Composite particles

Abstract

This disclosure relates to a method for producing composite particles. The method comprises contacting catalyst particles, an oxidizing agent, and a plurality of monomers. The oxidizing agent is a peroxide, a persulfate, or ozone, and the catalyst particles are or comprise a material having Fenton or Fenton-like catalytic activity. The catalyst particles catalyze the disproportionation of the oxidizing agent to produce oxygen-radical species, which then initiate a polymerization reaction, thereby polymerizing the plurality of monomers, thereby forming a polymer shell around the catalyst particles. The present invention extends to composite particles produced by the method, an apparatus containing the composite particles, and the use thereof.

Description

[Technical Field]

[0001] The present invention relates to a method for producing composite particles. The present invention extends to the composite particles themselves and methods for using them. [Background technology]

[0002] In several fields, inorganic nanoparticles are technically important. In many applications, micro and nanomaterials possess properties not evident in bulk-phase materials, such as variable plasmon resonance, magnetic and optical properties, and others known to those skilled in the art. This leads to their use in diagnostic applications such as assays (e.g., magnetic bead assays, colorimetric and fluorescence assays, microarray assays, Luminex® Multiplex assays, and / or Beckman-Coulter assays), surface plasmon resonance (SPR), and electrochemical detectors. Furthermore, due to their small size and mobility, nanomaterials and microparticles can access sites such as tissues and even intracellular regions that would otherwise be inaccessible.

[0003] However, the applications of unmodified inorganic micro and nanoparticles are limited by several factors, which must be overcome in order to bring out the unique properties of these materials.

[0004] Micro and nanoparticles may be prepared by several methods. Regardless of how nanoparticles are prepared, a common characteristic of unmodified nanomaterials is their strong tendency to aggregate in solution due to their high surface energy and large interfacial surface area. This, along with poor biocompatibility and / or toxicity associated with their size, shape, and surface chemistry, as well as poor stability in complex biological environments, limits the possibilities for the application of nanomaterials, for example, in vitro in immunoassays and lateral flow diagnostics, and in vivo in applications such as drug delivery, bioimaging, and therapeutic applications.

[0005] Therefore, coating inorganic micro and nanoparticles with a layer of additional material to prevent aggregation by electrostatic or steric stabilization is advantageous or necessary. Coating nanoparticles can also allow the surface to be adapted to the specific application in which its use is intended. Furthermore, the coating may also act as a carrier for other components. There are various strategies that involve coating micro and nanoparticles, including silica, lipids, peptides, and polymers (Nune SK, Gunda P., Thallapally PK, Lin Y.-Y., Forrest ML, Berkland CJ, Nanoparticles for biomedical imaging. Expert Opin. Drug Deliv., 2009, 6, 1175~1194). The addition of such coatings is a means of providing stabilization against aggregation, giving substrate functionality suitable for immobilizing other components, enabling adapted recognition properties, or immobilizing polymers, with organic polymers being particularly preferred in the majority of applications.

[0006] There are many methods for coating micro and nanoparticles with robust, insoluble polymers to alter their physical and chemical properties, particularly to change their dispersion and prevent aggregation. This may be achieved by treating synthesized nanoparticles with a pre-formed polymer that partially or completely replaces a low-molar mass stabilizer, such as a citrate or surfactant, which is typically used during the initial nanoparticle synthesis process. In other cases, the polymer forms a secondary shell on top of the first stabilizing layer, instead of replacing it (Pellegrino T., Manna L., Kudera S., Liedl T., Koktysh D., Rogach AL, Keller S., Radler J., Natile G., Parak WJ: Hydrophobic nanocrystals coated with an amphiphilic polymer shell: a general route to water-soluble nanocrystals. Nano Lett. 2004, 4, 703-707). Functional core-shell nanoparticles with controlled thickness were prepared by a multilayer deposition technique using polymeric polymer electrolytes (Asapu R., Claes N., Bals S. et al. Silver-polymer core-shell nanoparticles for ultrastable plasmon-enhanced photocatalysis. Applied Catalysis B:Environmental. 2017, 200, 31-38). The polymer components may be strongly adsorbed onto the nanoparticle surface so as not to be released into solution, or chemically bonded, or crosslinked by physical, photochemical, or chemical treatment to form a stable coating. Alternatively, the polymer coating may be formed by a polymerization process from monomers in the presence of nanoparticles, or from a mixture of monomers in the presence of micro and nanoparticles.However, the latter process may result in a mixture of products, such as free polymers formed in solution that are not associated with nanoparticles, which would then necessitate the application of physical (dialysis), chemical (reduction, precipitation), and / or chromatographic separation techniques to separate the intended product. Furthermore, this process excludes the use of several monomers, particularly crosslinkers, because polymer formation in free solution may form insoluble gels and larger composite particles in addition to the intended nanocomposite.

[0007] A far better approach is to ensure that polymers form only on or in close proximity to the surface of nanoparticles. This can be achieved in several ways, for example, by using stabilizing ligands that also function as monomers capable of polymerization by free radical polymerization, RAFT polymerization, addition polymerization, metathesis polymerization, Diels-Alder reaction, polycondensation, or any other process known to those skilled in the art. Alternatively, ligands may be used that combine stabilizing functionality with functional groups capable of initiating polymerization by thermal, physical, chemical, or photochemical activation, so that the polymerization site is limited to the surface of the nanoparticles. However, both of these methods are limited because both act as stabilizers and generally require the synthesis of new reagents containing the desired polymerizable or initiating functional groups. This is expensive and time-consuming.

[0008] However, in some cases where the polymerization chemistry is limited to the micro and nanoparticle surfaces by eliciting the physical or chemical properties of the nanomaterial, such that the physical or chemical conditions favorable for polymerization exist only on or in close proximity to the nanoparticle surface, but extend to the bulk phase, and the initiator, which is soluble unless immobilized, is immobilized on the surface of the nanoparticles, then soluble monomers or mixtures of monomers may be used. In this case, commercially available monomers or mixtures of monomers, as well as custom synthetic monomers that impart specific properties to the polymer, can be used.

[0009] Micro and nanoparticles can be physically captured by polymers using the "graft from" method (Pereira SO, Barros-Timmons A, Trindade T. Polymer@gold nanoparticles prepared via RAFT polymerization for opto-biodetection. Polym., 2018, 10, 189). Grafting polymers from the surface of nanoparticles can be achieved chemically or photochemically. Activation of nanomaterials by visible or UV irradiation can induce polymerization on their surfaces (Liu MS, Peng TY, Li HN et al. Photoresponsive nanostructure assisted green synthesis of organics and polymers. Appl. Catal. B-Environ. 2019, 249, 172~210). Visible light irradiation was used to polymerize methyl methacrylate, 2-(tert-butylamino)ethyl methacrylate, and ethylene glycol dimethacrylate on the surface of carbon-doped TiO2 nanoparticles (Wang X., Song X., Lin M. et al., Du. Surface initiated graft polymerization from carbon-doped TiO2 nanoparticles under sunlight illumination. Polymer, 2007, 48, 5834~5838; Kong H., Song J., Jang J. Photocatalytic antibacterial capabilities of TiO2-biocidal polymer nanocomposites synthesized by a surface-initiated photopolymerization. Environ. Sci. Technol., 2010, 44, 5672~5676). As shown by TGA analysis, the polymers contained on the surface of the TiO2 particles have high thermal stability.Beyazit et al. reported the construction of crosslinked polymer coatings on the surface of upconverting nanoparticles (UCNPs) during NIR irradiation (Beyazit S., Ambrosini S., Marchyk N. et al., Versatile synthetic strategy for coating upconverting nanoparticles with polymer shells through localized photopolymerization by using the particles as internal light sources. Angew. Chem. Int. Ed. Engl., 2014;53, 8919~23). Because the emission from UCNPs is weaker compared to direct light, polymerization proceeds only in limited proximity on the UCNP surface, thus resulting in the formation of a core / shell structure. Ding et al. reported SPR-induced radical polymerization via plasmon-"hot" electrons using Au NPs (Ding T., Mertens J., Lombardi A. et al., Light-directed tuning of plasmon resonances via plasmon-induced polymerization using hot electrons. ACS Photonics, 2017, 4, 1453~1458). It has been proposed that, upon absorption of visible photons, a strong SPR localized electric field causes the transfer of hot electrons from the AuNP surface to the monomer, forming initiating free radicals for polymer growth. However, photopolymerization is not feasible for coating nanomaterials that strongly absorb light, preventing irradiation from reaching all particles in the bulk volume.

[0010] For these reasons, chemical activation of polymerization reactions on the surface of nanomaterials is preferable to photochemical options for preparing polymer-coated nanoparticles in industrial-scale quantities. Polymer grafts can benefit from the fact that many known nanomaterials are catalytically active. Various nanoscale materials such as cerium oxide nanoparticles, Au, Pt, Pd nanoparticles, V2O5, Fe3O4, graphene oxide, and carbon nanotubes have been found to be catalytically active (Lin Y., Ren J., Qu X. Catalytically active nanomaterials: a promising candidate for artificial enzymes. Acc. Chem. Res. 2014, 47, 1097~1105). Polymerization can proceed on the surface of catalytic nanoparticles without the addition of an initiator. Therefore, Pd nanoparticles were used as an active catalyst to polymerize C2H2 to trans-polyacetylene, making their surfaces hydrophobic and preventing aggregation in organic solvents (Dai Y., Liu S., Zheng N. C2H2 treatment as a facile method to boost the catalysis of Pd nanoparticulate catalysts. J.Am.Chem.Soc.2014, 136, 5583~5586).

[0011] The formation of nanoparticles using polymer coatings (formed by UV or chemical polymerization) typically requires two separate steps. Therefore, pre-fabricated organic or inorganic nanoparticles were employed in all previous examples. This is not ideal, as it raises the problem of how to prevent aggregation of the nanoparticles prior to functionalization using polymer coatings. One option to solve this problem is to consolidate the nanoparticle formation using polymerization reactions into a single-step process. In one such example, in redox-oxidation polymerization with HAuCl4, noble metal ions were reduced to nanocrystals by dopamine (Fang QL, Zhang JF, Bai LF et al., In situ redox-oxidation polymerization for magnetic core-shell nanostructure with polydopamine-encapsulated-Au hybrid shell. J. Hazard. Mat. 2019, 367, 15-25). However, this process is limited to a very specific class of monomers, such as dopamine, aniline, and phenol, which readily undergo oxidative polymerization. [Overview of the project]

[0012] A method for producing composite particles is provided according to a first aspect of the present invention, comprising contacting catalyst particles, an oxidizing agent, and a plurality of monomers, wherein the oxidizing agent is a peroxide, a persulfate, or ozone, and the catalyst particles are a material having Fenton or Fenton-like catalytic activity, or comprising the same, wherein the catalyst particles catalyze the disproportionation of the oxidizing agent to produce oxygen-radical species, thereby initiating a polymerization reaction, which in turn polymerizes the plurality of monomers, thereby forming a polymer shell around the catalyst particles.

[0013] Advantageously, the method produces composite particles that may be used in several different applications. The method results in high yield, efficient atomic transmutation in the product, and a uniform and homogeneous collection of particles. The method can be modified, for example, by the selection of catalyst particles and / or polymers, to produce composite particles with various properties.

[0014] Since the polymerization reaction is catalyzed by catalyst particles, the polymer will form on or in close proximity to the surface of the catalyst particles. Because the polymer forms around the catalyst particles, the catalyst particles are more uniform than catalyst particles made using pre-formed polymers. Additionally, each catalyst particle will contain a polymer shell, avoiding the formation of clusters of catalyst particles encapsulated in the polymer coating. Furthermore, the formation of free polymer not attached to the catalyst particles is also avoided.

[0015] Additionally, the reaction can be carried out under mild conditions (e.g., at or near neutral pH) and can be used to produce a wide range of different polymer shells. The thickness and density of the polymer shell may be controlled by controlling the polymerization time, as well as by controlling the monomer concentration and / or the oxidizing agent concentration.

[0016] The composite particles of the present invention may be known as "nanozymes," i.e., artificial enzymes that possess catalytic activity and are made of nanomaterials. The composite particles may be capable of mimicking the function of natural enzymes and can be designed to be more stable, cost-effective, and efficient than their bioequivalents.

[0017] The composite particles or nanozymes of the present invention have several potential applications in diagnostic assays. For example, they can be used as labels in diagnostic tests to amplify the signal of target molecules and make them more detectable. They can also be used to catalyze reactions in diagnostic assays, such as the conversion of non-fluorescent molecules to fluorescent molecules that can be detected using a fluorescence reader.

