Photonic platform for detection of a biothreat

Abstract

The present disclosure provides a photonic crystal comprising a plurality of mesoscopic particles, an additive, and a moiety specific to a biothreat-marker. The disclosure extends to a kit for detecting a biothreat-marker and a method of determining whether a biothreat-marker of interest is present in a sample.

Description

[0001] Photonic Platform for Detection of a Biothreat

[0002] The present invention relates to a photonic crystal functionalised for biothreat detection.

[0003] Biothreats present a unique risk to public health bodies and security services worldwide.

[0004] Detection and identification of biothreats can be achieved via the detection of biothreat-markers that are produced exclusively by certain biological agents such as bacterial cells, spores, viruses, fungal cells and biotoxins.

[0005] Additionally, detection and identification of biothreats can be achieved via the detection of biothreat-markers associated with pharmaceutical-based agents (PBAs). PBAs are a distinct subset of biothreats, posing unique challenges compared to traditional biothreats. Their potential for large-scale industrial production, coupled with their extreme potency, allows for severe effects or fatalities with minimal quantities. Furthermore, the legitimate medical applications of some PBAs increase the risk of their diversion and accessibility. Their rapid incapacitating or lethal effects make them attractive for malicious use.

[0006] The detection of biothreats is a unique challenge and there is a present need for the development of improved detection techniques (Walper et al., ACS Sensors, 2018, 3, 1894-2024). The detection of biothreats is uniquely challenging for a number of reasons. In particular, threats can be deployed in almost any location and through several routes; a large number of potential threats exist (NATO have identified at least 39 potential threats); many threats also exist as naturally occurring materials so can be easily masked; threats can be engineered to hamper detection; and significant amounts of background biological matter exists which can cloud detection.

[0007] Furthermore, biothreats can be used in low quantities, this adds further complexity as an effective detection system would need to be able to detect low concentration of biothreat-markers. For example, a gram of anthrax possesses the same lethality as a tonne of sarin or sulphur-mustard gas. As such, limits of detection in the range of <1 ng / ml are desirable.

[0008] Current detection methods include enzyme-linked immunosorbent assays (ELISA) (Engvall et al., J Immunol, 1972, 109, 129-135), and those based upon ELISAs such as electrochemiluminescence assays (ECL) (Forster et al., Annu. Rev. Anal. Chem., 2009, 2, 359-385); immune-chip protein assays; and immunochromatographic assays (FLT). Detection methods also include molecular methods such as polymerase chain reaction (PCR) tests and DNA-based micro assays or gene chips. Further detection techniques include the chemical and physical identification of biothreats through mass spectrometry, UV-Visible spectrophotometry, Raman spectroscopy, and surface- enhanced Raman scattering (SERS) (Fleischmann et al., Chem. Phys. Lett., 1974, 26, 163-166).

[0009] Chemical and physical identification routes, although sensitive, require skilled users, specialised equipment, and laboratories. This incurs a high cost, a slow turnaround time which is often in the order of days, and ultimately limited deployment. To facilitate faster responses, and therefore better protections, there is a need for an accessible, affordable, and non-invasive biothreat detection platform which gives the user a rapid response in non-laboratory environments (urban, home, office, etc).

[0010] ELISA based techniques although faster, typically suffer from a lack of specificity and sensitivity. In particular, these immunoassays typically offer sensitivity thresholds that are 100- to 1000-fold below the minimum infectious dose (Zhang et al., Analyst, 2014, 139, 439-445).

[0011] A further problem associated with existing detection techniques and solutions is that they are rarely presented as multiplexed systems. Therefore, due to the wide number of biothreats, these systems are insufficient to effectively protect users and inform decision makers. Additionally, many techniques fail to differentiate live from dead biological threats and have a high probability of false alarm rates. Further drawbacks include the lack of long-term stability of reagents, complex operation which requires trained users, and the standard one-agent-per-test design.

[0012] Lateral flow tests (LFTs) overcome some challenges by offering a simple test that can be conducted in about 15 minutes. However, the readout is a single red line which a user arbitrarily reads by eye, followed by a technician who assigns a 1-5 level of redness to determine the threat level.

[0013] It is apparent that there is a requirement for a simple and multiplexed test which ensures biothreat detection is quantitative and not subjective.

[0014] The present invention has arisen from the inventors' work in attempting to overcome the problems associated with the prior art. In accordance with a first aspect of the invention, there is provided a kit for detecting a biothreat-marker in a sample, the kit comprising: a photonic crystal comprising a plurality of mesoscopic particles and an additive, wherein the additive comprises a zero-dimensional (0D) material, a onedimensional (ID) material and / or a two-dimensional (2D) material; and a moiety specific to the biothreat-marker.

[0015] Advantageously, the photonic crystal will change colour if exposed to the biothreatmarker. Advantageously, the colour change may be a visible colour change. Furthermore, the degree of colour change will also vary depending upon the concentration of the biothreat-marker. Accordingly, the kit of the first aspect may not only be used to determine the presence or absence of a biothreat-marker in a sample but may also be used to determine the concentration thereof.

[0016] The dry glass transition temperature (Tg) of the photonic crystal may be determined by differential scanning calorimetry (DSC). The photonic crystal preferably has a dry glass transition temperature (Tg) of at least 5°C, at least 15°C, at least 25°C, and more preferably at least 30°C, at least 35°C, at least 40°C or at least 50°C. The photonic crystal preferably has a dry glass transition temperature (Tg) of between 5°C and 50°C, between 15°C and 45°C, between 25°C and 40°C, between 30°C and 38°C and more preferably between 33°C and 35°C.

[0017] The photonic crystal may comprise at least 0.005 wt%, at least 0.01 wt%, at least 0.02 wt%, at least 0.05 wt% or at least 0.1 wt% additive. The photonic crystal may comprise between 0.001 and 0.5 wt%, between 0.002 and 0.2 wt%, between 0.005 and 0.1 wt%, between 0.01 and 0.05 wt%, between 0.009 and 0.02 wt% or between 0.0095 and 0.015 wt% additive.

[0018] The weight ratio of the mesoscopic particles to the additive may be between 100:0.001 and 100:0.1, between 100:0.002 and 100:0.08, more preferably between 100:0.004 and 100:0.06, between 100:0.006 and 100:0.04 or between 100:0.007 and 100:0.02, and most preferably between 100:0.008 and 100:0.015 or between 100:0.009 and 100:0.0125 mesoscopic particles:additive.

[0019] Alternatively, or additionally, the volumetric ratio of the mesoscopic particles to the additive may be between 100:0.001 and 100:0.01, more preferably between 100:0.002 and 100:0.08 or between 100:0.003 and 100:0.007, and most preferably between 100:0.004 and 100:0.006 mesoscopic particles:additive. A 0D material may be understood to be a material where all three dimensions of the material are in the nanoscale range. Similarly, a ID material may be understood to be a material where two of the dimensions of the material are in the nanoscale range, and one dimension is above the nanoscale range. A 2D material may be understood to be a material where one dimension is in the nanoscale range and two dimensions are above the nanoscale range.

[0020] A dimension may be understood to be in the nanoscale range if it is less than 200 nm or less than 100 nm. In some embodiments, a dimension may be understood to be in the nanoscale range if it is less than 10 nm. A dimension may be understood to be in the nanoscale range if it is between 0.01 and 200 nm, between 0.1 and 100 nm or between 1 and 10 nm.

[0021] The additive may comprise a 2D material. The additive may consist of a 2D material.

[0022] A 2D material may be understood to have a plate-like shape.

[0023] The term "2D material" can refer to a material with a thickness of a few nanometres or less. Accordingly, the material could have a thickness of 10 nm or less, 5 nm or less or 2 nm or less. The 2D material may comprise of a single layer of atoms. It may be appreciated that a single layer could comprise multiple strata. For instance, molybdenum disulphate comprises a plane of molybdenum ions sandwiched between two planes of sulphide ions. Alternatively, all of the carbon atoms in a layer of graphene are disposed in the same plane, so a single layer of graphene may be viewed as having one stratum. Accordingly, a single layer could comprise between 1 and 5 strata, preferably between 1 and 3 strata. An atom within the single layer of atoms may be covalently bonded to one or more other atoms within the single layer of atoms. In embodiments where the single layer comprises multiple strata, an atom may be covalently bonded to one or more atoms in a different stratum within the single layer of atoms. However, an atom within the single layer of atoms may not be covalently bonded to a further atom with is not in the single layer of atoms.

[0024] Accordingly, the 2D material may comprise a plurality of layers. The plurality of layers may be adjacent to each other. The plurality of layers may not be connected by covalent bonds.

[0025] The 2D material preferably comprises a plurality of particles. Alternatively, or additionally, the plurality of particles may have a mean number of layers between 1 and 20, more preferably between 5 and 15 or between 7 and 13 and most preferably between 9 and 11. The mean number of layers may be determined using UV-Visible spectrophotometry. Alternatively, the mean number of layers may be determined by atomic force microscopy (AFM).

[0026] The plurality of particles may comprise a largest lateral dimension with a mean size of less than 50 pm, less than 30 pm, less than 20 pm, less than 15 pm or less than 10 pm, more preferably a mean size of less than 5 pm or less than 2 pm, and most preferably less than 1 pm. In some embodiments, the plurality of particles may comprise a largest lateral dimension with a mean size of less than 800 nm, less than 600 nm, less than 500 nm, less than 400 nm, less than 300 nm, less than 200 nm or less than 100 nm. The plurality of particles may comprise a largest lateral dimension with a mean size of at least 0.1 nm, at least 1 nm, at least 2 nm, at least 4 nm, at least 6 nm, at least 8 nm or at least 10 nm. The plurality of particles may comprise a largest lateral dimension with a mean size of between 0.1 nm and 50 pm, between 1 nm and 1 pm, between 2 and 800nm, between 4 and 600nm, between 6 and 400nm, between 8 and 200nm or between 10 and 100 nm. It may be appreciated that the lateral dimension is a dimension perpendicular to the thickness of the particle. The lateral dimension may be measured with an atomic force microscope.

[0027] The 2D material may be selected from the group consisting of graphene, graphene oxide (GO), hexagonal boron nitride (h-BN), a transition metal dichalcogenide, an oxide of a transition metal dichalcogenide and combinations thereof. It may be appreciated that a transition metal dichalcogenide may have general formula MX2 where M is a transition metal and X is a chalcogen. The transition metal dichalcogenide may be molybdenum disulphide (M0S2), tungsten disulphide (WS2), molybdenum diselenide (MoSe2), tungsten diselenide (WSe2) or molybdenum(IV) telluride (MoTe2). It may be appreciated that an oxide of a transition metal dichalcogenide may be an oxide of molybdenum disulphide (M0S2), tungsten disulphide (WS2), molybdenum diselenide (MoSe2), tungsten diselenide (WSe2) or molybdenum(IV) telluride (MoTe2).

[0028] The additive may comprise a one-dimensional (ID) material. The additive may consist of a ID material. The ID material may be a nanotube, a nanofiber, a nanohorn, a nano rod or a nanowire.

[0029] Accordingly, the additive may comprise a nanotube, and more preferably comprises or consists of a plurality of nanotubes.

[0030] The, or each, nanotube may be a single-wall nanotube, a double-wall nanotube or multi-wall nanotube. The or each nanotube may be unfunctionalized or functionalised. The nanotube may be selected from the group consisting of a carbon nanotube, an aluminosilicate nanotube and combinations thereof. The aluminosilicate nanotube nanotube may be a halloysite (e.g. Al2Si2Os(OH)4).

[0031] The plurality of nanotubes may comprise a plurality of carbon nanotubes. The plurality of nanotubes may consist of a plurality of carbon nanotubes

[0032] The plurality of nanotubes may comprise a plurality of aluminosilicate nanotubes. The plurality of nanotubes may consist of a plurality of aluminosilicate nanotubes.

[0033] The additive may comprise or consist of a 0D nanomaterial, and more preferably comprise or consist or a plurality of 0D nanomaterials.

[0034] The 0D material may be selected from the group consisting of a metal nanoparticle, a quantum dot, a fullerene, a polymer dot and combinations thereof.

[0035] The fullerene may be a single-layer fullerene or a multi-layered fullerene (i.e. a carbon nano-onion).

[0036] The quantum dot may be selected from the group consisting an inorganic quantum dot, a graphene quantum dot and a carbon quantum dot.

[0037] The inorganic quantum dot may be a transition metal dichalcogenide (TMD) quantum dot, a boron nitride (h-BN) quantum dot or a semiconductor quantum dot. The semiconductor quantum dot may comprise or consist of a group II-VI semiconductor material, a group I-III-VI semiconductor material, a group IV semiconductor material and / or a group III-V semiconductor material. For instance, a group II-VI semiconductor material is CdS or ZnS. An example of a group I-III-VI semiconductor material is CuGaC . A group IV semiconductor material may be an elemental semiconductor, e.g. Si or Ge, or a compound semiconductor, e.g. SiC. The group III- V semiconductor material may be InAs or GaAs.

[0038] The 0D material may have a diameter of between 1 and 200 nm, between 2 and 100 nm, between 3 and 75 nm, between 4 and 60 nm, or between 5 and 50 nm.

[0039] The 0D material may be unfunctionalized or functionalised.

[0040] In a preferred embodiment the 0D material comprises plurality of quantum dots, and more preferably a plurality of carbon quantum dots.

[0041] Preferably, the or each quantum dots is fluorescent. The or each quantum dot may have an excitation wavelength of between 200 and 500 nm, between 300 and 400 nm, between 340 and 390 nm, between, 350 and 380 nm, between 360 and 375 nm, and preferably between 365 and 371 nm, and more preferably between 367 and 369 nm. The or each quantum dot may have an fluorescence wavelength of between 300 and 600 nm, between 350 and 530 nm, between 400 and 480 nm, between, 420 and 460 nm, between 430 and 450 nm, and preferably between 435 and 445 nm, and more preferably between 439 and 441 nm.

[0042] Preferably, the or each quantum dot is quasi-spherical.

[0043] The quantum dots may have a diameter of less than 50 nm, less than 25 nm, less than 20 nm, less than 15 nm and preferably less than 10 nm.

[0044] The additive may be functionalised or unfunctionalized. Advantageously, functionalisation of the additive may vary a chemical or electronic property of the additive. Functionalisation of the additive may vary or improve its selectivity or sensitivity. Functionalisation of the additive may vary or increase the active site density.

[0045] The additive may be functionalised by doping with a functionalisation substance. The additive may be doped with a P or an N type dopant. Alternatively, the additive may be functionalised by decorating with a functionalisation substance.

[0046] Alternatively, the additive may be functionalised by coating with a functionalisation substance. For instance, the 0D material may be functionalised by coating with a functionalisation substance. The functionalised substance may be a polyacrylate coating. The additive may be functionalised with oxygen, a metal or metal containing species, a polymer, an organic molecule or a functional group. In some embodiments, the 0D material, the ID material or the 2D material is functionalised with oxygen, a metal or metal containing species, a polymer, an organic molecule or a functional group.

[0047] The functional group may be -NH2, -COOH, -OH or -SH.

[0048] The organic molecule may be polyethylene glycol (PEG).

[0049] The metal may be or comprise a metal oxide or a noble metal. The metal may be a nanoparticle. Accordingly, the additive may be functionalised with a plurality of metal nanoparticles. The or each metal nanoparticle may have an average diameter of between 1 and 500 nm, between 10 and 400 nm, between 20 and 300 nm, between 25 and 200 nm, between 30 and 100 nm, between 35 and 75 nm, between 40 and 60 nm, or between 45 and 55 nm.

[0050] In one preferred embodiment, the additive is functionalised by doping with oxygen. For instance, the ID material or the 2D material may functionalised by doping with oxygen.

