Conjugated polymer nanoparticle biosensors

Abstract

Compositions comprising conjugated polymer nanoparticles for the detection and identification of target entities; substrates and lateral flow devices comprising the same; and methods of using the compositions to detect target entities.

Description

[0001] CONJUGATED POLYMER NANOPARTICLE BIOSENSORS

[0002] FIELD OF THE INVENTION

[0003] The invention relates to compositions and methods for the detection of target entities.

[0004] BACKGROUND

[0005] There is a substantial challenge to detect both biological and chemical matter with specificity, sensitivity, and in a timely manner. Examples of this matter commonly in need of detection include microorganisms such as bacteria and viruses, various proteins that could be diagnostic markers or allergens, bioagents which concern human health, and small molecules such as drugs. Rapid identification of matter which may be of health, clinical, or chemical interest serves to quicken outcomes and decisions. These outcomes and decisions could be for reaching a medical diagnosis to provide rapid treatment, a criminal forensics team learning of the presence of a drug or biological material, or identifying an agent used in a biological attack to allow effective management, resolution, and lower mortality rates.

[0006] There are several methods currently available for the identification of biological and chemical matter. The more traditional methods for detecting and identifying biological and chemical matter lack the speed and sensitivity required to be used in the field, as sometimes results can take hours to days to obtain. Analytical methods such as mass spectrometry can be used alone or in combination with chromatography to identify specific biological and chemical matter but involves a lengthy protocol that must be conducted in a laboratory. Polymerase chain reaction (PCR) is another method regularly used for various applications, including the identification of genes and diseases. PCR is dependent on using primers which determine the specificity of the method. Small amounts of contaminating DNA can be amplified, which lead to misleading or ambiguous results. The major limitations of current biological detection systems are that they are complex, expensive, subject to false alarms, and require samples to be taken to a laboratory for analysis.

[0007] Therefore, there is need for a simple, portable, cost-effective and rapid method of sensing target analytes. This would be of great use for scientific research, public health, security, and defence.

[0008] SUMMARY OF INVENTION

[0009] The present invention is predicated on the development of a conjugated polymer nanoparticle complex that can identify a target entity rapidly and with a distinct fluorescent signal. Unlike prior detection systems, the composition and method of the present invention has higher absorption capabilities, brightness, sensitivity, and stability than comparable technologies, and can be configured with a variety of surface chemistries to permit different linkages to targeting molecules to allow the detection of a large number of target entities. It is expected that this will provide the ability to detect numerous biological and non-biological targets, those of which may be hazardous, narcotic, or of interest in research or health. The present invention would therefore provide an invaluable resource for healthcare, environmental monitoring, and defence.

[0010] In a first aspect of the invention, there is provided a composition for detecting the presence of a target entity, the composition comprising a molecular complex comprising a conjugated polymer nanoparticle (CPN) capable of fluorescence, a targeting molecule which has affinity towards a target entity, and a quencher molecule, which together form the complex, such that the quencher molecule quenches fluorescence from the CPN, and either the quencher molecule or CPN can be displaced from the molecular complex when brought into contact with a target entity, such that in the absence of the quencher molecule, the fluorescence of the CPN can be visualised.

[0011] In a second aspect of the invention, there is composition for detecting the presence of a target entity, the composition comprising a conjugated polymer nanoparticle (CPN) capable of fluorescence bound to a targeting molecule which has affinity towards a target entity, the composition further comprising a quencher molecule.

[0012] In a third aspect of the invention, there is provided a composition for detecting the presence of a target entity in a sample, the composition comprising: a first molecular complex comprising a first conjugated polymer nanoparticle (CPN) capable of fluorescence, and a first targeting molecule which has affinity towards a control entity; a second molecular complex comprising a second CPN capable of quenching the fluorescence of the first CPN, and a second targeting molecule which has affinity towards a target entity.

[0013] In a fourth aspect of the invention, there is provided a method for detecting the presence of a target entity in a sample, the method comprising the steps: contacting the sample with a composition comprising the molecular complex as defined in the first aspect of the invention, whereby if present, the target entity displaces either the quencher molecule or the CPN from the molecular complex and replaces it, and in the absence of the quencher molecule, the fluorescence of the CPN can be visualised, thereby providing a positive indication of the presence of the target entity.

[0014] In a fifth aspect of the invention, there is provided a method for detecting the presence of a target entity in a sample, the method comprising the steps: contacting the sample with a composition as defined in the second aspect of the invention, whereby if present, the target entity binds to the quencher molecule, and the targeting molecule binds the targeting entity, bringing the quencher molecule in proximity to the CPN, resulting in a decrease in fluorescence due to quenching.

[0015] In a sixth aspect of the invention, there is provided a method for detecting the presence of a target entity in a sample, the method comprising the steps: contacting the sample first with a quencher molecule as defined in the second aspect of the invention, whereby if present, the target entity binds to the quencher molecule, and the subsequent addition of the sample and quencher molecule to the CPN bound to a targeting molecule of the composition as defined in second aspect of the invention, wherein the targeting molecule binds the targeting entity, bringing the quencher molecule in proximity to the CPN, resulting in a decrease in fluorescence due to quenching.

[0016] In a seventh aspect of the invention, there is provided a kit of parts for detecting the presence of a target entity in a sample, the kit comprising any composition as herein defined in the invention.

[0017] In an eighth aspect of the invention, there is a lateral flow device for detecting the presence of a target entity in a sample, the lateral flow device comprising a composition as herein defined in the first or third aspects of the invention.

[0018] BRIEF DESCRIPTION OF DRAWINGS

[0019] The present invention is described with reference to the following drawings, wherein:

[0020] Figure 1 shows the membrane layout and 610 Emission image showing the successful conjugation of CPN610 to ovalbumin (OV1) antibody.

[0021] Figure 2 shows the membrane layout and results for absorbance and emission signals.

[0022] Figure 3 shows the results from DotBlot experiments comparing the antiovalbumin antibodies.

[0023] Figure 4 shows the DotBlots showing the successful conjugation of various anti- Bacillus atrophaeus antibodies to CPN610.

[0024] Figure 5 shows the DotBlot to confirm that Bradford Reagent-stained ovalbumin can still bind to the CPN610 conjugate.

[0025] Figure 6 shows the DotBlot to compare the denaturing effect of different volumes of Bradford Reagent used during the staining process. Figure 7 shows the DotBlot to confirm that Bradford Reagent-stained Bacillus atrophaeus can still bind to the CPN610 conjugate.

[0026] Figure 8 shows the absorbance image to confirm that the Bacillus atrophaeus ± Bradford Reagent are of similar concentration.

[0027] Figure 9 shows a bar chart comparing the emission signal of CPN610 when ovalbumin ± Bradford Reagent is added, and then the ability to restore fluorescence after quenching by the addition of unstained ovalbumin.

[0028] Figure 10 shows a bar chart showing several different experiments comparing the emission signal of CPN610 when ovalbumin ± Bradford Reagent is added, and then the ability to restore fluorescence after quenching by the addition of unstained ovalbumin.

[0029] Figure 11 shows a bar chart comparing the emission signal of CPN610 when different Bradford Reagent + ovalbumin solutions are used to quench the fluorescence and then the emission signal following the addition of ovalbumin to restore emission signal.

[0030] Figure 12 shows images of polyether sulfone syringe filter showing quenching of fluorescence upon addition of Bradford Reagent + ovalbumin.

[0031] Figure 13 shows images of polyether sulfone syringe filter showing quenching of fluorescence upon addition of Bradford Reagent + ovalbumin and the restoration of fluorescence upon subsequent addition of unstained ovalbumin.

[0032] Figure 14 shows the proposed molecular principle of a composition of the “Fluorescence Safe, Dark Detection / Threat” configuration.

[0033] Figure 15 shows the proposed molecular principle of a composition in the “Dark Safe, Fluorescence Detection / Threat” configuration.

[0034] Figure 16 shows a working example of the “AWD Quenched CPN” LFT format. Figure 17 shows a proof-of-concept study which led to the development of the “AWD Quenched CPN” LFT format.

[0035] Figure 18 shows a working example of the “Quenched CPN (2x CPN Concept)” LFT format.

[0036] Figure 19 shows a proof-of-concept study which led to the development of the “Quenched CPN (2x CPN Concept)” LFT format.

[0037] DETAILED DESCRIPTION

[0038] In a first aspect of the invention, there is provided a composition for detecting the presence of a target entity, the composition comprising a molecular complex comprising a conjugated polymer nanoparticle (CPN) capable of fluorescence, a targeting molecule which has affinity towards a target entity, and a quencher molecule, which together form the complex, such that the quencher molecule quenches fluorescence from the CPN, and either the quencher molecule or CPN can be displaced from the molecular complex when brought into contact with a target entity, such that in the absence of the quencher molecule, the fluorescence of the CPN can be visualised.

[0039] The composition of this aspect of the invention can be referred to as the “Dark Safe, Fluorescence Detection / Threat” composition, or more simply, the molecular complex of the present invention. There are two fluorescent states for the molecular complex. The first state is the “dark” or “safe” state wherein the fluorescence of the CPN is quenched by the quencher molecule since they are initially in proximity. The CPN therefore displays little or no fluorescence. The second state is the “fluorescence”, “detection”, or “threat” state wherein either the quencher molecule or CPN (depending on the configuration of the complex) has been displaced from the molecular complex when brought into contact with a target entity, allowing the CPN to emit fluorescence since the quencher molecule is no longer in proximity. In one embodiment the molecular complex is configured such that the quencher molecule can be displaced from the molecular complex when brought into contact with a target entity, thereby reducing the components of the complex to only the targeting molecule and CPN. In the absence of the quencher molecule, the CPN can fluoresce. In a second embodiment, the molecular complex is configured such that the CPN can be displaced from the molecular complex when brought into contact with a target entity, thereby reducing the components of the complex to only the targeting molecule and quencher molecule. Again, in the absence of the quencher molecule, the CPN can emit a detectable fluorescence.

[0040] The displacement of either the quencher molecule or the CPN is possible due to the configuration of the molecular complex, where one of either the CPN or the quencher will be immobilised (directly or indirectly) on the targeting molecule with the other being removably bound in the complex in the absence of the target entity. For example, the quencher molecule or CPN may be bound to a molecule having affinity towards the targeting molecule, said molecule being part of the complex, but removed or displaced when the target entity is brought into contact. Alternatively, either the quencher molecule or CPN has affinity towards the targeting molecule itself, and is removably bound by the targeting molecule in the absence of the target entity. When the target entity is brought into contact there is displacement of the quencher molecule (or CPN), resulting in the quencher molecule and CPN being no longer in proximity, allowing the CPN to emit a detectable fluorescence.

[0041] Accordingly, in one embodiment, the molecular complex comprises a CPN bound (non-removably) to the targeting molecule, preferably by covalent bonding. In this embodiment, the quencher molecule is formed in the complex such that it can be displaced from the complex when the complex is brought into contact with a targeting entity. The quencher molecule may have direct affinity towards the targeting molecule or may be bound to a separate molecule that itself has affinity towards the targeting molecule and which may be removed / displaced if the complex is brought into contact with a target entity. In a separate embodiment, the quencher molecule is bound (non-removably) to the targeting molecule, preferably by covalent bonding. In this embodiment, the CPN is formed in the complex such that it can be displaced from the complex when the complex is brought into contact with a targeting entity. The CPN may have direct affinity towards the targeting molecule, or may be bound to a separate molecule that itself has affinity towards the targeting molecule and which may be removed / displaced if the complex is brought into contact with a target entity.

[0042] The present invention is intended for the detection of a target entity. As used herein, the term “target entity” is intended to refer to any biological or chemical entity, the detection of which is desirable. The skilled person will appreciate the wide variety of biological and chemical entities that may be the target for detection. Suitable examples are disclosed below.

[0043] The present invention makes use of a conjugated polymer nanoparticle. As used herein, the term “conjugated polymer nanoparticle” refers to highly fluorescent nanoparticles with light emitting polymer cores. The emitted light may be in the ultraviolet, visible light, or infra-red regions, but will typically be in the range 300nm to 1500nm of the electromagnetic spectrum. The term “polymer” refers to a compound comprising at least 5 monomer units, including homopolymers and copolymers. The term “conjugated polymer” refers to any polymer that has alternating single and double bonds between carbon atoms on a polymer backbone. These polymers give the CPNs their fluorescent properties that significantly exceed those of other molecular probes. A “nanoparticle” refers to an entity having dimensions most conveniently measured in nanometres. CPNs are commercially available (for example, see https: / / www.streambio.co.uk / products / ), and can come with a number of surface chemistries including a carboxyl surface, maleimide, and alkyne to fit desired linkage preferences. The nanoparticles can be readily conjugated to a variety of biomolecules including antibodies via suitable linkers, including using N-ethyl-N’ dimethylaminopropyl-carbodiimide (EDC) chemistry, as will be appreciated by the skilled person. CPNs are bioimaging molecular probes with superior properties to a variety of other reagents and can be used in numerous applications where visual, fluorescent, and infrared signal is required. CPNs come in a range of sizes (30nm-150nm) with a standard size of 70nm-80nm, but can also be made to bespoke sizes of 300nm or greater. CPNs may also have a hydrophobic polymer core surrounded with a capping agent to make it dispersible in water and to facilitate a variety of surface chemistries and conjugation methods. The range of colours cover the visible and near infra-red spectrum, (420nm-1000nm) with the oranges I reds (CPN 610 / 660 / 680) and livid (CPN 770 / 820 / 830 / 840) giving the highest fluorescent and visible contrasts. The extinction coefficients of CPN are extremely high which gives the particles a 40% higher absorption capability than colloidal gold. For applications where the fluorescent properties are important, CPNs are up to 1000x brighter than Quantum dots and 100x more sensitive than europium chelates when used in in vitro diagnostic devices such as lateral flow tests. Furthermore, they have incredible stability (measured in months / years) across a wide range of temperatures and pH’s. Further still, commercially available conjugated polymers are relatively cheap, and the resulting material can be used in labelling, detection and / or separation experiments without the need to alter microscope optics or labelling techniques or protocols.

[0044] In an embodiment of the invention, the CPN has maximal emission within the range 300nm to 1500nm, more preferably of at least: 420 nm, 435 nm, 475 nm, 510 nm, 530 nm, 550 nm, 580 nm, 610 nm, 660 nm, 680 nm, 770 nm, 820 nm, 830 nm, 840 nm or 1000 nm. The term “maximal emission” refers to the wavelength of light at which the fluorescence output of a fluorophore is most likely to occur. This wavelength is the emission maximum for that fluorophore. The excited fluorophore can also emit light at wavelengths near the emission maximum, as shown. However, this light may be less intense.

[0045] The skilled person will appreciate suitable conjugated polymers that emit fluorescence for use in the invention. In an embodiment of the invention, the polymer of the conjugated polymer nanoparticle is selected from: Poly [9 , 9-d i (3' , 7'- dimethyloctyl)fluoren-2,7-yleneethynylene], Poly[9,9-didodecylfluroenyl-2,7- yleneethylnylene], Poly[9,9-di(2'-ethylhexyl)fluoren-2,7-yleneethynylene], Poly(9,9-dioctylfluorenyl-2,7-yleneethynylene), Poly[9,9-bis-(2-ethylhexyl)-9H- fluorene-2,7-diyl], Poly(9,9-dihexyl-9H-fluorene-2,7-diyl), Poly(9,9-di-(2- ethylhexyl)-9H-fluorene-2,7-diyl), Poly[(9,9-di-(2-ethylhexyl)-9H-fluorene-2,7- diyl)-co-(1-methoxy-4-(2-ethylhexyloxy)-2,5-phenylenevinylene)], Poly[(9,9- dihexyl-9H-fluorene-2,7-diyl)-co-(1-methoxy-4-(2-ethylhexyloxy)-2,5- phenylenevinylene)], Poly(2,5-bis(1 ,4,7,10-tetraoxaundecyl)-1 ,4- phenylenevinylene), Poly (2 , 5-diocty 1-1 ,4-phenylenevinylene), Poly(2,5- dioctylphenylene-1 ,4-ethynylene),Poly[1 ,2-bis(benzylthio)acetylene], Poly[1 ,2- bis(ethylthio)acetylene], Poly[bis(methylthio)acetylene], Poly(3,5 pyridine), Poly(3-(2-methoxyethoxy)ethoxymethylthiophene-2, 5-diyl), 5,5'-Dibromo-2,2'- bithiophene, Poly(3-butylthiophene-2, 5-diyl), Poly(3-cyclohexyl-4- methylthiophene-2,5-diyl), Poly(3-cyclohexylthiophene-2, 5-diyl), Poly(3- decylthiophene-2, 5-diyl), Poly(3-dodecylthiophene-2, 5-diyl), Poly(3- hexylthiophene-2, 5-diyl), Poly(3-octylthiophene-2, 5-diyl), Thiophene

[0046] Oligothiophenes, 5,5'-Dibromo-2,2'-bithiophene, 2,2',5',2",5",2"'-Quaterthiophene, a-Sexithiophene, 311073 2,2':5',2"-Terthiophene, Poly(thiophene-2, 5-diyl), bromine terminated powder, 2,3-Dihydrothieno[3,4-b]-1 ,4-dioxin, 3,2':5',3"- Terthiophene, 5-Hexyl-2,2'-bithiophene, 22,2'-Bithiophene, Thiophene, 5,5""- Dihexyl-2,2':5',2":5",2"':5"',2"":5"",2 -sexithiophene, Poly(styrenesulfonate) / poly(2,3-dihydrothieno(3,4-b)-1 ,4-dioxin), Poly(3,4- ethylenedioxythiophene)-block-poly(ethylene glycol), Poly(3,4- ethylenedioxythiophene), tetramethacrylate endcapped, Poly((9,9-dihexyl-9H- fluorene-2,7-diyl)-co-(1-methoxy-4-(2-ethylhexyloxy)-2,5-phenylenevinylene)), Poly((9,9-dihexyl-9H-fluorene-2,7-diyl)-co-(1-methoxy-4-(2-ethylhexyloxy)-2,5- phenylenevinylene), Poly(1-methoxy-4-(3-propyloxy-heptaisobutyl-PSS)-2,5- phenylenevinylene)-co-(1-methoxy-4-(2-ethylhexyloxy)-2,5-phenylenevinylene), Poly(2,5-bis(3-sulfonatopropoxy)-1 ,4-phenylene, disodium salt-alt-1 ,4- phenylene), Poly(2,5-dihexyloxy-1 ,4-phenylenevinylene), Poly (2 , 5-diocty 1-1 ,4- phenylenevinylene), Poly(2,6-naphthalenevinylene), Poly[5-methoxy-2-(3- sulfopropoxy)-1 ,4-phenylenevinylene] Potassium salt, Poly(p-xylene tetrahydrothiophenium chloride), Poly[(m-phenylenevinylene)-alt-(2-methoxy-5- octyloxy-p-phenylenevinylene)] , Poly[(m-phenylenevinylene)-alt-(2,5-dihexyloxy- p-phenylenevinylene)], Poly[(m-phenylenevinylene)-alt-(2-methoxy-5-(2-ethyl- hexyloxy)-p-phenylenevinylene)], Poly[(m-phenylenevinylene)-co-(2,5-dioctoxy- p-phenylenevinylene)], Poly[(m-phenylenevinylene)-alt-(2,5-dibutoxy-p- phenylenevinylene)], Poly[(o-phenylenevinylene)-alt-(2-methoxy-5-(2- ethylhexyloxy)-p-phenylenevinylene)], Poly[(p-phenylenevinylene)-alt-(2- methoxy-5-(2-ethyl-hexyloxy)-p-phenylenevinylene)], Poly[2-(2',5'-bis(2"- ethylhexyloxy)phenyl)-1 ,4-phenylenevinylene], Poly[2,5-bis(3' 7'- dimethyloctyloxy)-1 ,4-phenylenevinylene], Poly[(2,5-bisoctyloxy)-1 ,4- phenylenevinylene], Poly[2-methoxy-5-(2-ethylhexyloxy)-1 ,4-phenylenevinylene], Poly[2-methoxy-5-(3',7'-dimethyloctyloxy)-1 ,4-phenylenevinylene], Poly[5- methoxy-2-(3-sulfopropoxy)-1 ,4-phenylenevinylene], Poly[9-(2-ethylhexyl)-3,6- carbazolevinylene-alt-2,6-naphthalenevinylene], Poly{[2-[2',5'-bis(2"- ethylhexyloxy)phenyl]-1 ,4-phenylenevinylene]-co-[2-methoxy-5-(2'- ethylhexyloxy)-1 ,4-phenylenevinylene]}, Poly[(9,9-dioctylfluorenyl-2,7-diyl)-co- (1 ,4-phenylene)], Poly[(9,9-dioctylfluorenyl-2,7-diyl)-co-(4,4'-(N-(4-sec- butylphenyl)diphenylamine)], Poly(2,5-di(3',7'-dimethyloctyl)phenylene-1 ,4- ethynylene), Poly[(9,9-dioctyl-2,7-divinylenefluorenylene)-alt-co-{2-methoxy5-(2- ethylhexyloxy)-1 ,4-phenylene}], Poly[(9, 9-diocty I-2 ,7-bis{2- cyanovinylenefluorenylene})-alt-co- (2-methoxy-5-{2-ethylhexyloxy}-1 ,4- phenylene)], Poly(9,9-dioctylfluorene-alt-benzothidiazole), Poly[2-methoxy-5-(2- ethylhexyloxy)-1 ,4-( 1 -cyanovinylene-1 ,4-phenylene)], Poly [{9, 9-dihexy I-2 , 7-bis( 1 - cyanovinylene)fluorenylene}-alt-co- {2, 5-bis(N,N’-diphenylamino)-1 ,4- phenylene}], Poly(4,4-dioctyldithieno(3,2-b:2',3'-d)silole)-2,6-diyl-alt-(2, 1 ,3- benzothiadiazole)-4,7-diyl), Poly[(5-fluoro-2, 1 ,3-benzothiadiazole-4,7-diyl)(4,4- dihexadecyl-4H- cyclopenta[2,1-b:3,4- b']dithiophene-2,6-diyl)(6-fluoro-2,1 ,3- benzothiadiazole- 4,7-diyl)(4,4-dihexadecyl-4H-cyclopenta[2, 1 -b:3,4- b']dithiophene-2,6-diyl)], Poly{2,2'-[(2,5-bis(2-hexyldecyl)-3,6-dioxo-2, 3,5,6- tetrahydropyrrolo[3,4-c ]pyrrole-1 ,4-diyl)dithiophene]- 5,5'-diyl-alt-thiophen-2 ,5- diyl}, Poly[[4,8-bis[(2-ethylhexyl)oxy]benzo[1 ,2-b:4,5-b']dithiophene-2,6-diyl][3- fluoro-2-[(2-ethylhexyl)carbonyl]thieno[3,4-b]thiophenediyl ]], Poly[2,6-(4,4-bis-(2- ethylhexyl)-4H-cyclopenta [2, 1 -b;3,4-b']dithiophene)-alt-4,7(2, 1 ,3- benzothiadiazole)], Poly[(4,4-bis(2-ethylhexyl)-dithieno[3,2-b:2',3'-d]silole)-2,6- d iy I -al t-(2 , 1 ,3-benzothiadiazole)-4,7-diyl], Poly[(9,9-bis(2-ethylhexyl)fluorenyl-2,7- diyl)-alt-(benzo[2, 1 ,3]thiadiazol-4,7-diyl)], or a combination of any two or more thereof.