[0018] The composite particles or nanozymes of the present invention may be used to catalyze the generation of redox-active species. For example, they may be used to catalyze the decomposition of hydrogen peroxide (H2O2) to hydroxyl radicals (HO·).

[0019] In addition, nanozymes can be used in biosensors for detecting various biomolecules such as glucose, cholesterol, and proteins. They can also be used in imaging applications such as photoacoustic imaging, where they can generate contrast by converting light energy into acoustic waves.

[0020] In general, nanozymes provide a promising platform for building new and improved diagnostic tools that have the potential to provide faster, more sensitive, and more accurate diagnoses of a variety of diseases.

[0021] While the first embodiment defines a method for producing one type of composite particle, it will be recognized that it can be used to produce multiple composite particles. When the method produces multiple composite particles, it will be recognized that the method will involve contacting multiple catalyst particles, an oxidizing agent, and multiple monomers. The method may also cause a polymer shell to form separately around each of the multiple catalyst particles.

[0022] The method may involve contacting catalyst particles, an oxidizing agent (e.g., a peroxide or persulfate), and a plurality of monomers in a solution. The solution may contain a solvent that is water, an organic solvent, or a combination thereof. The organic solvent may be an alcohol. The alcohol is C 1~5 Alcohol or C 1~3 Alcohol may also be used. In some embodiments, the preferred solution is an aqueous solution.

[0023] The solution may have a pH between 3 and 12 at 20°C, between 4 and 11 at 20°C, between 5 and 10 at 20°C, between 6 and 9 at 20°C, between 6.5 and 8.5 at 20°C, between 7 and 8 at 20°C, or between 7.4 and 7.6 at 20°C. Advantageously, the method may be carried out under mild reaction conditions and at a substantially neutral pH. It can be recognized that the temperature of 20°C is given to allow for the precise definition of the pH of the solution. However, the method may be carried out within the temperature range as defined herein. It should be understood that the description of temperature in relation to pH herein is not intended to limit the temperature at which the reaction may take place.

[0024] The solution may contain a buffer. The buffer may be configured to maintain the solution at a desired pH. The desired pH may be as defined above. Suitable buffers are known in the art, and any suitable buffer may be used. The buffer may be a phosphate buffer, an acetate buffer, or a borate buffer, or may contain a phosphate buffer, an acetate buffer, or a borate buffer. The buffer may be provided at concentrations between 0.05 and 1,000 mM, between 0.1 and 500 mM, between 0.5 and 100 mM, between 1 and 50 mM, between 2.5 and 20 mM, between 5 and 15 mM, between 7.5 and 12.5 mM, or between 9 and 11 mM.

[0025] Catalyst particles can be understood to have Fenton or Fenton-like catalytic activity if they are capable of catalyzing the disproportionation of an oxidizing agent to produce oxygen-radical species. Catalyst particles may also be capable of catalyzing the disproportionation of an oxidizing agent to produce two distinct radical species.

[0026] If a peroxide or persulfate is understood to have the general formula ROOR (wherein both R groups are the same or different and are any suitable groups that provide the peroxide or persulfate), then the oxygen-radical species is defined as ROOR. · and ROO ·It may have. If the peroxide is hydrogen peroxide, the oxygen-radical species is HO · and HOO · That's fine.

[0027] The catalyst particles can be understood as forming the core of the composite particles.

[0028] The composite particles may be composite nanoparticles or microparticles. A composite particle may be understood as a composite nanoparticle if it has dimensions or a diameter of less than 1 μm. A composite nanoparticle may be understood as a composite microparticle if it has dimensions or a diameter between 1 μm and 1000 μm. Preferably, this composite nanoparticle is a composite nanoparticle.

[0029] The composite particles may have dimensions or diameters of less than 5,000 nm, less than 2,000 nm, less than 1,500 nm, less than 1,250 nm, less than 1,000 nm, less than 900 nm, less than 800 nm, less than 700 nm, less than 600 nm, less than 500 nm, less than 400 nm, less than 300 nm, less than 200 nm, less than 150 nm, or less than 120 nm. The composite particles may have dimensions or diameters of at least 1 nm, at least 50 nm, at least 100 nm, at least 200 nm, at least 300 nm, or at least 400 nm. The composite particles may have dimensions or diameters between 1 and 5,000 nm, between 50 and 2,000 nm, between 100 and 1,500 nm, between 150 and 1,250 nm, between 200 and 1,000 nm, between 300 and 900 nm, or between 400 and 800 nm. The composite particles may have dimensions or diameters between 10 and 2,000 nm, 30 and 1,000 nm, 50 and 900 nm, 60 and 800 nm, 70 and 700 nm, 80 and 650 nm, 90 and 640 nm, or 95 and 630 nm. In some embodiments, the composite particles have dimensions or diameters between 10 and 600 nm, 30 and 500 nm, 50 and 400 nm, 60 and 300 nm, 70 and 200 nm, 80 and 175 nm, 90 and 150 nm, or 95 and 130 nm. The dimensions or diameters may be measured using dynamic light scattering (DLS) by transmission electron microscopy (TEM).

[0030] In embodiments in which the method includes producing a plurality of composite particles, the plurality of composite particles may have an average diameter as defined above.

[0031] The catalyst particles may be nanoparticles. For example, the catalyst particles may have dimensions or diameters of less than 1,000 nm, less than 900 nm, less than 800 nm, less than 700 nm, less than 600 nm, less than 500 nm, less than 400 nm, less than 300 nm, less than 200 nm, less than 150 nm, or less than 120 nm. The catalyst particles may have dimensions or diameters of at least 1 nm, at least 10 nm, at least 20 nm, at least 30 nm, at least 40 nm, at least 50 nm, at least 60 nm, at least 70 nm, at least 80 nm, at least 90 nm, or at least 100 nm. The catalyst particles may have dimensions or diameters between 1 and 1,000 nm, 10 and 900 nm, 20 and 800 nm, 25 and 700 nm, 25 and 600 nm, 30 and 500 nm, 30 and 400 nm, 35 and 300 nm, 35 and 200 nm, 40 and 150 nm, or 40 and 120 nm. The catalyst particles may have diameters between 1 and 100 nm, 2 and 80 nm, 3 and 60 nm, 4 and 40 nm, or 5 and 20 nm. The dimensions or diameters may be measured using dynamic light scattering (DLS) by transmission electron microscopy (TEM).

[0032] In embodiments of the method that use multiple catalyst particles, the multiple catalyst particles may have an average diameter as defined above.

[0033] In some embodiments, the catalyst particles are not bound. Therefore, in embodiments in which the catalyst particles, an oxidizing agent (e.g., a peroxide or persulfate), and a plurality of monomers are in contact in solution, the catalyst particles may be provided as a suspension in solution.

[0034] In some embodiments, catalyst particles are bonded to a substrate. The substrate may be a solid substrate. The substrate may contain metals or metalloids, glass, and / or polymers, or may contain metals or metalloids, glass, and / or polymers. The metal or metalloid may be a pure metal, a pure metalloid, an impure metal, an impure metalloid, an alloy, a metal-containing compound, or a metalloid-containing compound. The substrate may include a coating. The catalyst may be bonded to the coating.

[0035] In one embodiment, the catalyst is bonded to a substrate, and the substrate is an optical fiber including a polymer coating.

[0036] The catalyst particles may include, or consist of, metals, metal alloys, metal oxides, metal salts, semiconductors, two-dimensional materials, fullerenes, carbon nanotubes, quantum dots, carbon nanodots, carbides, and / or nitrides. The carbon nanotubes may be single-walled carbon nanotubes or multi-walled carbon nanotubes.

[0037] The metal may be gold, silver, platinum, palladium, iron, nickel, zinc, chromium, titanium, zirconium, hafnium, vanadium, niobium, tantalum, molybdenum, or tungsten. The metal alloy may contain iron, copper, chromium, nickel, titanium, zirconium, hafnium, vanadium, niobium, tantalum, chromium, molybdenum, and / or tungsten. The metal oxide may be iron oxide, aluminum oxide, titanium oxide, cerium oxide, zirconium oxide, hafnium oxide, vanadium oxide, niobium oxide, tantalum oxide, chromium oxide, molybdenum oxide, or tungsten oxide. The metal salt may be organic or inorganic. The metal salt may be iron salt, copper salt, titanium salt, zirconium salt, hafnium salt, vanadium salt, niobium salt, tantalum salt, chromium salt, molybdenum salt, or tungsten salt. The semiconductor may be silicon, gallium, or germanium, or may contain silicon, gallium, or germanium. The two-dimensional material may be graphene, or a graphene derivative such as graphene oxide. The fullerene may contain trapped ions. The trapped ions may be metal ions. The quantum dot may be a carbon quantum dot, or a semiconductor quantum dot. The semiconductor quantum dot may contain, or consist of, a semiconductor as defined above. The carbide may be titanium carbide, zirconium carbide, hafnium carbide, vanadium carbide, niobium carbide, tantalum carbide, chromium carbide, molybdenum carbide, or tungsten carbide. The nitride may be titanium nitride, zirconium nitride, hafnium nitride, vanadium nitride, niobium nitride, tantalum nitride, chromium nitride, molybdenum nitride, or tungsten nitride.

[0038] In some embodiments, the catalyst particles contain or consist of gold. In alternative embodiments, the catalyst particles contain or consist of iron oxide.

[0039] The method may include preparing catalyst particles.

[0040] The method may include a first step of preparing catalyst particles as defined above, and a second separate step of contacting the catalyst particles, an oxidizing agent, and a plurality of monomers. Thus, in this embodiment, the method may be seen as a two-step or two-pot synthesis. The catalyst particles may be prepared using standard methods known in the art.

[0041] The method may include stabilizing the catalyst particles to prevent aggregation. Therefore, the method may include preparing catalyst particles in the presence of a stabilizing ligand. The stabilizing ligand may be a citrate, phosphate, sulfate, borate, ascorbate, ketone, or surfactant. The method may include replacing the stabilizing ligand prior to or following contact of the catalyst particles, oxidizing agent, and multiple monomers. Alternatively, the multiple monomers may polymerize, thereby forming a polymer shell around the catalyst particles and stabilizing ligand.

[0042] In a preferred embodiment, the method comprises, in a single step, preparing catalyst particles and contacting the catalyst particles with an oxidizing agent and a plurality of monomers. In this embodiment, the method may be considered a one-step or one-pot synthesis.

[0043] Therefore, the method may include contacting a catalyst particle precursor, an oxidizing agent, and a plurality of monomers, wherein the catalyst particle precursor is configured to produce catalyst particles. Once formed, the catalyst particles come into contact with the oxidizing agent and the plurality of monomers, catalyzing the disproportionation of the oxidizing agent to produce oxygen-radical species, which then initiate a polymerization reaction, thereby polymerizing the plurality of monomers and forming a polymer shell around the catalyst particles. Therefore, once the catalyst particles are formed, the polymerization reaction may occur spontaneously.

[0044] It can be recognized that the method may include contacting a catalyst particle precursor, an oxidizing agent, and a plurality of monomers in a solution.

[0045] The catalyst particle precursor may include a reagent that can be oxidized, reduced, or hydrolyzed to produce catalyst particles, and an oxidizing agent, reducing agent, or hydrolyzing agent. The reagent that can be oxidized, reduced, or hydrolyzed to produce catalyst particles may be an inorganic salt, acid, or hydroxide. The inorganic salt, acid, or hydroxide may include a metal cation. The metal cation may be a gold cation, iron cation, silver cation, platinum cation, palladium cation, titanium cation, zirconium cation, hafnium cation, vanadium cation, niobium cation, tantalum cation, chromium cation, molybdenum cation, or tungsten cation. In some embodiments, the cation is Au 3+ It is a cation. Therefore, the acid may be hydrogen tetrachloroauro(III) or a solvate thereof. The solvate may be hydrogen tetrachloroauro(III) trihydrate or hydrogen tetrahydrate tetrahydrate. The reducing agent may be an organic compound containing a carboxyl group, a ketone group, and / or a hydroxyl group. In some embodiments, the reducing agent is a ketone. The ketone may be cyclohexanone or acetone.

[0046] Metal cations may be present in the solution at concentrations between 0.0001 and 5 mM, between 0.0005 and 1 mM, between 0.001 and 0.5 mM, between 0.005 and 0.25 mM, between 0.01 and 0.10 mM, between 0.02 and 0.08 mM, or between 0.04 and 0.05 mM.