[0051] Doping of the additive with oxygen may result in generation of an oxide species. The additive may be doped with oxygen such that it has an oxide content of between 1 and 50%, between 2 and 40%, between 3 and 30%, between 4 and 25%, or between 5 and 20%.

[0052] In another preferred embodiment, the additive is functionalised by doping with a high surface plasmon resonance material. For instance, the ID material or the 2D material may functionalised by doping with a high surface plasmon resonance material.

[0053] The high surface plasmon resonance material may be a noble metal such as ruthenium, rhodium, palladium, silver, osmium, iridium, platinum, or gold. Preferably, the additive is functionalised with gold or silver. Functionalisation of the additive with gold or silver is advantageous due to their chemical inertness and high SPR response curves. The weight ratio of the additive to the functionalisation substance may be between 100: 1 and 100:50, between 100:2 and 100:30, between 100:3 and 100:25, between 100:4 and 100:20, or between 100:5 and 100: 15 additive:functionalisation substance.

[0054] In embodiments where the additive is a 2D material, the 2D material may be functionalised in or on its basal plane. Alternatively, the 2D material may be functionalised in or on its edge plane. The 2D material may functionalised using a photocata lytic process; a chemical reduction method; a co-evaporation chemical vapor deposition method; or an electrochemical reduction technique. See Chia et al., Doping and Decorating 2D Materials for Biosensing: Benefits and Drawbacks. Adv. Funct. Mater. 2021, 31, 2102555, incorporated by reference in its entirety, for further examples of how the 2D material may be functionalised.

[0055] See Karakoti et al., Surface functionalization of quantum dots for biological applications, Adv. Coll. Int. Sci., 2015, 215, 28-45 for examples of how 0D material may be functionalised.

[0056] The photonic crystal preferably comprises an ordered array of mesoscopic particles.

[0057] The mesoscopic particles may be understood to be mesoscopic if they have a particle size or diameter of between 10 and 1,000 nm.

[0058] The inventors have found that to produce a photonic crystal with a visible colour, a particle size of between approximately 180 and 350 nm is required. Preferably, the plurality of mesoscopic particles have an average particle size of between 50 nm and 1,000 nm or between 100 nm and 500 nm, more preferably between 150 nm and 450 nm, and most preferably is between 160 nm and 400 nm, between 170 nm and 370 nm, between 180 nm and 350 nm, between 190 nm and 330 nm or between 200 nm and 300 nm. The particle size may be determined using a dynamic light scattering technique and / or by atomic force microscopy.

[0059] The inventors have found that if the plurality of mesoscopic particles is substantially monodisperse it can ensure an ordered self-assembly and ensure that the photonic crystal has a sharp stopband. Accordingly, the plurality of mesoscopic particles may be substantially monodisperse.

[0060] The plurality of mesoscopic particles may be viewed as being substantially monodisperse if they have a Polydispersity Index (PDI) from dynamic light scattering (DLS) of less than 0.4, more preferably less than 0.3 or less than 0.2, and most preferably less than 0.1, less than 0.08, less than 0.06 or less than 0.05. The calculation of the PDI from DLS is provided in the ISO standard document ISO 22412:2017.

[0061] Alternatively, or additionally, the plurality of mesoscopic particles may be viewed as being substantially monodisperse if they have a percentage polydispersity of less than 30%, more preferably less than 25%, and most preferably less than 20%. The percentage polydispersity is derived from the PDI.

[0062] The inventors have found that if the plurality of mesoscopic particles comprise some structural disorder, it can have the effect of broadening the photonic crystal stopband and removing iridescence. Accordingly, in some embodiments the plurality of mesoscopic particles may comprise some structural disorder. The plurality of mesoscopic particles may be viewed as comprising some structural disorder if they have a Polydispersity Index (PDI) from dynamic light scattering (DLS) of more than 0.001, more than 0.003, more than 0.006, more than 0.01, more than 0.02, more than 0.03 or more than 0.05.

[0063] Preferably the plurality of mesoscopic particles have a PDI from DLS of between 0.001 and 0.5, between 0.003 and 0.4, between 0.006 and 0.3, between 0.01 and 0.2, between 0.02 and 0.1, between 0.03 and 0.08 or between 0.05 and 0.06.

[0064] In some embodiments, each of the mesoscopic particles is substantially spherical.

[0065] It may be appreciated that the plurality of mesoscopic particles may define a three- dimensional (3D) array. Accordingly, the photonic crystal may be considered a 3D photonic crystal.

[0066] Preferably, the plurality of mesoscopic particles define a close packed structure, and more preferably a hexagonal close packed structure, within the photonic crystal.

[0067] The plurality of mesoscopic particles may define a structure comprising voids. These voids may be configured such that a liquid can diffuse through the photonic crystal. Diffusion of a liquid through the photonic crystal may be enabled by the action of capillary forces. Advantageously, the ability of liquids to diffuse and to flow through the photonic crystal can facilitate an ability to absorb a biothreat-marker within the matrix of the photonic crystal and to modify its spectral response according to the sensitising agent used. The, or each, mesoscopic particle may be or comprise an organic particle or inorganic particle.

[0068] The inorganic particle may be or comprise silica.

[0069] The organic particle may be or comprise a polymer. The polymer may be or comprise a latex polymer.

[0070] In some embodiments, the polymer comprises a carboxylic acid group, a carboxyl group, a carbonate group, an aryl group and / or an alkyl group.

[0071] The carboxyl group may be a C1-12 carboxyl group or a C1-6 carboxyl group. The aryl group may be a Ce-Cio aryl group, and may be phenyl. The alkyl group may be a C1-12 alkyl group, a C1-6 alkyl group or a C1-3 alkyl group.

[0072] The polymer may be a homopolymer made from a plurality of monomers. Alternatively, the polymer may be a copolymer made from a plurality of monomers. In some embodiments, the polymer is a random copolymer made from a plurality of monomers.

[0073] The plurality of monomers may comprise a monomer comprising a carboxylic acid group. The monomer comprising a carboxylic acid group may be an unsaturated carboxylic acid.

[0074] The plurality of monomers may comprise a monomer comprising an aryl group. The aryl group may be as defined above.

[0075] The plurality of monomers may comprise a monomer comprising a carboxyl group.

[0076] The monomer comprising a carboxyl group is preferably an unsaturated ester.

[0077] Accordingly, the plurality of monomers may comprise a compound of formula (I): , wherein R3is H or an optionally substituted C1-C20 straight or branched chain alkyl; and

[0078] R4is H or an optionally substituted C1-C20 straight or branched chain alkyl.

[0079] The alkyl may be unsubstituted or substituted with an optionally substituted carboxyl group and / or an oxo group. The carboxyl group may be unsubstituted or substituted with an oxo group. The carboxyl group may be an optionally substituted C1-12 carboxyl group or an optionally substituted C1-6 carboxyl group.

[0080] R3may H or an optionally substituted C1-C15 straight or branched chain alkyl, more preferably H or an optionally substituted C1-C10 straight or branched chain alkyl and most preferably H or an optionally substituted C1-C5 straight or branched chain alkyl. Accordingly, R3may be H, methyl, ethyl, propyl, butyl or pentyl. In some embodiments, R3is H or methyl.

[0081] R4may be an optionally substituted C1-C15 straight or branched chain alkyl, more preferably an optionally substituted C1-C10 straight or branched chain alkyl and most preferably an optionally substituted C1-C5 straight or branched chain alkyl.

[0082] Accordingly, R4may be an optionally substituted methyl, an optionally substituted ethyl, an optionally substituted propyl, an optionally substituted butyl or an optionally substituted pentyl. In some embodiments, R4is methyl. In some embodiments, R4is butyl. In some embodiments, R4is substituted ethyl.

[0083] The plurality of monomers may comprise methacrylic acid (MAA), acrylic acid (AA), butyl acrylate, methyl methacrylate (MAA), 2-(acetoacetoxy)ethyl methacrylate (AAEM), styrene or a combination thereof.

[0084] In some embodiments, the polymer is a copolymer made from a plurality of monomers comprising acrylic acid and styrene.

[0085] The photonic crystal preferably comprises a surfactant. The surfactant preferably comprises a non-ionic surfactant. Accordingly, the non-ionic surfactant may comprise a structure of formula (III):

[0086] R!-R2

[0087] (HI)

[0088] , wherein R1is a hydrophobic group; and R2is a hydrophilic group. R1may be an optionally substituted Ce-Cio aryl, optionally substituted 5 to 10 membered heteroaryl, an optionally substituted C3-6 cycloalkyl, an optionally substituted 3 to 6 membered heterocycle, an optionally substituted C1-30 alkyl, an optionally substituted C2-30 alkenyl or an optionally substituted C2-30 alkynyl, wherein the alkyl, alkenyl or alkynyl chain is optionally interrupted by one or more heteroatoms. The one or more heteroatoms may be O, S or NH. The optionally substituted Ce-Cio aryl may be an optionally substituted phenyl. Alternatively, R1may be an optionally substituted C10-20 alkyl, an optionally substituted C10-20 alkenyl or an optionally substituted C10-20 alkynyl group.

[0089] The alkyl, alkenyl or alkynyl may be unsubstituted or substituted with a halogen, an oxo group or an OH group.

[0090] The aryl, heteroaryl, cycloalkyl or heterocycle may be unsubstituted or substituted with a halogen, an optionally substituted C1-C20 alkyl, an optionally substituted C2-30 alkenyl and / or an optionally substituted C2-30 alkynyl, wherein the alkyl, alkenyl or alkynyl chain is optionally interrupted by one or more heteroatoms. The one or more heteroatoms may be O, S or NH. The alkyl, alkenyl or alkynyl may be unsubstituted or substituted with a halogen, an oxo group or an OH group. Preferably, the aryl, heteroaryl, cycloalkyl or heterocycle is substituted with a C2-C15 straight or branched chain alkyl, and most preferably with a C3-C10 straight or branched chain alkyl.

[0091] In a preferred embodiment,

[0092] R2may be an optionally substituted C 1-40 alkyl, an optionally substituted C2-40 alkenyl, an optionally substituted C2-40 alkynyl, a substituted Ce-Cio aryl, a substituted 5 to 10 membered heteroaryl, a substituted C3-6 cycloalkyl or a substituted 3 to 6 membered heterocycle, wherein the aryl, heteroaryl, cycloalkyl or heterocycle is substituted with an optionally substituted C 1-40 alkyl, an optionally substituted C2-40 alkenyl and / or an optionally substituted C2-40 alkynyl, wherein the backbone of the alkyl, alkenyl or alkynyl chain is interrupted by one or more heteroatoms and / or the alkyl, alkenyl or alkynyl is substituted with one or more of OH, NH2 or SH. More preferably, R2is an optionally substituted C5-30 alkyl, an optionally substituted C5-30 alkenyl or an optionally substituted C5-30 alkynyl, wherein the backbone of the alkyl, alkenyl or alkynyl chain is interrupted by one or more heteroatoms and / or the alkyl, alkenyl or alkynyl is substituted with one or more of OH, NH2 or SH. Most preferably, R2is an optionally substituted C8-20 alkyl, an optionally substituted C8-20 alkenyl or an optionally substituted C8-20 alkynyl, wherein the backbone of the alkyl, alkenyl or alkynyl chain is interrupted by one or more heteroatoms and / or the alkyl, alkenyl or alkynyl is substituted with one or more of OH, NH2 or SH. The heteroatoms which interrupt the backbone of alkyl, alkenyl or alkynyl may be O, S or NH.

[0093] Preferably, R2comprises oxygen, and more preferably . R2preferably is

[0094] “ , wherein n is an integer between 1 and 50. The non-ionic surfactant may comprise a plurality of molecules of formula (I). Accordingly, n may vary within the plurality of molecules. Preferably, the mean value of n is between 2 and 40 or between 3 and 30, and most preferably is between 5 and 15 or between 7.5 and 12.5.

[0095] Accordingly, in one embodiment, the non-ionic surfactant may comprise The non-ionic surfactant may comprise triton X-100 and / or polysorbate 80. In a preferred embodiment, the non-ionic surfactant comprises triton X-100.

[0096] The weight ratio of the plurality of mesoscopic particles to the non-ionic surfactant may be between 100:0.001 and 100:0.1, between 100:0.002 and 100:0.08, more preferably between 100:0.003 and 100:0.06 or between 100:0.004 and 100:0.04, and most preferably between 100:0.005 and 100:0.015 or between 100:0.009 and 100:0.011 mesoscopic particles: non-ionic surfactant.

[0097] The photonic crystal may comprise a further surfactant. The further surfactant may be configured to stabilise the plurality of mesoscopic particles. Suitable surfactants for use in stabilising mesoscopic particles and / or polymer particles are well known in the art. The further surfactant could be a non-ionic or an anionic surfactant. For instance, examples of surfactants used for emulsion polymerisation include alkyldiphenyloxide disulfonate, alkylphenol ethoxylate, sodium lauryl sulphate and sodium lauryl ether sulphate. The photonic crystal may comprise water. The photonic crystal may comprise at least 0.01 wt%, at least 0.1 wt%, at least 1 wt%, at least 2 wt%, at least 3 wt%, at least 4 wt%, at least 5 wt%, at least 6 wt%, at least 7 wt% at least 8 wt% or at least 9 wt% water. The photonic crystal may comprise less than 30 wt%, less than 25 wt%, less than 20 wt%,less than 18 wt%, less than 16 wt%, less than 14 wt%, less than 12 wt% or less than 11 wt% water. The photonic crystal may comprise between 0.1 and 40 wt% water, between 1 and 35 wt % water, between 2 and 30 wt% water, between 3 and 25 wt% water, between 4 and 20 wt% water, between 5 and 18 wt% water, between 6 and 16 wt% water, between 7 and 14 wt% water, between 8 and 12 wt% water or between 9 and 11 wt% water.

[0098] It may be understood that the term "dry photonic crystal" refers to a dehydrated photonic crystal. The photonic crystal may be understood to be dehydrated if it contains less than contain 10 wt% or less than 5 wt%, and preferably less than 2 wt%, and more preferably less than 1 wt% water. Alternatively, or additionally, the photonic crystal may be understood to be dehydrated if it is substantially transparent.

[0099] The inventors have found that to produce a photonic crystal which undergoes a visible colour change when exposed to the biothreat-marker, a certain thickness of dry photonic crystal is required. The dry photonic crystal may have a thickness of at least 5 pm, at least 7 pm, at least 10 pm, at least 15 pm, at least 20 pm, at least 25 pm, at least 30 pm, at least 40 pm or at least 50 pm. In some embodiments, the dry photonic crystal has a thickness of at least 60 pm, at least 70 pm or at least 80 pm.

[0100] In some embodiments, the dry photonic crystal has a thickness of between 5 pm and 10 mm, between 10 pm and 1 mm, between 20 and 500 pm, between 30 and 300 pm, between 50 and 200 pm, between 60 and 150 pm, between 70 and 125 pm, between 80 and 100 pm, or between 85 and 95 pm. In some embodiments, the dry photonic crystal has a thickness of about 90 pm.

[0101] The plurality of mesoscopic particles may define a bulk section of the photonic crystal. The bulk section of the photonic crystal may comprise the plurality of mesoscopic particles arranged in a 3D array.

[0102] The 3D array of mesoscopic particles may have a close packed structure, and more preferably a hexagonal close packed structure. The 3D array of mesoscopic particles may define interstitial sites between the mesoscopic particles.

[0103] In some embodiments, the bulk section of the photonic crystal comprises the additive. In some embodiments, the additive is disposed at interstitial sites in the bulk section of the photonic crystal.