[0047] Preferably, the polymer of the conjugated polymer nanoparticle is selected from: Poly[(9,9-dioctylfluorenyl-2,7-diyl)-co-(1 ,4-phenylene)], Poly[(9,9-dioctylfluorenyl-

[0048] 2.7-diyl)-co-(4,4'-(N-(4-sec-butylphenyl)diphenylamine)], Poly(2,5-di(3',7'- dimethyloctyl)phenylene-1 ,4-ethynylene), Poly[(9,9-dioctyl-2,7- divinylenefluorenylene)-alt-co-{2-methoxy5-(2-ethylhexyloxy)-1 ,4-phenylene}], Poly[(9,9-dioctyl-2,7-bis{2-cyanovinylenefluorenylene})-alt-co- (2-methoxy-5-{2- ethylhexyloxy}-1 ,4-phenylene)], Poly(9,9-dioctylfluorene-alt-benzothidiazole), Poly[2-methoxy-5-(2-ethylhexyloxy)-1 ,4-( 1 -cyanovinylene-1 ,4-phenylene)], Poly[{9,9-dihexyl-2,7-bis(1-cyanovinylene)fluorenylene}-alt-co- {2,5-bis(N,N’- diphenylamino)-1 ,4-phenylene}], Poly(4,4-dioctyldithieno(3,2-b:2',3'-d)silole)-2,6- d iy I -al t-(2 , 1 ,3-benzothiadiazole)-4,7-diyl), Poly[(5-fluoro-2, 1 ,3-benzothiadiazole-

[0049] 4.7-diyl)(4,4-dihexadecyl-4H- cyclopenta[2,1-b:3,4- b']dithiophene-2,6-diyl)(6- fluoro-2, 1 ,3-benzothiadiazole- 4,7-diyl)(4,4-dihexadecyl-4H-cyclopenta[2, 1 -b:3,4- b']dithiophene-2,6-diyl)], Poly{2,2'-[(2,5-bis(2-hexyldecyl)-3,6-dioxo-2, 3,5,6- tetrahydropyrrolo[3,4-c ]pyrrole-1 ,4-diyl)dithiophene]- 5,5'-diyl-alt-thiophen-2 ,5- diyl}, Poly[[4,8-bis[(2-ethylhexyl)oxy]benzo[1 ,2-b:4,5-b']dithiophene-2,6-diyl][3- fluoro-2-[(2-ethylhexyl)carbonyl]thieno[3,4-b]thiophenediyl ]], Poly[2,6-(4,4-bis-(2- ethylhexyl)-4H-cyclopenta [2, 1 -b;3,4-b']dithiophene)-alt-4,7(2, 1 ,3- benzothiadiazole)], Poly[(4,4-bis(2-ethylhexyl)-dithieno[3,2-b:2',3'-d]silole)-2,6- diyl-alt-(2, 1 ,3-benzothiadiazole)-4,7-diyl], or Poly[(9,9-bis(2-ethylhexyl)fluorenyl-

[0050] 2.7-diyl)-alt-(benzo[2, 1 , 3]thiadiazol-4, 7-diy I)] .

[0051] In an embodiment of the invention, a biocompatible surfactant and / or lipid is used to either partly or entirely encase the molecular complex to form a micelle. The molecular complex of the present invention may not be or be only partly water- soluble, and therefore may require the use of surfactants to either impart or enhance water-solubility. To achieve this, a plurality of surfactants and / or lipids may be used together, such as one, two, three or four surfactants and / or lipids forming the shell of the micellar structure. The emission signal strength from conjugated polymer nanoparticles enveloped in micelles is only slightly reduced compared to a non-encapsulated conjugated polymer nanoparticle, which would still allow for efficient detection.

[0052] The surfactants used may be either monomeric or polymeric to best achieve the desired solubility properties of the molecular complex. A polymeric surfactant comprises a hydrophilic polymer which can comprise at least two monomers, of which the number of individual monomers is adjusted to form a micelle of sufficient diameter to encapsulate the molecular complex. Surfactants used may be of either anionic (based on sulfate, sulfonate, or carboxylate anions), cationic (based on quaternary ammonium cations), zwitterionic or non-ionic nature.

[0053] The lipids used may be phospholipids, wherein the hydrocarbon chain may be saturated or unsaturated, straight-chain or branched, substituted or unsubstituted. Saturated or unsaturated hydrocarbon chain examples include n-dodecyl, n- tetradecyl, n-hexadecyl, n-octadecyl, and n-icosyl, oleyl, linoleyl, linolenyl and eleostearyl. Substituted hydrocarbons are preferably substituted by a C1-C4 alkyl, alkenyl, or alkynyl group, such as methyl, ethyl, propyl, isopropyl, n-butyl, t- butyl, isobutyl, ethenyl, propenyl, butadienyl, isobutenyl, ethynyl, propynyl or butynyl. Branched hydrocarbon chain examples include 3,7,11 ,15- tetramethy I hexadecyl and cis or trans 3,7,11 , 15-tetramethyl-2-hexadecenyl.

[0054] Any of the carbon atoms in any of the hydrocarbon chains described above may further comprise a hydrocarbon ring structure, which may be saturated or unsaturated. Non-limiting examples of hydrocarbon rings include cyclopentyl, cyclopentenyl, cyclohexyl, and phenyl, and an example of hydrocarbon chains further comprising hydrocarbon rings includes 1-butyl-4-cyclohexyl- 12-dodecyl.

[0055] The skilled person will appreciate suitable biocompatible surfactants to be used in this embodiment. In an embodiment of the invention, the biocompatible surfactant is selected from: polystyrene maleic anhydride, pluronic F127, sodium dodecyl sulfate, ammonium lauryl sulfate, alkyl sulfate salts, sodium laureth sulfate, sodium lauryl ether sulfate, alkyl benzene sulfonate, sodium cholate, sodium deoxycholate, N-lauroylsarcosine sodium salt, lauryldimethylamine-oxide, bis(2- ethylhexyl) sulfosuccinate sodium salt, cholic acid sodium salt, dodecyl sulfate (lithium or sodium salts), soaps, fatty acid salts, cetyl trimethylammonium bromide, hexadecyl trimethyl ammonium bromide, alkyltrimethylammonium salts, cetylpyridinium chloride, polyethoxylated tallow amine, benzalkonium chloride, benzethonium chloride, palmityl trimethylammonium bromide, (2- hydroxyethyl)trimethylammonium chloride, dodecyl betaine, dodecyl dimethylamine oxide, cocamidopropyl betaine, coco ampho glycinate, polyethylene glycol, alkyl polyethylene oxide), copolymers of poly (ethylene oxide) and polypropylene oxide), alkyl polyglucosides, octyl glucoside, decyl maltoside, fatty alcohols, cetyl alcohol, oleyl alcohol, cocamide MEA, cocamide DEA, cocamide TEA, bishydroxyethylether, N,N-dimethyldecylamine-N-oxide, N,N-dimethyldodecylamine-N-oxide, 1 ,6-hexanediol, or ethylene glycol octyl phenyl ether, or a combination of any two or more thereof.

[0056] Preferably, the suitable biocompatible surfactant is selected from polystyrene maleic anhydride or pluronic F127.

[0057] The skilled person will appreciate suitable biocompatible lipids to be used in this embodiment. In an embodiment of the invention, the biocompatible lipid is selected from: phospholipids, dipalmitoylphosphatidylcholine, phosphatidylethanolamine, n-poly(ethylene glycol) phosphatidylethanolamine, phosphatidylcholine, phosphatidic acids, phosphatidylglycerol, phosphatidylserine, phosphatidylinositol, bis(monoacylglycero)phosphate, cardiolipin, ether lipids, plasmalogens, or sphingolipids.

[0058] Preferably, the suitable biocompatible lipid is dipalmitoylphosphatidylcholine.

[0059] As used herein, the term “micelle” refers to a substance comprising a polymer which is partly or entirely encased within a biocompatible surfactant and / or lipid, to form a "core-shell" structure. In this structure, the molecular complex forms the core of the micelle and the biocompatible surfactant and / or lipid forms the encapsulating shell around the core.

[0060] As used herein, the term "biocompatible surfactant and / or lipid" refers to any surfactant or lipid which is substantially soluble in water or aqueous solution and does not cause toxic effects to biological material, such as but not limited to proteins, polypeptides, nucleic acids, cells, bacteria, fungi, and viruses.

[0061] The present invention makes use of two components, a CPN and quencher molecule, that can be bound to the targeting molecule in a variety of configurations to form the molecular complex. One of these two components is displaced upon the complex being brought into contact with the target entity, and the other remains on the targeting molecule. The loss in proximity between the CPN and the quencher molecule enables the CPN to fluoresce, generating a measurable signal. The component, whether it be the CPN or quencher molecule, that is bound in the complex such that it can be displaced from the complex when the complex is brought into contact with a targeting entity is preferably bound by electrostatic interactions but can be any kind of bond which allows the invention to function in this embodiment through the displacement of either the CPN or the quencher molecule. The component, whether it be the CPN or the quencher molecule, that remains bound to the targeting molecule during the displacement is preferably bound covalently but can be any kind of bond which allows the invention to function in this embodiment.

[0062] The bond or bonds formed between either the CPN and the targeting molecule, or the quencher molecule and the targeting molecule, can be any kind of bond which allows the invention to function in any embodiment through the displacement of one of two components, the CPN or the quencher molecule, and the retention of the remaining component.

[0063] The bond or bonds formed between either the CPN or quencher molecule and a molecule with affinity towards the targeting molecule can be any kind of bond which allows the invention to function in any embodiment through the displacement of the CPN or quencher molecule bound to molecule with affinity towards the targeting molecule.

[0064] As used herein, the term “covalent bonding” refers to the chemical bond that results from the sharing of an electron pair between two atoms. As used herein, the term “electrostatic interactions” refer to the forces between particles that are caused by their electric charges. Types of electrostatic interactions may include but are not limited to ionic bonding, hydrogen bonding, dipole-dipole interactions, n-effects and van der Waals forces. When the CPN (or quencher) is to be bound covalently to the targeting molecule this may be directly or via a suitable linker molecule. Suitable linkers that may be used in the invention will be apparent to the skilled person.

[0065] As used herein, the term “affinity” refers to the ability and strength of a molecule to bind a target due to various factors including but not limited to electrostatic interactions, stereochemistry, and contact surface area. Generally, this refers to the “binding affinity” between molecules, the strength of which can be measured as a dissociation constant (Kd). Both qualitative methods such as but not limited to enzyme-linked immunosorbent assay and quantitative methods such as but not limited to surface plasmon resonance, isothermal titration calorimetry, biolayer interferometry and fluorescence anisotropy.

[0066] The present invention makes use of a quencher molecule. As used herein, the term “quencher molecule” means any molecule that, when in proximity to the CPN, quenches or suppresses the fluorescence of the CPN.

[0067] As used herein, the term “quenching” refers to the physiochemical process that decreases the intensity of light emitted by a fluorescent molecule. Quenching can occur through a variety of processes known to the skilled person including but not limited to collisions, complex-formation, excited state reactions, and energytransfer. Suitable quencher molecules will be apparent to the skilled person. The quencher molecule can be any molecule that quenches the fluorescence from the CPN of the molecular complex. Specifically, the quencher molecule will absorb electromagnetic radiation of the same or similar wavelength as the CPN emits (i.e. the wavelength of the CPNs emission maxima). The skilled person will be able to identify and use appropriate quenching molecules based on the emission properties of the chosen CPN.

[0068] The quencher molecule may be selected from any one of: a dye, a dye quencher, acid wool dye, azo dye, cationic dye, anionic dye, dark quencher, or a CPN. In an embodiment of the invention, the quencher molecule is a dye or dye quencher. As used herein, the term “dye” refers to a coloured substance, often applied in an aqueous solution, that chemically bonds to a substrate. Dyes absorb certain wavelengths of light, hence appearing coloured in visible light. This property allows them to quench the fluorescence of other light-emitting compounds, such as CPNs, if the emitted wavelength is the same or similar to that absorbed by a dye. As used herein, the term “dye quencher” refers to a substance that can absorb wavelengths of light from fluorescent dyes and re-emit the absorbed energy as heat (dark quenchers) or light (fluorescent quenchers). As with dyes, this property allows dye quenchers to be used as quenchers of CPN fluorescence. Examples of dyes or dye quenchers selected include but are not limited to: acid wool dyes, azo dyes, cationic (basic) dyes, anionic (reactive) dyes, or dark quenchers.

[0069] As used herein, the term “acid wool dye” refers to dyes commonly used to dye protein fibres in acidic conditions. This is possible due to acidic groups such as - SO3H or -COOH being anionic and therefore able to form ionic bonds with other molecules. Targets are typically proteins, where the acid wool dye binds to the protonated amino groups of the protein via the anomic groups of the acid wool dye. Acid wool dyes may also bond to proteins via hydrogen bonding and Van der Waals forces. In a further embodiment of the invention, the acid wool dyes are selected from at least: Fast Green, Coomassie Blue, Erythrosine, Sunset Yellow, Tartrazine, Acid Red and Eriochrome Black.

[0070] As used herein, the term “azo dye” refers to a class of synthetic dyes containing the functional group R-N=N-R', wherein R is usually an aryl or substituted aryl group. Azo dyes can contain more than one azo group, such as diazo dyes, triazo dyes, and polyazo dyes. Examples of azo dyes include but are not limited to: Acid orange 5, Acid Orange 7, Acid orange 19, Acid orange 20, Acid Red 13, Acid red 88, Alcian yellow, Alizarine Yellow R, Allura Red AC, Congo red, Pigment Red 4, C.l. Direct Black 38, and C.l. Acid Red 114. As used herein, the term “cationic dye” refers to a class of dyes that are dissociated as positively charged ions in aqueous solution and can form bonds to the negatively charged groups of a substate. Types of cationic dyes can include azo dyes, triarylmethane dyes, anthraquinone dyes, and heterocyclic compounds. Examples of cationic dyes include but are not limited to: Basic Yellow 11 , Basic Yellow 28, Basic Red 29, Basic Orange 21 , Basic Blue 3, Basic Blue 4, Basic Blue 41 , Astrazon Pink FG, Astrazon Red 6B, and Astrazon Brilliant Red 4G.

[0071] As used herein, the term “anionic dye” refers to a class of dyes characterised by acidic groups, such as SO3H and COHOH, which can form bonds to the positively charged groups of a substrate. Types of cationic dyes can include azo dyes, triarylmethane dyes, anthraquinone dyes, and heterocyclic compounds. Examples of anionic dyes include but are not limited to: Acid Red 13, Acid Red 25, Acid Red 88, Acid Red 95, Acid Orange 3, Acid Orange 19, Acid Blue AS, Acid Fuchsin, and Alcian Blue 8GX, Alizarin yellow R, Alizarin Yellow GG, Alizarin Red S, Alizarin cyanin BSS, and Alizarin blue.

[0072] As used herein, the term “dark quencher” refers to a substance used to absorb the fluorescence of a fluorophore and re-emit this energy as heat. Examples of black hole quenchers include but are not limited to: Black Hole Quencher 1 , Black Hole Quencher 2, Black Hole Quencher 3, Iowa Black FQ, Iowa Black RQ, and IRDye QC-1.

[0073] In some embodiments, the quencher molecule may be a CPN.

[0074] When the quencher molecule is a CPN, the conjugated polymer nanoparticle (CPN) capable of fluorescence is a first CPN, and the quencher molecule is a second CPN. The second CPN will be capable of quenching the fluorescence of the first CPN, such as when they are both comprised within the molecular complex (i.e. due to their proximity).

[0075] Of these two CPNs, the first CPN may be used for visualisation, as in embodiments where the quencher molecule is not a CPN (i.e. when there is only one CPN comprised within the molecular complex). It will be understood that the fluorescence of the first CPN may be measured to detect whether the second CPN is within proximity to the CPN (and therefore quenching the fluorescence of the first CPN) or if the second CPN has been displaced from the first CPN (and is therefore no longer quenching the fluorescence of the first CPN). In embodiments where the quencher molecule is a CPN, the mechanism of target detection functions in the same way as the embodiments where the quencher molecule is not a CPN. Either the first (fluorescent) or second (quencher) CPN may be displaced from the molecular complex in the presence of a target entity, thereby abolishing quenching and allowing the first CPN to fluoresce.

[0076] Even though the second CPN may have fluorescent properties, its fluorescence may not be of consideration in detecting a target entity, and instead the fluorescence of the first CPN will be measured (i.e. measurement may be carried out in the part of the spectrum that the first CPN fluoresces). In these embodiments, the fluorescence of the second CPN may be measured separately to detect its presence in the molecular complex.

[0077] In this embodiment, the second CPN may be selected from the list of the polymers of the CPNs which the first CPN could comprise. Therefore, the polymer of the second CPN may be selected from: Poly[9,9-di(3',7'-dimethyloctyl)fluoren-2,7- yleneethynylene], Poly[9,9-didodecylfluroenyl-2,7-yleneethylnylene], Poly[9,9- di(2'-ethylhexyl)fluoren-2,7-yleneethynylene], Poly(9,9-dioctylfluorenyl-2,7- yleneethynylene), Poly[9,9-bis-(2-ethylhexyl)-9H-fluorene-2,7-diyl], Poly(9,9- dihexyl-9H-fluorene-2,7-diyl), Poly(9,9-di-(2-ethylhexyl)-9H-fluorene-2,7-diyl), Poly[(9,9-di-(2-ethylhexyl)-9H-fluorene-2,7-diyl)-co-(1-methoxy-4-(2- ethylhexyloxy)-2,5-phenylenevinylene)], Poly[(9,9-dihexyl-9H-fluorene-2,7-diyl)- co-(1-methoxy-4-(2-ethylhexyloxy)-2,5-phenylenevinylene)], Poly(2,5- bis( 1 ,4,7, 10-tetraoxaundecyl)-1 ,4-phenylenevinylene), Poly (2 , 5-d iocty I - 1 ,4- phenylenevinylene), Poly(2,5-dioctylphenylene-1 ,4-ethynylene), Poly [ 1 ,2- bis(benzylthio)acetylene], Poly[1 ,2-bis(ethylthio)acetylene],

