Molecularly imprinted polymers

Abstract

The present invention relates to molecularly imprinted polymers (MIPs), especially those formed around template molecules with magnetic supports (magnetically supported template molecules). The invent

Description

MOLECULARLY IMPRINTED POLYMERS INTRODUCTION

[0001] The present invention relates to molecularly imprinted polymers (MIPs) and their methods of production. In particular, the MIP template molecule (e.g. a protein) used to produce corresponding imprints is suitably tethered to a magnetic nanoparticle (MNP) during MIP production to facilitate recovery and reuse of the same MIP template in further MIP production rounds, thereby increasing MIP yields per unit mass of template molecule. The invention also relates to the use of such MIPs in the (electrochemical) detection and / or binding of target / template molecules. BACKGROUND

[0002] Molecularly Imprinted Polymers (MIPs) are polymers (often polymeric particles) bearing imprints (i.e. cavities) of a molecular template (or “template molecule”) around which they are built. MIPs are formed by polymerising monomers (typically along with other crosslinking monomers) in the presence of template molecules to yield polymers bearing imprints / cavities binding their complementary template molecules. After extracting the original template molecules, the resulting MIPs bear binding sites (i.e. imprints / cavities) having selective affinity for the template molecule itself or any “target molecule” with a bindable part that complements the molecular imprints within the MIPs. In general, the MIP imprints will be sterically complementary (e.g. in terms of size and shape) to the or a relevant part of the relevant template molecule, but additional complementarities may also prevail, such as electrostatic, hydrogen bonding, Van der Waals forces, and hydrophobic interactions. As such, affinities between the polymeric imprints and template (or target) may be considered similar to interactions between antibodies and antigens (or epitopes thereof). As such, the imprints or “binding sites” of these MIPs can be extremely selective. MIPs are thus powerful analytical, sensory, diagnostic, and therapeutic tools.

[0003] MIPs also have a variety of other well-known uses (e.g. in fields such a chemistry, biology, biochemistry, bioengineering, engineering, food science, and more). By way of example: ^ MIPs may be used as an affinity material in sensors or binding assays (e.g. “molecularly imprinted assays”), for instance, for the detection of specific molecules, such as antimicrobial agents, dyes, residues in foods, and the like. ^ MIPs are ideal as a chromatographic stationary phase for solid phase extraction of target molecules. Similarly, MIPs may be useful in removing byproducts in chemical reaction mixtures. ^ MIPs may be used to facilitate protein crystallisation (e.g. after isolating the relevant protein). ^ MIPs may be used as artificial antibodies or to create enzyme-like binding sites to facilitate drug development and screening. ^ MIPs may be biological receptor mimetics and thus be used therapeutically to reduce receptor overloads. ^ MIPs may (especially where binding between an MIP and a target is less strong) be components of controlled release drugs. ^ MIPs may be utilised as drug monitoring devices, or drug (e.g. narcotic) testing devices. ^ MIPs are, in theory, simpler and cheaper to produce and purify than proteins and antibodies, which may be more vulnerable to denaturing (e.g. through exposure to heat, extremes of pH, etc.), but in practice commercialisation of MIPs does currently present problems owing to costs of production. ^ MIPs may, more easily than antibodies and proteins, be “labelled” – e.g. radio-labelled or fluorescent-labelled, to facilitate assaying.

[0004] Acrylamide-based polymer hydrogels have been extensively researched as protein-selective MIPs1. They can be produced with cheap reagents, in less than a day, in a one-pot synthesis. These MIPs can be micron-sized particles2, thin- film3and nanoscale particles (50-200 nm)4. Recently evaluated and demonstrated were: virus imprinted MIPs to selectively capture and neutralise an animal virus5; a novel microwave method to rapidly produce monodisperse functionalised iron oxide magnetic nanoparticles (MNP) for selective MIP extraction6; a working MIP-based electrochemical sensor strategy for the antibody-free determination of SARS-CoV-2 in saliva7. The electrochemically produced MIP (E-MIP) technology has also been demonstrated for sub-nanomolar determination of proteins.

[0005] Recently, MIP syntheses have been performed in the presence of trypsin and pepsin protein templates tethered to a functionalised silica coating of a magnetic nanoparticle,8to yield an MIP-template conjugate that is heated to release the template molecules and ultimately deliver the free MIPs. Following their heat extraction, and inevitable and H. El Sharif, D. Hawkins, D. Stevenson and S. Reddy, Physical chemistry chemical physics : PCCP, 2014, 16. M. Kempe and K. Mosbach, JOURNAL OF CHROMATOGRAPHY A, 1995, 691, 317-323 A. Stephen, S. Dennison, M. Holden and S. Reddy, The Analyst, 2023, 148 F. Canfarotta, A. Poma, A. Guerreiro and S. Piletsky, Nat Protoc, 2016, 11, 443-455 S. Graham, H. El Sharif, S. Hussain, R. Fruengel, R. McLean, P. Hawes, M. Sullivan and S. Reddy, Frontiers in Bioengineering and Biotechnology, 2019, 7 M. Sullivan, W. Stockburn, P. Hawes, T. Mercer and S. Reddy, Nanotechnology, 2020, 32, 095502 H. El Sharif, S. Dennison, M. Tully, S. Crossley, W. Mwangi, D. Bailey, S. Graham and S. M. Reddy, Analytica Chimica Acta, 2022, 1206, 339777 R. Mahajan, M. Rouhi, S. Shinde, T. Bedwell, A. Incel, L. Mavliutova, S. Piletsky, I. A. Nicholls, B. Sellergren, Angew. Chem. Int. Ed.2019, 58, 727.consequential denaturing, these redundant protein with their magnetic tether) are discarded having served their purpose.

[0006] All of the aforementioned MIP syntheses suffer from the problem that expensive template molecules, such as proteins, are routinely discarded and thus arguably wasted. In an attempt to address this problem, researchers have synthesised MIPs around template molecules that are tethered to glass beads (solid supports).9Unfortunately, yields are very low, and the process is slow and practically cumbersome.

[0007] In light of the above, there remains a knowledge vacuum in respect of the scaling-up of MIP production for viable commercial use.

[0008] It is therefore an object of the present invention to provide an alternative or improved method of providing MIPs. Producing higher yields faster is a particular goal, as is reducing process complexity.

[0009] Moreover, it is an object of the present invention to provide alternative or improved MIPs, especially compared to those of the prior art.

[0010] An object of the invention is to provide a means of recycling a template molecule in the production of MIPs, thereby improving the yield of MIPs per unit template molecule (especially where said template substrate is reused).

[0011] Finally, an object of the present invention is to provide an alternative or improved implementation of MIPs, MIP production, and / or MIP usage. SUMMARY OF THE INVENTION Functionalised Magnetic Support (or Functionalised Magnetic Particle)

[0012] A functionalised magnetic support (or functionalised magnetic particle) comprises or consists of a magnetic core (or magnetic particle) bearing, preferably on the surface thereof, a reactive functional group (e.g. aldehyde group, carboxylate group, or amine group). Suitably, the magnetic support comprises or consists of a magnetic core (or magnetic particle) bearing, preferably on the surface thereof, one or more reactive functional groups. The magnetic support may comprise or consist of a magnetic core (or magnetic particle) bearing, preferably on the surface thereof, a plurality of reactive functional groups. Whilst, in theory, a functionalised magnetic support may comprise a plurality of magnetic cores, generally the functionalised magnetic support will have only a single magnetic core. A) Aspects of a functionalised magnetic support and manufacturing methods therefor

[0013] The present invention provides at least the following aspects relating to a functionalised magnetic support (or functionalised magnetic particle) and methods of manufacturing the same: A1. A method of manufacturing a functionalised magnetic support (or functionalised magnetic particle), the method comprising: forming a magnetic core (suitably from one or more magnetic core-forming components) bearing, preferably on the surface thereof, a reactive functional group (e.g. aldehyde group, carboxylate group, or amine group) (suitably derived from one or more functionalising molecules). Such a magnetic core (e.g. a paramagnetic metal-containing core or paramagnetic metal-containing particle) may be formed in the presence of a functionalising molecule (e.g. glutaraldehyde) so that the resulting magnetic support (or magnetic particle) bears a reactive functional group (e.g. aldehyde group, carboxylate group, or amine group), suitably on the surface thereof. More preferably, the functionalised magnetic support is a magnetic core / particle bearing a plurality of reactive functional groups on the surface thereof. A2. A method of manufacturing a functionalised magnetic support (or functionalised magnetic particle), the method comprising: forming a magnetic core (or magnetic particle) (e.g. a superparamagnetic metal oxide-containing core / particle, for instance an iron oxide-based core / particle) in the presence of a functionalising molecule (e.g. glutaraldehyde). A3. A method of manufacturing a functionalised magnetic support (or functionalised magnetic particle), the method comprising: i) Forming a magnetic core from one or more magnetic core-forming components; and ii) Functionalising the magnetic core with one or more reactive functional groups; optionally wherein steps i) and ii) are performed simultaneously (i.e. in a one-pot procedure). A4. A method of manufacturing a functionalised magnetic support (or functionalised magnetic particle), the method comprising mixing together one or more magnetic core-forming components with a functionalising molecule, and optionally further processing, to form a functionalised magnetic support comprising a magnetic core, derived from the magnetic core-forming components, the magnetic core bearing, at the surface thereof, one or more reactive functional group(s) derived from the functionalising molecule. A5. A method of manufacturing a functionalised magnetic support, the method comprising: mixing together one or more magnetic core-forming components with a functionalising molecule, and optionally further processing, to Canfarotta, F., Poma, A., Guerreiro, A. et al. Solid-phase synthesis of molecularly imprinted nanoparticles. Nat Protoc 11, 443–455 (2016). https: / / doi.org / 10.1038 / nprot.2016.030form a functionalised magnetic support a magnetic core, derived from the magnetic core-forming components, the magnetic core bearing, at the surface thereof, one or more reactive functional group(s) derived from the functionalising molecule; wherein the magnetic core is free of a silica coating or functionalised silica; and the one or more reactive functional groups are directly attached to the magnetic core. A6. A functionalised magnetic support (or functionalised magnetic particle) obtainable by, obtained by, or directly obtained by a method of manufacturing a functionalised magnetic support (or functionalised magnetic particle) as defined herein or as otherwise directly and unambiguously derivable from the present disclosure. A7. A functionalised magnetic support (or functionalised magnetic particle) comprising a magnetic core (or magnetic particle) bearing, preferably on the surface thereof, a reactive functional group (e.g. aldehyde group, carboxylate group, or amine group). Preferably, the magnetic core / particle bears a plurality of reactive functional groups on the surface thereof. Such a magnetic core (or magnetic particle) is preferably a paramagnetic metal-containing core / particle, for instance, an iron oxide-based or iron-oxide-containing core / particle (e.g. Fe2O3, Fe3O4, or a mixture thereof). Preferably the functionalised magnetic support comprises a magnetic core (preferably comprising or consisting of a paramagnetic metal oxide) with one or more (preferably a plurality of) reactive functional groups directly attached to the magnetic core. The magnetic core is preferably uncoated (e.g. is free of a silica-coating, especially free of a functionalised silica coating which bears the one or more reactive functional groups). Preferably, the functionalised magnetic support is free of silica or functionalised silica. A8. A functionalised magnetic support as defined herein (or comprising functionalised magnetic particles as defined herein).

[0014] Features and sub-features of any of aspects A1-A8 are applicable to each other and indeed to any functionalised magnetic supports and methods of manufacturing the same described here or elsewhere herein.

[0015] The term “functionalised magnetic support” may be used interchangeably with the term “functionalised magnetic particle” (or “functionalised magnetic particles”). Magnetically Supported Template Molecule (or Magnetic-Template Particle)

[0016] A magnetically supported template molecule (or a magnetic-template particle) comprises or consists of a magnetic core (or magnetic particle) coupled or tethered to a (or one or more of, preferably a plurality of) template molecule(s). Suitably, the magnetically supported template molecule comprises or consists of a magnetic core (or magnetic particle) coupled or tethered to one or more template molecule(s). The magnetically supported template molecule may comprise or consist of a magnetic core (or magnetic particle) coupled or tethered to a plurality of template molecule(s). Template molecules may be coupled or tethered to the magnetic core via a (preferably via one or more of, preferably via a plurality of) reactive functional group(s) (e.g. aldehyde group, carboxylate group, or amine group) borne by, preferably on the surface of, the magnetic core / particle. B) Aspects of a magnetically supported template molecule and manufacturing methods therefor

[0017] The present invention provides at least the following aspects relating to a magnetically supported template molecule and methods of manufacturing the same: B1. A method of manufacturing a magnetically supported template molecule (or a magnetic-template particle), the method comprising: providing a functionalised magnetic support (e.g. via one of the aforesaid methods) and coupling a template molecule to said functionalised magnetic support, suitably via a (preferably direct) chemical reaction between the template molecule and a reactive functional group borne on the surface of the functionalised magnetic support. Preferably the method comprises coupling a plurality of template molecules with a single functionalised magnetic support involves reacting the template molecule with the functionalised magnetic support, preferably directly. B2. A method of manufacturing a magnetically supported template molecule (or a magnetic-template particle), the method comprising: forming a magnetic core (or magnetic particle) (e.g. a superparamagnetic metal oxide- containing core / particle, for instance, an iron oxide-based core / particle) in the presence of a functionalising molecule (e.g. glutaraldehyde) and a template molecule. Preferably this method may be conducted under the same conditions as a method of manufacturing a functionalised magnetic support as defined herein. This advantageous because it allows a one-pot synthesis of the magnetically supported template molecule (or a magnetic-template particle). B3. A method of manufacturing a magnetically supported template molecule, the method comprising: providing a functionalised magnetic support by, or obtained by, the method of manufacturing a functionalised magnetic support as claimed in claim 43, and coupling a template molecule to said functionalised magnetic support via a (preferably direct) chemical reaction between the template molecule and a reactive functional group borne on the surface of the functionalised magnetic support; wherein the functionalised magnetic support comprises a magnetic core (suitably derived from one or more magnetic core-forming components) bearing, at the surface thereof, one or more reactive functional group(s) (suitably derived from a functionalising molecule), wherein the magnetic core is free of a silica coating or functionalised silica; and the one or more reactive functional groups are directly attached to the magnetic core.B4. A magnetically supported template molecule (or magnetic-template particle) obtainable by, obtained by, or directly obtained by a method of manufacturing a magnetically supported template molecule as defined herein or as otherwise directly and unambiguously derivable from the present disclosure. B5. A magnetically supported template molecule (or a magnetic-template particle) comprising a magnetic core (or magnetic particle) coupled to a (or one or more of, preferably a plurality of) template molecule(s) via a (preferably via one or more of, preferably via a plurality of) reactive functional group(s) (e.g. aldehyde group, carboxylate group, or amine group) borne by, preferably on the surface of, the magnetic core / particle. Such a magnetic particle is preferably a paramagnetic metal-containing particle, for instance, an iron oxide-based or iron-oxide-containing particle (e.g. Fe2O3, Fe3O4, or a mixture thereof). Preferably the magnetically supported template molecule comprises a magnetic core (preferably comprising or consisting of a paramagnetic metal oxide) that is attached directly (preferably without any intervening coating, preferably especially without any intervening functionalised silica coating), via one or more (preferably a plurality of) reactive functional groups directly attached to the magnetic core, to one or more (preferably a plurality of template molecules). The magnetic core is preferably uncoated (e.g. is free of a silica-coating, especially free of a functionalised silica coating). Preferably, the magnetically supported template molecule is free of silica or functionalised silica.

[0018] Features and sub-features of any of aspects B1-B5 are applicable to each other and indeed to any magnetically supported template molecules and methods of manufacturing the same described here or elsewhere herein.

[0019] The term “magnetically supported template molecule” may be used interchangeably with the term “magnetic- template particle” (or “magnetic-template particles”). Magnetic-Template-Bound Molecularly Imprinted Polymer (Magnetic-MIP conjugate)

[0020] A magnetic template-bound molecularly imprinted polymer (or magnetic-MIP conjugate, or magnetic-MIP conjugate particle) comprises or consists of a magnetically supported template molecule (or magnetic-template particle) and a molecularly imprinted polymer (e.g. a polymer formed in the presence of a magnetically supported template molecule), preferably wherein the or a part of the magnetically supported template molecule (most preferably the or a part of the template molecule of the magnetically supported template molecule) is bound to or within the molecularly imprinted polymer (preferably bound to or within a complementary imprint or cavity of the molecularly imprinted polymer).

[0021] Suitably, the magnetic template-bound molecularly imprinted polymer (or magnetic-MIP conjugate, or magnetic- MIP conjugate particle) may comprise or consist of a (or one or more) magnetically supported template molecule(s) and a (or one or more) molecularly imprinted polymer(s), preferably wherein the magnetically supported template molecule(s), or part(s) thereof, are bound to or within the molecularly imprinted polymer(s) (preferably bound to or within complementary imprints or cavities of the molecularly imprinted polymers or part(s) thereof).

[0022] Suitably, the or each magnetic template-bound molecularly imprinted polymer (or magnetic-MIP conjugate, or magnetic-MIP conjugate particle) may comprise or consist of a plurality of magnetically supported template molecules for each molecularly imprinted polymer, preferably wherein the plurality of magnetically supported template molecules, or part(s) thereof, are bound to or within the or each molecularly imprinted polymer (preferably bound to or within complementary imprints or cavities of the or each molecularly imprinted polymer). Suitably, the or each magnetic template-bound molecularly imprinted polymer may comprise a plurality of molecularly imprinted polymers for each magnetically supported template molecule, preferably wherein the or part(s) of the or each magnetically supported template molecule is bound to or within (preferably within complementary imprints) a plurality of molecularly imprinted polymer molecules.

[0023] The magnetically supported template molecule used in this section is preferably a magnetically supported template molecule in accordance with the invention (suitably as defined herein, e.g. with a molecular template directly coupled to a magnetic core via reactive functional group(s) borne on the surface thereof, where there is no silica or functionalised-silica coating upon the magnetic core), but it may (especially where it is recycled) be a magnetically supported template molecule of the prior art.

[0024] Preferably, a “polymer” referred to in this section (where it is a polymer formed in the presence of a template molecule or magnetically supported template molecule) is classed as a molecularly imprinted polymer. C) Aspects of a magnetic template-bound molecularly imprinted polymer, manufacturing methods therefor, and recycling magnetically supported template molecules

[0025] The present invention provides at least the following aspects relating to a magnetic template-bound molecularly imprinted polymer, methods of manufacturing the same, and recycling magnetically supported template molecules: C1. A method of manufacturing a magnetic template-bound molecularly imprinted polymer (or magnetic-MIP conjugate, or magnetic-MIP conjugate particle), the method comprising: forming a polymer (or polymer particle) (which becomes a molecularly imprinted polymer) in the presence of a magnetically supported template molecule (or magnetic-template particle), preferably a magnetically supported template molecule (or magnetic-template particle) as defined herein. Preferably, after production of the template-bound molecularly imprinted polymer (or magnetic-MIP conjugate, or magnetic-MIP conjugate particle), the magnetically supported template molecule (or magnetic-template particle) is bound within the polymer (which is suitably a molecularly imprinted polymer), preferably as a consequence of a cavity (or imprint) harnessing the template molecule or a part thereof. Themagnetically supported template molecule (or template particle) may be a recycled magnetically supported template molecule (or recycled magnetic-template particle) – i.e. one that has already been previously used in a (preferably the same) method of manufacturing a magnetic template-bound molecularly imprinted polymer (or magnetic-MIP conjugate) – whilst the recycled magnetically supported template molecule (or recycled magnetic-template particle) is preferably a recycled form of a magnetically supported template molecule (or a magnetic-template particle) as defined herein, it may in principle be any recycled form of a magnetically supported template molecule (or a magnetic-template particle), including a magnetically supported template molecule (or a magnetic-template particle) of the prior art. C2. A method of manufacturing a magnetic template-bound molecularly imprinted polymer, which comprises or consists of the (or one or more of the, preferably a plurality of) magnetically supported template molecule(s) bound to a molecularly imprinted polymer via a (or one or more, preferably a plurality of) corresponding complementary imprint(s) within the molecularly imprinted polymer(s), the method comprising: polymerising one or more monomer(s) (preferably at least one backbone-forming monomer and a cross-linking monomer) in the presence of a magnetically supported template molecule (preferably as defined herein). C3. A method of recycling a (or a method of forming a recycled) magnetically supported template molecule (or magnetic-template particle), comprising recovering (e.g. isolating) a magnetically supported template molecule (or magnetic-template particle) from a magnetic template-bound molecularly imprinted polymer (or magnetic-MIP conjugate, or magnetic-MIP conjugate particle) as a recycled magnetically supported template molecule (or recycled magnetic-template particle). C4. A method of recycling a (or a method of forming a recycled) magnetically supported template molecule (or magnetic-template particle), comprising separating a magnetically supported template molecule (or magnetic- template particle) from a molecularly imprinted polymer of a magnetic template-bound molecularly imprinted polymer (or magnetic-MIP conjugate, or magnetic-MIP conjugate particle) as a recycled magnetically supported template molecule (or recycled magnetic-template particle). C5. A method of recycling a (or a method of forming a recycled) magnetically supported template molecule (or magnetic-template particle), comprising: i) forming a polymer (or polymer particle) (which becomes a molecularly imprinted polymer) in the presence of a magnetically supported template molecule (or magnetic-template particle), preferably a magnetically supported template molecule (or magnetic-template particle) as defined herein, so as to produce a magnetic template-bound molecularly imprinted polymer (or magnetic-MIP conjugate, or magnetic-MIP conjugate particle); ii) recovering (e.g. isolating) the magnetically supported template molecule (or magnetic-template particle) from the magnetic template-bound molecularly imprinted polymer (or magnetic-MIP conjugate, or magnetic-MIP conjugate particle) as a recycled magnetically supported template molecule (or recycled magnetic-template particle). C6. A method of recycling a (or a method of forming a recycled) magnetically supported template molecule (or magnetic-template particle), comprising: i) forming a polymer (or polymer particle) (which becomes a molecularly imprinted polymer) in the presence of a magnetically supported template molecule (or magnetic-template particle), preferably a magnetically supported template molecule (or magnetic-template particle) as defined herein, so as to produce a magnetic template-bound molecularly imprinted polymer (or magnetic-MIP conjugate, or magnetic-MIP conjugate particle); ii) separating the magnetically supported template molecule (or magnetic-template particle) from the molecularly imprinted polymer of the magnetic template-bound molecularly imprinted polymer (or magnetic- MIP conjugate, or magnetic-MIP conjugate particle) as a recycled magnetically supported template molecule (or recycled magnetic-template particle). C7. A recycled magnetically supported template molecule (or recycled magnetic-template particle) obtainable by, obtained by, or directly obtained by a method of recycling a (or a method of forming a recycled) magnetically supported template molecule (or magnetic-template particle) as defined herein or as otherwise directly and unambiguously derivable from the present disclosure. C8. A method of manufacturing a magnetic template-bound molecularly imprinted polymer (or magnetic-MIP conjugate, or magnetic-MIP conjugate particles), the method comprising: i) providing a recycled magnetically supported template molecule (or recycled magnetic-template particle(s)), suitably as defined herein (e.g. with a molecular template directly coupled to a magnetic core via reactive functional group(s) borne on the surface thereof, where there is no silica or functionalised-silica coating upon the magnetic core) or via the method of recycling a (or a method of forming a recycled) magnetically supported template molecule (or magnetic-template particle) as defined herein - however, the recycled magnetically supported template molecule (or magnetic-template particle) may be otherwise, for instance, derived from a magnetically supported template molecule (or magnetic-template particle) of the prior art; and ii) forming a polymer (or polymer particle) (which becomes a molecularly imprinted polymer) in the presence of the recycled magnetically supported template molecule (or recycled magnetic-template particle(s)),preferably derived from a magnetically template molecule (or magnetic-template particle) as defined herein (e.g. with a molecular template directly coupled to a magnetic core via reactive functional group(s) borne on the surface thereof, where there is no silica or functionalised-silica coating upon the magnetic core). However, the recycled magnetically supported template molecule (or magnetic-template particle) may be otherwise, for instance, derived from a magnetically supported template molecule (or magnetic-template particle) of the prior art. C9. A method of manufacturing a magnetic template-bound molecularly imprinted polymer (or magnetic-MIP conjugate, or magnetic-MIP conjugate particles), the method comprising: i) forming a polymer (or polymer particle) (which becomes a molecularly imprinted polymer) in the presence of a magnetically supported template molecule (or magnetic-template particle), preferably a magnetically supported template molecule (or magnetic-template particle) as defined herein, so as to produce a magnetic template-bound molecularly imprinted polymer (or magnetic-MIP conjugate, or magnetic-MIP conjugate particle); ii) separating the magnetically supported template molecule (or magnetic-template particle), as a recycled magnetically supported template molecule, from the molecularly imprinted polymer of the magnetic template- bound molecularly imprinted polymer (or magnetic-MIP conjugate, or magnetic-MIP conjugate particle) and isolating: a. the recycled magnetically supported template molecule (or recycled magnetic-template particle); and suitably also b. the molecularly imprinted polymer; iii) forming a polymer (or polymer particle) (which becomes a molecularly imprinted polymer) in the presence of the recycled magnetically supported template molecule (or recycled magnetic-template particle), preferably a recycled magnetically supported template molecule (or recycled magnetic-template particle) derived from a magnetically supported template molecule (or magnetic-template particle) as defined herein (e.g. with a molecular template directly coupled to a magnetic core via reactive functional group(s) borne on the surface thereof, where there is no silica or functionalised-silica coating upon the magnetic core). However, the recycled magnetically supported template molecule (or magnetic-template particle) may be otherwise, for instance, derived from a magnetically supported template molecule (or magnetic-template particle) of the prior art. C10. A magnetic template-bound molecularly imprinted polymer (or magnetic-MIP conjugate, or magnetic-MIP conjugate particles) obtainable by, obtained by, or directly obtained by a method of manufacturing a magnetic template-bound molecularly imprinted polymer (or magnetic-MIP conjugate, or magnetic-MIP conjugate particles) as defined herein or as otherwise directly and unambiguously derivable from the present disclosure. C11. A magnetic template-bound molecularly imprinted polymer (or magnetic-MIP conjugate, or magnetic-MIP conjugate particles) comprising a magnetically supported template molecule (or a magnetic-template particle), preferably as defined herein, bound to (or within) a polymer (or polymer particle) (i.e. a molecularly imprinted polymer) built around the magnetically supported template molecule (or magnetic-template particle). The magnetically supported template molecule (or magnetic-template particle) is suitably as defined in accordance with the present invention (e.g. with a molecular template directly coupled to a magnetic core via reactive functional group(s) borne on the surface thereof, where there is no silica or functionalised-silica coating upon the magnetic core). However, where the magnetically supported template molecule (or magnetic-template particle) is a recycled magnetically supported template molecule (or magnetic-template particle), it may be a magnetically supported template molecule (or magnetic-template particle) of (or derived from) the prior art.

[0026] Features and sub-features of any of aspects C1-C11 are applicable to each other and indeed to any magnetic template-bound molecularly imprinted polymer, recycled magnetically supported template molecule, and methods of manufacturing the same, described here or elsewhere herein.

[0027] Preferably, “forming a polymer” comprises polymerising one or more monomers (or optionally copolymerising two or more monomers, preferably wherein at least one of the two or more monomers is a cross-linking monomer) in the presence of a magnetically supported template molecule (preferably as defined herein). Optionally one or more of the monomers may comprise a detectable moiety, such as a fluorescent moiety, though alternatively a one or more of the monomers may comprise reactive moieties that may be coupled to a detectable moiety after the polymer has been formed.

[0028] Preferably, the aforesaid methods of manufacturing a magnetic template-bound molecularly imprinted polymer (or magnetic-MIP conjugate, or magnetic-MIP conjugate particles) further comprise separating (and preferably isolating) the produced magnetic template-bound molecularly imprinted polymer from non-imprinted polymers (NIPs). NIPs suitably do not comprise any molecular imprints and suitably do not have a magnetically supported template molecule, recycled or otherwise, bound thereto or therewithin and, as such, are not functionally useful in the context of the present invention. This “separating” preferably involves applying a magnetic field to magnetically constrain (e.g. magnetically attract, localise, and / or harness) the magnetic template-bound molecularly imprinted polymer whilst non-imprinted polymers (NIPs) are otherwise separated therefrom (e.g. by washing, pipetting, decanting, pumping, etc.).

[0029] The term “magnetic template-bound molecularly imprinted polymer” may be used interchangeably with the term “magnetic-MIP conjugate”, “magnetic-MIP conjugate particle” (or “magnetic-MIP conjugate particles”).

[0030] In the context of a molecularly imprinted polymer , the term “polymer” may be used interchangeably with the term “polymer particle”, especially “MIP particle”. The “polymer” is preferably a molecularly imprinted polymer (MIP). Free (Magnetic Template-Unbound) Molecularly Imprinted Polymers (MIPs)

[0031] A molecularly imprinted polymer is a polymer comprising an imprint of a template molecule or a part thereof, which imprint suitably has an affinity for (i.e. binding affinity for or propensity to associate with) the template molecule. Suitably, the molecularly imprinted polymer may comprise or consist of one or more imprints of the template molecule or a part thereof. The molecularly imprinted polymer may comprise or consist of a plurality of imprints of the template molecule or one or more parts thereof. The imprint or one or more imprints is preferably formed, in accordance with the invention, by building a polymer around a magnetically supported template molecule – in this manner, the imprints are preferably samples of the template molecule (which is tethered to the magnetic support) or one or parts of said template molecule.

