GB2526112A

GB2526112A - Means and method for detection of analytes

Info

Publication number: GB2526112A
Application number: GB1408537.7A
Authority: GB
Inventors: Steffen Ramell
Original assignee: R Biopharm AG
Current assignee: R Biopharm AG
Priority date: 2014-05-14
Filing date: 2014-05-14
Publication date: 2015-11-18
Also published as: GB201408537D0

Abstract

A solution, lateral flow assay and a method for detection of at least 2 analytes in vitro. The solution comprises a mixture of at least two or more types of nanoparticles differing in their optical properties and/or being differently shaped, wherein said nanoparticles are coated with capture molecules sensitive for capturing specific analytes. In particular the particles amy be nanorods or nanospheres which have absorption maxima at approximately 520 nm and 625 nm respectively. The nanoparticle labelled capture molecule may be bound to a solid surface to provide a lateral flow or strip assay.

Description

Title: Means and method for detection of analytes

Description

Field of the invention

[0001] The present invention relates to a solution, lateral flow assay and a method for deter-mining the concentration of at least two analytes in a probe.

Background of the invention

[0001] For diagnostic purposes, it is often necessary to determine whether a macromolecule or an analyte is present in a probe. Test have been developed comprising a capture probe for binding the analyte, which should be detected. The capture probe is connected to a reporter capable of being perceived visually. Such reporter can be chromogens, luminescent com-pounds, radioactive compounds or any other visual labels allowing for the detection of the analvte, [0002] In case that the assay for determining the presence or concentration of an analyte in a solution is based on the use of an antibody or immunoglobulin, such assays are also designat-ed as immunoassay.

[0003] In order to provide tests, which are easy to handle, especially in home testing, lateral flow tests have been developed. A lateral flow test or lateral flow assay designates simple devices for the application of a probe in which an analyte should be determined. After the probe is applied, it migrates through capillary flow through a test line and a control line. The test line comprises immobilized capture probes sensitive for the analyte, which should be de-tected.

[0004] Document EP 1 794 592 B1 discloses a lateral flow assay for the diagnosis of large pathogens like Cant/ida a/b/cans. A conjugate pad of a device of this document is located upstream of a detection zone and has detection probes with specific binding members for the analyte comprising nanoparticles. A detection zone comprises an immobilized first capture agent, which is able to bind at least a portion of an analyte conjugate complex to generate a detection signal. Detection is achieved by using labels capable of being perceived visually.

[0005] As it is quite often necessary to determine not only one analyte in a probe but at least two or more analytes, there is a need for an assay which is suitable to detect more that one analyte in one assay.

Brief summary of the invention

[0006] It is an object of the present disclosure to provide means for determining the concen-tration of at least two or more analytes in a probe within one single test line.

[0007] The invention provides a solution for determining the concentration of at least two analytes in a probe, comprising a mixture of at least two or more types of nanoparticles differ-ing in their optical properties and/or being differently shaped, wherein said nanoparticles are linked to or coated with capture molecules sensitive for capturing specific analytes in the probe.

[0008] It is intended that one or more types of nanoparticle can be spherical and the other type can be rod shaped. It is obvious for a person ordinary skilled in the art that any other geomet-rical form of nanoparticles is within the scope of the present invention.

[0009] It is envisaged that spherical nanoparticles may have an absorption maximum at ap- proximately 520 nm and the rod-shaped nanoparticles at approximately 520 nm and approxi-mately 625 nm, [00 10] The spherical nanoparticles may have a diameter of approximately 1 to 1000 nm and the rod shaped nanoparticle have a width of about ito 100 nm and a length of about 2 to 1000 mm [00111 The capture molecule may be selected from the group comprising peptides, proteins, carbohydrates, antibodies, synthetic molecules and polymers.

[0012] It is further intended that the capture molecule can be directly or indirectly bound to a solid surface selected from the group comprising a column, a matrix, beads, glass including modified or functionalized glass, silica or silica-based materials including silicon and modi-fied silicon, plastics comprising polypropylene, polyethylene, polystyrene and copolymers of styrene and other materials, acrylics, polybutylene, polyurethanes, nylon or nitrocellulose, resins, polysaccharides, carbon as well as inorganic glasses and plastics.

