Classifications

• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
  • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
  • G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
  • G01N33/54346—Nanoparticles

Definitions

• 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.
• 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.
• 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.
• Figure 1 A) Illustrative diagram of spheroidal vs non-spheroidal particles 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.
• the test device further comprises a plastic support (7).
• FIG. 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.
• FIG. 4 Absorbance spectrum of nanoparticles encompassed by the present invention. These nanoparticles were synthesized in accordance with the method disclosed in Example 1.
• FIG. 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).
• AuNPs spheroidal nanoparticles of the prior art
• AuNTs nanoplate having a substantially triangular shape in accordance with the present invention
• 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.
• 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.
• 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).
• 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
• 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.
• kits 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.
• body 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).
• affinity substance refers to a molecule which has a measurable affinity for and preferentially binds a target.
• 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.
• 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.
• 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).
• DARPin designed ankyrin repeat proteins
• detection section refers to a site where the presence of a target analyte is visually detected.
• edge refers to a particular type of line segment joining two vertices in a polygon or polyhedron.
• an edge is a line segment on the boundary, and is often called a side.
• an edge is a line segment where two faces meet.
• 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).
• 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.
• isolated refers to anything which has been previously extracted from its natural setting.
• 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.
• 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.
• 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,
• heteroatoms e.g., nitrogen, oxygen, sulfur, etc.
• 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.
• 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.
• pH-sensitive linkers protease cleavable peptide linkers
• nuclease sensitive nucleic acid linkers include 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.
• 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).
• 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).
• 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).
• 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.
• 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).
• 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).
• 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).
• 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.
• 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,
• 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).
• 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.
• SMART nucleobase refers to an aldehyde-modified natural nucleobase (Bowler et al., 2010. Angew Chem Int Ed Engl. 49(10): 1809-12).
• a polyhedron can be defined as a solid in three dimensions with flat polygonal faces, straight edges and sharp comers or vertices.
• 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.
• 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.
• a substance e.g., molecule, protein, peptide, miRNA, DNA, virus, whole cell, bacteria, etc.
• vertex refers to the point where two or more edges meet.
• 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 nanoparticle is a nanoplate having a substantially polygonal shape comprising at least three vertices.
• 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.
• 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.
• 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.
• 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.
• 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.
• 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,
• (ii) is a nanoplate having substantially the same shape as a triangle, quadrilateral, pentagon, hexagon, heptagon, octagon, nonagon, decagon, undecagon or dodecagon.
• the nanoparticle :
• (ii) is a nanoplate having substantially the same shape as a triangle, quadrilateral, pentagon or hexagon.
• 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.
• 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.
• 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.
• 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.
• 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.
• 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.
• 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.
• the labeled detection substance further comprises a linker between the nanoparticle and the affinity substance.
• the linker comprises thymidine and/or adenine.
• 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.
• 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.
• 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.
• 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.
• 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.
• 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.
• 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.
• 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.
• 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.
• suitable support membranes include nitrocellulose, polyvinylidene difluoride (PVDF), and cellulose acetate.
• 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.
• 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).
• 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.
• 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.
• the lateral flow test device is the lateral flow test device of the present invention.
• the nanoparticle is a nanoplate having a substantially polygonal shape comprising at least three vertices.
• 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, tetrahemihexa
• (ii) is a nanoplate having substantially the same shape as a triangle, quadrilateral, pentagon, hexagon, heptagon, octagon, nonagon, decagon, undecagon or dodecagon.
• the nanoparticle :
• (ii) is a nanoplate having substantially the same shape as a triangle, quadrilateral, pentagon or hexagon.
• 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.
• 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.
• 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.
• 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.
• the present invention provides a kit comprising an affinity substance and a gold nanoparticle having a substantially polyhedron shape comprising at least three vertices.
• the nanoparticle is a nanoplate having a substantially polygonal shape comprising at least three vertices.
• 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, tetrahemihexa
• (ii) is a nanoplate having substantially the same shape as a triangle, quadrilateral, pentagon, hexagon, heptagon, octagon, nonagon, decagon, undecagon or dodecagon.
• the nanoparticle :
• (ii) is a nanoplate having substantially the same shape as a triangle, quadrilateral, pentagon or hexagon.
• 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.
• 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.
• 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.
• 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.
• 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.
• 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 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.
• a lateral flow test device comprising:
• 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]
• 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]
• a kit comprising:
• a gold nanoparticle having a substantially polyhedron shape comprising at least three vertices.
• the nanoparticle is a nanoplate having a substantially polygonal shape comprising at least three vertices, wherein, optionally, the nanoplate has a substantially triangular shape.
• Example 1 Synthesis of nanoplates having a substantially triangular shape
• CTAC cetyltrimethylammonium chloride
• 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
• 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.
• 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.
• 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).
• 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
• the pads were assembled onto two different test devices as explained previously and tested with different concentrations of miRNA.
• the strips containing AuNPs were assembled onto two different test devices as explained previously and tested with different concentrations of miRNA.
• the strips containing AuNPs were assembled onto two different test devices as explained previously and tested with different concentrations of miRNA.
• test line (TL) was also visible at concentrations over 10 ng/mL of miRNA.
• the device has a lower detection limit of around 10 ng/mL.
• TL is also visible at concentrations over 0.1 ng/mL of miRNA.
• the device has a lower detection limit of around 1 ng/mL.
• 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.
• TL intensity (a.u.) value is calculated using the following formula:
• [IMG] is calculated as 255 minus the value provided by the software for each TL
• [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.
• 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.
• blocking agents e.g. BSA, OVA or casein
• conjugate pads were prepared following the protocol described in example 3, but omitting the protecting step of the nanoparticles (the addition of BSA):
• TCEP tris(2- carboxyethyl)phosphine
• 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
• 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.
• 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.

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