[0032] A free molecularly imprinted polymer (a free MIP) is a molecularly imprinted polymer that is separated from the template molecule (i.e. in the context of the invention, separated from a magnetically supported template molecule) around which the molecularly imprinted polymer is formed / built. The free molecularly imprinted polymer (a free MIP) is suitably a molecularly imprinted polymer that is separated, isolated, or recovered from its corresponding / complementary magnetic template-bound molecularly imprinted polymer (or magnetic-MIP conjugate, or magnetic-MIP conjugate particles). A free molecularly imprinted polymer (free MIP) may be simply termed a molecularly imprinted polymer (MIP). D) Aspects of a (free) molecularly imprinted polymer, manufacturing methods therefor, and recycling magnetically supported template molecules

[0033] The present invention provides at least the following aspects relating to a (free) molecularly imprinted polymer (including plurality of batches thereof), methods of manufacturing the same, recycling magnetically supported templates, recovering a recycled magnetically supported template molecule, and relevant compositions: D1. A method of manufacturing a (free) molecularly imprinted polymer (MIP), the method comprising: providing a magnetic template-bound molecularly imprinted polymer (or magnetic-MIP conjugate, or magnetic-MIP conjugate particle) as defined herein (i.e. a magnetic template-bound molecularly imprinted polymer comprising or consisting of a magnetically supported template molecule and a molecularly imprinted polymer) or by a method of manufacturing a magnetic template-bound molecularly imprinted polymer (or magnetic-MIP conjugate, or magnetic-MIP conjugate particle) as defined herein; separating the molecularly imprinted polymer from the magnetically supported template molecule of the magnetic template-bound molecularly imprinted polymer; and optionally thereafter isolating the molecularly imprinted polymer. D2. A method of recovering a recycled magnetically supported template molecule from a magnetic template-bound molecularly imprinted polymer, wherein the magnetic template-bound molecularly imprinted polymer (which is suitably a magnetic template-bound molecularly imprinted polymer as defined herein or as provided by a method of manufacturing a magnetic template-bound molecularly imprinted polymer as defined herein) comprises or consists of a magnetically supported template molecule and a molecularly imprinted polymer; the method comprising: separating the molecularly imprinted polymer from the magnetically supported template molecule of the magnetic template-bound molecularly imprinted polymer; and thereafter isolating the magnetically supported template molecule as a recycled magnetically supported template molecule (which may optionally be further processed and / or purified). The molecularly imprinted polymer may also be separately isolated during the same method. The recycled magnetically supported template molecule may be fed back into the same or a similar method as defined herein. The recycled magnetically supported template molecule may be fed into any method described herein involving a magnetically supported template molecule as an input material. D3. A method of manufacturing a (free) molecularly imprinted polymer (MIP), the method comprising: providing a magnetic template-bound molecularly imprinted polymer (or magnetic-MIP conjugate, or magnetic-MIP conjugate particle) as defined herein (i.e. a magnetic template-bound molecularly imprinted polymer comprising or consisting of a magnetically supported template molecule and a molecularly imprinted polymer) or by a method of manufacturing a magnetic template-bound molecularly imprinted polymer (or magnetic-MIP conjugate, or magnetic-MIP conjugate particle) as defined herein; separating the magnetically supported template molecule (or magnetic-template particle), optionally as a recycled magnetically supported template molecule, from the molecularly imprinted polymer of the magnetic template-bound molecularly imprinted polymer (or magnetic-MIP conjugate, or magnetic-MIP conjugate particle); and isolating: a. the molecularly imprinted polymer; and optionally also b. the recycled magnetically supported template molecule (or recycled magnetic-template particle). D4. A method of manufacturing a (free) molecularly imprinted polymer (MIP), the method comprising: i) providing a magnetically supported template molecule (or a magnetic-template particle) as defined herein (whilst this may be a recycled magnetically supported template molecule, in which case preferably one that has been further processed and / or purified, it is preferably non-recycled, i.e. newly-produced) or via a method of manufacturing a magnetically supported template molecule as defined herein;ii) using the magnetically supported template of step i) to form a magnetic template-bound molecularly imprinted polymer (or magnetic-MIP conjugate, or magnetic-MIP conjugate particle) via a method of manufacturing a magnetic template-bound molecularly imprinted polymer as defined herein, wherein the magnetic template-bound molecularly imprinted polymer comprises or consists of: the magnetically supported template molecule and a molecularly imprinted polymer; iii) separating the magnetically supported template molecule from the molecularly imprinted polymer of the magnetic template-bound molecularly imprinted polymer; and iv) isolating: a. the molecularly imprinted polymer; and optionally also b. the magnetically supported template molecule as a recycled magnetically supported template molecule (or recycled magnetic-template particle); and optionally repeating steps ii)-iv) by reusing the recycled magnetically supported template molecule (or recycled magnetic-template particle) isolated in step iv). D5. A method of manufacturing a (free) molecularly imprinted polymer (MIP), the method comprising: i) providing a magnetic template-bound molecularly imprinted polymer (or magnetic-MIP conjugate, or magnetic-MIP conjugate particle) comprising or consisting of: a recycled magnetically supported template molecule (or recycled magnetic-template particle), suitably obtained by a method as defined herein, and a molecularly imprinted polymer, wherein the magnetic template-bound molecularly imprinted polymer is suitably provided by a method of manufacturing a magnetic template-bound molecularly imprinted polymer as defined herein (which involves a recycled magnetically supported template molecule); ii) separating the recycled magnetically supported template molecule (or recycled magnetic-template particle) from the molecularly imprinted polymer of the magnetic template-bound molecularly imprinted polymer; and iii) isolating: a. the molecularly imprinted polymer; and optionally also b. the recycled magnetically supported template molecule (or recycled magnetic-template particle); and optionally repeating steps i)-iii) by reusing the recycled magnetically supported template molecule (or recycled magnetic- template particle) isolated in step iii). D6. A method of manufacturing a (free) molecularly imprinted polymer (MIP), the method comprising: i) providing a magnetic template-bound molecularly imprinted polymer by, or obtained by, a method as defined herein; ii) separating the magnetically supported template molecule from the molecularly imprinted polymer of the magnetic template-bound molecularly imprinted polymer; iii) isolating: a. the molecularly imprinted polymer; and optionally also b. the magnetically supported template molecule as a recycled magnetically supported template molecule; and optionally repeating steps i)-iii) by reusing the recycled magnetically supported template molecule isolated in step iii). D7. A method of manufacturing a (free) molecularly imprinted polymer (MIP), the method comprising: i) providing a magnetically supported template molecule, which comprises or consists of a magnetic core coupled or tethered to one or more (preferably a plurality) of template molecule(s), suitably via a method of manufacturing a magnetically supported template molecule as defined herein; ii) forming a magnetic template-bound molecularly imprinted polymer, which comprises or consists of the (or one or more of the, preferably a plurality of) magnetically supported template molecule(s) bound to a molecularly imprinted polymer via a (or one or more, preferably a plurality of) corresponding complementary imprint(s) within the molecularly imprinted polymer(s), by a method comprising: polymerising one or more monomer(s) (preferably at least one backbone-forming monomer and a cross-linking monomer) in the presence of the magnetically supported template molecule (or a recycled magnetically supported template molecule, when this step is repeated); iii) unbinding and separating the magnetically supported template molecule from the molecularly imprinted polymer of the magnetic template-bound molecularly imprinted polymer, to yield a (free) molecularly imprinted polymer and a recycled magnetically supported template molecule; iv) repeating, one or more times, steps ii)-iii) using the recycled magnetically supported template molecule of step iii) (or of repeated step iii)) as the magnetically supported template molecule for repeated step ii). D8. A method of manufacturing a plurality of batches of a (free) molecularly imprinted polymer (MIP), the method comprising: i) providing a first batch of magnetic template-bound molecularly imprinted polymer (or magnetic-MIP conjugate, or magnetic-MIP conjugate particle) comprising or consisting of: a magnetically supported template molecule (or magnetic-template particle), suitably obtained by a method as defined herein, and a molecularly imprinted polymer, wherein the magnetic template-bound molecularly imprinted polymer issuitably provided by a method of a magnetic template-bound molecularly imprinted polymer as defined herein; ii) separating the magnetically supported template molecule (or magnetic-template particle) from the molecularly imprinted polymer of the magnetic template-bound molecularly imprinted polymer; iii) isolating: a. the molecularly imprinted polymer as a first batch of molecularly imprinted polymer; and b. the magnetically supported template molecule (or magnetic-template particle) as a once-recycled magnetically supported template molecule; iv) forming a polymer (or polymer particle) (which becomes a molecularly imprinted polymer) in the presence of the (or a portion of the) (once-) recycled magnetically supported template molecule (or recycled magnetic- template particle(s)), to furnish a second batch of magnetic template-bound molecularly imprinted polymer (or magnetic-MIP conjugate, or magnetic-MIP conjugate particle) comprising or consisting of: the (once-) recycled magnetically supported template molecule and a (further) molecularly imprinted polymer; v) separating the (once-) recycled magnetically supported template molecule (or recycled magnetic-template particle) from the (further) molecularly imprinted polymer of the second batch of magnetic template-bound molecularly imprinted polymer; vi) isolating: a. the (further) molecularly imprinted polymer as a second batch of molecularly imprinted polymer; and optionally also b. the (once-) recycled magnetically supported template molecule (or magnetic-template particle) as a twice-recycled magnetically supported template molecule; optionally wherein steps iv) to vi) are repeated one or more times (using the twice- and optionally further-recycled magnetically supported template molecule as feedstocks) to furnish third and optionally further batches of molecularly imprinted polymer, suitably up to a maximum of 4 to 10 batches thereof; optionally wherein batches of molecularly imprinted polymer are combined; optionally wherein the (once-, twice-, and / or further-) recycled magnetically supported template molecule are further processed and / or purified between steps iii) and iv) and / or between cycles of steps iv) to vi). D9. A method of manufacturing a plurality of batches of a (free) molecularly imprinted polymer (MIP), the method comprising: i) providing a magnetically supported template molecule (or a magnetic-template particle) as defined herein (whilst this may be a recycled magnetically supported template molecule, in which case preferably one that has been further processed and / or purified, it is preferably non-recycled, i.e. newly-produced) or via a method of manufacturing a magnetically supported template molecule as defined herein; ii) using the magnetically supported template molecule of step i) to form a first batch of magnetic template- bound molecularly imprinted polymer (or magnetic-MIP conjugate, or magnetic-MIP conjugate particle) via a method of manufacturing a magnetic template-bound molecularly imprinted polymer as defined herein, wherein the first batch of magnetic template-bound molecularly imprinted polymer comprises or consists of: the magnetically supported template molecule and a molecularly imprinted polymer; iii) separating the magnetically supported template molecule (or magnetic-template particle) from the molecularly imprinted polymer of the magnetic template-bound molecularly imprinted polymer; iv) isolating: a. the molecularly imprinted polymer as a first batch of molecularly imprinted polymer; and b. the magnetically supported template molecule (or magnetic-template particle) as a once-recycled magnetically supported template molecule; v) forming a polymer (or polymer particle) (which becomes a molecularly imprinted polymer) in the presence of the (or a portion of the) (once-) recycled magnetically supported template molecule (or recycled magnetic- template particle(s)), to furnish a second batch of magnetic template-bound molecularly imprinted polymer (or magnetic-MIP conjugate, or magnetic-MIP conjugate particle) comprising or consisting of: the (once-) recycled magnetically supported template molecule and a (further) molecularly imprinted polymer; vi) separating the (once-) recycled magnetically supported template molecule (or recycled magnetic-template particle) from the (further) molecularly imprinted polymer of the second batch of magnetic template-bound molecularly imprinted polymer; vii) isolating: a. the (further) molecularly imprinted polymer as a second batch of molecularly imprinted polymer; and optionally also b. the (once-) recycled magnetically supported template molecule (or magnetic-template particle) as a twice-recycled magnetically supported template molecule;optionally wherein steps v) to vii) are repeated one or times (using the twice- and optionally further-recycled magnetically supported template molecule as feedstocks) to furnish third and optionally further batches of molecularly imprinted polymer, suitably up to a maximum of 4 to 10 batches thereof; optionally wherein batches of molecularly imprinted polymer are combined; optionally wherein the (once-, twice-, and / or further-) recycled magnetically supported template molecule are further processed and / or purified between steps iv) and v) and / or between cycles of steps v) to vii). D10. A method of manufacturing a (free) molecularly imprinted polymer (MIP) comprising an imprint of a template molecule (or a part thereof), the method comprising: i) providing a supported molecular template comprising, consisting of, or otherwise characterised by the template molecule conjugated with a support; ii) performing a polymerisation reaction in the presence of the supported molecular template to form a template- bound molecularly imprinted polymer comprising a molecularly imprinted polymer bound to the supported molecular template; and iii) separating the molecularly imprinted polymer from the supported molecular template; iv) optionally recycling the same supported molecular template for reuse in steps i)-iii); v) optionally isolating the molecularly imprinted polymer. D11. A method of manufacturing MIPs, the method comprising performing a polymerisation reaction in the presence of a magnetically supported template molecule, and releasing any magnetically supported template molecule bound within the MIPs (e.g. by heat and / or sonication, preferably not by heat, most preferably by sonication). D12. A method of manufacturing MIPs, the method comprising performing a polymerisation reaction in the presence of a magnetically supported template molecule, and extracting (preferably via sonication and magnetisation) the magnetically supported template molecule from magnetic template-bound molecularly imprinted polymers, preferably directly from the reaction mixture or bulk solution within which the MIPs are formed. D13. A method of manufacturing MIPs, the method comprising performing a polymerisation reaction in the presence of a magnetically-tethered template molecule, thereafter extracting (suitably by magnetic extraction) the magnetically-tethered template molecule, and subsequently reusing the magnetic-tethered template molecule in further MIP production (optionally via this same method). D14. A (free) molecularly imprinted polymer (MIP) obtainable by, obtained by, or directly obtained by a method of manufacturing a (free) molecularly imprinted polymer as defined herein. D15. A (free) molecularly imprinted polymer (MIP), wherein the molecularly imprinted polymer comprises one or more imprints of a magnetically supported template molecule (suitably as defined herein). Suitably the (free) molecularly imprinted polymer (MIP) comprises a plurality of imprints of a magnetically supported template molecule (suitably as defined herein). D16. A composition comprising or consisting of a molecularly imprinted polymer (MIP) as defined (or as obtained by a method defined) herein.

[0034] Features and sub-features of any of aspects D1-D16 are applicable to each other and indeed to any (free) molecularly imprinted polymer, methods of manufacturing the same, and recycling magnetically supported templates, and related compositions described here or elsewhere herein.

[0035] “Separating” a magnetically supported template molecule (or magnetic-template particle) from a molecularly imprinted polymer to which it is found within a magnetic template-bound molecularly imprinted polymer (or magnetic-MIP conjugate, or magnetic-MIP conjugate particle) preferably involves: i) Unbinding the magnetically supported template molecule from the molecularly imprinted polymer, preferably by releasing an “unbound” magnetically supported template molecule from its complementary imprint (or binding site) within the molecularly imprinted polymer; and ii) Applying a magnetic field to magnetically constrain (e.g. magnetically attract, localise, and / or harness) unbound magnetically supported template molecules whilst the molecularly imprinted polymer is otherwise separated therefrom (e.g. by washing, pipetting, decanting, pumping, etc.); optionally thereafter isolating the molecularly imprinted polymer and optionally separately isolating the unbound magnetically supported template molecules (after removing the magnetic field).

[0036] “Unbinding” the magnetically supported template molecule from the molecularly imprinted polymer, preferably by releasing an “unbound” magnetically supported template molecule from its complementary imprint (or binding site) within the molecularly imprinted polymer, may be affected by a variety of methods known in the art. Preferably, however, to avoid or mitigate adverse effects on the template molecule tethered to the magnetic support, unbinding involves mild unbinding conditions, preferably avoiding elevated temperatures (especially elevating temperatures above 40oC). Preferably unbinding comprises sonication, and suitably sonication for less than 30 minutes, more suitably less than 20 minutes, most preferably less than 10 minutes (e.g. using a VWR ultrasonicator (600W, 45kHz); max 5 minutes) - longer sonication times can undermine the stability of a template molecule (especially a biological molecule such as a protein).

[0037] As explained herein, the method of manufacturing (free) MIPs may involve one or more monomers that comprise reactive moieties that are coupled to a detectable moiety after the polymer has been formed, and that the (free) molecularlyimprinted polymer (MIP) may be labelled, for instance or fluorescent-labelled. As such, it will be understood by those skilled in the art that references herein to a molecularly imprinted polymer, especially a free molecularly imprinted polymer (MIP), includes derivatives thereof (e.g. conjugates, post-functionalised, or post-labelled versions thereof), and that methods of manufacturing a (free) molecularly imprinted polymer (MIP) may optionally further comprise a step of derivatising (e.g. conjugating, post-functionalising, post-labelling). Derivatising preferably comprises coupling at least one derivatising molecule (e.g. an enzyme, a radioactive label, a fluorescent label, or redox label) to the initially-obtained (free) molecularly imprinted polymer (MIP), preferably via a reactive moiety (or functionalisable group) provided by one or more of the monomers used in the formation of the molecularly imprinted polymer. The functionalisable group may be, for instance, be a pendent amino group (-NH2) or carboxylate group (-CO2H). An APMA monomer can provide a pendent amino group to which a derivatising molecule may be coupled by means well known in the art. Such derivatising affords a derivatised molecularly imprinted polymer. A derivatised molecularly imprinted polymer may be especially useful in assaying. For instance, an enzyme-labelled MIP can be deployed in a manner similar to an enzyme-labelled antibody. For instance, an enzyme-labelled MIP may be used in immunoassays (e.g. ELISA - Enzyme-Linked Immunosorbent Assay).

[0038] Any of methods D1-D13 (and any corresponding sub-definitions or modifications thereof defined elsewhere herein) may comprise further processing the (free) molecular imprinted polymer after its formation (suitably after its unbinding and separation). Such further processing may, for instance, involve further modifying the (free) molecular imprinted polymer, be it physically and / or chemically. However, the methods of D1-D13 are in any case intended to encompass and accommodate such further processing, and the (free) molecular imprinted polymer of D14 and D15, and the (free) molecular imprinted polymer contained within the composition of D16, are intended to encompass any such further processed (free) molecular imprinted polymers. The (free) molecular imprinted polymer of D14 and D15, and the (free) molecular imprinted polymer contained within the composition of D16, may as such have been manufactured by said methods.

[0039] Any of methods D1-D13 (and any corresponding sub-definitions or modifications thereof defined elsewhere herein) may further comprise coupling a detectable moiety to reactive moieties of one or more of the monomers after the (free) molecular imprinted polymer has been formed. This detectable moiety may be or may be part of a label (e.g. radio- label, fluorescent-label, enzyme-label, or redox label). The detectable moiety may be or may comprise an enzyme. The (free) molecular imprinted polymer of D14 and D15, and the (free) molecular imprinted polymer contained within the composition of D16, may have been manufactured by said methods.

[0040] Any of methods D1-D13 (and any corresponding sub-definitions or modifications thereof defined elsewhere herein) may further comprise derivatising, functionalising, or conjugating the (free) molecular imprinted polymer to yield a derivatised (free) molecular imprinted polymer. However, the methods of D1-D13 are in any case intended to encompass such eventualities, and the (free) molecular imprinted polymer of D14 and D15, and the (free) molecular imprinted polymer contained within the composition of D16, are intended to encompass any such derivatised (free) molecular imprinted polymers. Derivatising suitably comprises coupling a derivatising molecule (e.g. a molecule being or comprising a radio- label, fluorescence label, enzyme, and / or other molecule, including another or the same MIP) to the (free) molecular imprinted polymer, be it directly or indirectly (e.g. via a cross-linker or further functional molecule).

[0041] Any of methods D1-D13 (and any corresponding sub-definitions or modifications thereof defined elsewhere herein) may further comprise labelling the (free) molecular imprinted polymer to yield a labelled (free) molecular imprinted polymer. However, the methods of D1-D13 are in any case intended to encompass such eventualities, and the (free) molecular imprinted polymer of D14 and D15, and the (free) molecular imprinted polymer contained within the composition of D16, are intended to encompass any such labelled (free) molecular imprinted polymers. Labelling suitably comprises coupling a label molecule (e.g. a molecule being or comprising a radio-label, fluorescence label, enzyme label, and / or other label, including another or the same MIP) to the (free) molecular imprinted polymer, be it directly or indirectly (e.g. via a cross-linker or further functional molecule). Any of methods D1-D13 (and any corresponding sub-definitions or modifications thereof defined elsewhere herein) may further comprise conjugating (or coupling, be it directly or indirectly) the (free) molecular imprinted polymer with an enzyme to yield an enzyme-MIP conjugate. However, the methods of D1-D13 are in any case intended to encompass such eventualities, and the (free) molecular imprinted polymer of D14 and D15, and the (free) molecular imprinted polymer contained within the composition of D16, are intended to encompass any such enzyme-MIP conjugates. Such enzyme-MIP conjugates may be particularly advantageous for use in assays, such as ELISAs. Uses of Molecularly imprinted polymers

[0042] A variety of uses of molecularly imprinted polymers are well known in the art. According to an aspect of the present invention, there is provided a use of a (free) molecularly imprinted polymer (preferably formed by a method of manufacturing a molecularly imprinted polymer as defined herein, or a method of manufacturing a plurality of batches of a molecularly imprinted polymer as defined herein), suitably wherein the use is as defined herein (including any uses described in the “BACKGROUND” section of this disclosure). E) Aspects of a Use of a (free) molecularly imprinted polymer

[0043] The present invention provides at least the following aspects relating to use of a (free) molecularly imprinted polymer (or a plurality of batches thereof), wherein the (free) molecularly imprinted polymer is suitably as defined herein or provided by a method of manufacturing the same as defined herein:E1. Use of a (free) molecularly imprinted polymer as material in sensors or binding assays (e.g. “molecularly imprinted assays”), for instance, for the detection of specific molecules, such as antimicrobial agents, dyes, and / or residues in foods. E2. Use of a (free) molecularly imprinted polymer as a chromatographic stationary phase for solid phase extraction of target molecules. E3. Use of a (free) molecularly imprinted polymer in removing byproducts in chemical reaction mixtures. E4. Use of a (free) molecularly imprinted polymer in isolating a protein. E5. Use of a (free) molecularly imprinted polymer to facilitate protein crystallisation (e.g. after isolating the relevant protein). E6. Use of a (free) molecularly imprinted polymer as an artificial antibody. E7. Use of a (free) molecularly imprinted polymer as artificial enzyme-like binding site to facilitate drug development and screening. E8. Use of a (free) molecularly imprinted polymer as a biological receptor mimetic, and suitably thus a therapeutic use to reduce receptor overloads. E9. Use of a (free) molecularly imprinted polymer for the temporary binding of and controlled release of drugs. E10. Use of a (free) molecularly imprinted polymer in drug monitoring or drug detection (e.g. narcotic drug testing, or testing for drug use in sports). E11. Use of a (free) molecularly imprinted polymer in assays, for instance, immunoassays. The assay may be an ELISA (Enzyme-Linked Immunosorbent Assay), especially where the (free) molecularly imprinted polymer is linked to an enzyme (which may be coupled or conjugated to a (free) MIP after its formation). The assay may be a Western Blot assay. The assay may be an immunofluorescence assay. The assay may be a rapid antigen assay / test (e.g. lateral flow test). E12. Use of a (free) molecularly imprinted polymer in the detection of biomarkers (e.g. in a blood sample, or sample from the body). E13. Use of a (free) molecularly imprinted polymer in facilitating assays (especially where e.g. radio-labelled or fluorescent-labelled). E14. Use of a (free) molecularly imprinted polymer in lateral flow tests. E15. Use in the production of MIP-bearing electrodes, which are suitably further used or usable as defined herein. E16. Use of a (free) molecularly imprinted polymer (MIP) to bind a target substrate (which is suitably identical or similar to, or has one or more parts identical or similar to, the or part(s) of the template molecule used to produce the MIP). E17. Use of a (free) molecularly imprinted polymer (MIP) to detect a target substrate / molecule (which is suitably identical or similar to, or has one or more parts identical or similar to, the or part(s) of the template molecule used to produce the MIP). E18. Use of a (free) molecularly imprinted polymer (MIP) in a method of diagnosing a disease or disorder in a subject. E19. Use of a (free) molecularly imprinted polymer (MIP) in a method of diagnosing a disease or disorder in a subject, wherein the method of diagnosing the disease or disorder comprises detecting a target substrate / molecule (which is suitably identical or similar to, or has one or more parts identical or similar to, the or part(s) of the template molecule used to produce the MIP) that serves as a biomarker for said disease or disorder. E20. Use of a (free) molecularly imprinted polymer (MIP) in a method of treating a disease or disorder in a subject. E21. Use of a (free) molecularly imprinted polymer (MIP) in a method of treating a disease or disorder in a subject, wherein the method of treating the disease or disorder administering a therapeutically effective amount of the MIP to a subject. E22. A method of treating a disease or disorder in a subject, the method comprising administering a therapeutically effective amount of the MIP to the subject. E23. An MIP for use in the treatment of a disease or disorder in a subject. E24. Use of an MIP (preferably as defined or as obtainable by a method defined herein) in the manufacture of a medicament for treating a disease or disorder in a subject. E25. A pharmaceutical composition (whether injectable, orally-ingestible / orally-administrable, etc.) comprising an MIP, suitably for use in the treatment of a disease or disorder in a subject. The pharmaceutical composition may suitably further comprise one or more pharmaceutically acceptable excipient(s), diluent(s), carrier(s), or any mixture thereof.

[0044] Suitably, an immunoassay, as defined herein, may include ELISA, Western Blot, Immunofluorescence assay, rapid antigen tests (e.g. lateral flow tests), and the like.

[0045] Features and sub-features of any of aspects E1-E25 are applicable to each other and indeed to any (free) molecularly imprinted polymer, uses thereof, and related compositions described here or elsewhere herein.Production of MIP-bearing Electrode

[0046] An MIP-bearing electrode is an electrode bearing on the surface thereof (or within a coating upon the surface thereof) an MIP (suitably as defined herein, or provided by a method of manufacturing the same as defined herein). Such an electrode may be a cyclic voltammetry electrode. An MIP-bearing electrode can be particularly useful in the electrochemical detection of target molecules that resemble (or comprise a part that resembles) the template molecule (or a part thereof) used in the production of the relevant MIP. Even very low concentrations of such a target (or template) molecule can be detected, especially using the electrode in electrochemical impedance spectroscopy (EIS), but also using cyclic voltammetry (CV).

[0047] A skilled person understands how to incorporate an initially uncoated electrode within an electrochemical cell in order to facilitate production of the MIP-bearing electrode. F) Aspects of an MIP-bearing Electrode

[0048] The present invention provides at least the following aspects relating to an MIP-bearing electrode, wherein the MIP is suitably a (free) molecularly imprinted polymer as defined herein or as provided by a method of manufacturing the same as defined herein: F1. A method of manufacturing a molecularly imprinted polymer-bearing electrode (or molecularly imprinted polymer- impregnated electrode), the method comprising forming a coating upon an electrode, which coating comprises a (free) molecularly imprinted polymer. F2. A method of manufacturing a molecularly imprinted polymer-bearing electrode, the method comprising forming a polymeric coating upon an electrode, wherein the polymeric coating comprises a (free) molecularly imprinted polymer (suitably as defined herein, suitably obtained by a method of manufacturing an MIP as defined herein). F3. A method of manufacturing a molecularly imprinted polymer-bearing electrode, the method comprising: providing a (free) molecularly imprinted polymer; providing an electrode; forming a coating polymer upon the surface of the electrode (suitably through electrochemically initiated and / or propagated polymerisation of one or more corresponding coating monomers) in the presence of the molecularly imprinted polymer (suitably to trap the molecularly imprinted polymer within the coating polymer as the coating polymer is formed upon the surface of the electrode, suitably trapping within layers thereof, though preferably at least some MIP(s) or at least some complementary imprints of the MIP(s) are exposed at the surface of the coating polymer and thereby available for binding). F4. A method of manufacturing a molecularly imprinted polymer-bearing electrode, the method comprising: providing an electrode; and forming a molecularly imprinted polymer upon the surface of the electrode (suitably through electrochemically initiated and / or propagated polymerisation of one or more corresponding coating monomers) in the presence of a (preferably magnetically-) supported template molecule (suitably as defined herein). F5. A molecularly imprinted polymer-bearing electrode obtainable by, obtained by, or directly obtained by a method of manufacturing a molecularly imprinted polymer-bearing electrode as defined herein. F6. A molecularly imprinted polymer-bearing electrode comprising an electrode with a coating, the coating comprising a molecularly imprinted polymer as defined herein (or a molecularly imprinted polymer provided by a method of manufacturing a molecularly imprinted polymer as defined herein). F7. A molecularly imprinted polymer-bearing electrode comprising an electrode coated with a coating polymer and a molecularly imprinted polymer trapped between (but not covalently bonded to) one or more layers of the coating polymer, though preferably at least some MIP(s) or at least some complementary imprints of the MIP(s) are exposed at the surface of the coating polymer and thereby available for binding.

[0049] The molecularly imprinted polymer is suitably embedded within (or within layers of) any coating (upon the electrode), suitably as localised islands of molecularly imprinted polymer, though preferably at least some MIP(s) or at least some complementary imprints of the MIP(s) are exposed at the surface of the coating polymer and thereby available for binding. Any coating (upon the electrode) suitably comprises the molecularly imprinted polymer and at least one (other) coating polymer. The coating polymer may be formed upon the electrode by (e.g. electrochemically initiated and / or electrochemically propagated) polymerisation of one or more corresponding coating monomers, suitably in the presence of the molecularly imprinted polymer. In preferred embodiments the molecularly imprinted polymer is pre-formed and thus separate from the coating polymer. However, in some embodiments, the coating polymer may be formed in the presence of a (preferably magnetically-) supported template molecule (suitably as defined herein) such that the coating polymer is and / or comprises the molecularly imprinted polymer, suitably formed in situ e.g. by electrochemically initiated and / or electrochemically propagated polymerisation of one or more corresponding coating monomers).

[0050] Preferably, where present following MIP-bearing electrode production (e.g. as per F4 above), the (preferably magnetically-) supported template molecule is separated from the molecularly imprinted polymer-bearing electrode (e.g. by sonication and / or magnetic attraction). The (preferably magnetically-) supported template molecule may then be reused / recycled, suitably as defined herein or in further rounds of manufacturing MIP-bearing electrodes. Whilst direct MIPproduction, in situ on the electrode, has been , this has not been previously achieved or even contemplated with a (preferably magnetically-) supported template molecule.

[0051] Features and sub-features of any of aspects F1-F7 are applicable to each other and indeed to any MIP-bearing electrode, and method of manufacturing the same, described here or elsewhere herein. Use of MIP-bearing Electrode

[0052] MIP-bearing electrodes have a variety of uses. A skilled person understands how to incorporate such electrodes within an electrochemical cell to facilitate its use. G) Aspects of a Use of An MIP-bearing Electrode

[0053] The present invention provides at least the following aspects relating to a use of an MIP-bearing electrode, wherein the MIP is suitably a (free) molecularly imprinted polymer as defined herein or as provided by a method of manufacturing the same as defined herein: G1. Use of an MIP-bearing electrode (as defined herein, or obtained by a method as defined herein) in the detection, characterisation, and / or quantification of a target molecule (e.g. a template molecule). G2. A molecular detector (or molecular detector device) comprising an MIP-bearing electrode (as defined herein, or obtained by a method as defined herein), wherein the molecular detector is operable to electrochemically detect a target molecule (e.g. a template molecule).

[0054] Features and sub-features of any of aspects G1-G2 are applicable to each other and indeed to any use of an MIP- bearing electrode described here or elsewhere herein. General Points Regarding these Aspect of the Invention

[0055] As a skilled person will readily appreciate, any of the aforementioned methods may be concatenated. A skilled person will also appreciate that inputs for downstream methods may be provided by any upstream methods or any combination of upstream methods that furnish such inputs. Whilst the novelty and inventiveness of downstream methods may stem from features of certain upstream methods (e.g. recycling of certain materials) and whilst, in the absence of the present disclosure, it may have been far from obvious that such features of certain upstream methods would produce viable inputs for the downstream methods, the skilled person would nonetheless understand, from the present disclosure, that downstream methods can be implemented using any of the described upstream methods – i.e. in light of information provided in the present disclosure, upstream methods of furnishing downstream inputs are neither essential nor indispensable to the functioning of downstream methods that utilise said inputs, and modifications to downstream methods are not required to compensate for variations in the upstream methods described herein.