[0047] The reducing agent may be present in the solution at concentrations between 0.005 and 10 M, between 0.01 and 5 M, between 0.05 and 2.5 M, between 0.1 and 1 M, between 0.2 and 0.8 M, and between 0.4 and 0.5 M.

[0048] The molar ratio of the metal cation to the reducing agent may be between 1:1 and 1:1,000,000, between 1:100 and 1:100,000, between 1:500 and 1:50,000, between 1:1,000 and 1:25,000, between 1:5,000 and 1:20,000, between 1:7,500 and 1:15,000, between 1:9,000 and 1:12,500, or between 1:9,500 and 1:10,000.

[0049] The peroxide may be hydrogen peroxide, a peroxy acid, a typical group peroxide, or an organic peroxide. The persulfate may be ammonium persulfate.

[0050] In some preferred embodiments, the oxidizing agent is hydrogen peroxide.

[0051] The oxidizing agent may be present in the solution at concentrations between 0.005 and 10 M, between 0.01 and 5 M, between 0.05 and 2.5 M, between 0.1 and 1 M, between 0.2 and 0.8 M, or between 0.4 and 0.5 M.

[0052] Preferably, the multiple monomers are multiple monomers capable of undergoing radical addition polymerization. Therefore, the multiple monomers can be understood as comprising multiple molecules, each molecule containing one or more unsaturated carbon-carbon bonds. It will be recognized that the phrase “multiple monomers” may be used to refer to multiple monomer molecules.

[0053] Multiple monomers may consist of a single chemical species. Alternatively, multiple monomers may contain two or more different chemical species.

[0054] The monomers may include acrylic acid, esters and / or amides of acrylic acid, methacrylic acid, esters and / or amides of methacrylic acid, vinyl ethers, vinyl esters, vinyl aromatic compounds, or combinations thereof.

[0055] In one embodiment, the plurality of monomers comprises one or more chemical species containing functional groups configured to impart desired properties to the polymer.

[0056] The functional groups may be configured to allow for the attachment of further species. For example, the functional groups may include carboxyl groups, amine groups, or glycidyl groups. The functional groups may also allow for the attachment of further species by chemical reactions or bioconjugation methods known to those skilled in the art.

[0057] Further species may include inorganic ions, nucleic acids, cells, spores, viruses, microorganisms, tissue samples, carbohydrates, peptides, proteins, drugs, drug intermediates, drug precursors, hormones, vitamins, biomarkers, toxins, insecticides, herbicides, explosives, nerve agents, contaminants, endocrine disruptors, nucleotides, nucleosides, metabolites, epitopes, antigens, receptors, receptor fragments, antibodies, antibody fragments, dyes, indicators, radionuclides, or any other components known to those skilled in the art. Carbohydrates may be oligosaccharides or polysaccharides. Polysaccharides may be glycosaminoglycans. Proteins may be nucleoproteins, mucoproteins, lipoproteins, synthetic proteins, or glycoproteins. Drugs may be steroids, immunosuppressants, heparin, or antibiotics. Biomarkers may be biomarkers of pathological or diseased conditions. Nucleic acids may be oligonucleotides, DNA, or RNA. Metabolites may be drug metabolites and / or secondary metabolites.

[0058] Alternatively or additionally, the functionality may be configured such that the polymer is fluorescent, for example, the functional group may include fluorescein, rhodamine, dansyl, Texas Red, Alexa Fluor, Cy5, or green fluorescent protein (GFP).

[0059] Alternatively or additionally, the functionality may be configured such that the polymer becomes electroactive. For example, the functional group may be an iron derivative such as ferricyanide, ferrocyanide, or ferrocene, 3+ / 2+ a ruthenium derivative such as [Ru(NH3)6],

[0060] methylene blue, porphyrin, or a metallocene. Alternatively or additionally, the plurality of monomers may include one or more chemical species having additional functionality. For example, the one or more chemical species having additional functionality may include an amino acid derivative, nucleoside, nucleotide, carbohydrate, styrene or its derivative, acrylamide or its derivative, butadiene, acrylonitrile, vinyl acetate, vinyl monomer, allyl monomer, acetylene, acrylate, methacrylate, acrylamide, methacrylamide, chloroacrylate, itaconate, or trifluoromethyl acrylate. The derivative of styrene may be divinylbenzene.

[0061] Alternatively or additionally, the plurality of monomers may include a polymerizable dye or a polymerizable reactive species. Advantageously, the polymer shell will enable detection, grafting, therapeutic action, diagnosis, and additional chemical modification.

[0062] Thus, the plurality of monomers has the formula (I):

[0063]

Chemical formula

[0064] (wherein R 1 ~R 4 are independently H, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted cycloalkyl, optionally substituted aryl, optionally substituted heterocyclyl, optionally substituted heteroaryl, COOR 5 , or CONR 5 R6 And, R 5 and R 6 (which independently comprises one or more chemical species of H, an optionally substituted alkyl, an optionally substituted alkenyl, or an optionally substituted alkynyl.)

[0065] The alkyl, alkenyl, or alkynyl in question may be optionally substituted with C 1~12 Alkyl, optionally substituted C 2~12 C replaced by an alkenil or of any choice 2~12 It may also be an alkynyl. More preferably, the alkyl, alkenyl, or alkynyl is optionally substituted with C 1~6 Alkyl, optionally substituted C 2~6 Alkenil, or optionally replaced C 2~6 It is alkinyl.

[0066] The alkyl, alkenyl, or alkynyl in question is either unsubstituted or oxo, OR 7 , NR 7 R 8 COOR 7 OCOR 7 CONR 7 R 8 , or NR 7 COR 8 One or more of these may be substituted, and in the formula, R 7 and R 8 H and C are independent of each other. 1~12 Alkyl, C 2~12 Alkenyl, or C 2~12 It is alkinyl.

[0067] The optionally substituted cycloalkyl group is the optionally substituted C 3~12 It may also be a cycloalkyl. The optionally substituted aryl is the optionally substituted C 6~10It may also be an aryl. An optionally substituted heteroring may be an optionally substituted 3- to 12-membered heteroring. An optionally substituted heteroaryl may be an optionally substituted 5- to 10-membered heteroaryl. In a polycyclic structure, if one ring is aromatic, the entire ring structure may be called an aryl, or if one of the ring atoms is a heteroatom, it may be called a heteroaryl. In a polycyclic structure, if one ring atom is a heteroatom, and none of the rings are aromatic, the entire structure may be called a heterocyclic formula, or if one or more of the rings are aromatic, it may be called a heteroaryl.

[0068] Cycloalkyl, aryl, heterocyclic, or heteroaryl are unsubstituted or oxo, OR 7 , NR 7 R 8 COOR 7 OCOR 7 CONR 7 R 8 , or NR 7 COR 8 One or more of these may be substituted, and in the formula, R 7 and R 8 H and C are independent of each other. 1~12 Alkyl, C 2~12 Alkenyl, or C 2~12 It is alkinyl.

[0069] Preferably, R 1 H is H.

[0070] Preferably, R 2 H is H.

[0071] Preferably, R 3 H is replaced by C of any choice. 1~6 Alkyl, optionally substituted C 2~6 Alkenil, or optionally replaced C 2~6 It is an alkynyl. More preferably, R 3 is H, or C 1~3 Alkyl, C 2~3 Alkenyl, or C2~3 It is alkynnyl. Most preferably, R 3 It is either H or methyl.

[0072] Preferably, R 4 COOR 5 or CONR 5 R 6 That is the case.

[0073] Preferably, R 5 and R 6 These are independently H and C, which are substituted by any choice. 1~12 Alkyl, optionally substituted C 2~12 Alkenil, or optionally replaced C 2~12 It is an alkynyl. More preferably, R 5 and R 6 These are independently H and C, which are substituted by any choice. 1~6 Alkyl, optionally substituted C 2~6 Alkenil, or optionally replaced C 2~6 It is alkynnyl. Most preferably, R 5 and R 6 These are independently H, methyl, ethyl, i-propyl, t-butyl, CH2CH2OH, or CH2CH2CH2NH2.

[0074] Alternatively, R 4 This is C, which has been replaced by an optional substitution. 3~12 Cycloalkyl, optionally substituted C 6~10 The aryl group may be an aryl group, a 3- to 12-membered heterocisyl group that has been optionally substituted, or a 5- to 10-membered heteroaryl group that has been optionally substituted. 4 R may be optionally substituted phenyl or optionally substituted 5- or 6-membered heteroaryl. 4 It may also be pyridine.

[0075] Alternatively, R 4 This is C, which has been replaced by an optional substitution. 1~12 Alkyl, optionally substituted C 2~12 Alkenil, or optionally replaced C2~12 may be alkynyl. More preferably, R 4 is optionally substituted C 1~6 alkyl, optionally substituted C 2~6 alkenyl, or optionally substituted C 2~6 alkynyl. Most preferably, R 4 is optionally substituted C 1~3 alkyl, optionally substituted C 2~3 alkenyl, or optionally substituted C 2~3 alkynyl. R 4 may be CH2NH2.

[0076] Thus, the plurality of monomers may include one or more chemical species selected from the group consisting of acrylic acid, acrylamide, methacrylamide, N-isopropylacrylamide, N-tert-butylacrylamide, hydroxyethyl methacrylate, methacrylic acid, N-3-aminopropyl methacrylamide, N,N'-diethylacrylamide, vinylpyridine, and allylamine.

[0077] The plurality of monomers may include one or more crosslinking agents. The or each crosslinking agent may be a molecule containing two or more unsaturated carbon-carbon bonds. The or each crosslinking agent has the formula (II):

[0078]

Chemical formula

[0079] (wherein R 9 ~R 14 are independently H, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, COOR 5 , or CONR 5 R 6 and R<00​​These are independently H, an optionally substituted alkyl, an optionally substituted alkenyl, or an optionally substituted alkynyl. It may also contain the chemical species L (where L is a linker).

[0080] Alkyl, alkenyl, or alkynyl may be as defined above.

[0081] R 9 ~R 14 Preferably, these are H and C, which is optionally replaced by H. 1~6 Alkyl, optionally substituted C 2~6 Alkenil, or optionally replaced C 2~6 It is an alkynyl. More preferably, R 9 ~R 14 is H, or C 1~3 Alkyl, C 2~3 Alkenyl, or C 2~3 It is alkynnyl. Most preferably, R 9 ~R 14 It is either H or methyl.

[0082] The linker may be one or more heteroatoms, an optionally substituted carbon chain, or a combination thereof. The optionally substituted carbon chain may incorporate one or more cyclic groups. The one or more cyclic groups may be optionally substituted C 3~12 Cycloalkylene, optionally substituted C 6~10 Arirenes may be selected from arrinenes, optionally substituted 3- to 12-membered heterocyclenes, and optionally substituted 5- to 10-membered heteroarrinenes.

[0083] Cycloalkylenes, arylenes, heterocyclenes, or heteroarylenes are unsubstituted or oxo, OR 7 , NR 7 R 8 COOR 7 OCOR 7 CONR 7 R 8 , or NR 7 COR8 (In the formula, R 7 and R 8 H and C are independent of each other. 1~12 Alkyl, C 2~12 Alkenyl, or C 2~12 It may be replaced by one or more of the following (which are alkynnyls):

[0084] The heteroatom in question or each of them is NR 5 The group may be selected from the group consisting of , O, and S, where R 5 R is defined above. 5 H is replaced by C of any choice. 1~6 Alkyl, optionally substituted C 2~6 Alkenil, or optionally replaced C 2~6 It may also be an alkynyl. More preferably, R 5 H is replaced by C of any choice. 1~3 Alkyl, optionally substituted C 2~3 Alkenil, or optionally replaced C 2~3 It is alkinyl. Therefore, R 5 This can be H or -CH2CHCH2.

[0085] The carbon chain is either unsubstituted or oxo, OR 7 , NR 7 R 8 COOR 7 OCOR 7 CONR 7 R 8 , NR 7 COR 8 , may be substituted with one or more of optionally substituted alkyls, optionally substituted alkenyls, or optionally substituted alkynyls, in the formula, R 7 and R 8 The above definition applies. Optionally substituted alkyls, optionally substituted alkenyls, or optionally substituted alkynyls are either unsubstituted or oxo, OR 7 , NR 7 R 8 COOR 7OCOR 7 CONR 7 R 8 , and NR 7 COR 8 It may be replaced with one or more of the following:

[0086] Preferably, the linker is an optionally substituted carbon chain optionally interrupted by one or more heteroatoms. 3~9 Cycloalkylene, optionally substituted C 6~10 The carbon chain may incorporate one or more of the following: arylene, optionally substituted 3- to 8-membered heterosisylene, and / or optionally substituted 5- to 8-membered heteroarylene groups. The carbon chain may be substituted with one or more oxo groups, one or more OH groups, and / or one or more methyl groups.