[0013] Another object of the present invention is a lateral flow assay for determining the con-centration of at least two or more analytes in a probe, comprising a sample pad comprising a mixture of at least two or more types of nanoparticles differing in their optical properties and/or being differently shaped, wherein each nanoparticle is coated with a first capture mole-cule sensitive for capturing a specific analyte in the probe, a solid surface arranged between sample pad and a wicking pad for transporting the probe from the sample pad to the wicking pad, a test line with a second capture probe sensitive for binding an analyte in the probe ar-ranged on the solid surface, a control line with an unspecific capture molecule.

[0014] It is envisaged that in a lateral of the invention at least two or more types of differently colored and/or differently shaped nanoparticles may be used. Furthermore, spherical nanopar- tides may have an absorption maximum at approximately 520 nm and the rod-shaped nano-particles at approximately 520 nm and 625 nm.

[0015] The spherical nanoparticles may have a diameter of approximately I to 1000 nm and the rod shaped nanoparticle may have a width of approximately I to 100 nm and a length of approximately 2 to 1000 nm.

[0016] The capture molecule shall be selected from the group comprising peptides, proteins, carbohydrates, antibodies, synthetic molecules and polymers.

[0017] The solid surface of a lateral flow assay may be selected from the group comprising a column, a matrix, beads, glass including modified or functionalized glass, silica or silica-based materials including silicon and modified silicon, plastics comprising polypropylene, polyethylene, polystyrene and copolymers of styrene and other materials, acrylics, polybutyl- ene, polyurethanes, nylon or nitrocellulose, resins, polysaccharides, carbon as well as inorgan-ic glasses and plastics.

[0018] It is envisaged that the first and second capture molecule are identical or different.

[00t9] A further object of the invention is all assay comprising a solution for determining the concentration of at least two analytes in a probe, comprising a mixture of at least two or more types of nanoparticles differing in their optical properties and/or being differently shaped, wherein said nanoparticles are linked to or coated with capture molecules sensitive for captur-ing specific analytes in the probe.

[0020] A further object of the present invention is a method for determining the concentration of at least two or more analytes in a probe, comprising the steps of a. Bringing a probe in contact with a mixture of at least two or more types of nanoparticles with a first capture molecules sensitive for capturing specif-ic analytes in the probe, wherein the at least two types of nanoparticles have different optical properties; b. Separating the different types of nanoparticles captured specifically ana-lytes within one single test line by immobilized second capture molecules on the test line which are also able to bind the analytes specifically; c. Determining the optical density at approximately 520 nm and approxi-mately 625 nm of captured nanoparticles on the test line; d. Calculating the portion of each captured nanoparticle, and e. Comparing the calculated portion with a predetermined standard curve to read out the concentration of the at least two or more analytes.

[0021] It is intended that at least two or more types of nanoparticles having the same and/or different shapes maybe used in a method of the present invention.

[0022]lt is further intended that that one type of nanoparticles used may be spherical and the respective other type used may be rod shaped.

[0023] The method of one of claims 14 to 16, wherein the spherical nanoparticles have an absorption maximum at approximately 520 nm and the rod-shaped nanoparticles at approximately 520 nm and 625 nm and the amount of nanoparticles is determined at these wavelengths.

[0024] The standard curves for each analyte can be prepared under identical conditions to the method for determining the concentration of the at least two or more analytes.

[0025] It is further intended that the captured nanoparticles may be immobilised on a surface via a second capture molecule sensitive for binding the analyte captured to a nanoparticle, wherein the second capture probe may be immobilised on a solid surface.

[0026] The solid surface used in a method of the present invention may be selected from the group comprising a column, a matrix, beads, glass including modified or functional-ized glass, silica or silica-based materials including silicon and modified silicon, plastics comprising polypropylene, polyethylene, polystyrene and copolymers of styrene and other materials, acrylics, polybutylene, polyurethanes, nylon or nitrocellulose, resins, polysaccharides, carbon as well as inorganic glasses and plastics.

[0027] The first and second capture molecules used in a method of the present inven-tion may be are identical or different.

