile phone 22 and the flow plate 20, there is an optical unit 24, which contains a microscopic lens 27 that makes the camera suitable for taking a sharp close-up image of the imaging area containing the indicator zones of the flow plate 20 and optionally also comprises a light source capable of illuminating the indicator zones. In general, the camera 28 and the microscopic lens 27 work together so that a large enough portion of the plate 20 can be captured to a single image. The closer together the indicator zones are on the plate, the smaller the imaging area can be.

(32) In one embodiment, the camera 28, the optical unit 24 and the plate 20 are during imaging stacked firmly against each other such that the imaging situation is geometrically as constant as possible. According to one embodiment, the indicator zones of the plate 20 are symmetrically positioned with respect to the optical axis of the system such that they are evenly imaged.

(33) FIG. 2B shows an exemplary implementation of the optical device in more detail. There is provided an imaging panel 21, such as a CCD or CMOS panel, and imaging optics 23 of the camera 28 of the mobile phone 22. The lens of the optical unit is denoted with reference numeral 25. The lens contains a magnifying central portion, herein implemented as convex-concave lens with the convex surface facing the camera and concave surface facing the flow plate 20. There is also provided a light source 26, such a LED or laser source, whose light is directed to a fringe area of the lens 25, from which its is reflected by total internal reflection from a slanted portion of the lens 25 to the imaging area of the flow plate 20. The illumination angle at can be e.g. 45-85 degrees, in particular 60-80 degrees.

(34) One exemplary conversion unit which is optically suitable to be used in the present invention is the KeepLoop unit provided by KeepLoop Oy, Finland.

(35) In particular when fluorescence-based imaging is taken advantage of, there may also be provided a selective filter, such as a bandpass filter or band-reject filter, between the lens of the optical unit and the camera of the mobile phone for filtering out undesired bandwidths of light, in particular the illumination wavelength. This prevents any illumination light directly entering the imaging panel because of reflections in the lens or at the plate, for example. The filter is shown as an element 29 in FIG. 2B.

(36) A filter is not necessarily needed in particular if a monochromatic light source is used for illumination of the plate or if the emission bandwith of the light source is otherwise narrow. It is also proposed that the plate is in this case manufactured from a low-autofluorescence material, such as glass.

(37) It should be noted that in particular when using a dedicated reader, no imaging lenses are necessarily needed at all, but the imaging can be made using a direct image forming at the imaging sensor.

(38) FIG. 2C shows a docking station 34 comprising on its upper surface a first docking zone for a mobile phone 32 and a second docking zone for a lateral flow plate 30. The optical unit is positioned inside the body of the station 34 between the first and second docking zones. The first docking zone is capable of immobilizing the phone 32 such that its camera is aligned in a predefined position. In particular, there may be members holding the phone 32 laterally in a fixed position such that the camera of the phone is aligned with the optical unit built in the docking station. The second docking zone is also designed to immobilize the lateral flow plate 20 such that its imaging area is aligned with the camera and the optical unit. In the illustrated example, the second docking zone is implemented using frame 35 placed below the first docking zone and having a recess for the plate 30. The frame 35 also comprises a mechanism allowing the plate 30 to be inserted and removed conveniently. In this case, the frame 35 is hinged to the main body of the docking station 34 such that during imaging, the frame 35 is closed towards the body and during insertion and removal of the plate 20, the frame is in an opened position.

(39) FIG. 2C shows only one example of the docking station. There are numerous other mechanical implementation options available too. For example, there may be provided a slot where the flow plate 30 can be slid.

(40) Once the analysis image containing the two or more indicator zones has been captured, a processor of the mobile phone is used to derive a parameter depicting the composition of the sample based on pixel or subpixel (color) intensities of the analysis image using an analysis algorithm defined in a software executed in the mobile phone.