[0087] In some embodiments, C is optionally replaced. 6~10 Arylene is phenylene that has been optionally substituted.

[0088] In some embodiments, the optionally substituted 3- to 8-membered heterosisylene is a 5- or 6-membered heterosisylene. The optionally substituted heterosisylene may be an optionally substituted piperidinylene or an optionally substituted piperazinerene.

[0089] The linker may have lengths of atoms between 1 and 50, between 2 and 30, between 2 and 20, between 3 and 10, between 3 and 7, or between 4 or 6. In embodiments in which the linker incorporates a cyclic group, the length of the linker may be understood to be counted as the shortest possible length from end to end.

[0090] Therefore, the linker,

[0091] [ka]

[0092] (wherein n is an integer of at least 1. n may be an integer between 1 and 100, between 2 and 50, between 3 and 25, or between 4 and 10.)

[0093] The crosslinking agent may be N,N-methylenebisacrylamide, N,N'-diallyl tartardiamide, 2,2-bis[4-(2-hydroxy-3-methacryloxypropoxy)phenyl]propane, tricyclodecanedimethanol diacrylate, 1,4-butanediol diacrylate, tetraethylene glycol diacrylate, polyethylene glycol dimethacrylate, N,N-diallylacrylamide, trimethylpropane trimethylacrylate, N,N'-bis(acryloyl)cystamine, N,N'-bis(acryloyl)ethylenediamine, divinylbenzene, or N,N'-bisacryloylpiperazine.

[0094] Multiple monomers may be present in solution at concentrations between 0.0005 and 1,000 mM, between 0.001 and 500 mM, between 0.005 and 100 mM, between 0.01 and 50 mM, between 0.05 and 25 mM, between 0.1 and 10 mM, or between 0.2 and 5 mM. If multiple monomers contain more than one chemical species, the concentration may be understood as the combined concentration of the more than one chemical species.

[0095] The molar ratio of one or more crosslinking agents to the remaining monomers may be between 1:1 and 1:500, between 1:5 and 1:250, between 1:10 and 1:100, between 1:20 and 1:75, between 1:25 and 1:50, or between 1:30 and 1:40. The remaining monomers can be understood as all of the monomers other than those from one or more crosslinking agents.

[0096] The method may involve contacting catalyst particles, an oxidizing agent, and a plurality of monomers in the presence of a template species such that the polymer shell is or contains a molecularly imprinted polymer (MIP).

[0097] The template species may be one or more of the following: ions, metal complexes, organic compounds, carbohydrates, oligosaccharides, polysaccharides, drugs, toxins, insecticides, biomarkers, peptides, proteins, nucleoproteins, mucoproteins, lipoproteins, synthetic proteins, glycoproteins, nucleic acids, biological receptors, receptor fragments, cells, cell fragments, tissue fragments, bacteria, viruses, microorganisms, tissue samples, inorganic crystals, protein crystals, glucosaminoglycans, steroids, immunosuppressants, hormones, heparin, antibiotics, and / or vitamins. In principle, any small molecule can serve as a template. For example, an organic compound may have a molecular weight of at least 20 g / mol, at least 50 g / mol, at least 75 g / mol, at least 100 g / mol, at least 125 g / mol, at least 150 g / mol, or at least 175 g / mol. The organic compound may have a molecular weight of less than 1,000 g / mol, less than 500 g / mol, less than 300 g / mol, less than 250 g / mol, less than 200 g / mol, less than 175 g / mol, or less than 150 g / mol. The organic compound may have a molecular weight between 20 and 1,000 g / mol, between 50 and 500 g / mol, between 75 and 300 g / mol, between 100 and 250 g / mol, or between 125 and 200 g / mol. For example, the organic compound may be 4-nitrophenol, o-nitrophenol, amphetamine, citric acid, hydrocortisone, cocaine, tetrahydrocannabinol (THC), or fentanyl.

[0098] The template species may be bonded to a substrate. The substrate may be a solid substrate. The substrate may contain metals or metalloids or their oxides, glass, or polymers, or metals or metalloids or their oxides, glass, or polymers. The metal or metalloid may be a pure metal, a pure metalloid, an impure metal, an impure metalloid, an alloy, a metal-containing compound, or a metalloid-containing compound. Therefore, the substrate may be iron oxide, or may contain iron oxide. The substrate may be in the form of beads. Therefore, the template species may be bonded to glass beads. It should be recognized that multiple template species may be bonded to multiple solid substrates. Therefore, multiple template species may be bonded to multiple beads.

[0099] Alternatively, the template species may be dissolved in a solution. Therefore, in some embodiments, the template species is not bound to the substrate.

[0100] The template species may be present in the solution at concentrations between 0.01 and 1,000 mM, between 0.05 and 500 mM, between 0.1 and 100 mM, between 0.5 and 50 mM, between 1 and 25 mM, between 2 and 10 mM, or between 4 and 8 mM.

[0101] The molar ratios of multiple monomers to the template species may be between 10:1 and 1:10, between 5:1 and 1:5, between 2:1 and 1:3, between 1.5:1 and 1:2, or between 1:1 and 1:1.5.

[0102] The method may include contacting catalyst particles, an oxidizing agent, and a plurality of monomers in the presence of a chain transfer agent. The chain transfer agent may be optionally substituted mercaptans, alcohols, amines, silanes, halogens, halogenated alkanes, aromatic hydrocarbons, or carbon disulfides. The mercaptan may be optionally substituted C 1~20It may be a mercaptan, and may be 2-mercaptoethanol, dodecyl mercaptan, thioglycolic acid, or 4-methylbenzenethiol. The alcohol is C 1~20 Alcohol, C 2~10 Alcohol, or C 3~8 It may be an alcohol, and may be isopropanol or benzyl alcohol. The amine is C 1~10 or C 3~8 It may be an amine, and may be triethylamine or diisopropylamine. Silane is C 1~10 or C 2~8 It may be a silane, and may be trimethylsilane or triethylsilane. The halogen may be chlorine or bromine. The halide alkane is C 1~10 , C 1~5 , or C 1~2 The halogenated alkane may also be carbon tetrachloride. The aromatic hydrocarbon may be pentaphenylethane.

[0103] The molar ratio of multiple monomers to the chain transfer agent may be between 10,000:1 and 1:1, between 5,000:1 and 2:1, between 2,500:1 and 5:1, or between 1,000:1 and 10:1.

[0104] In some embodiments, it may be recognized that the present invention describes the application of living polymerization for producing soluble or colloidal MIP particles. It may be recognized that any known living free radical polymerization method, such as inducer polymerization, nitroxide-mediated radical polymerization, atom transfer radical polymerization (ATRP), and reversible addition-fraction chain transfer (RAFT) polymerization, may be applied to the method of the first embodiment. These open up new pathways for the synthesis of polymers with controlled and relatively low molecular weights. Controlled / living polymerization methods are based on a delicate equilibrium between resting and active species, effectively reducing the concentration of free radicals in the system and minimizing the degree of termination. Living polymerization can be free from side reactions such as termination and chain transfer, and therefore can produce polymers with a clearly defined molecular weight distribution and structure. The same techniques can be applied to copolymers, and therefore it is possible to produce block copolymers by free radical polymerization with a suitable sequence of monomer additions.

[0105] Typically, the reaction is terminated early, producing polymers with molecular weights of 500–1,000,000 Da. This can be recognized as an inherent characteristic of living polymerization. Other ways of terminating the reaction early may include stopping irradiation in the case of light-induced reactions, or flushing with oxygen in the case of chemical initiation.

[0106] The method may be carried out at temperatures between -25 and 100°C, between 0 and 80°C, between 5 and 60°C, between 10 and 40°C, between 15 and 30°C, or between 20 and 25°C. Advantageously, the method may be carried out at room temperature.

[0107] The method may include contacting catalyst particles, an oxidizing agent, and multiple monomers for a period of time between 1 minute and 72 hours, between 15 minutes and 48 hours, between 30 minutes and 24 hours, between 45 minutes and 12 hours, between 1 to 6 hours, between 2 to 4 hours, or between 2.5 to 3.5 hours. In some embodiments, it may be recognized that the method may include contacting catalyst particle precursors, an oxidizing agent, and multiple monomers for a period of time between 1 minute and 72 hours, between 15 minutes and 48 hours, between 30 minutes and 24 hours, between 45 minutes and 12 hours, between 1 to 6 hours, between 2 to 4 hours, or between 2.5 to 3.5 hours.

[0108] The method may be carried out in a batch process or a continuous process. Therefore, the method may be carried out in a microfluidic reactor or a flow reactor.

[0109] The method may include contacting catalyst particles, an oxidizing agent, and multiple monomers under UV light. The catalyst particles, oxidizing agent, and multiple monomers may be contacted under UV light for at least 1 second, at least 15 seconds, at least 30 seconds, at least 45 seconds, or at least 1 minute. The catalyst particles, oxidizing agent, and multiple monomers may be contacted under UV light for between 15 seconds and 30 minutes, between 30 seconds and 10 minutes, between 45 seconds and 5 minutes, or between 1 and 3 minutes. Contacting the catalyst particles, oxidizing agent, and multiple monomers under UV light may be recognized as initiating a reaction.

[0110] The method may include isolating the composite particles.

[0111] The composite particles may be isolated from one or more unreacted low-molar components and / or one or more reaction byproducts. The composite particles may be isolated by affinity, magnetic separation, dialysis, extraction, filtration, or centrifugation. Filtration may be ultrafiltration or nanofiltration. The composite particles may be isolated from the solid phase. Therefore, the composite particles may be isolated by solid-phase extraction.

[0112] The method may include conjugating the composite particles with further species. This may be done after the composite particles have been isolated. The further species may be as defined above. The method may also include conjugating the polymer shell with further species. Advantageously, the resulting nanocomposites may be used in diagnostics and pharmaceuticals.

[0113] The composite particles produced using the method of the first embodiment are considered to be novel and inventive in themselves.

[0114] Accordingly, in a second embodiment, composite particles are provided that can be obtained or obtained by the method of the first embodiment.

[0115] The composite particles produced by the method of the first embodiment may be used in a variety of applications. These applications are considered novel and inventive in themselves.

[0116] Accordingly, a third embodiment provides an use of composite particles in detecting a target molecule, wherein the composite particles include a polymer shell surrounding catalyst particles.

[0117] Preferably, the use is the use of composite particles in detecting the concentration of a target molecule in a solution.

[0118] The composite particles may be used to detect target molecules using surface plasmon resonance (SPR), localized surface plasmon resonance (LSPR), optical density, electrochemical methods such as voltammetry, amperometry, or coulometry, or impedance, resistance measurement, lateral flow detectors, or in homogeneous system assays.

[0119] A method for detecting a target molecule in a solution according to a fourth aspect, The process involves bringing a solution into contact with composite particles, wherein the composite particles include a polymer shell surrounding the catalyst particles. The optical properties of catalyst particles are detected, thereby enabling the detection of target molecules in the solution. A method including this is provided.

[0120] Preferably, the composite particles are manufactured using the method of the first embodiment or are composite particles of the second embodiment.

[0121] Preferably, the composite particles are as described above. Preferably, the polymer shell comprises a molecularly imprinted polymer (MIP). Preferably, the MIP has a target molecule imprinted on it.

[0122] The optical properties may be surface plasmon resonance (SPR) absorbance, localized surface plasmon resonance (LSPR) absorbance, or fluorescence. Preferably, the method includes determining values ​​related to the optical properties.

[0123] Preferably, the method includes calculating the concentration of the target molecule in the solution. The concentration of the target molecule in the solution may be calculated by comparing the optical properties of the composite particles in the solution with a calibration curve that includes optical property values ​​for various known concentrations of the target molecule in the solution, thereby determining the concentration of the target molecule in the solution.

[0124] The target molecule may be one or more of the following: ions, metal complexes, organic compounds, drugs, toxins, insecticides, biomarkers, peptides, proteins, receptors, receptor fragments, cells, cell fragments, tissue fragments, bacteria, viruses, inorganic crystals, and / or protein crystals. The organic compound may be the same as those defined above in relation to the template species. The organic compound may be 4-nitrophenol, o-nitrophenol, pentaerythritol tetranitrate, amphetamine, citrate, hydrocortisone, cocaine, or fentanyl.

[0125] A method for detecting a target molecule in a solution according to a fifth aspect, The arrangement involves placing an electrode with immobilized composite particles and a further electrode in a solution, wherein the composite particles include a polymer shell around catalyst particles. Applying a voltage to the electrodes, By measuring electric current, the target molecule in the solution is detected. A method including this is provided.

