EP4070105A1

EP4070105A1 - Lateral flow assays comprising non-spheroidal gold nanoparticles

Info

Publication number: EP4070105A1
Application number: EP20816505.0A
Authority: EP
Inventors: Daniel QUESADA; Marc GALLEGOS
Original assignee: Paperdrop Diagnostics SL
Current assignee: Paperdrop Diagnostics SL
Priority date: 2019-12-04
Filing date: 2020-12-04
Publication date: 2022-10-12
Also published as: WO2021110958A1

Abstract

Spherical gold nanoparticles are the most used label in lateral flow test devices because it is easy to conjugate biological molecules onto gold nanoparticles, they have a strong red color and they are relatively stable. However, there is a need to improve the sensitivity of lateral flow devices to decrease their lower detection limit. The present invention aims to improve the sensitivity of lateral flow devices by using nanoparticles having a substantially polyhedron shape and stronger plasmon/color.

Description

Lateral flow assays comprising non-spheroidal gold nanoparticles

Technical field

The present invention can be included in the field of lateral flow test devices. Lateral flow test devices can be used to detect a target analyte in a sample, for example, for diagnostic purposes or in the food industry.

Background art

Biosensors which can be classified as “ lateral flow ” devices typically comprise a paper substrate onto which a sample pad (where the liquid sample is loaded), a conjugate pad (where the labeled detection substance is stored), a support membrane and an absorption pad are affixed. The support membrane typically comprises at least a detection section wherein an affinity substance is immobilized. Such a typical lateral flow device is depicted in Quesada-Gonzalez & Merkoci. 2015. Biosens Bioelectron. 73:47-63. According to the World Health Organization, lateral flow devices represent the most promising point-of-care tool in developing countries due to their low production cost, robustness and ease of use.

Most commercial lateral flow devices use colored particles as labels to provide a qualitative result (positive or negative). Many publications highlight the advantages of using nanoparticles instead of microparticles. For example, it is known that nanoparticles per se are colored and often result in a stronger signal than microparticles.

Due to the characteristically strong color of nanomaterials (due, at least in part, to the plasmonic resonance effect), it is sometimes possible to obtain semiquantitative data from a lateral flow device by measuring the color intensity at the detection section. This can be achieved by using a colorimetric reader or even a mobile phone.

Spherical gold nanoparticles are the most used label in lateral flow test devices because it is easy to conjugate biological molecules onto gold nanoparticles, they have a strong red color and they are relatively stable (Quesada-Gonzalez & Merkoci. 2015. Biosens Bioelectron. 73:47-63). However, there is a need to improve the sensitivity and limit of detection of lateral flow devices.

The present invention therefore aims to provide labeled detection substances which result in a higher sensitivity and lower detection limit when used in a lateral flow test device

Figures

Figure 1: A) Illustrative diagram of spheroidal vs non-spheroidal particles binding to a target analyte on a lateral flow test device. B) Illustrative diagram of non-spheroidal particles coated indiscriminately with affinity substances vs non-spheroidal particles labeled at the vertices binding to a target analyte on a lateral flow test device. Figure 2: Diagram of a lateral flow test device. A) Bird’s-eye view of the test device in the form of a strip. The test device comprises the following elements: an absorbent material (1), a support membrane (2) with a detection section (3) and a control section (4), a sample addition section (5) and a section of the device which comprises the labeled detection substance (6). The arrow indicates the flow direction. B) Side-view of the test device in the form of a strip. The test device further comprises a plastic support (7). (c) Angled side-view of the immunochromatographic test device.

Figure 3: Transmission electron microscopy image of nanoparticles encompassed by the present invention. These nanoparticles were synthesized in accordance with the method disclosed in Example 1.

Figure 4: Absorbance spectrum of nanoparticles encompassed by the present invention. These nanoparticles were synthesized in accordance with the method disclosed in Example 1.

Figure 5: Transmission electron microscopy image of nanoparticles encompassed by the present invention. These nanoparticles were synthesized in accordance with the method disclosed in Example 2.

Figure 6: Measuring the intensity of the detection section of a lateral flow test device comprising spheroidal nanoparticles of the prior art (AuNPs) or a nanoplate having a substantially triangular shape in accordance with the present invention (AuNTs).

Figure 7: Calibration curves modeled using the data depicted in Figure 6.

Figure 8: Images of lateral flow test devices exposed to sodium chloride as described in Example 5. The test devices were imaged after 30 seconds and after 600 seconds.

Summary of the invention

The present application discloses that using triangular gold nanoplates in lateral flow test devices instead of the well-known spherical nanoparticles of the prior art results in a device with a lower detection limit and higher sensitivity. It is plausible that this effect applies to any nanoparticles having a substantially polyhedron shape because, without being bound to a particular theory, such shapes exhibit, in most cases, a stronger plasmon (color) per unit of nanoparticle than a spherical nanoparticle with the same diameter and chemical composition.

The increase in sensitivity which results from using non-spheroidal nanoparticles can be further increased by only labeling the vertices of the nanoparticles. By only labeling the vertices of the nanoparticles, one increases the nanoparticle: detection substance ratio, which will be higher as less vertices (thus, less detection substances) the nanoparticle has. Therefore, nanoplates that are, for example, triangular in shape exhibit higher sensitivity increments.