[0056] Any features, including optional, suitable, and preferred features, described in relation to any particular aspect of the invention may also be features, including optional, suitable and preferred features, of any other aspect of the present invention unless incompatible therewith. BRIEF DESCRIPTION OF THE DRAWINGS

[0057] For a better understanding of the invention, and to show how embodiments of the same are put into effect, reference is now made, by way of example, to the following diagrammatic drawings, in which:

[0058] Figure 1 is a schematic diagram illustrating a process for producing NanoMIPs (nano-molecularly imprinted polymers) by: (A) coupling protein molecules with functionalised magnetic nanoparticles (MNPs) to produce (B) a magnetically supported protein template particles (MNP@Protein), which are then (C) mixed with monomers that polymerise around the protein templates of the MNP@Protein particles which, whereafter the MNP@Protein particles are released from the resulting polymers to yield (D) NanoMIPs which are then harvested.

[0059] Figure 2 is a schematic diagram illustrating (A) forming a layer containing entrapped NanoMIPs of Figure 1 upon the surface of an electrode to produce a protein-specific detector electrode; and (B) using said protein-specific detector electrode to selectively bind and detect the specific protein.

[0060] Figure 3 shows a DLS spectrum of the MNP@CHO produced when using a microwave synthesis ramp time of 18 °C / min over 10 min, with overall synthesis being terminated after a further 20 min, and an average particle size of 120 nm is indicated.

[0061] Figure 4 shows SEM images of the MNP@CHO produced when using a microwave synthesis ramp time of 18 °C / min over 10 min, with overall synthesis being terminated after a further ~ 20 min, and particle size was determined to be 60-80 nm (white circles represent individual MNP particles within multiple clumped particles).

[0062] Figure 5 is a graph showing the degree of protein functionalisation / coupling of MNP@CHO over time – circles are for bare MNPs (which obviously don’t couple to protein), and squares are for MNP@CHO. A 1mg / mL BHb starting solution was used and amount adsorbed with time was determined by subtraction of amount of BHb remaining in solution (measured A. Stephen, S. Dennison, M. Holden and S. Reddy, The Analyst, 2023, 148using UV / Vis spectroscopy) during the protein conjugation Fifteen minutes was determined to be the minimum time required to complete the conjugation process.

[0063] Figure 6 shows the DLS spectrum for MNP@BHb. The particles are again monodisperse but have now more thandoubled in size to approximately 330 nm.

[0064] Figure 7 shows a Randles Equivalent Circuit model used to determine charge transfer resistance (RCT) from electrochemical impedance spectra of bare and MIP-loaded disposable BT-Au SPCEs.

[0065] Figure 8 shows AFM images of (a) bare, (b) electrochemically grown thin film layer, and (c) E-layer-entrapped nanoMIPs. All samples were immersed in PBS, which were imaged using a Bruker Dimension Icon® AFM with a NanoScope 6 controller in Peak Force Tapping™ mode with silicon nitride cantilevers (SNL-10, nominal spring constant 0.35 Nm-1and SCANASYST-FLUID, nominal spring constant 0.7 Nm-1).

[0066] Figure 9 is a graph showing EIS determination of protein binding on E-layer entrapped nanoMIPs. Plot for BHb concentration vs ΔRCTusing the nanoMIPs. The dynamic linear range was 100 fM to 10 nM with a limit of quantitation of 10 fM.

[0067] Figure 10 shows NanoNIP islands linear range (this is control polymer (nanoNIP) and gives very little signal; a few ohms compared to 100s of ohms with nanoMIP).

[0068] Figure 11 shows BHb nanoMIP islands cross-bound with bovine serum albumin, a non-target protein and gives very little signal; a few ohms compared to 100s of ohms with target protein binding.

[0069] Figure 12 is a bar chart showing % change (% yield loss) in the production yield of nanoMIPs between subsequent uses of same MNP@protein particles (i.e. cycles 2 to 5).

[0070] Figure 13 is a bar chart showing a summary of CD spectra data demonstrating that 5 cycles of nanoMIP production does not change BHb structure on MNP@BHb.

[0071] Figure 14 is a bar chart demonstrating that each batch / cycle retained its affinity for target protein (BHb) and no signal for non-target (BSA) (both at 1 nM).

[0072] Figure 15 is a bar chart showing the effect of microwave temperature ramp time, during MNP production, on ultimate nanoMIP island electrochemical signal.

[0073] Figure 16 is a plot showing the effect of ramp time (taken from 20 to 200 °C) on MNP particle size. Ramp time, R=600s is equivalent 18 °C / min ramping over 10 min; R = 120s is equivalent to 90 °C / min ramping over 2 min.

[0074] Figure 17 shows EIS data showing the trend of BHb rebinding to E-layer of nanoMIP islands. The plots for “post polymerisation”, “100 fM” and “1 pM” merge into visibly indistinguishable lines.

[0075] Figure 18 shows cyclic voltammetry (CV) plots used to deposit E-layers entrapping nanoMIP islands.

[0076] Figure 19 shows the change in ferricyanide (redox marker) CV response: before, after E-layer with entrapped nanoMIP islands and after 1 nM target protein reloading. CV is not sensitive enough to pick up on subtle changes following 1nM protein addition, hence why EIS was used.

[0077] Figure 20 shows full CD data comparing individual batches / cycles to a control protein.

[0078] Figure 21 shows an AFM image of densely packed nanoMIP islands on a gold electrode.

[0079] Figure 22 is a bar chart showing how the microwave ramp time (up to 200oC) affects the average hydrodynamic size (nm) of the MNP – the longer the ramp time the larger the size.

[0080] Figure 23 is a bar chart showing how the microwave ramp time (up to 200oC) affects the average hydrodynamic size (nm) of the MNP – this shows that if the ramp time is too long (here at 15 min ramp time), the method is less likely to provide useful material.

[0081] Figure 24 is a TEM image of MNPs produced with a ramp time of 2 mins.

[0082] Figure 25 is a TEM image of MNPs produced with a ramp time of 6 mins.

[0083] Figure 26 is a TEM image of MNPs produced with a ramp time of 8 mins.

[0084] Figure 27 is a zoomed in TEM image of MNPs produced with a ramp time of 10 mins.

[0085] Figure 28 is a zoomed out TEM image of MNPs produced with a ramp time of 10 mins.

[0086] Figure 29 shows TEM images, A and B, of MNP@CHO produced using microwave synthesis with a 10-minute ramp time at 18 °C / min followed by a 20 min dwell time at 200 °C. The average cluster size (A) was determined to be 91 ±15 nm and the average core size in (B) was determined by TEM to be approximately 18 ±5 nm.

[0087] Figure 30 shows a bar chart showing the change in production yield of nanoMIPs with 5 successive cycles of re- using MNP@BHb.

[0088] Figure 31 shows a graph of ^RCT(^) against BHb concentration for BHb nanoMIPs formed using 10% APMA. DETAILED DESCRIPTION OF THE INVENTION DEFINITIONS

[0089] Unless otherwise stated, the following terms used in the specification and claims have the following meanings set out below.

[0090] Throughout the description and claims of this the words “comprise” and “contain” and variations of them mean “including but not limited to”, and they are not intended to (and do not) exclude other moieties, additives, components, integers or steps. Throughout the description and claims of this specification, the singular encompasses the plural unless the context otherwise requires. In particular, where the indefinite article is used, the specification is to be understood as contemplating plurality as well as singularity, unless the context requires otherwise.

[0091] Features, integers, characteristics, compounds, chemical moieties or groups described in conjunction with a particular aspect, embodiment or example of the invention are to be understood to be applicable to any other aspect, embodiment or example described herein unless incompatible therewith. All of the features disclosed in this specification (including any accompanying claims, abstract and drawings), and / or all of the steps of any method or process so disclosed, may be combined in any combination, except combinations where at least some of such features and / or steps are mutually exclusive. The invention is not restricted to the details of any foregoing embodiments. The invention extends to any novel one, or any novel combination, of the features disclosed in this specification (including any accompanying claims, abstract and drawings), or to any novel one, or any novel combination, of the steps of any method or process so disclosed.

[0092] Compatible features defined at the same level of preference (e.g. “suitably”, “preferably”, “more preferably”, “most preferably”) are especially combinable.

[0093] The reader’s attention is directed to all papers and documents which are filed concurrently with or previous to this specification in connection with this application and which are open to public inspection with this specification, and the contents of all such papers and documents are incorporated herein by reference.

[0094] For the avoidance of doubt, it is hereby stated that the information disclosed earlier in this specification under the heading “Background” is relevant to the invention and is to be read as part of the disclosure of the invention.

[0095] Unless stated otherwise, any reference herein to an “average” value is intended to relate to the mean value.

[0096] Unless otherwise stated, wherever a dependent claim / paragraph depends upon a stipulated claim / paragraph “…or any other claims / paragraphs dependent thereon” (or similar such language, e.g. “the composition as defined in paragraph A1 or any preceding paragraphs dependent thereon”), such “other claims / paragraphs” suitably include any claims / paragraphs that depend directly or indirectly (i.e. via one or more other dependencies) on the stipulated claim / paragraph. In such a manner, numbered paragraphs, much like patent claims, may be multiply dependent.

[0097] It will be readily understood that numbered paragraphs can include alphanumerically-“numbered” paragraphs (e.g. A1-A12, which are sequentially numbered 1-12 each with the prefix “A”). Wherever a range of numbered paragraphs is designated, for example “the composition of A1-A12”, as with patent claims this discloses each numbered paragraph within that range – in the case of “the composition of B1-B6”, this includes “the composition” as defined in any of paragraphs B1, B2, B3, B4, B5, and B6.

[0098] It is to be appreciated that references to “treating” or “treatment” include prophylaxis as well as the alleviation of established symptoms of a condition. “Treating” or “treatment” of a state, disorder or condition therefore includes: (1) preventing or delaying the appearance of clinical symptoms of the state, disorder or condition developing in a human that may be afflicted with or predisposed to the state, disorder or condition but does not yet experience or display clinical or subclinical symptoms of the state, disorder or condition, (2) inhibiting the state, disorder or condition, i.e., arresting, reducing or delaying the development of the disease or a relapse thereof (in case of maintenance treatment) or at least one clinical or subclinical symptom thereof, or (3) relieving or attenuating the disease, i.e., causing regression of the state, disorder or condition or at least one of its clinical or subclinical symptoms.

[0099] Suitably, unless stated otherwise, where reference is made to a parameter (e.g. pH, pKa, etc.) or state of a material (e.g. liquid, gas, etc.) which may depend on pressure and / or temperature, suitably in the absence of further clarification such a reference refers to said parameter at standard ambient temperature and pressure (SATP). SATP is a temperature of 298.15 K (25 °C, 77 °F) and an absolute pressure of 100 kPa (14.504 psi, 0.987 atm).

[0100] Herein, the term “particle size” or “pore size” refers respectively to the length of the longest dimension of a given particle or pore. Commonly, a (average) particle size may be the (average) diameter of the particle. Particle and pore sizes may be measured using methods well known in the art, including a laser particle size analyser and / or electron microscopes (e.g. transmission electron microscope, TEM, scanning electron microscope, SEM), dynamic light scattering (DLS), atomic force microscopy (AFM), and so forth. Generally, unless stated otherwise, particle sizes defined herein are determined or determinable via DLS. As such, particle sizes defined herein may suitably be hydrodynamic diameters. Such particle sizes may therefore be cluster (i.e. agglomerate) sizes rather than individual core sizes.

[0101] Where a composition is said to comprise a plurality of stipulated ingredients (optionally in stipulated amounts of concentrations), said composition may optionally include additional ingredients other than those stipulated. However, a composition said to comprise a plurality of stipulated ingredients may in fact consist essentially of or consist of all the stipulated ingredients, optionally in the amounts specified, optionally (where compatible with the applicable context) with a solvent system (e.g. water). In either circumstance, an individual component may itself comprise, consist essentially of, or consist of a sub-component or one or more sub-components. Herein, wherever the term “comprise” is used it may (whether in the context of a composition or component / ingredient thereof), where compatible within the applicable context, be replaced by “consists essentially of” or “consists of”.

[0102] Amounts given herein in respect of any given ingredient class will, unless specified to the contrary, be applicable to any specific ingredient within said ingredient class.

[0103] Herein, amounts stipulated for components and whether specified in terms of “parts”, ppm (parts per million), percentages (%, e.g. wt%), or ratios, are intended to be by weight, unless stated otherwise.

[0104] Herein, where something (e.g. a mixture, such as a reaction mixture) is said to be “exposed to a magnetic field” (or similar such wording), suitably this means purposefully bringing a magnet to or generating a magnetic field (e.g. via an electromagnet) near to the same – i.e. it does not include naturally prevailing magnetic fields. GENERAL POINTS AND ADVANTAGES REGARDING THE INVENTION

[0105] The present application describes elegant and surprising solutions to the problems mentioned in the “BACKGROUND” section above. In particular, downstream methods and products (including inventive aspects thereof) benefit significantly from the inventive aspects of the upstream methods and products. In particular, the inventive functionalised magnetic support and magnetically support template molecules derived therefrom afford excellent quality and excellent yields of downstream MIPs, and consequently excellent downstream electrodes derived therefrom. The inventive functionalised magnetic support and magnetically support template molecules allow the magnetically support template molecules to be recycled during MIP production, which would not be otherwise feasible. Such inventive functionalised magnetic support and magnetically support template molecules also enable the production of MIPs with particularly advantageous properties, including multiple imprints and a diversity of imprint structures within the same MIP, thus allowing MIPs to bind different parts of the same target / template molecule. This in turn augments downstream applications.

[0106] The methodologies elucidated in the present application unlock rapid, low cost, high yield, and high quality industrial production of MIPs imprinted by costly template molecules, thus making a huge technical contribution to the art. A) FUNCTIONALISED MAGNETIC SUPPORT (OR FUNCTIONALISED MAGNETIC PARTICLE)

[0107] Aspects and features of the invention relating to a functionalised magnetic support (or functionalised magnetic particle) are described above in the relevant sub-section of the “SUMMARY OF THE INVENTION” and, in particular in numbered paragraphs A1-A8. Further features and sub-features of the same are elaborated below, all of which are applicable to any of the aforesaid aspects and to any other compatible aspects and embodiments described elsewhere herein.

[0108] A functionalised magnetic support (or functionalised magnetic particle) comprises or consists of a magnetic core (or magnetic particle) bearing, preferably on the surface thereof, a reactive functional group (e.g. aldehyde group, carboxylate group, or amine group) or two or more such reactive functional groups. The magnetic core may be produced in the presence of a functionalising molecule so that the magnetic core and reactive functional group(s) thereof are produced in situ – i.e. in a one-pot reaction.

[0109] A variety of different magnetic cores and different reactive functional groups, and combinations thereof, are possible without departing from the invention. It will, however, be self-evident that some functionalised magnetic supports (and configurations thereof) are more preferred than others for downstream use (e.g. in the production of MIPs, especially in multi-batch MIP production).

[0110] The functionalised magnetic support (or functionalised magnetic particle) suitably has an average particle size between 1 and 1000 nm, or an average particle size between 100 and 300 nm.

[0111] Preferably the functionalised magnetic support (or functionalised magnetic particle) has an average particle size between 5 and 500 nm, more preferably between 10 and 300 nm, more preferably between 20 and 200 nm, most preferably between 50 and 180 nm.

[0112] A functionalised magnetic support particle may in fact be an individual cluster (i.e. agglomerate). As such, the (average) particle size may be an (average) cluster size. The particle size (and thus suitably cluster size) may be determined by DLS and / or SEM (and / or TEM). DLS is suitably the default method of measuring particle sizes - this generally measures cluster size.

[0113] The size of an individual functionalised magnetic support particle (i.e. as distinct from the size of a cluster thereof) is more difficult to establish but may be separately determined, for instance, by reference to TEM images or magnetic measurement techniques – i.e. via TEM or magnetic measurement techniques the size of an individual functionalised magnetic support particles, generally within a cluster but also on its own, may suitably be determined. Preferably, the average size of individual functionalised magnetic support particles (as distinct from clusters thereof), is between 0.5 and 100 nm (suitably corresponding to an average cluster size of 5 and 500 nm), more preferably between 1 and 50 nm (suitably corresponding to an average cluster size of 10 and 300 nm), more preferably between 3 and 20 nm (suitably corresponding to an average cluster size of 20 and 200 nm), most preferably between 5 and 15 nm (suitably corresponding to an average cluster size of 50 and 180 nm).

[0114] Whilst the magnetic core, preferably as defined below, is suitably functionalised with one or more reactive functional group(s), preferably the magnetic core is otherwise uncoated. Preferably the magnetic core, and indeed the functionalised magnetic support as a whole, is free of silica or functionalised silica. Preferably the magnetic core, and indeed the functionalised magnetic support as a whole, is free of functionalised silica coating that bears one or more reactive functional groups. In particular, the functionalised magnetic support is not configured as per the functionalised-silica-coated particles described in R. Mahajan, M. Rouhi, S. Shinde, T. Bedwell, A. Incel, L. Mavliutova, S. Piletsky, I. A. Nicholls, B. Sellergren, Angew. Chem. Int. Ed.2019, 58, 727 (Mahajan et al). Whilst the magnetic particles of Mahajan et al may beused in downstream methods such as the batch-wise manufacturing methods that recycle / reuse corresponding magnetically supported template molecules, such “functionalised silica coated” magnetic particles are significantly less effective in such methods than the uncoated functionalised magnetic supports described herein because: a) Having a template molecule (e.g. protein) more tightly bound around the magnetic core, as with uncoated functionalised magnetic supports, appears to better facilitate MIP cavity production and subsequent disengagement of the template molecule (the latter of which can be affected under milder conditions, such as sonication); b) Mahajan et al particles generally require heat to dissociate them from MIPs, which is detrimental to the stability of the template molecules they carry (especially complex molecules, such as proteins which readily denature with heat); c) Mahajan et al particles are less responsive to magnetic fields, perhaps owing to shielding by their silica coatings, and thus more difficult to attract / separate using magnetism; and d) Disengaging uncoated functionalised magnetic particles from MIPs can be affected under even milder conditions, for instance, via mild agitation methods (e.g. sonication) optionally in combination with magnetic attraction (thanks to their greater magnetic efficacy) – indeed the disengageability and magnetic efficacy of particles of the invention facilitates the use of milder separation techniques, which is helpful in the context of complex biological template molecules.

[0115] Since Mahajan only earmarks its magnetic particles for single use, its disadvantages (especially damaging the appended template molecule) are not relevant in the Mahajan context, but are in the context of downstream methods of the invention, especially those which seek to recycle / reuse undamaged magnetically supported template molecules.

[0116] The functionalised magnetic support (or functionalised magnetic particle) is suitably defined by Formula I: of to or or or or be the L,

[0118] An example functionalised magnetic support of Formula Ia with X=aldehyde (-CH(O)), and n=4 is:by a Magnetic Core / Particle

[0120] The magnetic core / particle may be any suitable magnetic or magnetisable core or particle. The magnetic core is suitably physically perturbable by (or physically attractable to) a magnetic force. The magnetic core may be magnetically polarisable – i.e. magnetisable so that it thereafter responds to (or is perturbed by, or attracted to) a magnetic force. Most preferably the magnetic core / particle is a magnetic nanoparticle (MNP). Magnetic nanoparticles are well known in the art, as are their methods of production.

[0121] Preferably the magnetic core comprises or consists of a magnetic or magnetisable metal or metal compound, more preferably a magnetic or magnetisable d-metal or d-metal compound. More preferably the magnetic core comprises or consists of a magnetic or magnetisable metal compound, more preferably a magnetic or magnetisable d-metal compound. Most preferably, the magnetic core comprises or consists of a magnetic or magnetisable metal oxide compound, more preferably a magnetic or magnetisable d-metal oxide compound, optionally comprising one or more ligands (e.g. glutaraldehyde, ethylene glycol, acetate). Such metal compounds may contain a plurality of different metals (or ions of a plurality of different metals). Such metal compounds may contain a single metal in a plurality of oxidation states. Such metal compounds may contain a single metal in a single oxidation state. Such metal oxide compounds may be mixed oxide compounds (e.g. comprising ions of the same metal but in different oxidation states) , optionally comprising one or more ligands (e.g. acetate). Preferred d-metals are iron, nickel, and cobalt, but most preferably iron.

[0122] Most preferably, the magnetic core / particle comprises or consists of iron oxide(s), preferably Fe2O3, Fe3O4, or a combination thereof, optionally comprising one or more ligands (e.g. glutaraldehyde, ethylene glycol, acetate). The magnetic core may be an iron oxide nanoparticle, and may preferably be an iron oxide nanoparticle with a crystal structure of maghemite and / or magnetite, optionally comprising one or more ligands (e.g. glutaraldehyde, ethylene glycol, acetate).

[0123] Whilst described as a “magnetic core”, the magnetic core (or magnetic particle) said core / particle may in fact be magnetic or magnetisable by virtue of its shell – e.g. the magnetic core / particle may itself comprise or consist of a non- magnetic / non-magnetisable inner core (e.g. a silica core) surrounded by a magnetic / magnetisable shell (e.g. iron oxide(s) shell).

[0124] The magnetic core, by itself (i.e. not accounting for pendent reactive functional group(s)), suitably has an average particle size between 1 and 1000 nm, or an average particle size between 100 and 300 nm.

[0125] Preferably the magnetic core by itself has an average particle size between 5 and 500 nm, more preferably between 10 and 300 nm, more preferably between 20 and 200 nm, most preferably between 50 and 180 nm. The size of the magnetic core is very similar in size (suitably within 10%, more suitably within 5%) to that of the entire functionalised magnetic support (or functionalised magnetic particle) and, as such it is straightforward to assess the size of the magnetic core – alternatively the magnetic core / particle may be produced using the same conditions but in the absence of a functionalising molecule and the size thereof determined. The aforementioned particle sizes of the magnetic core may in fact relate to an individual cluster thereof (as mentioned above in relation to particle sizes of a functionalised magnetic support). As such, the (average) particle size of the magnetic core may be its (average) cluster size. Particle size (and thus the cluster size) may be determined by DLS and / or SEM (and / or TEM). DLS is suitably the default method of measuring particle sizes - this generally measures cluster size.

[0126] The size of an individual magnetic core (i.e. as distinct from the size of a cluster of magnetic cores) is more difficult to establish but may be separately determined, for instance, by reference to TEM images or magnetic measurementtechniques – i.e. via TEM or magnetic measurement the size of an individual magnetic core, generally within a cluster but also on its own, may suitably be determined. Preferably, the average size of individual magnetic cores (as distinct from clusters thereof), is between 0.5 and 100 nm, more preferably between 1 and 50 nm, more preferably between 3 and 20 nm, most preferably between 5 and 15 nm. Reactive Functional Groups / Functionalising Molecules

[0127] The functionalised magnetic support (or functionalised magnetic particle) suitably comprises or consists of a magnetic core (or magnetic particle) bearing, preferably on the surface thereof, one or more reactive functional group(s) (e.g. aldehyde group, carboxylate group, or amine group), more suitably two or more reactive functional groups. Where there are two or more reactive functional groups (or a plurality of reactive functional groups), such reactive functional groups may be the same or different, most preferably the same. The magnetic core may be produced in the presence of a functionalising molecule (or one or more functionalising molecules, or even two or more different functionalising molecules) so that the magnetic core and reactive functional group(s) thereof are produced in situ – i.e. in a one-pot reaction.

[0128] Whilst the surface of the magnetic core suitably “bears” the one or more reactive functional group(s), the association between (e.g. mode of bonding between) the reactive functional group(s) and the magnetic core may be covalent or non-covalent, most preferably non-covalent. Importantly, the reactive functional group(s) are permanently associated with the magnetic core and not readily detached therefrom.

[0129] The one or more reactive functional group(s) may be attached to or associated with the magnetic core via an optional intervening linker. The linker may comprise or be formed from an additional functionalising molecule, or may be pre-attached to the reactive functional group(s). Reactive Functional Groups

[0130] An X group as defined herein (e.g. in Formula I, Ia, and Ib above) suitably corresponds to any definition of a reactive functional group given herein, in particularly as set forth below.

[0131] A skilled person will appreciate that a variety of different reactive functional groups, and thus functionalising molecules, may be used. The reactive functional groups may be judiciously chosen to complement the template molecules to be attached in subsequent steps, such as those described in aspects B1-B5. For instance, if the (free) uncoupled template molecule comprises a nucleophilic moiety (such as an amine group), it may be desirable for the reactive functional groups to comprise an electrophilic moiety (such as an aldehyde or ketone group). Alternatively, if the (free) uncoupled template molecule comprises an electrophilic moiety (such as an aldehyde or ketone group), it may be desirable for the reactive functional groups to comprise a nucleophilic moiety (such as an amine group). In either fashion, the template molecule may be coupled to the reactive functional groups via nucleophilic addition or substitution reactions. A skilled person is capable of determining an appropriate match.

[0132] The reactive functional group(s) is / are suitably selected from nucleophilic functional groups(s) (especially where the template molecule comprises reactive electrophilic groups or groups that can be transformed into reactive electrophilic groups), electrophilic functional group(s) (especially where the template molecule comprises reactive nucleophilic groups or groups that can be transformed into reactive nucleophilic groups), or any combination thereof, though most preferably the reactive functional group(s) is / are of a single class, either electrophilic or nucleophilic.

[0133] Where the reactive functional group(s) is / are electrophilic groups, the reactive functional group(s) is / are preferably selected from the group consisting of aldehyde(s), ketone(s), activated carboxylic acid(s) (e.g. activated with a coupling agent, such as above), activatable carboxylic acid(s) (a carboxylic acid that is yet to be activated, be it with a coupling agent or by transformation into a an acid halide or similar), acid halide(s), acid chloride(s), acid bromide(s), and any combination thereof (though preferably only one of the aforesaid). Most preferably, where the reactive functional group(s) is / are electrophilic groups, the reactive functional group(s) is / are aldehyde(s), ketone(s), or a combination thereof (especially in molecules where tautomerism furnishes equilibrium concentrations of both aldehyde and ketone forms), most preferably aldehyde(s).

[0134] Where the reactive functional group(s) is / are nucleophilic groups, the reactive functional group(s) is / are preferably selected from the group consisting of amine(s) (e.g. primary or secondary amine(s), preferably primary amine(s)), alcohol(s) (most preferably primary alcohol(s)), thiol(s) (most preferably primary thiol(s)), and any combination thereof (though preferably only one of the aforesaid). Most preferably, where the reactive functional group(s) is / are nucleophilic groups, the reactive functional group(s) is / are amine groups, most preferably primary amine groups.

[0135] Preferably, the reactive functional group(s) is / are preferably selected from the group consisting of aldehyde(s), ketone(s), activated carboxylic acid(s) (e.g. activated with a coupling agent, such as above), activatable carboxylic acid(s) (a carboxylic acid that is yet to be activated, be it with a coupling agent or by transformation into a an acid halide or similar), acid halide(s), acid chloride(s), acid bromide(s), amine(s) (e.g. primary or secondary amine(s), preferably primary amine(s)), alcohol(s) (most preferably primary alcohol(s)), thiol(s) (most preferably primary thiol(s)), and any combination thereof (though preferably only one of the aforesaid). More preferably, the reactive functional group(s) is / are selected from the group consisting of aldehyde(s), ketone(s), and amine(s). Most preferably, the reactive functional group(s) is / are selected from the group consisting of aldehyde(s) and ketone(s), most preferably aldehyde(s).

[0136] Preferably, the functionalised magnetic support a plurality of reactive functional groups – i.e. each magnetic core is, on average, associated with more than 1, preferably two or more reactive functional group. Optional Linker Groups an be are toDistance of Reactive Functional Groups from the Magnetic Core

[0139] The number of intervening atoms (of a chain, e.g. atoms of an optionally substituted carbon chain) between the reactive functional group(s) and magnetic core is suitably at most 20 intervening atoms, more preferably at most 10 intervening atoms, and most suitably at most 8 intervening atoms. The number of intervening atoms is usually determinable from the functionalising molecule used to furnish the reactive functional groups. For instance, in addition to the reactive functional group (aldehyde), glutaraldehyde has 5 chain atoms (4 carbon and one additional aldehydic oxygen), so functionalised magnetic supports formed with glutaraldehyde as the only functionalising molecule exhibit 5 intervening atoms between the reactive functional group(s) (aldehyde) and the magnetic core. By contrast, if 1,6-hexyldiamine (hexane-1,6- diamine) is used as the only functionalising molecule when forming the functionalised magnetic support, there would be 7 intervening atoms (6 carbon, and one nitrogen) between the reactive functional group (primary amine) and the magnetic core. Balance of Factors

[0140] The inventors have surprisingly found that a good balance between particle size, average number (and distribution) of reactive functional groups per magnetic core, and distance of the reactive groups from the surface of the magnetic core, facilitates the efficacy of the downstream chemistry.

[0141] As such, in a preferred embodiment, the functionalised magnetic support (or functionalised magnetic particle) has an average particle size of 50-300 nm, an sufficient average number of reactive functional groups per magnetic core (at least 3, though it may in practice be orders of magnitude higher, since only a certain number of template molecules will ultimately react) for coupling at least 3 template molecules in downstream chemical reactions, and the number of intervening atoms (of a chain, e.g. atoms of an optionally substituted carbon chain) between the reactive functional group(s) and magnetic core is suitably at most 20 intervening atoms.

[0142] In a more preferred embodiment, the functionalised magnetic support (or functionalised magnetic particle) has an average particle size of 50-180 nm, an sufficient average number of reactive functional groups per magnetic core (at least 10, though it may in practice be orders of magnitude higher, since only a certain number of template molecules will ultimately react) for coupling at least 3 template molecules in downstream chemical reactions, and the number of intervening atoms (of a chain, e.g. atoms of an optionally substituted carbon chain) between the reactive functional group(s) and magnetic core is suitably at most 10 intervening atoms.

[0143] The magnetic core of these embodiments is more preferably an iron oxide magnetic core (preferably comprising Fe2O3, Fe3O4, or a combination thereof), optionally comprising one or more ligands (e.g. acetate).

[0144] It will, however, be understood that advantages of the invention may still be enjoyed outside of the limitations of these specific embodiments. Manufacturing of the Functionalised Magnetic Support

[0145] Methods of manufacturing a functionalised magnetic support (or functionalised magnetic particle) are outlined above in numbered paragraphs A1-A5 of the “SUMMARY OF THE INVENTION” section.