Brief description of the figures

[0028] Figure 1 Schematic depiction of a lateral flow assay according to the disclosure [0029] Figure 2 Absorption spectra of mixtures of red spherical nanopartides and blue rod-shaped nanoparticles and mixtures thereof [0030] Figure 3 Comparison between calculated OD values and theoretical OD values

Detailed description of the invention

[003 t] The present invention provides a solution, a lateral flow assay and a method for de-termining the concentration of at least two or more analytes in a probe within one single test line.

[0032] The disclosure is based on a solution comprising two types of nanoparticles with dif- ferent colors results in a mixed color. Different color within the meaning of the present dis-closure means that nanoparticles themselves have a different color, emit light or signals with a different color or even emit signals that can be differentiated from each other. The problem with such a mixture is, whether it will be possible to determine the portion of at least two col-ored nanoparticles contributing to the mixed color and deduce the concentration of at least two analytes bound to the colored nanoparticles via a capture molecule, which can be immo- bilized on a surface (e.g. nitrocellulose membrane). The colored nanoparticles can be immobi-lized and arranged on one single test line of a lateral flow assay.

[0033] Experiments of the applicant revealed that the use of nanoparticles with different shapes and different absorption spectra enables the determination of the concentration of at least two analytes in a probe. One type of nanoparticles may be spherical and the other rod-shaped.

[0034] Reading out the mixed colored test line at a wavelength of approximately 520 nm and approximately 625 nm for red and blue nanoparticles showed good results for calculating the respective portion of each nanoparticle (comp. fig 3).

[0035] Figure la shows a schematic depiction of a lateral flow assay for determining the con- centration of at least two analytes in a probe. Such a test has a zone for capillary flow ar-ranged between a conjugate pad and a wicking pad.

[0036] The conjugate pad comprises two types of nanoparticles with eventually different shapes and colors (open circle arid circle with cross) attached to different specific anti-analyte-A-antibodies, for instance mouse-anti-analyte A-antibodies (filled Y) and specific anti-Analyte B-antibodies (open Y).

[0037] Other than the previously mentioned specific antibodies against analyte A and B are immobilized on one single test line. The control line comprises immobilized anti-mouse-antibodies for detecting binding to anti-mouse-antibodies.

[0038] Applying a probe (figure lb; arrow with open and filled diamonds) results in binding of the respective analyte by the respective specific anti-mouse-analyte-antibody, compare figure Ic. The conjugates migrate through the membrane and the analytes will be bound by the respective mouse-anti-analyte-antibody on the test line.

[0039] The test line will be read-out at wavelengths of approximately 520 nm (figure Id) and approximately 625 nm (figure le). Based on the results of the read-out of the mixed colored test line it is possible to calculate the portion of each colored nanoparticle.

[0040] A predefined standard curve allows for determining the concentration of both analytes, The standard curve was prepared using probes with different known concentrations of both analytes and mixtures of these analvtes. The known concentrations of both analytes (a and b) are detected by the nanoparticles (e.g. analyte a by the red-and analyte b by blue-particle), [0041] Figure 2 shows the absorption spectra of different mixtures of red spherical and bluerod-shaped nanoparticles. As can be taken from Figure 2 the respective absorption spectra of a solution depend on the mixture of the nanoparticles contributing to the mixed color.

Thus, it turned out, that it is possible to derive from the mixed color the respective portion of each nanoparticle.

[0042] Transferring this results to a lateral flow assay means that the mixed color of the test line can be used to determine the concentration of an analyte through calculating the portion the respective nanoparticle which is specifically bound to an analyte, [0043] Table 1 shows the results of photometric measurements of the respective nanoparticles (lower line) in comparison to the theoretically known portion of each nanoparticle (upper line) in the solution.

[0044] Table 1: Photometric detection of known portions of nanoparticles in solution Red parti-cle%/BIue par-90/10 75/25 50/50 25/75 10/90 0/100 tOO/0 ticle% Red particle% 90,2 75,3 50,2 24,9 10,2 0,00 00 Blue particle% 10,0 24,9 49,6 74,8 89,5 100 0,00 1 1 1 0,9999 0,9999 1 1 [0045] As can be taken from Table 1 there seems to be no interference of the red and blue nanoparticles so that they are suitable for calculating and determining the concentration of an analyte which is bound to a nanoparticle based on determining the portion of a nanoparticle of a mixed color.