Without being limited by a particular theory, the use of nanoparticles having a substantially polyhedron shape results in a higher ratio of nanoparticle: target analyte and that this ratio may be further increased by increasing the nanoparticle: detection substance ratio through selective labeling at the vertices (see Figure 1).

Thus, the present invention provides a labeled detection substance comprising one or more affinity substances attached to a gold nanoparticle characterized in that the nanoparticle has a substantially polyhedron shape comprising at least three vertices.

The present invention also provides a lateral flow test device comprising: (a) a support membrane, (b) the labeled detection substance of the present invention, and (c) one or more affinity substances immobilized on the support membrane

Further, the present invention also provides a method for detecting an analyte in an isolated sample which comprises contacting the sample with the test device of the present invention.

The present invention also provides the use of a gold nanoparticle having a substantially polyhedron shape comprising at least three vertices for the manufacture of a lateral flow test device.

A kit comprising an affinity substance and a gold nanoparticle having a substantially polyhedron shape comprising at least three vertices is provided by the invention. The use of the kit of the present invention for the manufacture of the labeled detection substance of the present invention or the lateral flow test device of the present invention is also provided.

Detailed description of the invention

Definitions

The term “affibody” refers to a protein that is derived from the Z domain of protein A and that been engineered to bind to a specific target (see Frejd & Kim, 2017. Exp Mol Med. 49(3): e306).

The term “affinity substance” refers to a molecule which has a measurable affinity for and preferentially binds a target. In some embodiments, the affinity substance is a single-stranded DNA molecule, nucleic acid aptamer, peptide aptamer, anticalin, repebody, monobody, scFv, antibody, affibody, fynomer, DARPin, peptide nucleic acid, SMART nucleobase, locked nucleic acid or nanobody. As used herein, the term "antibody" encompasses intact polyclonal antibodies, intact monoclonal antibodies, bivalent antibody fragments (such as F(ab')2), multispecific antibodies such as bispecific antibodies, chimeric antibodies, humanized antibodies, human antibodies, and any other modified immunoglobulin molecule comprising two antigen binding sites.

An antibody can be of any the five major classes (isotypes) of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, or subclasses thereof (e.g. IgGl, IgG2, IgG3, IgG4, IgAl and IgA2), based on the identity of their heavy-chain constant domains referred to as alpha, delta, epsilon, gamma, and mu, respectively. The different classes of immunoglobulins have different and well-known subunit structures and three- dimensional configurations. Antibodies can be naked or conjugated to other molecules such as therapeutic agents or diagnostic agents to form immunoconjugates.

The term “anticabn” refers to a protein that is derived from the lipocalin and that been engineered to bind to a specific target (see Skerra, 2008. FEBS J. 275(11):2677-83).

The term “designed ankyrin repeat proteins” or “DARPin” refers to a protein that is derived from an ankyrin repeat that has been engineered to bind to a specific target (see Pliickthun, 2015. Annu Rev Pharmacol Toxicol. 55:489-511).

As used herein, "detection section" refers to a site where the presence of a target analyte is visually detected.

The term “edge” refers to a particular type of line segment joining two vertices in a polygon or polyhedron. In a polygon, an edge is a line segment on the boundary, and is often called a side. In a polyhedron or more generally a polytope, an edge is a line segment where two faces meet.

The term “fynomer” refers to a protein that is derived from the SH3 domain of human Fyn kinase that has been engineered to bind to a specific target (see Bertschinger et ah, 2007. Protein Eng Des Sel. 20(2): 57- 68).

As used herein, the term "immobilize" refers to attaching an affinity substance on a support such as a membrane so that the affinity substance can no longer move from its position on the support. The immobilized affinity substance is a capture affinity substance and constitutes a detection section by being immobilized on the support.

The term “isolated” refers to anything which has been previously extracted from its natural setting. For example, the expression “isolated blood sample” refers to a sample of blood that has previously been extracted from a patient and now exists in an ex vivo setting. The expression also encompasses samples which are not derived from a patient such as a water or food sample. As used herein, the term “linker” refers to a carbon chain that can contain heteroatoms (e.g., nitrogen, oxygen, sulfur, etc.) and which may be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49,

50 atoms long. Linkers may be substituted with various substituents including, but not limited to, hydrogen atoms, alkyl, alkenyl, alkynl, amino, alkylamino, dialkylamino, trialkylamino, hydroxyl, alkoxy, halogen, aryl, heterocyclic, aromatic heterocyclic, cyano, amide, carbamoyl, carboxylic acid, ester, thioether, alkylthioether, thiol, and ureido groups. Those of skill in the art will recognize that each of these groups may in turn be substituted. Examples of linkers include, but are not limited to, pH-sensitive linkers, protease cleavable peptide linkers, nuclease sensitive nucleic acid linkers, lipase sensitive lipid linkers, glycosidase sensitive carbohydrate linkers, hypoxia sensitive linkers, photo-cleavable linkers, heat-labile linkers, enzyme cleavable linkers (e.g., esterase cleavable linker), ultrasound-sensitive linkers, and x-ray cleavable linkers.

The term “locked nucleic acid” refers to an oligonucleotide that contains one or more nucleotide building blocks in which an extra methylene bridge fixes the ribose moiety either in the C3'-endo (beta-D-LNA) or C2'-endo (alpha-L-LNA) conformation (Griinweller & Hartmann, 2007. BioDrugs. 21(4):235-43).