[0146] The method suitably involves providing: i) the magnetic core (suitably derived from one or more magnetic core-forming components); and ii) one or more reactive functional group(s) (suitably derived from one or more functionalising molecules) borne by the magnetic core (preferably borne upon the surface of the magnetic core); wherein i) and ii) are either provided simultaneously or sequentially, most preferably simultaneously (i.e. in one-pot).

[0147] As described above, a skilled person may form a variety of different magnetic cores using their common general knowledge. A skilled person can also select appropriate functionalising molecule(s) to furnish desired reactive functional groups. However, the magnetic core is preferably formed in accordance with the features outlined above in the sub-sectionby or Magnetic core-forming components

[0149] Magnetic core-forming components are suitable used for forming the magnetic core, which may in principle be formed with or without reactive functional groups.

[0150] The one or more magnetic core-forming components suitably comprise a metal-containing (preferably a d-metal- containing) precursor to the magnetic or magnetisable metal or metal compound (preferably magnetic or magnetisable d- metal or d-metal compound) of the magnetic core as described above in the sub-section entitled “Magnetic Core / Particle”. Preferably, the one or more magnetic core-forming components comprise a metal-containing (preferably a d-metal- containing) precursor to the magnetic or magnetisable metal oxide compound (preferably magnetic or magnetisable d-metal oxide compound) of the magnetic core as described above in the sub-section entitled “Magnetic Core / Particle”. More preferably, the one or more magnetic core-forming components comprise an iron-, nickel-, or cobalt- containing precursor to the iron-, nickel-, or cobalt- oxide compound of the magnetic core as described above in the sub-section entitled “Magnetic Core / Particle”. Most preferably, the one or more magnetic core-forming components comprise an iron-containing precursor to the iron-oxide compound of the magnetic core as described above in the sub-section entitled “Magnetic Core / Particle”. Such precursors are suitably a corresponding metal halide (e.g. iron halide, preferably iron(III) halide), preferably a corresponding metal chloride (e.g. iron chloride, preferably iron(III) chloride). A precursor may advantageously be provided in a hydrated form (e.g. FeCl3.6H2O) to facilitate ultimate oxide formation.

[0151] Preferably such precursors comprise the corresponding metal in an oxidation state that is already magnetic or magnetisable or is otherwise capable of becoming magnetic or magnetisable (with an appropriate crystal structure or metal- complex geometry) without any change in oxidation state – e.g. FeCl3fulfils such a criterion, because Fe3+is capable of being magnetic or magnetisable). However, the oxidation state of precursors (or a portion thereof) may be changed, for instance when mixed with one or more further oxidising or reducing magnetic core-forming components – this may result in previously non-magnetic / non-magnetisable metal species becoming magnetic or magnetisable following oxidation or reduction, or it may result in the formation of an advantageous mixture of oxidation states which together afford the magnetic core. As such, the one or more magnetic core-forming components may comprise a metal-containing precursor (preferably one of the preferred examples suggested above) and a redox agent (reducing or oxidising, preferably reducing). An example of a redox agent, which may be used alongside a metal compound already in its highest oxidation state (e.g. FeCl3) is ethylene glycol, which may reduce part of the metal within the metal compound to yield a mixture of oxidation states – FeIII / FeII– suitably in addition to its role as a solvent.

[0152] The one or more magnetic core-forming components may further comprise (in addition to a precursor as defined above, and optionally also in addition to a redox agent) one or more core-forming reactants. Such core-forming reactants may be an acidic or basic compound, most preferably a basic compound. The core-forming reactants (or even solvent(s) or functionalising molecule(s)) may provide ligands which ultimately become a part of the magnetic core. In a particular embodiment, the core-forming reactant is sodium acetate. Where acetate is used, it may become one of the ligands within the ultimate magnetic core, though preferably it does not (or preferably it is eliminated, optionally during a high temperature dwell time). References to metal-oxide magnetic cores still include those containing ligands other than oxygen, such as acetate, functionalising molecule(s) (or derivatives thereof), or even solvent molecule(s).

[0153] In a particular embodiment, the core-forming components comprise a metal-containing precursor (preferably as defined above, most preferably iron(III) chloride), a core-forming reactant (preferably as defined above, most preferably sodium acetate). Such core-forming components may further comprise a redox agent (e.g. ethylene glycol).

[0154] The terms “precursor”, “metal-containing precursor”, “metal-containing (preferably a d-metal-containing) precursor to the magnetic or magnetisable metal or metal compound (preferably magnetic or magnetisable d-metal or d-metal compound) of the magnetic core” may be used interchangeable. The “precursor” may also be referred to as a “metal source” or “magnetic or magnetisable metal source”.

[0155] The solvent used in this method of manufacture may be a redox agent (even if only mildly so), such as ethylene glycol. Functionalising Molecule

[0156] Functionalising molecules are used to install the reactive functional group(s) of the functionalised magnetic support. Preferred reactive functional groups are described above, and a skilled person is well able to determine functionalising molecules that can be utilised to furnish such reactive functional groups.

[0157] The magnetic core is preferably produced in presence of a functionalising molecule (or one or more functionalising molecules, or even two or more different functionalising molecules) so that the magnetic core and reactive functional group(s) thereof are produced in situ – i.e. in a one-pot reaction. Where there are two or more different functionalising molecules, these may lead to the formation of a plurality (two or more) different reactive functional groups or, especially where the different functionalising molecules reactive together, a single reactive functional group.

[0158] The or at least one of the functionalising molecules, preferably comprises a (desired) reactive functional group(s) (e.g. aldehyde). However, the desired reactive functional group(s) may be formed following transformation of the functionalising molecule(s) or following a transformative reaction between two or more different functionalising molecules – by way of example, one functionalising molecule may comprise a carboxylic acid group and another (companion) functionalising molecule may be a transformative molecule which, for example, transforms the carboxylic acid group into a form that is more reactive with amines (e.g. N-terminus or amine groups within a protein). This companion functionalising molecule may be a coupling agent, for example, carbonyldiimidazole (CDI), DCC, dicyclohexylcarbodiimide (DCC), diisopropylcarbodiimide (DIC), ethyl-(N’,N’-dimethylamino)propylcarbodiimide hydrochloride (EDC), or any other coupling agent well known in the art. N-hydroxysuccinimide (NHS) is another useful coupling agent well known in the art. Further functionalising molecules may also be involved to facilitate transformations (e.g. catalytic 4-(N,N-dimethylamino)pyridine, DMAP) and / or mitigate problems (such as racemisation), e.g.1-hydroxybenzotriazole (HOBt).

[0159] Where two or more different functionalising molecules are utilised, one functionalising molecule may be a linker molecule. Such linkers may permit template molecules to be coupled more distal to the magnetic core. However, linkers are less desirable as it is ultimately preferable for the template molecule to be as close to the magnetic core as possible.

[0160] Preferably, a single functionalising molecule (which is preferably relatively short – see below) comprising a / the reactive functional group(s) (e.g. aldehyde) is used, for simplicity. The (or a) functionalising molecule preferably already comprises the desired reactive functional group(s) without further transformation(s). However, the functionalising molecule may be, or otherwise form (e.g. during formation of the functionalised magnetic support), an oligomer or polymer – e.g. the functionalising molecule may be glutaraldehyde, an oligomer (or polymer) of glutaraldehyde, or a combination thereof.

[0161] Where the reactive functional group(s) is / are electrophilic group(s), preferably the corresponding functionalising molecule comprises said electrophilic reactive functional group(s). Where the reactive functional group(s) is / are nucleophilic group(s), preferably the corresponding functionalising molecule comprises said nucleophilic reactive functional group(s).

[0162] As such, the (or one of the) functionalising molecule(s) comprises one or more reactive functional group(s) selected from the group consisting of aldehyde(s), ketone(s), activated carboxylic acid(s) (e.g. activated with a coupling agent, such as above), activatable carboxylic acid(s) (a carboxylic acid that is yet to be activated, be it with a coupling agent or by transformation into a an acid halide or similar), acid halide(s), acid chloride(s), acid bromide(s), amine(s) (e.g. primary or secondary amine(s), preferably primary amine(s)), alcohol(s) (most preferably primary alcohol(s)), thiol(s) (most preferably primary thiol(s)), and any combination thereof (though preferably only one of the aforesaid).

[0163] More preferably, the (or one of the) functionalising molecule(s) comprises one or more reactive functional group(s) selected from the group consisting of aldehyde(s), ketone(s), and amine(s), and any combination thereof (though suitably only one of the aforesaid, though aldehydes and ketones may exist in equilibrium via tautomerism). Most preferably, the (or one of the) functionalising molecule(s) comprises one or more reactive functional group(s) selected from aldehyde(s) and / or ketone(s), most preferably aldehyde(s).

[0164] In a particularly preferred embodiment, the functionalising molecule is glutaraldehyde – this suitably provides aldehyde reactive functional groups. The glutaraldehyde may be any form of glutaraldehyde, whether free glutaraldehyde, partially or fully hydrated glutaraldehyde (e.g. with either or both aldehyde groups forming a geminal diol), an oligomer (or polymer) of glutaraldehyde, or a combination thereof. Free or (optionally partially-) hydrated glutaraldehyde may forming oligomeric (or polymeric) glutaraldehyde in situ during the formation of the functionalised magnetic support.

[0165] In an alternative embodiment, the functionalising molecule may be 1,6-hexyldiamine (hexane-1,6-diamine) – this suitably provides primary amine reactive functional groups.

[0166] Functionalising molecules, including those bearing the reactive functional group(s) (especially where the functionalising molecule comprises two or more such reactive functional groups), may react with and / or bond to the magnetic core or a part thereof. For instance, the functionalising molecule partake in dative bonding with the magnetic core – e.g. in iron-oxide magnetic cores: ^ One aldehyde group of a glutaraldehyde functionalising molecule may participate in direct or indirect bonding (potentially in an acetal-like form derived from reaction with oxygen species of the iron oxide) leaving the other aldehyde group as a free reactive functional group capable of reacting with nucleophiles (e.g. the amine group of a protein template molecule). ^ One amine group of a hexane-1,6-diamine functionalising molecule may be involved in direct dative bonding, leaving the other amine group as a free reactive functional group capable of reacting with electrophiles (e.g. activated carboxylate groups of a protein template molecule).

[0167] However, functionalising molecules may instead be simply physically trapped within the magnetic core, without having formed any covalent bonds therebetween. Without wishing to be bound by theory, the inventors believe, in the case of glutaraldehyde (and possibly other dialdehydes or diketones), that glutaraldehyde polymerises with itself (e.g. when aged or heated) to form glutaraldehyde oligomers (still exhibiting aldehyde groups, such as terminal aldehyde) which become entrapped as magnetic nanoparticles are forming, thereby allowing glutaraldehyde groups (or the aldehyde moieties thereof)to cover the MNPs. The magnetic particles (or magnetic thus be considered coated with a functionalised molecule (or reacted form(s) thereof). Solvent

[0168] Preferably methods of manufacturing a functionalised magnetic support take place in a solvent (or solvent system comprising one or more solvents). Suitably, a solvent (or solvent system) is chosen to facilitate the transformation of the one of more core-forming components into a magnetic core, suitably bearing (on the surface thereof) one or more reactive functional groups.

[0169] Preferably, the one or more magnetic core-forming components are mixed with the one or more (preferably just one) functionalising molecule within the solvent (or solvent system), suitably so that a one-pot reaction may occur to simultaneously facilitate both the magnetic core and reactive functional group(s) borne by the surface thereof.

[0170] Suitably, at least one (preferably the precursor to the magnetic or magnetisable metal or metal compound) of the one or more magnetic core-forming components are soluble or partially soluble within the solvent or solvent system.

[0171] The product (functionalised magnetic support) is preferably insoluble in the solvent or solvent system.

[0172] The solvent or solvent system is preferably a solvent system that is fully miscible with water, suitably even if the solvent system itself is substantially free of water or contains only a relatively small quantity thereof. The solvent or solvent system is suitably an organic solvent or solvent system, suitably comprising a heteroatom-bearing (e.g. nitrogen- and / or oxygen-hearing) organic solvent.

[0173] The solvent or solvent system suitably has a boiling point (at SATP) of at least 70oC, preferably at least 95oC, more preferably at least 130oC, most preferably at least 170oC.

[0174] The solvent may be multifunctional – for example, it may also be a redox agent (even if only mildly so), such as ethylene glycol.

[0175] Preferably the solvent system is ethylene glycol. Exclusions

[0176] The method of manufacturing a functionalised magnetic support suitably affords a functionalised magnetic support as described herein, preferably with the aforementioned exclusions (e.g. free of silica or functionalised silica, preferably free of coatings, preferably without any intervening coating or shell between the reactive functional groups and magnetic core). If there is any “coating” at all around the magnetic core, preferably this is only a coating comprising a functionalising molecule (as defined herein) or a direct product formed therefrom during formation of the functionalised magnetic support. As such, preferably the method of manufacturing a functionalised magnetic support excludes any, some, or all (most preferably all) of the following extra steps: a) Forming a silica shell around the magnetic core (especially forming a silica shell around a pre-formed magnetic core), for instance via aqueous hydrolysis of tetraethyl orthosilicate (TEOS) in the presence of the magnetic core; b) Functionalising a / the silica shell formed around the magnetic core, for instance via amine-functionalisation with an aminosilane such as (3-Aminopropyl) trimethoxysilane (APTMS), optionally followed by further functionalisation of the installed amine group with glutaraldehyde (to ultimately present aldehyde reactive functional groups borne by the silica shell); c) Functionalising the magnetic core with reactive functional group(s) via an additional functionalising step performed after the magnetic core has been produced; d) Forming a functionalised magnetic support with a shell or coating intervening between the reactive functional groups (and optionally their linkers) and the magnetic core, optionally with the exception of an intervening shell or coating formed directly from a functionalising molecule as defined herein; and / or e) Coupling a template molecule with a / the functionalised shell (especially a functionalised silica shell) around the magnetic core. Reaction Conditions / Heating

[0177] The method(s) of manufacturing a functionalised magnetic support (or functionalised magnetic particle) described herein, including as outlined above in numbered paragraphs A1-A5 of the “SUMMARY OF THE INVENTION” section and preferably as refined by any one or more of the aforesaid sub-features, in particular any step of forming the magnetic core (preferably simultaneously with forming the reactive functional group(s) on the surface of the magnetic core), preferably comprise heating – e.g. heating input components (suitably to facilitate formation of the magnetic core, preferably simultaneously with formation of any reactive functional group(s) borne on the surface thereof). Such methods, thus, preferably comprise heating input components, where said input components are suitably mixed together. The method(s) thus preferably comprise heating a reaction mixture (initially formed by mixing together the input components). Naturally the composition of the reaction mixture progresses as a reaction takes place. However, “input components” and “reaction mixture” may be use interchangeably and references herein to any processing of the “input components” may suitably correspond to the same processing of the reaction mixture.

[0178] The input components suitably comprise or of all necessary components required to form the magnetic core (e.g. one or more magnetic core-forming components and suitably also a solvent or solvent system, as defined herein) of the functionalised magnetic support, and more preferably comprise or consist of all necessary components required to form the functionalised magnetic support (e.g. one or more magnetic core-forming components, one or more functionalising monomer(s), and suitably also a solvent or solvent system, as defined herein).

[0179] The input components suitably comprise or consist of one or more magnetic core-forming components (preferably including at least a precursor to the magnetic or magnetisable metal or metal compound of the magnetic core) as defined herein, suitably along with a solvent or solvent system as defined herein. Preferably, the input components comprise or consist of one or more magnetic core-forming components (preferably including at least a precursor to the magnetic or magnetisable metal or metal compound of the magnetic core) as defined herein, and a (or one or more) functionalising molecule(s) as defined herein, suitably along with a solvent or solvent system as defined herein.

[0180] The input components are preferably mixed to form a / the reaction mixture.

[0181] Before any heating is commenced, preferably the input components are mixed (e.g. via stirring or any other form of agitation), more preferably homogenised, and most preferably dissolved.

[0182] The heating is preferably microwave heating. The heating, whether microwave heating or otherwise (though preferable microwave heating), may suitably comprise solvothermal heating (e.g. heating in a sealed vessel at a temperature higher than the boiling temperature of the solvent or solvent system).

[0183] Preferably, the heating culminates in heating at a maximum temperature for a maximum temperature holding period. The heating at the maximum temperature is suitably sufficient for solvothermal heating (e.g. higher than the SATP boiling point of the solvent or solvent system).

[0184] The heating may comprise heating (e.g. isothermal heating), preferably solvothermal heating, at a (substantially) fixed temperature (e.g. at a temperature between 50 and 250oC, or between 80 and 180oC, for instance at 50oC, 100oC, or 150oC) for a fixed temperature holding period. In such circumstances, the fixed temperature holding period may suitably be between 5 min and 48 hours, preferably between 20 min and 24 hours, more preferably between 40 min and 12 hours, most preferably between 1 hour and 6 hours.

[0185] Preferably the heating comprises ramped or gradient heating (e.g. gradually raising temperature, at a ramp rate, from a starting temperature to a maximum temperature, and optionally thereafter holding at a maximum temperature), preferably ramped or gradient heating of the reaction mixture. As such, ramped (or gradient) heating may suitably comprise: a) Starting (time zero, t0) at a start temperature (Tmin) (which may be room temperature or standard temperature, preferably 25oC + / - 10oC), and commencing heating; b) Heating, over a predetermined ramp time (tramp), to increase the temperature by a predetermined ramp temperature (^Tramp) – e.g. heating at a ramp rate (Rramp) of ^Tramp / tramp; c) Repeating heating step b) until a predetermined maximum temperature (Tmax) is reached.

[0186] Temperature is preferably the temperature of (or target temperature for) the components (e.g. input components) being (or to be) heated – i.e. temperature of the reaction mixture. Temperatures of the reaction mixture may be measured directly or presumed based on tried-and-tested target temperatures.

[0187] In principle, the start temperature of step a) may be any temperature that is lower than the maximum temperature, though it is preferably a temperature at which no magnetic core or functionalised magnetic support is formed, and is preferably 25oC + / - 25oC, more preferably 25oC + / - 10oC.

[0188] The difference between the start temperature (Tmin) and maximum temperature (Tmax) is suitably between 30 and 400oC, preferably between 50 and 300oC, more preferably between 70 and 250oC, most preferably between 100 and 200oC.

[0189] Suitably the maximum temperature is between 60 and 350oC, preferably between 100 and 300oC, and more preferably between 150oC and 250oC, most preferably between 180oand 220oC (e.g. about 200oC).

[0190] The time taken between step a) (when heating first begins) and the end of step c) (reaching the maximum temperature) is the total ramp time (ttotal). The total ramp time is suitably between 1 and 60 mins, preferably between 2 and 30 minutes, more preferably between 5 and 20 minutes, more preferably between 5 and 14 minutes, most preferably between 8 and 12 minutes (e.g. about 10 mins).

[0191] Step b) (ramp heating) may include holding a temperature for a predetermined intermediate temperature holding period.

[0192] Suitably, the ramp rate is between 2oC / min (2 degrees Celsius per minute) and 150oC / min. Preferably, the ramp rate is between 5oC / min and 100oC / min, more preferably between 8oC / min and 50oC / min, most preferably between 10oC / min and 30oC / min (e.g. about 18oC / min).

[0193] Following step c), heating may continue at the maximum temperature for a predetermined maximum temperature holding period. Suitably, the maximum temperature holding period is between 1 and 60 min, preferably between 2 and 40 min, more preferably between 5 and 30 min, most preferably between 15 and 25 min. Preferably, however, most of the reaction has already taken place during the ramp period (i.e. during the total ramp time).

[0194] Preferably the heating comprises heating input components (i.e. the reaction mixture) in a sealed vessel, such as a microwave reaction vial (MRV). As such, during the course of the heating (optionally including heating at the maximum temperature for a predetermined maximum temperature holding period) suitably a maximum pressure is reached. The20 or 50 a or 70 the (or or (s) by the by of (or v) Optionally washing the solid product(s);or the (or or of 70 Particle Size and Tuning

[0210] The particle size of the magnetic core(s) and thus also the functionalised magnetic support (or functionalised magnetic particles) are suitably tuneable by altering heating protocols. In particular it has been found that judicious tuning of ramp rate (Rramp) can provide a desired average particle size. Preferably such tuning involves tuning ramp rate (Rramp), total ramp time (ttotal), maximum temperature, and the difference between the start temperature (Tmin) and maximum temperature (Tmax). In general, a slower ramp rate and longer total, ramp time is preferred in order to provide larger particle sizes, preferably a particle size between 10 and 300 nm, more preferably between 20 and 200 nm, most preferably between 50 and 180 nm. To achieve such desired particle sizes, the preferred reaction conditions set forth above in the section “Reaction Conditions / Heating” may be used, in particular: ^ The difference between the start temperature (Tmin) and maximum temperature (Tmax) is preferably between 70 and 250oC; ^ The maximum temperature is between 100 and 300oC; ^ The total ramp time (ttotal) is 5 and 60 minutes; and ^ The ramp rate is between 8oC / min and 50oC / min.

[0211] As explained elsewhere herein, larger average functionalised magnetic support particle sizes are preferred because these inter alia afford higher yields in downstream method steps, and facilitate recyclability of magneticallysupported template molecules formed therefrom. That can be achieved whilst still enjoying the benefits of such fast MNP production provides significant benefits, especially in the scale-up of MIP production. B) MAGNETICALLY SUPPORTED TEMPLATE MOLECULE

[0212] Aspects and features of the invention relating to a magnetically supported template molecule are described above in the relevant sub-section of the “SUMMARY OF THE INVENTION” and, in particular in numbered paragraphs B1-B5. Further features and sub-features of the same are elaborated below, all of which are applicable to any of the aforesaid aspects and to any other compatible aspects and embodiments described elsewhere herein.

[0213] The functionalised magnetic support (or functionalised magnetic particle) used in this section is preferably as defined and / or formed as defined in the above section entitled “FUNCTIONALISED MAGNETIC SUPPORT (OR FUNCTIONALISED MAGNETIC PARTICLE)”.

[0214] A magnetically supported template molecule (or a magnetic-template particle) comprises or consists of a magnetic core (or magnetic particle) coupled or tethered to one or more (preferably a plurality) of template molecule(s). The one or more (preferably a plurality) of template molecule(s) are preferably coupled or tethered to the magnetic core via one or more reaction functional groups borne on the surface of the magnetic core. As such, the reactive functional groups intervening between the magnetic core and template molecule(s) are transformed into a post-reacted form. For instance, where the amine of a template molecule reacts with an aldehyde reactive functional group borne by the magnetic core, the reactive functional group becomes a corresponding imine, enamine, or hemiaminal.

[0215] As such, the magnetically supported template molecule is preferably formed by reacting a functionalised magnetic support (or functionalised magnetic particle), preferably as defined and / or formed as defined in the above section entitled “FUNCTIONALISED MAGNETIC SUPPORT (OR FUNCTIONALISED MAGNETIC PARTICLE)”. Preferably the functionalised magnetic support inherits one or more of the aforementioned preferred features, especially with respect to particle size. A functionalised magnetic support having a preferred (e.g. relatively large) particle size (e.g.5-500 nm, 10- 300 nm, 20-200 nm, or 50-180 nm) is suitably sterically capable of being coupled to a plurality of template molecules (potentially at least 100, preferably at least 1000), which provides several downstream advantages, including higher MIP yields, better MIP cavity production, and more effective disengagement of the template molecule under mild conditions that do not compromise the template molecule (thus allowing recycling and reuse thereof).

[0216] Any suitable template molecule may be used in conjunction with the invention. As explained above in the section “FUNCTIONALISED MAGNETIC SUPPORT (OR FUNCTIONALISED MAGNETIC PARTICLE)”, the reactive functional group(s) of the functionalised magnetic support can be chosen to complement the template molecule, in particular the reactive functional group(s) of the functionalised magnetic support an be match to complementary reactive groups of the template molecule.

[0217] The magnetically supported template molecule is suitably defined by Formula II, and is suitably formed by reacting a template molecule (T) with a functionalised magnetic support of Formula I: (T) is an m is of thedefined by Formula IIa:by

[0220] The magnetically supported template molecule suitably has an average particle size that is larger that the functionalised magnetic support used in its production. The size of the functionalised magnetic support suitably increases as template molecule(s) (T) are coupled therewith. Suitably the average particle size of the magnetically supported template molecule is between 2 and 2000 nm, or an average particle size between 50 and 600 nm.

[0221] Preferably the magnetically supported template molecule has an average particle size between 10 and 1000 nm, more preferably between 20 and 600 nm, more preferably between 50 and 500 nm, most preferably between 100 and 400. The (average) particle size may be determined by DLS and / or SEM, with DLS suitably being the default.

[0222] Suitably the average particle size of the magnetically supported template molecule is larger than its corresponding functionalised magnetic support by a factor of at least 1.1, preferably by a factor of 1.5, more preferably by a factor of at least 2, most preferably by a factor of at least 2.5.

[0223] Suitably, the ratio of average particle size of the functionalised magnetic support (used to form the magnetically supported template molecule) and the average particle size of the template molecule is between 1:1 and 1000:1, preferably between 2:1 and 200:1, more preferably between 5:1 and 50:1, most preferably between 10:1 and 30:1.

[0224] Suitably, the average number of template molecules coupled to each functionalised magnetic support (or magnetic core) is between 2 and 3000. Preferably the average number of template molecules coupled to each functionalised magnetic support (or magnetic core) is between 10 and 2000. More preferably the average number of template molecules coupled to each functionalised magnetic support (or magnetic core) is between 50 and 1000. Most preferably the average number of template molecules coupled to each functionalised magnetic support (or magnetic core) is between 100 and 900.

[0225] It was somewhat surprising to find that a magnetically supported template molecule having multiple template molecules per magnetic core, rather than a typical 1:1 ratio as per the prior art, afforded better quality and greater yields of MIPs in downstream methods. Without wishing to be bound by theory, it is thought that MIP templating around multiple template molecules clustered around a relatively large magnetic core, may still form excellent molecular imprints. This in turn also reduces erroneous harvesting of MIPs still bound to magnetic supports. This ease of disengagement also advantageously feeds into the ability to recycle and reused magnetically supported template molecules in subsequent rounds of MIP production. As such, the magnetically supported template molecule described in this section may be a magnetically supported template molecule recovered from an MIP during MIP production.

[0226] As explained in the section entitled “FUNCTIONALISED MAGNETIC SUPPORT (OR FUNCTIONALISED MAGNETIC PARTICLE)” in relation to functionalised magnetic supports used to produce the magnetically supported template molecules described in this section, desirably the magnetically supported template molecule is likewise free of silica or functionalised silica and is preferably also categorised by the same exclusions.Functionalised Magnetic Support

[0227] The functionalised magnetic support used to form the magnetically supported template molecule is suitably as defined in the section entitled “FUNCTIONALISED MAGNETIC SUPPORT (OR FUNCTIONALISED MAGNETIC PARTICLE)”, and its reactive functional group(s) are preferably chosen to match relevant reactive group(s) of the template molecule intended for coupling therewith.

[0228] It is particularly preferred for the functionalised magnetic support to bear aldehyde (or ketone) reactive functional group(s), because these can react particularly well with amine group(s), especially a terminal amine group, or biological molecules such as proteins (which are preferred template molecules). Template Molecule

[0229] Whilst any suitable template molecule may be used to form the magnetically supported template molecules of the invention, preferably the template molecule is a molecule of interest, or is or comprises representative parts of a molecule of interest (target molecule), in respect of downstream uses of MIPs. In particular, therefore, the template molecule is preferably identical to, similar to, or comprises a part that is identical to or similar to a or a part of a downstream target molecule (e.g. to be sequestered or detected in downstream methods or with downstream detection devices that incorporate MIPs of the invention). Where the target molecule is a target protein molecule (e.g. an enzyme, receptor, cytokine, etc.) and the template molecule is a protein template molecule, preferably the protein template molecule has at least 80% sequence identity with the protein target molecule, more preferably at least 90% sequence identity therewith, most preferably at least 95% sequence identity therewith. Alternatively, where downstream MIPs Iare intended to bind a part of the target protein molecule (e.g. akin to an antibody binding an epitope), preferably the corresponding part of a protein template molecule has at least 80% sequence identity with the corresponding part of the protein target molecule, more preferably at least 90% sequence identity therewith, most preferably at least 95% sequence identity therewith.

[0230] The template molecule suitably has a molecular weight between 100 and 100,000,000 g / mol (i.e. 0.1 kDa to 100,000 kDa).

[0231] The template molecule suitably has an average particle size between 0.001 nm (2 pm) and 1000 nm, or 0.1 nm to 100 nm. Preferably, however, the template molecule has an average particle size between 0.2 and 60 nm, more preferably between 0.6 and 20 nm, most preferably between 1.5 and 7.5 nm.

[0232] The template molecule suitably has a non-spherical shape. Suitably the template molecule is asymmetric, suitably asymmetric across a number of planes.

[0233] The template molecule may be a single molecule, which may be referred to as a simple template molecule. The simple template molecule may be any suitably simple template molecule. The simple template molecule may be a drug molecule, antibiotic, pesticide, antigen, toxin, or protein (e.g. an enzyme, a receptor, cytokine, etc.). Where the template molecule is a simple template molecule, preferably the simple template molecule has a molecular weight 100 and 3,000,000 g / mol (between 0.2 and 3000 kDa). Where the template molecule is a simple template molecule, the simple template molecule suitably has an average particle size between 0.5nm and 20 nm.

[0234] However, the template molecule may in fact be a complex of molecules (i.e. the template molecule may comprise a plurality of associated molecules), which may be referred to as a complex template molecule. Examples of a complex template molecule include, for example: ^ A protein composed of multiple subunits (i.e. a protein having a quaternary structure), for example haemoglobin, insulin, alcohol dehydrogenase, DNA polymerase, ribosomes and antibodies; ^ Proteins binding a substrate; ^ DNA and RNA which may comprise complementary strands; and ^ Viruses, for example influenza, coronavirus, rhinovirus, norovirus.

[0235] Where the template molecule is a complex template molecule, suitably the entire complex template molecule is bound to the functionalised magnetic support, following an appropriate coupling reaction to produce a corresponding magnetically supported template molecule.

[0236] Where the template molecule is a complex template molecule, preferably the complex template molecule has a molecular weight between 10kDa and 100,000 kDa. Where the template molecule is a complex template molecule, the complex template molecule suitably has an average particle size between 2.5nm and 80 nm.

[0237] Preferably, the template molecule is biological molecule. Most preferably, the template molecule is a protein (i.e. protein template molecule).

[0238] The protein template molecule suitably has a molecular weight between 1 and 3000 kDa, preferably between 3 and 2000 kDa, more preferably between 10 kDa and 1000 kDa, and most preferably between 15 and 100 kDa. The protein template molecule suitably has an average particle size between 1 and 20 nm, preferably between 1.5 and 17 nm, more preferably between 2.5 and 15 nm, most preferably between 3 and 7 nm.