[0046] Figure 3 shows a graphical representation of photometrically measured OD values.

Again, it can be observed that the determined values are in close proximity to the diagonal representing the line, where calculated on theoretical values coincide.

[0047] The above-described principle can be extended to all kind of colored particles. Conse-quently, it will be possible to determine several parameters in a standard lateral flow test strip with one single test line only with such color combinations.

[0048] The size of the nanoparticles should be in the range of approximately ito 1000 nm, it is intended that the nanoparticles are bound to specific receptor molecules, for instance anti-bodies, which bind specifically the desired analyte in a probe. The analyte will be bound to a test strip or any other solid surface through a second captured molecule, e.g. a different anti-body bound to the nanoparticle, which is immobilized on the solid surface.

[0049] Such assays as described-above can be read out by usually known devices having the appropriate filters or diodes at the appropriate wavelength for determining the optical density of the respective colored nanoparticle. Any other labeling of the nanoparticles making them suitable for detection by fluorescence, phosphorescence or luminescence is also within the scope of the present invention. It is preferred that a dye or reporter is not linked to the nano- particle, but is an integral part of the nanoparticle, for instance of the outer shell of the nano-particle.

[0050] It is a prerequisite for the disclosed method that prior to determining the concentration of multiple analytes in a probe, a standard curve for each analyte is prepared using probes with known concentrations and mixtures of different analytes under comparable conditions.

Such standard curves for each analyte are used for determining the concentration of an analyte from the calculated portion of each nanoparticle of the mixed color, which is immobilized and bound to the desired analyte, [0051] It is also within the scope of the invention to have an aray of different combinations of immobilized second captured molecu'es for binding the analyte bound to the colored nano- particle. An automatic read-out of such an array is also within the scope of the present inven-tion.

[0052] The present disclosure provides a solution, a lateral flow assay and a method, which are suitable for easy and reliable detection of at least two or more analytes in a probe on the same test line. It is an advantage that no complicated and time-consuming methods have to be applied in order to visualize or detect an anlyte. Commonly used devices for reading-out the signal line by particle specific wavelength via e. g. reflectometric measurment can be used to determine the concentration of the analyte.

[0053] Another advantage is that the capture probe is bound to a nanoparticle, which itself is suitable for the detection of captured nanoparticles, No further addition of an enzyme for con-version of a chemical or radioactive labelling is necessary. It was completely surprising that coloured nanoparticles are suitable for an accurate determination of the concentration of at least two analytes in a probe. It was not possible to anticipate that there will be no interference of the different colours so that an absorption spectrum of a test line could not be used to de-termine the portion of the nanoparticles contributing to the mixed colour and further deduce the concentration of an analyte from the determined portion.