[0078] Poly[bis(methylthio)acetylene], Poly(3,5 pyridine), Poly(3-(2- methoxyethoxy)ethoxymethylthiophene-2,5-diyl), 5,5'-Dibromo-2,2'-bithiophene, Poly(3-butylthiophene-2,5-diyl), Poly(3-cyclohexyl-4-methylthiophene-2,5-diyl), Poly(3-cyclohexylthiophene-2,5-diyl), Poly(3-decylthiophene-2,5-diyl), Poly(3- dodecylthiophene-2,5-diyl), Poly(3-hexylthiophene-2,5-diyl), Poly(3- octylthiophene-2,5-diyl), Thiophene Oligothiophenes, 5,5'-Dibromo-2,2'- bithiophene, 2,2',5',2",5",2"'-Quaterthiophene, a-Sexithiophene, 3110732,2':5',2"- Terthiophene, Poly(thiophene-2,5-diyl), bromine terminated powder, 2,3- Dihydrothieno[3,4-b]-1 ,4-dioxin, 3,2':5',3"-Terthiophene, 5-Hexyl-2,2'-bithiophene, 22,2'-Bithiophene, Thiophene, 5,5""-Dihexyl-2,2':5',2":5",2"':5"',2"":5"",2 - sexithiophene, Poly(styrenesulfonate) / poly(2,3-dihydrothieno(3,4-b)-1 ,4-dioxin), Poly(3,4-ethylenedioxythiophene)-block-poly(ethylene glycol), Poly(3,4- ethylenedioxythiophene), tetramethacrylate endcapped, Poly((9,9-dihexyl-9H- fluorene-2,7-diyl)-co-(1-methoxy-4-(2-ethylhexyloxy)-2,5-phenylenevinylene)), Poly((9,9-dihexyl-9H-fluorene-2,7-diyl)-co-(1-methoxy-4-(2-ethylhexyloxy)-2,5- phenylenevinylene), Poly(1-methoxy-4-(3-propyloxy-heptaisobutyl-PSS)-2,5- phenylenevinylene)-co-(1-methoxy-4-(2-ethylhexyloxy)-2,5-phenylenevinylene), Poly(2,5-bis(3-sulfonatopropoxy)-1 ,4-phenylene, disodium salt-alt-1 ,4- phenylene), Poly(2,5-dihexyloxy-1 ,4-phenylenevinylene), Poly (2 ,5-diocty 1-1 ,4- phenylenevinylene), Poly(2,6-naphthalenevinylene), Poly[5-methoxy-2-(3- sulfopropoxy)-1 ,4-phenylenevinylene] Potassium salt, Poly(p-xylene tetrahydrothiophenium chloride), Poly[(m-phenylenevinylene)-alt-(2-methoxy-5- octyloxy-p-phenylenevinylene)] , Poly[(m-phenylenevinylene)-alt-(2,5-dihexyloxy- p-phenylenevinylene)], Poly[(m-phenylenevinylene)-alt-(2-methoxy-5-(2-ethyl- hexyloxy)-p-phenylenevinylene)], Poly[(m-phenylenevinylene)-co-(2,5-dioctoxy- p-phenylenevinylene)], Poly[(m-phenylenevinylene)-alt-(2,5-dibutoxy-p- phenylenevinylene)], Poly[(o-phenylenevinylene)-alt-(2-methoxy-5-(2- ethylhexyloxy)-p-phenylenevinylene)], Poly[(p-phenylenevinylene)-alt-(2- methoxy-5-(2-ethyl-hexyloxy)-p-phenylenevinylene)], Poly[2-(2',5'-bis(2"- ethylhexyloxy)phenyl)-1 ,4-phenylenevinylene], Poly[2,5-bis(3' 7'- dimethyloctyloxy)-1 ,4-phenylenevinylene], Poly[(2,5-bisoctyloxy)-1 ,4- phenylenevinylene], Poly[2-methoxy-5-(2-ethylhexyloxy)-1 ,4-phenylenevinylene], Poly[2-methoxy-5-(3',7'-dimethyloctyloxy)-1 ,4-phenylenevinylene], Poly[5- methoxy-2-(3-sulfopropoxy)-1 ,4-phenylenevinylene], Poly[9-(2-ethylhexyl)-3,6- carbazolevinylene-alt-2,6-naphthalenevinylene], Poly{[2-[2',5'-bis(2"- ethylhexyloxy)phenyl]-1 ,4-phenylenevinylene]-co-[2-methoxy-5-(2'- ethylhexyloxy)-1 ,4-phenylenevinylene]}, Poly[(9,9-dioctylfluorenyl-2,7-diyl)-co- (1 ,4-phenylene)], Poly[(9,9-dioctylfluorenyl-2,7-diyl)-co-(4,4'-(N-(4-sec- butylphenyl)diphenylamine)], Poly(2,5-di(3',7'-dimethyloctyl)phenylene-1 ,4- ethynylene), Poly[(9,9-dioctyl-2,7-divinylenefluorenylene)-alt-co-{2-methoxy5-(2- ethylhexyloxy)-1 ,4-phenylene}], Poly[(9, 9-diocty I-2 ,7-bis{2- cyanovinylenefluorenylene})-alt-co- (2-methoxy-5-{2-ethylhexyloxy}-1 ,4- phenylene)], Poly(9,9-dioctylfluorene-alt-benzothidiazole), Poly[2-methoxy-5-(2- ethylhexyloxy)-1 ,4-( 1 -cyanovinylene-1 ,4-phenylene)], Poly [{9, 9-dihexy I-2 , 7-bis( 1 - cyanovinylene)fluorenylene}-alt-co- {2, 5-bis(N,N’-diphenylamino)-1 ,4- phenylene}], Poly(4,4-dioctyldithieno(3,2-b:2',3'-d)silole)-2,6-diyl-alt-(2, 1 ,3- benzothiadiazole)-4,7-diyl), Poly[(5-fluoro-2, 1 ,3-benzothiadiazole-4,7-diyl)(4,4- dihexadecyl-4H- cyclopenta[2,1-b:3,4- b']dithiophene-2,6-diyl)(6-fluoro-2,1 ,3- benzothiadiazole- 4,7-diyl)(4,4-dihexadecyl-4H-cyclopenta[2, 1 -b:3,4- b']dithiophene-2,6-diyl)], Poly{2,2'-[(2,5-bis(2-hexyldecyl)-3,6-dioxo-2, 3,5,6- tetrahydropyrrolo[3,4-c ]pyrrole-1 ,4-diyl)dithiophene]- 5,5'-diyl-alt-thiophen-2 ,5- diyl}, Poly[[4,8-bis[(2-ethylhexyl)oxy]benzo[1 ,2-b:4,5-b']dithiophene-2,6-diyl][3- fluoro-2-[(2-ethylhexyl)carbonyl]thieno[3,4-b]thiophenediyl ]], Poly[2,6-(4,4-bis-(2- ethylhexyl)-4H-cyclopenta [2, 1 -b;3,4-b']dithiophene)-alt-4,7(2, 1 ,3- benzothiadiazole)], Poly[(4,4-bis(2-ethylhexyl)-dithieno[3,2-b:2',3'-d]silole)-2,6- d iy I -al t-(2 , 1 ,3-benzothiadiazole)-4,7-diyl], Poly[(9,9-bis(2-ethylhexyl)fluorenyl-2,7- diyl)-alt-(benzo[2, 1 , 3]thiadiazol-4, 7-diy I)] , Poly[2,6-(4,4-bis(2-ethylhexyl)-4H- cyclopenta-[2,1-b:3,4-b']dithiophene-alt-4,7-bis(thiophen-2-yl)benzo-2,1 ,3- thiadiazole], Poly[2, 1 ,3-benzothiadiazole-4,7-diyl[4,4-bis(2-ethylhexyl)-4H- silolo[3,2-b:4,5-b']dithiophene-2,6-diyl]], Poly[2,7-(9,9-di-octyl-fluorene)-alt-4,7- bis(thiophen-2-yl)benzo-2,1 ,3-thiadiazole] or a combination of any two or more thereof.

[0079] Preferably, the polymer of the second CPN is selected from: Poly[(9,9- dioctylfluorenyl-2,7-diyl)-co-(1 ,4-phenylene)], Poly[(9,9-dioctylfluorenyl-2,7-diyl)- co-(4,4'-(N-(4-sec-butylphenyl)diphenylamine)], Poly(2,5-di(3',7'- dimethyloctyl)phenylene-1 ,4-ethynylene), Poly[(9,9-dioctyl-2,7- divinylenefluorenylene)-alt-co-{2-methoxy5-(2-ethylhexyloxy)-1 ,4-phenylene}], Poly[(9,9-dioctyl-2,7-bis{2-cyanovinylenefluorenylene})-alt-co- (2-methoxy-5-{2- ethylhexyloxy}-1 ,4-phenylene)], Poly(9,9-dioctylfluorene-alt-benzothidiazole), Poly[2-methoxy-5-(2-ethylhexyloxy)-1 ,4-( 1 -cyanovinylene-1 ,4-phenylene)], Poly[{9,9-dihexyl-2,7-bis(1-cyanovinylene)fluorenylene}-alt-co- {2,5-bis(N,N’- diphenylamino)-1 ,4-phenylene}], Poly(4,4-dioctyldithieno(3,2-b:2’,3’-d)silole)-2,6- d iy I -al t-(2 , 1 ,3-benzothiadiazole)-4,7-diyl), Poly[(5-fluoro-2, 1 ,3-benzothiadiazole- 4,7-diyl)(4,4-dihexadecyl-4H- cyclopenta[2,1-b:3,4- b’]dithiophene-2,6-diyl)(6- fluoro-2, 1 ,3-benzothiadiazole- 4,7-diyl)(4,4-dihexadecyl-4H-cyclopenta[2, 1 -b:3,4- b’]dithiophene-2,6-diyl)], Poly{2,2'-[(2,5-bis(2-hexyldecyl)-3,6-dioxo-2, 3,5,6- tetrahydropyrrolo[3,4-c ]pyrrole-1 ,4-diyl)dithiophene]- 5,5'-diyl-alt-thiophen-2 ,5- diyl}, Poly[[4,8-bis[(2-ethylhexyl)oxy]benzo[1 ,2-b:4,5-b’]dithiophene-2,6-diyl][3- fluoro-2-[(2-ethylhexyl)carbonyl]thieno[3,4-b]thiophenediyl ]], Poly[2,6-(4,4-bis-(2- ethylhexyl)-4H-cyclopenta [2, 1 -b;3,4-b']dithiophene)-alt-4,7(2, 1 ,3- benzothiadiazole)], Poly[(4,4-bis(2-ethylhexyl)-dithieno[3,2-b:2',3'-d]silole)-2,6- d iy I -al t-(2 , 1 ,3-benzothiadiazole)-4,7-diyl], Poly[(9,9-bis(2-ethylhexyl)fluorenyl-2,7- diyl)-alt-(benzo[2, 1 , 3]thiadiazol-4, 7-diy I)] , Poly[2,6-(4,4-bis(2-ethylhexyl)-4H- cyclopenta-[2,1-b:3,4-b']dithiophene-alt-4,7-bis(thiophen-2-yl)benzo-2,1 ,3- thiadiazole], Poly[2, 1 ,3-benzothiadiazole-4,7-diyl[4,4-bis(2-ethylhexyl)-4H- silolo[3,2-b:4,5-b']dithiophene-2,6-diyl]] or Poly[2,7-(9,9-di-octyl-fluorene)-alt-4,7- bis(thiophen-2-yl)benzo-2, 1 ,3-thiadiazole].

[0080] Preferably, the first and second CPNs are different to one another.

[0081] In an embodiment, the second CPN has maximal emission of at least: 420 nm, 435 nm, 475 nm, 510 nm, 530 nm, 550 nm, 580 nm, 610 nm, 660 nm, 680 nm, 770 nm, 820 nm, 830 nm, 840 nm or 1000 nm.

[0082] The first and second CPNs may have different maximal emissions. The second CPN may absorb electromagnetic radiation of the same or similar wavelength as the first CPN emits (i.e. the wavelength of the emission maxima of the first CPN). The skilled person will be able to select such pairs of first and second CPNs that exhibit this property.ln preferred embodiments, the polymer of the first CPN and the polymer of the second CPN are selected from: (i) Poly[2-methoxy-5-(2-ethylhexyloxy)-1 ,4-( 1 -cyanovinylene-1 ,4- phenylene)] and Poly[2,6-(4,4-bis(2-ethylhexyl)-4H-cyclopenta-[2, 1 - b:3,4-b']dithiophene-alt-4,7-bis(thiophen-2-yl)benzo-2,1 ,3-thiadiazole];

[0083] (ii) Poly[2-methoxy-5-(2-ethylhexyloxy)-1 ,4-(1 -cyanovinylene- 1 ,4- phenylene)] and Poly[[4,8-bis[(2-ethylhexyl)oxy]benzo[1 ,2-b:4,5- b']dithiophene-2,6-diyl][3-fluoro-2-[(2-ethylhexyl)carbonyl]thieno[3,4- b]thiophenediyl]];

[0084] (iii) Poly[(9,9-bis(2-ethylhexyl)fluorenyl-2,7-diyl)-alt- (benzo[2,1 ,3]thiadiazol-4,7-diyl)] and Poly[2,7-(9,9-di-octyl-fluorene)-alt- 4,7-bis(thiophen-2-yl)benzo-2, 1 , 3-th i ad iazole] ;

[0085] (iv) Poly(2,5-di(3',7'-dimethyloctyl)phenylene-1 ,4-ethynylene) and Poly[2- methoxy-5-(2-ethylhexyloxy)-1 ,4-(1 -cyanovinylene- 1 ,4-phenylene)]; or

[0086] (v) Poly[2,7-(9,9-di-octyl-fluorene)-alt-4,7-bis(thiophen-2-yl)benzo-2, 1 ,3- thiadiazole] and Poly[2, 1 ,3-benzothiadiazole-4,7-diyl[4,4-bis(2- ethylhexyl)-4H- silolo[3,2-b:4,5-b']dithiophene-2,6-diyl]] respectively.

[0087] In an embodiment of the invention, the quencher is bound to a molecule that is intended to have binding affinity with the targeting molecule, to thereby hold the quencher in proximity to the CPN. The quencher can be conjugated to the to this molecule covalently (such as a dye to a small molecule, such as a drug), or coat the target entity via non-covalent interactions (such as an acid wool dye to a protein). The skilled person will be able to identify and use appropriate quenching molecules based on the chemistry required to conjugate to or coat a molecule with binding affinity with the targeting molecule. This allows the complexing of the quencher molecule to the molecular complex, increasing the absorbance of the fluorescent light from the CPN, thereby quenching it. Furthermore, this can decrease the binding affinity of the molecule with the targeting molecule compared to a molecule without the quencher molecule bound. By decreasing the affinity of the molecule (to which the quencher is bound) for the targeting molecule, this allows the displacement of the molecule from the targeting molecule when the target entity is brought into contact. In an embodiment, the quencher molecule is bound to a molecule which is structurally identical or structurally similar to the target entity.

[0088] In an embodiment, the acid wool dye (quencher) is bound to a molecule that is intended to have binding affinity with the targeting molecule, to thereby hold the quencher in proximity to the CPN. In a further embodiment, the molecule the acid wool dye is bound to is a protein. Acid wool dyes can be bonded to proteins by the process of staining, due to the properties of acid wool dyes and proteins as explained herein. As used herein, the term “staining” refers to the application of dyes to a specimen, such as but not limited to a biological specimen.

[0089] The present invention makes use of a targeting molecule. As used herein, the term “targeting molecule” refers to any molecule able to specifically bind to a target entity. As such it can be an antibody, a protein, a nucleic acid, a lipid, an affimer, an aptamer, a molecularly imprinted polymer, a small molecule, or an antigen. The skilled person will be able to identify and use appropriate targeting molecules, based on conventional rapid diagnostic tests.

[0090] Preferably, the targeting molecule is an antibody. In one embodiment, the antibody is selected from is either a full-length antibody, an antibody fragment containing antigen binding domains, or a modified antibody or modified antibody fragment containing substitutions or modifications of the amino acid residues.

[0091] As used herein, the term "antibody" refers to a protein, or polypeptide sequence derived from an immunoglobulin molecule which specifically binds with an antigen. Antibodies can be monoclonal or polyclonal, and be of multiple or single chain types. They can be derived from both natural sources and recombinant sources. Antibodies can be tetramers of immunoglobulin molecules. The antibody may be humanized, non-human, or chimeric. Antibodies suitable for use will also be readily apparent to the skilled person.

[0092] As used herein, the term “antibody fragment” refers to a fragment of an antibody that retains the ability to interact specifically with an antigen epitope. These fragments include but are not limited to Fab, F(ab')2, monospecific Fab2, bispecific Fab2, trispecific Fab2, linear antibodies, single domain antibodies, scFv, and these fragments can also be incorporated into single domain antibodies, minibodies, maxibodies, nanobodies, intrabodies, diabodies, triabodies, and tetrabodies.

[0093] In one embodiment, a CPN is bound to the antibody, preferably covalently, but can be any kind of bond which allows the invention to function in this embodiment through the displacement of the quencher molecule. In this embodiment, the target entity would be an antigen, and the quencher molecule will be removably bound within the complex, eg the quencher may have affinity towards the antibody or be bound to a molecule with affinity towards the antibody. In one embodiment, the quencher molecule could be bound to an antigen, wherein the antigen has affinity towards the targeting molecule. In one embodiment, the quencher molecule is an acid wool dye. In this embodiment, the antibody (the targeting molecule) would contact a target entity, which would bind the antibody, displacing the quencher molecule. Since the quencher-bound antigen in the molecular complex has decreased affinity towards the antibody (due to quencher binding), this allows the displacement of it from the molecular complex in favour of the target entity, such as an antigen not bound by a quencher molecule. In the absence of the quencher molecule, the fluorescence of the CPN can be visualised. The CPN would be in a complex with the target entity.

[0094] Alternatively, in a separate embodiment, a quencher molecule is bound to the antibody, preferably covalently, but can be any kind of bond which allows the invention to function in this embodiment through the displacement of the CPN. In this embodiment, the target entity would be an antigen, and the CPN would have affinity towards the antibody or be bound to a molecule with affinity towards the antibody. In one embodiment, the CPN could be bound to an antigen, wherein the antigen has affinity towards the targeting molecule. In one embodiment, the quencher molecule is an acid wool dye. In this embodiment, the antibody (the targeting molecule) would contact a target entity, which would bind the antibody, displacing the CPN. Since the quencher-bound antigen in the molecular complex has decreased affinity towards the antibody (due to quencher binding), this allows the displacement of it from the molecular complex in favour of the target entity, such as an antigen not bound by a quencher molecule. In the absence of the quencher molecule, the fluorescence of the CPN can be visualised. The CPN would not be in a complex with the target entity.

[0095] As used herein, the term “visualised” refers to the identification of fluorescence through a suitably visual format. For example, this could be via illumination using a suitable light source of any particular wavelength and the subsequent identification either by eye or through a suitable imaging camera equipped with a sensor such as a CCD or sCMOS.

[0096] In one embodiment, the targeting molecule is an affimer.

[0097] As used herein, the term “affimer” refers to novel protein binders that have high specificity against a target entity. Affimers are selected for their high specificity against the target protein and have benefits over antibodies like batch-to-batch reproducibility and are stable across a wide range of chemical conditions.

[0098] In one embodiment, a CPN is bound to the affimer, preferably covalently, but can be any kind of bond which allows the invention to function in this embodiment through the displacement of the quencher molecule. In this embodiment, the target entity would be a protein, and the quencher molecule will be removably bound within the complex, eg the quencher may have affinity towards the affimer or be bound to a molecule with affinity towards the affimer. In one embodiment, the quencher molecule could be bound to a protein, wherein the protein has affinity towards the targeting molecule. In one embodiment, the quencher molecule is an acid wool dye. In this embodiment, the affimer (the targeting molecule) would contact a target entity, which would bind the affimer, displacing the quencher molecule. Since the quencher-bound protein in the molecular complex has decreased affinity towards the affimer (due to quencher binding), this allows the displacement of it from the molecular complex in favour of the target entity, such as a protein not bound by a quencher molecule. In the absence of the quencher molecule, the fluorescence of the CPN can be visualised. The CPN would be in a complex with the target entity. Alternatively, in a separate embodiment, a quencher molecule is bound to the affimer, preferably covalently, but can be any kind of bond which allows the invention to function in this embodiment through the displacement of the CPN. In this embodiment, the target entity would be a protein, and the CPN would have affinity towards the affimer or be bound to a molecule with affinity towards the affimer. In one embodiment, the CPN could be bound to a protein, wherein the protein has affinity towards the targeting molecule. In one embodiment, the quencher molecule is an acid wool dye. In this embodiment, the affimer (the targeting molecule) would contact a target entity, which would bind the affimer, displacing the CPN. Since the quencher-bound protein in the molecular complex has decreased affinity towards the affimer (due to quencher binding), this allows the displacement of it from the molecular complex in favour of the target entity, such as a protein not bound by a quencher molecule. In the absence of the quencher molecule, the fluorescence of the CPN can be visualised. The CPN would not be in a complex with the target entity.

[0099] In one embodiment, the targeting molecule is an aptamer.

[0100] As used herein, the term “aptamer” refers to synthetic molecules comprising DNA, RNA, or peptides capable of binding moieties with high affinity and specificity. They can be used in similar applications as to antibodies, and can be engineered to bind moieties such as but not limited to cells, proteins, small molecules, heavy metal ions, and other ligands.

[0101] In one embodiment, a CPN is bound to the aptamer, preferably covalently, but can be any kind of bond which allows the invention to function in this embodiment through the displacement of the quencher molecule. In this embodiment, the target entity would be selected from a suitable aptamer ligand, such as a cell, protein, small molecule, or heavy metal ion, and the quencher molecule will be removably bound within the complex, eg the quencher may have affinity towards the aptamer or be bound to a molecule with affinity towards the aptamer. In one embodiment, the quencher molecule could be bound to a suitable aptamer ligand, wherein the suitable aptamer ligand has affinity towards the targeting molecule. In one embodiment, the quencher molecule is an acid wool dye. In this embodiment, the aptamer (the targeting molecule) would contact a target entity, which would bind the aptamer, displacing the quencher molecule. Since the quencher-bound suitable aptamer ligand in the molecular complex has decreased affinity towards the aptamer (due to quencher binding), this allows the displacement of it from the molecular complex in favour of the target entity, such as a suitable aptamer ligand not bound by a quencher molecule. In the absence of the quencher molecule, the fluorescence of the CPN can be visualised. The CPN would be in a complex with the target entity.

[0102] Alternatively, in a separate embodiment, a quencher molecule is bound to the aptamer, preferably covalently, but can be any kind of bond which allows the invention to function in this embodiment through the displacement of the CPN. In this embodiment, the target entity would be selected from a suitable aptamer ligand, such as a cell, protein, small molecule, or heavy metal ion, and the CPN would have affinity towards the aptamer or be bound to a molecule with affinity towards the aptamer. In one embodiment, the CPN could be bound to a suitable aptamer ligand, wherein the suitable aptamer ligand has affinity towards the targeting molecule. In one embodiment, the quencher molecule is an acid wool dye. In this embodiment, the aptamer (the targeting molecule) would contact a target entity, which would bind the aptamer, displacing the CPN. Since the quencher-bound suitable aptamer ligand in the molecular complex has decreased affinity towards the aptamer (due to quencher binding), this allows the displacement of it from the molecular complex in favour of the target entity, such as a suitable aptamer ligand not bound by a quencher molecule. In the absence of the quencher molecule, the fluorescence of the CPN can be visualised. The CPN would not be in a complex with the target entity.

[0103] In one embodiment, the targeting molecule is a molecularly imprinted polymer.

[0104] As used herein, the term “molecularly imprinted polymer” refers to a polymer that has been produced using Molecular Imprinting Technology to create artificial recognition sites in polymeric matrices. This is achieved through the polymerisation of monomers around a chosen “template” molecule which generates complimentary cavities in the polymer. This polymer therefore has high affinity binding sites for the chosen “template” molecule.

[0105] In one embodiment, a CPN is bound to the molecularly imprinted polymer, preferably covalently, but can be any kind of bond which allows the invention to function in this embodiment through the displacement of the quencher molecule. In this embodiment, the target entity would be the template molecule of the molecularly imprinted polymer, and the quencher molecule will be removably bound within the complex, eg the quencher may have affinity towards the molecularly imprinted polymer or be bound to a molecule with affinity towards the molecularly imprinted polymer. In one embodiment, the quencher molecule could be bound to the template molecule of the molecularly imprinted polymer, wherein the template molecule has affinity towards the targeting molecule. In one embodiment, the quencher molecule is an acid wool dye. In this embodiment, the molecularly imprinted polymer (the targeting molecule) would contact a target entity, which would bind the molecularly imprinted polymer, displacing the quencher molecule. Since the quencher-bound template molecule of the molecularly imprinted polymer in the molecular complex has decreased affinity towards the molecularly imprinted polymer (due to quencher binding), this allows the displacement of it from the molecular complex in favour of the target entity, such as a template molecule of the molecularly imprinted polymer not bound by a quencher molecule. In the absence of the quencher molecule, the fluorescence of the CPN can be visualised. The CPN would be in a complex with the target entity.

[0106] Alternatively, in a separate embodiment, a quencher molecule is bound to the molecularly imprinted polymer, preferably covalently, but can be any kind of bond which allows the invention to function in this embodiment through the displacement of the CPN. In this embodiment, the target entity would be the template molecule of the molecularly imprinted polymer, and the CPN would have affinity towards the molecularly imprinted polymer or be bound to a molecule with affinity towards the molecularly imprinted polymer. In one embodiment, the CPN could be bound to the template molecule of the molecularly imprinted polymer, wherein the template molecule has affinity towards the targeting molecule. In one embodiment, the quencher molecule is an acid wool dye. In this embodiment, the molecularly imprinted polymer (the targeting molecule) would contact a target entity, which would bind the molecularly imprinted polymer, displacing the CPN. Since the quencher-bound template molecule of the molecularly imprinted polymer in the molecular complex has decreased affinity towards the molecularly imprinted polymer (due to quencher binding), this allows the displacement of it from the molecular complex in favour of the target entity, such as a template molecule of the molecularly imprinted polymer not bound by a quencher molecule. In the absence of the quencher molecule, the fluorescence of the CPN can be visualised. The CPN would not be in a complex with the target entity.ln one embodiment, the targeting molecule is a small molecule.