[0239] The protein template molecule preferably reacts with the functionalised magnetic support to furnish a magnetically supported template molecule, which may be described as a magnetically supported protein template molecule. As discussed above, whilst template molecules may react with functionalised magnetic supports in a variety of ways, most preferably anucleophilic moiety (preferably an amine moiety, most a terminal amine moiety) of the protein template molecule reacts with an electrophilic reactive functional group(s) borne by the magnetic core of the functionalised magnetic support, most preferably an aldehyde (or ketone) reactive functional group(s). As such, the product of such a reaction is preferably an imine or enamine. However, an amine side chain of a non-terminal amine acid (e.g. lysine, and possible arginine which may react via its guanidine moiety) may also react and thereby couple to the functionalised magnetic support. Variance in the way the template molecule binds to the functionalised magnetic support, variance which may increase by virtue of having multiple template molecules coupled to the same functionalised magnetic support, may in fact increase the richness (and variance) is MIP structures which may be permit MIP binding to different parts of a template molecule (like and antibody that targets various different epitopes).

[0240] The protein template molecule is suitably selected from the group consisting of haemoglobin, myoglobin, SARS- Cov-II, and bovine serum albumin. However, any suitable protein may be used.

[0241] The suitable and preferred average particle sizes mentioned above suitably complement and are suitably combinable with suitable and preferred average particle sizes of the functionalised magnetic support elaborated in the section entitled “FUNCTIONALISED MAGNETIC SUPPORT (OR FUNCTIONALISED MAGNETIC PARTICLE)” and of the magnetically supported template molecule elaborated in the section entitled “B) MAGNETICALLY SUPPORTED TEMPLATE MOLECULE”, the ratio of average particle size of the functionalised magnetic support elaborated in “B) MAGNETICALLY SUPPORTED TEMPLATE MOLECULE”, and the factor by which the magnetically supported template molecule is larger than the corresponding functionalised magnetic support as also elaborated in “B) MAGNETICALLY SUPPORTED TEMPLATE MOLECULE”.

[0242] Importantly, the template molecule is capable of reaction (and coupling) with the functionalised magnetic support based on reactive moieties within the template molecule and complementary reactive functional group(s) of the functionalised magnetic support. Manufacturing of the Magnetically Supported Template Molecule

[0243] Methods of manufacturing a magnetically supported template molecule are outlined above in numbered paragraphs B1-B3 of the “SUMMARY OF THE INVENTION” section.

[0244] The method suitably involves coupling a template molecule with a functionalised magnetic support to provide a magnetically supported template molecule. The method suitably involves reacting the template molecule with the functionalised magnetic support. Preferably, the coupling involves a reactive moiety (e.g. amine group) of the template molecule reacts with a reactive functional group (e.g. aldehyde, or ketone) of the functionalised magnetic support. The functionalised magnetic support is suitably as defined herein, or suitably obtained by a method of manufacturing a functionalised magnetic support as defined herein. Preferably the functionalised magnetic support conforms with the exclusions set forth herein (e.g. is free of silica or functionalised silica, especially a silica or functionalised silica coating). Reaction Conditions

[0245] The functionalised magnetic support may be pre-processed by forming a suspension thereof, in a solvent or solvent system (e.g. water), centrifuging the suspension (e.g. at 1000-50000 rpm, preferably about 15000 rpm), and removing the liquid phase (supernatant). The pre-processed functionalised magnetic support may then be used to form a reaction mixture with the template molecule.

[0246] The method suitably involves forming a reaction mixture (preferably in a reaction vessel, suitably not a column to be eluted) comprising the functionalised magnetic support (suitably a suspension thereof) and a template molecule (suitably each as defined herein), suitably in a solvent or solvent system. The reaction mixture may comprise further reagents (e.g. a coupling agent, a catalyst), for instance, to assist in coupling the template molecule with the functionalised magnetic support.

[0247] The reaction mixture is suitably agitated (e.g. by sonication, shaken, and / or stirred) to ensure that the functionalised magnetic support is sufficiently dispersed.

[0248] The reaction mixture may be heated, but preferably a temperature of 25oC + / - 25oC, more preferably 25oC + / - 10oC is maintained. When coupling a biological template molecule (e.g. a protein template molecule) it is preferable to maintain a temperature that mitigates denaturing thereof.

[0249] A coupling reaction between the template molecule and functionalised magnetic support is allowed to ensue within the reaction mixture, optionally with or without agitation, suitably for a sufficient period of time (e.g. 1 min to 24 hours, preferably 10-60 mins), suitably at a temperature of 25oC + / - 25oC, more preferably 25oC + / - 10oC . In process checks, well known to the skilled chemist, may be used to establish when the reaction is sufficiently complete – for instance, the concentration of unreacted template molecule may be measured by spectrophotometry, e.g. using a UV / visible spectrometer, and the reaction may proceed until no further concentration decrease thereof is observed). Preferably sufficient template molecule is used to saturate the functionalised magnetic support (i.e. so that no further coupling of template molecule is possible). As such, suitably the template molecule is used in excess and, as such, suitably unreacted template molecule remains once the reaction is complete.

[0250] The reaction preferably does not involve eluting a column, containing the functionalised magnetic support, with a template molecule, but instead involves forming a reaction mixture in a reaction vessel.

[0251] Once the reaction is complete, the reaction comprises a solid product(s), and suitably also a liquid product(s). The solid product(s) are preferably separated from the liquid products (e.g. via filtration, decanting, pipetting, centrifugation), most preferably by centrifugation. The solid products may be washed (suitably washed a plurality of times) with a solvent or solvent system (suitably a buffered solvent or solvent system, e.g. where unreacted protein template molecules are to be removed), suitably to further remove unreacted template molecule from the solid product(s). The amount of unreacted protein template molecule may be determined in the separated liquid phase, optionally with any washes, and the amount of coupled template molecule determined / calculated therefrom.

[0252] The solid product(s) are suitably the magnetically supported template molecule as defined herein. The solid product(s) may be dried (optionally via heating and / or evacuation), but suitably the solid product(s) may be stored wet, preferably under refrigeration (2-8oC) or frozen (-100 to 0oC). Magnetically supported template molecules, especially those comprising biological template molecules (e.g. protein template molecules) of the invention exhibit excellent storage stability. One-pot synthesis when forming a functionalised magnetic support

[0253] Where the template molecule is sufficiently robust (e.g. preferably where the template molecule is not a biological template molecule that is susceptible to degradation, e.g. by denaturation), such as a drug template molecule, it is possible to form a magnetically supported template molecule in situ during formation of the functionalised magnetic support. The relevant method is set forth in numbered paragraph B2 of the “SUMMARY OF THE INVENTION” section. Essentially, such a method simply involves adding / including a template molecule to / in relevant reaction mixtures of the methods of manufacturing a functionalised magnetic support (or functionalised magnetic particle) defined in numbered paragraphs A1- A5 of the “SUMMARY OF THE INVENTION” section, suitably as further defined in sub-section “Manufacturing of the Functionalised Magnetic Support” of the section entitled “FUNCTIONALISED MAGNETIC SUPPORT (OR FUNCTIONALISED MAGNETIC PARTICLE)”. In this manner, a magnetically supported template molecule may be produced (and isolated) using a method of manufacturing a functionalised magnetic support as defined herein. C) MAGNETIC-TEMPLATE-BOUND MOLECULARLY IMPRINTED POLYMER

[0254] Aspects and features of the invention relating to a magnetic template-bound molecularly imprinted polymer are described above in the relevant sub-section of the “SUMMARY OF THE INVENTION” and, in particular in numbered paragraphs C1-C11. Further features and sub-features of the same are elaborated below, all of which are applicable to any of the aforesaid aspects and to any other compatible aspects and embodiments described elsewhere herein.

[0255] The magnetically supported template molecule used in this section is preferably as defined and / or formed as defined in the above section entitled “B) MAGNETICALLY SUPPORTED TEMPLATE MOLECULE”. in – a to in if is ii). Bymolecules of just one magnetically supported template molecule, thereby leading to arrangement iii). However,arrangements ii) or iii), especially arrangement iii), may to non-zero quantities (and possibly large quantities in the case of arrangement iii) of arrangement iv) since a magnetically supported template molecule may bridge between two or more molecularly imprinted polymer molecules and a molecularly imprinted polymer molecule may bridge between two or more magnetically supported template molecules. What is perhaps of more importance is the relative ratios of magnetically supported template molecule(s) to molecularly imprinted polymer molecule(s) per magnetic template-bound molecularly imprinted polymer.

[0260] Preferably, there are a plurality of (i.e. at least two) molecularly imprinted polymer molecules per magnetically supported template molecule, suitably be it through arrangement iii) or iv) of the magnetic template-bound molecularly imprinted polymer. More preferably, there are at least three molecularly imprinted polymer molecules per magnetically supported template molecule, suitably be it through arrangement iii) or iv) of the magnetic template-bound molecularly imprinted polymer. Most preferably, there are at least four molecularly imprinted polymer molecules per magnetically supported template molecule, suitably be it through arrangement iii) or iv) of the magnetic template-bound molecularly imprinted polymer.

[0261] Suitably, each magnetic template-bound molecularly imprinted polymer molecule (or the MIP thereof) comprises one or more complementary imprints (also referred to herein as complementary cavities). Preferably, each magnetic template-bound molecularly imprinted polymer molecule (or the MIP thereof) comprises a plurality of (i.e. two or more) complementary imprints. More preferably, each magnetic template-bound molecularly imprinted polymer molecule (or the MIP thereof) comprises at least 10 complementary imprints. Most preferably, each magnetic template-bound molecularly imprinted polymer molecule (or the MIP thereof) comprises at least 20 complementary imprints.

[0262] A magnetic template-bound molecularly imprinted polymer is suitably a particle (magnetic-MIP conjugate particle).

[0263] Whilst the magnetic template-bound molecularly imprinted polymer may be isolated, in practice it is usually processed immediately to produce a (free) molecularly imprinted polymer, and suitably also to recover the magnetically supported template molecule as a recycled magnetically supported template molecule in readiness to produce further magnetic template-bound molecularly imprinted polymer molecule. Magnetically supported template molecule

[0264] The magnetically supported template molecule may be any suitably magnetically supported template molecule.

[0265] Where said magnetically supported template molecule is destined to be recycled / reused (i.e. to be or become a “recycled magnetically supported template molecule”), even magnetically supported template molecules of the prior art may be used (e.g. functionalised-silica-coated particles as described in R. Mahajan, M. Rouhi, S. Shinde, T. Bedwell, A. Incel, L. Mavliutova, S. Piletsky, I. A. Nicholls, B. Sellergren, Angew. Chem. Int. Ed.2019, 58, 727 (Mahajan et al)), since it would come as a surprise to a skilled person that such magnetically supported template molecules could be recycled, especially given that prior art methods of using such magnetically supported template molecules would render them unrecyclable.

[0266] However, most preferably, whenever defined in connection with the magnetic template-bound molecularly imprinted polymer (and its formation), whether in this section, hereinbefore, or hereinafter, the magnetically supported template molecule is suitably: a) A “new” (or non-recycled) magnetically supported template molecule produced by a method of manufacturing a magnetically supported template molecule as defined herein, suitably as defined by aspects B1-B3 or any sub- definitions thereof, such as those elaborated in the section entitled “B) MAGNETICALLY SUPPORTED TEMPLATE MOLECULE”; and / or b) A recycled magnetically supported template molecule, suitably as defined in any of aspects C1-C11 or any sub- definitions thereof, including as set forth below in the sub-section entitled “Recycling Magnetically Supported Template Molecule”.

[0267] Preferably the magnetically supported template molecule used in methods of manufacturing a magnetic template- bound molecularly imprinted polymer is either a) or b) above (with a) being used first and b) being used in subsequent recycling / reuse steps), but optionally it may be a mixture thereof. For instance, a batch of recycled magnetically supported template molecule may be doped with “new” magnetically supported template to offset any degradation in physical and / or chemical form thereof – this may be particularly helpful for batches that have already undergone multiple recycling rounds).

[0268] In the most preferred methods of the invention, a recycled magnetically supported template molecule is used at least once in the formation of magnetic template-bound molecularly imprinted polymers. Polymer, Monomer(s), and Cross-linking Monomer(s)

[0269] Any suitable polymer-forming components may be used to form a magnetic template-bound molecularly imprinted polymer or to form the molecularly imprinted polymer (MIP) of the magnetic template-bound molecularly imprinted polymer, and indeed a number of options are common general knowledge to the person skilled in the art.

[0270] The molecularly imprinted polymer (MIP) of the magnetic template-bound molecularly imprinted polymer preferably comprises (or is formed from) one or more monomers, wherein at least one of the monomers is a backbone- forming monomer and optionally another of the monomers is a cross-linking monomer. The molecularly imprinted polymer (MIP) of the magnetic template-bound molecularly imprinted polymer preferably comprises (or is formed from) one or more backbone-forming monomers, and optionally one or more cross-linking monomers. Preferably, the molecularly imprintedpolymer (MIP) of the magnetic template-bound polymer comprises at least one backbone-forming monomer and at least one cross-linking monomer.

[0271] The molecularly imprinted polymer (MIP) of the magnetic template-bound molecularly imprinted polymer may be a simple polymer (e.g. formed from a single backbone-forming monomer), a simple cross-linked polymer (e.g. formed from a single backbone-forming monomer and one or more single cross-linking monomers), a co-polymer (e.g. formed from two or more different backbone-forming monomers), or a cross-linked co-polymer (e.g. formed from two or more different backbone-forming monomers and one or more cross-linking monomers).

[0272] The molecularly imprinted polymer (MIP) of the magnetic template-bound molecularly imprinted polymer preferably comprises one or more, more preferably a plurality (see above for further preferred values), complementary imprints. Preferably, the molecularly imprinted polymer (MIP) of the magnetic template-bound molecularly imprinted polymer comprises at least two different complementary imprints (e.g. imprints that sample different parts of a template molecule). The complementary imprint(s) are suitably characterised by a plurality of different monomers. Preferably these different monomers are characterised by a difference in hydrophilicity and hydrophobicity. The complementary imprint(s) are suitably characterised by a plurality of different backbone-forming monomers. Preferably these different backbone-forming monomers are characterised by a difference in hydrophilicity and hydrophobicity.

[0273] Complementary imprints (of the MIP of the magnetic template-bound molecularly imprinted polymer) may be optimised for a given template molecule of the magnetically supported template molecule (e.g. in terms of template / target molecule affinity, template / / target molecule selectivity, diversity of imprints for a given template / target molecule, and / or ease of dissociation of magnetically supported template molecule from the MIP) through judicious selection of monomers, such as backbone-forming monomer(s) and optional cross-linking monomer(s), and, where relevant, relative amounts (or relative monomer ratios) thereof. It may be unwise to use exclusively hydrophobic monomers to form MIPs around a hydrophilic template molecule, or to use exclusively hydrophilic monomers to form MIPs around a hydrophobic template molecule, because mutual aversion would compromise templating and result in poor yields of complementary imprints. It may be possible to use monomer(s) that are neither too hydrophilic or too hydrophobic, especially where a template molecule contains a mixture of hydrophilic and hydrophobic moieties.

[0274] Since polymer production around the magnetically supported template molecule is “templated”, when using a mixture of monomer(s) (especially when using a mixture of backbone-forming monomer(s)), the monomer sequence characterising the complementary imprints may be statistically less randomised than other parts of the MIP owing to a templating effect. As such, a suitable balance and mix of hydrophobic, hydrophilic, ionised / ionisable, non-ionised / non- ionisable, hydrogen-bonding, non-hydrogen-bonding monomers (especially backbone-forming monomers) may be used, suitably alongside appropriate crosslinking monomer(s). Well-known molecular modelling, computational or otherwise, may be used to design optimal complementary imprints and prescribe appropriate monomer(s) or monomer mixtures for magnetic template-bound molecularly imprinted polymer production.

[0275] The one or more backbone-forming monomers are suitably selected from the group consisting of optionally substituted acrylamide(s), optionally substituted alkacrylamide(s) (e.g. optionally substituted methacrylamides), optionally substituted acrylic acid(s), optionally substituted alkacrylic acid(s) (e.g. optionally substituted methacrylic acids), optionally substituted acrylate ester(s), optionally substituted alkacrylate ester(s) (e.g. optionally substituted methacrylate esters), or any combination thereof (suitably a combination of two or more thereof where plurality of backbone-forming monomers are used). Preferably, the one or more backbone-forming monomers are selected from the group consisting of N- hydroxmethylacrylamide (NHMA), N-isopropylacrylamide (NIPAm), N-t-butylacrylamide (TBAm), N-(3- Aminopropyl)methacrylamide (APMA) (or a salt thereof), N-[Tris(hydroxymethyl)methyl]acrylamide (TrisAm), 2-Acrylamido- 2-methylpropane sulfonic acid (AMPS), fluorescein acrylamide, fluorescein o-acrylate, acrylic acid, methacrylic acid, methyl acrylate, methyl methacrylate, or any combination thereof. Most preferably, the one or more backbone-forming monomers comprise (or more preferably consist of) N-hydroxmethylacrylamide (NHMA).

[0276] The one or more cross-linking monomers are suitably selected from polyvinyl monomers (a monomer with two or more vinyl moieties), more suitably divinyl monomers (i.e. a monomer with two alkene moieties). Preferably, the one or more cross-linking monomers are suitably selected from the group consisting of N,N’methylenebisacrylamide (BIS, MBAm), allyl methacrylate (AM), divinylbenzene, ethyleneglycol dimethacrylate, dipentaerythritol pentaacrylate, pentaerythritol tetraacrylate, and any combination (though preferably only a single cross-linking monomer). Most preferably the one or more cross-linking monomers comprise (or more preferably consist of) N,N’methylenebisacrylamide (BIS, MBAm),

[0277] The polymer (MIP) may comprise one or more detector moieties, suitably thereby allowing the polymer to act as a molecular sensor. Physical and / or chemical properties (e.g. fluorescence) of the detector moieties are suitably sensitive (and thus susceptible to change) to changes in the molecular-binding status of complementary imprint(s) – i.e. the physical and / or chemical properties (e.g. fluorescence) of the detector moieties change when complementary imprint(s) bind a molecule, as compared to when the complementary imprint(s) are unbound. As such, physical and / or chemical properties of said detector moieties may be used to determine whether or not, and / or the extent to which, molecules are bound by or within complementary imprint(s) (e.g. target molecules or indeed magnetically supported template molecules). This can be useful for detecting the presence or otherwise, or concentration (relative or absolute depending on calibration), of “target molecules” (see later uses of MIPs). This may also be used to detect whether or not magnetically supported template molecules have been successfully removed, thus serving as a means to perform in-process check checks.

[0278] As such, one or more of the monomer(s), suitably forming monomer(s), used to form the polymer (MIP) may suitably comprise one or more detector monomer(s) comprising one or more of the aforesaid detector moieties. The relative concentration of the detector monomer(s) may be selected to suit the application. However, in general detector monomer(s) constitute less than or equal to 10 mol% of all of the monomers used to form the polymer (i.e. less than or equal to 10 moles of detection monomer per hundred moles of all monomers, or 10:90 molar ratio of detector monomer to other monomer(s)), preferably less than or equal to 2 mol%, more preferably less than 1 mol%. It will be understood that detector monomer(s) may be monomer(s) (e.g. with one or more pendent reactive functional groups, such as amino and / or carboxylate groups and the like) that can be post-functionalised with a detector moiety or otherwise post-coupled with a derivatising molecule comprising a detector moiety.

[0279] A variety of detector moieties are well known in the art, and desired MIP polymers can be produced accordingly. The detector moieties preferably comprise or consist of electromagnetic radiation detector moieties (i.e. moieties whose absorption and / or emission of electromagnetic radiation changes in response to differential binding of complementary imprints), radioactive detector moieties (i.e. moieties whose radioactivity changes in response to differential binding of complementary imprints), or electrochemical detector moieties (i.e. moieties whose electrochemical properties change in response to differential binding of complementary imprints). For instance, a fluorescent detector moiety may fluoresce differently when a molecule is bound within a complementary imprint compared to when the same complementary imprint is empty. An example of a suitable fluorescent detector monomer with a fluorescent detector moiety is fluorescein acrylamide, fluorescein o-acrylate or Nile blue acrylamide. The detector moiety may be an enzyme. For instance, an enzyme-labelled MIP may be used in immunoassays (e.g. ELISA - Enzyme-Linked Immunosorbent Assay).

[0280] It will be understood that MIP polymers may be optionally derivatised after their initial production (e.g. after producing a free molecularly imprinted polymer) to furnish derivatised (free) molecularly imprinted polymers, and that these are duly encompassed by the general term molecularly imprinted polymer, especially free molecularly imprinted polymer, used herein. Derivatisation may include, for instance, conjugation (with another molecule), functionalisation, and / or labelling. As such, An MIP may be derivatised with detector moieties, including those mentioned above in relation to monomers that contain detector moieties. This can be readily achieved by initially forming MIPs with at least some monomers that bear a functionalisable group, such as a pendent amino group (-NH2) or carboxylate group (-CO2H), to which another molecule (e.g. detector molecule with a detector moiety) may be coupled. An example includes an APMA monomer which can be used, suitably alongside other monomers, to afford an MIP bearing amino moieties which may thereafter be derivatised by conjugation, functionalisation, or labelling. It will be understood that references herein to a molecularly imprinted polymer, especially a free molecularly imprinted polymer, include such derivatives.

[0281] Suitably, each MIP of the magnetic template-bound molecularly imprinted polymer comprises one or more complementary imprints, preferably a plurality of complementary imprints. More preferably, each MIP comprises at least 10 complementary imprints, most preferably at least 20 complementary imprints. These numbers of complementary imprints may be an average.

[0282] Suitably, MIPs with a plurality of complementary imprints comprise one or more different complementary imprints (e.g. differently-shaped complementary imprints). Different complementary imprints may be a consequence of from polymerisation around different parts of the template molecule of the magnetically supported template molecule. Manufacturing of the Magnetic Template-Bound Molecularly Imprinted Polymer

[0283] Methods of manufacturing a magnetic template-bound molecularly imprinted polymer are outlined above in numbered paragraphs C1, C2, C8, and C9 of the “SUMMARY OF THE INVENTION” section. Such methods also foreshadow methods of recycling the magnetically supported template molecule, as outlined in C3-C6, and the recycled magnetically supported template molecule itself in C7, which may be reused in further rounds of manufacturing a magnetic template-bound molecularly imprinted polymer.

[0284] The method suitably involves forming a polymer (or polymerising one or more monomers or co-monomers) in the presence of a magnetically supported template molecule (as defined herein). The polymer is suitably an MIP, and the monomer(s) and / or co-monomer(s) (including any cross-linking monomer(s) and / or detector monomer(s)) are suitably as defined above in the section entitled “Polymer, Monomer(s), and Cross-linking Monomer(s)”.

[0285] Needless to say, the magnetically supported template molecule used in methods of manufacturing a magnetic template-bound molecularly imprinted polymer may be a recycled magnetically supported template molecule. Reaction Conditions

[0286] The method of manufacturing the magnetic template-bound molecularly imprinted polymer suitably comprises mixing together input components in a solvent or solvent system to form a reaction mixture. After mixing the input components, a reaction (e.g. polymerisation reaction) suitably takes places or a reaction is otherwise initiated. The solvent or solvent system suitably comprises water, and is suitably a buffered solvent or solvent system (e.g. at a pH between 5 and 10, preferably at a pH between 6 and 9, more preferably at a pH between 7 and 8, most preferably a pH of about 7.4). The buffered solvent or solvent system suitably comprises a buffer system, suitably phosphate-buffered saline (PBS).

[0287] The input components comprise the magnetically supported template molecule and one or more monomers, suitably as defined above in the section entitled “Polymer, Monomer(s), and Cross-linking Monomer(s)”. As explained in thesame section, the one or more monomers preferably at least one backbone-forming monomer and at least one cross-linking monomer. Also as explained above, optimal complementary imprinted may be obtained by using monomers, or a combination thereof, which complements the nature of the magnetically supported template molecule.

[0288] The input components preferably also comprise an initiator or initiator system, suitably an initiator system comprising a redox couple, most preferably ammonium persulfate (APS) and N,N,N’,N’-tetramethylethylenediamine (TEMED). The input component may preferably also comprise a surfactant (preferably an anionic surfactant, most preferably sodium dodecyl sulphate (SDS)), especially where it is desirable to use a surfactant to better support the sustained dispersion of magnetically supported template molecule (e.g. MNPs) during this magnetic template-bound molecularly imprinted polymer formation process.

[0289] The reaction mixture is suitably located within a reaction vessel. Preferably, all of the input components (or at least the solutes thereof) are dissolved, dispersed, or suspended within the reaction mixture. Preferably, the reaction does not take place in the presence of a column or packed column of the magnetically support template molecule.

[0290] The reaction mixture is preferably agitated (e.g. stirred) – suitably this facilitates polymerisation (i.e. formation of MIPs) at the surface of the magnetically supported template molecule(s). Even larger magnetically support template molecules are surprisingly robust upon agitation and stirring.

[0291] During the reaction, the reaction mixture is suitably maintained at a temperature of 25oC + / - 25oC, more preferably 25oC + / - 10oC.

[0292] The reaction is suitably left for a sufficient time period for a reaction to take place. The time period is suitably 1 to 120 mins, suitably 5 to 30 minutes, suitably 10 to 20 mins.

[0293] In process checks (IPCs) may be used to determine whether or not the reaction is complete.

[0294] Preferably the reaction is quenched, suitably after sufficient reaction has taken place or after a suitable period of reaction time, such as mentioned above. The reaction is preferably quenched with a quenching agent that suitably quenches polymerisation reactions, suitably radical polymerisation reactions. Methylhydroquinone (MHQ) is a suitable quenching agent.

[0295] If a reaction is allowed to proceed for too long, the MIPs can become too large, rendering them less dispersible and thus less useful in downstream methods.

[0296] Using magnetically supported template molecules with template molecules tightly bound to the surface of the magnetic cores facilitates optimal complementary imprints which are selective and yet more easily dissociated form the magnetically supported template molecules. This allows for excellent MIPs and also optimal recycling of magnetically supported template molecules that can then be reused to produce more MIPs. Overall this may increase MIP yield per template molecule. Separating molecularly imprinted polymer (MIP) from non-imprinted polymer (NIP)

[0297] Suitably, after the reaction is deemed complete, sufficiently complete, or has been left for a sufficient time period (e.g. as per above), and / or after the reaction has been quenched, the reaction mixture comprises magnetic template-bound molecularly imprinted polymer. The reaction mixture may suitably also comprise non-imprinted polymer (NIP), which are polymers that have (substantially) no complementary imprints (e.g. polymers that formed away from the surface of the magnetically supported template molecule), and are thus have no magnetically supported template molecule(s) bound thereto or therein. Preferably, non-imprinted polymer (NIP) is separated from the magnetic template-bound molecularly imprinted polymer, suitably by magnetic separation (since magnetic template-bound molecularly imprinted polymer is suitably attracted to a magnetic field whereas NIP is not). As such, after the reaction is deemed complete, sufficiently complete, or has been left for a sufficient time period (e.g. as per above), and / or after the reaction has been quenched, the reaction mixture is preferably exposed to a magnetic field (e.g. to magnetically constrain magnetic template-bound molecularly imprinted polymer) whilst non-imprinted polymer (NIP) is separated therefrom.

[0298] After the reaction is deemed complete, sufficiently complete, or has been left for a sufficient time period (e.g. as per above), and / or after the reaction has been quenched, the reaction mixture is preferably exposed to a magnetic field and, whilst the magnetic template-bound molecularly imprinted polymer is constrained by the magnetic field, supernatant is removed and the magnetic template-bound molecularly imprinted polymer is optionally washed with further solvent or solvent system (which is optionally the same or a different solvent or solvent system to that used in the reaction mixture). This process suitably also removes any unreacted monomers and reagents.

[0299] Magnetic template-bound molecularly imprinted polymer may thereafter be recovered and suitably further processed in downstream method steps, such as a method of manufacturing a (free) molecularly imprinted polymer (MIP). Recycling Magnetically Supported Template Molecule

[0300] Methods of recycling a magnetically supported template molecule, used in a method of manufacturing a template- bound molecularly imprinted polymer, are outlined in numbered paragraphs C3-C6 of the “SUMMARY OF THE INVENTION” section, and yield the recycled magnetically supported template molecule as per paragraph C7 of the same section, which may be reused in further rounds of manufacturing a magnetic template-bound molecularly imprinted polymer.

[0301] Recycling a magnetically supported template preferably occurs during the formation of a (free) molecularly imprinted polymer (MIP), as outlined in paragraphs D1-D13 of the “SUMMARY OF THE INVENTION” section, since forming a (free) MIP suitably involves separating (or unbinding) the magnetically supported template molecule from MIP to which the magnetically supported template molecule is bound within the magnetic template-bound molecularly imprinted polymer.

[0302] As such, recycling a magnetically supported template molecule suitably involves separating (or unbinding) the magnetically supported template molecule from the MIP to which the magnetically supported template molecule is bound within the magnetic template-bound molecularly imprinted polymer, and suitably thereafter isolating the recycled / recovered magnetically supported template molecule. Suitably, during separating / unbinding, the recycled / recovered magnetically supported template molecule is unbound (or dissociated) from complementary imprints within the MIP.

[0303] Separating the magnetically supported template molecule (as a recycled magnetically supported template molecule) from the molecularly imprinted polymer of the magnetic template-bound molecularly imprinted polymer is preferably performed under mild conditions, suitably to avoid damaging or degrading the template molecule, especially where the template molecule is a biological molecule, such as a protein, that is capable of denaturing (e.g. unfolding / degradation). As such, such separating is preferably performed at a temperature between -50oC and 80oC, more preferably between 0oC and 70oC, more preferably 25oC + / - 25oC, most preferably 25oC + / - 10oC.

[0304] Separating preferably comprises agitating the magnetic template-bound molecularly imprinted polymer, preferably in a solvent or solvent system (preferably comprising or consisting of water). Agitating preferably comprises sonication (or sonicating) – i.e. application of ultrasound. Sonication is suitably performed at ultrasound frequencies between 2 and 18,000 kHz, preferably between 5 and 2,000 kHz, more preferably between 10 and 200 kHz, most preferably between 20 and 100 kHz. Sonication is suitably performed at an ultrasound power between 5 and 10,000 Watts, preferably between 10 and 3,000 Watts, more preferably between 50 and 1,000 Watts, most preferably between 400 and 800 Watts.

[0305] Separating (especially where agitation, such as the aforesaid sonication, is used) suitably proceeds, or agitation (or sonication) suitably proceeds, over a “separating time period”. The “separating time period” is suitably between 10 seconds and 120 minutes, preferably between 30 seconds and 30 minutes, more preferably between 1 minute and 15 minutes, most preferably between 2 minutes and 10 minutes. Longer separating time periods may lead to degradation of the recycled magnetically supported template molecule.