The term “monobody” refers to a protein that is derived from a fibronectin type III domain that has been engineered to bind to a specific target (see Koide et ah, 2013. J Mol Biol. 415(2):393-405).

The term “nanobody” refers to a protein comprising the soluble single antigen-binding V-domain of a heavy chain antibody, preferably a camelid heavy chain antibody (see Bannas et ah, 2017. Front Immunol. 8:1603).

The term “nanoparticle” refers to a nano-object with all external dimensions in the nanoscale where the lengths of the longest and the shortest axes of the nano-object do not differ significantly. If the dimensions differ significantly (typically by more than 3 times), terms such as nanofiber or nanoplate should be used (ISO/TS 80004-2:2015).

The term “nanoplate” refers to a nano-object with one external dimension in the nanoscale, wherein the other two external dimensions are significantly larger (typically three times larger). This is the standardized definition according to ISO/TS 80004-2:2015.

The term “nucleic acid aptamer” refers to a short synthetic single-stranded oligonucleotide that specifically binds to various molecular targets (see Ni et ak, 2011. Curr Med Chem. 18(27):4206-4214). The term “peptide aptamer” refers to a short, 5-20 amino acid residue sequence that can bind to a specific target (see Reverdatto et al., 2015. Curr Top Med Chem. 15(12): 1082-101).

The term “peptide nucleic acid” refers to a polymer wherein the deoxyribose phosphate backbone of DNA is replaced with an achiral polyamide backbone (see Nielsen et al., 1991. Science. 254(5037): 1497-500).

The term “polygon” refers to a plane figure that is described by a finite number of straight-line segments connected to form a closed polygonal chain or polygonal circuit. Exemplary polygons include the triangle, quadrilateral, pentagon, hexagon, heptagon, octagon, nonagon, decagon, undecagon and dodecagon. A triangular shape is preferred because it is the shape with the least number of vertices.

The term “polyhedron” refers to a solid in three dimensions with flat polygonal faces, straight edges and sharp comers or vertices. Exemplary polyhedrons include the tetrahedron, triangular prism, truncated tetrahedron, truncated cube, truncated dodecahedron, cube, pentagonal prism, hexagonal prism, octagonal prism, decagonal prism, dodecagonal prism, truncated octahedron, truncated cuboctahedron, dodecahedron, truncated icosahedron, octahedron, square antiprism, pentagonal antiprism, hexagonal antiprism, octagonal antiprism, decagonal antiprism, dodecagonal antiprism, cuboctahedron, rhombicuboctahedron, icosidodecahedron, icosahedron, snub cube, octahemioctahedron, tetrahemihexahedron, cubohemioctahedron, great dodecahedron, pentagrammic prism, heptagrammic prism, pentagrammic antiprism, pentagrammic crossed-antiprism, heptagrammic antiprism, heptagrammic crossed-antiprism, octogrammic antiprism, octogrammic crossed-antiprism, small stellated dodecahedron, great stellated dodecahedron, ditrigonal dodeca-dodecahedton, small ditrigonal icosidodecahedron, stellated truncated hexahedron, great rhombihexahedron, and great cubicuboctahedron.

The term “repebody” refers to a protein that is derived from a leucine-rich repeat module and that been engineered to bind to a specific target (see Lee et al., 2012. PNAS. 109(9): 3299-3304).

The term “single-chain variable fragment” or “scFv” refers to a fusion protein comprising the variable domains of the heavy chain and light chain of an antibody linked to one another with a peptide linker.

The term “SMART nucleobase” refers to an aldehyde-modified natural nucleobase (Bowler et al., 2010. Angew Chem Int Ed Engl. 49(10): 1809-12).

The term “substantially” has been used when the strict mathematical definition of a geometrical term cannot be applied in a real-world setting. The use of the term “substantially” means that on a visual level, the shape seen on an electron microscopy image resembles a certain geometrical shape. For example, a polyhedron can be defined as a solid in three dimensions with flat polygonal faces, straight edges and sharp comers or vertices. However, it is very difficult to obtain a molecular object in the real world with a completely flat face and completely straight edges. Thus, in this example, the term “substantially” is included to encompass shapes that look like a polyhedron when viewed on an electron microscopy image but may, for example, have a face that is not completely flat or an edge that is not completely straight. The term “substantially” may be omitted from any embodiment disclosed herein. However, omission of this term may not indicate that strict compliance with the mathematical definition is a requisite of the invention.

The term “target analyte” refers to a substance (e.g., molecule, protein, peptide, miRNA, DNA, virus, whole cell, bacteria, etc.) of interest present in a sample which the lateral flow test device is configured to detect.

As used herein, the term “vertex” refers to the point where two or more edges meet.

Labeled detection substance

The present invention provides a labeled detection substance comprising one or more affinity substances attached to a gold nanoparticle characterized in that the nanoparticle has a substantially polyhedron shape comprising at least three vertices.

In some embodiments, the nanoparticle is a nanoplate having a substantially polygonal shape comprising at least three vertices.

In some embodiments, at least 80% of the affinity substances attached to the nanoparticles are attached to the vertices. In some embodiments, at least 90% of the affinity substances attached to the nanoparticles are attached to the vertices. In some embodiments, the affinity substances are only attached to the vertices of the nanoparticle. Methods of site-selectively attaching affinity substances onto gold nanoparticles are known in the art ( see Chen et al., 2019. Nat Mater. 18(2): 169-174).