[0104] In some embodiments, the photonic crystal comprises one or more additive layers, wherein the or each additive layer is disposed on a surface of the bulk section of the photonic crystal. The or each additive layer comprises or consists of the additive. Advantageously, the additive in the or each additive layer is accessible to further components, such as a capture moiety and / or biothreat-marker of interest, which might bind to the additive.

[0105] It may be appreciated that in embodiments where the photonic crystal comprises one or more additive layers, there will be a certain coverage of the additive on one or more surfaces of the bulk section of the photonic crystal.

[0106] The coverage of the additive on a surface of the bulk section of the photonic crystal may be between 0.5 and 100 pg / cm2, between 2 and 80 pg / cm2, between 3 and 70 pg / cm2, between 4 and 60 pg / cm2, between 5 and 50 pg / cm2, between 7.5 and 40 pg / cm2, between 10 and 30 pg / cm2, between 15 and 25 pg / cm2, between 18 and 22 pg / cm2, or between 19 and 21 pg / cm2.

[0107] In some embodiments, the bulk section comprises the additive and the photonic crystal does not comprise an additive layer. It may be appreciated in this embodiment, the additive may only be present in the bulk section.

[0108] In some embodiments, the bulk section comprises the additive, and the photonic crystal further comprises one or more additive layers. It may be appreciated in this embodiment, the additive is present in both the bulk section and in the or each additive layer.

[0109] In further embodiments, the bulk section does not comprise the additive, and the photonic crystal comprises one or more additive layers. It may be appreciated in this embodiment, the additive may only be present in the or each additive layer.

[0110] In some embodiments the photonic crystal comprises one additive layer. In some embodiments, the photonic crystal comprises two or more additive layers.

[0111] Each additive layer may be disposed on a corresponding surface of the bulk section. It may be appreciated that the different additive layers may be disposed on different surfaces of the bulk section to each other.

[0112] In some embodiments, the photonic crystal comprises two additive layers. The first additive layer may be disposed on a first surface. The second additive layer may be disposed on a second surface, which may be different to the first surface. The first and second surfaces may be disposed on opposite sides of the bulk section.

[0113] In embodiments where the photonic crystal comprises water, the bulk section may comprise the water.

[0114] In embodiments where the photonic crystal comprises a surfactant, the bulk section may comprise the surfactant.

[0115] In embodiments where the photonic crystal comprises a further surfactant, the bulk section may comprise the further surfactant.

[0116] In some embodiments, the kit may be configured to detect two or more biothreatmarkers in a sample. Accordingly, the kit may comprise two or more photonic crystals; and two or more binding moieties specific to a biothreat-marker, wherein each moiety is specific to a different biothreat-marker.

[0117] The moiety specific to the biothreat-marker may comprise or be an antibody, or a biothreat-marker-binding fragment thereof, or an aptamer.

[0118] In some embodiments, the moiety specific to the biothreat-marker comprises or is an aptamer.

[0119] The term "aptamer" is used herein to refer to a small artificial ligand, comprising DNA, RIMA or modifications thereof, capable of specifically binding to a biothreat-marker with high affinity and specificity.

[0120] The aptamer may be a DNA aptamer. For example, the aptamer may be formed from single-stranded DNA (ssDNA). Alternatively, the aptamer may be an RNA aptamer. For example, the aptamer can be formed from single-stranded RNA (ssRNA). In some embodiments, the moiety specific to the biothreat-marker is an antibody, or a biothreat-marker-binding fragment thereof.

[0121] The antibody, or biothreat-marker-binding fragment thereof, may be monovalent, divalent or polyvalent. Monovalent antibodies are dimers (HL) comprising a heavy (H) chain associated by a disulphide bridge with a light chain (L). Divalent antibodies are tetramers (H2L2) comprising two dimers associated by at least one disulphide bridge. Polyvalent antibodies may also be produced, for example by linking multiple dimers. The basic structure of an antibody molecule consists of two identical light chains and two identical heavy chains which associate non-covalently and can be linked by disulphide bonds. Each heavy and light chain contains an amino-terminal variable region of about 110 amino acids, and constant sequences in the remainder of the chain. The variable region includes several hypervariable regions, or Complementarity Determining Regions (CDRs), that form the biothreat-marker-binding site of the antibody molecule and determine its specificity for the biothreat-marker. On either side of the CDRs of the heavy and light chains is a framework region, a relatively conserved sequence of amino acids that anchors and orients the CDRs. Antibody fragments may include a bi-specific antibody (BsAb) or a chimeric antigen receptor (CAR).

[0122] The heavy chain constant region typically comprises three domains, CHI, CH2, and CHS. Each light chain typically comprises a light chain variable region (VL) and a light chain constant region. The light chain constant region typically comprises one domain, abbreviated CL.

[0123] Each heavy chain and light chain generally comprise three CDRs and four FRs, arranged in the following order (from N-terminus to C-terminus): FR1 - CDR1 - FR2 - CDR2 - FR3 - CDR3 - FR4. The CDRs are involved in biothreat-marker binding and confer biothreat-marker specificity and binding affinity to the antibody. See Kabat et al., Sequences of Proteins of Immunological Interest 5th ed. (1991) Public Health Service, National Institutes of Health, Bethesda, MD, incorporated by reference in its entirety.

[0124] Preferably, the antibody or biothreat-marker-binding fragment thereof is isolated or purified. The term "biothreat-marker-binding fragment" can mean a region of the antibody having specific binding affinity for its target biothreat-marker, or a variant or fragment thereof.

[0125] One skilled in the art knows that the exact boundaries of a fragment of an antibody are not important, so long as the fragment maintains a functional activity. Using well- known recombinant methods, one skilled in the art can engineer a polynucleotide sequence to express a functional fragment with any endpoints desired for a particular application. A functional fragment of the antibody may comprise or consist of a fragment with substantially the same heavy and light chain variable regions as the human antibody.

[0126] The biothreat-marker-binding fragment thereof may comprise or consist of any one of the biothreat-marker binding region sequences of the VL, any one of the biothreatmarker binding region sequences of the VH, or a combination of VL and VH biothreatmarker binding regions of an antibody. The appropriate number and combination of VH and VL biothreat-marker binding region sequences may be determined by those skilled in the art depending on the desired affinity and specificity and the intended use of the biothreat-marker-binding fragment. Functional fragments or biothreat-markerbinding fragments of antibodies may be readily produced and isolated using methods well known to those skilled in the art. Such methods include, for example, proteolytic methods, recombinant methods and chemical synthesis. Proteolytic methods for the isolation of functional fragments comprise using human antibodies as a starting material. Enzymes suitable for proteolysis of human immunoglobulins may include, for example, papain, and pepsin. The appropriate enzyme may be readily chosen by one skilled in the art, depending on, for example, whether monovalent or bivalent fragments are required. For example, papain cleavage results in two monovalent Fab' fragments that bind antigen and an Fc fragment. Pepsin cleavage, for example, results in a bivalent F (ab') fragment. An F (ab')2 fragment of the invention may be further reduced using, for example, DTT or 2-mercaptoethanol to produce two monovalent Fab' fragments.

[0127] Functional or biothreat-marker-binding fragments of antibodies produced by proteolysis may be purified by affinity and column chromatographic procedures. For example, undigested antibodies and Fc fragments may be removed by binding to protein A. Additionally, functional fragments may be purified by virtue of their charge and size, using, for example, ion exchange and gel filtration chromatography. Such methods are well known to those skilled in the art.

[0128] The antibody or biothreat-marker-binding fragment thereof may be produced by recombinant methodology. Preferably, one initially isolates a polynucleotide encoding desired regions of the antibody heavy and light chains. Such regions may include, for example, all or part of the variable region of the heavy and light chains. Preferably, such regions can particularly include the biothreat-marker-binding regions of the heavy and light chains, preferably the biothreat-marker-binding sites, most preferably the CDRs.

[0129] The polynucleotide encoding the antibody or biothreat-marker-binding fragment thereof according to the invention may be produced using methods known to those skilled in the art. The polynucleotide encoding the antibody or biothreat-markerbinding fragment thereof may be directly synthesized by methods of oligonucleotide synthesis known in the art. Alternatively, smaller fragments may be synthesized and joined to form a larger functional fragment using recombinant methods known in the art.

[0130] It may be appreciated that the term "biothreat" may refer to the threat posed by a harmful biological agent or a pharmaceutical-based agent (PBA). The biothreat may be a biothreat identified by NATO or the United States Centers for Disease Control and Prevention (CDC).

[0131] A biothreat may be a threat posed by a pharmaceutical-based agent (PBA). The PBA may be a chemical based on a pharmaceutical compound. The PBA may cause illness or death when misused. The PBA may have rapid incapacitating or lethal effects. The PBA may have a legitimate human or veterinary medical application. The PBA may be a human or veterinary drug.

[0132] The PBA may be a synthetic opioid, medetomidine, 3-MeO-PCE (N-ethyl-l-(3- methoxyphenyl)cyclohexanamine)), ketamine or a ketamine analogue.

[0133] The synthetic opioid may be fentanyl a fentanyl analogue or a nitazine.

[0134] Fentanyl is a synthetic, lipophilic phenylpiperidine opioid agonist with potent analgesic and anaesthetic properties. The fentanyl analogue may be 2-fluorofentanyl, acetylfentanyl, acrylfentanyl, carfentanil, cyclopropylfentanyl, tetrahydrof uranylfentanyl, furanylfentanyl, ocfentanil, and valerylfentanyl. Nitazenes are a class of synthetic opioids, often significantly more potent than fentanyl, and pose an extreme risk.

[0135] The ketamine analogue may be ethoxetamine ((RS)-2-(ethylamino)-2-(3- methoxyphenyl)cyclohexanone).

[0136] 3-MeO-PCE (N-ethyl-l-(3-methoxyphenyl)cyclohexanamine)) is a dissociative anaesthetic with analgesic and hallucinogenic properties, and has the ability to induce rapid incapacitation.

[0137] A biothreat may be a threat posed by a biological agent. The biological agent may be easily disseminated or transmitted from person to person. For instance, the biological agent may be transmitted in an aerosol. The biological agent may still pose a threat when in an aerosolised form.

[0138] The biothreat may result in high mortality rates. The biothreat may have the potential for major public health impact. The biothreat might cause public panic and / or social disruption. Alternatively or additionally, the biothreat may require special action for public health preparedness.

[0139] The biological agent may be a pathogen or a biotoxin. The pathogen may be a bacterium, a fungus or a virus. It may be appreciated that a "biotoxin" may refer to a toxin produced by an organism. The organism may be a bacteria, a fungus, a plant or an animal.

[0140] The biothreat may be the threat of a disease or poisoning. Accordingly, the biological agent may have the potential to cause a disease or poisoning. The disease or poisoning may be in a human or an animal. The disease may be a contagious disease.

[0141] For example, the disease may be anthrax, botulism, brucellosis, cholera, melioidosis, pneumonic plague, shigella, tularaemia, typhoid fever, typhus, Q fever, rocky mountain spotted fever, scrub fever, psittacosis, coccidioidomycosis, histoplasmosis, a viral haemorrhagic fever, chikungunya fever, dengue fever, eastern equine encephalitis, influenza, rift valley fever, Russian spring-summer encephalitis, smallpox, Venezuelan equine encephalitis, yellow fever, pan-Orthopox virus, Hendra virus or Nipah virus. Typhus may be epidemic typhus, scrub typhus or murine typhus. In some embodiments, typhus is epidemic typhus. A viral haemorrhagic fever may be Argentina haemorrhagic fever, Bolivian haemorrhagic fever (black typhus), Crimean Congo haemorrhagic fever, Ebola, Korean haemorrhagic fever, Lassa, Marburg virus disease (MVD),Omsk haemorrhagic fever, Venezuelan haemorrhagic fever, Brazilian haemorrhagic fever or Chapare haemorrhagic fever.

[0142] The poisoning may be caused by a botulinum toxin, a perfringens toxin, a trichothecene mycotoxin, a palytoxin, ricin, saxitoxin, a staphylococcal enterotoxin, tetrodotoxin, Abrin toxin, tetanus toxin or Shiga toxin.

[0143] It may be appreciated that the term "biothreat-marker" may refer to a measurable indicator of a biothreat. The biothreat-marker may be the biological agent or pharmaceutical-based agent (PBA) which causes the biothreat, or a marker associated therewith. In some embodiments, the biothreat-marker may be transported by air or an aerosol. Suitable biothreat-markers are known in the art.

[0144] The marker associated with a PBA may be a fragment of the PBA. Accordingly, in some embodiments, the biothreat-marker may be a PBA or fragment thereof. Preferably, when the biothreat is associated with a PBA, the bio-threat marker is the PBA.

[0145] The marker associated with a biological agent may be produced by the biological agent. In some embodiments, the marker associated with the biological agent may not be a marker produced by the human body.

[0146] For instance, the biothreat-marker may be an organism, a cell, a virus, a biotoxin, a biological entity produced thereby, or a fragment or indicator thereof. The organism may be a bacterium, a fungus, a plant or an animal. The animal may not be a human. The animal may be a marine animal or an amphibian. The animal may be a coral, a fish, a shellfish, an octopus or an amphibian. The cell may be a bacterial cell, a fungal cell, a plant cell, an animal cell or a spore. In some embodiments, the organism, the cell or the virus may be the biological agent which causes the biothreat. Alternatively, in embodiments where the biological agent which causes the biothreat is a biotoxin, the organism or the cell may produce the biotoxin.

[0147] It may be appreciated that a "spore" may refer to a reproductive cell produced by a fungus, bacteria or plant.

[0148] The biological entity may be an organic molecule. In some embodiments, the biological entity may be a protein or peptide. In further embodiments, the biological entity may be a carbohydrate, phospholipid or nucleic acid molecule. Accordingly, the biothreat-marker may be an antigen.

[0149] In some embodiments, the biothreat-marker is a bacterium, a biological entity produced by the bacterium or a fragment or indicator of the bacterium. The bacterium may be a Bacillus anthracis, Brucella melitensis, Vibrio cholerae, Burkholderia pseudomallei, Yersinia pestis, Shigella dysenteriae, Francisella tularensis, Salmonella typhi, Rickettsia prowazekii, Coxiella burnetiid, Rickettsia rickettsia, Orientia tsutsugamushi, Chlamydophila psittaci, Clostridium botulinum, Clostridium perfringens or Staphylococcus aureus cell.

[0150] In some embodiments, the biothreat-marker is a plant, a plant cell, a biological entity produced by the plant or a fragment or indicator of the plant. The plant may be Ricinus communis.

[0151] In some embodiments, the biothreat-marker is a virus, a biological entity produced by the virus, or a fragment or indicator of the virus. The virus may be Junin virus, Machupo virus, chikungunya virus, CCHF virus, dengue virus, an Ebola virus, eastern equine encephalitis virus (EEEV), influenza virus, hantavirus, Lassa virus, Omsk haemorrhagic fever virus, rift valley fever virus, tick-borne encephalitis virus, variola major, Venezuelan equine encephalitis virus, yellow fever virus, pan-Orthopox virus, Hendra virus, Nipah virus, Chapare virus, Guanarito virus or Sabia virus .

[0152] In some embodiments, the biothreat-marker is a fungus, a fungal cell, a biological entity produced by the fungus or a fragment or indicator of the fungus. The fungus may be Coccidioides immitis or Histoplasma capsulatum.

[0153] In some embodiments, the biothreat-marker is a biotoxin or fragment thereof. The bioxtoxin may be a chemical compound. The biotoxin may be a botulinum toxin, a perfringens toxin, a trichothecene mycotoxin, palytoxin, ricin, saxitoxin, a staphylococcal enterotoxin or tetrodotoxin.

[0154] In some embodiments, the biothreat-marker is a spore.