[0306] Separating may comprise (preferably in combination with one or more other separating conditions, such as agitating, especially sonicating), exposing the magnetic template-bound molecularly imprinted polymer, preferably in a solvent or solvent system, to a magnetic field. Application of such a magnetic field, during the separating step, may further assist separation, and may permit even milder separating conditions to be used. The magnetic field may be a pulsed or resonating magnetic field (e.g. the strength of the magnetic field changes over time, suitably at an appropriate pulse or resonant frequency).

[0307] Separating (or unbinding) suitably yields a mixture of (unbound) recycled magnetically supported template molecule and (free) MIP. After separating (or unbinding) the recycled magnetically supported template molecule from the MIP to which the magnetically supported template molecule is bound within the magnetic template-bound molecularly imprinted polymer, the recycled magnetically supported template molecule is preferably magnetically separated from the MIP, suitably by exposing the mixture of (unbound) recycled magnetically supported template molecule and (free) MIP to a magnetic field and then separating the recycled magnetically supported template molecule (which is suitably magnetically constrained by the magnetic field) from the (free) MIP, suitably by removing supernatant containing the MIP and optionally further washing (suitably magnetically constrained) the recycled magnetically supported template molecule. The recycled magnetically supported template molecule may then be recovered, isolated, and optionally dried (e.g. in vacuo), though preferably the recycled magnetically supported template molecule is not exposed to heat.

[0308] The recycled magnetically supported template molecule may then be reused, and optionally recycled again for further reuse, in further methods of manufacturing a magnetic template-bound molecularly imprinted polymer and further methods of manufacturing a (free) molecularly imprinted polymer.

[0309] Preferably a magnetically supported template molecule is recycled at least once – i.e. recovered and reused at least once in a method of manufacturing a magnetic template-bound molecularly imprinted polymer (or a method of manufacturing an MIP). More preferably a magnetically supported template molecule is recycled at least twice – i.e. is recovered after having been already reused as a recycled magnetically supported template molecule and then further reused. Most preferably a magnetically supported template molecule is recycled at least three times. Preferably a magnetically supported template molecule is recycled at most 20 times, more preferably at most 10 times, most preferably at most 6 times. So long as mild conditions are used in the recycling of magnetically supported template molecules, the template molecule will not (substantially) degrade (as suggested by CD analysis) and, even if it does, when using magnetically supported template molecules of the correct size, a sufficient number of useful complementary imprints can still be generated within MIPs. The more a magnetically supported template molecule is recycled, the more likely it can become contaminated, e.g. with unbound MIPs (which are thus still magnetically attracted), which can render it less dispersible during formation of MIPs. This can be mitigated by “doping” the recycled magnetically supported template molecule with a “new” (or less recycled) magnetically supported template molecule.

[0310] Recycling of the magnetically supported template molecule can significantly increase yields of MIP per template molecule, and it avoids wasting of precious and potentially expensive template molecules.D) FREE (MAGNETIC TEMPLATE-UNBOUND) MOLECULARLY IMPRINTED POLYMERS (MIPS)

[0311] Aspects and features of the invention relating to a (free) molecularly imprinted polymer, methods of manufacturing the same, recycling magnetically supported templates, and related compositions, are described above in the relevant sub- section of the “SUMMARY OF THE INVENTION” and, in particular in numbered paragraphs D1-D16. Further features and sub-features of the same are elaborated below, all of which are applicable to any of the aforesaid aspects and to any other compatible aspects and embodiments described elsewhere herein.

[0312] The magnetic template-bound molecularly imprinted polymer used in this section is preferably as defined and / or formed as defined in the above section entitled “C) MAGNETIC-TEMPLATE-BOUND MOLECULARLY IMPRINTED POLYMER”.

[0313] The (free) molecularly imprinted polymer (MIP) is preferably the MIP of the magnetic template-bound molecularly imprinted polymer described in the previous section entitled “C) MAGNETIC-TEMPLATE-BOUND MOLECULARLY IMPRINTED POLYMER”, and the polymer and monomer(s) described in the sub-section thereof entitled “Polymer, Monomer(s), and Cross-linking Monomer(s)” preferably also define the MIP of this section. The above sub-section entitled “Recycling Magnetically Supported Template Molecule" describes separating the MIP from the magnetically supported template molecule in the context of recovering a recycled magnetically supported template molecule, but the MIP may be recovered similarly by processing the components separated from the recycled magnetically supported template molecule and isolating the MIP.

[0314] The magnetic template-bound molecularly imprinted polymer (MIP-MNP@Template, e.g. MIP-MNP@Protein) is suitably an intermediate formed enroute to the (free) molecularly imprinted polymer (e.g. MIP or NanoMIP) described in this section. The MIP is preferably formed by separating (or unbinding) the MIP from the magnetically supported template molecule (MNP@Template) of the magnetic template-bound molecularly imprinted polymer (MIP-MNP@Template) and thereafter isolating the MIP. As described herein, the magnetically supported template molecule may also be isolated as a recycled magnetically supported template molecule (rMNP@Template) which may be reused as the magnetically supported template molecule in further MIP production rounds.

[0315] The molecularly imprinted polymer is preferably a polymer, co-polymer, cross-linked polymer, or cross-linked co- polymer as described in the sub-section entitled “Polymer, Monomer(s), and Cross-linking Monomer(s)” of the previous section entitled “C) MAGNETIC-TEMPLATE-BOUND MOLECULARLY IMPRINTED POLYMER”, the only difference being that the molecularly imprinted polymer is free and unbound. That said, where imperfect or incomplete “unbinding” has occurred, the molecularly imprinted polymer may comprise traces of magnetically supported template molecules bound therewith, and references herein to a (free) molecularly imprinted polymer do not exclude the possibility that some magnetically supported template molecules remain bound – importantly at least one or some of the complementary imprints are “free” and unbound. Suitably at least 50% of the complementary imprint(s) of the (free) molecularly imprinted polymer are vacated (or unbound to a magnetically supported template molecule), preferably at least 70% vacated, more preferably at least 90% vacated, most preferably 100% vacated. The higher the level of vacation, the easier the MIPs are magnetically separated from unbound (or recycled) magnetically supported template molecules.

[0316] Preferably the molecularly imprinted polymer comprises molecularly imprinted polymer (or batches thereof) formed using a recycled magnetically supported template molecule.

[0317] The molecularly imprinted polymer is a polymer comprising one or more imprints (also referred to herein as “complementary imprints”) of a template molecule or a part(s) thereof. Such imprints preferably have a binding affinity for the template molecule or another target molecule comprising part(s) that are the same or similar to a part of the template molecule from which the imprint was formed. The molecularly imprinted polymer may comprise a plurality of imprints of the template molecule (or part(s)) thereof. One or more of the plurality of imprints may be different, suitably as a consequence of imprinting different parts of a template molecule.

[0318] Suitably, the MIP comprises one or more complementary imprints, preferably (suitably on average) a plurality of complementary imprints. More preferably, the MIP comprises (on average) at least 10 complementary imprints, most preferably at least 20 complementary imprints. The multiplicity of complementary imprints is suitably facilitated by the particle size of and relatively large number of pendant template molecules around the magnetically supported template molecule. Suitably, the MIP comprise one or more different complementary imprints (e.g. differently-shaped complementary imprints). Different complementary imprints suitably mirror different parts of the template molecule of the magnetically supported template molecule.

[0319] The (free) MIP is preferably a particulate solid.

[0320] Suitably, the average particle size of the (free) MIP is between 3 and 1500 nm, or between 50 and 500 nm. Preferably, the (free) MIP has an average particle size between 10 and 1000 nm, more preferably between 20 and 500 nm, most preferably between 100 and 400 nm. The (average) particle size may be determined by DLS and / or SEM. DLS is suitably the default method of measuring (average) particle size.

[0321] Preferably, the (free) MIP has an average particle size between 30 and 500 nm, more preferably between 40 and 400 nm, most preferably between 80 and 300 nm. The average particle size of the (free) MIP may depend on the corresponding template molecule (e.g. target protein), and may in particular depend on the size of the corresponding template molecule (e.g. size of the target protein).

[0322] Suitably, ratio of average particle size of the supported template molecule (used to form the MIP, i.e. the MIP of the magnetic template-bound molecularly imprinted polymer) to the average particle size of the (free) MIP is 0.2- 20 : 1, preferably 0.5-10 : 1, more preferably 0.75-5 : 1, most preferably 1-2 : 1. Preferably the average particle size of the magnetically supported template molecule is greater than or equal to, most preferably greater than, the average particle size of the (free) MIP.

[0323] Preferably, the ratio of average particle size of the magnetically supported template molecule to the average particle size of the (free) MIP is 0.5-15 : 1, preferably 0.75-10 : 1, more preferably 1-5 : 1, most preferably 2-3 : 1.

[0324] The MIP is preferably a hydrogel, and is thus preferably formed from monomers that yield a hydrogel. A hydrogel MIP facilitates transport to and from imprints (i.e. binding sites).

[0325] The magnetically supported template molecule used in this section (and that used in the formation of the magnetic template-bound molecularly imprinted polymer from which the MIPs of this section are isolated) is preferably a magnetically supported template molecule in accordance with the invention (suitably as defined herein, e.g. with a molecular template directly coupled to a magnetic core via reactive functional group(s) borne on the surface thereof, where there is no silica or functionalised-silica coating upon the magnetic core), but it may (especially where it is recycled) be a magnetically supported template molecule of the prior art.

[0326] As per aspect D16 of the relevant sub-section of the “SUMMARY OF THE INVENTION”, the present invention provides a composition comprising a (free) MIP. Such a composition is suitably a solid composition. Alternatively, the composition may be a liquid composition, for instance, with the (free) MIP dispersed or suspended within a solvent or solvent system.

[0327] An aspect of the invention thus provides a solid composition comprising MIP particles (suitably obtained by any method of manufacturing a (free) MIP as defined herein).

[0328] An aspect of the invention also provides a particulate dispersion or suspension of (free) MIP particles (suitably obtained by any method of manufacturing a (free) MIP as defined herein).

[0329] As will be readily appreciated by a person skilled in the art, methods pertaining to the manufacture of a (free) MIP may be preceded by any method pertaining to the manufacture of a magnetic template-bound molecularly imprinted polymer (used in such methods), which in turn may be optionally preceded by any method pertaining to the manufacture of a magnetically supported template molecule (used in said methods), which in turn may be optionally preceded by any method pertaining to the manufacture of a functionalised magnetic support (used in said methods). Manufacturing of the (free) Molecularly Imprinted Polymer Unbinding and Separating

[0330] The (free) MIP is preferably formed by unbinding and separating the MIP from the magnetically supported template molecule (MNP@Template) of the magnetic template-bound molecularly imprinted polymer (MIP-MNP@Template). Such unbinding and separating may optionally proceed as described in the previous section entitled “C) MAGNETIC-TEMPLATE- BOUND MOLECULARLY IMPRINTED POLYMER”, and in particular the sub-section thereof entitled “Recycling Magnetically Supported Template Molecule". Likewise, methodologies outlined in this section may be applied to those in the sub-section entitled “Recycling Magnetically Supported Template Molecule".

[0331] Preferably, the magnetic template-bound molecularly imprinted polymer is subjected to “unbinding conditions” to unbind (or dissociate) the MIP from the magnetically supported template molecule with which it is bound (or associated) within the magnetic template-bound molecularly imprinted polymer. Any suitable unbinding conditions may be used, and many are known in the art, including inter alia solvent extraction (e.g. using hot solvent to wash out the magnetically supported template molecule) such as: ^ Soxhlet extraction; ^ Incubation (e.g. insolvents that cause MIP swelling and dissociation of the magnetically supported template molecule from the MIP); ^ Ultrasound-assisted extraction (i.e. sonication); ^ Microwave-assisted extraction; ^ Mechanical extraction; ^ Subcritical fluid (e.g. subcritical water) extraction; and / or ^ Supercritical fluid (e.g. supercritical carbon dioxide) extraction.

[0332] However, where the intention is to recycle and reuse the magnetically supported template molecule, many of these methods may be too aggressive, especially where the template molecule is unstable (e.g. protein template molecules). MIPs with imprints of complex molecules (e.g. protein template molecules) may also be damaged if unbinding conditions are too harsh – the imprint region may in particular be vulnerable and suffer loss of selectivity.

[0333] Preferably the “unbinding conditions” comprise agitating the magnetic template-bound molecularly imprinted polymer, preferably in a solvent or solvent system (preferably comprising or consisting of water). The solvent or solvent system may suitably include a buffer, such as phosphate-buffered saline (e.g. at a pH between 4-8) to mitigate degradation of protein template molecules. Agitating preferably comprises sonication (or sonicating) – i.e. application of ultrasound.Sonication is suitably performed at ultrasound frequencies 2 and 18,000 kHz, preferably between 5 and 2,000 kHz, more preferably between 10 and 200 kHz, most preferably between 20 and 100 kHz. Sonication is suitably performed at an ultrasound power between 5 and 10,000 Watts, preferably between 10 and 3,000 Watts, more preferably between 50 and 1,000 Watts, most preferably between 400 and 800 Watts. The magnetically supported template molecule is suitably subjected to such unbinding conditions for a time period between 10 seconds and 120 minutes, preferably between 30 seconds and 30 minutes, more preferably between 1 minute and 15 minutes, most preferably between 2 minutes and 10 minutes.

[0334] The unbinding conditions may comprise (especially in combination with agitating, especially sonicating as per above), exposing the magnetic template-bound molecularly imprinted polymer, preferably in a solvent or solvent system, to a magnetic field. Application of such a magnetic field, during the unbinding step, may further assist unbinding, and may permit even milder unbinding conditions to be used. The magnetic field may be a pulsed or resonating magnetic field (e.g. the strength of the magnetic field changes over time, suitably at an appropriate pulse or resonant frequency).

[0335] The method may comprise implementing in-process checks to determine whether unbinding is (substantially or sufficiently) complete. If the polymer comprises detector monomers, these may be used (e.g. by measuring spectroscopic changes) to ascertain whether or not unbinding is complete.

[0336] After unbinding (e.g. after the magnetically supported template molecule is subjected to unbinding conditions for a sufficient time period) the (free) MIP is suitably magnetically separated from the magnetically supported template molecule magnetically. Preferably, magnetic separating comprises application of a magnetic field to magnetically constrain the magnetically supported template molecule, followed by removal / separating of the MIP (which is not magnetically constrained). The MIP is suitably separated from the magnetically supported template molecule within a solvent or solvent system (i.e. a supernatant or washing). Suitably the magnetically supported template molecule, still under magnetic constraint, may be washed (optionally a plurality of times) with a solvent or solvent system and suitably washings are combined (e.g. with the supernatant and / or other washings) to provide a liquid composition comprising the MIP. The MIP is suitably insoluble within the liquid composition (i.e. suitably as suspended or dispersed MIP particles).

[0337] Unbinding and separating are preferably performed at a temperature between -50oC and 80oC, more preferably between 0oC and 70oC, more preferably 25oC + / - 25oC, most preferably 25oC + / - 10oC.

[0338] If unbinding is incomplete, there can be a loss of MIP yield since the magnetic template-bound molecularly imprinted polymer is magnetic and will become magnetically constrained alongside unbound magnetically supported template molecule during magnetic separation. The methodology of the present invention, including qualities associated with the magnetically supported template molecule itself, facilitate such unbinding without compromising selectivity for target molecules in downstream MIP uses. Further Isolating the MIP

[0339] Isolating the MIP may simply comprise unbinding and separating the MIP from the magnetically supported template molecule. As such, the MIP may be stored and / or used immediately following the unbinding and separating of the MIP from the magnetically supported template molecule. For instance, the liquid composition comprising the MIP, described above, may be stored and / or used. However, the MIP (or composition thereof) may be further processed to isolate the MIP. For instance, after unbinding and separating the MIP from the magnetically supported template molecule, the MIP may be additionally isolated. Such isolating may comprise separating the solid matter of the aforementioned liquid composition from the liquid. For instance, isolating the MIP may comprise filtration (or centrifugation) and subsequent isolation of the solid as the MIP. Multi-batch MIP production by recycling / reusing magnetically-Supported Molecular Template

[0340] The methods of manufacturing outlined in aspects D4-D10, in particular those in D8 and D9,of the relevant sub- section of the “SUMMARY OF THE INVENTION” foreshadow multi-batch (free) MIP production involving recycling and reuse of the magnetically supported template molecule.

[0341] Multi-batch MIP production preferably comprises several MIP manufacturing rounds, at least one round of which uses a recycled magnetically supported template molecule.

[0342] As such, after unbinding and separating the MIP from the magnetically supported template molecule (MNP@Template), as per above, the magnetically supported template molecule is preferably isolated as a recycled magnetically supported template molecule. Separating and isolating the recycled magnetically supported template molecule is also described in the previous section entitled “C) MAGNETIC-TEMPLATE-BOUND MOLECULARLY IMPRINTED POLYMER”, and in particular the sub-section thereof entitled “Recycling Magnetically Supported Template Molecule".

[0343] Preferably, the aforesaid method of manufacturing a (free) MIP, suitably via a corresponding magnetic template- bound molecularly imprinted polymer, is repeated one or more times (through “one or more further manufacturing rounds”) with the recycled magnetically supported template molecule, suitably each time (with the optional exception of the final time) isolating further recycled magnetically supported template molecule. Suitably, before reuse, each (batch of) recycled magnetically supported template molecule is analysed (e.g. using visual inspection and / or circular dichroism (CD) spectroscopy) to confirm that it can be reused. Where a (batch of) recycled magnetically supported template molecule isdetermined to be unsuitable for reuse (e.g. due to chemical of the template molecule and / or degradation in the physical form of the recycled magnetically supported template molecule), optionally said (batch of) recycled magnetically supported template molecule may be further processed to purify the recycled magnetically supported template molecule and / or said (batch of) recycled magnetically supported template molecule may be doped with (suitably a purer and / or physically less degraded form of) magnetically supported template molecule.

[0344] Preferably a magnetically supported template molecule is recycled at least once – i.e. recovered and reused at least once in methods of manufacturing an MIP (suitably via the corresponding magnetic template-bound molecularly imprinted polymer). More preferably a magnetically supported template molecule is recycled at least twice – i.e. is recovered after having been already reused as a recycled magnetically supported template molecule and then further reused. Most preferably a magnetically supported template molecule is recycled at least three times. Preferably a magnetically supported template molecule is recycled at most 20 times, more preferably at most 10 times, most preferably at most 6 times. So long as mild conditions are used in the recycling of magnetically supported template molecules, the template molecule will not (substantially) degrade (as suggested by CD analysis) and, even if it does, when using magnetically supported template molecules of the correct size, a sufficient number of useful complementary imprints can still be generated within MIPs. The more a magnetically supported template molecule is recycled, the more likely it can become contaminated, e.g. with unbound MIPs (which are thus still magnetically attracted), which can render it less dispersible during formation of MIPs. This can be mitigated by “doping” the recycled magnetically supported template molecule with a “new” (or less recycled) magnetically supported template molecule.

[0345] Recycling of the magnetically supported template molecule can significantly increase yields of MIP per template molecule, and it avoids wasting of precious and potentially expensive template molecules.

[0346] Using larger magnetically supported template molecules (especially where the template molecule is tightly bound thereto with minimal linker(s)) in MIP production facilitates formation of multiple complementary imprints per MIP molecule, thereby providing multiple target binding sites per MIP. Using larger magnetically supported template molecules in MIP production facilitates formation of multiple different complementary imprints which may bind to different parts of a template or target molecule. Furthermore, using larger magnetically supported template molecules in MIP production facilitates formation of complementary imprints which offer an excellent balance of high selectivity and binding affinities alongside relatively facile unbinding of magnetically supported template molecules – this allows for milder unbinding conditions, thereby improving the recyclability of magnetically supported template molecules and consequently improving overall yields of MIP per unit mass of original template molecule.

[0347] Suitably, the method of manufacturing an MIP (suitably via a corresponding magnetic template-bound molecularly imprinted polymer), especially where the method involves the aforementioned recycling of the magnetically supported template molecule, yields between 0.1 and 100 MIP molecules per magnetically supported template molecule, preferably between 0.5 and 50, more preferably between 1 and 20, most preferably between 3 and 9.

[0348] Suitably, the method of manufacturing an MIP (suitably via a corresponding magnetic template-bound molecularly imprinted polymer), where the method involves protein template molecules (i.e. magnetically supported template molecules whose template molecules are protein template molecules, suitably as defined herein), yields at least 5 milligrams of MIP per milligram of protein template molecule, preferably at least 10 milligrams of MIP per milligram of protein template molecule, more preferably at least 50 milligrams of MIP per milligram of protein template molecule, most preferably at least 100 milligrams of MIP per milligram of protein template molecule.

[0349] Suitably, the method of manufacturing an MIP (suitably via a corresponding magnetic template-bound molecularly imprinted polymer), where the method involves protein template molecules (i.e. magnetically supported template molecules whose template molecules are protein template molecules, suitably as defined herein), yields between 5 and 500 milligrams of MIP per milligram of protein template molecule, preferably 10-400 mg / mg, more preferably 50-300 mg / mg, most preferably 100-200 mg / mg.

[0350] The aforementioned recycling methodology surprisingly provides a route to a rapid, low-cost, industrial scale production of MIPs, even when using protein template molecules which are typically more vulnerable to degradation and are less amenable to recycling.

[0351] As explained herein, the method of manufacturing (free) MIPs may involve one or more monomers that comprise reactive moieties that are coupled to a detectable moiety after the polymer has been formed, and that the (free) molecularly imprinted polymer (MIP) may be labelled, for instance radio-labelled or fluorescent-labelled. As such, it will be understood by those skilled in the art that references herein to a molecularly imprinted polymer, especially a free molecularly imprinted polymer (MIP), includes MIPs that have been subjected to further processing, such as derivatives thereof (e.g. conjugates, post-functionalised, or post-labelled versions thereof), and that methods of manufacturing a (free) molecularly imprinted polymer (MIP) may optionally further comprise a step of derivatising (e.g. conjugating, post-functionalising, post-labelling). Derivatising preferably comprises coupling at least one derivatising molecule (e.g. an enzyme, a radioactive label, a fluorescent label, or redox label) to the initially-obtained (free) molecularly imprinted polymer (MIP), preferably via a reactive moiety (or functionalisable group) provided by one or more of the monomers used in the formation of the molecularly imprinted polymer. The functionalisable group may be, for instance, be a pendent amino group (-NH2) or carboxylate group (-CO2H). An APMA monomer can provide a pendent amino group to which a derivatising molecule may be coupled by means well known in the art. Such derivatising affords a derivatised molecularly imprinted polymer. A derivatisedmolecularly imprinted polymer may be especially useful in For instance, an enzyme-labelled MIP can be deployed in a manner similar to an enzyme-labelled antibody. For instance, an enzyme-labelled MIP may be used in immunoassays. E) USES OF MOLECULARLY IMPRINTED POLYMERS

[0352] Aspects and features of the invention relating to a use of a (free) molecularly imprinted polymer (or a plurality of batches thereof) are described above in the relevant sub-section of the “SUMMARY OF THE INVENTION” and, in particular in numbered paragraphs E1-E25. Further features and sub-features of the same are elaborated below, all of which are applicable to any of the aforesaid aspects and to any other compatible aspects and embodiments described elsewhere herein.

[0353] The (free) molecularly imprinted polymer used in this section is preferably as defined and / or formed as defined in the above section entitled “D) FREE (MAGNETIC TEMPLATE-UNBOUND) MOLECULARLY IMPRINTED POLYMERS (MIPS)”.

[0354] Whilst MIPs of the invention are described herein in the context of MIP-bearing electrodes which may be used for detecting target molecules, MIPs of the invention may be used for detecting target molecules in a number of ways. For instance, where the MIP includes detection monomers (e.g. monomers with a fluorescent moiety), target molecules may be detected by measuring changes in fluorescence of the MIPs. The same applies for derivatised MIPs in which a detector moiety is introduced after initial MIP production by derivatising an MIP (e.g. by conjugation, functionalisation, and / or labelling). MIPs may be conjugated with one or more enzymes, and then used in immunoassays (or a method of assaying – e.g. ELISA - Enzyme-Linked Immunosorbent Assays).

[0355] Since biological samples, such a blood, are complex mixtures containing many molecules that may biofoul (and thus reduce the performance of) MIPs according to the invention, it is particularly preferably for the MIPs to be hydrogels, such as polyacrylamides, since such hydrogels more readily self-clean due to a water later which facilitates diffusion. Hydrogels, such as polyacrylamides, are also advantageously biocompatible. F) PRODUCTION OF MIP-BEARING ELECTRODE

[0356] Aspects and features of the invention relating to an MIP-bearing electrode are described above in the relevant sub-section of the “SUMMARY OF THE INVENTION” and, in particular in numbered paragraphs F1-F7. Further features and sub-features of the same are elaborated below, all of which are applicable to any of the aforesaid aspects and to any other compatible aspects and embodiments described elsewhere herein.

[0357] The (free) molecularly imprinted polymer (MIP) used in this section is preferably as defined and / or formed as defined in the above section entitled “D) FREE (MAGNETIC TEMPLATE-UNBOUND) MOLECULARLY IMPRINTED POLYMERS (MIPS)”.

[0358] An MIP-bearing electrode is an electrode bearing on the surface thereof (or within a coating upon the surface thereof) an MIP (suitably as defined herein, or provided by a method of manufacturing the same as defined herein). A skilled person understands how to incorporate an initially uncoated electrode within an electrochemical cell in order to facilitate production of the MIP-bearing electrode.

[0359] Whilst the methods according to numbered paragraphs F1-F4, and molecularly imprinted polymer-bearing electrodes of F5-F7, preferably use or incorporate MIPs of the invention (or manufactured in accordance with the invention), the method according to numbered paragraph F3 and molecularly imprinted polymer-bearing electrode of F7 may pertain to any MIP, including of the prior art, since the principle trapping pre-prepared MIPs within a polymer coating produced in situ upon an electrode is novel even in its broadest sense. Moreover, whilst the method according to numbered paragraph F4 (and its dependent molecularly imprinted polymer-bearing electrode of F5) preferably utilises a magnetically supported template molecule according to the invention (or manufactured in accordance with the invention), it may alternatively utilise any supported template molecule (albeit preferably a magnetically supported template molecule), including those of the prior art, because the principle of forming a molecularly imprinted polymer upon the surface of the electrode (through electrochemically initiated and / or propagated polymerisation of one or more corresponding coating monomers) in the presence of a (preferably magnetically-) supported template molecule is novel even in its broadest sense.

[0360] The method of manufacturing a molecularly imprinted polymer-bearing electrode preferably involves forming a coating polymer (preferably formed of one or more coating monomers) upon the surface of the electrode in the presence of a molecularly imprinted polymer (MIP), preferably a (pre-formed) MIP of the invention formed as described herein (e.g. using preferred magnetically supported template molecules, etc.).

[0361] The electrode may be any suitable electrode, though preferably the electrode is associated with other electrodes (e.g. as part of an electrode device) that together permit cyclic voltammetry (CV) and / or electrochemical impedance spectroscopy (EIS). As such, references herein to “electrode” may suitably be replaced by a reference to an “electrode device comprising one or more electrodes”. As such, preferably the electrode (or electrode device) is a CV electrode (or electrode device) and / or an EIS electrode (or electrode device). Preferably, therefor the electrode (or electrode device) comprises a working electrode, a counter electrode, and a reference electrode. The working electrode is preferably a gold working electrode. The counter electrode is preferably a platinum counter electrode. The reference electrode is preferably a silver reference electrode. The electrode to be coated is preferably the working electrode.

[0362] Preferably the electrode (or electrode device) is a screen-printed electrodes (SPE), most preferably a disposable SPE. Preferably the SPE comprises a gold working electrode (suitably with a diameter between 0.05 and 20 mm, preferably 0.1-10 mm, more preferably 2-5 mm), a gold counter electrode and silver reference electrode.

[0363] The method preferably involves contacting the electrode (or electrode device, preferably contacting all three of a working electrode, counter electrode, and reference electrode of an electrode device) with a reaction mixture, which initially comprises the MIP (suitably dispersed or suspended) and the one or more coating monomers (dissolved or dispersed) in a solvent or solvent system, and electrochemically initiating (e.g. by passing an electric current through the electrode or electrode device) and propagating a polymerisation reaction to cause a polymer or co-polymer comprising (or formed from) the one or more coating monomers to form on the surface of the electrode (and / or electrode device) producing a polymer coating entrapping or incorporating MIP (which are suitably not covalently bonded to the polymer coating) therein. Preferably localised islands of MIP are trapped within the polymer coating. Preferably at least some MIP(s) or at least some complementary imprints of the MIP(s) are exposed at the surface of the polymer coating and thereby available for binding. The reaction mixture may additionally comprise one or more reagents (e.g. to facilitate polymerisation). Such one or more reagents may include an initiator or initiator system. For instance, the initiator system may comprise a redox agent, for instance, potassium persulphate (KPS). Such one or more reagents may include a supporting electrolyte (or electrolytic agent or salt), for instance, sodium nitrate.

[0364] Electrochemical initiation and propagation (and the polymerisation reaction in general) is suitably caused by cyclic voltammetry (CV), suitably by cycling the electrochemical potential (i.e. cycling the electrochemical potential of the working electrode with respect to the reference electrode). Suitably the polymerisation reaction is monitored by cyclic voltammetry (e.g. by observing changes or a lack thereof in oxidation and reduction traces). Suitably, cycling of the electrochemical potential is performed until both the oxidation and reduction CV traces remain substantially unchanged, which is preferably after at least 2 cycles, more preferably after at least 4 cycles, most preferably after at least 6 cycles. The polymerisation reaction suitably takes place at a temperature of 25oC + / - 25oC, more preferably 25oC + / - 10oC. A person skilled in the art is capable of selecting appropriate CV conditions.

[0365] The electrode (or electrode device) is suitably relatively small. As such, the reaction mixture may be a mere droplet. For instance, the volume of the reaction mixture is suitably between 1 and 1000 μL, preferably between 5 and 500 μL, more preferably between 10 and 100 μL, most preferably between 20 and 80 μL.