(41) An exemplary analysis algorithm for fluorescence-based assays proceeds as follows: 1. Selecting a desired region of interest (ROI) from the analysis image, containing at least a portion of one indicator zone. 2. Forming a single-channel (image of ROI). If green (G) excitation light is used, for example, red (R) channel can be chosen to avoid excitation light from appearing (in particular if a physical filter is not used or as an additional filter). 3. Calculating a histogram of the single-channel image and from the histogram at least one of the following values Mean value, Median value, Mode value (most common value), 95 percentile (depicting well the maximum intensity) 97 percentile (depicting well the maximum intensity) 4. Comparing the value with a reference curve obtained using known concentrations of the analyte and thus obtaining an estimate for the concentration of the measured analyte.

(42) Other non-illustrated examples of the invention include competitive immunoassay (the amount of analyte in the sample is inversely related to the amount of label measured in the indicator zone) and microarray. As a deviation from the description above it is also possible according to the novel concept to measure decrease of signal as in competitive immunoassay or multiple spots (array) at same time.

EXAMPLE

(43) Suitability of the present invention for THC immunoassays has been demonstrated. The test setup is briefly presented below.

(44) The lateral flow devices used comprised a meandering-channel chips of the type shown in principle in FIG. 1A and as a photograph in FIG. 3A. The test zone was situated closer to the injection zone and the reference zone close to the waste reservoir. The test zone was provided with antibody sensitive to THC-immunocomplex in serum. The control zone contained a non-specific antibody as negative control. Alexa Fluor 546 labeled anti-THC antibody was mixed with the sample to form THC-anti-THC immunocomplex.

(45) The chips were provided with four serum samples with 0, 50, 100 and 250 ng/ml THC concentration. Images of chips with 0 and 250 ng/ml concentration are shown in FIGS. 5A and 5B.

(46) Imaging was carried out using a test setup with a mobile phone, the setup corresponding to that schematically shown in FIGS. 2A-2C. Roithner 532 nm lasermodules were used to illuminate the sample and to excite the fluorophore. Chroma ZET405/473/532/640m was used to block undesired reflections of excitation light. An iPhone 5S was used as the imaging device. FIG. 5 shows the absorption and emission spectra of Alexa fluor 546 and the filter spectrum of FF01-593/40.

(47) A reference system was also used, comprising a commercial fluorometer. Results of the reference setup measurement are shown in FIG. 4A. It can be seen that the fluorescence signal measured increases roughly linearly as the THC concentration increases.

(48) Results of the test setup measurement are shown in FIG. 4B. The graph comprises Mean, Median and Mode curves of THC concentration using steps 1-3 of the exemplary analysis algorithm described above.

(49) FIG. 4C shows an image of a test chip with 0 ng/ml THC concentration and FIG. 4D with 250 ng/ml THC concentration. The difference of the optical response signal of the test zone can be clearly seen.

(50) It can be seen, that the THC concentration can be well detected and the response signal of the test setup corresponds roughly to that of the reference setup. It can be concluded that the test measurement has been reliable and thus the curves of FIG. 4B could be used as reference curves for further experiments with unknown samples using steps 1 to 4 of the exemplary analysis algorithm described above.

(51) It should be noted that the reference zone adjacent to the test zone is used to ascertain that the signal originates from the immunoreaction as opposed to being a reflection or another error signal. The reference zone therefore contained an antibody, which should not attract THC and thus represents a negative control.

(52) It can be well assumed that the invention works as well in other immunoassays with suitable fluorescent antibodies.

(53) TABLE-US-00001 LIST OF REFERENCE NUMERALS Number Part 10 flow plate 11 support structure 11A bottom section 11B intermediate section 11C top section 12 injection zone 13 flow channel 14A first portion 14B second portion 15 turning portion 15 branching portion 16 reference zone 19 imaging area 20 flow plate 21 imaging panel 22 mobile phone 23 imaging optics 24 optical unit 25 lens of optical unit 26 light source 97 lens 28 camera 29 filter 30 flow plate 32 mobile phone 34 docking station 35 frame 56 indicator zone