[0155] In some embodiments, the biothreat may be a Class A biothreat. Class A biothreats are high-priority agents which pose the highest risk. Class A biothreats may typically be characterised by their ease of person-to-person transmission, high mortality rates, potential for major public health impacts, ability to cause panic and social disruption and their requirement for special action for public health preparedness. In one embodiment, the biothreat is anthrax, pneumonic plague, botulinum toxin poisoning (i.e. botulism), smallpox, tularaemia or a viral haemorrhagic fever.

[0156] In embodiments where the biothreat is anthrax, the biothreat-marker may be Bacillus antrantcis or a fragment or indicator thereof. The biothreat-marker may be anthrax protective antigen (PA).

[0157] In embodiments where the biothreat is pneumonic plague, the biothreat-marker may be Yersinia pestis or a fragment or indicator thereof. The biothreat-marker may be Yersinia pestis fraction 1 (Fl) antigen.

[0158] In embodiments where the biothreat is botulinum toxin poisoning, the biothreatmarker may be Clostridium botulinum or a fragment or indicator thereof. The biothreat-marker may be botulinum neurotoxin type A (BotNT A), botulinum neurotoxin type B (BotNT B), botulinum neurotoxin type C (BotNT C), botulinum neurotoxin type D (BotNT D), botulinum neurotoxin type E (BotNT E) or botulinum neurotoxin type F (BotNT F).

[0159] In embodiments where the biothreat is smallpox, the biothreat-marker may be Variola major or a fragment or indicator thereof. The biothreat-marker may be Variola major outer surface protein membrane A27.

[0160] In embodiments where the biothreat is tularaemia, the biothreat-marker may be Francisella tularensis or a fragment or indicator thereof. The biothreat-marker may be Francisella tularensis lipopolysaccharide (LPS).

[0161] In embodiments where the biothreat is a viral haemorrhagic fever, then the viral haemorrhagic fever may be Ebola. Accordingly, the biothreat-marker may be an Ebola virus or a fragment or indicator thereof. The biothreat-marker may be Ebola virus glycoprotein (GP) integral membrane protein or an Ebola virus structural viral protein (VP). The Ebola virus structural viral protein may be VP40 or VP24.

[0162] In embodiments where the kit is configured to detect two or more biothreat-markers in a sample, each biothreat-marker may be a measurable indicator for the same biothreat. Advantageously, the kit may therefore provide a level of self-validation. Alternatively, each biothreat-marker may be a measurable indicator for a different biothreat.

[0163] The moiety specific to the biothreat-marker may comprise or be a detection moiety. In embodiments where the kit is be configured to detect two or more biothreatmarkers, the kit may comprise two or more detection moieties, wherein each detection moiety is specific to a different biothreat-marker.

[0164] A detection moiety may be understood to comprise or be a moiety specific to a biothreat-marker, as defined above, which may bind specifically to a biothreat-marker immobilised in or on the photonic crystal.

[0165] In some embodiments, the detection moiety comprises or is an antibody, or a biothreat-marker-binding fragment thereof. In some embodiments, the detection moiety comprises or is an aptamer.

[0166] In some embodiments, the detection moiety is specific for ketamine or fentanyl.

[0167] Accordingly, the detection moiety may be ketamine monoclonal antibody (4E2) or a fentanyl monoclonal antibody.

[0168] In some embodiments, the detection moiety is specific for anthrax protective antigen (PA), Yersinia pestis fraction 1 (Fl) antigen, BotNT A, BotNT B, BotNT C, BotNT D, BotNT E, BotNT F, Variola major outer surface protein membrane A27, Francisella tularensis lipopolysaccharide (LPS), an Ebola virus glycoprotein (GP) integral membrane protein or an Ebola virus structural viral protein (VP).

[0169] Accordingly, the detection moiety may be an anti-PA antibody, an anti-Fl antibody, an anti-BotNT A antibody, an anti-BotNT B antibody, an anti-BotNT C antibody, an anti- BotNT D antibody, an anti-BotNT E antibody, an anti-BotNT F antibody, an antivaccina antibody, an anti-E tularensis antibody, an F. tularensis IgG, IgM or IgA antibody, or a VP40 antibody.

[0170] Antibodies specific to biothreats are known in the art.

[0171] In particular, antibodies, and biothreat-marker-binding fragments thereof, specific for anthrax protective antigen (PA), Yersinia pestis Fl antigen, botulinum neurotoxin type A, B, C, D, E and F (BotNT A-F), Variola major outer surface protein membrane A27, Francisella tularensis lipopolysaccharide (LPS), Ebola virus glycoprotein (GP) integral membrane proteins, and Ebola virus structural viral proteins (VP) VP40,VP24, ketamine and fentanyl are known in the art.

[0172] The kit may comprise a detection moiety solution comprising the detection moiety. In embodiments where the kit comprises two or more detection moieties, the detection moiety solution may comprise the two or more detection moieties. Alternatively, the kit may comprise two or more detection moiety solutions, wherein each detection moiety solution comprises a different detection moiety.

[0173] The detection moiety solution may comprise between 0.5 and 3.5 pg / mL, between 1 and 3 pg / mL, between 1.5 and 2.5 pg / mL or between 1.75 and 2.25 pg / mL of the or each detection moiety.

[0174] The detection moiety solution may comprise a buffer configured to stabilise the moiety specific to the biothreat-marker. The buffer may be or comprise phosphate-buffered saline (PBS).

[0175] The detection moiety may comprise a detection label. For example, the detection moiety may comprise an antibody, or a biothreat-marker-binding fragment thereof, or an aptamer conjugated to the detection label.

[0176] The detection label may be or comprise a detection enzyme, a fluorophore and / or a metal nanoparticle.

[0177] The detection enzyme may be an enzyme configured to catalyse the reaction of a chromogenic substrate. In some embodiments, the detection enzyme may be configured to catalyse the oxidation of a chromogenic substrate. Accordingly, the detection enzyme may be horseradish peroxidase (HRP) or alkaline phosphatase (Alk- Phos).

[0178] The metal nanoparticle may be a gold nanoparticle.

[0179] The kit may comprise a secondary detection reagent configured to react with or in the presence of the detection moiety. For instance, the detection moiety may be configured to catalyse a reaction of the secondary detection reagent. The secondary detection reagent may be configured to undergo a colour change upon contact with the detection moiety. The secondary detection reagent may be a chromogenic substrate configured to undergo a colour change upon contact with the detection moiety.

[0180] The secondary detection reagent may be specific to the detection moiety. The secondary detection reagent may be specific to the detection label. In some embodiments, the secondary detection reagent is specific to the detection enzyme.

[0181] In some embodiments, the secondary detection reagent is a chromogenic substrate, which is a substrate for the detection enzyme.

[0182] In some embodiments, the chromogenic substrate may be a substrate for horseradish peroxidase (HRP). Accordingly, the chromogenic substrate may be 3, 3', 5,5'- tetramethylbenzidine (TMB), o-phenylenediamine dihydrochloride (OPD) or 2,2'-azino- di-[3-ethyl-benzothiazoline-6 sulfonic acid] diammonium salt (ABTS). In some embodiments, the chromogenic substrate is 3,3',5,5'-tetramethylbenzidine (TMB). In alternative embodiments, the chromogenic substrate may be a substrate for alkaline phosphatase. Accordingly, the chromogenic substrate may be p-nitrophenyl phosphate (PnPP).

[0183] In some embodiments, the kit may comprise a secondary detection solution comprising the secondary detection reagent. The secondary detection solution may comprise a solvent. The solvent may be water.

[0184] The secondary detection solution may further comprise an oxidizing agent. The oxidising agent may be hydrogen peroxide.

[0185] In some embodiments, the chromogenic substrate is p-nitrophenyl phosphate (PnPP) or 3,3',5,5'-tetramethylbenzidine (TMB), o-phenylenediamine dihydrochloride (OPD) 2,2'-azino-di-[3-ethyl-benzothiazoline-6 sulfonic acid] diammonium salt (ABTS) .

[0186] Alternatively, or additionally, the moiety specific to the biothreat-marker may comprise or be a capture moiety. In embodiments where the kit is configured to detect two or more biothreat-markers, the kit may comprise two or more capture moieties wherein each capture moiety is specific to a different biothreat-marker.

[0187] A capture moiety may be understood to comprise or be a moiety specific to a biothreat-marker, as defined above, which may be immobilised in or on the photonic crystal. In some embodiments, the capture moiety comprises or is an antibody, or a biothreatmarker-binding fragment thereof. In some embodiments, the capture moiety comprises or is an aptamer.

[0188] In embodiments where the kit is configured to detect two or more biothreat-markers in a sample, the kit may comprise two or more capture moieties, wherein each capture moiety is specific to a different biothreat-marker. The different biothreatmarkers may be the same biothreat-markers that the detection moieties, are specific to.

[0189] In a preferred embodiment, the kit comprises a capture moiety specific to the biothreat-marker and a detection moiety specific to the biothreat-marker. It may be understood that the capture moiety and the detection moiety may be specific to the same biothreat-marker. In embodiments where the kit is be configured to detect two or more biothreat-markers, the kit may comprise two or more pairs of capture and detection moieties, wherein of the capture and detection moieties in each pair are specific to the same biothreat-marker as each other and to a different biothreatmarker to the or each other pair of capture and detection moieties.

[0190] As noted above, antibodies, and biothreat-marker-binding fragments thereof, specific for biothreat-markers are known in the art.

[0191] In some embodiments, the photonic crystal comprises the capture moiety. In embodiments where the kit is configured to detect two or more biothreat-markers in a sample, the two or more photonic crystals may comprise the two or more capture moieties, wherein each photonic crystal comprises a different capture moiety.

[0192] The capture moiety is preferably bound to the additive in the photonic crystal.

[0193] The photonic crystal may comprise at least 0.001 wt %, at least 0.005 wt%, at least 0.01 wt%, at least 0.012 wt% or at least 0.015 wt% capture moiety. The photonic crystal may comprise between 0.001 and 0.05 wt%, between 0.005 and 0.03 wt%, between 0.01 and 0.025 wt% or between 0.016 and 0.02 wt% capture moiety.

[0194] Alternatively, the kit may comprise a capture moiety solution comprising the capture moiety. In embodiments where the kit comprises two or more capture moieties, the kit may comprise two or more capture moiety solutions, wherein each capture moiety solution comprises a different capture moiety.

[0195] The capture moiety solution may comprise between 1 and 20 pg / mL, between 5 and 15 pg / mL, between 8 and 12 pg / mL or between 9 and 11 pg / mL of the or each capture moiety.

[0196] The capture moiety solution may comprise a buffer configured to stabilise the capture moiety. The buffer may be or comprise phosphate-buffered saline (PBS).

[0197] Preferably, the kit further comprises a blocking agent. In some embodiments, the photonic crystal comprises the capture moiety and the blocking agent. In embodiments where the kit comprises two or more photonic crystals, each photonic crystal may comprise the blocking agent. Preferably, the blocking agent is bound to the additive. The blocking agent may be configured to block binding sites on the additive.

[0198] In embodiments in which the photonic crystal comprises the capture moiety and the blocking agent, the concentration of blocking agent in the photonic crystal may be between 0.001 and 0.5 wt% between 0.005 and 0.2 wt%, between 0.01 and 0.1 wt% or between 0.03 and 0.05 wt%.

[0199] In some embodiments, the blocking agent is or comprises a protein and / or a polysorbate. The protein may be or comprise bovine serum albumin. The polysorbate may be or comprise polysorbate 20.

[0200] Alternatively, the kit may comprise a blocking solution comprising the blocking agent. The blocking solution may comprise at least 0.01% (w / v), at least 0.1% (w / v), at least 0.2% (w / v), at least 0.4% (w / v) or at least 0.5% (w / v) blocking agent. The blocking solution may comprise between 0.01 and 25% (w / v), between 0.05 and 10% (w / v), between 0.1 and 5% (w / v), between 0.2 and 2% (w / v), between 0.4 and 1.5% (w / v), or between 0.5 and 1% (w / v) blocking agent.

[0201] Preferably, the kit further comprises a washing solution.

[0202] The washing solution may comprise a detergent. The detergent may be a polysorbate, e.g. polysorbate 20. The washing solution may comprise at least 0.0001% (w / v), at least 0.001% (w / v), at least 0.005% (w / v), at least 0.01% (w / v), at least 0.05% (w / v) or at least 0.08% (w / v) detergent. The washing solution may comprise between 0.0001 and 10% (w / v), between 0.001 and 5% (w / v), between 0.005 and 1% (w / v), between 0.01 and 0.5% (w / v), between 0.05 and 0.2% (w / v) or between 0.08 and 0.15% (w / v) detergent.

[0203] The washing solution may further comprise a buffer configured to stabilise the moiety specific to the biothreat-marker. The buffer may comprise phosphate-buffered saline (PBS).

[0204] In some embodiments, the kit further comprises a stop solution. The stop solution may comprise a reagent configured to inhibit interaction between the detection moiety and the secondary detection reagent. For example, in embodiments where the detection moiety comprises a detection enzyme, the stop solution may comprise a reagent configured to inhibit activity of the detection enzyme. In some embodiments, the reagent may be or comprise an acid or a base.

[0205] For instance, if the detection enzyme is horseradish peroxidase (HRP) the stop solution may comprise an acid. The acid may be sulfuric acid. Alternatively, if the detection enzyme is alkaline phosphatase (Alk-Phos), the stop solution may comprise a base. The base may be sodium hydroxide.

[0206] The kit may further comprise a substrate. The substrate may comprise a polymer. The polymer may be a thermoplastic polymer. The thermoplastic polymer may be a polycarbonate, poly(methyl methacrylate), poly(ethylene terephthalate), polypropylene, acrylonitrile butadiene styrene or poly(lactic acid) . In some embodiments, the substrate is poly(ethylene terephthalate).

[0207] The substrate may define a sheet or film. The substrate may have a thickness of between 0.05 and 0.3 mm, between 0.1 and 0.25 mm, between 0.15 and 0.2 mm, between 0.16 and 0.19 mm or between 0.17 and 0.18 mm.

[0208] Preferably, the substrate is transparent.

[0209] The photonic crystal may be disposed on the substrate.

[0210] In embodiments where the photonic crystal comprises an additive layer, the additive layer of the photonic crystal may be disposed between the substrate and the bulk section. Accordingly, the additive layer may be disposed adjacent to the substrate. Alternatively, the bulk section may be disposed adjacent to the substrate.

[0211] Accordingly, the bulk section may be disposed between the substrate and the additive layer. The additive layer may be disposed on a first side of the bulk section and a second opposing side of the bulk section may be disposed adjacent to the substrate.

[0212] In embodiments where the photonic crystal comprises two or more additive layers, a first additive layer may be disposed on a first side of the bulk section; and a second additive layer may be disposed on a second opposing side of the bulk section. The second additive layer may be disposed between the substrate and the bulk section.

[0213] The kit may comprise a test strip comprising: the photonic crystal; an input port configured to receive a liquid; and a first channel extending between the input port and the photonic crystal.

[0214] The first channel may be configured to transport a liquid from the input port to the photonic crystal.

[0215] The first channel may be a microfluidic channel.

[0216] The input port may comprise a sample pad. The sample pad may be configured to act as a filter to aid flow of the sample. The sample pad may be treated to adjust a property of the sample, e.g. pH or viscosity.

[0217] The test strip may further comprise an adsorption pad and a second channel extending between the photonic crystal and the adsorption pad. The adsorption pad is preferably configured to absorb fluid, and more preferably a liquid. The adsorption pad preferably comprises a porous material.