[0366] The coating polymer, and the corresponding one or more coating monomers, is suitably characterised by a polymer, monomer(s), and optional cross-linking monomer(s) as set forth in the sub-section “Polymer, Monomer(s), and Cross-linking Monomer(s)” of the section entitled “C) MAGNETIC-TEMPLATE-BOUND MOLECULARLY IMPRINTED POLYMER”. The monomer(s) used to form the coating monomer may be exactly the same as those used to form the MIP. However, the, some or all of the monomer(s) used to form the coating polymer may be different to used to form the MIP but may nonetheless be independently characterised by those set forth in the sub-section “Polymer, Monomer(s), and Cross- linking Monomer(s)” of the section entitled “C) MAGNETIC-TEMPLATE-BOUND MOLECULARLY IMPRINTED POLYMER”. Preferably at least one monomer, suitably a monomer that constitutes at least 10 wt% of the MIP and at least 10 wt% of the coating polymer (preferably at least 20 wt% and 20 wt%, more preferably at least 30 wt% and 30 wt%, most preferably at least 40 wt% and 40 wt% respectively), is the same in both the MIP and the coating polymer. Preferably, however, in the context of an MIP-bearing electrode, whilst the coating polymer may comprise any monomer(s) (including backbone-forming monomer(s) and optional cross-linking monomer(s)) set forth in the aforesaid sub-section, the coating polymer is free of any detector monomers described in the aforesaid sub-section (this is because the applications of such MIP-bearing electrodes suitably involve electrochemical detection of analytes rather than any other forms of detection of analytes). G) USE OF MIP-BEARING ELECTRODE

[0367] Aspects and features of the invention relating to a use of an MIP-bearing electrode are described above in the relevant sub-section of the “SUMMARY OF THE INVENTION” and, in particular in numbered paragraphs G1-G2. Further features and sub-features of the same are elaborated below, all of which are applicable to any of the aforesaid aspects and to any other compatible aspects and embodiments described elsewhere herein.

[0368] The MIP-bearing electrode (or electrode device) used in this section is preferably as defined and / or formed as defined in the above section entitled “F) PRODUCTION OF MIP-BEARING ELECTRODE”.

[0369] The molecularly imprinted polymer-bearing electrode (which suitably comprises MIPs of the invention) may be used in the detection, characterisation, and / or quantification of a target molecule (preferably when the target molecule is dissolved, dispersed, or suspended in a solvent or solvent system). The molecularly imprinted polymer-bearing electrode is preferably used in the detection and / or quantification of the target molecule, most preferably in the detection of the target molecule. Suitably, when a target molecule binds to a molecularly imprinted polymer (suitably via complementary imprints thereof) of the molecularly imprinted polymer-bearing electrode, the state or properties of the molecularly imprinted polymer- bearing electrode change in an electrochemically detectable manner.

[0370] Detection, characterisation, and / or quantification of the target molecule may be performed by cyclic voltammetry and / or electrochemical impedance spectroscopy, most preferably by electrochemical impedance spectroscopy (EIS).

[0371] Target molecules may be detected even at very low concentrations. Suitably, the target molecule is dissolved, dispersed, or suspended in a solvent or solvent system), and the target molecule is present at a concentration between 1 fM (1 femtomolar) to 500 µM (100 micromolar), preferably between 100 fM to 100 µM. The target molecule is suitably at aconcentration below 100 µM, suitably below 10 µM, 1 µM, suitably below 100 nM, suitably below 100 pM, suitably below 1 pM.

[0372] Since the (complementary) imprints of the MIP are imprints of a template molecule, preferably the target molecule binds or at least comprises a part that binds to (or within) said imprints. Suitably the target molecule, or a part thereof, is (substantially) identical to the template molecule, or a part thereof, used in the manufacture of the molecularly imprinted polymer of the molecularly imprinted polymer-bearing electrode.

[0373] Suitably both the target molecule and the template molecule may be proteins – i.e. the target molecule is a target protein molecule and the template molecule is a protein template molecule. Preferably, the protein template molecule has at least 80% sequence identity with the protein target molecule, more preferably at least 90% sequence identity therewith, most preferably at least 95% sequence identity therewith. Where downstream MIPs Iare intended to bind a part of the target protein molecule (e.g. akin to an antibody binding an epitope), preferably the corresponding part of a protein template molecule has at least 80% sequence identity with the corresponding part of the protein target molecule, more preferably at least 90% sequence identity therewith, most preferably at least 95% sequence identity therewith.

[0374] Preferably, the target molecule is identical to the template molecule used in the manufacture of the molecularly imprinted polymer of the molecularly imprinted polymer-bearing electrode.

[0375] An aspect of the invention provides a detection device comprising a molecularly imprinted polymer-bearing electrode. The detection device is suitably operable to use the molecularly imprinted polymer-bearing electrode in the aforementioned manner. The detection device is suitably operable to detect, characterise, and / or quantify a target molecule. SPECIFIC EMBODIMENTS

[0376] In a particular embodiment, providing a functionalised magnetic support comprises: mixing together one or more magnetic core-forming components with a (or one or more) functionalising molecule(s), and further processing, to form a functionalised magnetic support comprising a magnetic core, derived from the magnetic core-forming components, the magnetic core bearing, at the surface thereof, one or more reactive functional group(s) derived from the functionalising molecule; wherein: the further processing comprises ramped (or gradient) heating (preferably by microwave heating) comprising: a. Starting (time zero, t0) at a start temperature (Tmin) (which may be room temperature or standard temperature, preferably 25oC + / - 25oC), and commencing heating; b. Heating, over a predetermined ramp time (tramp), to increase the temperature by a predetermined ramp temperature (^Tramp) – e.g. heating at a ramp rate (Rramp) of ^Tramp / tramp; c. Repeating heating step b) until a predetermined maximum temperature (Tmax) is reached; the difference between the start temperature (Tmin) and maximum temperature (Tmax) is between 50 and 300oC (preferably between 70 and 250oC); the maximum temperature is between 60 and 350oC (preferably between 150oC and 250oC); the total ramp time (ttotal) (between step a), when heating first begins, and the end of step c), reaching the maximum temperature) is between 2 and 60 mins (preferably between 5 and 20 mins, more preferably between 5 and 14 mins); the ramp rate is between 5oC / min and 100oC / min (preferably between 8oC / min and 30oC / min); preferably the magnetic core comprises a magnetic or magnetisable iron-, nickel-, or cobalt- oxide compound and the one or more magnetic core-forming components comprise an iron-, nickel-, or cobalt- containing precursor (preferably a halide thereof, most preferably chloride) to said iron-, nickel-, or cobalt- oxide compound of the magnetic core; the magnetic core, and indeed the functionalised magnetic support as a whole, is free of silica or functionalised silica; preferably the one or more reactive functional group(s) is / are selected from the group consisting of aldehyde(s), ketone(s), and / or amine(s) (preferably selected from aldehyde(s) and / or ketone(s)), and the functionalising molecule(s) comprises one or more (preferably at least two) of said reactive functional group(s); the functionalised magnetic support has an average particle size between 10 and 300 nm (preferably between 20 and 200 nm).

[0377] In a particular embodiment, providing a magnetically supported template molecule comprises: coupling a template molecule to the functionalised magnetic support via a (preferably direct) chemical reaction between the template molecule and a reactive functional group borne on the surface of the functionalised magnetic support;wherein: preferably the template molecule is a protein template molecule, suitably having an average particle size between 1 and 20 nm (preferably between 2.5 and 15 nm); preferably the average number of template molecules coupled to each functionalised magnetic support (or magnetic core) is between 10 and 2000 (preferably between 50 and 1000); the magnetically supported template molecule has an average particle size between 20 and 600 nm (preferably between 50 and 500 nm).

[0378] In a particular embodiment, providing a magnetic template-bound molecularly imprinted polymer comprising one or more magnetically supported template molecule(s) and one or more molecularly imprinted polymer(s) (MIP(s)), comprises: forming a polymer (preferably a hydrogel) by polymerising one or more monomer(s) (preferably at least one backbone-forming monomer and a cross-linking monomer) in the presence of the magnetically supported template molecule (optionally a recycled magnetically supported template molecule, especially when repeating this step); wherein: the magnetically supported template molecule(s) is / are bound to the molecularly imprinted polymer(s) via a (or one or more) complementary imprint(s) in the molecularly imprinted polymer(s), said complementary imprint(s) being complementary to the or a part of the template molecule(s); preferably, each magnetic template-bound molecularly imprinted polymer molecule (or the MIP thereof) comprises at least 2 (preferably at least 10) complementary imprints; preferably there are at least two (preferably at least three) molecularly imprinted polymer molecules per magnetically supported template molecule; forming a polymer is suitably performed at a temperature of 25oC + / - 25oC (more preferably 25oC + / - 10oC).

[0379] In a particular embodiment, providing a (free) molecularly imprinted polymer (MIP) comprises: unbinding and separating the molecularly imprinted polymer from the magnetically supported template molecule of the magnetic template-bound molecularly imprinted polymer; wherein: preferably unbinding comprises sonicating (i.e. application of ultrasound) the magnetic template- bound molecularly imprinted polymer, preferably in a solvent or solvent system, suitably for a time period between 30 seconds and 30 minutes (preferably between 1 minute and 15 minutes); separating comprises magnetically separating the unbound molecularly imprinted polymer from the unbound magnetically supported template molecule; unbinding and separating is performed at a temperature between 0oC and 70oC (preferably 25oC + / - 25oC); the average particle size of the (free) MIP is between 10 and 1000 nm (preferably between 50 and 500) nm; preferably the ratio of average particle size of the magnetically supported template molecule (used to form the MIP) to the average particle size of the (free) MIP is 0.5-10 : 1 (preferably 0.75-5 : 1), though most preferably the average particle size of the magnetically supported template molecule is greater than the average particle size of the (free) MIP; preferably the MIP is a hydrogel.

[0380] In a particular embodiment, providing a (free) molecularly imprinted polymer (MIP) comprises: unbinding and separating the molecularly imprinted polymer from the magnetically supported template molecule of the magnetic template-bound molecularly imprinted polymer; wherein: preferably unbinding comprises sonicating (i.e. application of ultrasound) the magnetic template- bound molecularly imprinted polymer, preferably in a solvent or solvent system, suitably for a time period between 30 seconds and 30 minutes (preferably between 1 minute and 15 minutes); separating comprises magnetically separating the unbound molecularly imprinted polymer from the unbound magnetically supported template molecule; unbinding and separating is performed at a temperature between 0oC and 70oC (preferably 25oC + / - 25oC); the average particle size of the (free) MIP is between 30 and 500 nm (preferably between 80 and 300) nm; preferably the ratio of average particle size of the magnetically supported template molecule (used to form the MIP) to the average particle size of the (free) MIP is 0.75-10 : 1 (preferably 1-5 : 1),though most preferably the size of the magnetically supported template molecule is greater than the average particle size of the (free) MIP; preferably the MIP is a hydrogel.

[0381] In a particular embodiment, a method of manufacturing a plurality of batches of a (free) molecularly imprinted polymer (MIP) comprises: i) providing a functionalised magnetic support, by or obtained by a method of manufacturing a functionalised magnetic support comprising: mixing together one or more magnetic core-forming components with a (or one or more) functionalising molecule(s), and further processing, to form a functionalised magnetic support comprising a magnetic core, derived from the magnetic core-forming components, the magnetic core bearing, at the surface thereof, one or more reactive functional group(s) derived from the functionalising molecule; wherein: the further processing comprises ramped (or gradient) heating (preferably by microwave heating) comprising: a. Starting (time zero, t0) at a start temperature (Tmin) (which may be room temperature or standard temperature, preferably 25oC + / - 25oC), and commencing heating; b. Heating, over a predetermined ramp time (tramp), to increase the temperature by a predetermined ramp temperature (^Tramp) – e.g. heating at a ramp rate (Rramp) of ^Tramp / tramp; c. Repeating heating step b) until a predetermined maximum temperature (Tmax) is reached; the difference between the start temperature (Tmin) and maximum temperature (Tmax) is between 50 and 300oC (preferably between 70 and 250oC); the maximum temperature is between 60 and 350oC (preferably between 150oC and 250oC); the total ramp time (ttotal) (between step a), when heating first begins, and the end of step c), reaching the maximum temperature) is between 2 and 60 mins (preferably between 5 and 20 mins, more preferably between 5 and 14 mins); the ramp rate is between 5oC / min and 100oC / min (preferably between 8oC / min and 30oC / min); preferably the magnetic core comprises a magnetic or magnetisable iron-, nickel-, or cobalt- oxide compound and the one or more magnetic core-forming components comprise an iron-, nickel-, or cobalt- containing precursor (preferably a halide thereof, most preferably chloride) to said iron-, nickel-, or cobalt- oxide compound of the magnetic core; the magnetic core, and indeed the functionalised magnetic support as a whole, is free of silica or functionalised silica; preferably the one or more reactive functional group(s) is / are selected from the group consisting of aldehyde(s), ketone(s), and / or amine(s) (preferably selected from aldehyde(s) and / or ketone(s)), and the functionalising molecule(s) comprises one or more (preferably at least two) of said reactive functional group(s); the functionalised magnetic support has an average particle size between 10 and 300 nm (preferably between 20 and 200 nm); ii) providing a magnetically supported template molecule, preferably by or obtained by a method of manufacturing a magnetically supported template molecule comprising: coupling a template molecule to the functionalised magnetic support via a (preferably direct) chemical reaction between the template molecule and a reactive functional group borne on the surface of the functionalised magnetic support; wherein: preferably the template molecule is a protein template molecule, suitably having an average particle size between 1 and 20 nm (preferably between 2.5 and 15 nm); preferably the average number of template molecules coupled to each functionalised magnetic support (or magnetic core) is between 10 and 2000 (preferably between 50 and 1000); the magnetically supported template molecule has an average particle size between 20 and 600 nm (preferably between 50 and 500 nm); iii) providing a magnetic template-bound molecularly imprinted polymer comprising one or more magnetically supported template molecule(s) and one or more molecularly imprinted polymer(s) (MIP(s)), by or obtained by a method of manufacturing a magnetic template-bound molecularly imprinted polymer comprising: forming a polymer (preferably a hydrogel) by polymerising one or more monomer(s) (preferably at least one backbone-forming monomer and a cross-linking monomer) in the presence of the magneticallysupported template molecule a recycled magnetically supported template molecule, especially when repeating this step); wherein: the magnetically supported template molecule(s) is / are bound to the molecularly imprinted polymer(s) via a (or one or more) complementary imprint(s) in the molecularly imprinted polymer(s), said complementary imprint(s) being complementary to the or a part of the template molecule(s); preferably, each magnetic template-bound molecularly imprinted polymer molecule (or the MIP thereof) comprises at least 2 (preferably at least 10) complementary imprints; preferably there are at least two (preferably at least three) molecularly imprinted polymer molecules per magnetically supported template molecule; forming a polymer is suitably performed at a temperature of 25oC + / - 25oC (more preferably 25oC + / - 10oC); iv) providing a (free) molecularly imprinted polymer (MIP), by or obtained by a method of manufacturing a (free) molecularly imprinted polymer (MIP) comprising: unbinding and separating the molecularly imprinted polymer from the magnetically supported template molecule of the magnetic template-bound molecularly imprinted polymer; wherein: preferably unbinding comprises sonicating (i.e. application of ultrasound) the magnetic template- bound molecularly imprinted polymer, preferably in a solvent or solvent system, suitably for a time period between 30 seconds and 30 minutes (preferably between 1 minute and 15 minutes); separating comprises magnetically separating the unbound molecularly imprinted polymer from the unbound magnetically supported template molecule; unbinding and separating is performed at a temperature between 0oC and 70oC (preferably 25oC + / - 25oC); the average particle size of the (free) MIP is between 10 and 1000 nm (preferably between 50 and 500) nm; preferably the ratio of average particle size of the magnetically supported template molecule (used to form the MIP) to the average particle size of the (free) MIP is 0.5-10 : 1 (preferably 0.75-5 : 1), though most preferably the average particle size of the magnetically supported template molecule is greater than the average particle size of the (free) MIP; preferably the MIP is a hydrogel; v) optionally recovering the unbound and separated magnetically supported template molecule as a recycled magnetically supported template molecule, and repeating steps iii)-iv) and optionally also v) with the recycled magnetically supported template molecule.

[0382] In a particular embodiment, a method of manufacturing a plurality of batches of a (free) molecularly imprinted polymer (MIP) comprises: i) providing a functionalised magnetic support, by or obtained by a method of manufacturing a functionalised magnetic support comprising: mixing together one or more magnetic core-forming components with a (or one or more) functionalising molecule(s), and further processing, to form a functionalised magnetic support comprising a magnetic core, derived from the magnetic core-forming components, the magnetic core bearing, at the surface thereof, one or more reactive functional group(s) derived from the functionalising molecule; wherein: the further processing comprises ramped (or gradient) heating (preferably by microwave heating) comprising: a. Starting (time zero, t0) at a start temperature (Tmin) (which may be room temperature or standard temperature, preferably 25oC + / - 25oC), and commencing heating; b. Heating, over a predetermined ramp time (tramp), to increase the temperature by a predetermined ramp temperature (^Tramp) – e.g. heating at a ramp rate (Rramp) of ^Tramp / tramp; c. Repeating heating step b) until a predetermined maximum temperature (Tmax) is reached; the difference between the start temperature (Tmin) and maximum temperature (Tmax) is between 50 and 300oC (preferably between 70 and 250oC); the maximum temperature is between 60 and 350oC (preferably between 150oC and 250oC);the total ramp time (ttotal) a), when heating first begins, and the end of step c), reaching the maximum temperature) is between 2 and 60 mins (preferably between 5 and 20 mins, more preferably between 5 and 14 mins); the ramp rate is between 5oC / min and 100oC / min (preferably between 8oC / min and 30oC / min); preferably the magnetic core comprises a magnetic or magnetisable iron-, nickel-, or cobalt- oxide compound and the one or more magnetic core-forming components comprise an iron-, nickel-, or cobalt- containing precursor (preferably a halide thereof, most preferably chloride) to said iron-, nickel-, or cobalt- oxide compound of the magnetic core; the magnetic core, and indeed the functionalised magnetic support as a whole, is free of silica or functionalised silica; preferably the one or more reactive functional group(s) is / are selected from the group consisting of aldehyde(s), ketone(s), and / or amine(s) (preferably selected from aldehyde(s) and / or ketone(s)), and the functionalising molecule(s) comprises one or more (preferably at least two) of said reactive functional group(s); the functionalised magnetic support has an average particle size between 10 and 300 nm (preferably between 20 and 200 nm); ii) providing a magnetically supported template molecule, preferably by or obtained by a method of manufacturing a magnetically supported template molecule comprising: coupling a template molecule to the functionalised magnetic support via a (preferably direct) chemical reaction between the template molecule and a reactive functional group borne on the surface of the functionalised magnetic support; wherein: preferably the template molecule is a protein template molecule, suitably having an average particle size between 1 and 20 nm (preferably between 2.5 and 15 nm); preferably the average number of template molecules coupled to each functionalised magnetic support (or magnetic core) is between 10 and 2000 (preferably between 50 and 1000); the magnetically supported template molecule has an average particle size between 20 and 600 nm (preferably between 50 and 500 nm); iii) providing a magnetic template-bound molecularly imprinted polymer comprising one or more magnetically supported template molecule(s) and one or more molecularly imprinted polymer(s) (MIP(s)), by or obtained by a method of manufacturing a magnetic template-bound molecularly imprinted polymer comprising: forming a polymer (preferably a hydrogel) by polymerising one or more monomer(s) (preferably at least one backbone-forming monomer and a cross-linking monomer) in the presence of the magnetically supported template molecule (optionally a recycled magnetically supported template molecule, especially when repeating this step); wherein: the magnetically supported template molecule(s) is / are bound to the molecularly imprinted polymer(s) via a (or one or more) complementary imprint(s) in the molecularly imprinted polymer(s), said complementary imprint(s) being complementary to the or a part of the template molecule(s); preferably, each magnetic template-bound molecularly imprinted polymer molecule (or the MIP thereof) comprises at least 2 (preferably at least 10) complementary imprints; preferably there are at least two (preferably at least three) molecularly imprinted polymer molecules per magnetically supported template molecule; forming a polymer is suitably performed at a temperature of 25oC + / - 25oC (more preferably 25oC + / - 10oC); iv) providing a (free) molecularly imprinted polymer (MIP), by or obtained by a method of manufacturing a (free) molecularly imprinted polymer (MIP) comprising: unbinding and separating the molecularly imprinted polymer from the magnetically supported template molecule of the magnetic template-bound molecularly imprinted polymer; wherein: preferably unbinding comprises sonicating (i.e. application of ultrasound) the magnetic template- bound molecularly imprinted polymer, preferably in a solvent or solvent system, suitably for a time period between 30 seconds and 30 minutes (preferably between 1 minute and 15 minutes); separating comprises magnetically separating the unbound molecularly imprinted polymer from the unbound magnetically supported template molecule; unbinding and separating is performed at a temperature between 0oC and 70oC (preferably 25oC + / - 25oC);the average particle size of the MIP is between 30 and 500 nm (preferably between 80 and 300) nm; preferably the ratio of average particle size of the magnetically supported template molecule (used to form the MIP) to the average particle size of the (free) MIP is 0.75-10 : 1 (preferably 1-5 : 1), though most preferably the average particle size of the magnetically supported template molecule is greater than the average particle size of the (free) MIP; preferably the MIP is a hydrogel; v) optionally recovering the unbound and separated magnetically supported template molecule as a recycled magnetically supported template molecule, and repeating steps iii)-iv) and optionally also v) with the recycled magnetically supported template molecule.

[0383] In an embodiment: the magnetic core, and indeed the functionalised magnetic support as a whole, is free of silica or functionalised silica; the functionalised magnetic support has an average particle size between 10 and 300 nm (preferably between 20 and 200 nm); the template molecule is a protein template molecule, suitably having an average particle size between 1 and 20 nm (preferably between 2.5 and 15 nm); the magnetically supported template molecule has an average particle size between 20 and 600 nm (preferably between 50 and 500 nm); and the ratio of average particle size of the functionalised magnetic support (used to form the magnetically supported template molecule) and the average particle size of the template molecule is between 5:1 and 50:1 (preferably between 10:1 and 30:1). EXAMPLES Materials and Equipment

[0384] N-hydroxymethylacrylamide (NHMA, 48% w / v), N,N′-methylenebisacrylamide (MBAm), ethylene glycol, iron chloride (FeCl3ˑ6H2O), methylhydroquinone, sodium dodecylsulphate (SDS), sodium acetate (NaOAc), phosphate buffered saline tablets (PBS, 10 mM, pH 7.4 ± 0.2), potassium ferricyanide (K3Fe(CN)6), potassium chloride (KCl), sodium nitrate (NaNO3), ammonium persulphate (APS), potassium peroxydisulfate (KPS), haemoglobin from bovine blood (BHb), bovine serum albumin (BSA), chicken egg lysozyme (Lys) and glutaraldehyde (25% v / v)) were used as received from Merck. Recombinant nucleocapsid protein for SARS-CoV-2 was purchased from BioservUK Ltd (Rotherham, UK). Buffers were prepared in MilliQ water (resistivity 18.2 ± 0.2 MΩ.cm). DropSens disposable screen-printed electrodes (Au-BT) comprising a gold working electrode (0.4 cm diameter), a platinum counter electrode and silver reference electrode were purchased from Metrohm (Runcorn, Cheshire, UK).

[0385] BioDrop μLITE UV / visible spectrometer was purchased from Biochrom Ltd Cambridge, UK. Nicolet AVATAR 330 FTIR spectrophotometer with Pike MIRacle accessory and FEI Tecnai 12 TEM at 100 kV with a Tietz F2142k × 2k CCD camera were purchased from Thermo Fisher Scientific, Loughborough, UK. Anton Paar monowave 200 microwave oven was purchased from Anton Paar Ltd Hertfordshire, UK. SLS Lab basics centrifuge was purchased from Scientific Laboratory Supplies, Nottingham, UK. Analytical Protocols Yield Determination:

[0386] The nanoMIP solution within the Eppendorf microfuge was first flash frozen using a CHRIST Alpha 2-4 LDplus freeze-dryer. Then, with the cap open on the Eppendorf tube, Parafilm® was then placed over the Eppendorf mouth and pierced to allow the water to escape during the freeze-drying process. The Eppendorf was then placed in the freeze dryer at -90 °C and at low pressure (0.011 mbar) for a minimum of 16 hr until a fine fluffy off-white powder was produced. The mass of the lyophilised powder was then determined. The nanoNIPs produced were also taken through a similar process. Characterisation and Sizing:

[0387] The sizing of the nanoparticles was characterized using a Zetasizer Nano ZS. The produced MNPs / nanoMIPS / NanoNIPs were suspended in a PBS. The sample was loaded into a disposable cuvette with the refractive index set to 1.32. The solution was equilibrated for 60 seconds before the measurement was taken. Measurements were formed in triplicate.°C, 1´ 0.1 5 for of to a a 5 μL theGeneral Scheme

[0391] Figure 1 is a schematic diagram illustrating a process for producing NanoMIPs (nano-molecularly imprinted polymers) by: (A) coupling protein molecules with functionalised magnetic nanoparticles (MNPs) to produce (B) a magnetically supported protein template particles (MNP@Protein), which are then (C) mixed with monomers that polymerise around the protein templates of the MNP@Protein particles which, whereafter the MNP@Protein particles are released from the resulting polymers to yield (D) NanoMIPs which are then harvested.

[0392] Figure 2 is a schematic diagram illustrating (A) forming a layer containing entrapped NanoMIPs of Figure 1 upon the surface of an electrode to produce a protein-specific detector electrode; and (B) using said protein-specific detector electrode to selectively bind and detect the specific protein.

[0393] Specific examples of this general scheme are discussed below. Example 1 – Magnetic Nanoparticle (MNP) and Functionalised MNP Production

[0394] Bare and aldehyde functionalised magnetic particles were produced following our previously published solvothermal microwave method6. Briefly, 0.5 g of FeCl3ˑ6H2O and 1.8 g of NaOAc were dissolved in 15 ml of ethylene glycol in a 30 ml Anton Parr G30 microwave reaction vial (MRV). Glutaraldehyde (3.5 ml) was then added to the resulting solution with stirring for a further 5 min. The stirrer bar was then removed and the MRV was placed into an Anton Paar monowave 200 microwave oven and the reaction was heated up to a temperature of 200 °C with a ramp time of 18 °C / min (over 10 min). The reaction was held at 200 °C for 20 min under pressure (9 bar). The resulting composite products were allowed to cool for 10 min, washed five times with deionised water followed by two washes of ethanol, and then collected with a magnet and finally oven dried (110 °C for 2 days) before further use. The method was repeated, but in the absence of gluataraldehyde, to produce bare MNPs.

[0395] This example provides an input material for A of Figure 1.

[0396] The inventors have previously reported6an effective solvothermal microwave synthesis method for rapid (20 min) production of magnetic nanoparticles. In this study the ramp time was fixed at 90 °C / min, resulting in MNPs with an average size of 7 ± 2 nm. Now the inventors have altered the ramp time, and found to there surprise that in so doing they can control the size of the particles and yield, thereby increasing the surface area per particle which could in turn affect the number of target protein molecules that could be subsequently conjugated to each particle. This was an important development towards a method that can efficiently produce anywhere from one to multiple nanoMIPs per MNP.

[0397] The inventors investigated slow (10 mins; 18 °C / min) and fast (2 mins; 90 °C / min) ramp times. The corresponding yields for slow and fast ramp times were 330 mg and 300 mg respectively for a 30 mL reaction solution. Bare MNPs were also produced as a comparison with yields of 280 mg for a 30 mL reaction solution.

[0398] Figure 3 shows a DLS spectrum of the MNP@CHO produced when using a microwave synthesis ramp time of 18 °C / min over 10 min, with overall synthesis being terminated after a further 20 min, and an average particle size of 120 nm is indicated. M Sullivan, S Dennison, G Archontis, SM Reddy, JM Hayes, J. Phys Chem B., 2019, 123, 5432-5443 Whitmore, L.; Wallace, B. A. Protein secondary structure analyses from circular dichroism spectroscopy: methods and reference databases. Biopolymers 2008, 89, 392‐400

[0399] Figure 4 shows SEM images of the MNP@CHO when using a microwave synthesis ramp time of 18 °C / min over 10 min, with overall synthesis being terminated after a further ~ 20 min, and particle size was determined to be 60-80 nm (white circles represent individual MNP particles within multiple clumped particles).

[0400] Figure 3 and Figure 4 thus show DLS and SEM of MNP@CHO produced at the slow ramp time. Whereas DLS produced in e-pure water indicates monodispersed particles sized at 122 ±49 nm, SEM suggests particles are typically 60- 80 nm. The discrepancy can be due to the DLS presenting a hydrodynamic radius for the wet particles whereas SEM, operating under a vacuum, is representing dried particles which have also apparently clumped together. Further information from this Example

[0401] Figure 22 is a bar chart showing how the microwave ramp time (up to 200oC) affects the average hydrodynamic size (nm) of the MNP – the longer the ramp time the larger the size.

[0402] Figure 23 is a bar chart showing how the microwave ramp time (up to 200oC) affects the average hydrodynamic size (nm) of the MNP – this shows that if the ramp time is too long (here at 15 min ramp time), the method is less likely to provide useful material.

[0403] Figure 24 is a TEM image of MNPs produced with a ramp time of 2 mins. Figure 25 is a TEM image of MNPs produced with a ramp time of 6 mins. Figure 26 is a TEM image of MNPs produced with a ramp time of 8 mins. Figure 27 is a zoomed in TEM image of MNPs produced with a ramp time of 10 mins. Figure 28 is a zoomed out TEM image of MNPs produced with a ramp time of 10 mins. The particles increase in cluster size with increasing ramp time.

[0404] Figure 29 shows TEM images, A and B, of MNP@CHO produced using microwave synthesis with a 10-minute ramp time at 18 °C / min followed by a 20 min dwell time at 200 °C. The average cluster size (A) was determined to be 91 ±15 nm and the average core size in (B) was determined by TEM to be approximately 18 ±5 nm.

[0405] Table 1 below shows how particle size changes with ramp time using different analytical techniques. The average particle sizes recorded by TEM and DLS generally relate to cluster sizes. An “individual magnetic core size” may be determined, by TEM, by measuring individual cores within a cluster. However, cluster sizes are generally more informative for the purposes of the present invention.

[0406] The individual magnetic core size was determined via a dry measurement technique involving magnetic measurements – this is distinct from measuring general particle size (i.e. cluster size). The TEM sizing of MNP@CHO are on average smaller than the DLS sizes. Whereas DLS sizing is conducted in aqueous media, and therefore represent a hydrodynamic diameter, the TEM measurements are conducted in vacuo and in a dried state. In general, and unless stated otherwise, average particle sizes set forth herein are determined by DLS.

[0407] It is believed that in the context of this method ethylene glycol is primarily solvent, but can act as a mild reducing agent resulting in the production of Fe2+ions en route to producing Fe3O4.

[0408] It is believed that acetate helps prevent particle agglomeration during MNP synthesis and possibly aids the production of Fe(OH)3and subsequently maghemite and magnetite formation.

[0409] It is believed that, at the 200 °C dwell temperature, elimination of acetate (as part of iron complexes) occurs through the direct thermal decomposition of iron acetate salts.