In some embodiments, a maximum of 1 to n affinity substances are attached to a single nanoparticle, wherein n is the number of vertices present on the nanoparticle. When the nanoparticle is not a nanoplate, the number of total vertices is derivable from the shape that the particle takes. For example, if the nanoparticle has substantially the shape of a tetrahedron it will have four vertices and will therefore have a maximum of 1 to 4 affinity substances attached to it. If the nanoparticle is a nanoplate having a substantially polygonal shape, then the number of vertices present in the nanoparticle is derived from the number of vertices of the polygonal shape. For example, if the nanoparticle is a nanoplate having a substantially triangular shape it will have three vertices and will therefore have a maximum of 1 to 3 affinity substances attached to it. Thus, in some embodiments, the nanoparticle is a nanoplate having a substantially polygonal shape comprising at least three vertices and 1 to n affinity substances are attached to the nanoplate, wherein n is the number of vertices of the polygonal shape.

In some embodiments, the nanoparticle: (i) has substantially the same shape as a tetrahedron, triangular prism, truncated tetrahedron, truncated cube, truncated dodecahedron, cube, pentagonal prism, hexagonal prism, octagonal prism, decagonal prism, dodecagonal prism, truncated octahedron, truncated cuboctahedron, dodecahedron, truncated icosahedron, octahedron, square antiprism, pentagonal antiprism, hexagonal antiprism, octagonal antiprism, decagonal antiprism, dodecagonal antiprism, cuboctahedron, rhombicuboctahedron, icosidodecahedron, icosahedron, snub cube, octahemioctahedron, tetrahemihexahedron, cubohemioctahedron, great dodecahedron, pentagrammic prism, heptagrammic prism, pentagrammic antiprism, pentagrammic crossed-antiprism, heptagrammic antiprism, heptagrammic crossed-antiprism, octogrammic antiprism, octogrammic crossed- antiprism, small stellated dodecahedron, great stellated dodecahedron, ditrigonal dodeca-dodecahedton, small ditrigonal icosidodecahedron, stellated truncated hexahedron, great rhombihexahedron, or great cubicuboctahedron; or

(ii) is a nanoplate having substantially the same shape as a triangle, quadrilateral, pentagon, hexagon, heptagon, octagon, nonagon, decagon, undecagon or dodecagon.

In some embodiments, the nanoparticle:

(i) has substantially the same shape as a cube; or

(ii) is a nanoplate having substantially the same shape as a triangle, quadrilateral, pentagon or hexagon.

In some embodiments, the size of the nanoparticle is 10-50 nm. In some embodiments, the size of the nanoparticle is 20-50 nm. In some embodiments, the nanoparticle is a nanoplate having a substantially polygonal shape, wherein the length of the edges found in the plane of the two larger external dimensions is 20-50 nm, preferably 20-26 nm. In some embodiments, the nanoparticle is a nanoplate having a substantially triangular shape, wherein the length of the edges found in the plane of the two larger external dimensions is 20-50 nm, preferably 20-26 nm.

In some embodiments, the nanoparticle is a nanoplate having a substantially polygonal shape comprising at least three vertices, wherein the thickness of the nanoplate is less than 2, 3, 4 or 5 nm, preferably less than 2 nm.

In some embodiments, the nanoparticle has a maximum absorbance somewhere between 500 and 650 nm. In some embodiments, the maximum absorbance of the nanoparticle is somewhere between 520 and 630 nm, preferably 540 and 600 nm, more preferably 550 and 575 nm, and most preferably 555 and 565 nm.

In some embodiments, the nanoparticle has at least two maximum absorbance peaks somewhere between 500 and 650 nm. In some embodiments, the nanoparticle has at least two maximum absorbance peaks somewhere between 510 and 590 nm. In some embodiments, the affinity substance is a nucleic acid and/or a polypeptide. In some embodiments, the affinity substance is a single-stranded DNA molecule, nucleic acid aptamer, peptide aptamer, anticalin, repebody, monobody, scFv, antibody, affibody, fynomer, DARPin, peptide nucleic acid, SMART nucleobase, locked nucleic acid or nanobody.

In some embodiments, the affinity substance is a nucleic acid. In some embodiments, the affinity substance is a single-stranded DNA molecule, nucleic acid aptamer, peptide nucleic acid, SMART nucleobase, or locked nucleic acid.

In some embodiments, the labeled detection substance comprises on or more single-stranded DNA molecules attached to a gold nanoplate having a substantially triangular shape, wherein the length of the edges found in the plane of the two larger external dimensions is 20-50 nm, preferably 20-26 nm.

In some embodiments, the labeled detection substance further comprises a linker between the nanoparticle and the affinity substance. In some embodiments, the linker comprises thymidine and/or adenine. Some reports disclose that long adenine chains can be attached to gold via electrostatic interactions (Liu & Liu, 2017. Anal Methods. 9(18): 2633-43).

In some embodiments, the affinity substance is attached to the nanoparticle through a gold-sulfur bond. This attachment could be a direct gold-sulfur bond between the affinity substance and the nanoparticle or via a linker. For example, in the latter case the linker could be a polyethylene glycol moiety comprising a sulfhydryl group and a further reactive group that can react and bind to the affinity substance.

Lateral flow test device

The present invention provides a lateral flow test device comprising: (a) a support membrane, (b) the labeled detection substance of the present invention, and (c) one or more affinity substances immobilized on the support membrane.