[0218] The second channel may be configured to transport a liquid from the photonic crystal to the adsorption pad. The wicking pad may be disposed downstream of the photonic crystal. The second channel may be a microfluidic channel. The photonic crystal may be disposed between the input port and the adsorption pad. The input port may be disposed substantially adjacent a first end of the test strip and the adsorption pad may be disposed substantially adjacent to a second end of the test strip, wherein the second end is opposite the first end. The first and second channels may be configured to enable or cause a liquid to flow in a direction substantially from the first end to the second end.

[0219] The inventors believe that their photonic crystal is novel and inventive per se.

[0220] Accordingly, in accordance with a second aspect, there is provided a photonic crystal comprising a plurality of mesoscopic particles, an additive and a capture moiety specific to a biothreat- marker, wherein: the additive comprises a zero-dimensional (0D) material, a one-dimensional (ID) material and / or a two-dimensional (2D) material; and the capture moiety specific to the biothreat-marker is bound to the additive.

[0221] The photonic crystal, plurality of mesoscopic particles, additive, capture moiety and biothreat-marker may be defined as described in relation to the first aspect.

[0222] The photonic crystal may further comprise a blocking agent. The blocking agent may be defined as described in relation to the first aspect.

[0223] Advantageously, the inventors have found that the photonic effects of photonic crystals can be used to aid the detection of biothreat-markers. In particular, binding of substrates to a photonic crystal can result in a change in the effective refractive index, and therefore visible colour, of the photonic crystal. Advantageously, such visible colour changes can be detected using direct and simple detection means.

[0224] Accordingly, in accordance with a third aspect, there is provided a method of determining whether a biothreat-marker of interest is present in a sample, the method comprising: providing a photonic crystal, wherein the photonic crystal comprises a plurality of mesoscopic particles and an additive, wherein the additive comprises a zerodimensional (0D) material, a one-dimensional (ID) material and / or a two- dimensional (2D) material;

[0225] - contacting the photonic crystal and the sample;

[0226] - further contacting the photonic crystal with a detection moiety, wherein the detection moiety is configured to bind specifically to the biothreat-marker of interest; and determining whether or not the detection moiety has bound to the biothreatmarker of interest, and thereby determining whether or not the biothreatmarker of interest is present in the sample.

[0227] The photonic crystal, plurality of mesoscopic particles, additive, detection moiety and biothreat-marker of the third aspect may be defined as described in relation to the first aspect.

[0228] Determining whether or not the detection moiety has bound to the biothreat-marker of interest may comprise determining whether or not a colour change has occurred. If a colour change is observed in the photonic crystal, it may be appreciated that the biothreat-marker of interest is present in the sample.

[0229] In some embodiments, the colour is a visible colour change.

[0230] In some embodiments, the colour change does not comprise fluorescence.

[0231] In accordance with a fourth aspect, there is provided a method of determining whether a biothreat-marker of interest is present in a sample, the method comprising: providing a photonic crystal, wherein the photonic crystal comprises a plurality of mesoscopic particles, an additive and a capture moiety, wherein the additive comprises a zero-dimensional (0D) material, a one-dimensional (ID) material and / or a two-dimensional (2D) material and the capture moiety is configured to bind specifically to the biothreat-marker of interest;

[0232] - contacting the photonic crystal and the sample; and determining whether or not the capture moiety has bound to the biothreatmarker of interest, and thereby determining whether or not the biothreatmarker of interest is present in the sample.

[0233] The photonic crystal, plurality of mesoscopic particles, additive, capture moiety and biothreat-marker of the fourth aspect may be defined as described in relation to the first aspect.

[0234] The methods of the third and fourth aspects may be conducted using the kit of the first aspect and / or the photonic crystal of the second aspect.

[0235] Determining whether or not the capture moiety has bound to the biothreat-marker of interest may comprise determining whether or not a colour change has occurred. If a colour change is observed in the photonic crystal, it may be appreciated that the biothreat-marker of interest is present in the sample.

[0236] In some embodiments, the colour is a visible colour change.

[0237] In some embodiments, the colour change does not comprise fluorescence.

[0238] In a preferred embodiment, the methods of the third and fourth aspect may be combined. Accordingly, the method may comprise: providing a photonic crystal, wherein the photonic crystal comprises a plurality of mesoscopic particles, an additive and a capture moiety, wherein the additive comprises a zero-dimensional (0D) material, a one-dimensional (ID) material and / or a two-dimensional (2D) material and the capture moiety is configured to bind specifically to the biothreat-marker of interest;

[0239] - contacting the photonic crystal and the sample;

[0240] - further contacting the photonic crystal with a detection moiety, wherein the detection moiety is configured to bind specifically to the biothreat-marker of interest; and determining whether or not the capture and / or detection moieties have bound to the biothreat-marker of interest, and thereby determining whether or not the biothreat-marker of interest is present in the sample.

[0241] The method may comprise determining whether two or more biothreat-markers of interest are present in the sample. Accordingly, the method may comprise: providing two or more photonic crystals, each photonic crystal comprising a plurality of mesoscopic particles, an additive and a capture moiety, wherein the additive comprises a zero-dimensional (0D) material, a one-dimensional (ID) material and / or a two-dimensional (2D) material and the capture moiety is configured to bind specifically to the biothreat- marker of interest and the two or more photonic crystals each comprise a different capture moiety, wherein each different capture moiety is configured to bind specifically to a different biothreat-marker of interest to the other capture moieties;

[0242] - contacting each photonic crystal with the sample;

[0243] - further contacting the two or more photonic crystals with two or more detection moieties, wherein each detection moiety is configured to bind specifically to a different biothreat-marker of interest; and determining whether or not the capture and / or detection moieties have bound to the biothreat-marker of interest, and thereby determining whether or not each biothreat-marker of interest was present in the sample.

[0244] The sample preferably is a fluid. It will be appreciated that "fluid" may refer to a liquid, gas or aerosol. In some embodiments, the sample is a liquid sample. The liquid sample may be a solution or a suspension. In some embodiments, the sample is an aerosol sample.

[0245] In some embodiments, the sample may be or comprise an environmental sample. For example, the sample may be or comprise a soil sample, a water sample, an air sample, a food sample or other environmental material.

[0246] In some embodiments, the sample may be or comprise a biological sample. It will be appreciated that "biological sample" may refer to sample collected from an organism. The organism may be a human or animal. In some embodiments, the organism is an animal. In some embodiments, the organism is not a human. For example, the sample may be or comprise a blood sample, a urine sample, a tissue sample, a sweat sample, a saliva sample, a semen sample, a faeces sample, a vaginal fluid or tissue sample.

[0247] In some embodiments, the sample does not comprise a biological sample.

[0248] Alternatively, the method may comprise preparing a liquid sample. The liquid sample may be prepared from a primary sample using methods known in the art. Once prepared, the liquid sample may be used in the above method. In particular, the liquid sample may be contacted with the photonic crystal, as described above.

[0249] The liquid sample may be prepared by contacting a primary sample and a solvent to thereby extract a biothreat or biothreat-marker, if present, from the primary sample into the liquid, and thereby provide the liquid sample. Alternatively, the liquid sample may be prepared by condensing or centrifuging the primary sample to provide the liquid sample. In a further alternative, the liquid sample may be prepared by filtering the primary sample, contacting a residue obtained during the filtration step and a liquid, and thereby providing the liquid sample. The primary sample may be a solid, a gas, an aerosol or a liquid. In some embodiments, the primary sample is a gas or an aerosol. The primary sample may be an environmental sample or a biological sample, as defined above. The sample and / or the primary sample may be collected using methods known in the art. For example, in embodiments where the sample is or comprises a food sample, the food sample may be collected by wipe sampling or swab sampling. In embodiments where the sample comprises an aerosol sample, the sample may be collected using an impingement, centrifugal, or impaction method. It may be appreciated that samplers are known in the art and are commercially available.

[0250] In embodiments where the photonic crystal comprises a capture moiety. The photonic crystal may further comprise a blocking agent. The capture moiety and the blocking agent may be as defined in relation to the first aspect.

[0251] The method may comprise contacting the photonic crystal and the capture moiety. In some embodiments, the method may comprise contacting the photonic crystal and a capture moiety solution. The capture moiety solution may be as defined in relation to the first aspect. The capture moiety may bind to the additive in the photonic crystal.

[0252] In embodiments where the method comprises determining whether two or more biomarkers of interest are present in the sample, the method may comprise contacting two or more photonic crystals with two or more capture moieties, wherein each photonic crystal is contacted with a different capture moiety, and each different capture moiety is specific to a different biomarker.

[0253] The method may comprise contacting the photonic crystal and the capture moiety for at least 1 minute, at least 5 minutes, at least 10 minutes, at least 20 minutes, at least 30 minutes or at least 40 minutes. The method may comprise contacting the photonic crystal and the capture moiety for between 1 minute and 24 hours, between 5 minutes and 12 hours, between 10 minutes and 6 hours, between 20 minutes and 2 hours, between 30 and 60 minutes or between 40 and 45 minutes.

[0254] Subsequent to contacting the photonic crystal and the capture moiety, the method may comprise contacting the photonic crystal and the blocking agent. The method may comprise contacting the photonic crystal and a blocking agent solution. The blocking agent solution may be as defined in relation to the first aspect. In embodiments where the method comprises providing two or more photonic crystals, the method may comprise contacting each photonic crystal and the blocking agent. The blocking agent may bind to the additive. The method may comprise contacting the photonic crystal and the blocking agent for at least 1 minute, at least 5 minutes, at least 10 minutes, at least 20 minutes or at least 30 minutes. The method may comprise contacting photonic crystal and the blocking agent for between 1 minute and 24 hours, between 5 minutes and 12 hours, between 10 minutes and 3 hours, between 20 minutes and 1 hours or between 30 and 45 minutes.

[0255] Between contacting the photonic crystal and the capture moiety and contacting photonic crystal and the blocking agent, the method may comprise rinsing the photonic crystal. Rinsing the photonic crystal may comprise contacting the photonic crystal with a washing solution. The washing solution may be as defined in relation to the first aspect. The method may comprise removing the washing solution from the photonic crystal. In embodiments where the method comprises providing two or more photonic crystals, the method may comprise rinsing each photonic crystal.

[0256] Between contacting the photonic crystal and the blocking agent and contacting the photonic crystal and the sample, the method may comprise rinsing the photonic crystal. Rinsing the photonic crystal may be as defined above.

[0257] The photonic crystal and the sample may be contacted for at least 1 minute, at least 5 minutes, at least 10 minutes, at least 30 minutes, at least 45 minutes or at least 1 hour. The photonic crystal and the sample may be contacted for between 1 minutes and 24 hours, between 5 minutes and 12 hours, between 10 minutes and 6 hours, between 30 minutes and 2 hours or between 45 and 75 minutes.

[0258] If present in the sample, a biothreat-marker of interest may bind to the capture moiety, in embodiments where a capture moiety is present. Alternatively, in embodiments where a capture moiety is not used, the biothreat-marker of interest, if present in the sample, may bind to the additive.

[0259] Prior to contacting the photonic crystal and the sample, the photonic crystal may be dehydrated. It may be understood that the term "dry photonic crystal" refers to a dehydrated photonic crystal. The photonic crystal may be understood to be dehydrated if it contains less than contain 5-10 wt% or less than 5 wt%, and preferably less than 2 wt%, and more preferably less than 1 wt% water. Alternatively, or additionally, the photonic crystal may be understood to be dehydrated if it is substantially transparent. Contacting the photonic crystal and the sample may cause the photonic crystals to be rehydrated, and thereby cause a colour change. The method may comprise checking that a colour change occurs when the photonic crystal and the sample are contacted. If no colour change occurs, the method may be terminated at this stage as being invalid.

[0260] Between contacting the photonic crystal and the sample and the photonic crystal and the detection moiety, the method may comprise contacting the photonic crystal and a blocking agent. The method may comprise contacting the photonic crystal and the blocking agent subsequent to contacting the photonic crystal and the sample in embodiments where the photonic crystal does not comprise a capture moiety and the method does not comprise contacting the photonic crystal and a capture moiety. Contacting the photonic crystal and the blocking agent may be as defined above.

[0261] Between contacting the photonic crystal and the sample and contacting the photonic crystal and the blocking agent or the detection moiety, the method may comprise rinsing the photonic crystal. Rinsing the photonic crystal may be as defined above.

[0262] It may be appreciated that if the biothreat-marker of interest is present it may have bound to either the capture moiety (in embodiments where it is present) or the additive (in embodiments where the capture moiety is not present). Accordingly, the photonic crystal may continue to comprise the biothreat-marker of interest after the rinsing step.

[0263] Between contacting the photonic crystal and the blocking agent and contacting the photonic crystal and the detection moiety, the method may comprise rinsing the photonic crystal. Rinsing the photonic crystal may be as defined above.

[0264] In some embodiments, the method may comprise contacting the photonic crystal and a detection moiety solution. The detection moiety solution may be as defined in relation to the first aspect. The detection moiety may bind to the biothreat-marker of interest if it is present.

[0265] The photonic crystal and the detection moiety may be contacted for at least 1 minute, at least 5 minutes, at least 10 minutes, at least 30 minutes, at least 45 minutes or at least 1 hour. The photonic crystal and the detection moiety may be contacted for between 1 minutes and 24 hours, between 5 minutes and 12 hours, between 10 minutes and 6 hours, between 30 minutes and 2 hours or between 45 and 75 minutes. Between contacting the photonic crystal and the detection moiety and determining whether or not the capture and / or detection moieties have bound to the biothreatmarker of interest, the method comprise rinsing the photonic crystal. Rinsing the photonic crystal may be as defined above.

[0266] It may be appreciated that if the photonic crystal comprises the biothreat-marker of interest (due to it having been present in the sample) then the detection moiety will bind to it when the photonic crystal and the detection moiety are contacted. As noted above, in embodiments where it is present, the biothreat-marker of interest may be either bound to the capture antibody or to the additive. This may prevent removal of the detection moiety in a rinsing step. Accordingly, the detection moiety may still be present after a rinsing step. Conversely, if the biothreat-marker of interest is not present in the photonic crystal then a rinsing step may remove the detection moiety therefrom.

[0267] Accordingly, determining whether or not the capture and / or detection moieties have bound to the biothreat-marker of interest may comprise determining whether or not the photonic crystal comprises the detection moiety after the rinsing step.

[0268] Determining whether or not the photonic crystal comprises the detection moiety may comprise determining whether or not a colour change has occurred in the photonic crystal.

[0269] In some embodiments, the colour change is a visible colour change.

[0270] In some embodiments, the colour change does not comprise fluorescence.

[0271] In embodiments where the detection moiety comprises a detection label, determining whether or not the photonic crystal comprises the detection moiety may comprise determining whether or not the photonic crystal comprises the detection label. For instance, in embodiments where the detection label comprises a fluorophore the method may comprise determining whether or not the photonic crystal fluoresces.

[0272] The method may comprise contacting the contacting the photonic crystal and a secondary detection reagent. The method may comprise contacting the photonic crystal and a secondary detection reagent subsequent to contacting the photonic crystal and the detection moiety. The method may comprise rinsing the photonic crystal between contacting the photonic crystal and the secondary detection reagent subsequent and contacting the photonic crystal and the detection.

[0273] The secondary detection reagent may be configured to react with or in the presence of the detection moiety. In particular, the detection moiety may comprise a detection enzyme and the secondary detection reagent may be configured to react with or in the presence of the detection enzyme. Determining whether or not the photonic crystal comprises the detection label may comprise determining whether or not the secondary detection reagent has reacted. The secondary detection reagent may undergo a colour change when it reacts with or in the presence of the detection enzyme. Accordingly, determining whether or not the secondary detection reagent has reacted may comprise determining whether or not a colour change has occurred in the photonic crystal. If a colour change has been detected this may be understood to mean that the secondary detection reagent has reacted. Accordingly, detecting a colour change may be understood to mean that the biothreat-marker of interest is present in the sample.