[0107] As used herein, the term “small molecule” refers to a low molecularweight organic compound which is often involved in biological processes, either as a substrate or product. Small molecules used as targeting molecules would need to have specificity and affinity towards an agent of interest. Examples of such small molecules include but are not limited to any pharmacologically active molecule, biotin, a drug with affinity towards a specific protein, or a metabolite with affinity towards a certain protein.

[0108] In one embodiment, a CPN is bound to the small molecule, preferably covalently, but can be any kind of bond which allows the invention to function in this embodiment through the displacement of the quencher molecule. In this embodiment, the target entity would be a substance wherein the small molecule is a ligand of, and the quencher molecule will be removably bound within the complex, eg the quencher may have affinity towards the small molecule or be bound to a molecule with affinity towards the small molecule. In one embodiment, the quencher molecule could be bound to a substance wherein the small molecule is a ligand of, wherein the substance has affinity towards the targeting molecule. In one embodiment, the quencher molecule is an acid wool dye. In this embodiment, the small molecule (the targeting molecule) would contact a target entity, which would bind the small molecule, displacing the quencher molecule. Since the quencher-bound substance in the molecular complex has decreased affinity towards the small molecule (due to quencher binding), this allows the displacement of it from the molecular complex in favour of the target entity, such as a substance wherein the small molecule is a ligand of not bound by a quencher molecule. In the absence of the quencher molecule, the fluorescence of the CPN can be visualised. The CPN would be in a complex with the target entity.

[0109] Alternatively, in a separate embodiment, a quencher molecule is bound to the small molecule, preferably covalently, but can be any kind of bond which allows the invention to function in this embodiment through the displacement of the CPN. In this embodiment, the target entity would be a substance wherein the small molecule is a ligand of, and the CPN would have affinity towards the small molecule or be bound to a molecule with affinity towards the small molecule. In one embodiment, the CPN could be bound to a substance wherein the small molecule is a ligand of, wherein the substance has affinity towards the targeting molecule. In one embodiment, the quencher molecule is an acid wool dye. In this embodiment, the small molecule (the targeting molecule) would contact a target entity, which would bind the small molecule, displacing the CPN. Since the quencher-bound substance in the molecular complex has decreased affinity towards the small molecule (due to quencher binding), this allows the displacement of it from the molecular complex in favour of the target entity, such as substance wherein the small molecule is a ligand of not bound by a quencher molecule. In the absence of the quencher molecule, the fluorescence of the CPN can be visualised. The CPN would not be in a complex with the target entity. In the embodiment wherein the targeting molecule is an antigen, this embodiment is similar to the embodiment wherein the targeting molecule is an antibody.

[0110] As used herein, the term “antigen” refers to a molecule that binds specifically to the antigen-binding domain of any antigen receptor. This could be the antigenbinding site of an immunoglobulin of fragment thereof, comprising the VL and VH domains. In the context where the quencher molecule can be displaced from the molecular complex, and the target entity is intended to be an antibody, the CPN is bound (preferably covalently) to an antigen with affinity for the target antibody. The quencher molecule will be removably bound within the complex, eg the quencher may have affinity towards the antigen or be bound to a molecule with affinity towards the antigen.

[0111] In the context where the CPN can be displaced from the molecular complex, and the target entity is intended to be an antibody, the quencher molecule is bound (preferably covalently) to an antigen with affinity for the target antibody. The CPN will be removably bound within the complex, eg the CPN may have affinity towards the antigen or be bound to a molecule with affinity towards the antigen.

[0112] The conjugated polymer nanoparticle complex of the present invention can be engineered to identify and therefore target a wide variety of target entities. The targeting ability of the conjugated polymer nanoparticle complex is due to the targeting molecule, which has affinity towards at least one target entity. Targeting molecules can be selected based on the target entity in need of detection and identification. In an embodiment of the invention, the targeting molecule has affinity towards a biological or chemical target. The target may be any suitable biological entity, the detection of which is desirable. The skilled person will appreciate the types of biological entities that are desirable to detect. Suitable examples include biological toxins, bacteria, viruses, fungi, proteins, biomarkers, or nucleic acids. The target may also be a chemical entity. Again, the skilled person will appreciate chemical entities which are desirable to detect. Suitable chemical entities include chemical toxins, narcotics, harmful chemical agents, residues of explosives, etc.

[0113] As used herein, the term “toxin” refers to an organic poison produced by a living organism. Toxins can comprise small molecules, enzymes, or fragments thereof, and have bacterial, fungal, plant, or animal origin. An example includes anthrax toxin, a three-protein exotoxin secreted by virulent strains of Bacillus anthracis. As used herein, the term “bacteria” refers to prokaryotic single-celled microorganisms of the domain Bacteria. This includes gram-positive and gramnegative bacteria. Examples include: Bacillus anthracis, Escherichia coli, Staphylococcus aureus, Salmonella, Streptococcus pyogenes, Clostridium botulinum, Clostridium difficile, Mycobacterium tuberculosis, Helicobacter pylori, Neisseria gonorrhoeae, Chlamydia trachomatis, Vibrio cholerae, Listeria monocytogenes, Pseudomonas aeruginosa, Mycobacterium leprae and Yersinia pestis among others.

[0114] As used herein, the term “virus” refers to a submicroscopic infectious agent capable of replication inside living cells. Examples include: influenza virus, rhinovirus, enterovirus, human immunodeficiency virus, coronavirus, herpes simplex virus, rotavirus, Ebola virus and Dengue virus among others.

[0115] As used herein, the term “fungi” refers to eukaryotic organisms from the groups such as yeasts and moulds. Examples include: Candida albicans, aspergillus fumigatus and cryptococcus neoformans among others.

[0116] As used herein, the term “protein” refers to a biological macromolecule comprising one or more polypeptide chains. Examples of protein classes include, but are not limited to: structural proteins, enzymes, hormones, and antibodies.

[0117] As used herein, the term “nucleic acid” refers to a biopolymer of nucleotide monomers. Examples include, but are not limited to: DNA, RNA and artificial nucleic acid analogues.

[0118] In one embodiment of the invention, the protein is an allergen. It is envisaged that the allergen is selected from at least: milk protein, egg protein, peanut protein, soy protein, wheat protein, tree nut protein, shellfish protein, fish protein, sesame protein, celery protein, gluten protein, lupin protein or mustard protein.

[0119] As used herein, the term “allergen” refers to a substance capable of inducing an allergic reaction. Examples of allergens include, but are not limited to: milk protein, egg protein, peanut protein, soy protein, wheat protein, tree nut protein, shellfish protein, fish protein, sesame protein, celery protein, gluten protein, lupin protein or, mustard protein.

[0120] In an embodiment of the invention, the chemical entity is a drug. As used herein, the term “drug” refers to a substance that has a physiological effect when introduced into an organism. These substances can be, but are not limited to, small molecule or proteins. Classes of drugs include stimulants, opioids, depressants, hallucinogens, inhalants, dissociataives, and encompass narcotics and psychoactive drugs. Drugs of interest may be prescribed drugs, or drugs not available for prescription. Examples of drugs include, but are not limited to fentanyl, acetylfentanyl, xylazine, heroin, morphine, opium, methadone, hydrocodone, buprenorphine, oxycodone, alprazolam, tetrahydrocannabinol, methamphetamine, cocaine, lysergic acid diethylamide, 3,4- Methylenedioxymethamphetamine, phencyclidine, psilocybin, psilocin, N,N- Dimethyltryptamine, amphetamine, codeine, cannabinol, cannabis, cannabidiol, synthetic cannabinoids, ketamine, methaqualone, 4-Methylmethcathinone, methylphenidate, alprazolam, flunitrazepam, 4-Hydroxy-n-butyric acid, nitrous oxide, mephedrone, sotonitazene, metonitazene, etonitazene or, protonitazene. Preferably, the drug is fentanyl.

[0121] As used herein, the term “analog” means a substance that is structurally or functionally similar to another drug. These analogs may be intentionally designed and synthesized for various purposes, including medical, scientific, or forensic applications. A structural analog has a similar chemical structure to a reference drug, but it may have slight modifications. These modifications can affect the drug's pharmacological properties, such as its potency, efficacy, or safety profile. A functional analog has a different chemical structure but produces similar biological effects or acts on the same target as the reference drug. Functional analogs may have distinct chemical structures but share certain pharmacological properties with the original drug. An “analog” may also refer to substances that are chemically similar to illegal drugs but have been designed to evade legal restrictions. These substances are sometimes referred to as “designer drugs” or “research chemicals”. They are created by modifying the chemical structure of known illegal drugs in an attempt to produce substances with similar psychoactive effects but that are not explicitly covered by existing drug laws.

[0122] As used herein, the term “metabolite” refers to any substance that is produced or consumed during cellular metabolism. This can include endogenous compounds naturally occurring in cells, or compounds of extracellular origin such as drugs.

[0123] In one embodiment, the molecular complex may further comprise a metal oxide, which may have magnetic properties. These properties are useful because they make it possible for a magnet or magnetic field to be used to purify or separate bound cells, proteins, or other moieties from a mixed population or sample, or to move them.

[0124] As used herein, the term “metal oxide” refers to entities consisting of any ferromagnetic, paramagnetic, or superparamagnetic materials, which are able to be wholly or partly encapsulated in the nanoparticle of the present invention. Such materials include, but are not limited to, iron oxide (FeO, Fe2O3, Fe3O4), including both superparamagnetic iron oxide (SPIO) and ultrasmall super-paramagnetic iron oxide (USPIO); other metal oxides and metal oxide heterostructures such as MnO, M-Fe3O4 (M= Fe, Au, Ag) or MFe2O4 (M = Ni, Co, Mn); metal particles such as Fe, FePt, Co and FeCo; gadolinium complexes such as Gd2O3, GdF3and NaGdF4. In many instances metal oxides are used, in particular iron oxides such as Fe3O4.

[0125] The metal oxide may comprise a magnetic material or a plurality of magnetic materials used in combination, for instance, there may be one, two, three or four different magnetic materials present in the CPN of the invention. In some embodiments of the invention the core of the CPN will include a metal oxide, and a second (or a plurality of) magnetic materials. This second magnetic material may be selected from gadolinium, iron, or manganese complexes such as those listed above.

[0126] The metal oxides may be randomly distributed throughout the polymer core or may be distributed within the core such that it is generally present towards the outer surface of the core. In some instances, the magnetic material will be present in a layer around the polymer core, in some cases the magnetic material could be said to encapsulate the core, with the surfactant and / or lipid encapsulating the core of magnetic material and water-insoluble conjugated polymer. It has been observed, however, that regardless of the internal structure of the core, the luminescence / fluorescence properties of the conjugated polymer are not impaired by the presence of the magnetic material and vice versa.

[0127] The molecular complex of the present invention is envisaged to be configured in a variety of modalities to detect target entities in different environments, whether that be in a laboratory, in the field, or in vivo. In a further embodiment of the invention, the molecular complex is arranged on a substrate. As used herein, the term “substrate” means a substance, surface, or layer upon which an entity can be placed. In an embodiment of the invention, the substrate is filter paper. In another embodiment of the invention, the substrate is a porous or solid support. As used herein, the term “solid support” means a material or structure that provides a solid surface for the attachment, immobilisation, or support of a substance. Examples include, but are not limited to, beads and membranes. In a further embodiment of the invention, the substrate is ceramic. In a further embodiment of the invention, the substrate is borosilicate glass. In yet a further embodiment of the invention, the substrate is plastic. As used herein, the term “plastic” refers to synthetic or semi-synthetic material made from polymers, which are large molecules composed of repeating structural units. These structural units, called monomers, are chemically bonded together to form long chains or networks, creating the characteristic properties of plastics. Plastics can be moulded, shaped, and hardened into a wide variety of forms, making them versatile materials with diverse applications. Preferably, the plastic is polyethersulfone.

[0128] The molecular complex may need to be configured in such a way to be applied to a particular substrate effectively. In a further embodiment of the invention, the molecular complex is configured for application to a substrate. It is envisaged that the molecular complex will be configured to be applied to a wide variety of substrates, including substrates that are both homogenous and heterogenous in character. In this embodiment, detection of the target entity would be possible due to a target entity contacting the molecular complex arranged on a substrate. Samples which may contain target entities could be collected and applied to the substrate. Alternatively, if the target entity was present in the air, such as in the form of an aerosol, the exposure of the substrate to the air will allow for detection of the target entity without the need for collecting and applying a sample to the substrate.

[0129] In yet a further embodiment of the invention, the molecular complex may be suspended in a liquid. It is envisaged that this liquid comprising the molecular complex could be used in an assay, wherein a colour change would occur on the detection of a target entity. The molecular complexes of the liquid would be prequenched, and a sample would be added to the liquid. The presence of target entities in the sample would facilitate the displacement of the quencher molecules, allowing a fluorescent signal or colour change to be detected. As used herein, the term “liquid” refers to a suitable solvent capable of forming a solution of molecular complexes of the present invention. Examples may include, but are not limited to water, alcohols, acids and bases, organic reagents, oils, and ionic liquids.

[0130] The ability to detect multiple targets at once is also of high interest, as this would reduce resources used and quicken the analysis pipeline through multiple detections of target entities in parallel. The present invention could be worked in two different ways to allow the detection of multiple targets at once. The first involves using different molecular complexes at once, their variations resulting from changing the targeting molecule. Each molecular complex would fluoresce at the same or similar wavelength, but each would be specific towards one target entity. The second involves using different molecular complexes at once, their variations resulting from changing the targeting and changing the CPN. Each molecular complex would fluoresce at different wavelengths, and each would be specific towards one target entity. In one embodiment, the molecular complex is configured as a plurality of unique molecular complexes with different targeting molecules arranged in discrete areas on a support material such that they can recognise multiple target entities through multiple affinities to target entities. It is envisaged that many molecular complexes featuring different targeting molecules for the target entities of interest could be arranged on a substrate in discrete ‘zones’, allowing the identification of the target entity by matching the location of the resultant fluorescence with the initial location of the application of the molecular complex to the substrate. In this embodiment, each molecular complex may fluoresce at similar wavelengths due to the same quenching molecule being used (positive identification being confirmed by the position of fluorescence on the substrate) or each discrete zone on the substrate may have a unique fluorescence signal.

[0131] In another embodiment, a plurality of unique molecular complexes with different targeting molecules and different CPNs can recognise multiple target entities by fluorescing at different wavelengths. This differs from the previous embodiment, wherein only the targeting molecule is changed between different molecular complexes. Unique molecular complexes of the present invention can be made by taking different targeting molecules, each with a specific affinity towards a target entity, and conjugating a different CPN to each one. Therefore, each molecular complex would be specific to a target entity and would generate a unique signal due to each CPN fluorescing at a different wavelength. The additional property of this embodiment therefore negates the requirement for a discrete arrangement of the unique molecular complexes on a support material as in the previous embodiment. This is because detection of each CPN would no longer require the separation of each molecular complex (due to each CPN being of the same identity and fluorescing at the same or similar wavelengths), but instead could be detected through different wavelengths of light (due to each CPN being of different identity and fluorescing at different wavelengths). The composition of the present embodiment could still be applied to a support material, but could be applied uniformly rather than in discrete zones by molecular complex type. The composition of this embodiment could also be suspended in a liquid. To detect each unique molecular complex, the absorption and emission maxima of each CPN in each complex would be known, and different wavelengths of excitation light would be applied, and the corresponding wavelengths of emission light would be detected for each CPN. It is envisaged that various combinations of conjugated polymer nanoparticles and quenching molecules could be created to form a library of signal colours. This library may be created by combining different CPNs with acid wool dyes (quencher molecules). These combinations of conjugated polymer nanoparticles and quenching molecules can be applied to specific targeting molecules to form many molecular complexes of the present invention, each with unique target specificities and unique signal colours (resulting from their unique maximal emission wavelengths). As such, the detection of multiple unique target entities simultaneously would be possible. It is envisaged that this library of signal colours could be applied to sets of target entities which are of related analytical interest. For example, a set of chemical entities, such as drugs, could be simultaneously targeted to provide a profile on the presence of such chemical entities.

[0132] It is also envisaged that the molecular complex of the present invention could be used as a surface contact disclosure fluid. As used herein, the term “surface contact disclosure fluid” refers to the molecular complex of the present invention suspended in a liquid that when applied to a surface may allow the detection of a target entity. In an embodiment of the invention, the molecular complex is formulated for delivery as a spray or aerosol. The molecular complexes of the invention may be pre-quenched in the surface contact disclosure fluid. It is envisaged that the surface contact disclosure fluid could be applied to any physical surface that contains or is thought to contain at least one target entity. This would allow for target entity detection through the molecular complexes detecting the target entity leading to the displacement of the quencher particle or CPN from the from the molecular complex and the molecular complex becoming bound to the areas where the target is present. In the embodiment of the invention wherein the CPN is bound to the targeting molecule and the quencher molecule is displaced from the targeting molecule when brought into contact with a targeting entity, the contaminated areas would be differentiated from safe areas by the fluorescent signal from the molecular complex which would be visible due to the reversal of the quenching effect. It is envisaged that the target entity for the surface contact disclosure fluid would preferably be small molecules or drugs. The surface contact disclosure fluid could be used to detect drugs of abuse, with a fluorescent signal indicating that the drug is present. It may also be used in a hospital setting to detect possible pathogens. The application of this invention in this way would similarly negate the requirement of collecting, extracting, and analysing samples using laborious techniques as discussed. Identification of any target entities would be rapid, following the application of the surface contact disclosure fluid and following visualisation.

[0133] It is also envisaged that the molecular complex of the present invention could be used for in vivo applications. The molecular complex of the present invention generates a detectable signal upon encountering a target entity. For example, the detection of an agent of interest, such as an antigen expressed by a cancerous cell, could be achieved by using a formulation of the molecular complex of the present invention, such as the surface contact disclosure fluid described herein, and would negate the need for lengthy and complex ex vivo or in vitro assays. Since the molecular complex is initially quenched, no signal is provided until a target entity is located. This would be advantageous in the context where a fluorescent signal is not desired until a target entity has been produced or needs to be identified or detected. As described herein, many different types of molecular complexes can be configured, each specific to a target entity and fluorescing at a different wavelengths. This could be used in an in vivo context through the identification of multiple target entities, wherein the presence of a single entity may not be sufficient for a characterising a medical indication. Furthermore, the molecular complex of the present invention could be used for in vivo fluorescent imaging. Since tissue transparency is highest in the near-infrared window (700-1700nm), CPNs fluorescing in this wavelength range could be used for deep tissue imaging and would elucidate both the presence and location of target entities of interest.

[0134] In a second aspect of the invention, there is composition for detecting the presence of a target entity, the composition comprising a conjugated polymer nanoparticle (CPN), capable of fluorescence, bound to a targeting molecule which has affinity towards a target entity, the composition further comprising a quencher molecule.

[0135] This aspect of the invention can be referred to as the “Fluorescence Safe, Dark Detection / Threat” composition. As with the molecular complex, or the “Dark Safe, Fluorescence Detection / Threat” composition as described herein, there are two fluorescent states of this composition. The first state is the “fluorescence” or “safe” state wherein the CPN is bound to a targeting molecule and can freely fluorescence. The second state is the “dark”, “detection”, or “threat” state, wherein the quencher molecule is in proximity to the CPN, and quenches the fluorescence of the CPN, resulting in a decrease in fluorescence.

[0136] The CPN of this composition is defined as in the composition of the first aspect of the invention, the quencher molecule of this composition is defined as in the composition of the first aspect of the invention, and the targeting molecule of this composition is defined as in the composition of the first aspect of the invention.

[0137] In one embodiment, the quencher molecule has affinity towards the target entity. The detection of the presence of a target entity by the “Fluorescence Safe, Dark Detection / Threat” composition is possible due to the decrease in fluorescence due to the quencher molecule being in proximity to the CPN. Since the CPN-bound targeting molecule and quencher molecule do not have affinity towards each other, the two are brought in proximity via the target entity. The quencher molecule has affinity for and can bind to the target entity. The targeting molecule has affinity for the target entity. Since the target entity has affinity for both the quencher molecule and the targeting molecule, the two can be brought in proximity resulting in a decrease in fluorescence from the CPN.

[0138] In a third aspect of the invention, there is provided composition for detecting the presence of a target entity in a sample, the composition comprising: a first molecular complex comprising a first conjugated polymer nanoparticle (CPN) capable of fluorescence, and a first targeting molecule which has affinity towards a control entity; a second molecular complex comprising a second CPN capable of quenching the fluorescence of the first CPN, and a second targeting molecule which has affinity towards a target entity.

[0139] In this aspect of the invention, the polymer of the first CPN (i.e. the CPN capable of fluorescence) may be any of those defined in the first aspect of the invention.

[0140] In this aspect of the invention, the polymer of the second CPN (i.e. acting as a quencher molecule) may be any of those defined in the first aspect of the invention.

[0141] Preferably, the first and second CPNs are different to one another.

[0142] In this aspect of the invention the first targeting molecule or second targeting molecule may be any of the targeting molecules defined in the first aspect of the invention. Preferably, the first and second targeting molecules are different to one another.

[0143] As used herein, a “control entity” refers to an entity which may be targeted by a targeting molecule which is distinct from that targeting the target entity (i.e. different targeting molecules are required to target the control entity and the target entity). Therefore, the control entity and target entity may be different, or they may be identical. In the embodiment where the control entity and target entity are identical, targeting molecules binding to two distinct regions of the control / target entity may be used to discern the two. Suitable identities of the control entity include any of the target entities as described herein, or any other entity that may be targeted by a targeting molecule. The control entity will not be the subject of identification, unlike the target entity. The use of both a control entity and a target entity allows the two molecular complexes of this aspect of the invention to have independent affinitities towards a target.