In a test device of the present invention, a liquid sample dropped onto the sample addition section of the device wicks towards a section of the device which comprises the labeled detection substance, and the mixture of the sample and the labeled detection substance migrate through the support membrane, and the signal develops at the detection site (see Figure 2). The target analyte and the labeled detection substance form a complex when the sample contains the target analyte. At the detection section, the immobilized affinity substance captures the complex through a non-covalent interaction, and the conjugate accumulates and develops color. The presence or absence of target analyte in the sample can then be determined by visually checking the extent of the color at the detection section. When the support has a control section, the test device may further comprise a control labeled substance in or adjacent to the section of the device which comprises the labeled detection substance. The control labeled substance can be captured by a substance that can bind the control labeled substance at the control section, and the control labeled substance accumulates and develops color. When the labeled detection substance is also used as a control labeled substance, the residual labeled detection substance that did not form a complex with the target analyte in the sample passes through the detection site and is captured by a substance that is immobilized at the downstream control section. The labeled detection substance accumulates and develops color.

In an alternative test device of the present invention, a liquid sample dropped onto the sample addition section of the device wicks towards a section of the device which comprises the labeled detection substance, and the mixture of the sample and the labeled detection substance migrate through the support membrane, and the signal develops at the detection site (see Figure 2). The target analyte and the labeled detection substance form a complex when the sample contains the target analyte. At the detection section, immobilized target analyte or target analyte analogue captures non-complexed labeled detection substances through a non-co valent interaction. A further site (which may be a control section) downstream of the detection site comprises an immobilized affinity substance that can bind complexed and non-complexed labeled detection substances. The presence of the analyte results in a stronger color development at the further site (which may be a control section) downstream of the detection site than at the detection site.

The support membrane may be of any material which allows an affinity substance to be immobilized through electrostatic interactions, hydrophobic interactions or chemical coupling and on which substances such as the sample and the labeled detection substance can move to the detection site. Examples of suitable support membranes include nitrocellulose, polyvinylidene difluoride (PVDF), and cellulose acetate.

In some embodiments, the support membrane comprises a control section for checking whether the sample has developed properly. A substance that can to bind to a control substance may be immobilized on the control section. The locations of the detection section and the control section on the support are not particularly limited. Typically, the control section is downstream of the detection section.

In some embodiments, the lateral flow test device does not comprise silver nanoparticles. In some embodiments, the lateral flow test device is not suitable for the detection of more than one target analyte in a sample (i.e., not suitable for concurrent multi-analyte detection). In some embodiments, the lateral flow test device is monochromatic (only nanoparticle labels of one color are used).

Method

The present invention provides a method for detecting an analyte in an isolated sample which comprises contacting the sample with the test device of the present invention. The sample may be contacted with the test device of the present invention by dropping the sample on the device or by dipping the device in the sample. Uses of nanoparticles

The present invention provides the use of a gold nanoparticle having a substantially polyhedron shape comprising at least three vertices for the manufacture of a lateral flow test device. In some embodiments, the lateral flow test device is the lateral flow test device of the present invention.

In some embodiments, the nanoparticle:

(i) has substantially the same shape as a tetrahedron, triangular prism, truncated tetrahedron, truncated cube, truncated dodecahedron, cube, pentagonal prism, hexagonal prism, octagonal prism, decagonal prism, dodecagonal prism, truncated octahedron, truncated cuboctahedron, dodecahedron, truncated icosahedron, octahedron, square antiprism, pentagonal antiprism, hexagonal antiprism, octagonal antiprism, decagonal antiprism, dodecagonal antiprism, cuboctahedron, rhombicuboctahedron, icosidodecahedron, icosahedron, snub cube, octahemioctahedron, tetrahemihexahedron, cubohemioctahedron, great dodecahedron, pentagrammic prism, heptagrammic prism, pentagrammic antiprism, pentagrammic crossed-antiprism, heptagrammic antiprism, heptagrammic crossed-antiprism, octogrammic antiprism, octogrammic crossed- antiprism, small stellated dodecahedron, great stellated dodecahedron, ditrigonal dodeca-dodecahedton, small ditrigonal icosidodecahedron, stellated truncated hexahedron, great rhombihexahedron, or great cubicuboctahedron; or

In some embodiments, the nanoparticle:

(i) has substantially the same shape as a cube; or

In some embodiments, the nanoparticle is a nanoplate having a substantially polygonal shape comprising at least three vertices, wherein the thickness of the nanoplate is less than 2, 3, 4 or 5 nm, preferably less than 2 nm. In some embodiments, the nanoparticle has a maximum absorbance somewhere between 500 and 650 nm. In some embodiments, the maximum absorbance of the nanoparticle is somewhere between 520 and 630 nm, preferably 540 and 600 nm, more preferably 550 and 575 nm, and most preferably 555 and 565 nm.

In some embodiments, the affinity substance is a nucleic acid and/or a polypeptide. In some embodiments, the affinity substance is a single-stranded DNA molecule, nucleic acid aptamer, peptide aptamer, anticalin, repebody, monobody, scFv, antibody, affibody, fynomer, DARPin, peptide nucleic acid, SMART nucleobase, locked nucleic acid or nanobody.

Kit

The present invention provides a kit comprising an affinity substance and a gold nanoparticle having a substantially polyhedron shape comprising at least three vertices. In some embodiments, the nanoparticle is a nanoplate having a substantially polygonal shape comprising at least three vertices.