Claims

Title: Means and method for detection of analytes Claims 1. A solution for determining the concentration of at least two analytes in a probe, comprising a mixture of at least two or more types of nanopartides differing in their optical properties and/or being differently shaped, wherein said nanoparti- des are linked to or coated with capture molecules sensitive for capturing specif-ic analytes in the probe.
2. The solution of claim 1, wherein one or more types of nanoparticle is spherical and the other type is rod shaped.
3. The solution of one of claims 1 or 2, wherein the spherical nanoparticles have an absorption maximum at approximately 520 nm and the rod-shaped nanoparticles at approximately 520 nm and approximately 625 nm.
4. The solution of one of claims 1 to 3, wherein the spherical nanoparticles have a diameter of approximately 1 to 1000 nm and the rod shaped nanoparticle have a width of about 1 to 100 nm and a length of about 2 to 1000 nm.
5. The solution of one of claims 1 to 4, wherein the capture molecule is selected from the group comprising peptides, proteins) carbohydrates, antibodies, syn-thetic molecules and polymers.
6. The solution of one of claims 1 to 5, wherein the capture molecule is directly or indirectly bound to a solid surface selected from the group comprising a column, a matrix, beads, g'ass including modified or functionalized glass, silica or silica- based materials including silicon and modified silicon, plastics comprising poly- propylene, polyethylene, polystyrene and copolymers of styrene and other mate- rials, acrylics, polybutylene, polyurethanes, nylon or nitrocellulose, resins, poly-saccharides, carbon as well as inorganic glasses and plastics.
7. An assay comprising a solution for determining the concentration of at least two analytes in a probe, comprising a mixture of at least two or more types of nano-particles differing in their optical properties and/or being differently shaped, wherein said nanoparticles are linked to or coated with capture molecules sensi-tive for capturing specific analytes in the probe.
8. A lateral flow assay for determining the concentration of at least two or more an-alytes in a probe, comprising a. a sample pad comprising a mixture of at least two or more types of nano-particles differing in their optical properties and/or being differently shaped nanoparticles, wherein each nanoparticle is coated with a first capture molecule sensitive for capturing a specific analyte in the probe; b. a solid surface arranged between sample pad and a wicking pad for trans-porting the probe from the sample pad to the wicking pad; c. a test line with a second capture probe sensitive for binding an analyte in the probe arranged on the solid surface; d. a control line with an unspecific capture molecule.
9. The lateral flow assay of claim 7, wherein at least two or more types differently colored and/or differently shaped nanoparticles are used.
10. The lateral flow assay of one of claims 7 or 8, wherein the spherical nanoparticles have an absorption maximum at approximately 520 nm and the rod-shaped na-nopartides at approximately 520 nm and 625 nm.liThe lateral flow assay of one of claims 7 toY, wherein the spherical nanoparticles have a diameter of approximately 1 to 1000 nm and the rod shaped nanoparticle have a width of approximately 1 to 100 nm and a length of approximately 2 to 1000 nm.12.The lateral flow assay of one of claims 7 to 10, wherein the capture molecule is selected from the group comprising peptides, proteins, carbohydrates, antibod-ies, synthetic molecules and polymers.13.The lateral flow assay of one of claims 7 to 11, wherein the solid surface is select-ed from the group comprising a column, a matrix, beads, glass including modified or functionalized glass, silica or silica-based materials including silicon and modi-fied silicon, plastics comprising polypropylene, polyethylene, polystyrene and copolymers of styrene and other materials, acrylics, polybutylene, polyurethanes, nylon or nitrocellulose, resins, polysaccharides, carbon as well as inorganic glass-es and plastics.14. The lateral flow assay of one of claims 7 to 12, wherein first and second capture molecule are identical or different 15.A method for determining the concentration of at least two or more analytes in a probe, comprising the steps of: a. Bringing a probe in contact with a mixture of at least two or more types of nanoparticles with a first capture molecules sensitive for capturing specif-ic analytes in the probe, wherein the at least two types of nanoparticles have different optical properties; b. Separating the different types of nanoparticles captured specifically ana-lytes within one single test line by immobilized second capture molecules on the test line which are also able to bind the analytes specifically; c. Determining the optical density at approximately 520 nm and approxi-mately 625 nm of captured nanoparticles on the test line; d. Calculating the portion of each captured nanoparticle, and e. Comparing the calculated portion with a predetermined standard curve to read out the concentration of the at least two or more analytes.16. The method of claim 14, wherein the at least two or more types of nanoparticles have a the same and/or different shapes.17. The method of one of claims 14 or 15, wherein one type of nanoparticles is spher-ical and the respective other type is rod shaped.18. The method of one of claims 14 to 16, wherein the spherical nanoparticles have an absorption maximum at approximately 520 nm and the rod-shaped nanopar-tides at approximately 520 nm and 625 nm and the amount of nanoparticles is determined at these wavelengths.19. The method of one of claims 14 to 17, wherein the standard curves for each ana-lyte were prepared under identical conditions to the method for determining the concentration of the at least two or more analytes.20.The method of one of claims l4to 18, wherein captured nanoparticles are immo- bilised on a surface via a second capture molecule sensitive for binding the ana-lyte captured to a nanoparticle, wherein the second capture probe is immobilised on a solid surface.21.The method of claim 19, wherein the solid surface selected from the group com-prising a column, a matrix, beads, glass including modified or functionalized glass, silica or silica-based materials including silicon and modified silicon) plas- tics comprising polypropylene, polyethylene, polystyrene and copolymers of sty- rene and other materials, acrylics, polybutylene, polyurethanes, nylon or nitrocel-lulose, resins, polysaccharides, carbon as well as inorganic glasses and plastics.22.The method of one of claims 14 or 20, wherein first and second capture mole-cules is identical or different.