[0144] Without wishing to be bound by theory, it is thought that this aspect of the invention functions by having two separate molecular complexes, which comprise a first and second targeting molecule which have affinities for different entities (the control entity and the targeting entity). Since the first molecular complex can be quenched by the fluorescence of the second molecular complex, when they are in proximity to one another, the quenching effect will take place. This may occur when the first molecular complex and the second molecular complex are in solution together. Alternatively, this may occur when the first molecular complex and the second molecular complex are together comprised on a surface or substrate. In this embodiment, the first molecular complex and the second molecular complex are present on the surface or substrate such that the second CPN can quench the fluorescence of the first CPN. This may be accomplished by a portion of a surface or substrate comprising the control entity and the target entity, which would allow the first molecular complex and second molecular complexes to find to the surface or substrate. The control entity and the target entity will be present on the surface or substrate in close enough proximity to facilitate quenching when the first molecular complex and the second molecular complex are bound. When a plurality of control entities and the target entities are present on the surface or substrate, they will exist on the surface or substrate in a density sufficient to facilitate quenching enough so that a detectable change may be measured in comparison to measuring the unquenched first molecular complex (i.e. a plurality of pairs of control entities and the target entities will be present on the surface or substrate in close enough proximity to facilitate quenching when the first molecular complex and the second molecular complex are bound).

[0145] In this embodiment, when a target entity is present in the sample and contacted with the second molecular complex, the second molecular complex will bind the target entity via its targeting molecule. Since the second targeting molecule of the second molecular complex is now bound to a target entity from the sample, when the first molecular complex and second molecular complex contacted with a surface or substrate comprising the control entity and the target entity, the first molecular complex will bind to the control entity, however the second molecular complex will no longer bind the targeting entity of the substrate or strip due to already being bound by the target entity of the test sample. Therefore, the first molecular complex not bound to the surface or substrate may be separated from the second molecular complex bound to the surface or substrate, and therefore will fluoresce, as the first CPN is no longer within proximity to the second CPN. In the case where the sample did not comprise the target entity, the second molecular complex will instead bind to the target entity comprised on the surface or substrate, and therefore will be brought within proximity of the second molecular complex (which bound the control entity on the surface or substrate), thereby having the fluorescence of the first CPN quenched by the fluorescence of the second CPN.

[0146] In an embodiment, the first and second targeting molecules of the first and second molecular complexes can bind, on the portion of the surface, to the control entity and the target entity respectively, such that when the first and second targeting molecules of the first and second molecular complexes are bound to said surface, the second CPN of the second molecular complex can quench the fluorescence of the first CPN of the first molecular complex, such that, upon the addition of the sample comprising the target entity, the second targeting molecule of the second molecular complex binds the target entity of the sample, and is therefore prevented from binding to the target entity of the surface so that the fluorescence of the first CPN can be visualised.

[0147] Suitable polymer pairs comprised within the first and second CPNs will be selected based on the ability of the second CPN to quench the fluorescence of the first CPN. In an embodiment, the polymer of the first CPN and the polymer of the second CPN are selected from:

[0148] (i) Poly[2-methoxy-5-(2-ethylhexyloxy)-1 ,4-( 1 -cyanovinylene-1 ,4- phenylene)] and Poly[2,6-(4,4-bis(2-ethylhexyl)-4H-cyclopenta-[2, 1 - b:3,4-b']dithiophene-alt-4,7-bis(thiophen-2-yl)benzo-2,1 ,3-thiadiazole];

[0149] (ii) Poly[2-methoxy-5-(2-ethylhexyloxy)-1 ,4-(1 -cyanovinylene- 1 ,4- phenylene)] and Poly[[4,8-bis[(2-ethylhexyl)oxy]benzo[1 ,2-b:4,5- b']dithiophene-2,6-diyl][3-fluoro-2-[(2-ethylhexyl)carbonyl]thieno[3,4- b]thiophenediyl]];

[0150] (iii) Poly[(9,9-bis(2-ethylhexyl)fluorenyl-2,7-diyl)-alt- (benzo[2,1 ,3]thiadiazol-4,7-diyl)] and Poly[2,7-(9,9-di-octyl-fluorene)-alt- 4,7-bis(thiophen-2-yl)benzo-2, 1 ,3-thiadiazole]; (iv) Poly(2,5-di(3',7'-dimethyloctyl)phenylene-1 ,4-ethynylene) and Poly[2- methoxy-5-(2-ethylhexyloxy)-1 ,4-(1 -cyanovinylene- 1 ,4-phenylene)]; or

[0151] (v) Poly[2,7-(9,9-di-octyl-fluorene)-alt-4,7-bis(thiophen-2-yl)benzo-2, 1 ,3- thiadiazole] and Poly[2, 1 ,3-benzothiadiazole-4,7-diyl[4,4-bis(2- ethylhexyl)-4H- silolo[3,2-b:4,5-b']dithiophene-2,6-diyl]] respectively.

[0152] In a fourth aspect of the invention, there is provided a method for detecting the presence of a target entity in a sample, the method comprising the steps: contacting the sample with a composition comprising the molecular complex as defined in the first aspect of the invention, whereby if present, the target entity displaces either the quencher molecule or the CPN from the molecular complex and replaces it, and in the absence of the quencher molecule, the fluorescence of the CPN can be visualised, thereby providing a positive indication of the presence of the target entity.

[0153] In a fifth aspect of the invention, there is provided a method for detecting the presence of a target entity in a sample, the method comprising the steps: contacting the sample with a composition as defined in the second aspect of the invention, whereby if present, the target entity binds to the quencher molecule, and the targeting molecule binds the targeting entity, bringing the quencher molecule in proximity to the CPN, resulting in a decrease in fluorescence due to quenching.

[0154] In this aspect, the method comprises a single step of adding a sample to the composition as defined in the second aspect of the invention. Since the quencher molecule and CPN are present initially, a low concentration of quencher is required to lessen any initial quenching. A sample would be added to the composition, and if a target entity was present, the quencher molecule would be brought within proximity to the CPN, decreasing the fluorescence emitted. This would be due to the target entity first binding the targeting molecule and the quenching molecule then binding the target entity, or alternatively the quencher molecule would first bind the target entity and the target entity then binding the targeting molecule. In a sixth aspect of the invention , there is provided a method for detecting the presence of a target entity in a sample, the method comprising the steps: contacting the sample first with the quencher molecule of the composition as defined in the second aspect of the invention, whereby if present, the target entity binds to the quencher molecule, and the subsequent addition of the sample and quencher molecule to the CPN bound to a targeting molecule of the composition as defined in second aspect of the invention, wherein the targeting molecule binds the targeting entity, bringing the quencher molecule in proximity to the CPN, resulting in a decrease in fluorescence due to quenching.

[0155] In this aspect, the method comprises two steps. First, the sample is added to the quencher molecule. If the target entity is present, then the quencher molecule will bind to the target entity. This complex is then added to the CPN-targeting molecule, wherein the target entity, carrying the quencher molecule, binds to the targeting molecule, and quenches the fluorescence of the CPN. This method differs from the previous method as initially, quencher molecules are not present with the CPN, allowing a stronger initial fluorescence signal to be measured. Furthermore, due to the two-step process, after the initial binding of the target entity to the quencher molecule, any unbound quencher molecules could be separated from those that have bound to a target entity. Combined, this would increase the sensitivity of detecting the decrease in fluorescence.

[0156] Also provided herein is a method for detecting the presence of a target entity in a sample, the method comprising the steps: contacting the sample with a composition of the third aspect of the invention, whereby if present in the sample, the target entity binds to the second targeting molecule of the second molecular complex, and the control entity binds to the first targeting molecule of the first molecular complex, such that the second molecular complex is separated from the first molecular complex, thereby resulting in fluorescence of the first CPN.

[0157] In an embodiment of the method, the fluorescence of the first CPN results from the second targeting molecule of the second molecular complex being unable to bind to a further target entity located at a portion of a test surface, said portion of the test surface further comprising the control entity.

[0158] This method relies upon the ability of the second molecular complex to be separated from the first molecular complex in the case of a positive test due to the target entity comprised within in the sample binding the second targeting molecule of the second molecular complex, and thereby inhibiting any further binding to any additional target entities encountered. This separation abolishes any quenching caused by the second CPN to the first CPN. The separation may occur due to contacting the composition with a surface or substrate which comprises a control antigen and a target antigen, as defined in the third aspect of the invention. For the negative result, there is no target antigen comprised within the sample for the second targeting molecule of the second molecular complex to bind to, and therefore the first and second molecular complexes, when contacted with surface or substrate which comprises a control antigen and a target antigen, may bind to this and are hence within proximity, so the second CPN can quench the fluorescence of the first CPN.

[0159] For the methods of the fifth and sixth aspects of the invention, the decrease in fluorescence could be measured through the change in magnitude of the maximal emission of the CPN after the addition of the sample (and also the quencher molecule in the method of the sixth aspect) to the CPN-bound targeting molecule. In one embodiment, the decrease in maximal emission is at least: 1 %, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100%.

[0160] The target entity could include anything as defined in the description as such. In one embodiment, the method of detecting the presence of a target entity is applied to a biological or chemical entity, eg at least one of: a toxin, a bacteria, a virus, a fungi, a protein, a biomarker, or a chemical entity. In a further embodiment of the invention, the protein is an allergen. In a further embodiment of the invention, the allergen is selected from at least: milk protein, egg protein, peanut protein, soy protein, wheat protein, tree nut protein, shellfish protein, fish protein, sesame protein, celery protein, gluten protein, lupin protein or mustard protein. In an embodiment of the invention, the method of detecting the presence of a target entity is applied to a chemical entity, wherein the chemical entity is a drug. It is envisaged that the drug is selected from at least: fentanyl, acetylfentanyl, xylazine, 2heroin, morphine, opium, methadone, hydrocodone, buprenorphine, oxycodone, alprazolam, tetrahydrocannabinol, methamphetamine, cocaine, lysergic acid diethylamide, 3,4-Methylenedioxymethamphetamine, phencyclidine, psilocybin, psilocin, N,N-Dimethyltryptamine, amphetamine, codeine, cannabinol, cannabis, cannabidiol, synthetic cannabinoids, ketamine, methaqualone, 4- Methylmethcathinone, methylphenidate, alprazolam, flunitrazepam, 4-Hydroxy-n- butyric acid, nitrous oxide, mephedrone, sotonitazene, metonitazene, etonitazene, protonitazene or analogs and metabolites thereof. Preferably, the drug is fentanyl.

[0161] As used herein, “detecting” refers to the ascertainment of the location of bound fluorescent ligand-binding molecules in the device. Detection can be via illumination with an ultraviolet light source and subsequent visualisation by eye or CCD, or it can represent a measurement by a spectrophotometer, densitometer, or other appropriate apparatus; it may be automated or manual, carried out in ambient or artificially produced light of any wavelength, or carried out in the form of any other method which allows the presence or absence of the label in any region of the diagnostic device to be ascertained.

[0162] As used herein, the term “sample” can refer to any tissue, solid, or fluid whether taken from a person, another animal, a plant, an environmental source such as seawater or soil, or any other place, in which the presence or absence of a particular ligand is of interest. The sample is preferably taken from a person, and may be blood, any extract thereof such as serum; saliva, or cerebrospinal fluid.

[0163] In an embodiment of the invention, the sample is selected from at least blood, saliva, semen, mucus, urine, interstitial fluid. In this way, the sample may be able to identify a person when contacted with the molecular complex of the present invention.

[0164] In an embodiment of the invention, the sample is sweat. Sweat is the ultrafiltrate of blood plasma, containing inorganic ions, lactate, urea and amino acids and these species are therefore present within a freshly deposited fingerprint. In addition, it is known that orally ingested and metabolised drugs are excreted in sweat. These drugs have been measured in sweat using collection devices, such as patches of adsorbent cotton, followed by extraction and subsequent analysis using techniques such as gas chromatography coupled with mass spectrometry (GC-MS) detection. However, the methods are laborious, require a large amount of sweat collected over a period of time and are therefore not suitable for rapid analysis, for example roadside testing of persons suspected of driving under the influence of drugs. The detection of substances in fingerprints has not been possible using the methods of the prior art because of the small quantity of the substances in the fingerprint. It is envisaged that the present invention would be able to detect these small quantities rapidly and in the field.

[0165] The structure or pattern of the deposition of certain samples may be of analytical interest, not just the chemical composition of such depositions. In an embodiment of the invention, a fingerprint can be visualized from the sample, wherein the sample is sweat. As used herein, the term “fingerprint” refers to the unique pattern of ridges and grooves on the skin of the palms and fingers of a person. Each skin ridge has a single row of pores, through which sweat is excreted and deposited on the surface of the skin. When a finger touches a surface, sweat is deposited leaving an impression of the finger's ridge pattern, referred to as a latent fingerprint. Such fingerprints are considered 'invisible prints' as they require physical or chemical treatments to enable visualisation. In this embodiment, targeting molecules would be used towards any target entity that may be deposited in a fingerprint, such as those found in sweat. Upon detection, the fluorescence of such deposited matter would elucidate a fingerprint, allowing subsequent identification of the individual.

[0166] In a seventh aspect of the invention, there is provided a kit of parts for detecting the presence of a target entity in a sample, the kit comprising any composition as herein defined in the invention. As used herein, the term “kit of parts” refers to a collection of discrete components that are designed to be assembled in a variety of ways to define a method for detecting the presence of a target entity in a sample.

[0167] In an eighth aspect of the invention, there is a lateral flow device for detecting the presence of a target entity in a sample, comprising the composition as herein defined in the first or third aspects of the invention. As used herein, the term “lateral flow device” refers to a type of diagnostic test that is designed for rapid and simple detection of specific analytes, such as biomarkers or pathogens, in a sample. Lateral flow devices are also commonly known as lateral flow tests, lateral flow assays, or immunochromatographic assays. They are widely used in various fields, including medical diagnostics, environmental testing, and food safety. Components often include but are not limited to: a test strip (often made of nitrocellulose or porous material), a conjugate pad, a sample application zone, a reaction zone, a control zone and an absorbent pad.

[0168] The sandwich assay is a common assay used for lateral flow tests, wherein two targeting molecules are required. In the context where antibodies are used to target an analyte, a first antibody is used to bind the analyte and can provide a visual signal (through tagging with colloidal gold), and a second antibody is used to bind the analyte-antibody-tag complex to concentrate it to show this signal. The molecular complex of the present invention only requires one targeting molecule to function, as the visual signal is achieved by the loss in proximity between the CPN and the quencher molecule which is part of the molecular complex, and only required the target entity, not bound to anything else, to displace either the quencher molecule or the CPN from the molecular complex.

[0169] In one embodiment of the invention, there is lateral flow device wherein the quenching molecule is displaced from the targeting molecule when brought into contact with a targeting entity. In this embodiment, the molecular complex of the first aspect of the invention would be immobilised on a suitable membrane, wherein the CPN is bound to the targeting molecule, preferably covalently, but can be any kind of bond which allows the invention to function in this embodiment through the displacement of the quencher molecule, and the quencher molecule has affinity towards the targeting molecule or is bound to a molecule with affinity towards the targeting molecule. This would therefore leave a population of molecular complexes fixed on the membrane wherein the CPN can fluoresce and be detected as a test line.

[0170] In an embodiment, the control line comprises a control antibody and the test line comprises a mixture of (i) the quencher molecule (for example, a target entity stained with a molecule capable of quenching) which is removably bound to a targeting molecule, and (ii) a CPN which is non-removably bound to the targeting molecule. This forms the molecular complex which is initially in a quenched state. Preferably, the polymer of the CPN in this embodiment is Poly[2-methoxy-5-(2- ethylhexyloxy)-1 ,4-( 1 -cyanovinylene-1 ,4-phenylene)]. This embodiment functions by the quencher molecule being displaced from the molecular complex in the presence of a (non-quencher stained) target entity, thereby restoring fluorescence of the CPN, which will be displayed on the test line. When no target entity is present in a sample, the molecular complex still comprises the quencher molecule, and hence the fluorescence of the CPN remains quenched.

[0171] In an alternative embodiment of the invention, there is a lateral flow device wherein the CPN is displaced from the targeting molecule when brought into contact with a targeting entity. In this embodiment, the molecular complex would be immobilised on a suitable membrane, wherein the quencher molecule is bound to the targeting molecule, preferably covalently, but can be any kind of bond which allows the invention to function in this embodiment through the displacement of the CPN, and the CPN has affinity towards the targeting molecule, or is bound to a molecule with affinity towards the targeting molecule. This would therefore generate a population of free CPNs on the membrane that would be deposited at the end of the lateral flow device wherein the CPN can fluoresce and be detected.

[0172] In an alternative embodiment of the invention, there is provided a lateral flow device comprising the composition according to the third aspect of the invention. This embodiment concerns the application of the composition of the third aspect of the invention to a lateral flow device to detect a target entity.

[0173] In an embodiment, a portion of a surface of the device comprises a control entity (as defined in the third aspect of the invention) and a target entity.

[0174] In an embodiment, the first and second targeting molecules of the first and second molecular complexes can bind, on the portion of the surface, to the control entity and the target entity respectively, such that when the first and second targeting molecules of the first and second molecular complexes are bound to said surface, the quencher molecule of the second molecular complex can quench the fluorescence of the CPN of the first molecular complex, such that, upon the addition of the sample comprising the target entity, the second targeting molecule of the second molecular complex binds the target entity of the sample, and is therefore prevented from binding to the target entity of the surface so that the fluorescence of the CPN can be visualised.

[0175] In an embodiment, the control line comprises a control entity and the test line comprises the control entity and the target entity. In an embodiment, the lateral flow test further comprises a conjugate pad which comprises the composition according to the third aspect of the invention (i.e. (i) a first molecular complex comprising a first conjugated polymer nanoparticle (CPN) capable of fluorescence, and a first targeting molecule which has affinity towards a control entity; and (ii) a second molecular complex comprising a second CPN capable of quenching the fluorescence of the first CPN, and a second targeting molecule which has affinity towards a target entity).

[0176] In this embodiment, the first molecular complex will bind to both the control and test strips. This embodiment functions in the case of a positive test by the target entity in the sample first binding the second molecular complex, thereby preventing this complex from binding the target entity on the test strip. Therefore, the test strip fluoresces due to the first CPN-targeting molecule complex binding the control entity (i.e. on both the control line and test line). When the target entity is not present in the sample, the both the first molecular complex and the second molecular complex will bind at the test strip (the first molecular complex to the control entity and the second molecular complex to the target entity). Since the second CPN is able to quench the fluorescence of the first CPN, the fluorescence of the first CPN is quenched on the test strip, indicating a negative result.

[0177] The composition of the present invention can be well adapted for optimisation. For example, the ability of the quencher molecule to absorb the CPN fluorescence and the ability to be displaced in the presence of the target entity to maximise the restoration of fluorescence can be modulated by changing the ratio of dye to protein in the protein-dye quencher complex. An improvement in these factors would enhance the signal difference between a dark safe signal to a fluorescent signal when a threat is present. If more than one molecular complex is being used in an embodiment of the invention, then additional optimisation strategies exist, including but not limited to: varying the ratio of multiple molecular complexes depending on their sensitivity towards the target entities, and varying the acid wool dye used in the quencher molecule of each molecular complex to increase detection sensitivity for more than one target entity.

[0178] EXAMPLES

[0179] In the following examples, CPNs were successfully conjugated to antibodies and conjugation reactions were validated using a DotBlot. Interaction between the target particle and the respective antibody conjugated to a CPN led to an emission and absorbance signal characteristic of the chosen CPN. These signals were visualised and imaged by Cytiva’s ImageQuant.

[0180] Since CPNs have a natural affinity to PES (polyether sulfone) membranes, CPN conjugates were affixed to this substrate as a basis for a reporter system.

[0181] A visual transition from dark to a fluorescent signal upon the presence of the simulant antigen was seen in multiple experimental set-ups. The CPNs fluorescence was prequenched using the simulant antigen bound to Bradford Reagent (Coomassie brilliant blue G-250) in a protein-dye complex. This interaction denatures the dyed protein to a degree proportional to the amount of staining and therefore reduces the affinity of the protein to the detection antibody in the same proportional manner.

[0182] This quenching effect was shown quantitatively via a well plate assay, where the endpoint emission signal measured at 610 nm reduces numerically following the addition of the quencher protein-dye complex. Upon the addition of the unstained simulant antigen the endpoint emission signal increases showing some restoration of fluorescence has taken place. This effect can also be visually seen through the PES syringe experiments, in which several absorbance and emission channels were imaged to monitor CPN610 absorbance and emission, and Bradford Reagent absorbance.

[0183] Example 1 : Conjugation of CPNs to Antibodies Against Simulant Agents

[0184] Conjugated polymer nanoparticles (CPNs) can be conjugated to targeting molecules, for example antibodies, which have specificity to target entities (or simulant agents). Specifically, CPN610 was chosen to be conjugated to antibodies. CPN610 is predominantly the polymer MEH-CN-PPV (Poly[2- methoxy-5-(2-ethylhexyloxy)-1 ,4-(1 -cyanovinylene- 1 ,4-phenylene)]) which can be obtained from various suppliers (for example, American Dye Source #ADS110RE). The biocompatible surface is polystyrene maleic anhydride, which can also be obtained from various suppliers (for example, Sigma #442399). CPN610 can be synthesised using nanoprecipitation synthesis and miniemulsion using a combination of Fe3O4, poly[2-methoxy-5-(2-ethylhexyloxy)-1 ,4-(1- cyanovinylene-1 ,4-phenylene)] and PSMA. CPN610 has excitation maximum of 480nm and an emission maximum of 610nm, which means it is compatible with instruments with 488nm laser excitation or standard filter sets for dyes with similar fluorescent properties.

[0185] The conjugation of the chosen antibodies against simulant agents to CPN610 comprises of an N-(3-Dimethylaminopropyl)-N’-ethylcarbodiimide hydrochloride / N-Hydroxysuccinimide (EDC / NHS) activation step to prepare the CPN surface for the addition of antibodies. Following an incubation period with the antibody, the remaining carboxylic acid sites on the CPN surface were blocked with glycine and then a stabilisation buffer was added. The CPN conjugates were then purified to remove any remaining unreacted antibodies.

[0186] Specifically, the conjugation of CPN610 to chosen antibodies used the following materials and steps. The antibodies used were prepared in-house, but antibodies to both Bacillus atrophaeus and ovalbumin are commercially available and could be used instead. All samples were supplied frozen and were aliquoted after their first use. Thawed proteins were kept at 4°C when not being used.

[0187] Table 1. Table of materials used.

[0188] 1 . Diluted antibodies to 1 mg / mL by the addition of HEPES / PEG buffer (20 mM HEPES / 0.1 % PEG).

[0189] 2. To 150 pL pre-activated CPN610 (~60 nm, -6E+10 p / mL), 10 pL of the chosen antibody (1 mg / mL) was added.

[0190] 3. Left solution to incubate at room temperature for 30 minutes.

[0191] 4. To this 6 pL Glycine (1 mg / mL) was added and the mixture was left to incubate at room temperature for a further 5 minutes.

[0192] 5. Following this, 6 pL stabilisation buffer (5% BSA / 1% Tween-20 / 20 mM HEPES / 0.1 % PEG) was added.