In some embodiments, the nanoparticle:

(i) has substantially the same shape as a cube; or

The present invention also provides the use of the kit of the present invention for the manufacture of the labeled detection substance of the present invention or the lateral flow test device of the present invention.

Items of the invention

The present invention also provides the following items which may be combined with any one or more of the embodiments described above:

[1] A labeled detection substance comprising one or more affinity substances attached to a gold nanoparticle characterized in that the nanoparticle has a substantially polyhedron shape comprising at least three vertices.

[2] The labeled detection substance of item [1], wherein the one or more affinity substances are only attached to the vertices of the nanoparticle.

[3] The labeled detection substance of any one of items [l]-[2], wherein the nanoparticle is a nanoplate having a substantially polygonal shape comprising at least three vertices.

[4] The labeled detection substance of item [3], wherein the nanoplate has a substantially triangular shape.

[5] The labeled detection substance of any one of items [l]-[4], wherein the size of the nanoparticle is

20-50 nm. [6] The labeled detection substance of any one of items [l]-[5], wherein the affinity substance is a nucleic acid and/or a polypeptide.

[7] The labeled detection substance of item [6], wherein the affinity substance is a single-stranded DNA molecule, nucleic acid aptamer, peptide aptamer, anticalin, repebody, monobody, scFv, antibody, affibody, fynomer, DARPin, peptide nucleic acid, SMART nucleobase, locked nucleic acid or nanobody.

[8] The labeled detection substance of any one of items [l]-[7], wherein the affinity substance is attached to the nanoparticle through a gold-sulfur bond.

[9] A lateral flow test device comprising:

(a) a support membrane;

(b) the labeled detection substance of any one of items [l]-[8]; and

(c) one or more affinity substances immobilized on the support membrane.

[10] The device of item [9], wherein the support membrane consists of nitrocellulose, polyvinybdene difluoride or cellulose acetate.

[11] A method for detecting an analyte in an isolated sample which comprises contacting the sample with the test device of any one of items [9]-[10]

[12] Use of a gold nanoparticle having a substantially polyhedron shape comprising at least three vertices for the manufacture of a lateral flow test device, wherein, optionally, the lateral flow test device is the device of any one of items [9]-[10]

[13] A kit comprising:

(i) an affinity substance; and

(ii) a gold nanoparticle having a substantially polyhedron shape comprising at least three vertices.

[14] The kit of item [13], wherein the nanoparticle is a nanoplate having a substantially polygonal shape comprising at least three vertices, wherein, optionally, the nanoplate has a substantially triangular shape.

[15] Use of the kit of item [13] or [14] for the manufacture of the labeled detection substance of any one of items [l]-[8] or the lateral flow test device of any one of items [9]-[10] Examples

Example 1 : Synthesis of nanoplates having a substantially triangular shape

1.6 mL of 0.1 M cetyltrimethylammonium chloride (CTAC) are slowly added to 8 mL of double distilled water, avoiding the production of foam due to the CTAC. Then, 75 pL of 0.01 M KI are added and the solution is carefully shaken, no more than 5 seconds, to homogenize it (during all the steps, being careful to not produce foam). Then, 80 pF of 25 mM HAuCTp followed by 20.3 pL of 0.1 M NaOH, are added to the solution and it is shaken again until the yellow color is homogeneous in all the liquid. 80 pL of 0.064 M ascorbic acid are added and the solution is carefully shaken until all the color is lost (if red color appears, the solution is then discarded). Once the solution is completely colorless and in rest, 10 pL of 0.1 M NaOH are added. A red mist should appear in the solution and, in that moment, it should be shaken 1 or 2 seconds to disperse it. The solution cannot be moved or shaken for 20 minutes (and foam should not have been produced). Gold nanotriangles (AuNTs) should have a dark blue-violet color and can be stored at 4 °C.

The resulting gold nanoplates were substantially triangular and had a length of 22-24 nm from one vertex to another when measured using a transmission electron microscope (see Figure 3). Further, the thickness of the nanoplate was estimated to be less than about 2 nm using the transmission electron microscope. The substantially triangular nanoplates had an absorption maximum at 562 nm (see Figure 4).

Example 2: Synthesis of triangular nanoplates with an edge-length of around 40 nm The synthesis disclosed in Example 1 can also be performed by using type 1 ultrapure water instead of double distilled water (resistance of ultrapure water greater than 18.6 MW). If ultrapure water is used, the resultant nanoplates are substantially triangular and have a length of around 40 nm from one vertex to another (see Figure 5). These larger nanoplates have a clear blue color.

Example 3: Construction of a lateral flow test device

We have developed a lateral flow test device for the detection of a certain miRNA, but the device could be adapted for any other protein, DNA structure, molecule or even whole cells and bacteria. The affinity substances used in the present example are single stranded DNAs, but other affinity substances such as antibodies, nanobodies or aptamers could be used instead. The three single stranded DNAs used were: signaling DNA (are attached to the AuNTs with a thiol group in one of the ends of its chain; the thiol group is "protected", the manufacturer, Integrated DNA Technologies, provides the DNA with the thiol in an oxidized form, with S=S bond), capture DNA (for the detection section, with biotin in one of the ends of its chain) and control DNA (for the control section, with biotin in one of the ends of its chain).