[0126] Preferably, the composite particles are manufactured using the method of the first embodiment or are composite particles of the second embodiment.

[0127] Preferably, the composite particles are as described above. Preferably, the polymer shell comprises a molecularly imprinted polymer (MIP). Preferably, the MIP has a target molecule imprinted on it.

[0128] The target molecule may be one or more of the following: ions, metal complexes, organic compounds, drugs, toxins, insecticides, biomarkers, peptides, proteins, receptors, receptor fragments, cells, cell fragments, tissue fragments, bacteria, viruses, inorganic crystals, and / or protein crystals. The organic compound may be the same as those defined above in relation to the template species.

[0129] The method may include immobilizing composite particles on an electrode. Immobilizing composite particles on an electrode may include covalently immobilizing the composite particles on the electrode. Immobilization can be carried out using techniques known in the art, such as the EDC / NHS (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide / N-hydroxysuccinimide) activation mechanism, which uses a mercaptan compound or a silane compound containing an amino or carboxyl group.

[0130] Preferably, applying voltage to two electrodes and measuring current involves applying differential pulsed voltammetry (DPV). Thus, the method may include applying a first pulse having a ground potential to the electrodes, and applying further pulses to the electrodes such that the potential increases with each pulse. Preferably, the potential increases by the same amount with each pulse, i.e., the potential increases in a linear ramp. Preferably, the current is measured before pulse application and at the end of the pulse, and the difference between them is recorded.

[0131] Preferably, the method includes calculating the concentration of the target molecule in the solution. The concentration of the target molecule in the solution may be calculated by comparing a calibration curve of the current response at a given voltage with the current response at a given voltage when the electrode is placed in solutions containing various known concentrations of the target molecule, thereby determining the concentration of the target molecule in the solution.

[0132] The target molecule may be as defined above.

[0133] In a sixth embodiment, a use of composite particles in a lateral flow detector is provided, wherein the composite particles include a polymer shell surrounding catalyst particles.

[0134] In a seventh embodiment, a lateral flow detector is provided that includes composite particles, wherein the composite particles include a polymer shell surrounding catalyst particles.

[0135] It should be recognized that lateral flow detectors may also be known as lateral flow tests (LFTs), lateral flow devices (LFDs), or lateral flow immunochromatography assays.

[0136] The lateral flow detector may include a sandwich assay or a competitive assay.

[0137] Preferably, the composite particles are manufactured using the method of the first embodiment or are composite particles of the second embodiment.

[0138] Preferably, the composite particles are as described above. The polymer shell may contain a molecularly imprinted polymer (MIP). Preferably, the MIP has a target molecule imprinted on it.

[0139] The target molecule may be one or more of the following: ions, metal complexes, organic compounds, drugs, toxins, insecticides, biomarkers, peptides, proteins, receptors, receptor fragments, cells, cell fragments, tissue fragments, bacteria, viruses, inorganic crystals, and / or protein crystals. The organic compound may be the same as those defined above in relation to the template species. The target molecule may be a protein, biomarker, drug, toxin, or hormone. The drug may be an abuse drug.

[0140] Alternatively, the polymer shell may have further species attached thereto. These further species may be functional groups (e.g., PEG) that have fluorescent properties and / or can reduce nonspecific binding.

[0141] The lateral flow detector may include a sample receiving section configured to receive the sample to be tested, a test section containing one or more test lines, and optionally a control line, and the lateral flow detector may be configured to allow the sample to flow from the sample receiving section to the test section. The sample may flow by capillary flow.

[0142] The sample may be understood as a liquid sample. The sample may contain body fluids, or may be body fluids.

[0143] The sample receiving section may include a sample pad. The sample pad may be configured to act as a filter to help the sample flow. The sample pad may be treated to adjust the properties of the sample, such as pH or viscosity.

[0144] The lateral flow detector may include a conjugate section positioned between the sample receiving section and the test section. The conjugate section may include a conjugate release pad. The conjugate section preferably includes one or more conjugates. Preferably, the conjugate release pad is configured to release one or more conjugates so that one or more conjugates flow into the test section with the sample. The one or more conjugates may include one or more conjugates configured to bind to one or more target molecules in the sample. Alternatively or additionally, the one or more conjugates may include one or more conjugates configured to bind to a control line. The one or more conjugates may include one or more antibodies, one or more MIPs, one or more aptamers and / or composite particles. Each of the one or more conjugates may be configured to bind to a target molecule and / or antibody.

[0145] The test section may include a membrane. The membrane may be a nitrocellulose membrane.

[0146] In one embodiment, one or more test lines are configured to capture one or more target molecules. This may be recognized as a sandwich assay. Preferably, one or more test lines include immobilized molecules configured to conjugate with one or more target molecules, and the one or more immobilized molecules define the line. Preferably, the one or more immobilized molecules define the line across the test section. Preferably, the line is substantially perpendicular to the direction of sample flow. The one or more immobilized molecules may be antibodies and / or composite particles, or may include antibodies and / or composite particles.

[0147] In an alternative embodiment, one or more test lines contain immobilized molecules, the immobilized molecules being one or more target molecules or analogs thereof. This may be recognized as a competitive assay. Preferably, one or more immobilized molecules define a line across the test section. Preferably, the line is substantially perpendicular to the direction of sample flow.

[0148] The control line may be configured to capture one or more conjugates configured to bind to it. Preferably, the control line includes an immobilized molecule configured to conjugate with one or more conjugates configured to bind to the control line. Preferably, one or more immobilized molecules define a line across the test section. Preferably, the line is substantially perpendicular to the direction of sample flow. One or more immobilized molecules may be antibodies and / or composite particles, or may comprise antibodies and / or composite particles.

[0149] The lateral flow detector may include a wicking pad. The wicking pad may be located adjacent to the test section. The test section may be located between the sample receiving section and the wicking pad. The test section may be located between the conjugate section and the wicking pad. Thus, the lateral flow detector may outline the strip, the sample receiving section may be located substantially adjacent to the first end of the strip, and the wicking pad may be located substantially adjacent to the second end of the strip, the second end being on the opposite side of the first end. The lateral flow detector may be configured to allow the sample to flow substantially from the first end to the second end.

[0150] Preferably, the wicking pad is configured to absorb fluid. Preferably, the wicking pad contains a porous material.

[0151] A method for detecting a target molecule in a sample is provided according to the eighth aspect, comprising bringing the sample into contact with a lateral flow detector according to the seventh aspect, and observing whether or not a test line appears on the lateral flow detector, thereby detecting the target molecule in the sample.

[0152] Advantageously, the composite particles of the present invention can be used in diagnostic tests.

[0153] In the ninth embodiment, a method for detecting a target molecule in a sample, The process involves contacting a spectrophotometric reagent, composite particles, an oxidizing or reducing agent, and a sample, wherein the composite particles include a polymer shell surrounding the catalyst particles. The absorbance of the resulting composition is measured to detect the target molecule in the sample. A method including this is provided.

[0154] Preferably, the composite particles are manufactured using the method of the first embodiment or are composite particles of the second embodiment.

[0155] Preferably, the polymer shell comprises a molecularly imprinted polymer (MIP). Preferably, the MIP has a target molecule imprinted on it.

[0156] The target molecule may be one or more of the following: ions, metal complexes, organic compounds, drugs, toxins, insecticides, biomarkers, peptides, proteins, receptors, receptor fragments, cells, cell fragments, tissue fragments, bacteria, viruses, inorganic crystals, and / or protein crystals. The organic compound may be the same as those defined above in relation to the template species.

[0157] Preferably, the catalyst particles contain or consist of a material capable of catalyzing a bleaching or color-enhancing reaction between a spectrophotometric reagent and an oxidation or reducing agent. Therefore, the catalyst particles may contain or consist of a magnetic material such as gold, silver, or iron oxide.

[0158] It may be recognized that the above method may also be described as a homogeneous assay.

[0159] The spectrophotometric reagent may be a redox-sensitive dye. In some embodiments, the spectrophotometric reagent may be bromopyrogallol red, 3,3',5,5'-tetramethylbenzidine (TMB), or 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS).

[0160] The oxidizing agent may be a peroxide, persulfate, or ozone. The peroxide may be hydrogen peroxide. The persulfate may be ammonium persulfate.

[0161] The sample may contain a buffer solution. The buffer solution may be phosphoric acid, acetic acid, or borate buffer.

[0162] The method may include contacting a spectrophotometric reagent, an oxidizing or reducing agent, composite particles, and a sample for a predetermined time prior to measuring the absorbance of the resulting composition. The predetermined time may be determined by those skilled in the art. For example, the predetermined time may be between 15 seconds and 24 hours, between 30 seconds and 12 hours, between 1 minute and 6 hours, between 5 minutes and 2 hours, between 10 and 90 minutes, between 15 and 60 minutes, between 20 and 45 minutes, between 25 and 35 minutes, or between 28 and 32 minutes.

[0163] Preferably, the method includes calculating the concentration of the target molecule in the sample. The concentration of the target molecule in the sample may be calculated by comparing the measured absorbance of the resulting composition with a calibration curve of absorbance for a composition containing a reference sample having a known concentration of the target molecule, thereby determining the concentration of the target molecule in the sample.

[0164] In a tenth embodiment, a use of composite particles in a catalytic reaction is provided, wherein the composite particles include a polymer shell surrounding the catalyst particles.

[0165] Preferably, the composite particles are manufactured using the method of the first embodiment or are composite particles of the second embodiment.

[0166] Their use may involve catalyzing oxidation or hydrolysis reactions. They can also be used to catalyze reactions in diagnostic assays, such as the conversion of non-fluorescent molecules to fluorescent molecules that can be detected using a fluorescence reader.

[0167] Advantageously, polymer coatings can help enhance reaction specificity by restricting access to catalytic sites for specific compounds of appropriate size and charge. Alternatively, polymer coatings can aid in pre-concentration of the substrate, increasing the yield of the catalytic reaction.

[0168] In an eleventh embodiment, a pharmaceutical composition is provided comprising composite particles and a pharmaceutically acceptable carrier, wherein the composite particles comprise a polymer shell surrounding catalyst particles.

[0169] In an eleventh embodiment, a composite particle or a pharmaceutical composition of the eleventh embodiment is provided for use in a method of treatment or diagnosis, wherein the composite particle comprises a polymer shell surrounding a catalyst particle.

[0170] In a twelfth embodiment, a composite particle or a pharmaceutical composition of an eleventh embodiment is provided for use in imaging methods, wherein the composite particle comprises a polymer shell surrounding a catalyst particle.

[0171] Preferably, the composite particles are manufactured using the method of the first embodiment or are composite particles of the second embodiment.

[0172] The composite particles may contain an antibody or aptamer conjugated to a polymer shell. Alternatively or additionally, the polymer shell may contain a MIP. The antibody, aptamer, or MIP may be configured to deliver the composite particles to a specific target. Preferably, the specific target is present in the body of the patient to whom the composite particles are administered. The target may be a membrane protein or a cell.

[0173] Advantageously, composite particles with a high contrast ratio can be specifically delivered to targets in the patient's body due to antibodies, aptamers, or MIPs. For example, composite particles with an iron oxide core can be understood to have a high contrast ratio.

[0174] In a thirteenth embodiment, a composite particle or a pharmaceutical composition of an eleventh embodiment is provided for use in drug delivery, wherein the composite particle comprises a polymer shell surrounding a catalyst particle.

[0175] Preferably, the composite particles are manufactured using the method of the first embodiment or are composite particles of the second embodiment.

[0176] The composite particles may contain an antibody or aptamer conjugated to a polymer shell. Alternatively or additionally, the polymer shell may contain a MIP. The antibody, aptamer, or MIP may be configured to deliver the composite particles to a specific target. Preferably, the specific target is present in the body of the patient to whom the composite particles are administered. The target may be a membrane protein or a cell.

[0177] The composite particles may contain drug molecules adsorbed to them.

[0178] The catalyst particles may also contain magnetic materials such as iron oxide.

[0179] Advantageously, after the composite particles are delivered to the patient, they can be heated in a magnetic field, causing them to release drug molecules adsorbed adjacent to the target.

[0180] According to the 14th aspect, Substrate and Multiple nanoparticles arranged on a substrate, Multiple composite particles arranged on a substrate, wherein the composite particles include a polymer shell surrounding the catalyst particles. A detector including the following is provided.