[0193] 6. The 610+Ab solution was added to a pre-blocked 300 kDA PES spin filter and made up the volume to 500 pL with HEPES / PEG buffer. 7. Centrifuged for 2 minutes at 2,000 xg and then disposed of supernatant.

[0194] 8. Made up retentate to 500 pL with HEPES / PEG buffer and centrifuge, repeated once more.

[0195] 9. Removed retentate from PES spin filter column and made up solution to 150 pL using HEPES / PEG buffer.

[0196] Confirmation of a successful conjugation reaction between the CPNs and antibodies was shown by a DotBlot. The target and controls were spotted onto the nitrocellulose and then incubated with a blocking buffer to reduce non-specific binding. This was then probed with the CPN conjugate, washed, and imaged using Cytiva’s ImageQuant.

[0197] Example 2: Anti-Ovalbumin (OV) Antibodies and Staining of Simulant Agents

[0198] Antibodies were chosen as the targeting molecule with CPNs conjugated thereto, and the guenching effect was investigated using a target entity (or simulant agent) and a guencher molecule. The affinity of the CPN-antibody conjugate towards both a target entity (or simulant agent) and guencher-bound target entity was also investigated.

[0199] Anti-OV antibody OV1 was initially chosen to demonstrate a successful conjugation to CPN610.

[0200] Validation of the conjugation is demonstrated by a DotBlot and is imaged using Cytiva’s ImageQuant to visualise CPN610’s fluorescence via its emission signal.

[0201] Acid wool dyes were selected as the guencher molecules in the CPN610-antibody conjugates. Specifically, Ovalbumin (acting as the target entity or simulant agent) was stained with Bradford Reagent (BR).

[0202] Bradford Reagent is an acidified solution of Coomassie Brilliant Blue G-250; the dye is primarily doubly protonated and red / brown in colour. When it comes into contact with a protein, the first electron is donated to charged groups on the protein. This disrupts the protein structure, resulting in the exposure of hydrophobic pockets. The dye is able to bind to these pockets with the sulfonic acid groups on the Bradford Reagent binding to positive amines present on the protein. There are also relatively weak electric forces present in the form of Van der Waals forces. The stably bound Coomassie G-250 is the blue unprotonated form and has an absorbance maximum of 595 nm.

[0203] The staining protocol consisted of the following steps:

[0204] 1 . 40 pL of chosen simulant agent added to X pL Bradford Reagent add. (X was varied depending on experiment - see Table 2)

[0205] 2. Left to incubate in the fridge for 30 minutes.

[0206] 3. Centrifuged for 5 minutes at 15,000 xg.

[0207] 4. Removed excess Bradford Reagent supernatant.

[0208] 5. Resuspended blue pellet in 40 pL HEPES / PEG buffer.

[0209] Table 2. Table indicating volume of Bradford Reagent used during the staining process of simulant agents.

[0210] Both unstained and Bradford Reagent stained Ovalbumin samples were spotted on the nitrocellulose membrane alongside a negative control of 1x PBS.

[0211] These samples were visualised via DotBlot using the following steps:

[0212] 1. Spotted 2x 1 pL of protein±BR onto nitrocellulose (Vivid™ 120 Nitrocellulose, Lot#1207005621), and 2x 1 pL negative control PBS.

[0213] 2. Blocked for 30 minutes using 1 mL 2% Milk in PBST. 3. Washed 3x with PBST.

[0214] 4. Incubated with 1 mL diluted 610+Ab (1 :99 in 2% Milk in PBST) for 30 minutes.

[0215] 5. Washed 3x with PBST.

[0216] 6. Waited for DotBlot to dry and then imaged using Cytiva’s ImageQuant using the following filter sets:

[0217] Table 3. Table of ImageQuant settings used to image CPN610 Absorbance / Emission and Bradford Reagent Absorbance.

[0218] As seen in Figure 1 , the spots where the unstained OV is present had an emission signal. This indicated that the conjugation reaction has been a success and that the CPN610+OV1 conjugate was capable of binding to the OV protein. At the BR+OV spots there was some CPN610 emission visible, but this was significantly reduced compared to the OV signal.

[0219] This could either be due to a quench in fluorescence due to the Bradford Reagent or a reduction in OV binding efficiency after the staining process. To decipher which effect is witnessed, the CPN610 absorbance signal, which is directly proportional to the amount of CPN610 present, can be used as a control: if there is a matching amount of CPN610 absorbance but a reduction in emission this indicates that the fluorescence has been quenched. Whereas if there is a reduction in the CPN610 absorbance signal it suggests that there is less CPN610 bound and therefore the binding efficiency has been reduced. For future experiments the CPN610 absorbance images were also collected. The negative control spots had no emission signal visible, indicating that the fluorescence seen at the OV±BR spots is due to targeted binding and not as a result of non-specific binding of the CPNs.

[0220] To evaluate which Anti-OV antibody has the best signal-to-noise to quench effect, DotBlots were set up to be probed with the different CPN610 conjugates. Figure 2 shows the membrane layout and the expected results of absorbance and emission signal when looking at the different filter sets. It is expected that CPN610 absorbance and emission signals will be present at the OV and BR+OV spots; and BR absorbance signals seen at the BR+OV and BR+Casein spots.

[0221] Casein had been introduced as a ‘null’ protein into this study, the CPN610 conjugates should not bind to either the unstained or stained Casein spots as they are not selective to this protein. The BR+Casein spots had also been introduced to confirm that the signal seen for CPN610 absorbance at BR+OV spots was due to genuine binding of the CPN610 conjugate and not as a result of bleed through from the Bradford Reagent.

[0222] All of the CPN610 conjugates showed the desired results with both absorbance and emission signals visible at the expected spots on the membrane (Figure 3). All the DotBlots show a reduced CPN610 absorbance signal at the BR+OV spots in comparison to the unstained OV spots, this suggests that there was a reduction in affinity between the BR+OV and CPN610 conjugates as a result of the Bradford Reagent staining. The most intense binding with the best signal to noise appears to be the CPN610+OV2 conjugate and therefore was the chosen antibody for further studies.

[0223] Example 3: Anti-Bacillus atrophaeus (BG) Antibodies

[0224] In addition to CPN610 + anti-OV conjugates, conjugations between CPN610 and anti-BG antibodies were also validated by DotBlot (Figure 4). Several DotBlots were prepared, each to be probed with a different CPN610 conjugate to evaluate which antibody has the best signal to noise. All of the CPN610 conjugates appear to have bound to the BG spots on the nitrocellulose, the efficiency of this binding varies depending on the conjugate. The CPN610 conjugate of anti-BG antibody BG2 showed very poor binding with a barely visible emission signal and a comparatively weak absorbance signal. Aside from this DotBlot, the CPN610+BG2 struggled to remain in solution and showed significant aggregation after purification. This may have contributed to its poor binding to the BG during the DotBlot experiment. The most consistent binding and the best signal-to-noise for both absorbance and emission can be seen for the CPN610+BG1 conjugate, therefore this antibody was chosen for further studies.

[0225] Example 4: Staining of Ovalbumin

[0226] The effect of staining Ovalbumin with Bradford Reagent on the CPN610 anti-OV conjugate was investigated and was shown to decrease its affinity towards the CPN610 anti-OV conjugate.

[0227] Upon the addition of Ovalbumin to Bradford Reagent, there was an immediate colour change from red / brown to blue indicating that a protein-dye complex has formed. After an incubation period of 30 minutes, the BR+OV mixture was centrifuged, leaving a blue pellet present. The supernatant containing excess Bradford Reagent was removed and the pellet was resuspended in a resuspension buffer. The DotBlot in Figure 5 had both unstained and Bradford Reagent stained Ovalbumin spotted on the nitrocellulose membrane, alongside a negative control. An absorbance and emission signal was visible at the OV spots showing that the CPN610 conjugate had successfully bound here. The BR+OV spots had a CPN610 absorbance signal that was significantly reduced as is the CPN610 emission signal. This indicates that the staining of the quencher particle had resulted in a reduction of its affinity towards the anti-OV antibody present on the CPN.

[0228] It was proposed that the ratio of Bradford Reagent to protein present during the protein staining process was proportional to the reduction in affinity of the stained protein to the detection antibody. To investigate this, a set volume and concentration of Ovalbumin was stained with varying volumes of Bradford Reagent as described in Table 4.

[0229] Table 4. Table indicating volume of Bradford Reagent used during the staining process of Ovalbumin

[0230] An experiment to observe the denaturing effect of the various BR+OV solutions involved a DotBlotwith each of the BR+OV solutions spotted, alongside a positive control (OV) and negative control (1x PBS).

[0231] Binding between the CPN610 conjugate and the OV±BR spots was apparent due to the CPN610 absorbance signal seen for all of the spots described above. As with previous DotBlots the BR+OV spots had significantly less CPN610 conjugate bound resulting in a reduction of the CPN610 absorbance signal when compared directly to the OV signals. When focusing on just the BR+OV spots it appeared that BR+OV(0) had the poorest binding, this is the Ovalbumin stained in the lowest volume of Bradford Reagent. The absorbance seen for BR+OV(1) and BR+OV(2) was comparable (Figure 6).

[0232] Example 5: Staining of Bacillus atrophaeus

[0233] The effect of staining Bacillus atrophaeus (BG) with Bradford Reagent on the CPN610 anti-OV conjugate was investigated and was shown to decrease its affinity towards the CPN610 anti-OV conjugate.

[0234] An almost immediate colour change from red / brown to blue was witnessed when Bacillus atrophaeus is added to Bradford Reagent. After an incubation period of 30 minutes and subsequent centrifugation, a blue pellet was visible. When the unreacted Bradford Reagent was removed, the blue pellet was resuspended in a resuspension buffer, forming a blue solution. To investigate the effect of staining the BG with Bradford Reagent and to ensure that it was still able to recognise and bind to the CPN610 anti-BG conjugate, a DotBlot was set up. As shown in Figure 7, BR+BG was still able to bind 610+BG1 , but similarly to the results seen for BR+OV there was a reduction in the amount of 610 conjugate bound. With less 610 bound to the spot there was also a comparatively low signal from the CPN610 emission at the BR+OV spots too.

[0235] The BG and BR+BG solutions used in this experiment were at the same concentration so it can be concluded that the reduction in CPN610 absorbance signal was due to a reduced binding affinity of the CPN610 conjugate for the BG with Bradford Reagent, i.e. the bind of the Bradford reagent to the BG has changed BG’s conformation, reducing the strength of the interaction between the BR+BG and the CPN610 conjugate. To confirm that both BG solutions were at the same concentration the BR absorbance signal of 1 pL BR+BG solution was compared to 1 pL of the unstained BG then probed with Bradford Reagent. As shown in Figure 8 the absorbance signals of the two are comparable therefore suggesting that the concentration had minimal difference.

[0236] Example 6: Well Plate Assay - Quenching and Restoration of Fluorescence Unstained Ovalbumin was shown to displace Bradford Reagent stained Ovalbumin, facilitating the restoration of fluorescence by loss in proximity between the CPN and the quencher.

[0237] The DotBlot experimental set-up was transferred to a Well Plate Assay format to allow for monitoring of the quenching effect and subsequent restoration of fluorescence in a quantitative manner. This assay used a commercial protein A / G plate (Reacti-Bind™ Protein A / G Coated Strip Plate, REF#1858788, Lot#VA291466), with the protocol consisting of the following steps:

[0238] 1. To wells 100 pL 2% Milk Powder added and left to incubate for 1 hour at room temperature under agitation using a plate rocker.

[0239] 2. Tipped solution out of the wells and washed 3x with PBS.

[0240] 3. Added 100 pL of diluted CPN610+Ab (1 :9 in 2% Milk Powder) and left to incubate for 2 hours at room temperature under agitation (plate rocker). 4. Recorded fluorescent endpoint reading using SpectraMax (ex 480 nm, em 610 nm)

[0241] 5. Tipped solution out of wells and washed with PBS, and then recorded fluorescent endpoint reading.

[0242] 6. Repeated for a further two washes / readings.

[0243] 7. Added 100 pL diluted OV±BR (1 :9 in 2% Milk Powder) and left to incubate for 1 hour at room temperature under agitation (plate rocker).

[0244] 8. Recorded fluorescent endpoint reading using SpectraMax (ex 480 nm, em 610 nm)

[0245] 9. Tipped solution out of wells and washed with PBS, and then recorded fluorescent endpoint reading.

[0246] 10. Repeated for a further two washes / readings.

[0247] 11. Added 100 pL diluted OV (1 :9 in 2% Milk Powder) and left to incubate for 1 hour at room temperature under agitation.

[0248] 12. Recorded fluorescent endpoint reading using SpectraMax (ex 480 nm, em 610 nm)

[0249] 13. Tipped solution out of wells and washed with PBS, and then recorded fluorescent endpoint reading.

[0250] 14. Repeated for a further two washes / readings.

[0251] CPN610+OV2 was able to bind to Protein A / G present at the bottom of the wells of commercial A / G plates. Protein A / G is a recombinant fusion protein combining the IgG binding domains of Protein A from Staphylococcus aureus and Protein G from Streptococcal bacteria. Protein A / G can bind all subclasses of both human IgGs and mouse IgGs, as well as other subclasses of human IgGs such as IgA, IgE, IgM, and IgD. After several wash steps the bound CPN conjugate gave emission endpoint readings of between 45-50 counts. After a set incubation period with BR+OV, these endpoint readings are seen to drop to -30% of their original emission value. In comparison the control experiments that had unstained Ovalbumin added instead during the incubation step all remain at 85-90% of their original emission value. This suggests that the drop in emission signal was due to quenching as a result of the presence of Bradford Reagent stained Ovalbumin and not due to the CPN610 conjugate coming unbound from the A / G plate and being removed during wash steps.

[0252] In wells where BR+OV had been incubated, unstained OV has been left for a set incubation period to try and displace the BR+OV bound to the CPN610 conjugate. Figure 9 shows that following the addition of OV there was a slight increase in signal seen at the emission endpoint to -20% above the quenched reading.

[0253] Table 5. Table showing the emission endpoint values at 610 nm after addition of OV±BR and then after unstained OV to restore fluorescence.

[0254] Further data collected on alternative batches of the CPN610+OV2 conjugate demonstrated the repeatability of this result (Figure 10). There was a consistent drop in emission signal witnessed upon the addition of BR+OV and then a subsequent restoration of emission signal following an incubation with unstained OV. This in combination with the control data collected using the same CPN conjugate batches and experimental conditions but using unstained OV in place of the BR+OV, showed that the quenching effect and fluorescent revival was only seen when the Ovalbumin was stained with Bradford Reagent and then it was displaced when the more favourable target of unstained OV was present. Table 6. Table showing the emission endpoint values at 610 nm after addition of OV±BR and then after unstained OV to restore fluorescence of the above experiments.

[0255] To optimise the recovery in fluorescence following the addition of unstained Ovalbumin, it was desirable to reduce the binding affinity of the quencher molecule to the CPN610 conjugate so that it could be more readily displaced in the presence of the target particle. A series of experiments was designed using Ovalbumin stained in various volumes of Bradford Reagent as described in Table 7 in order to monitor the difference in quenching effect and the subsequent displacement of the quencher by the target protein. 5 different volumes of Bradford Reagents were used to stain the same volume and concentration of Ovalbumin. All were left to incubate in the fridge for the 30 minutes and then excess Bradford Reagent was removed by centrifugation at 15,000 xg for a set period of 5 minutes. The blue pellets that were formed were all resuspended in a set volume of resuspension buffer to make up the various BR+OV solutions to their original protein concentration. These 5 different BR+OV solutions were then incorporated into the original Well Plate Assay and the quenching effect quantified. Following this, all experiments were subject to the same volume and concentration of unstained Ovalbumin for a set period of incubation time and the emission endpoint signal recorded to monitor the recovery in 610 fluorescence as a result of the displacement of quencher molecules by the unstained target. Table 7. Table indicating volume of Bradford Reagent used during the staining process of simulant agents.

[0256] From Figure 11 , it appeared that generally the quencher molecules with a higher volume of Bradford Reagent present during the protein staining process had a more efficient quenching effect. The experiment using BR+OV(4) had an emission value that dropped to 58% of its original emission signal. This contrasted with BR+OV(1) which saw the emission signal quenched to 85% of its original value.

[0257] Similarly for the restoration of fluorescence, the most impressive recovery was seen for those BR+OV quenchers that had a higher volume of Bradford Reagent present during staining. The best recovery of fluorescence saw the emission value increased by 64% for the BR+OV(4) experiment. This restored the signal to 96% of the original fluorescence prior to the quencher being added. Fluorescent recovery seen for BR+0V(1) only saw an emission signal increase of 1 % in comparison to the quenched signal.

[0258] Table 8. Table showing the emission endpoint values at 610 nm after the addition of different BR+OV solutions and then after the addition of unstained OV to restore fluorescence. Example 7: Transfer to PES Syringe Membrane

[0259] CPNs have a natural affinity for PES (polyether sulfone) membranes when no pacification process is implemented. This was utilised to enable the CPN610 conjugates to be anchored onto a PES membrane syringe surface in order to visualise and image the quenching effect and fluorescence recovery.

[0260] The CPN610 conjugate was allowed to deposit on the syringe membrane by gravitational force through a syringe barrel placed in the inlet of the syringe filter. After a wash step, the syringe filter was imaged using Cytiva’s ImageQuant using a variety of filter sets that allowed for an ambient lighting, CPN610 emission, CPN610 absorbance and Bradford Reagent absorbance image to be obtained (Figure 12).

[0261] Specifically, this protocol consisted of the following steps:

[0262] 1. Wetted the PES membrane with 1 mL PBS, initially inverted syringe membrane whilst inserting solution, until an air bubble appeared and solution flowed through, then returned syringe filter to upright position whilst flushing through the remainder of solution.

[0263] 2. Added 1 mL of diluted CPN610+OV2 (1 :2000 in PBS) to syringe membrane by inserting an empty syringe casing into the inlet of syringe filter and then pipetted the diluted solution into the empty syringe casing and allowed it to flow through the syringe filter by gravitational force.

[0264] 3. Once the diluted solution had soaked through the membrane washed with 1 mL PBS.

[0265] 4. Imaged on Cytiva’s ImageQuant using the filter sets listed in Table 8.

[0266] 5. Added 1 mL 5% BSAin PBS and let it flow through the syringe membrane using the method described above.

[0267] 6. Once the solution has flowed through the membrane, washed with 1 mL PBS.

[0268] 7. Imaged on the ImageQuant.

[0269] 8. Added 1 mL of diluted BR+OV(4) (1 :50 in PBS) and let it flow through the syringe membrane using the method described above.

[0270] 9. Once the solution has flowed through the membrane, washed with 1 mL PBS. 10. Imaged on the ImageQuant.

[0271] 11 . To the outlet of the syringe filter: parafilm it closed to prevent solution running through the syringe filter.

[0272] 12. Added 1 mL of 10 mg / mL OV to the empty syringe casing inserted in the syringe filter inlet and left for 16 hours.

[0273] 13. Removed the parafilm blocking the syringe outlet and allowed the solution to flow through the syringe membrane.

[0274] 14. Once the solution has flowed through the membrane, washed with 1 mL PBS.

[0275] 15. Imaged on the ImageQuant.

[0276] Confirmation that the CPN610 conjugate had been successfully deposited onto the membrane surface was indicated by the strong signal seen in both the CPN610 absorbance and emission. A blocking step had been introduced to ensure that the CPN610 conjugate remained bound to the PES membrane as previous iterations of the experiment showed that a fraction of the CPN610 conjugate was removed during the wash steps which in itself led to a reduction in fluorescence. Following the addition of the blocking step, the signal seen for the CPN610 absorbance and emission remained almost identical showing that the CPN610 conjugate remains bound to the PES membrane.

[0277] Upon the addition of BR+OV the syringe membrane appeared to have been stained a pale blue colour indicating the presence of Bradford Reagent, this was further supported by the signal seen for BR absorbance. Looking at the CPN610 emission images there was a reduction in the fluorescence signal, this quench correlated to the area of the membrane that also had a strong BR absorbance. The small patch of strong CPN610 emission on the left of the membrane was an area that has no BR absorbance signal, supporting the case that the reduction in fluorescence was due to the interaction of BR+OV. The CPN610 absorbance signal remained similar to what was seen prior to the BR+OV addition, indicating that the CPN610 conjugate was still bound to the syringe membrane.

[0278] The CPN610 emission image in combination with CPN610 absorbance image indicated that the drop in emission signal was as a result of quenching due to the presence of BR+OV and not due to the CPN610 conjugate being washed from the PES membrane surface.

[0279] The next step was to work towards reversing this quenching effect in order to restore the fluorescent signal in the presence of the target protein. A similar experiment was initiated which showed a similar reduction in CPN610 emission signal upon the addition of BR+OV whilst maintaining an almost identical CPN610 absorbance signal (Figure 13).

[0280] On a syringe filter membrane deposited with the CPN610+OV2 conjugate, following the addition of stained Ovalbumin (BR+OV), an excess of unstained Ovalbumin was left to incubate overnight on the syringe filter membrane which was then washed prior to imaging. The syringe filter membrane appeared to remain a pale blue colour and the Bradford Reagent was evidently still present as seen by the BR absorbance signal.

[0281] The most apparent change can be seen when looking at the CPN610 emission signal which showed that some of the previously quenched fluorescence had now been restored. The CPN610 absorbance signal remained consistent. The increase in fluorescence as a result of the incubation with unstained OV showed that a displacement of some of the BR+OV had occurred in the presence of the target.

[0282] Example 8: Different coloured CPN detectors

[0283] Different coloured signals can be generated from different combinations of CPNs and AWD (acid wool dye) quenchers listed In Table 9.

[0284] Table 9. Table of potential combinations of CPN-AWD reporter systems.

[0285] Example 9: Development of a Lateral Flow Test (LFT)

[0286] The invention was then applied to the development of LFTs.

[0287] A first LFT was designed, “AWD Quenched CPN” LFT, in which the test line comprised a mixture of quencher-stained simulant agent and a CPN610 + antisimulant agent antibody conjugate, and the control line comprised a control antibody.

[0288] When a sample positive for the simulant agent is flown through this LFT, it will displace the quencher-stained simulant agent from the CPN610 + anti-simulant agent antibody conjugate at the test line. This displacement will restore fluorescence to CPN610, and therefore the test line will fluoresce. When a sample negative for the simulant agent is flown through this LFT, the quencher-bound simulant agent will remain bound to the CPN610 + anti-simulant agent antibody conjugate at the test line. No displacement of the quencher-bound simulant agent occurs, and therefore CPN610 remains quenched at the test line. This format of LFT is further detailed in Figure 16.