100 pF of signaling DNA, at a concentration of 60 pg/mF, are mixed with 10 pF of 0.3 M tris(2- carboxyethyl)phosphine (TCEP) for 2 h at 80 °C while shaking at 650 rpm. Then, 1 mF of AuNTs (at the concentration obtained from the synthesis disclosed in Example 1) is added to the DNA and incubated overnight (22 °C, 650 rpm). The next day, 100 pF of a 0.1 mg/mF BSA solution was added and the mixture was incubated for 1 h. The mixture was centrifuged at 6000 rpm for 20 minutes and the pellet was reconstituted in a 2 mM solution of borate buffer, at pH 7.5, containing 10% (w/v) sucrose. The solution was quickly dispensed over the conjugate pad (glass fiber, Millipore GFDX0008000) and dried in a vacuum chamber for 4 h. This conjugation method results in a labeled detection substance wherein the affinity substance is attached at random locations thereby coating the nanoparticle.

Two lines of a 1 mg/mL solution of streptavidin solution were dispensed at 0.05 pL/mm, at 6 mm of distance of each other, using a lateral flow reagent dispenser manufactured by Imagene Technology on MDI nitrocellulose CNPC-SS 12 paper. The nitrocellulose membrane was attached to a Millipore laminated card HF000MC100. The nitrocellulose membrane was left to dry overnight. The next day, at each of the same positions where the streptavidin lines were dispensed, 1 mg/mL capture DNA or 1 mg/mL control DNA were dispensed at 0.05 pL/mm. The membrane was allowed to dry overnight and then a pad of cellulose fiber Millipore CFSP0017000 comprising the AuNT:ssDNA conjugate was assembled to overlap 1 mm with the nitrocellulose membrane at the end closest to the detection section.

A further cellulose fiber pad which constitutes the sample addition part was soaked with 10 mM PBS buffer at pH 7.5 containing 5% BSA and 0.05% Tween 20 and allowed dry overnight. The sample addition part was assembled to overlap 1 mm with the section comprising the conjugate.

The strips were cut to a width of 5 mm. Such strips are suitable for samples of up to 200 pL in volume.

Example 4: Comparison of a nanoparticle of the present invention with a spheroidal nanoparticle of the prior art

Two types of conjugate pads, one containing AuNPs (spheres made according to Quesada-Gonzalez et al., 2018. Sci Rep. 8: 16157) and the other AuNTs (substantially triangular nanoplates with an edge length of 22-24 nm), were prepared as described:

1) 100 pL ofthiolated DNA probe was mixed with 10 pL 0.3 M TCEP solution and incubated during 1 h at 650 rpm and 20 °C.

2) 1.2 mL of a 0.5 mM (concentration was estimated based on the quantity of HAuCL salt used to obtain the nanomaterial and the volume of the nanomaterial) suspension of nanomaterial (AuNPs or AuNTs) was added and the mixture was incubated for a further hour.

3) The conjugate suspension was centrifuged, the pellet reconstituted in 200 pL of 2 mM borate buffer at pH 7.5 with 10% sucrose, and the mixture was dispensed over a glass fiber pad and dried at room temperature overnight.

The pads were assembled onto two different test devices as explained previously and tested with different concentrations of miRNA. The strips containing AuNPs

-Control line (CL) was visible 2-3 minutes after the addition of the sample, in all the strips.

-Test line was visible 6-8 minutes after the addition of the sample, but only at concentrations over 100 ng/mL of miRNA.

-After 12-15 minutes, the test line (TL) was also visible at concentrations over 10 ng/mL of miRNA.

-After around 20 minutes, TL is visible (very tenuous) in all the strips (even in blank samples).

-Using only the naked-eye to look at the intensity of the TL, the device has a lower detection limit of around 10 ng/mL.

The strips containing AuNTs

-CL is visible 1 minute after the addition of the sample, in all the strips.

-TL is visible 3-6 minutes after the addition of the sample, but only at concentrations over 1 ng/mL of miRNA.

-After 8-9 minutes, TL is also visible at concentrations over 0.1 ng/mL of miRNA.

-After around 30 minutes, TL is visible (very tenuous) in all the strips (even in blank samples).

- Using only the naked-eye to look at the intensity of the TL, the device has a lower detection limit of around 1 ng/mL.

We estimate that at 10 min is the optimal moment to measure the strips.

The different strips were also imaged, and the resulting images were processed using Image! The images were transformed into a “black and white” 8-bit format. ImageJ then measured the average color intensity of the TL of three replicate test devices per miRNA concentration.

The TL intensity (a.u.) value is calculated using the following formula:

[IMG]-[BLK]

Wherein [IMG] is calculated as 255 minus the value provided by the software for each TL, and [BLK] is 255 minus the average value of the TLs of the blanks (0 ng/mL miRNA; i.e. only buffer solution). 255 is the value that the software would give to a pure white measurement, while 0 would be for pure black. The results of this analysis are provided in Figure 6.

The data were then plotted, and a calibration curve was modelled for each set of data (see Figure 7). According to the resulting models, test devices comprising AuNPs had a lower limit of detection of 2 ng/mL and test devices comprising AuNTs had a lower limit of detection of 0.3 ng/mL. Further, comparing the slopes of the two models indicated that the sensitivity obtained using AuNTs is around 250 % greater than when using AuNPs.