[0181] Preferably, the composite particles are manufactured using the method of the first embodiment or are composite particles of the second embodiment. Preferably, the composite particles are as described above. Preferably, the composite particles are arranged in layers covering the surface of the substrate. Preferably, the composite particles are arranged in layers covering the surface of the substrate and covering the nanoparticles.

[0182] Preferably, the polymer shell is as defined above. Preferably, the polymer shell comprises a molecularly imprinted polymer (MIP). Preferably, the MIP has a target molecule imprinted on it.

[0183] Preferably, the substrate contains or consists of a light-transmitting material. Preferably, the substrate contains or consists of a visible light-transmitting material. For example, the substrate may contain glass or a polymer, or may consist of glass or a polymer.

[0184] The multiple nanoparticles are preferably multiple metal nanoparticles, more preferably multiple silver and / or gold nanoparticles.

[0185] Preferably, the nanoparticles are randomly distributed. Preferably, the nanochip nanoparticles are arranged to enable surface plasmon resonance (SPR) or localized surface plasmon resonance (LSPR).

[0186] A method for detecting a target molecule in a fluid, according to the 15th aspect, To provide a detector of the 14th embodiment, The detector is brought into contact with the fluid, The optical properties of the detector are detected, thereby enabling the detection of target molecules in the fluid. A method including this is provided.

[0187] The fluid may be a liquid or a gas, or may contain a liquid or a gas. The liquid may be a solution or a suspension, or may contain a solution or a suspension. In some embodiments, the fluid is a gas. The gas may contain air.

[0188] The optical properties may be surface plasmon resonance (SPR) absorption, localized surface plasmon resonance (LSPR) absorption, or fluorescence. Preferably, the optical properties are localized surface plasmon resonance (LSPR) absorption. Therefore, the optical properties of the substrate may be determined using a photometer.

[0189] Preferably, the method includes determining values ​​relating to the optical properties of the detector. Determining values ​​relating to the optical properties may include removing background light signals. Removing background light signals may include removing light signals generated by a reference detector, the reference detector being in contact with a reference fluid, and the reference does not contain target molecules. Apart from the removal of target molecules, the reference fluid may be the same as a fluid containing target molecules.

[0190] Preferably, the method includes calculating the amount or concentration of target molecules in the fluid. The amount of target molecules in the fluid may be calculated by comparing the optical properties of a substrate in contact with the fluid with a calibration curve that includes optical property values ​​for various known amounts or concentrations of target molecules in the fluid.

[0191] The target molecule may be as described in the fourth embodiment.

[0192] In the 16th embodiment, An optical fiber extending between a first end and a second end, including a detection unit, wherein the detection unit is configured to enable the generation of an evanescent field, A composite particle arranged on the detection section of an optical fiber, wherein the composite particle includes a polymer shell surrounding the catalyst particle. A detector including the following is provided.

[0193] Preferably, the composite particles are manufactured using the method of the first embodiment or are composite particles of the second embodiment.

[0194] Preferably, the composite particles are as described above. Preferably, the polymer shell comprises a molecularly imprinted polymer (MIP). Preferably, the MIP has a target molecule imprinted on it.

[0195] The optical fiber may include a core extending between the first end and the second end. The core may include a synthetic resin, glass, or a combination thereof, or may consist of a synthetic resin, glass, or a combination thereof. The synthetic resin and / or glass may be doped.

[0196] The optical fiber may further include a cladding layer arranged circumferentially around the core. The cladding layer may extend between a first end and a second end. The cladding layer may include a synthetic resin, glass, or a combination thereof, or may consist of a synthetic resin, glass, or a combination thereof.

[0197] Preferably, the refractive index of the core is greater than the refractive index of the cladding layer.

[0198] The optical fiber may further include a buffer layer arranged circumferentially around the cladding layer.

[0199] It can be understood that the optical signal can propagate through the optical fiber within the core. The optical fiber detector can be configured such that an evanescent field is generated on the surface of the detector when the optical signal propagates through the optical fiber.

[0200] In some embodiments, the detection unit includes a tapered portion of the optical fiber. In particular, the tapered portion is understood to have a smaller diameter compared to the adjacent portion of the optical fiber.

[0201] Therefore, the tapered portion of the optical fiber may include a cladding layer having a reduced thickness compared to the adjacent portion. Alternatively, the detection portion of the optical fiber may not include a cladding layer.

[0202] The tapered portion may be positioned adjacent to the first end of the optical fiber. The adjacent portion may extend between the tapered portion and the second end of the optical fiber. Alternatively, the tapered portion may be positioned adjacent to the second end of the optical fiber. The adjacent portion may extend between the first end of the optical fiber and the tapered portion.

[0203] In some embodiments, the optical fiber includes a tapered region located between the first and second ends and spaced apart from the first and second ends. The optical fiber may include a first adjacent portion extending between the first end of the optical fiber and the tapered portion. The optical fiber may include a second adjacent portion extending between the tapered portion and the second end of the optical fiber. The first and second adjacent portions may have substantially the same diameter as each other.

[0204] Preferably, the detector further includes a light source capable of emitting an optical signal. The light source may include an LED (light-emitting diode) and / or a laser, or may be an LED (light-emitting diode) and / or a laser. The light source may be configured to provide an optical signal to the optical fiber. The light source may be configured to input an optical signal to the first end of the optical fiber. The light source may be connected to the first end of the optical fiber. Thus, the first end of the optical fiber may be called the input end of the optical fiber.

[0205] Preferably, the detector further includes a detector capable of detecting an optical signal. The detector may be an optical spectrometer or a photodiode. The detector may be configured to receive and detect an optical signal from an optical fiber, preferably from its second end. The detector may be connected to the second end of the optical fiber. The second end of the optical fiber may be called the output end of the optical fiber.

[0206] Therefore, the light source, detector, and optical fiber may be configured such that an optical signal is generated by the light source, propagated along the optical fiber, and detectable by the detector.

[0207] The detector may further include a processor configured to process the data generated by the detector.

[0208] A method for detecting a target molecule in a fluid, according to the 17th aspect, To provide a detector of the 16th embodiment, Bringing a fluid and composite particles into contact, An optical signal is input to the first end of the optical fiber so that the composite particles are at least partially positioned in the evanescent field, thereby generating an evanescent field adjacent to the detection section of the optical fiber. The optical signal or value output from the second end of the optical fiber is detected, and the output optical signal is compared with a reference optical signal or value to detect target molecules in the fluid. A method including this is provided.

[0209] The fluid may be defined as described in the 15th embodiment.

[0210] Preferably, the composite particles are manufactured using the method of the first embodiment or are composite particles of the second embodiment.

[0211] Preferably, the composite particles are as described above. Preferably, the polymer shell comprises a molecularly imprinted polymer (MIP). Preferably, the MIP has a target molecule imprinted on it.

[0212] Detecting the output optical value may include detecting the intensity of the optical signal at a given wavelength.

[0213] The reference signal may be an optical signal detected from the second end of the optical fiber in the absence of the target molecule. The reference value may be the intensity of the optical signal at a given wavelength detected from the second end of the optical fiber in the absence of the target molecule.

[0214] The method may include calculating the amount or concentration of target molecules in a fluid. The amount or concentration of target molecules in the fluid may be calculated by comparing the output optical signal or value with a further reference signal or value, the further reference signal or value being an optical signal detected from a second end of an optical fiber in the presence of various known amounts or concentrations of target molecules. The further reference value may be the intensity of the optical signal at a given wavelength detected from the second end of an optical fiber in the presence of various known amounts or concentrations of target molecules. Thus, the method may include calculating the amount or concentration of target molecules in the fluid by comparing a calibration curve of the reference value for various known amounts of target molecules in the fluid with the output optical value.

[0215] The target molecule may be as described in the fourth embodiment.

[0216] All features described herein (including any appended claims, abstracts, and drawings) and / or all steps of any method or process disclosed herein may be combined with any of the embodiments described herein in any combination except any combination in which at least some of such features and / or steps are mutually exclusive.

[0217] To better understand the present invention and to illustrate how its embodiments can be carried out, the accompanying drawings are referenced below as examples. [Brief explanation of the drawing]

[0218] [Figure 1]Figure showing transmission electron microscopy (TEM) images and Fourier transform infrared (FTIR) spectra of gold nanoparticles coated with polyacrylic acid. [Figure 2] TEM images and FTIR spectra of gold nanoparticles coated with molecularly imprinted polymer (MIP). [Figure 3] (A) Figure showing the localized surface plasmon resonance (LSPR) signal monitored over time as the concentration of the analyte present in a solution containing gold nanoparticles coated with MIP is increased, and (B) calibration plot showing absorbance versus concentration. [Figure 4] (A) Differential pulse voltammetry (DPV) responses of a gold-based detector modified with nano-MIP to hydrocortisone standard solutions at (a) 0, (b) 150, (c) 200, (d) 250, (e) 300, and (f) 350 nM in 5 mM PBS, and (B) calibration plots of the responses of a detector containing (a) gold nanoparticles modified with nano-MIP to hydrocortisone solution; and (b) a detector containing non-functionalized gold nanoparticles to hydrocortisone solution. [Figure 5] Figure showing a lateral flow device based on a pad adsorbent having a control line of hydrocortisone-bovine serum albumin (BSA). [Figure 6] Calibration plot showing bromopyrogallol red (BPR) absorbance versus amphetamine concentration. [Figure 7] (A) Plots of the BRP assay response (BRP reaction rate) using amphetamine concentrations in the range of 0 - 133 nM, and (B) plots of the BRP assay response using amphetamine concentrations in the range of 0 - 40 nM. [Figure 8] (A) Plots of the BRP assay response to amphetamine using Au / MIP and Au / NIP nanozymes in the amphetamine concentration range of 0 - 133 nM and (B) in the amphetamine concentration range of 0 - 40 nM. [Figure 9]Figure showing the plot of the BRP assay response to 0.3 - 40 nM of amphetamine, paracetamol, morphine, and cocaine. [Figure 10] Figure showing the plot of the BRP assay response to amphetamine, urine, and plasma in buffer (5 mM PBS, pH 7.0). [Figure 11] Figure showing the kinetic dependence of the absorbance of LSPR Au nanoplates coated with gold o - nitrophenol imprinted MIP composite nanoparticles upon exposure to air (0 g of o - nitrophenol) and o - nitrophenol vapor from a dry cotton swab containing approximately 1 femtogram of o - nitrophenol. [Figure 12] Figure showing an optical fiber type detector having a detector probe containing composite nanoparticles. [Figure 13] Figure showing the response plot of the optical fiber type detector upon contact with the target substance (cocaine).

Mode for Carrying Out the Invention

[0219] [Examples] Example 1. Preparation of Gold Nanoparticles Coated with Polyacrylic Acid 30 μL of a precursor solution (6.6 mg mL -1 of hydrogen tetrachloroaurate(III) trihydrate) was added to 10 mL of 10 mM Na - phosphate buffer pH 7.5, and mixed with 0.22 μL of acrylic acid, 500 μL of cyclohexanone, and 300 μL of a 50 wt% hydrogen peroxide stock solution. The mixture was incubated for 3 hours and the nanoparticles were purified by dialysis. The nanoparticles were characterized by TEM and IR spectroscopy (Figure 1). It is noted that in the TEM image, a dark - colored core of the inorganic material and a bright - colored shell of the polymer coating can be seen.

[0220] Example 2. Preparation of Gold Nanoparticles Coated with Molecular Imprinted Polymer (MIP) Dissolve 9 mg of template amphetamine, 3.8 mg of N-isopropylacrylamide, 3.3 mg of N-tert-butylacrylamide, 0.58 mg of N-3-aminopropylmethacrylamide hydrochloride, 0.22 μL of acrylic acid, and 0.2 mg of N,N-methylenebisacrylamide in 10 mL of 10 mM sodium phosphate buffer, pH 7.5, and add 30 μL of precursor solution (6.6 mg mL). -1 The tetrachloroano(III) hydrogen trihydrate was mixed with 500 μL of cyclohexanone and 300 μL of a commercially available 50% hydrogen peroxide stock solution. The mixture was incubated for 3 hours, and the nanoparticles were purified by dialysis. The nanoparticles were characterized by EM and IR spectroscopy (Figure 2). Again, in the TEM images, a dark core of inorganic material and a light shell of polymer coating can be seen.

[0221] Example 3. Detection of citric acid as an explosive-like substance. The method described in Example 2 was used, but amphetamine was replaced with citrate to prepare gold nanoparticles coated with MIP shells imprinted with citrate. It should be noted that citrate is an analogue for pentaerythritol tetranitrate.