[0289] The development of this first LFT was a result of a proof-of-concept study detailed in Figure 17. To initially test the interaction between CPN610 and the quencher- stained simulant agent, the following protocol was used:

[0290] 1. Wet a PES membrane with 1 mL 1x PBS.

[0291] 2. Add 1 mL diluted CPN610+anti-ovalbumin antibody in 1x PBS (1 :2000). 3. Wait for solution to seep through PES membrane then wash with 1 ml_ 1x PBS.

[0292] 4. Add 5% BSA in PBS.

[0293] 5. Wait for solution to seep through PES membrane then wash with 1 mL 1x PBS.

[0294] 6. Image on ImageQuant.

[0295] 7. Add diluted BR + OV in 1x PBS (1 :50).

[0296] 8. Wait for solution to seep through PES membrane then wash with 1 mL 1x PBS.

[0297] 9. Image on ImageQuant.

[0298] 10. Add 1 mL 10 mg / mL OV.

[0299] 11. Parafilm the outlet of the syringe closed and leave overnight.

[0300] From this, CPN610 fluorescence was reduced in the presence of BR + OV, thereby showing that the initial mixture of the quencher-stained simulant agent and a CPN610 + anti-simulant agent antibody conjugate produced quenching of fluorescence. In the presence of OV (the simulant agent), some of the fluorescence in the BR + OV and CPN610+anti-ovalbumin antibody mixture was restored, thereby showing that a simulant agent may displace the quencher- stained simulant from the CPN-bound targeting molecule.

[0301] A second LFT was designed, “Quenched CPN (2x CPN Concept)” LFT, in which the test line comprised a mixture of simulant agent and control antibody, and the control line comprised a control antibody. Here, the conjugate pad of the LFT comprises an CPN610 + anti-control antibody conjugate and a CPN830 + antisimulant agent antibody conjugate. When the test sample is added, the conjugate pad releases these two CPN-antibody complexes, which then produce differing results based on whether the simulant agent was present. In each case, CPN610 will bind to both the control and test lines regardless of whether the simulant agent is present due to it being bound to an antibody that is anti-control antibody.

[0302] CPN830 predominantly comprises the polymer PCPDTTBTT (Poly[2,6-(4,4-bis(2- ethylhexyl)-4H-cyclopenta-[2,1-b:3,4-b']dithiophene-alt-4,7-bis(thiophen-2- yl)benzo-2,1 ,3-thiadiazole]), and may be synthesised using the same method as for CPN610 (as detailed in Example 1), but instead using the PCPDTTBTT polymer. CPN830 has an excitation maximum of 610nm and an emission maximum of 830nm which means it may be excited by instruments with 610nm and 637nm laser excitation.

[0303] When a sample positive for the simulant agent is flown through this LFT, the CPN830 + anti-simulant agent antibody conjugate binds to the simulant agent in the sample, preventing it from binding the simulant target at the test line. Both the control line and test line will show fluorescence for CPN610. When a sample negative for the simulant agent is flown through this LFT, the CPN830 + antisimulant agent antibody conjugate binds to the simulant agent at the test line. Due to the presence of CPN830 in the test line, the fluorescence of CPN610 is quenched. This format of LFT is further detailed in Figure 18.

[0304] The development of this second LFT was a result of a proof-of-concept study detailed in Figure 19. To initially test the quenching of CPN610 by CPN830, the following protocol was used:

[0305] 1. Pre-mix 2 pL 1 mg / mL OV with 2 pL 1 mg / mL Anti-Rabbit.

[0306] 2. Spot 1 pL of this pre-mixed solution onto 2x dipstrips and allow to dry.

[0307] 3. Prepare a multi-well set-up with dipstrip buffer prepared by prepared by adding 10 pL buffer, 10 pL CPN conjugate, then 20 pL buffer to the well.

[0308] 4. In a first well, further add 1 pL CPN610 + anti-OV (10 pg). In a second well, further add 1 pL CPN610 + anti-OV (10 pg) and 8 pL CPN830+Anti-Goat (10 pg).

[0309] 5. Spot dipsticks into wells and leave to dry.

[0310] 6. Image on ImageQuant when dry.

[0311] From this, a clear quench in the CPN610 fluorescence was seen resulting from exposure to CPN830, thereby showing that the presence of a second CPN may quench the fluorescence of the first CPN in the case of a positive test of this LFT format.

Claims

CLAIMS1. A composition for detecting the presence of a target entity, the composition comprising: a molecular complex comprising a conjugated polymer nanoparticle (CPN) capable of fluorescence, a targeting molecule which has affinity towards a target entity, and a quencher molecule, which together form the complex, such that the quencher molecule quenches fluorescence from the CPN, and either the quencher molecule or CPN can be displaced from the molecular complex when brought into contact with a target entity, such that in the absence of the quencher molecule, the fluorescence of the CPN can be visualised.

2. The composition of claim 1 , wherein the CPN is bound to the targeting molecule, preferably by covalent bonding.

3. The composition of claim 1 or claim 2, wherein the quencher molecule is formed in the complex such that it can be displaced from the complex when the complex is brought into contact with a targeting entity.

4. The composition of any preceding claim, wherein the quencher molecule has affinity towards the targeting molecule or is bound to a molecule with affinity towards the targeting molecule.

5. The composition of claim 1 , wherein the quencher molecule is bound to the targeting molecule, preferably by covalent bonding.

6. The composition of claim 1 or claim 5, wherein the CPN is formed in the complex such that it can be displaced from the complex when the complex is brought into contact with a targeting entity.

7. The composition of claim 1 , claim 5, or claim 6, wherein the CPN has affinity towards the targeting molecule, or is bound to a molecule with affinity towards the targeting molecule.

8. The composition according to claim 1 , wherein the CPN has maximal emission of at least: 420 nm, 435 nm, 475 nm, 510 nm, 530 nm, 550 nm, 580 nm, 610 nm, 660 nm, 680 nm, 770 nm, 820 nm, 830 nm, 840 nm or 1000 nm.

9. The composition according to any preceding claim, wherein the polymer of the conjugated polymer nanoparticle is selected from: Poly[9,9-di(3',7'- dimethyloctyl)fluoren-2,7-yleneethynylene], Poly[9,9-didodecylfluroenyl-2,7- yleneethylnylene], Poly[9,9-di(2'-ethylhexyl)fluoren-2,7-yleneethynylene], Poly(9,9-dioctylfluorenyl-2,7-yleneethynylene), Poly[9,9-bis-(2-ethylhexyl)-9H- fluorene-2,7-diyl], Poly(9,9-dihexyl-9H-fluorene-2,7-diyl), Poly(9,9-di-(2- ethylhexyl)-9H-fluorene-2,7-diyl), Poly[(9,9-di-(2-ethylhexyl)-9H-fluorene-2,7- diyl)-co-(1-methoxy-4-(2-ethylhexyloxy)-2,5-phenylenevinylene)], Poly[(9,9- dihexyl-9H-fluorene-2,7-diyl)-co-(1-methoxy-4-(2-ethylhexyloxy)-2,5- phenylenevinylene)], Poly(2,5-bis(1 ,4,7,10-tetraoxaundecyl)-1 ,4- phenylenevinylene), Poly (2 , 5-diocty 1-1 ,4-phenylenevinylene), Poly(2,5- dioctylphenylene-1 ,4-ethynylene),Poly[1 ,2-bis(benzylthio)acetylene], Poly[1 ,2- bis(ethylthio)acetylene], Poly[bis(methylthio)acetylene], Poly(3,5 pyridine), Poly(3-(2-methoxyethoxy)ethoxymethylthiophene-2,5-diyl), 5,5'-Dibromo-2,2'- bithiophene, Poly(3-butylthiophene-2,5-diyl), Poly(3-cyclohexyl-4- methylthiophene-2, 5-diyl), Poly(3-cyclohexylthiophene-2,5-diyl), Poly(3- decylthiophene-2, 5-diyl), Poly(3-dodecylthiophene-2,5-diyl), Poly(3- hexylthiophene-2, 5-diyl), Poly(3-octylthiophene-2,5-diyl), ThiopheneOligothiophenes, 5,5'-Dibromo-2,2'-bithiophene, 2,2',5',2",5",2"'-Quaterthiophene, a-Sexithiophene, 311073 2,2':5',2"-Terthiophene, Poly(thiophene-2, 5-diyl), bromine terminated powder, 2,3-Dihydrothieno[3,4-b]-1 ,4-dioxin, 3,2':5',3"- Terthiophene, 5-Hexyl-2,2'-bithiophene, 22,2'-Bithiophene, Thiophene, 5,5""- Dihexyl-2,2':5',2":5",2"':5"',2"":5"",2 -sexithiophene, Poly(styrenesulfonate) / poly(2,3-dihydrothieno(3,4-b)-1 ,4-dioxin), Poly(3,4- ethylenedioxythiophene)-block-poly(ethylene glycol), Poly(3,4- ethylenedioxythiophene), tetramethacrylate endcapped, Poly((9,9-dihexyl-9H- fluorene-2,7-diyl)-co-(1-methoxy-4-(2-ethylhexyloxy)-2,5-phenylenevinylene)), Poly((9,9-dihexyl-9H-fluorene-2,7-diyl)-co-(1-methoxy-4-(2-ethylhexyloxy)-2,5- phenylenevinylene), Poly(1-methoxy-4-(3-propyloxy-heptaisobutyl-PSS)-2,5-phenylenevinylene)-co-(1-methoxy-4-(2-ethylhexyloxy)-2,5-phenylenevinylene), Poly(2,5-bis(3-sulfonatopropoxy)-1 ,4-phenylene, disodium salt-alt-1 ,4- phenylene), Poly(2,5-dihexyloxy-1 ,4-phenylenevinylene), Poly (2 ,5-diocty 1-1 ,4- phenylenevinylene), Poly(2,6-naphthalenevinylene), Poly[5-methoxy-2-(3- sulfopropoxy)-1 ,4-phenylenevinylene] Potassium salt, Poly(p-xylene tetrahydrothiophenium chloride), Poly[(m-phenylenevinylene)-alt-(2-methoxy-5- octyloxy-p-phenylenevinylene)] , Poly[(m-phenylenevinylene)-alt-(2,5-dihexyloxy- p-phenylenevinylene)], Poly[(m-phenylenevinylene)-alt-(2-methoxy-5-(2-ethyl- hexyloxy)-p-phenylenevinylene)], Poly[(m-phenylenevinylene)-co-(2,5-dioctoxy- p-phenylenevinylene)], Poly[(m-phenylenevinylene)-alt-(2,5-dibutoxy-p- phenylenevinylene)], Poly[(o-phenylenevinylene)-alt-(2-methoxy-5-(2- ethylhexyloxy)-p-phenylenevinylene)], Poly[(p-phenylenevinylene)-alt-(2- methoxy-5-(2-ethyl-hexyloxy)-p-phenylenevinylene)], Poly[2-(2',5'-bis(2"- ethylhexyloxy)phenyl)-1 ,4-phenylenevinylene], Poly[2,5-bis(3' 7'- dimethyloctyloxy)-1 ,4-phenylenevinylene], Poly[(2,5-bisoctyloxy)-1 ,4- phenylenevinylene], Poly[2-methoxy-5-(2-ethylhexyloxy)-1 ,4-phenylenevinylene], Poly[2-methoxy-5-(3',7'-dimethyloctyloxy)-1 ,4-phenylenevinylene], Poly[5- methoxy-2-(3-sulfopropoxy)-1 ,4-phenylenevinylene], Poly[9-(2-ethylhexyl)-3,6- carbazolevinylene-alt-2,6-naphthalenevinylene], Poly{[2-[2',5'-bis(2"- ethylhexyloxy)phenyl]-1 ,4-phenylenevinylene]-co-[2-methoxy-5-(2'- ethylhexyloxy)-1 ,4-phenylenevinylene]}, Poly[(9,9-dioctylfluorenyl-2,7-diyl)-co- (1 ,4-phenylene)], Poly[(9,9-dioctylfluorenyl-2,7-diyl)-co-(4,4'-(N-(4-sec- butylphenyl)diphenylamine)], Poly(2,5-di(3',7'-dimethyloctyl)phenylene-1 ,4- ethynylene), Poly[(9,9-dioctyl-2,7-divinylenefluorenylene)-alt-co-{2-methoxy5-(2- ethylhexyloxy)-1 ,4-phenylene}], Poly[(9, 9-diocty I-2 ,7-bis{2- cyanovinylenefluorenylene})-alt-co- (2-methoxy-5-{2-ethylhexyloxy}-1 ,4- phenylene)], Poly(9,9-dioctylfluorene-alt-benzothidiazole), Poly[2-methoxy-5-(2- ethylhexyloxy)-1 ,4-( 1 -cyanovinylene-1 ,4-phenylene)], Poly [{9, 9-dihexy I-2 , 7-bis( 1 - cyanovinylene)fluorenylene}-alt-co- {2, 5-bis(N,N’-diphenylamino)-1 ,4- phenylene}], Poly(4,4-dioctyldithieno(3,2-b:2',3'-d)silole)-2,6-diyl-alt-(2, 1 ,3- benzothiadiazole)-4,7-diyl), Poly[(5-fluoro-2, 1 ,3-benzothiadiazole-4,7-diyl)(4,4- dihexadecyl-4H- cyclopenta[2,1-b:3,4- b']dithiophene-2,6-diyl)(6-fluoro-2, 1 ,3-benzothiadiazole- 4,7-diyl)(4,4-dihexadecyl-4H-cyclopenta[2, 1 -b:3,4- b']dithiophene-2,6-diyl)], Poly{2,2'-[(2,5-bis(2-hexyldecyl)-3,6-dioxo-2, 3,5,6- tetrahydropyrrolo[3,4-c ]pyrrole-1 ,4-diyl)dithiophene]- 5,5'-diyl-alt-thiophen-2 ,5- diyl}, Poly[[4,8-bis[(2-ethylhexyl)oxy]benzo[1 ,2-b:4,5-b']dithiophene-2,6-diyl][3- fluoro-2-[(2-ethylhexyl)carbonyl]thieno[3,4-b]thiophenediyl ]], Poly[2,6-(4,4-bis-(2- ethylhexyl)-4H-cyclopenta [2, 1 -b;3,4-b']dithiophene)-alt-4,7(2, 1 ,3- benzothiadiazole)], Poly[(4,4-bis(2-ethylhexyl)-dithieno[3,2-b:2',3'-d]silole)-2,6- d iy I -al t-(2 , 1 ,3-benzothiadiazole)-4,7-diyl], Poly[(9,9-bis(2-ethylhexyl)fluorenyl-2,7- diyl)-alt-(benzo[2, 1 , 3]thiadiazol-4, 7-diy I)] , or a combination of any two or more thereof.

10. The composition according to claim 9, wherein the polymer of the conjugated polymer nanoparticle is selected from: Poly[(9,9-dioctylfluorenyl-2,7- diyl)-co-(1 ,4-phenylene)], Poly[(9,9-dioctylfluorenyl-2,7-diyl)-co-(4,4'-(N-(4-sec- butylphenyl)diphenylamine)], Poly(2,5-di(3',7'-dimethyloctyl)phenylene-1 ,4- ethynylene), Poly[(9,9-dioctyl-2,7-divinylenefluorenylene)-alt-co-{2-methoxy5-(2- ethylhexyloxy)-1 ,4-phenylene}], Poly[(9, 9-diocty I-2 ,7-bis{2- cyanovinylenefluorenylene})-alt-co- (2-methoxy-5-{2-ethylhexyloxy}-1 ,4- phenylene)], Poly(9,9-dioctylfluorene-alt-benzothidiazole), Poly[2-methoxy-5-(2- ethylhexyloxy)-1 ,4-( 1 -cyanovinylene-1 ,4-phenylene)], Poly [{9, 9-dihexy I-2 , 7-bis( 1 - cyanovinylene)fluorenylene}-alt-co- {2, 5-bis(N,N’-diphenylamino)-1 ,4- phenylene}], Poly(4,4-dioctyldithieno(3,2-b:2’,3’-d)silole)-2,6-diyl-alt-(2, 1 ,3- benzothiadiazole)-4,7-diyl), Poly[(5-fluoro-2, 1 ,3-benzothiadiazole-4,7-diyl)(4,4- dihexadecyl-4H- cyclopenta[2, 1 -b:3,4- b’]dithiophene-2,6-diyl)(6-fluoro-2, 1 ,3- benzothiadiazole- 4,7-diyl)(4,4-dihexadecyl-4H-cyclopenta[2, 1 -b:3,4- b’]dithiophene-2,6-diyl)], Poly{2,2'-[(2,5-bis(2-hexyldecyl)-3,6-dioxo-2, 3,5,6- tetrahydropyrrolo[3,4-c ]pyrrole-1 ,4-diyl)dithiophene]- 5,5'-diyl-alt-thiophen-2 ,5- diyl}, Poly[[4,8-bis[(2-ethylhexyl)oxy]benzo[1 ,2-b:4,5-b’]dithiophene-2,6-diyl][3- fluoro-2-[(2-ethylhexyl)carbonyl]thieno[3,4-b]thiophenediyl ]], Poly[2,6-(4,4-bis-(2- ethylhexyl)-4H-cyclopenta [2, 1 -b;3,4-b']dithiophene)-alt-4,7(2, 1 ,3- benzothiadiazole)], Poly[(4,4-bis(2-ethylhexyl)-dithieno[3,2-b:2',3'-d]silole)-2,6- diyl-alt-(2, 1 ,3-benzothiadiazole)-4,7-diyl], or Poly[(9,9-bis(2-ethylhexyl)fluorenyl- 2,7-diyl)-alt-(benzo[2, 1 , 3]thiadiazol-4, 7-diy I)] .11 . The composition according to any preceding claim, wherein the quencher molecule is a dye or dye quencher.

12. The composition according to claim 11 , wherein the dye is selected from an acid wool dye, azo dye, cationic dye, or anionic dye.

13. The composition according to claim 11 , wherein the dye quencher is a dark quencher.

14. The composition according to claim 12, wherein the acid wool dye is selected from at least: Fast Green, Coomassie Blue, Erythrosine, Sunset Yellow, Tartrazine, Acid Red, or Eriochrome Black.

15. The composition according to any of claims 1 -10, wherein the conjugated polymer nanoparticle (CPN) capable of fluorescence is a first CPN, and the quencher molecule is a second CPN.

16. The composition according to claim 15, wherein the second CPN has maximal emission of at least: 420 nm, 435 nm, 475 nm, 510 nm, 530 nm, 550 nm, 580 nm, 610 nm, 660 nm, 680 nm, 770 nm, 820 nm, 830 nm, 840 nm or 1000 nm.