Example 5: stability of AuNPs and AuNTs against aggregation in presence of NaCl It is well-known that spherical AuNPs aggregate in the presence of salts in the medium, like NaCl. Thus, it is often necessary to protect the AuNPs with blocking agents (e.g. BSA, OVA or casein) when they are meant to be applied in saline samples, like urine or marine water. Surprisingly, triangular gold nanoplates do not follow this trend.

To evaluate the stability of AuNTs and AuNPs, conjugate pads were prepared following the protocol described in example 3, but omitting the protecting step of the nanoparticles (the addition of BSA):

100 pL of signaling DNA, at a concentration of 60 pg/mL, are mixed with 10 pL of 0.3 M tris(2- carboxyethyl)phosphine (TCEP) for 2 h at 80 °C while shaking at 650 rpm. Then, 1 mL of AuNTs (at the concentration obtained from the synthesis disclosed in Example 1) or AuNPs is added to the DNA and incubated overnight (22 °C, 650 rpm). The next day, the mixture was centrifuged at 6000 rpm for 20 minutes and the pellet was reconstituted in a 2 mM solution of borate buffer, at pH 7.5, containing 10% sucrose and 2 M NaCl (big excess of salt in the medium). The solution was quickly dispensed over the conjugate pad (glass fiber, Millipore GFDX0008000) and dried in a vacuum chamber for 4 h.

The AuNT and AuNP conjugate pads were blue and red respectively. In barely 1 min AuNPs start aggregating and after 10 minutes the conjugate pads are completely black (Fig. 8). Instead, AuNTs remain blue even the day after.

Example 6: synthesis of AuNTs conjugated at the vertices

1 mL of AuNTs synthetized following protocol shown in example 1 were centrifuged and reconstituted in 1 mL of 1 mM cetyltrimethylammonium bromide (CTAB), centrifuged again and reconstituted in only 15 pL of 1 mM CTAB. Then, to the concentrate of AuNPs 203 pL of double distillated water, 581 pL of NN- Dimethylformamide (99% purity), 150 pL of 0.25 mM polystyrene-b-polyacrylic acid and 6 pL of 100 mM 2-methylaminoethanol were added. The mixture was incubated without being shaken for 10 minutes and then 60 pL of 2.74 mM l,2-dipalmytoyl-sn-glycero-3-phosphothioetanol were added. The mixture was heated at 105 °C for 90 min. Once the solution was cooled down, the white precipitate is removed. This step was repeated every 24h until no more white precipitate appeared. At this point, AuNTs were covered by polymer on their edges and planar faces, but not on the vertices. AuNTs in this state should be able to be conjugated with any type of probe (detection substance) and protocol, being that the probe will only be able to interact with the non-covered areas (i.e. the vertices). Example 7: comparison of vertex-labeled AuNTs (vAiiNTs) with “coated” AuNTs (cAiiNTs)

Two types of lateral flow strips are prepared as described on example 3, containing cAuNTs and vAuNTs. The cAuNTs are synthetized as explained in example 1. The vAuNTs have an additional treatment, described on example 6.

It is expected that vAuNTs will provide a better sensitivity than cAuNTs due to a larger nanoparticle: analyte ratio (see Figure IB).

Example 8: synthesis of other polygonal shapes Methods for synthesizing gold nanoparticles of other shapes are known in the art (Krajczewski et al., 2019. RSC Adv. 9(32): 18609-18; Kuo et al., 2004. Langmuir. 20(18):7820-4). The skilled person could readily synthesize gold nanoparticles with suitable shapes for use in the present invention.

Claims

1. A lateral flow test device comprising:

(a) a support membrane;

(b) a labeled detection substance comprising one or more affinity substances attached to a gold nanoparticle characterized in that the nanoparticle has a substantially polyhedron shape comprising at least three vertices, wherein the one or more affinity substances are only attached to the vertices of the nanoparticle; and

(c) one or more affinity substances immobilized on the support membrane.

2. The test device of claim 1, wherein the nanoparticle is a nanoplate having a substantially polygonal shape comprising at least three vertices.

3. The test device of claim 2, wherein the nanoplate has a substantially triangular shape.

4. The test device of any one of claims 1-3, wherein the size of the nanoparticle is 20-50 nm.

5. The test device of any one of claims 1-4, wherein the affinity substance is a nucleic acid and/or a polypeptide.

6. The test device of claim 5, wherein the affinity substance is a single-stranded DNA molecule, nucleic acid aptamer, peptide aptamer, anticalin, repebody, monobody, scFv, antibody, affibody, fynomer, DARPin, peptide nucleic acid, SMART nucleobase, locked nucleic acid or nanobody.

7. The test device of any one of claims 1-6, wherein the affinity substance is attached to the nanoparticle through a gold-sulfur bond.

8. The test device of any one of claims 1-7, wherein the support membrane consists of nitrocellulose, polyvinylidene difluoride or cellulose acetate.

9. A method for detecting an analyte in an isolated sample which comprises contacting the sample with the test device of any one of claims 1-8.

10. Use of a gold nanoparticle having a substantially polyhedron shape comprising at least three vertices for the manufacture of the test device of any one of claims 1-8.

11. Use of a kit comprising:

(i) an affinity substance; and

(ii) a gold nanoparticle having a substantially polyhedron shape comprising at least three vertices for the manufacture of the test device of any one of claims 1-8.

12. The use of claim 11, wherein the nanoparticle is a nanoplate having a substantially polygonal shape comprising at least three vertices, wherein, optionally, the nanoplate has a substantially triangular shape.