[0274] Accordingly, in some embodiments, subsequent to contacting the photonic crystal and the detection moiety, the method comprises a step comprising contacting the photonic crystal and a secondary detection reagent. The secondary detection reagent may be as defined in the first aspect. Contacting the photonic crystal and the secondary detection reagent may comprise contacting the photonic crystal and a secondary detection reagent solution. The secondary detection reagent solution may be as defined in relation to the first aspect. The secondary detection reagent may react upon contact with the detection moiety.

[0275] The photonic crystal and the secondary detection reagent may be contacted for at least 1 minute, at least 5 minutes, at least 10 minutes, at least 30 minutes or at least 20 minutes. The photonic crystal and the secondary detection reagent may be contacted for between 1 minutes and 24 hours, between 5 minutes and 12 hours, between 10 minutes and 3 hours, between 15 minutes and 1 hour or between 20 and 45 minutes.

[0276] Between contacting the photonic crystal and the detection moiety and contacting the photonic crystal and the secondary detection reagent, the method may comprise rinsing the photonic crystal. Rinsing the photonic crystal may be as defined above. In some embodiments, the method comprising contacting the photonic crystal and a stop solution. The method may comprise contacting the photonic crystal and the stop solution subsequent to contacting the photonic crystal and the detection moiety.

[0277] Preferably, the method comprises contacting the photonic crystal and the stop solution subsequent to contacting the photonic crystal and the secondary detection reagent.

[0278] The stop solution may be as defined in the first aspect.

[0279] The photonic crystal and the stop solution may be contacted for at least 1 minute, at least 5 minutes or at least 10 minutes. The photonic crystal and the stop solution may be contacted for between 1 minute and 1 hour, between 5 and 30 minutes or between 8 and 15 minutes.

[0280] In some embodiments, detecting a colour change in the photonic crystal may comprise detecting a visible colour change.

[0281] In some embodiments, detecting a colour change in the photonic crystal does not comprise detecting fluorescence.

[0282] In some embodiments, detecting a colour change in the photonic crystal may comprise a user detecting a colour change in the photonic crystal by visual inspection.

[0283] In alternative embodiments, the method may comprise detecting a colour change in the photonic crystal by using a spectrometer. The spectrometer may be UV-Vis spectrometer.

[0284] In alternative embodiments, the method may comprise detecting a colour change in the photonic crystal by capturing an image of the photonic crystal and using an image processing device to determine if a colour change has occurred. The image processing device may be a smart phone. Accordingly, the method may comprise taking a photo of the photonic crystal with the smart phone and using an app to process the image to determine if the biothreat-marker of interest is present in the sample.

[0285] Detecting a colour change in the photonic crystal may comprise comparing the colour of the photonic crystal to a reference sample or samples. The reference sample or samples may define a calibration curve. The method may comprise determining a concentration of the biothreat-marker of interest, if present in the sample, by comparing the colour of the photonic crystal to a reference sample or samples. The method may be a method of detecting a specific biothreat.

[0286] The method may comprise implementing a treatment plan, a public health strategy and / or a security response if a biothreat is detected.

[0287] All of the features described herein (including any accompanying claims, abstract and drawings), and / or all of the steps of any method or process so disclosed, may be combined with any of the above aspects in any combination, except combinations where at least some of such features and / or steps are mutually exclusive.

[0288] For a better understanding of the invention, and to show how embodiments of the same may be carried into effect, reference will now be made, by way of example, to the accompanying Figures, in which:-

[0289] Figure 1 shows (A) dried photonic crystal (PC) films, with (from top to bottom) 0, 0.01 and 0.05 wt% M0S2; (B) as prepared 0.6 cm diameter PC samples with varying M0S2 content in their dry state; and (C) as prepared 0.6 cm diameter PC samples with varying M0S2 content in their hydrated state;

[0290] Figure 2 shows the conditions for constructive interference through a simplified depiction of Bragg-Snell law, an incident wave is reflected off a particle interface and if the path length between two rays is equal to 2d sin 0, constructive interference of the reflected light takes place;

[0291] Figure 3 shows (A) an atomic force microscopy (AFM) height micrograph showing an M0S2 flake (scale bar 1 pm); and (B) an AFM height micrograph showing an M0S2 flake integrated with a capture antibody (scale bar 1 pm); inset images show height line profiles corresponding to the blue line profiles;

[0292] Figure 4 shows an overview of the mechanism of photonic crystal detection for Agl and Ag2; if the sample is present (positive case) then with each additional stage the neff is modified, thus modifying the colour of the photonic crystal; to add further specificity in stage 4, a chromogenic substrate reacts with the detection antibody and enhances the colour change;

[0293] Figure 5 shows PC-M0S2 exposed to increasing concentrations of Agl;

[0294] Figure 6 shows (A) UV-Vis spectra of representative PC samples at varying concentrations of Agl taken in the in-well plate state; and (B) optical densities (ODs) taken at 650 nm of PC-M0S2 in the in-well plate state averaged across three samples for each condition;

[0295] Figure 7 shows PC-M0S2 exposed to increasing concentrations of Ag2; Figure 8 shows (A) UV-Vis spectra of representative PC samples at varying concentrations of Ag2; (B) ODs averaged over nine repeats taken at 405 nm; and (C) ODs averaged over nine repeats taken at 650 nm;

[0296] Figure 9 shows (A) PC-M0S2 exposed to increasing concentrations of Ag2 as viewed from within the well-plate; and (B) shows ODs taken at 650 nm of PC-M0S2;

[0297] Figure 10 shows PC-M0S2 exposed to increasing concentrations of Ag2, using PnPP as a chromogenic substrate;

[0298] Figure 11 shows how a smartphone camera can be used to enhance sample contrast to enable easier, or more sensitive readings; shown are three columns of PCs with no, low, and high Ag2 loading, rows are sample replicates; (A) shows a normal image of PCs after an Ag2 test; (B) shows the same image with a "cool" filter applied, enhancing the contrast and saturation; and finally (C) shows that by increasing the contrast significantly, the differences between samples can be artificially adjusted to white-black;

[0299] Figure 12 shows (A) a smartphone photograph of PCs after Ag2 testing; and (B) a plot produced by first measuring the brightness in three areas in each PC, shown by yellow circles in Figure 12A, and then averaging the values;

[0300] Figure 13 shows (A) PC-M0S2 exposed to increasing concentrations of Ag2 as viewed from within the well-plate produced using a one-sided antibody detection method; (B) UV-Vis spectra of PC samples produced using a one-sided antibody detection method at varying concentrations of Ag2; (C) ODs at 405 nm averaged over 9 samples for each Ag2 concentration;

[0301] Figure 14 shows the potential design of a kit for biothreat-marker detection from a liquid sample; and

[0302] Figure 15 shows an atomic force microscope (AFM) micrograph of a photonic crystal produced from cast films of latex formulation SI with 200 pm wet thickness.

[0303] Examples

[0304] Example 1 - Incorporating M0S2 into Photonic Crystals

[0305] Photonic crystal (PC) films incorporating different M0S2 contents were synthesised as described in the Methods section (below). Figure 1 shows the appearance of these films in their dry and hydrated states. The as-dried PC films have a transparent appearance, see Figure 1 (A) and (B). However, once hydrated by water or similar (e.g. sample diluent, PBS, TBS, etc.) the PC films rapidly develop (<5 seconds) a bright blue iridescent appearance as shown in Figure 1 (C).

[0306] The assembly of polymer particles into an ordered nano-structure of face-centred cubic packing gives rise to the photonic effects of the PCs. The particles have diameters approximately half the wavelength of visible light. This ensures the effects are in the visible wavelength region and gives the PCs structural colouration. Figure 2 depicts a simplified diagram of this mechanism. Constructive and destructive interference of diffracted light off particle interfaces leads to wavelengths that are forbidden from propagating through the structure. For wavelengths leading to constructive interference, the light is diffracted backwards, causing a reflectance maximum, or Bragg peak, in the reflectance spectra and giving rise to a stopband.

[0307] The colour of a PC can be predicted by the determining the maximum reflected wavelength, Bragg wavelength Asragg. The Bragg-Snell law, Equation 1, is used to find this wavelength, using the spacing between the planes within the PC, d; the angle of incident light with respect to the normal, 0; and neff , the effective refractive index.

[0308] The effective refractive index, neff, is defined in Equation 2 and is dependent on r and <l>i which are the refractive index and volume fraction of each constituent in the system.

[0309] Example 2 - Antibody Binding to M0S2 flakes

[0310] The inventors wanted to see if M0S2 flakes, like those incorporated into PCs in example 1, could be modified to bind with a capture antibody (Ab).

[0311] Figure 3 (A) shows an atomic force microscopy (AFM) height micrograph of an M0S2 dispersion drop coated onto a mica substrate. This shows that the M0S2 flakes have a flat plane surface, as expected for a 2D layered material.

[0312] The substrate-bound M0S2 was subsequently mixed in a solution comprising a capture antibody (Ab), as described in the methods section (below), and an AFM height micrograph for the resulting material is shown in Figure 3 (B). This shows that the M0S2 flakes mixed in capture Ab solution have a rounded dome like surface, indicating that the Ab is bound to the surface of the flakes. Example 3 - Concept of Biothreat-Marker Detection

[0313] The inventors sought to develop a method of biothreat-marker detection which utilises the ability of the MoSz-containing PCs to bind biomarkers, as discussed in example 2.

[0314] In particular, the inventors noted that the Bragg-Snell law (Equation 1) states that the colour of the crystal is dependent upon, amongst other parameters, the effective refractive index of the material, neff. The neff is the weighted sum of all the refractive indices of each constituent material within the crystal, such as polymer, water, and M0S2. Thus, if the PCs comprise a capture antibody specific to a biothreat-marker and were modified, due to the addition of the biothreat-marker, the effective refractive index would change and would alter the colour of the PC. In light of this, the inventors hypothesised that if the PCs of Example 2 were exposed to a sample with a biothreat-marker present, it would alter the appearance of the PC.

[0315] In light of this, the inventors devised a method of biothreat-marker detection. A brief overview of their method is provided in Figure 5 and summarised as follows:

[0316] • Stage 1 : Capture Ab configured to capture a specific biothreat-marker of interest is bound to M0S2 flakes within a PC. As explained in Example 2, the capture Ab is selectively bound to the M0S2 flakes.

[0317] • Stage 2: A sample comprising the biothreat-marker of interest is added to the PC matrix. The biothreat-marker will then bind to the capture Ab.

[0318] • Stage 3: A detection Ab, specific to the biothreat-marker of interest, conjugated to an enzyme (e.g. horseradish peroxidase (HRP) or alkaline phosphatase) is added to the PC matrix. The detection Ab will specifically bind to the biothreat-marker.

[0319] • Stage 4: A chromogenic substrate (e.g. p-nitrophenyl phosphate (PnPP) or 3,3',5,5'-tetramethylbenzidine (TMB)) is added to the matrix, where it reacts with the conjugated enzyme, resulting in a colour change.

[0320] With each stage from stage 2 onwards, the addition and binding of additional material increases the / of the PC, which will vary the reflected colour. However, in negative cases (no biothreat-marker present in the sample) no additional material will be added to the PC matrix and thus the neff, and hence colour, will remain unchanged from stage 1. Thus, by stage 4, in the positive case (when the biothreat-marker sample is present) the colour will be developed by both the dye and the significantly varied neff which modifies the reflected colour of the PC. Example 4 - Antigen 1 (Agl) Detection

[0321] The detection of prostate specific antigen (PSA), identified herein as Antigen 1 (Agl), was conducted as per the protocol outlined in the Methods section. After testing, the colour of each well-plate was dependent on the concentration of Agl used. As shown in Figure 5, the strength of the observed colour in each well-plate increased with increased Agl concentration. There was little to no visible colour appearance at 0 ng / ml Agl, showing that the background colour in this test was low. In addition to readings being taken within the well, the PCs were removed from the well plate and allowed to dehydrate. As shown in Figure 5, the dehydrated PCs removed from the well plate appeared increasingly strong in colour with increasing Agl concentration.

[0322] In addition to visual analysis of the PCs, UV-Vis spectra were taken of the samples prior to the removal of the liquid and PCs from the well. As shown in Figure 6(A), the optical density peaks at 450 and 650 nm increased in intensity with increasing Agl concentration. These peaks are consistent with the visible blue colour produced by the HRP-TMB reaction. In addition, the peak at 405 nm significantly overlaps with the stopband of the PC, leading to an enhanced superimposed peak. Figure 6(B) shows the OD taken at 650 nm and it can be seen that the OD at this wavelength increases with Agl concentration. The peak at 650 nm was chosen to analyse the Agl concentration due to the superposition of the shoulders of the stopband and chromogenic dye peaks at 405 nm.

[0323] Example 5 - Antigen 2 (Ag2) Detection

[0324] The detection of engrailed-2 (EN2), identified herein as Antigen 2 (Ag2), using M0S2 functionalised PCs was conducted as described in the Methods section. As was observed for the Agl test described in Example 4, the colour of the well-plate was dependent on the concentration of Ag2 sample used. As shown in Figure 7, the strength of the observed blue colour in each well increased with increased Ag2 concentration. The background colour in Ag2 case was marginally higher than for Agl as seen by the light blue appearance of the PC at 0 ng / ml. Just as for Agl, the colour shift can be visually detected both in the well-plate with the PC and liquid present, and in the dry PCs alone.

[0325] In addition to the visual analysis of the PCs, UV-Vis spectra of the samples prior to the removal of the liquid and PCs from the well-plate. As shown in Figure 8(A), the peaks at 450 and 650 nm increased with the Agl concentration. These peaks are consistent with the blue colour produced by the HRP-TMB reaction. In addition, the peak at 405 nm significantly overlaps with the stopband of the PC, leading to an enhanced superimposed peak. Figures 8(B) and (C) show the peaks measured at 405 nm and 650 nm, and it can be seen that the ODs increase with increased Ag2 concentration. Both 405 nm and 650 nm were used to analyse the impact of Ag2 concentration, since the high background OD led to the stopband effects being less prominent. It is noted that since the background OD was high, the OD at the peak exceeded the spectrometer range and hence has OD = 4 at its maximum. A Gaussian fitting was applied to the peaks that exceeded the range to determine OD and extrapolation from analysis of both peaks provided additional reliability.

[0326] To further probe the sensitivity of the PC platform, reduced Ag2 concentrations were tested. To increase the likelihood of distinguishing between the low concentrations of 0, 1 and 10 ng / ml Ag2, the background OD in the negative case (0 ng / ml) needed to be reduced from that observed in Figure 7. As shown in Figure 9, it was possible to detect Ag2 concentrations of 1 ng / ml.

[0327] Comparison of the Ag2 and Agl test results shows that in negative controls (when the concentration of biomarkers is 0 ng / ml) the Ag2 detection PCs have a higher background colour than the Agl detection PCs. This is attributed to the fact that the detection antibodies used for the Ag2 testing system are likely either physically larger or more prone to non-specific binding to the PC and thus are harder to remove by washing.

[0328] Example 6 - Varying the Chromogenic Dve for Ag2 Detection

[0329] In addition to using the chromogenic dye TMB to produce an increasing blue colour with increasing Ag2 concentration, the dye PnPP was also tested. The protocol used was the same as described for Ag2 detection in the Methods section, except that the detection antibody was conjugated to Alk-Phos following the instructions from the Lightning Link Alk-Phos Conjugation Kit to provide a secondary conjugated (detector) antibody and PnPP was used instead of TMB.