[0222] Nanoparticles were mixed in an optical cell while increasing the concentration of the target molecule (citric acid). The MIP imprinted with respect to citric acid showed a linear decrease in localized surface plasmon resonance (LSPR) absorbance (Panel a, Figure 3), and these data were used to create a calibration plot (Panel b, Figure 3), allowing the user to calculate the concentration of an unknown solution.

[0223] Therefore, the inventors have demonstrated that the nanoparticles of the present invention can be used to determine the concentration of a target molecule in a solution. Example 4: Use of gold-MIP composite nanoparticles for hydrocortisone detection We synthesized composite nanoparticles using gold cores and MIP shells imprinted with hydrocortisone.

[0224] Dissolve 3.8 mg of N-isopropylacrylamide, 3.3 mg of N-tert-butylacrylamide, 0.58 mg of N-3-aminopropylmethacrylamide hydrochloride, 0.22 μL of acrylic acid, and 0.2 mg of N,N-methylenebisacrylamide in 5 mL of water, and then add 30 μL of precursor solution (6.6 mg mL) -1 Hydrogen tetrachloroferrous(III) trihydrate was added, followed by 300 μL of a commercially available 50% hydrogen peroxide stock solution and 500 μL of an acetone / acetylacetone mixture in a 2:1 volume ratio. For control, nanoparticles with a gold core and a non-imprinted polymer (NIP) shell were prepared using a similar but template-free method.

[0225] Two detectors were fabricated by covalently immobilizing nanoparticles onto graphite electrodes: one containing gold-MIP nanoparticles and the other containing gold-NIP nanoparticles. As shown in Figure 4A, the DPV response of the detector containing gold-MIP nanoparticles was measured at 0.59 V (relative to Ag / AgCl).

[0226] As shown in Figure 4B, the detector containing gold-MIP nanoparticles (represented by line (a)) is significantly more sensitive to changes in hydrocortisone concentration than the detector containing gold-NIP nanoparticles (represented by line (b)).

[0227] Example 5: Application of composite nanoparticles in lateral flow detectors For this study, gold coated with a non-imprinted polymer and gold coated with a hydrocortisone-imprinted polymer were tested in a lateral flow format using strips with a hydrocortisone-bovine serum albumin (BSA) control line. The upper strip shows the elution of gold-NIP without binding at the control line, while gold-MIP produced a red control line (Figure 5, lower portion).

[0228] Example 6. Homogeneous assay based on composite nanoparticles The assay was prepared by preparing a mixture of bromopyrogallol red (BPR), the gold-MIP nanoparticles from Example 2 in phosphate-buffered saline (PBS), and hydrogen peroxide in wells. 200 μL of PBS containing gold-MIP nanoparticles, 100 μL of H2O2, and 200 μL of bromopyrogallol red solution were used. The sample to be analyzed, in this case a solution of amphetamine with a concentration range of 9–1000 nM, was prepared in 10 mM PBS. The sample was added to a separate well and incubated for 30 minutes. The absorbance was then measured, and the results are shown in Figure 6.

[0229] The dispersion of absorbance is due to the fact that MIP-coated gold nanoparticles act as artificial enzyme nanozymes in the colorimetric oxidation of BPR dyes by peroxides. The mechanism inherent in these nanozymes is rooted in a Fenton-like reaction and encompasses two key components. First, the gold core of the nanozyme catalyzes the decomposition of hydrogen peroxide (H2O2) to hydroxyl radicals (HO·) on their surface, and this decomposition can be detected via a colorimetric reaction. Second, the analyte bound to the MIP induces structural changes in the polymer, mimicking the "inducible fit" action characteristic of enzymes and natural receptors. Specifically, this involves swelling of the nanozyme shell, exposing sections of the gold core available for catalytic reaction. As a result, when a target such as amphetamine binds to the MIP, the latter undergoes conformational changes, influencing the diffusion of the substrate to the catalytic gold surface, thereby increasing the rate of BPR oxidation. Therefore, the absorbance of BPR is directly related to the presence of a target (e.g., amphetamine) in the solution.

[0230] Example 7. Homogeneous assay based on composite nanoparticles Iron oxide magnetic nanoparticles were prepared in this manner. 50 mg of FeCl36H2O and 50 mg of FeSO4 were dissolved in H2O, then treated with NH4OH (commercial 30% solution), and the resulting particles were collected using a magnet and dissolved in deionized H2O.

[0231] Next, Fe2O3-MIP composite nanoparticles were prepared as follows. In 5 mL of water, 3.8 mg of N-isopropylacrylamide, 3.3 mg of N-tert-butylacrylamide, 0.58 mg of N-3-aminopropylmethacrylamide hydrochloride, 0.22 μL of acrylic acid, and 0.2 mg of N,N-methylenebisacrylamide were dissolved, followed by 300 μL of a commercially available 50% hydrogen peroxide stock solution and 50 μL of the iron oxide magnetic nanoparticles prepared as described above.

[0232] Next, an assay was prepared by preparing a mixture of bromopyrogallol red (BPR) and Fe2O3-MIP nanoparticles as follows. 200 uL of Fe2O3-MIP nanoparticles, 100 μL of H2O2, and 200 μL of bromopyrogallol red solution were used. The sample was added to a separate well and incubated for 30 minutes. Then, the absorbance was measured (results not shown).

[0233] Example 8 - Investigation of the Specificity of Binding Sites on Composite Nanoparticles (Nanozymes) for Use in Homogeneous Assays Composite nanoparticles (Au / MIP nanozymes) with gold cores and amphetamine-imprinted MIP shells were synthesized using the following protocol. The following monomers—N-3-aminopropyl methacrylamide (3 mg, 17 μmol), N-isopropylacrylamide (19.5 mg, 172 μmol), N-tert-butylacrylamide solution (400 μL, 236 μM in ethanol), acrylic acid (50 μL, 313 μM in water), and N,N'-methylenebisacrylamide (400 μL, 38 μM in water)—were dissolved together in PBS (1 mL, 100 mM, pH 7.5). The monomer / PBS solution was mixed with 150 μL of gold nanoparticle stock solution (20 nm in diameter, 0.06 μM, in 5 mM PBS, pH 7.5). The monomer solution was then sonicated under nitrogen for 20 minutes. 30 g of template glass beads modified with amphetamine were added to the monomer solution. Polymerization was initiated by adding hydrogen peroxide (50% w / v, 600 μL, 14.7 mM). The resulting mixture was shaken for 2 hours during polymerization. A control polymer was prepared using the same procedure by replacing the amphetamine-modified glass beads with unmodified glass beads (silanization only, without amphetamine). After polymerization, the solution was discarded, and the solid phase was transferred to a solid-phase extraction (SPE) cartridge and washed with water at 4°C (40 mL x 2 times). The resulting Au / MIP nanozyme was then eluted from the solid phase by washing first with hot water (60°C, 30 mL x 2 times) and then with hot ethanol (60°C, 30 mL x 2 times). Finally, the Au / MIP nanozyme was dialyzed using a 10 kDa cutoff snakeskin dialysis membrane (72 hours), with the water changed every 4 hours.

[0234] The catalytic activity of the Au / MIP nanozyme may be tuned by its binding interaction with a specific target molecule (in this case, amphetamine). As described in Example 6, the analyte bound to the MIP induces a structural change in the polymer, thereby exposing a section of the gold core available for catalytic reaction.

[0235] To illustrate the induced adaptation phenomenon observed with respect to the constructed nanozymes, their hydrodynamic sizes were measured using dynamic light scattering (DLS) in the presence of various targets. Au / MIP nanozymes in solution exhibited a size of 124.5 ± 1.8 nm (PDI, 0.112). Upon exposure to amphetamine, the Au / MIP nanozymes swelled, increasing in size by approximately 53%. Conversely, in response to the same concentration of paracetamol, the Au / MIP nanozymes showed minimal change in size, increasing by only about 6%. This result highlights that the specific behavior of the nanozymes occurs exclusively in the presence of the target analyte. Control experiments using Au / NIP showed no significant change (<0.8%).

[0236] Example 9 - Investigation of the effect of gold core diameter on composite nanoparticles (nanozymes) for use in homogeneous assays. As described in Example 8, composite nanoparticles having a gold core and an imprinted MIP shell using amphetamine were prepared. Au nanoparticles with various diameters (5, 20, 50, and 100 nm) were added to the polymer mixture to thus produce composite nanoparticles (nanozymes) with gold cores of various diameters. Non-imprinted polymers with gold nanoparticles (AuNPs / NIP) were used as a control.

[0237] The catalytic properties of synthesized Au / MIP nanozymes and AuNP / NIP were evaluated by a colorimetric assay using BRP. The BRP-based colorimetric assay was performed using the following assay conditions: each well contained 10 μL of Au / MIP nanozyme (0.4 mg mL-1), 135 μL of 10 nM BPR in PBS (5 mM, pH 7.0), 135 μL of H2O2 (50% w / v), and 20 μL of amphetamine (66.7 nM).

[0238] Overall, the assay results summarized in Table 1 show that MIP1 and MIP2, which have gold cores with diameters of 5 nm and 20 nm, respectively, yielded the highest V for these particles. maxAs demonstrated by the values, these particles showed the highest reaction rate for the oxidation of BPR dyes. These particles also showed the greatest response to amphetamine, as demonstrated by these particles having the lowest limit of detection (LOD). Therefore, there is a clear relationship between the size of the gold core and the catalytic activity of the Au / MIP nanozyme.

[0239] [Table 1]

[0240] Example 10 - Investigation of the detection range of a homogeneous assay based on composite nanoparticles (nanozymes) As described in Example 8, composite nanoparticles (i.e., MIP3) having a 50 nm gold core and an imprinted MIP shell using amphetamine were prepared. A colorimetric assay using BRP was performed using various concentrations of amphetamine (0.3–133 nM) under the conditions described in Example 9. The results are shown in Figure 7.

[0241] Importantly, this detection range of the assay falls within a clinically relevant range for forensic applications, ranging from 20 to 100 ng mL in urine. -1 Amphetamine concentrations in the range of up to (equivalent to 0.148 nM to 0.74 nM) are considered to fall within the therapeutic range (Drugs UNOo, Laboratory C, Section S. Recommended Methods for the Identification and Analysis of Amphetamine, Methamphetamine and Their Ring-substituted Analogues in Seized Materials: Manual for Use by National Drug Testing Laboratories. New York: United Nations (2006)); 100 ng mL in urine -1Concentrations exceeding 0.74 nM may indicate potential drug abuse; levels exceeding 2500 ng mL-1 (18.5 nM) are toxic and potentially life-threatening (Moeller KE, Kissack JC, Atayee RS, Lee KC. Clinical interpretation of urine drug tests: what clinicians need to know about urine drug screens. In: Mayo Clinic Proceedings; Elsevier (2017)).

[0242] Example 11 - Investigation of the specificity of a homogeneous assay based on composite nanoparticles (nanozymes) As described in Example 8, composite nanoparticles (i.e., MIP1) having a 5 nm gold core and an imprinted MIP shell with amphetamine were prepared. A colorimetric assay using BRP was performed with various concentrations (0.3–40 nM) of amphetamine or other drugs, as described in Example 9. Non-imprinted polymers decorated with gold nanoparticles (AuNP / NIP) were used as controls.

[0243] First, assay specificity was analyzed by comparing the responses of Au / MIP and Au / NIP nanozymes to amphetamine. As shown in Figure 8, no significant response to amphetamine was detected with respect to Au / NIP nanozymes. This result demonstrates the specificity of the constructed nanozyme assay for the corresponding target.

[0244] The assay specificity for amphetamine was further investigated by performing assays in the presence of various drugs. Separate assays were performed in the presence of paracetamol, morphine, and cocaine. As shown in Figure 9, no cross-reactivity was practically observed with respect to these interfering molecules, demonstrating the excellent specificity of the Au / MIP nanozyme for the corresponding target.

[0245] Example 12 - Homogeneous assay based on composite nanoparticles for use in various media and storage conditions To demonstrate possible applications, nanozyme assays were employed to detect amphetamine levels in both urine and plasma. Colorimetric assays using BRP were performed with composite nanoparticles having a 5 nm gold core (i.e., MIP1) and amphetamine at various concentrations (0.3–40 nM), as described in Example 9. Assays were performed using different media (PBS, plasma, and urine).

[0246] As shown in Figure 10, the assay remains usable even within these biological matrices. The limit of detection (LOD) of the assay was determined to be 0.17 nM in buffer, 5.1 nM in urine, and 23.9 nM in plasma.