17. The composition according to claim 15 or 16, wherein the polymer of the second CPN is selected from: Poly[9,9-di(3',7'-dimethyloctyl)fluoren-2,7- yleneethynylene], Poly[9,9-didodecylfluroenyl-2,7-yleneethylnylene], Poly[9,9- di(2'-ethylhexyl)fluoren-2,7-yleneethynylene], Poly(9,9-dioctylfluorenyl-2,7- yleneethynylene), Poly[9,9-bis-(2-ethylhexyl)-9H-fluorene-2,7-diyl], Poly(9,9- dihexyl-9H-fluorene-2,7-diyl), Poly(9,9-di-(2-ethylhexyl)-9H-fluorene-2,7-diyl), Poly[(9,9-di-(2-ethylhexyl)-9H-fluorene-2,7-diyl)-co-(1-methoxy-4-(2- ethylhexyloxy)-2,5-phenylenevinylene)], Poly[(9,9-dihexyl-9H-fluorene-2,7-diyl)- co-(1-methoxy-4-(2-ethylhexyloxy)-2,5-phenylenevinylene)], Poly(2,5- bis( 1 ,4,7, 10-tetraoxaundecyl)-1 ,4-phenylenevinylene), Poly (2 , 5-d iocty I - 1 ,4- phenylenevinylene), Poly(2,5-dioctylphenylene-1 ,4-ethynylene), Poly [ 1 ,2- bis(benzylthio)acetylene], Poly[1 ,2-bis(ethylthio)acetylene],Poly[bis(methylthio)acetylene], Poly(3,5 pyridine), Poly(3-(2- methoxyethoxy)ethoxymethylthiophene-2,5-diyl), 5,5'-Dibromo-2,2'-bithiophene,Poly(3-butylthiophene-2,5-diyl), Poly(3-cyclohexyl-4-methylthiophene-2,5-diyl), Poly(3-cyclohexylthiophene-2,5-diyl), Poly(3-decylthiophene-2,5-diyl), Poly(3- dodecylthiophene-2,5-diyl), Poly(3-hexylthiophene-2,5-diyl), Poly(3- octylthiophene-2,5-diyl), Thiophene Oligothiophenes, 5,5'-Dibromo-2,2'- bithiophene, 2,2',5',2",5",2"'-Quaterthiophene, a-Sexithiophene, 3110732,2':5',2"- Terthiophene, Poly(thiophene-2,5-diyl), bromine terminated powder, 2,3- Dihydrothieno[3,4-b]-1 ,4-dioxin, 3,2':5',3"-Terthiophene, 5-Hexyl-2,2'-bithiophene, 22,2'-Bithiophene, Thiophene, 5,5""-Dihexyl-2,2':5',2":5",2"':5"',2"":5"",2 - sexithiophene, Poly(styrenesulfonate) / poly(2,3-dihydrothieno(3,4-b)-1 ,4-dioxin), Poly(3,4-ethylenedioxythiophene)-block-poly(ethylene glycol), Poly(3,4- ethylenedioxythiophene), tetramethacrylate endcapped, Poly((9,9-dihexyl-9H- fluorene-2,7-diyl)-co-(1-methoxy-4-(2-ethylhexyloxy)-2,5-phenylenevinylene)), Poly((9,9-dihexyl-9H-fluorene-2,7-diyl)-co-(1-methoxy-4-(2-ethylhexyloxy)-2,5- phenylenevinylene), Poly(1-methoxy-4-(3-propyloxy-heptaisobutyl-PSS)-2,5- phenylenevinylene)-co-(1-methoxy-4-(2-ethylhexyloxy)-2,5-phenylenevinylene), Poly(2,5-bis(3-sulfonatopropoxy)-1 ,4-phenylene, disodium salt-alt-1 ,4- phenylene), Poly(2,5-dihexyloxy-1 ,4-phenylenevinylene), Poly (2 ,5-diocty 1-1 ,4- phenylenevinylene), Poly(2,6-naphthalenevinylene), Poly[5-methoxy-2-(3- sulfopropoxy)-1 ,4-phenylenevinylene] Potassium salt, Poly(p-xylene tetrahydrothiophenium chloride), Poly[(m-phenylenevinylene)-alt-(2-methoxy-5- octyloxy-p-phenylenevinylene)] , Poly[(m-phenylenevinylene)-alt-(2,5-dihexyloxy- p-phenylenevinylene)], Poly[(m-phenylenevinylene)-alt-(2-methoxy-5-(2-ethyl- hexyloxy)-p-phenylenevinylene)], Poly[(m-phenylenevinylene)-co-(2,5-dioctoxy- p-phenylenevinylene)], Poly[(m-phenylenevinylene)-alt-(2,5-dibutoxy-p- phenylenevinylene)], Poly[(o-phenylenevinylene)-alt-(2-methoxy-5-(2- ethylhexyloxy)-p-phenylenevinylene)], Poly[(p-phenylenevinylene)-alt-(2- methoxy-5-(2-ethyl-hexyloxy)-p-phenylenevinylene)], Poly[2-(2',5'-bis(2"- ethylhexyloxy)phenyl)-1 ,4-phenylenevinylene], Poly[2,5-bis(3' 7'- dimethyloctyloxy)-1 ,4-phenylenevinylene], Poly[(2,5-bisoctyloxy)-1 ,4- phenylenevinylene], Poly[2-methoxy-5-(2-ethylhexyloxy)-1 ,4-phenylenevinylene], Poly[2-methoxy-5-(3',7'-dimethyloctyloxy)-1 ,4-phenylenevinylene], Poly[5- methoxy-2-(3-sulfopropoxy)-1 ,4-phenylenevinylene], Poly[9-(2-ethylhexyl)-3,6-carbazolevinylene-alt-2,6-naphthalenevinylene], Poly{[2-[2',5'-bis(2"- ethylhexyloxy)phenyl]-1 ,4-phenylenevinylene]-co-[2-methoxy-5-(2'- ethylhexyloxy)-1 ,4-phenylenevinylene]}, Poly[(9,9-dioctylfluorenyl-2,7-diyl)-co- (1 ,4-phenylene)], Poly[(9,9-dioctylfluorenyl-2,7-diyl)-co-(4,4'-(N-(4-sec- butylphenyl)diphenylamine)], Poly(2,5-di(3',7'-dimethyloctyl)phenylene-1 ,4- ethynylene), Poly[(9,9-dioctyl-2,7-divinylenefluorenylene)-alt-co-{2-methoxy5-(2- ethylhexyloxy)-1 ,4-phenylene}], Poly[(9, 9-diocty I-2 ,7-bis{2- cyanovinylenefluorenylene})-alt-co- (2-methoxy-5-{2-ethylhexyloxy}-1 ,4- phenylene)], Poly(9,9-dioctylfluorene-alt-benzothidiazole), Poly[2-methoxy-5-(2- ethylhexyloxy)-1 ,4-( 1 -cyanovinylene-1 ,4-phenylene)], Poly [{9, 9-dihexy I-2 , 7-bis( 1 - cyanovinylene)fluorenylene}-alt-co- {2, 5-bis(N,N’-diphenylamino)-1 ,4- phenylene}], Poly(4,4-dioctyldithieno(3,2-b:2',3'-d)silole)-2,6-diyl-alt-(2, 1 ,3- benzothiadiazole)-4,7-diyl), Poly[(5-fluoro-2, 1 ,3-benzothiadiazole-4,7-diyl)(4,4- dihexadecyl-4H- cyclopenta[2,1-b:3,4- b']dithiophene-2,6-diyl)(6-fluoro-2,1 ,3- benzothiadiazole- 4,7-diyl)(4,4-dihexadecyl-4H-cyclopenta[2, 1 -b:3,4- b']dithiophene-2,6-diyl)], Poly{2,2'-[(2,5-bis(2-hexyldecyl)-3,6-dioxo-2, 3,5,6- tetrahydropyrrolo[3,4-c ]pyrrole-1 ,4-diyl)dithiophene]- 5,5'-diyl-alt-thiophen-2 ,5- diyl}, Poly[[4,8-bis[(2-ethylhexyl)oxy]benzo[1 ,2-b:4,5-b']dithiophene-2,6-diyl][3- fluoro-2-[(2-ethylhexyl)carbonyl]thieno[3,4-b]thiophenediyl ]], Poly[2,6-(4,4-bis-(2- ethylhexyl)-4H-cyclopenta [2, 1 -b;3,4-b']dithiophene)-alt-4,7(2, 1 ,3- benzothiadiazole)], Poly[(4,4-bis(2-ethylhexyl)-dithieno[3,2-b:2',3'-d]silole)-2,6- d iy I -al t-(2 , 1 ,3-benzothiadiazole)-4,7-diyl], Poly[(9,9-bis(2-ethylhexyl)fluorenyl-2,7- diyl)-alt-(benzo[2, 1 , 3]thiadiazol-4, 7-diy I)] , Poly[2,6-(4,4-bis(2-ethylhexyl)-4H- cyclopenta-[2,1-b:3,4-b']dithiophene-alt-4,7-bis(thiophen-2-yl)benzo-2,1 ,3- thiadiazole], Poly[2, 1 ,3-benzothiadiazole-4,7-diyl[4,4-bis(2-ethylhexyl)-4H- silolo[3,2-b:4,5-b']dithiophene-2,6-diyl]], Poly[2,7-(9,9-di-octyl-fluorene)-alt-4,7- bis(thiophen-2-yl)benzo-2, 1 ,3-thiadiazole], or a combination of any two or more thereof.

18. The composition according to claim 17, wherein the polymer of the second CPN is selected from: Poly[(9,9-dioctylfluorenyl-2,7-diyl)-co-(1 ,4- phenylene)], Poly[(9,9-dioctylfluorenyl-2,7-diyl)-co-(4,4'-(N-(4-sec- butylphenyl)diphenylamine)], Poly(2,5-di(3',7'-dimethyloctyl)phenylene-1 ,4-ethynylene), Poly[(9,9-dioctyl-2,7-divinylenefluorenylene)-alt-co-{2-methoxy5-(2- ethylhexyloxy)-1 ,4-phenylene}], Poly[(9, 9-diocty I-2 ,7-bis{2- cyanovinylenefluorenylene})-alt-co- (2-methoxy-5-{2-ethylhexyloxy}-1 ,4- phenylene)], Poly(9,9-dioctylfluorene-alt-benzothidiazole), Poly[2-methoxy-5-(2- ethylhexyloxy)-1 ,4-( 1 -cyanovinylene-1 ,4-phenylene)], Poly [{9, 9-dihexy I-2 , 7-bis( 1 - cyanovinylene)fluorenylene}-alt-co- {2, 5-bis(N,N’-diphenylamino)-1 ,4- phenylene}], Poly(4,4-dioctyldithieno(3,2-b:2’,3’-d)silole)-2,6-diyl-alt-(2, 1 ,3- benzothiadiazole)-4,7-diyl), Poly[(5-fluoro-2, 1 ,3-benzothiadiazole-4,7-diyl)(4,4- dihexadecyl-4H- cyclopenta[2, 1 -b:3,4- b’]dithiophene-2,6-diyl)(6-fluoro-2, 1 ,3- benzothiadiazole- 4,7-diyl)(4,4-dihexadecyl-4H-cyclopenta[2, 1 -b:3,4- b’]dithiophene-2,6-diyl)], Poly{2,2'-[(2,5-bis(2-hexyldecyl)-3,6-dioxo-2, 3,5,6- tetrahydropyrrolo[3,4-c ]pyrrole-1 ,4-diyl)dithiophene]- 5,5'-diyl-alt-thiophen-2 ,5- diyl}, Poly[[4,8-bis[(2-ethylhexyl)oxy]benzo[1 ,2-b:4,5-b’]dithiophene-2,6-diyl][3- fluoro-2-[(2-ethylhexyl)carbonyl]thieno[3,4-b]thiophenediyl ]], Poly[2,6-(4,4-bis-(2- ethylhexyl)-4H-cyclopenta [2, 1 -b;3,4-b']dithiophene)-alt-4,7(2, 1 ,3- benzothiadiazole)], Poly[(4,4-bis(2-ethylhexyl)-dithieno[3,2-b:2',3'-d]silole)-2,6- d iy I -al t-(2 , 1 ,3-benzothiadiazole)-4,7-diyl], Poly[(9,9-bis(2-ethylhexyl)fluorenyl-2,7- diyl)-alt-(benzo[2, 1 , 3]thiadiazol-4, 7-diy I)] , Poly[2,6-(4,4-bis(2-ethylhexyl)-4H- cyclopenta-[2,1-b:3,4-b']dithiophene-alt-4,7-bis(thiophen-2-yl)benzo-2,1 ,3- thiadiazole], Poly[2, 1 ,3-benzothiadiazole-4,7-diyl[4,4-bis(2-ethylhexyl)-4H- silolo[3,2-b:4,5-b']dithiophene-2,6-diyl]], or Poly[2,7-(9,9-di-octyl-fluorene)-alt-4,7- bis(thiophen-2-yl)benzo-2, 1 ,3-thiadiazole],19. The composition according to any of claims 15-18, wherein the polymer of the first CPN and the polymer of the second CPN are selected from:(i) Poly[2-methoxy-5-(2-ethylhexyloxy)-1 ,4-( 1 -cyanovinylene-1 ,4- phenylene)] and Poly[2,6-(4,4-bis(2-ethylhexyl)-4H-cyclopenta-[2, 1 - b:3,4-b']dithiophene-alt-4,7-bis(thiophen-2-yl)benzo-2, 1 ,3-thiadiazole]; or(ii) Poly[2-methoxy-5-(2-ethylhexyloxy)-1 ,4-(1 -cyanovinylene- 1 ,4- phenylene)] and Poly[[4,8-bis[(2-ethylhexyl)oxy]benzo[1 ,2-b:4,5- b']dithiophene-2,6-diyl][3-fluoro-2-[(2-ethylhexyl)carbonyl]thieno[3,4- b]thiophenediyl]]; or(iii) Poly[(9,9-bis(2-ethylhexyl)fluorenyl-2,7-diyl)-alt- (benzo[2,1 ,3]thiadiazol-4,7-diyl)] and Poly[2,7-(9,9-di-octyl-fluorene)-alt- 4,7-bis(thiophen-2-yl)benzo-2,1 ,3-thiadiazole]; or(iv) Poly(2,5-di(3',7'-dimethyloctyl)phenylene-1 ,4-ethynylene) and Poly[2- methoxy-5-(2-ethylhexyloxy)-1 ,4-(1 -cyanovinylene- 1 ,4-phenylene)]; or(v) Poly[2,7-(9,9-di-octyl-fluorene)-alt-4,7-bis(thiophen-2-yl)benzo-2, 1 ,3- thiadiazole] and Poly[2, 1 ,3-benzothiadiazole-4,7-diyl[4,4-bis(2- ethylhexyl)-4H- silolo[3,2-b:4,5-b']dithiophene-2,6-diyl]] respectively.

20. The composition according to any preceding claim, wherein the targeting molecule is at least one of: an antibody, a protein, a nucleic acid, a lipid, an affimer, an aptamer, a molecularly imprinted polymer, a small molecule, or an antigen.21 . The composition of claim 20, where the targeting molecule is an antibody, preferably wherein the antibody is selected from a full-length antibody or an antibody fragment containing antigen binding domains.

22. The composition according to any preceding claim, wherein the targeting molecule has affinity towards a toxin, a bacteria, a virus, a fungi, a protein, a biomarker, a nucleic acid, or a chemical entity.

23. The composition according to claim 22, wherein the protein is an allergen.

24. The composition according to claim 23, wherein the allergen is selected from at least: milk protein, egg protein, peanut protein, soy protein, wheat protein, tree nut protein, shellfish protein, fish protein, sesame protein, celery protein, gluten protein, lupin protein or mustard protein.

25. The composition according to claim 22, wherein the chemical entity is a drug.

26. The composition according to claim 25, wherein the drug is selected from at least: fentanyl, acetylfentanyl, xylazine, heroin, morphine, opium, methadone, hydrocodone, buprenorphine, oxycodone, alprazolam, tetrahydrocannabinol,methamphetamine, cocaine, lysergic acid diethylamide, 3,4- Methylenedioxymethamphetamine, phencyclidine, psilocybin, psilocin, N,N- Dimethyltryptamine, amphetamine, codeine, cannabinol, cannabis, cannabidiol, synthetic cannabinoids, ketamine, methaqualone, 4-Methylmethcathinone, methylphenidate, alprazolam, flunitrazepam, 4-Hydroxy-n-butyric acid, nitrous oxide, mephedrone, sotonitazene, metonitazene, etonitazene, protonitazene or analogs and metabolites thereof.

27. The composition according to claim 26, wherein the drug is fentanyl.

28. The composition according to any preceding claim, wherein a metal oxide is bound to the molecular complex.

29. The composition according to claim 28, wherein the metal oxide is iron oxide.

30. The composition according to any preceding claim, wherein the molecular complex is either partly or entirely encased by a biocompatible surfactant and / or lipid.

31. The composition according to claim 30, wherein the biocompatible surfactant is selected from polystyrene maleic anhydride or pluronic F127.

32. The composition according to claim 30, wherein the biocompatible lipid is dipalmitoylphosphatidylcholine.

33. The composition according to any preceding claim, wherein the molecular complex is arranged on a substrate.

34. The composition according to claims 1-33, wherein the molecular complex is configured for application to a substrate.

35. The composition according to claim 34, wherein the substrate is filter paper or a solid support.

36. The composition according to claim 35, wherein the solid support is selected from ceramic, plastic, or borosilicate glass.

37. The composition according to claim 36, wherein the plastic is preferably polyethersulfone.

38. The composition according to claim 33, wherein a plurality of unique molecular complexes with different targeting molecules are arranged in discrete areas and can recognise multiple target entities through multiple affinities to target entities.

39. A substrate comprising the composition of claims 1-33, wherein the substrate is selected from filter paper or a solid support.

40. The substrate according to claim 39, wherein the solid support is selected from ceramic, plastic, or borosilicate glass.

41. The substrate according to claim 40, wherein the plastic is preferably polyethersulfone.

42. The substrate according to claim 39, wherein a plurality of unique molecular complexes with different targeting molecules are arranged in discrete areas and can recognise multiple target entities through multiple affinities to target entities.

43. The composition according to claims 1-33, wherein the molecular complex is suspended in a liquid.

44. The composition according to any preceding claim, wherein a plurality of unique molecular complexes with different targeting molecules and different CPNs can recognise multiple target entities by fluorescing at different wavelengths.

45. The composition according to any preceding claim, wherein the molecular complex is formulated for delivery as a spray or aerosol.

46. A composition for detecting the presence of a target entity, the composition comprising: a conjugated polymer nanoparticle (CPN) capable of fluorescence bound to a targeting molecule which has affinity towards a target entity, the composition further comprising a quencher molecule.

47. The composition of claim 46, wherein the CPN is defined as in claims 8- 10, the quencher molecule is defined as in claims 11-19, the targeting molecule is defined as in claims 20-27.

48. The composition of claim 46 or 47, wherein the quencher molecule has affinity towards the target entity.

49. A composition for detecting the presence of a target entity in a sample, the composition comprising: a first molecular complex comprising a first conjugated polymer nanoparticle (CPN) capable of fluorescence, and a first targeting molecule which has affinity towards a control entity; a second molecular complex comprising a second CPN capable of quenching the fluorescence of the first CPN, and a second targeting molecule which has affinity towards a target entity.

50. The composition of claim 49, wherein the first CPN is defined as in claims 8-10 and the second CPN is defined as in claims 15-19.

51. The composition of claim 49 or claim 50, wherein the first targeting molecule or second targeting molecule is defined as in claims 20-27.

52. The composition of any of claims 49 to 51 , wherein the first CPN and second CPN are selected from:(i) Poly[2-methoxy-5-(2-ethylhexyloxy)-1 ,4-( 1 -cyanovinylene-1 ,4- phenylene)] and Poly[2,6-(4,4-bis(2-ethylhexyl)-4H-cyclopenta-[2, 1 - b:3,4-b']dithiophene-alt-4,7-bis(thiophen-2-yl)benzo-2,1 ,3-thiadiazole];(ii) Poly[2-methoxy-5-(2-ethylhexyloxy)-1 ,4-(1 -cyanovinylene- 1 ,4- phenylene)] and Poly[[4,8-bis[(2-ethylhexyl)oxy]benzo[1 ,2-b:4,5- b']dithiophene-2,6-diyl][3-fluoro-2-[(2-ethylhexyl)carbonyl]thieno[3,4- b]thiophenediyl]];(iii) Poly[(9,9-bis(2-ethylhexyl)fluorenyl-2,7-diyl)-alt- (benzo[2,1 ,3]thiadiazol-4,7-diyl)] and Poly[2,7-(9,9-di-octyl-fluorene)-alt- 4,7-bis(thiophen-2-yl)benzo-2, 1 , 3-th i ad iazole] ;(iv) Poly(2,5-di(3',7'-dimethyloctyl)phenylene-1 ,4-ethynylene) and Poly[2- methoxy-5-(2-ethylhexyloxy)-1 ,4-(1 -cyanovinylene- 1 ,4-phenylene)]; or(v) Poly[2,7-(9,9-di-octyl-fluorene)-alt-4,7-bis(thiophen-2-yl)benzo-2, 1 ,3- thiadiazole] and Poly[2, 1 ,3-benzothiadiazole-4,7-diyl[4,4-bis(2- ethylhexyl)-4H- silolo[3,2-b:4,5-b']dithiophene-2,6-diyl]] respectively.

53. The composition of any of claims 49 to 52, wherein the molecular complexes are arranged on a substrate according to claims 33-37.

54. A method for detecting the presence of a target entity in a sample, the method comprising the steps:Contacting the sample with a composition as defined in any of claims 1- 45 whereby if present, the target entity displaces the either the quencher molecule or the CPN from the molecular complex and replaces it, and in the absence of the quencher molecule, the fluorescence of the CPN can be visualised, thereby providing a positive indication of the presence of the target entity.

55. A method for detecting the presence of a target entity in a sample, the method comprising the steps:Contacting the sample with a composition as defined in any of claims 46- 48, whereby if present, the target entity binds to the quencher molecule, and the targeting molecule binds the targeting entity, bringing the quencher molecule in proximity to the CPN, resulting in a decrease in fluorescence due to quenching.

56. A method for detecting the presence of a target entity in a sample, the method comprising the steps:Contacting the sample first with the quencher molecule of the composition as defined in any of claims 46-48, whereby if present, the target entity binds to the quencher molecule, and the subsequent addition of the sample and quencher molecule to the CPN bound to a targeting molecule of the composition as defined in any of claims 46-48, wherein the targeting molecule binds the targeting entity, bringing the quencher molecule in proximity to the CPN, resulting in a decrease in fluorescence due to quenching.

57. A method for detecting the presence of a target entity in a sample, the method comprising the steps: contacting the sample with a composition as defined in any of claims 49- 53, whereby if present in the sample, the target entity binds to the second targeting molecule of the second molecular complex, and the control entity binds to the first targeting molecule of the first molecular complex, such that the second molecular complex is separated from the first molecular complex, thereby resulting in fluorescence of the first CPN.

58. The method of claim, wherein the fluorescence of the first CPN results from the second targeting molecule of the second molecular complex being unable to bind to a further target entity located at a portion of a test surface, said portion of the test surface further comprising the control entity.

59. The method according to claims 54-58, wherein the target entity is at least one of: a toxin, a bacteria, a virus, a fungi, protein, a biomarker, a nucleic acid, or a chemical entity.

60. The method according to claim 59, wherein the protein is an allergen.61 . The method according to claim 60, wherein the allergen is selected from at least: milk protein, egg protein, peanut protein, soy protein, wheat protein, tree nut protein, shellfish protein, fish protein, sesame protein, celery protein, gluten protein, lupin protein or mustard protein.

62. The method according to claim 59, wherein the chemical entity, is a drug.

63. The method according to claim 62, wherein the drug is selected from at least: fentanyl, acetylfentanyl, xylazine, heroin, morphine, opium, methadone, hydrocodone, buprenorphine, oxycodone, alprazolam, tetrahydrocannabinol, methamphetamine, cocaine, lysergic acid diethylamide, 3,4- Methylenedioxymethamphetamine, phencyclidine, psilocybin, psilocin, N,N- Dimethyltryptamine, amphetamine, codeine, cannabinol, cannabis, cannabidiol, synthetic cannabinoids, ketamine, methaqualone, 4-Methylmethcathinone, methylphenidate, alprazolam, flunitrazepam, 4-Hydroxy-n-butyric acid, nitrous oxide, mephedrone, sotonitazene, metonitazene, etonitazene, protonitazene or analogs and metabolites thereof.

64. The method according to claim 63, wherein the drug is fentanyl.

65. The method according to claim 54-58, wherein the sample is selected from at least: blood, saliva, semen, mucus, urine, or interstitial fluid.

66. The method according to claim 54-58, wherein the sample is sweat.

67. The method according to claims 54-58, wherein a fingerprint can be visualised from the sample.

68. The method according to claim 54 and 55, wherein the decrease in fluorescence is at least: 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100%.

69. A kit of parts for detecting the presence of a target entity in a sample, comprising the composition according to claims 1-53.

70. A lateral flow device for detecting the presence of a target entity in a sample, comprising the composition according to claims 1-40 or the composition according to any of claims 49-53.

71. The lateral flow device of claim 70, wherein the quenching molecule is displaced from the targeting molecule when brought into contact with a targeting entity.

72. The lateral flow device of claim 70, wherein the CPN is displaced from the targeting molecule when brought into contact with a targeting entity.

73. The lateral flow device of claim 70, wherein a portion of a surface of the device comprises the control entity and the target entity.

74. The lateral flow device of claim 73, wherein the first and second targeting molecules of the first and second molecular complexes can bind, on the portion of the surface, to the control entity and the target entity respectively, such that when the first and second targeting molecules of the first and second molecular complexes are bound to said surface, the second CPN of the second molecular complex can quench the fluorescence of the first CPN of the first molecular complex, such that, upon the addition of the sample comprising the target entity, the second targeting molecule of the second molecular complex binds to the target entity of the sample, and is therefore prevented from binding to the target entity of the surface so that the fluorescence of the first CPN can be visualised.

75. A method for detecting the presence of a target entity in a sample, the method comprising the steps: contacting the sample with a lateral flow device as defined in 73 or 74, whereby if present, the target entity binds to the second targeting molecule of the second molecular complex and prevents the second targeting molecule of the second molecular complex from binding to the target entity of the surface so that the fluorescence of the CPN can be visualised.

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