[0330] It was found that use of PnPP produced an increased yellow colour with increased Ag2 concentration. The PnPP produced a higher background colour in the well-plate when compared to the TMB. As such, the visual colour change was hard to distinguish in the well plate. However, as shown in Figure 10, upon the removal of the PC from the well plate, the colour change became more evident as a darkening, or yellowing, of the PC with increasing Ag2 concentration. The HRP-TMB system produced clear blue colours at different Ag2 concentrations, while the PnPP system produced a consistent yellow colour at all concentrations. This was attributed to the HRP conjugated detection antibody being easier to wash away than that in the PnPP system. Although the PnPP wells all appeared yellow, it was possible to determine the changes in Ag2 concentration more accurately after the samples were removed from the well plate. This indicates that the photonic effects of the PCs are key for biothreat-markers detection. In the positive case (where Ag2 is present), the PCs are loaded with additional materials (capture Ab, Ag2, detection Ab, and PnPP), which modify and increase the neff. This causes a shift in the photonic band gap, which can be detected as a change in colour. The negative case (where Ag2 is not present) only has capture Ab present, so the neff is not modified and there is no shift in the photonic band gap. This is significant because it suggests that the photonic effects of PCs can be used to detect bio-markers and or biothreat-markers even when the chromogenic dye system does not produce a clear colour difference.

[0331] Example 7 - Digital Enhancement and Analysis

[0332] As described in Examples 4 to 6, the colour changes of functionalized PCs in the presence of biomarkers were detectable by the naked eye and spectrometry. However, the inventors also investigated if image enhancement using a standard smartphone camera was possible. Figure 11 presents a series of images, starting with (A), a standard photograph (iPhone 11) of functionalized PCs exposed to different Ag2 loadings taken under ambient lighting. Image (B) shows that applying a cool filter to the image enhances the contrast between the cases. In image (A), the three columns appear similar, but in image (B), the first column appears transparent (no Ag2 loading), the middle column shows a yellow-green tint (low Ag2 loading), and the final column (high Ag2 loading) exhibits bright yellow coloured PCs. Image (C) demonstrates the principle at the maximum, where the image has been saturated with maximum contrast to create a stark difference between white and black, unlike the relatively similar appearance observed in image (A).

[0333] In addition to smartphones being able to digitally "enhance" the contrast between samples, the images can also be used to measure the amount of Ag2 concentration. This is demonstrated in Figure 12. As shown in image (A), three regions in each sample were measured and the brightness of each sample averaged. This average brightness was then measured in ImageJ between 0 (black) and 255 (white) which in turn was normalised between 0 (black) and 1 (white). The data points were then plotted, as shown in image (B). The inventors envisage that by incorporating a reference in the sample reading, an image can be taken and a brightness measured, and then normalised by the reference card. The brightness could then be compared to a known curve and a cut-off point used to determine whether the test is positive or negative. Advantageously, this test would also indicate the specific Ag2 concentration.

[0334] This shows that the method described can be used to determine the concentration of a specific antigen. Thus, it is clear that the test could be used to determine the concentration of biothreat-marker. In turn, this information could be used to determine the severity of the threat and what response should be taken.

[0335] Example 8 - One-Sided Ag2 Detection

[0336] The inventors conducted further studies to optimize the Ag2 detection system and investigate alternative approaches. These included testing a one-sided detection method which uses a single detection antibody, as opposed to a capture antibody and a detection antibody. The protocol for this method is described under the heading "One-Sided EN2 Detection" in the Methods Section.

[0337] As shown in Figure 13, the intensity of the observed blue coloration increased proportionally with the Ag2 loading. This change was readily detectable using UV-Vis spectroscopy, as evidenced by the increasing intensity of the peak at 405 nm with increasing Ag2 loading (image B). Averaging the optical density (OD) measurements at 405 nm for 9 samples revealed a clear increase in OD with increasing Ag2 concentration (image C).

[0338] In the presence of Ag2, a substantial rise in OD was observed. In the negative control samples, a comparatively higher background and greater fluctuation were evident compared to the two-antibody tests, as expected from this faster but less sensitive testing approach. Despite the saturation of the response between the 100 and 1000 ng / ml Ag2 concentrations, the results demonstrate the versatility of the PC technology to adapt to different protocols.

[0339] Employing a single primary antibody (APS2) conjugated with HRP and eliminating the secondary antibody step offered several advantages, including reduced complexity, shorter test time, lower material consumption, and a lower cost. However, while this method proved successful, it exhibited reduced sensitivity compared to the tests described in Examples 4 to 6. Example 9 -Device Design

[0340] The inventors envisage that a simple testing platform may include the delivery of a sample container to a user. A sample may be collected by the user and transported to a lab. The PC biothreat test may then carried out by trained users. This approach would enable the sample to be obtained "in the field", but would allow the test to be conducted in a safer and / or more controlled setting.

[0341] It is possible that samples are collected by users and sent to laboratories for tests to be conducted in controlled settings. This allows for samples to be collected from a range of different sites.

[0342] An in situ-test is also envisaged by the inventors for "in the field" testing. Figure 14 shows one potential test kit. This kit comprises a sample pot 2, designed to receive a sample 4. The kit further comprises a sample loader 6 partially filled with a buffer 8. This ensures that when the sample 4 is taken up into the sample loader 6 the resulting solution 10 has an acceptable pH. The kit further comprises a multi-syringe 12, which is configured to receive the sample and buffer solution 10 and is also pre-filled with a detection moiety solution 14 and two wash buffer solutions 16a and 16b. The kit may be used for the detection of multiple biothreat-markers. Consequently, the multisyringe 12 may contain a detection Ab solution comprising multiple different detection Abs, wherein each different detection Ab is specific to a different biothreat-marker of interest. Alternatively, the syringe may comprise multiple detection Ab solutions, each detection Ab solution contained in a separate internal chamber thereof.

[0343] The test kit further comprises a test strip 18 comprising an input port 20 configured to receive a sample, a number of PCs A to E comprising different capture Abs for different biothreat-markers, microfluidic channels 22 extending between the input port 20 and the PCs A to E. The test strip 18 further comprises adsorption pads 24 downstream of the photonic crystals A to E and further microfluidic channels 26 extending between the photonic crystals A to E and the adsorption pads 24.

[0344] The multi-syringe 12 may be used to deposit the combined liquid buffer and sample solution 10 into the input port. The microfluidic channels then carry the solution 10 to the PCs A to E. At this stage the PCs may be checked to ensure proper hydration, this is visible as the PCs will turn from transparent to a reflective blue. This acts as a negative control and if any of the PCs do not turn reflective blue, the test is failed. If the test passes this stage, the test strip 18 is then incubated for one hour at room temperature. Then the first wash buffer 16a is released into the input port 20, followed by the detection antibody 14. The test strip 18 is then incubated for a further hour at room temperature. Subsequently, the final wash buffer 16b is released into the input port 20.

[0345] The final component of the test kit is a syringe 28 comprising a solution comprising a chromatic substrate 30. In the final stage, the syringe 28 is used to inject the solution 30 into the input port 20. Whether or not the biothreat-marker is present may then be determined based upon whether or not a colour change is observed. As explained above, a smart phone or other electronic device could be used to determine the concentration of the biothreat-marker.

[0346] It is noted that, at each stage, excess liquid is carried to the adsorbent pads 24 and adsorbed.

[0347] The PCs A-E can be designed to suit the various biothreat-markers that would be investigated, with at least one sample acting as a positive control. The positive control sample would contain a specific biomarker that is always positive in the tested biological background or is present in the buffer solution 8, and thus will change colour if the test has been successful.

[0348] Example 10 - Photonic crystal film comprising a surface layer of additive

[0349] As explained in the methods section, the photonic crystals described in the above examples were produced using the methods provided in WO 2020 / 115486 Al, the contents of which are incorporated herein by reference. In particular, these PCs were produced by mixing an M0S2 dispersion and a water-borne latex formulation (SI). Accordingly, these PCs comprised both additive (M0S2) and mesoscopic particles (latex) throughout the bulk of their structures.

[0350] Additionally, the inventors have found that it is possible to spray M0S2 directly onto the surface of a photonic crystal film. The following method may be used.

[0351] M0S2 dispersion is placed into a dual-action gravity fed airbrush with a 0.3 mm needle. The airbrush is pressurised by an FD-196 air compressor. The M0S2 dispersion is then sprayed directly onto the surface of the dried photonic crystal at a distance of 100 mm at 4 PSI. The airbrush is moved over the photonic crystal film to create an even coverage of M0S2 on the crystal surface. The M0S2 is sprayed from a 1 mg / ml dispersion onto the photonic crystal surface. The volume and sprayed area are predetermined to ensure that the surface coverage of M0S2 is 20 pg / cm2, (for example spraying a volume of 1 ml of dispersion at a concentration of 1 mg / ml over a fixed area of 50 cm2). It is noted that a coverage of between 0.5 and 100 pg / cm2would be workable.

[0352] The additive (M0S2) can be sprayed from a water-based dispersion. Alternatively, the additive can be sprayed from a solvent-based dispersion (such as ethanol, acetone, isopropanol, N-Methyl-2-pyrrolidone, tetra hydrofuran, or methyl ethyl ketone (butanone)). The solvent can evaporate during the spray surface, so there is no damage to the crystal surface from solvent exposure. Additionally, in the final coated film the solvent will not be present due to evaporation.

[0353] The inventors note that the additive can be sprayed onto a photonic crystal film, which may or may not comprise the additive within its bulk material.

[0354] Furthermore, the additive can be sprayed onto a substrate (such as polycarbonate) and the photonic crystal subsequently film cast over the additive-coated substrate, forming a base layer of the additive.

[0355] Conclusions

[0356] In-place of testing using a biothreat-marker, the examples above test the inventors' proposed system using prostate specific antigen (PSA), identified herein as Antigen 1 or Agl, engrailed-2 (EN2), identified herein as Antigen 2 or Ag2. While PSA and EN2 are not biothreat-markers, it will be appreciated that these experiments provide proof of concept that the inventors system can be used to detect biomarkers at low concentrations. Biothreat-markers which may be detected to determine the presence of a biothreat are known, and the system and methods described in the examples could readily be adapted to detect these markers.

[0357] In particular, the above examples show that the photonic crystal (PC) platform has been shown to be a capable method of detection for both Agl and Ag2, and thus can be used for the effective detection of biomarkers. Having proved their capability to act as a platform for the detection of two biomarkers, it is envisaged that the PCs can act as a wider platform for detection of numerous unique biothreat-markers, allowing this technology to be employed in the detection and identification of biothreats. In particular, by changing the detection and / or capture moieties from ones specific to PSA or EN2 to ones specific to a certain biothreat or biothreats, then the same procedure may be applied for the detection of biothreats. The underlying capture and detection system for biomarkers and biothreat-markers is the same.

[0358] There are a number of advantages associated with the photonic crystal detection platform generally. In particular, as demonstrated in Example 5, the platform can detect a biomarker at a concentration of 1 ng / ml, indicating that it is highly sensitive. Furthermore, the protocol of the PC diagnostic test would permit the detection of biothreat-markers from multiple sources.

[0359] The thin polymer latex films are produced from widely available low-cost latex with scalable techniques, ensuring that this device can be offered cheaply.

[0360] Additionally, the PC platform can be used in a wet or dry state, as shown in Figures 5 and 7. The PC responses are visible both in and out of the well plate. This is advantageous as it is envisaged that this will allow users to take a reading at the site or location of concern and then send the sample back to a lab for further analysis if necessary. This can therefore provide users with both an immediate response and, if needed, a more detailed follow-up response. Additionally, the natural colour development of PCs with hydration permits the in-built use of both a positive and negative control.

[0361] Furthermore, the PC biothreat-markers detection platform can make beneficial use of digital imaging. Imaging with a smartphone camera could be implemented as described in Example 7. Inclusion of a reference colour pattern alongside the sample viewing window would allow digital analysis of brightness. The images could then be exposed to a pre-determined filter to maximise the colour differences. This could be advantageous for users with varying colour perception e.g. those with colour vision deficiency, a condition which effects 8% of men in the UK. This may be a vital tool to allow detection of biothreat-markers with a PC system, as colour changes may be less significant or occur at very low biothreat-markers concentrations. The ability to simply enhance the output signal with an everyday item such a smartphone is a unique strength of the platform.

[0362] The system's dual capability of being read by eye and digitally offers other advantages. For example, in some instances the system may be deployed in locations where a positive or negative result is required immediately and there is no additional equipment available to assist with a digital reading. However, the full capability of the equipment can be extracted when used in locations where more infrastructure is available.

[0363] In some settings or future applications, an accompanying app or other system to measure the colour shift more specifically or discreetly may be employed. Incorporation of a digital platform may facilitate further analysis or automated detection, using artificial intelligence (Al) or simple algorithms to correlate the brightness with a specific result, concentration, or other metric. For example, some biothreats, e.g. influenza, may have a natural background level, thus the presence of a positive result may not necessarily be a reason for concern. However, this system, could allow the determination of the risk of an outbreak / attack from a single photograph, which could in turn revolutionize public health and security responses. The ability to infer the associated risks and expedite responses and avoid creating panic or wasting resources can significantly improve the outlook of public health.

[0364] The test described herein can be run in a few hours. This is a significant advantage over some current tests which can require days to correctly identify the biothreat after detection, e.g. cell culture, or mouse bioassay. Here, the PC platform can offer detection, identification, and concentration in a single package with a turnaround time in the range of hours not days.

[0365] Materials

[0366] The photonic crystal substrate used in the examples was made from polycarbonate which was procured from Goodfellow, product code CT30-FM-000110.

[0367] The latex formulation used in the examples (SI) was a styrene-acrylate with a Tgin the range 25 - 35 °C, a particle diameter of between 200 and 300 nm and a PDI of 0.015.

[0368] Bovine serum albumen (BSA), phosphate-buffered saline (PBS), and Tris-buffered saline (TBS), and para-nitrophenylphosphate (PnPP)were procured from Sigma- Aldrich.

[0369] Biotinylated anti-human PSA, recombinant human PSA standard (lyophilized), 20x wash buffer, assay diluent, and 3,3',5,5'-Tetramethylbenzidine (TMB) One-Step substrate reagent were procured from Abeam. EN2 testing

[0370] Synthetic protein corresponding to the C-terminal 100 amino acids of EN2 (full sequence available from https: / / www.ncbL nlm.nih.gov / nuccore / NM 001427) was procured from Genescript. The sequence procured used N-terminus H, C-terminus OH, and the following sequence was used :

[0371] PKKKNPNKEDKRPRTAFTAEQLQRLKAEFQTNRYLTEQRRQSLAQELSLNESQIKIWFQN KRAKIKKATGNKNTLAVLMAQGLYNHSTTAKEGKSDSE

[0372] SEQ ID No. 2

[0373] Antibodies were procured from applied protein synthesis, referred to as APS1 and APS2 for the detection and capture antibodies respectively. APS1 and APS2 were raised as described in Pandga et al. (BJU Int. 2012 Sep; 110(6 Pt B) : E287-92. doi : 10. llll / j.l464-41OX.2O12.11208.x. Epub 2012 May 15). APS1 and APS2 antibodies recognize the C-terminus of the 14 amino acids of EN2:

[0374] NHSTTAKEGKSDSE

[0375] SEQ ID No. 1

[0376] A Lightning Link Alkaline Phosphatase (Alk-Phos) Conjugation Kit, and a Lightning Link Horseradish Peroxidase (HRP) Conjugation Kit were procured from Abeam.

[0377] Methods

[0378] Photonic crystal production

[0379] Unless otherwise stated, photonic crystals were produced using the methods provided in WO 2020 / 115486 Al, the contents of which are incorporated herein by reference.