[0247] Additionally, the assay performance of Au / MIP nanozymes stored in a refrigerator at 5°C was measured over a period of 6 months. No significant variation in assay performance was observed for nanozymes stored under these conditions (results not shown), demonstrating the excellent stability of the synthesized material.

[0248] Example 13 - Detection of o-nitrophenol in the gas phase using LSPR Au nanochips coated with MIP Localized surface plasmon resonance (LSPR) Au nanochips are glass substrates on which randomly distributed gold nanostructures are deposited. These Au nanochips were coated with various gold MIP composite nanoparticles (NPs) including o-nitrophenol, citrate, and picric acid imprinted MIP NPs, and unmodified (non-imprinted) NIP NPs. The composite nanoparticles were synthesized as described in Example 2.

[0249] Next, the LSPR Au nanochip was placed in a spectrophotometric cuvette containing varying amounts of the solid explosive-like analyte o-nitrophenol, and then placed in the working channel of the photometer. The uncoated Au nanochip in the spectrophotometric cuvette was placed in the reference channel of the photometer. To reduce background noise, the cuvettes in both channels were closed with lids during the measurement.

[0250] The kinetic dependence of the absorbance shift for Au nanochips upon exposure to o-nitrophenol vapor from various amounts of explosive-like substances (powder samples) was recorded. The absorbance response was measured using femtogram amounts of explosive-like substance analytes (4.5 cm³). 3 The concentration-dependent behavior and signal-to-noise ratio were sufficient for detecting 10 femtograms of o-nitrophenol in a given volume. The light absorption response time was up to 5 minutes.

[0251] The inventors observed that Au nanochips coated with o-nitrophenol imprint MIP NPs exhibited a cumulative dose response.

[0252] Example 14 - Detection of o-nitrophenol in the gas phase using LSPR Au nanochips coated with composite nanoparticles in a "real-world" scenario. We examined realistic scenarios for detecting trace amounts of explosive-like substances on surfaces of interest.

[0253] To prepare a "realistic" model of a trace amount of explosive-like material, the following protocol was used: 1 femtogram of o-nitrophenol was dissolved in 10 μL of 2-propanol, dropped onto a glass surface, and allowed to dry completely for 1 minute. The dried droplet was then swiped using a clean, dry cotton swab and placed in a spectrophotometric cuvette. A gold nanochip coated with composite nanoparticles, imprinted with o-nitrophenol as the MIP, was prepared (as described in Example 13). The gold nanochip was placed in the spectrophotometric cuvette containing the cotton swab. The cuvette was closed with a lid, and measurements were taken using a 2-channel photometer.

[0254] The kinetic dependence of the absorbance shift for coated Au nanochips upon exposure to o-nitrophenol vapor from a dry cotton swab is shown in Figure 11. The absorbance response clearly shows an increase when trace amounts of o-nitrophenol are present near the Au nanochip, and therefore, a response time of several minutes and up to 5.10 -4 The light absorption response up to a certain magnitude confirms the ability to detect an explosive-like substance of approximately 1 femtogram in a real-world scenario.

[0255] Example 15 - Design of a Fiber Optic Detection System The inventors constructed experimental setups designed to reduce the cost of detectors while maintaining selectivity for target substances, as found using the LSPR photometer detection system described in Examples 13 and 14, which has a very low limit of detection (LOD).

[0256] As shown in Figure 12, the detector is based on an optical fiber connected to a portable spectrometer. The detector includes a light source 3, e.g., an LED powered by USB, a detection element 4 including an optical fiber on which composite nanoparticles are deposited, an optical detector 2, e.g., a portable spectrometer or photodiode, and an operating device 1, e.g., a laptop computer. Light is emitted from the light source, propagates through the optical fiber, and is collected by the optical detector. The gold MIP composite nanoparticles act as a detection element configured to detect the target substance 5. The spectrum of the Au core changes when the target molecule binds to the MIP.

[0257] This detection system was manufactured by coating the tapered portion of an optical fiber with gold nanoparticles. Next, an MIP coating was synthesized onto the gold nanoparticles.

[0258] The detection system was tested for its ability to detect the target substance, cocaine. Figure 13 shows the light signal generated in this test, demonstrating that its intensity increases upon exposure of the detector to the target substance.

[0259] As demonstrated in Figure 13, the target molecule interacts with the composite nanoparticles. The interaction between the target molecule and the composite nanoparticles has a direct and proportional effect on the propagated light signal. The optical detector then records this signal change to indicate the presence and / or concentration of the target molecule.

[0260] By using optical fibers instead of Au nanochips, significant cost reductions are possible while maintaining a low detection limit. Compared to LSPR used in the photometer detectors in Examples 13 and 14, this technique has the advantage of being more resistant to interference.

[0261] method Preparation of glass beads For solid-phase synthesis, glass beads were functionalized using amphetamine. To this end, 60 g of glass beads were incubated for 8 hours in amphetamine (65 mg, 0.481 μmol) in borate buffer (65 mL, 100 mM, pH 9.2), protected from light. To block unreacted iodine groups, mercaptoethanol (10 μL, 0.143 μmol) was added to the mixture and incubated for 2 hours. The glass beads were then washed with water (100 mL twice) and then with acetone (100 mL four times), and subsequently dried under reduced pressure.

[0262] Construction of a BPR assay For the measurements, a Corning® 96-well microplate (300 μL well volume) and a Hidex Sense 425-301 microplate reader were used. Nanozyme catalytic activity was evaluated using a colorimetric assay format, similar to other peroxidase-like activity assays, measuring the change in absorbance caused by the oxidation of bromopyrogallol red (BPR, a chromogenic substrate) with hydrogen peroxide (H2O2).

Claims

1. A method for producing composite particles, comprising contacting catalyst particles, an oxidizing agent, and a plurality of monomers, wherein the oxidizing agent is a peroxide, a persulfate, or ozone, and the catalyst particles are or comprise a material having Fenton or Fenton-like catalytic activity, wherein the catalyst particles catalyze the disproportionation of the oxidizing agent to produce oxygen-radical species, thereby initiating a polymerization reaction, which in turn polymerizes the plurality of monomers, thereby forming a polymer shell around the catalyst particles.

2. The method according to claim 1, comprising contacting the catalyst particles, the oxidizing agent, and the plurality of monomers in a solution.

3. The method according to claim 1 or claim 2, wherein the composite particles are nanoparticles and the catalyst particles are nanoparticles.

4. The method according to any one of claims 1 to 3, wherein the catalyst particles are not bound.

5. The method according to any one of claims 1 to 3, wherein the catalyst particles are bonded to a substrate, and preferably the substrate is an optical fiber including a polymer coating.

6. The method according to any one of claims 1 to 5, wherein the catalyst particles include or consist of a metal, a metal alloy, a metal oxide, a metal salt, a semiconductor, a two-dimensional material, a fullerene, a carbon nanotube, a quantum dot, a carbon nanodot, a carbide, and / or a nitride.

7. The method according to claim 6, wherein the catalyst particles contain or consist of a metal or a metal oxide, the metal being gold, silver, platinum, palladium, iron, nickel, zinc, chromium, titanium, zirconium, hafnium, vanadium, niobium, tantalum, molybdenum, or tungsten, and the metal oxide being iron oxide, aluminum oxide, titanium oxide, cerium oxide, zirconium oxide, hafnium oxide, vanadium oxide, niobium oxide, tantalum oxide, chromium oxide, molybdenum oxide, or tungsten oxide.

8. The method includes contacting a catalyst particle precursor, the oxidizing agent, and the plurality of monomers, wherein the catalyst particle precursor is configured to produce the catalyst particles. The method according to any one of claims 1 to 7, comprising, in a single step, producing catalyst particles and contacting the catalyst particles, the oxidizing agent, and the plurality of monomers.

9. The method according to claim 8, comprising a reagent capable of producing catalyst particles by oxidizing, reducing, or hydrolyzing the catalyst particle precursor, and an oxidizing agent, a reducing agent, or a hydrolyzing agent.

10. The method according to claim 9, wherein the reagent that can be oxidized, reduced, or hydrolyzed to produce the catalyst particles is an inorganic salt, an acid, or a hydroxide, and the inorganic salt, the acid, or the hydroxide contains a metal cation.

11. The method according to any one of claims 1 to 10, comprising contacting the catalyst particles, an oxidizing agent, and a plurality of monomers in the presence of a template species such that the polymer shell is or contains a molecularly imprinted polymer (MIP).

12. A composite particle that can be obtained or obtained by the method of any one of claims 1 to 11.

13. Use of composite particles in detecting target molecules, wherein the composite particles include a polymer shell surrounding catalyst particles.

14. A method for detecting target molecules in a solution, Bringing the solution and composite particles into contact, The optical properties of the catalyst particles are detected, thereby allowing the target molecules in the solution to be detected. Includes, A method wherein the composite particles include a polymer shell surrounding catalyst particles.

15. The method according to claim 12, wherein the optical properties are surface plasmon resonance (SPR) absorption, localized surface plasmon resonance (LSPR) absorption, or fluorescence.

16. A method for detecting target molecules in a solution, Placing the electrode with the composite particles immobilized and further electrodes in a solution, Applying a voltage to the electrode, The current is measured to detect the target molecule in the solution. Includes, A method wherein the composite particles include a polymer shell surrounding catalyst particles.

17. The method according to claim 16, wherein applying a voltage to the two electrodes and measuring the current includes applying differential pulse voltammetry (DPV).

18. Use of composite particles in a lateral flow detector, wherein the composite particles include a polymer shell surrounding catalyst particles.

19. A lateral flow detector comprising composite particles, wherein the composite particles include a polymer shell surrounding catalyst particles.

20. A method for detecting a target molecule in a sample, comprising: bringing the sample into contact with a lateral flow detector according to claim 19; and observing whether or not a test line appears on the lateral flow detector, thereby detecting the target molecule in the sample.

21. A method for detecting a target molecule in a sample, Contacting a spectrophotometric reagent, composite particles, an oxidizing or reducing agent, and the sample, The absorbance of the resulting composition is measured to detect the target molecule in the sample. Includes, A method wherein the composite particles include a polymer shell surrounding catalyst particles.

22. The method according to claim 21, wherein the spectrophotometric reagent is a redox-sensitive dye.

23. Use of composite particles in a catalytic reaction, wherein the composite particles include a polymer shell surrounding the catalyst particles.

24. A pharmaceutical composition comprising composite particles and a pharmaceutically acceptable carrier, wherein the composite particles include a polymer shell surrounding catalyst particles.

25. A composite particle for use in a method of treatment or diagnosis, or a pharmaceutical composition according to claim 24, wherein the composite particle comprises a polymer shell surrounding a catalyst particle.

26. A composite particle for use in an imaging method or the pharmaceutical composition according to claim 24, wherein the composite particle comprises a polymer shell surrounding a catalyst particle.

27. A composite particle for use in drug delivery or the pharmaceutical composition according to claim 24, wherein the composite particle comprises a polymer shell surrounding a catalyst particle.

28. The composite particle or pharmaceutical composition for use according to any one of claims 25 to 27, wherein the composite particle comprises a drug molecule adsorbed thereto.

29. The composite particle or pharmaceutical composition for use according to any one of claims 25 to 28, wherein the catalyst particles include a magnetic material.

30. The composite particle or pharmaceutical composition for use according to any one of claims 25 to 29, wherein the composite particle comprises an antibody or aptamer conjugated to the polymer shell, or the polymer shell comprises a MIP, and the antibody, aptamer, or MIP is configured to deliver the composite particle to a specific target.

31. Substrate and A plurality of nanoparticles arranged on the substrate, Multiple composite particles arranged on the substrate and Includes, The aforementioned composite particle is a detector comprising a polymer shell surrounding a catalyst particle.

32. A method for detecting target molecules in a fluid, To prepare the detector described in claim 31, The detector is brought into contact with the fluid, The optical properties of the detector are detected, thereby detecting the target molecules in the fluid. A method that includes this.

33. An optical fiber extending between a first end and a second end, including a detection unit, wherein the detection unit is configured to enable the generation of an evanescent field, Composite particles arranged on the detection section of the optical fiber and Includes, The aforementioned composite particle is a detector comprising a polymer shell surrounding a catalyst particle.

34. A method for detecting target molecules in a fluid, To prepare the detector described in claim 33, Bringing the fluid and the composite particles into contact, An optical signal is input to the first end of the optical fiber so that the composite particles are at least partially positioned in the evanescent field, thereby generating the evanescent field adjacent to the detection unit of the optical fiber. The optical signal or value output from the second end of the optical fiber is detected, and the output optical signal is compared with a reference optical signal or value to detect the target molecule in the fluid. A method that includes this.

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