[0380] Unless otherwise stated, in the examples described herein, PCs were produced using an M0S2 dispersion and a water-borne latex formulation (SI).

[0381] The M0S2 dispersion was prepared by liquid phase exfoliation. The final concentration of the dispersion was calculated using UV-Visible spectrophotometry and was found to be 1 gL-1with an average layer number of N ~ 10. The lateral dimensions of the M0S2 were measured with an atomic force microscope and found to be typically 10 - 100 nm.

[0382] The M0S2 dispersion was then added to the latex formulation, and sonicated at 5 W for 10 minutes (1 min per ml, if larger or smaller quantities were used). Following this the bottle was placed on a shaking plate and a plasticiser, butyl glycol, was pipetted into the container. The parameters used for the formulation of a standard blue photonic crystal using ~10 ml of latex are given in Table 1.

[0383] Table 1. Formulation parameters for the standard blue PCs.

[0384] PCs were produced by employing evaporation-driven self-assembly. An Elcometer 4340 automatic film applicator was used with a 200 pm cube applicator to produce films. A 200 pm thick layer of wet latex was applied to a polycarbonate substrate 0.175 mm thick (Goodfellow) and left to dry at 25 °C (touch-dry in 20 minutes). The final film thickness was estimated to be ~ 90 pm.

[0385] The film was then prepared into samples to be used in 96-well plates using a diecutting machine, circular discs of 6 mm in diameter were cut from the larger polycarbonate-latex film. To conduct a test, the latex-on-polycarbonate discs were placed in a transparent, flat-bottom 96-well plates (Thermo-Fisher Nunclon Delta™).

[0386] Photonic crystal imaging

[0387] Figure 15 shows an atomic force microscope (AFM) micrograph of an example of the ordered crystalline arrays of polymeric particles, produced from cast films of SI with 200 pm wet thickness. The inset fast Fourier transforms (FFTs) show the PC has a well-ordered hexagonal-close packed structure; this creates a repeating dielectric structure with a similar periodicity to that of the length-scale of visible light.

[0388] AFM imaging was performed using a Bruker Dimension Edge AFM operating in PeakForce Tapping mode. ScanAsyst-Air silicon nitride cantilevers with a nominal resonant frequency of 70 kHz and a tip radius of 2 nm were used. To correct for sample tilt and curvature, a polynomial levelling function was applied to the resulting AFM micrograph.

[0389] PSA Detection

[0390] Latex dispersion, nanomaterial inclusion and film casting was carried out as described above to produce blue latex with 0 wt%,.%, 0.01 wt%.% and 0.05 wt%.% M0S2. PC films were produced as follows: 6 mm diameter film was gently removed from the larger film with clean gloves and collected on a clean Petri dish, with the clean part facing up. Placed one by one in relevant wells by using a pair of clean fine tweezers.

[0391] 96-well plate arrangement: three replicates for each condition which are given in Table 2 were used, thus in total of 27 samples were tested.

[0392] Table 2. The tested experimental conditions in the 96-well plate.

[0393] The detection antibody was conjugated to HRP following the instructions from the Lightning Link HRP Conjugation Kit to provide a secondary (detector) antibody.

[0394] The following protocol was used, keeping testing tubes and reagent in ice for the duration of the experiment:

[0395] 1) Prepare testing tubes with samples and reagents

[0396] Primary (capture) antibody in PBS

[0397] Secondary conjugated (detector) antibody in PBS

[0398] PSA samples in sample dilutent

[0399] Washing buffer in distilled water

[0400] BSA 0.5% (w / v) (from pre-made stock)

[0401] TMB solution

[0402] 2) Mix PCs with primary antibody

[0403] Apply primary (capture) antibody to each well

[0404] Rinse each well with washing buffer

[0405] After washing crystals have a blue appearance (Figure 1)

[0406] 3) Block with 0.5% BSA (w / v)

[0407] Apply blocking solution to each well

[0408] Seal with parafilm to avoid evaporation

[0409] Incubate for 15 to 60 minutes

[0410] Rinse with washing buffer 4) Add biomarker sample to relevant wells (Opg / mL, 1 pg / mL or 10 pg / mL PSA) Apply biomarker sample to each well

[0411] Remove excess liquid from wells

[0412] 5) Incubate with the secondary conjugated antibody Apply secondary conjugated antibody to each well Seal with parafilm to avoid evaporation Incubate for 10 to 60 minutes

[0413] Rinse with washing buffer

[0414] 6) Add TMB Development Solution (from ELISA kit)

[0415] Add development solution to each well

[0416] Incubate at room temperature for 10 to 60 minutes

[0417] TMB turns blue in presence of HRP (obtain different shades of blue depending on the PSA concentration, Figure 1)

[0418] EN2 Detection

[0419] Latex dispersion, nanomaterial inclusion and film casting was carried out as described above for blue latex with 0.01 wt%.% M0S2.

[0420] PC films were produced as follows: 6 mm diameter gently removed from the larger film with clean gloves and collected on a clean Petri dish, with the clean part facing up. PC films placedPlaced one by one in relevant wells by using a pair of clean fine tweezers.

[0421] Table 3. The 96-well plate arrangement of experimental parameters.

[0422] The detection antibody was conjugated to HRP following the instructions from the Lightning Link HRP Conjugation Kit to provide a secondary conjugated (detector) antibody. 3 repeats were performed for each condition, giving a total of 27 wells tested. The following protocol was used:

[0423] 1) Prepare testing tubes with samples and reagents

[0424] Capture antibody (APS1) diluted in PBS

[0425] EN2 samples (prepared from 1 pg / mL stock)

[0426] PBST / Washing buffer

[0427] BSA 1% (w / v)

[0428] Detection antibody (APS2-HRP) diluted in PBS

[0429] TMB substrate or PnPP substrate

[0430] 2) Mix PCs in capture antibody (APS1)

[0431] Apply primary (capture) antibody to each well

[0432] Incubate for 15 to 60 minutes

[0433] Rinse with washing buffer / PBST

[0434] 3) Block with 1% BSA (from stock)

[0435] Apply blocking solution to each well

[0436] Incubate for 15 to 60 minutes

[0437] Rinse with washing buffer / PBST

[0438] 4) Add samples and detection antibody (0 ng / mL, 1 ng / mL, 10 ng / mL EN2) Add biomarker sample to each well

[0439] Incubate for 15 to 60 minutes

[0440] Add detection antibody (APS2-HRP) to each well

[0441] Wash with washing buffer / PBST

[0442] 4. Add TMB substrate or PnPP substrate

[0443] Add TMB or PnPP substrate to each well

[0444] Incubate for 15 to 60 minutes

[0445] Read absorbance at 405 nm using a plate reader

[0446] EN2 One-Sided Detection

[0447] Latex dispersion, nanomaterial inclusion and film casting was carried out as described above for blue latex with 0.01 wt%.% M0S2. PC samples: 6 mm diameter gently removed from the larger film with clean gloves and collected on a clean Petri dish, with the clean part facing up. PC films placedPlaced one by one in relevant wells by using a pair of clean fine tweezers.

[0448] Table 4. The 96-well plate arrangement of experimental parameters.

[0449] 96-well plate arrangement: 3 replicates for each condition for a total of 27 wells tested .

[0450] The following testing protocol was used:

[0451] 1) Prepare testing tubes with samples and reagents

[0452] EN2 samples

[0453] PBST / Washing buffer

[0454] BSA 1% (w / v)

[0455] Primary antibody (APS2-HRP)

[0456] TMB substrate

[0457] 2) Mix PCs with samples (0 ng / mL, 100 ng / mL, 1000 ng / mL EN2)

[0458] Apply sample to each well

[0459] Incubate for 15 to 60 minutes

[0460] 3) Block with 1% BSA (from stock)

[0461] Add blocking solution to each well

[0462] Incubate for 30 to 60 minutes

[0463] Rinse with washing buffer / PBST

[0464] 4) Add detection antibody

[0465] Add detection antibody (APS2-HRP) to each well

[0466] Incubate for 10 to 60 minutes

[0467] Wash with washing buffer / PBST on shaker

[0468] 5) Add TMB substrate

[0469] Add TBB substrate to each well Cover with foil to protect from light

[0470] Incubate for 15 to 30 minutes

[0471] Read absorbance at 405 nm using a plate reader

Claims

Claims1. A kit for detecting a biothreat-marker in a sample, the kit comprising: a photonic crystal comprising a plurality of mesoscopic particles and an additive, wherein the additive comprises a zero-dimensional (0D) material, a onedimensional (ID) material and / or a two-dimensional (2D) material; and a moiety specific to the biothreat-marker.

2. The kit according to claim 1, wherein the additive comprises a 2D material and the 2D material is selected from the group consisting of graphene, graphene oxide (GO), hexagonal boron nitride (h-BN), a transition metal dichalcogenide, an oxide of a transition metal dichalcogenide and combinations thereof.

3. The kit according to claim 2, wherein the 2D material is a transition metal dichalcogenide and the transition metal dichalcogenide is molybdenum disulphide (M0S2), tungsten disulphide (WS2), molybdenum diselenide (MoSe2), tungsten diselenide (WSe2) or molybdenum(IV) telluride (MoTe2).

4. The kit according to any preceding claim, wherein the plurality of mesoscopic particles have an average particle size of between 50 nm and 1,000 nm, between 100 nm and 500 nm, between 150 nm and 450 nm, between 160 nm and 400 nm, between 170 nm and 370 nm, between 180 nm and 350 nm, between 190 nm and 330 nm or between 200 nm and 300 nm.

5. The kit according to any preceding claim, wherein the, or each, mesoscopic particle is or comprises a polymer.

6. The kit according to any preceding claim, wherein the biothreat-marker is an indicator for a biothreat, and the biothreat is the threat of a disease or poisoning.

7. The kit according to claim 6, wherein the disease is anthrax, botulism, brucellosis, cholera, melioidosis, pneumonic plague, shigella, tularaemia, typhoid fever, typhus, Q fever, rocky mountain spotted fever, scrub fever, psittacosis, coccidioidomycosis, histoplasmosis, a viral haemorrhagic fever, chikungunya fever, dengue fever, eastern equine encephalitis, influenza, rift valley fever, Russian springsummer encephalitis, smallpox, Venezuelan equine encephalitis or yellow fever and / or the poisoning is poisoning caused by a botulinum toxin, a perfringens toxin, atrichothecene mycotoxin, a palytoxin, ricin, saxitoxin, a staphylococcal enterotoxin or tetrodotoxin.

8. The kit according to claim 6 or claim 7, wherein the biothreat-marker is selected from the group consisting of anthrax protective antigen (PA), Yersinia pestis fraction 1 (Fl) antigen, botulinum neurotoxin type A, botulinum neurotoxin type B, botulinum neurotoxin type C, botulinum neurotoxin type D, botulinum neurotoxin type E, botulinum neurotoxin type F, Variola major outer surface protein membrane A27, Francisella tularensis lipopolysaccharide (LPS), Ebola virus glycoprotein integral membrane protein, or an Ebola virus structural viral protein (VP).

9. The kit according to any one of claims 1 to 5, wherein the biothreat-marker is an indicator for a biothreat, and the biothreat is associated with a pharmaceuticalbased agent (PBA).

10. The kit according to any preceding claim, wherein the moiety specific to the biothreat-marker comprises or is an antibody, or a biothreat-marker-binding fragment thereof or an aptamer.

11. The kit according to any preceding claim, wherein the moiety specific to the biothreat-marker comprises or is a detection moiety.

12. The kit according to claim 11, wherein the detection moiety comprises a detection label, wherein the detection label is or comprises a detection enzyme, a fluorophore and / or a metal nanoparticle.

13. The kit according to claim 12, wherein the detection label is or comprises a detection enzyme and the detection enzyme is configured to react with or catalyse the reaction of a second detection reagent, and the kit comprises the second detection reagent.

14. The kit according to claim 13, wherein the detection enzyme is horseradish peroxidase (HRP) or alkaline phosphatase (Alk-Phos).

15. The kit according to claim 13 or 14, wherein the secondary detection reagent is a chromogenic substrate configured to undergo a colour change upon contact with the detection moiety.

16. The kit according to any preceding claim, wherein the moiety specific to the biothreat-marker comprises a capture moiety, wherein the capture moiety is immobilised in or on the photonic crystal.

17. The kit according to claim 16, wherein the capture moiety comprises or is an antibody, or a biothreat-marker-binding fragment thereof, or an aptamer.

18. The kit according to claim 16 or claim 17, wherein the moiety specific to the biomarker comprises a capture moiety specific to the biomarker and a detection moiety specific to the biomarker.

19. The kit according to any preceding claim, wherein the kit comprises a test strip comprising: the photonic crystal; an input port configured to receive a liquid; and a first channel extending between the input port and the photonic crystal.

20. The kit according to claim 19, wherein the test strip further comprises an adsorption pad and a second channel extending between the photonic crystal and the adsorption pad.

21. A photonic crystal comprising a plurality of mesoscopic particles, an additive and a capture moiety specific to a biothreat-marker, wherein: the additive comprises a zero-dimensional (0D) material, a one-dimensional (ID) material and / or a two-dimensional (2D) material; and the capture moiety specific to the biothreat-marker is bound to the additive.

22. A method of determining whether a biothreat- marker of interest is present in a sample, the method comprising: providing a photonic crystal, wherein the photonic crystal comprises a plurality of mesoscopic particles and an additive, wherein the additive comprises a zerodimensional (0D) material, a one-dimensional (ID) material and / or a two- dimensional (2D) material;- contacting the photonic crystal and the sample;- further contacting the photonic crystal with a detection moiety, wherein the detection moiety is configured to bind specifically to the biothreat-marker of interest; anddetermining whether or not the detection moiety has bound to the biothreatmarker of interest, and thereby determining whether or not the biothreatmarker of interest is present in the sample.

23. The method of claim 22, wherein the photonic crystal comprises a capture moiety configured to bind specifically to the biothreat-marker of interest, wherein the capture moiety is bound to the additive in the photonic crystal.

24. The method according to claim 22 or claim 23, wherein between contacting the photonic crystal and the detection moiety and determining whether or not the detection moiety has bound to the biothreat-marker of interest, the method comprises rinsing the photonic crystal, and determining whether or not the detection moiety has bound to the biothreat-marker of interest comprises determining whether or not the photonic crystal comprises the detection moiety after the rinsing step.

25. The method according to any one of claims 22 to 24, wherein the method comprises contacting the contacting the photonic crystal and a secondary detection reagent, wherein the detection moiety comprises a detection enzyme which reacts with or catalyses a reaction of the secondary detection reagent.

26. The method according to claim 25, wherein the secondary detection reagent undergoes a colour change when it reacts with or in the presence of the secondary detection reagent, and determining whether or not the biothreat-marker of interest is present in the sample thereby comprises determining whether or not a colour change has occurred in the photonic crystal.

27. The method according to claim 26, wherein the method comprises determining a concentration of the biothreat-marker of interest, if present in the sample, by comparing the colour of the photonic crystal to a reference sample or samples.

28. A method of determining whether a biothreat- marker of interest is present in a sample, the method comprising: providing a photonic crystal, wherein the photonic crystal comprises a plurality of mesoscopic particles, an additive and a capture moiety, wherein the additive comprises a zero-dimensional (0D) material, a one-dimensional (ID) material and / or a two-dimensional (2D) material and the capture moiety is configured to bind specifically to the biothreat-marker of interest and is bound to the additive in the photonic crystal;- contacting the photonic crystal and the sample; and determining whether or not the capture moiety has bound to the biothreatmarker of interest, and thereby determining whether or not the biothreatmarker of interest is present in the sample.

29. The method according to any one of claims 22 to 28, wherein the method is a method of detecting and identifying a biothreat.

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