[0410] Without wishing to be bound by theory, the following is postulated. We propose that the time taken to reach the microwave dwell temperature of 200 °C will influence the composition of the reaction mixture. The levels of acetate present may also influence final particle and aggregate sizes. At a fast (2 minute) temperature ramp (ie 90 °C / min) to the dwell temperature, there is less iron hydroxide produced during the ramping period. At a slow (10 minute) temperature ramp (ie 18 °C / min) to the dwell temperature, there is more time for iron acetate to be converted to iron hydroxide during the ramping period, resulting in more maghemite and magnetite production during the ramping phase. We believe the acetate is acting as a weak buffer to produce hydroxide ions in situ supporting the production of [Fe(OH)3] resulting in iron oxide precipitation and subsequent aggregation. Therefore, by altering the ramp time we control the degree of FeOAc conversion to Fe(OH)3in the early stages of MNP production which in turn controls the size of the initial particles produced. Slowing down the time at which acetate decomposition takes place leads to further precipitation and aggregation, and controlled production of larger magnetic nanoparticles.Example 2 – Coupling Protein Template to Functionalised MNP

[0411] A suspension (1ml) equivalent to 0.010 g of aldehyde functionalised magnetic nanoparticle (MNP@CHO) was placed in an Eppendorf tube and then centrifuged at 15000 rpm for five minutes to rapidly separate the magnetic nanoparticles form the solution. The supernatant was removed and replaced with 1 ml of a 1 mg / ml solution of bovine haemoglobin (BHb). The Eppendorf was then sonicated for 2 minutes followed by vigorous shaking and vortexing to ensure the nanoparticles were fully dispersed. The reaction mixture was left undisturbed at room temperature (22 °C) for 30 minutes allowing the protein to conjugate with the MNP@CHO. After 30 minutes, the particles were once again centrifuged and the supernatant exchanged with fresh buffer in triplicate to remove any non-conjugated protein. The amount of protein conjugated with the MNPs (functionalized and bare) was calculated through comparing the initial and final concentrations of protein remaining in the supernatant. The concentration of the non-adsorbed protein was measured by spectrophotometry (405 nm for Mb) using a BioDrop μLITE UV / visible spectrometer. The resulting MNP@CHO~protein particles thus produced were stored wet at 4 °C until further use.

[0412] This Example illustrates A and B of Figure 1.

[0413] As explained, the MNP@CHO (10 mg) obtained from “Example 1 – Magnetic Nanoparticle (MNP) and Functionalised MNP Production” were then resuspended in a 1 mL of e-pure water containing target protein, bovine haemoglobin (1 mg / mL final concentration) resulting in production of MNP@BHb conjugates. The extent of protein capture by the MNPs was determined by spectroscopically monitoring the depletion of protein in the reaction solution, after the MNP@BHb had been magnetically separated (Figure 5). Whereas the 7 nm MNP@CHO particles adsorbed 0.05mg / mL of protein, the 120 nm MNP@CHO particles adsorbed 12 times more protein (0.6 mg / mL). In contrast, the corresponding larger bare MNP control particles adsorbed only 0.02mg / mL of protein indicative of a very small element of non-specific binding.

[0414] Figure 5 is a graph showing the degree of protein functionalisation / coupling of MNP@CHO over time – circles are for bare MNPs (which obviously don’t couple to protein), and squares are for MNP@CHO. A 1mg / mL BHb starting solution was used and amount adsorbed with time was determined by subtraction of amount of BHb remaining in solution (measured using UV / Vis spectroscopy) during the protein conjugation reaction. Fifteen minutes was determined to be the minimum time required to complete the conjugation process.

[0415] Figure 6 shows the DLS spectrum for MNP@BHb. The particles are again monodisperse but have now more than doubled in size to approximately 330 nm.

[0416] CD spectroscopy was used to confirm that the protein retained its native state following MNP conjugation. Table 2 shows a comparison of % alpha helix and beta sheet character for native protein in solution and following MNP conjugation with protein. The method to be used to release subsequent (multi-cycle) production of nanoMIP on the MNP was sonication. In order to show that sonication did not affect protein structure on the MNP, the particles were exposed to sonication for 5 min and 1hr. There was no significant change to protein structure parameters after 5 min of sonication (in contrast to 1hr sonication). A sonication time of 5 min was selected to ensure that the protein would remain intact in between batches of nanoMIP production. Table 2 - Comparison of percentage of secondary structures of protein when attached to MNPs and after 5min and 1hr sonication Percentage Secondary structure Sample α-helical ß-sheet ß-turns Unordered Protein functionalised magnetic nanoparticles (MNP@BHb) 46.5 ±1.41 25±1.41 7 ±1.41 20.5±1.41 MNP@BHb after 5 min sonication 43 ± 0.071 29 ± 0.071 7 ± 0.071 21 ± 0.071 MNP@BHb after after 1 hour sonication 42.1 ± 0.071 18.2 ± 0.071 18.4 ± 0.071 21.4 ± 0.071

[0417] The chemically conjugated MNP@BHb particles were then taken forward for nanoMIP production. MNP@CHO particles were used as control. Example 3 – Batch production of NanoMIPs from MNP@Protein Particles

[0418] With sonication followed by vigorous shaking and votexing the magnetic nanoparticles (0.023 g) were resuspended in 906 µL of PBS (pH7.4) and transferred to a 15ml falcon tube. The tube was then placed into the thermos- mixer and set to mix at 400rpm at room temperature. The sample was then degassed using nitrogen for 15 minutes with the stirring. The nitrogen line was then removed, NHMA monomer (54 µg; though in a more refined reaction / experiment 77 µL of 48% w / v solution was used), and MBAm (0.006g), preferably (for a more refined reaction / experiment) along with thesurfactant SDS (0.0004 g) (to facilitate dispersion of immediately added to the reaction mixture, followed by 20 µL each of 20% TEMED solution (though for a more refined reaction / experiment 20 µL of 5% TEMED solution is preferably used) and 10% APS solution. A nitrogen headspace was then created, and the falcon tube sealed with the cap and then wrapped in parafilm. The solution left to stir at 400 rpm for 15 minutes to allow nanoMIP shells to be produced at the surface of each MNP@BHb particle.

[0419] At 15 minutes, the reaction was rapidly quenched with 1ml of 10 mM methylhydroquinone (MHQ). The solution was then resealed, and the tube placed on its side on a magnetic particle concentrator, allowing the magnetic particles to be separated without the need for centrifugation (2 minutes). The supernatant was then removed. This illustrates C of Figure 1.

[0420] The MNP@BHb~nanoMIP particles were dispersed in 600 µL of e-pure water and placed in a sonicator for 5 minutes at room temperature. The falcon tube was then once again placed in the magnetic nanoparticle collector and the supernatant now containing the released nanoMIPs were placed in a 1.5 mL volume Eppendorf and stored at 4 °C until further use. This illustrates D of Figure 1.

[0421] The preparation was repeated by using either bare MNP and MNP@CHO instead of MNP@BHb to produce non- imprinted control polymer (nanoNIP).

[0422] In further experiments, the MNP@BHb particles produced (ca.10 mg) were resuspended in a solution containing the pre-MIP monomer mix in a total solution volume of 1 mL. The reaction proceeded (under nitrogen atmosphere) after initiation with ammonium persulphate, and the mixture was continuously agitated to allow MNP@BHb particles to remain dispersed and to allow nanoMIP to form as a loosely bound shell around each particle. The reaction was terminated at different time points using a free radical quencher and the reaction solution exchanged three times with fresh PBS to remove any unreacted monomers and quencher. Sonication was used to detach the non-covalently bound nanoMIPs from the MNP@BHb particles. Post sonication, a magnet was then used to separate the recovered MNP@BHb from the solution containing nanoMIP. The nanoMIP solution was then lyophilised to concentrate the nanoMIP particles which produced a fluffy white powder. The yield was determined to be 20 mg ± 2 mg nanoMIP per 1 mL of MNP@BHb suspension (containing 10 mg of MNP@BHb). A comparison of large and small MNP@BHb (at equivalent mass) studied demonstrate that the yield is a function of MNP size. Whereas the larger (ca.330 nm) MNP@BHb particles produced a nanoMIP yield of 20 mg ± 2 mg per cycle the smaller (15 nm) MNPS@protein produced a nanoMIP yield of only 0.2 mg ± 0.1 mg. The smaller particles therefore were not efficient candidates for nanoMIP synthesis probably due to the very low density of protein initially attached to the smaller MNPs. NanoNIP was also produced using MNP@CHO as the synthesis surface. Table 3 compares size by DLS of the functionalised MNPs, nanoMIPs and nanoNIP control. Table 3 - DLS particle sizing of aldehyde functionalised MNP, subsequent protein functionalised MNP, and nanoMIP end product. NanoNIP control produced on MNP@CHO surface Material Average size (nm) SD MNP@CHO 122 49 MNP@BHb 331 72 NanoMIP 259 69 NanoNIP 171 22 However, in a further more refined reaction / experiment (see above for referenced experimental modifications), the following DLS particle sizes were obtained (Table 4): Table 4 - DLS particle sizing of aldehyde functionalised MNP, subsequent protein functionalised MNP, and nanoMIP end product. NanoNIP control produced on MNP@CHO surface. (SD is standard deviation; PDI is polydispersity index) Nanomaterial Average size (nm) SD PDI MNP@CHO 122 49 1.0 MNP@BHb 331 72 1.0Further

[0423] The impact of MNP size on subsequent nanoMIP particle size, yield, and affinity factors have been examined, with results set forth in Table 5 below. Table 5 – Impact of MNP size on nanoMIP factors (particle size, yield, and affinity) to of to its the theramp Example 4 – Electrochemical Deposition and Analysis of NanoMIPs

[0426] All electrochemical experiments were performed using a Metrohm Autolab PGSTAT204 potentiostat and NOVA2.1.4 software. NanoMIPs were eluted using sonication and were then entrapped within an electropolymerized layer (E-layer). E-Layers were fabricated directly onto BT-Au screen-printed electrodes (SPEs; Metrohm) using cyclic voltammetry (CV) largely following the literature procedure3, though this procedure was varied so as to entrap pre-formed nanoMIPs (as nanoMIP islands) within the non-templated E-layers. Briefly, a 50 μL solution in PBS comprising 1 mg / mL of nanoMIP, 640 mM of NHMA as the functional monomer, 41.5 mM MBAm as the cross-linker, 0.29 M NaNO3, 48.15 mM KPS was deposited onto the SPE. The potential was then cycled between −0.2 V and −1.4 V for 7 cycles at 50 mV s-1(10 min, RT, 22 ±2 °C) to produce the E-layer with entrapped nanoMIP. E-layers in the absence of nanoMIP were also produced as a control.

[0427] The E-layer comprising entrapped nanoMIP islands (E-NMI) or control E-layer were exposed to varying concentrations of target protein (haemoglobin) template solutions over a wide concentration range (1 fM to 100 µM) for a period of 5 minutes at each concentration and analysed using electrochemical impedance spectroscopy (EIS) post-rebinding and subsequent rinsing in order to determine the degree of target rebound to the nanoMIP islands.

[0428] Selective protein binding was tracked using electrochemical impedance spectroscopy (EIS) of an external 5 mM potassium ferricyanide solution containing 0.5 M KCl as supporting electrolyte. Electrochemical impedance spectroscopy(EIS) measurements were conducted at a standard of 0.1 V (± 0.01 V) with 10 scans of frequencies, and a sinusoidal potential peak-to-peak with amplitude 0.01 V in the 0.1 - 100000 Hz frequency range. A Randles equivalent circuit was fitted for all EIS experiments using the FRA32 module (see Figure 7).

[0429] Samples in PBS were imaged using a Bruker Dimension Icon® AFM with a NanoScope 6 controller in Peak Force Tapping™ mode with silicon nitride cantilevers (SNL-10, nominal spring constant 0.35 Nm-1 and SCANASYST-FLUID, nominal spring constant 0.7 Nm-1). Representative surface images of both bare and nanoMIP polymer entrapped gold electrodes, were obtained. The coated electrodes were prepared through electrochemical polymerisation (see E-MIP Production above). Roughness measurements were collected for a minimum of three 20 µm × 20 µm scans for each surface, and the reported average roughness (Ra) and RMS roughness (Rq) data were obtained following a 1st order plane fit of the raw data.

[0430] This example is illustrative of the Scheme shown in Figure 2.

[0431] In further experiments, lyophilised nanoMIP (1 mg) was reconstituted in 1 mL of PBS buffer comprising NHMA monomer, MBAm crosslinker and initiators as detailed in the experimental above. A 50 µL droplet was deposited on the surface of a BT-Au screen printed electrode covering all three electrodes. The potential was cycled allowing the entrapment of top surface exposed nanoMIP within the surrounding electrochemically polymerised NHMA layer. SEMs of polyNHMA entrapped nanoMIPs were not obtained as they would be an artefact of the actual shape and size due to dehydration of both pNHMA and hydrogel-based nanoMIPs in the instrument. Instead, AFM of pNHMA-NanoMIP samples while immersed in PBS were obtained.

[0432] Figure 8 shows AFM images of (a) bare, (b) electrochemically grown thin film layer, and (c) E-layer-entrapped nanoMIPs. All samples were immersed in PBS, which were imaged using a Bruker Dimension Icon® AFM with a NanoScope 6 controller in Peak Force Tapping™ mode with silicon nitride cantilevers (SNL-10, nominal spring constant 0.35 Nm-1and SCANASYST-FLUID, nominal spring constant 0.7 Nm-1). Whereas the former two present a smooth and homogeneous 10 by of E- to at11M. is to µM 1(selectivity factor) of 120:1 at 1 nM protein and an infinite selectivity factor between 0.1 fM to 0.1 nM protein. Example 5 – Scale-up of NanoMIP Production (Recycling / Re-using MNP@BHb)

[0438] In order for scale-up to be effective, and importantly, to conserve the amount of target protein required to mass produce nanoMIPs, the inventors investigated the re-cycling of MNP@BHb. It was crucial to first ascertain if the MNP@BHb showed any degradation following each use. CD spectroscopy was therefore used to test whether the protein remained pristine after each re-use (5 times in this study) of the same batch of MNP@BHb particles. An aliquot of the MNP particles were tested using CD spectroscopy after each cycle of production and compared this against native protein in solution.

[0439] Figure 12 is a bar chart showing % change (% yield loss) in the production yield of nanoMIPs between subsequent uses of same MNP@protein particles (i.e. cycles 2 to 5).

[0440] Figure 13 is a bar chart showing a summary of CD data demonstrating that 5 cycles of nanoMIP production does not change BHb structure on MNP@BHb.

[0441] Figure 12 shows the % change in the production yield of nanoMIPs and Figure 13 summarises the % alpha helix and beta sheet composition of conjugated protein after each subsequent re-use of MNP@BHb, showing little change in native protein structure. It is evident that nanoMIP production shows no significant hysteresis between batches with average yield per batch of 20 mg ± 2 mg.

[0442] Further a single concentration rebinding study at 1 nM of BHb demonstrated that each batch retained its affinity for target protein (Figure 14). Figure 14 is a bar chart demonstrating that each batch / cycle retained its affinity for target protein (BHb) and no signal for non-target (BSA) (both at 1 nM). Therefore, we demonstrate that by using only 0.6 mg of protein, five sequential cycles of MNP@BHb can produce 100 mg of nanoMIP within one day. This power ratio of protein used to produce high affinity nanoMIP yield in such a short timescale is unprecedented in the MIP field and opens opportunities for industry to realistically adopt such materials as an antibody replacement technology in diagnostics and therapeutics.

[0443] Figure 30 shows a bar chart showing the change in production yield of nanoMIPs with 5 successive cycles of re- using MNP@BHb. Example 6 – NanoMIP Production For different Protein Templates / Targets

[0444] We further demonstrated that our MNP-nanoMIP methodology is versatile by producing high affinity nanoMIPs for proteins ranging in molecular weight including albumin, lysozyme and SARS-CoV-2 recombinant nucleocapsid protein (N- protein). We tested their rebinding and affinity using our E-layer nanoMIP island method. The results and binding affinities are shown below in Table 6. Binding affinity was determined using the Hill-Langmuir adsorption isotherm model to analyse EIS calibration plots (ie KD = concentration of protein at Bmax / 2) for the nanoMIPs produced. We assumed the Hill coefficient is equal to 1, indicative of ligand (MIP) binding with no cooperativity to one site. Table 6 - NanoMIP KD values determined for a range of proteins NanoMIP Protein Molecular weight (KDa) KDBovine Serum Albumin 66.5 1.6 nM Bovine Haemoglobin 64.5 0.0347 nM Recombinant Nucleocapsid Protein 46 1.2 nM for SARS CoV 2 Chicken Egg Lysozyme 14 0.058 nM Further Results and Discussion

[0445] Figure 15 is a bar chart showing the effect of microwave temperature ramp time, during MNP production, on ultimate nanoMIP island electrochemical signal. Evidently, response is independent of ramp time. However, importantly, as 2 min ramp time (i.e.10 nm MNPs) only yields 0.1-0.3 mg nanoMIP compared to a 10 min ramp time (ie 120 nm MNPs) yielding 20 mg of nanoMIP, the larger MNP particles and corresponding higher nanoMIP yield produced by the longer ramp time are preferred. Without wishing to be bound by theory, it is believed that the larger MNP particles are the result of stable agglomeration of the smaller MNPs. Whereas the 10 nm MNP@CHO particles adsorbed 0.05 mg / mL of protein, the 120 nm MNP@CHO particles adsorbed 12 times more protein (0.6 mg / mL). The larger (10 min ramp time) MNP particles therefore are more efficient candidates for nanoMIP synthesis probably due to the higher density of protein initially attached to the larger MNPs.

[0446] Figure 16 is a plot showing the effect of ramp time (taken from 20 to 200 °C) on MNP particle size. Ramp time, R=600s is equivalent 18 °C / min ramping over 10 min; R = 120s is equivalent to 90 °C / min ramping over 2 min.

[0447] Figure 17 shows EIS data showing the trend of BHb rebinding to E-layer of nanoMIP islands.

[0448] Figure 18 shows cyclic voltammetry (CV) plots used to deposit E-layers entrapping nanoMIP islands.

[0449] Figure 19 shows the change in ferricyanide (redox marker) CV response: before, after E-layer with entrapped nanoMIP islands and after 1 nM target protein reloading. CV is not sensitive enough to pick up on subtle changes following 1nM protein addition, hence why EIS was used.

[0450] Figure 20 shows full CD data comparing individual batches / cycles to a control protein.

[0451] Figure 21 shows an AFM image of densely packed nanoMIP islands on a gold electrode. Theoretical Calculations for 10 min ramp time particles:

[0452] The MNP@CHO (120 nm), MNP@protein (330 nm) and nanoMIPs (260 nm) are modelled as spheres and their diameters are taken from DLS measurements. Assume protein has diameter 5 nm (BHb).Theoretical / Geometric considerations:

[0453] Assuming protein attachment to MNP@CHO gives a 100% monolayer, we can determine the number of protein molecules attached to each MNP surface Surface area (SA) of MNP@CHO (120 nm diameter and radius r1): SA = 4πr12Area (A) of protein (radius r2= 2.5 nm): πr22Total number of protein molecules = SA / A = 4r12 / r22= 4 x 900 = 2304

[0454] Therefore, theoretically, we can have 2304 protein molecules packed tightly on one MNP@CHO particle.

[0455] The obtained DLS data indicates that the hydrated MNP particle size increases to 330 nm upon protein attachment (MNP@protein; radius r3= 165 nm). NanoMIPs with particle size 260 nm (radius r4= 130 nm). Therefore we can theoretically determine how many nanoMIP particles each MNP@protein can produce: SA = 4πr32Area (A) of nanoMIP (radius r4= 130 nm): πr42Total number of protein molecules = SA / A = 4r32 / r42= 6.44

[0456] Therefore, theoretically, we can have 6.44 nanoMIP particles produced for every one MNP@protein particle. Experimental considerations: Magnetic nanoparticle volume in a 10 mg MNP@CHO batch:

[0457] From DLS (r1for MNP@CHO = 60 nm) Volume per MNP@CHO particle: V1= 4 / 3πr13= 2.16 x 10-22m3= 2.16 x 10-16cm3

[0458] Assuming the density of an iron oxide core nanoparticle to be 5.15 g / cm3, then 10 mg of MNP@CHO can be approximated to have a total volume of V2:V2= 0.01 / 5.15 = 0.001942 cm3

[0459] Therefore estimated no. of MNP@CHO particles in 10 mg = V2 / V10.001942 / 2.16 x 10-16= 8.990 x 1012particles.

[0460] With 6.44 nanoMIP particles theoretically being produced per MNP particle, we can then say that 10 mg in 1 mL suspension can generate: 6.44 x 8.990 x 1012= 5.789 x 1013nanoMIP particles / mL

[0461] However, our data suggests that only 0.6 mg of protein is taken up by 10 mg of MNP@CHO. No of protein molecules in 0.6mg of BHb = ((0.6 x10-3) / 64500) x 6.022 x 1023= 5.60279 x 1015protein molecules

[0462] Therefore, actual number of protein molecules adsorbed per particle = 5.60279 x 1015 / 8.990 x 1012= 623.25 protein molecules per MNP@CHO particle

[0463] Note: our geometric calculation for a tightly packed MNP sphere suggested: 2304 protein molecules per particle.

[0464] Therefore % actual protein coverage per MNP@CHO particle = (623.25 / 2304) x 100 = 27.05%

[0465] It is not unreasonable that each MNP is only 27% decorated with protein, when taking into account steric hindrance during protein conjugation reaction with MNP@CHO.

[0466] Further with 6.44 nanoMIP particles being produced per MNP@protein particle, we can say that each nanoMIP particle theoretically has 623.25 / 6.44 = 97 protein recognition (binding) sites ie. ratio of protein bound : nanoMIP = 100:1. Theoretical Calculations for 10 min ramp time particles in a more refined reaction / experiment:

[0467] The MNP@CHO (120 nm), MNP@protein (330 nm) and nanoMIPs (125 nm) are modelled as spheres and their diameters are taken from DLS measurements. Assume protein has diameter 5 nm (BHb). Theoretical / Particle Geometry considerations:

[0468] Assuming protein attachment to MNP@CHO gives a 100% monolayer, we can determine the number of protein molecules attached to each MNP surface Surface area (SA) of MNP@CHO (120 nm diameter with radius r1): SA = 4πr12Area (A) of protein r2= 2.5 nm): πr22Total number of protein molecules = SA / A = 4r12 / r22= 4 x 900 = 2304

[0469] Therefore, theoretically, we can have 2304 protein molecules packed tightly on one MNP@CHO particle.

[0470] Our DLS data, in the more-refined reactions / experiments indicates that the hydrated MNP particle size increases to 330 nm upon protein attachment (MNP@protein; radius r3= 165 nm). NanoMIPs with particle size 125 nm (radius r4= 63 nm). Therefore, we can theoretically determine how many nanoMIP particles each MNP@protein can produce: SA = 4πr32Area (A) of nanoMIP (radius r4= 130 nm): πr42Total number of protein molecules = SA / A = 4r32 / r42= = 27.4

[0471] Therefore, theoretically, we can have 27.4 nanoMIP particles produced for every one MNP@protein particle. Experimental considerations: Magnetic nanoparticle volume in a 10 mg MNP batch:

[0472] From DLS (r1for MNP@CHO = 60 nm) Volume per MNP@CHO particle: V1= 4 / 3πr13= 2.16 x 10-22m3= 2.16 x 10-16cm3

[0473] Assuming the density of an iron oxide core nanoparticle to be 5.15 g / cm3, then 10 mg of MNP@CHO can be approximated to have a total volume of V2:V2= 0.01 / 5.15 = 0.001942 cm3

[0474] Therefore estimated no. of MNP@CHO particles in 10 mg = V2 / V10.001942 / 2.16 x 10-16= 8.990 x 1012particles.

[0475] With 27.4 nanoMIP particles theoretically being produced per MNP particle, we can then say that 10 mg in 1 mL suspension can generate: 27.4 x 8.990 x 1012= 2.463 x 1014nanoMIP particles / mL.

[0476] However, our experimental data (Fig.3) suggests that only 0.6 mg of protein is taken up by 10 mg of MNP@CHO. No of protein molecules in 0.6mg of BHb = ((0.6 x10-3) / 64500) x 6.022 x 1023= 5.60279 x 1015protein molecules: Initial Conclusions

[0483] The inventors have produced high affinity nanoMIPs for the protein BHb. Affinity was determined electrochemically. When nanoMIP was integrated with an electropolymerized layer on a disposable electrode, EIS showed the device to be sensitive to BHb in the wide range 100fM to 10 nM, with a LOQ of 10 fM. The recycling of protein template is fundamental to economical scaling-up of nanoMIP production. The inventors demonstrate a method of economically producing aldehyde functionalised MNPs using a microwave method and subsequent conjugation with target protein. With the re-usability of target template (over at least 5 cycles), the inventors demonstrate the scaled-up mass production of nanoMIPs, with 10mg produced per cycle. This method offers a commercially viable route to bespoke nanoMIP production for a range of targets.

[0484] The invention is broadly applicable to any template molecule, and thus any protein. Other modes of MNP functionalisation, other than aldehyde-functionalisation, are self-evidently also possible, allowing for a variety of template tethering modes including aldehyde (-CHO), amine (-NH2), and carboxylate (-COOH).Example 7 – Nanoparticle tracking analysis (NTA) for BHb and SARS-CoV-2 NanoMIPs 3 – of 7 of 8

[0488] These results show “SARS-Cov-2 NanoMIPs” having a particle diameter ranging from 50-200 nm.

[0489] There appears to be a relationship between nanoMIP particle size and the protein target attached to MNP@CHO. Example 8 – Comparison of nanoMIP and antibody for the same protein target

[0490] As previously explained herein, “MIPs may be used as artificial antibodies or to create enzyme-like binding sites to facilitate drug development and screening”, and “MIPs may, more easily than antibodies and proteins, be “labelled” – e.g. radio-labelled or fluorescent-labelled, to facilitate assaying” (as per paragraph

[0003] above). Indeed, the present invention provides a “Use of a (free) molecularly imprinted polymer as an artificial antibody” as per “use” aspect E6.

[0491] Recent experiments have also shown that nanoMIPs of the invention can be as effective as biological antibodies directed towards the same target or molecular template. Example 9 – nanoMIPs with increased monomeric functionality and functionalisability

[0492] The “BHb nanoMIP” formations described in Example 3 were performed by doping-in an extra functional monomer, in this example N-(3-Aminopropyl)methacrylamide hydrochloride (APMA), to the polymerisation reactant mixture – in this example 10 wt% of the NHMA monomer used in Example 3 was replaced with APMA. The APMA-doped “BHb nanoMIP”product was then examined and compared to the undoped nanoMIP” product. It was noted that introduction of APMA monomeric units into the nonoMIP did not detrimentally impact the affinity (KD) of the nanoMIP. The extra functional groups (-NH2groups) introduced by the APMA monomer unlocks further functionalisation opportunities for the nanoMIPs – for instance, the ability to enzyme-label the nanoMIPs to allow them to behave like enzyme-labelled antibodies. Such enzyme- labelled nanoMIPs may then be applied to immunoassays (e,g, ELISA - Enzyme-Linked Immunosorbent Assays). Fluorescently-labelled nanoMIPs represent another amongst a host of functionalised nanoMIP derivatives.

[0493] Figure 31 shows a graph of ^RCT(^) against BHb concentration for BHb nanoMIPs formed using 10% APMA.

[0494] The inventors hereby demonstrate that it is possible to functionalise the nanoMIP with -NH2 (primary amine) groups through inclusion of co-monomer (APMA; 1-10% w / w) during the MIP production process. This enables the subsequent bioconjugation of enzymes and other proteins using, for example, the crosslinking agent glutaraldehyde or through EDC / NHS coupling chemistry. FURTHER NUMBERED PARAGRAPHS

[0495] Further aspects, embodiments and features of the invention are set forth in the following further numbered paragraphs: 1. A method of manufacturing a (free) molecularly imprinted polymer (MIP), the method comprising: i) providing a magnetically supported template molecule, which comprises or consists of a magnetic core coupled or tethered to one or more (preferably a plurality) of template molecule(s); ii) forming a magnetic template-bound molecularly imprinted polymer, which comprises or consists of the (or one or more of the, preferably a plurality of) magnetically supported template molecule(s) bound to a molecularly imprinted polymer via a (or one or more, preferably a plurality of) corresponding complementary imprint(s) within the molecularly imprinted polymer(s), by a method comprising: polymerising one or more monomer(s) (preferably at least one backbone-forming monomer and a cross-linking monomer) in the presence of the magnetically supported template molecule (or a recycled magnetically supported template molecule, when this step is repeated); iii) unbinding and separating the magnetically supported template molecule from the molecularly imprinted polymer of the magnetic template-bound molecularly imprinted polymer, to yield a (free) molecularly imprinted polymer and a recycled magnetically supported template molecule; iv) repeating, one or more times, steps ii)-iii) using the recycled magnetically supported template molecule of step iii) (or of repeated step iii)) as the magnetically supported template molecule for repeated step ii). 2. The method as defined in paragraph 2, wherein the (free) molecularly imprinted polymer is isolated (optionally in a solvent or solvent system) after each step iii) (including repeats of step iii)) in a plurality of batches of the (free) molecularly imprinted polymer, and the plurality of batches are combined. 3. The method as defined in any preceding paragraph, wherein step iv) comprises repeating, at least three times, steps ii)-iii) using the recycled magnetically supported template molecule of step iii) (or of repeated step iii)) as the magnetically supported template molecule for repeated step ii). 4. The method as defined in any preceding paragraph, wherein the recycled magnetically supported template molecule is analysed to confirm that it can be reused before the recycled magnetically supported template molecule is reused in repeating step iv). 5. The method as defined in any preceding paragraph, wherein the recycled magnetically supported template molecule is subjected to further processing before the recycled magnetically supported template molecule is reused in repeating step iv), said further processing comprising either or both purification and / or doping with a purer batch of magnetically supported template molecule. 6. The method as defined in any preceding paragraph, wherein the unbinding and separating of step iii) (and any repeated step iii)) comprises unbinding the magnetically supported template molecule from the molecularly imprinted polymer of the magnetic template-bound molecularly imprinted polymer by subjecting the magnetic template-bound molecularly imprinted polymer to unbinding conditions, said unbinding conditions comprising agitating the magnetic template-bound molecularly imprinted polymer in a solvent or solvent system. 7. The method as defined in paragraph 6, wherein agitating the magnetic template-bound molecularly imprinted polymer comprises sonicating (i.e. via the application of ultrasound) the magnetic template-bound molecularly imprinted polymer in a solvent or solvent system, suitably at an ultrasound frequency between 10 and 200 kHz and ultrasound power between 50 and 1,000 Watts for a time period between 1 minute and 15 minutes. 8. The method as defined in paragraph 6 or paragraph 7, wherein the unbinding conditions further comprise exposing the magnetic template-bound molecularly imprinted polymer to a magnetic field, optionally a resonating or pulsed magnetic field. 9. The method as defined in any preceding paragraph, wherein the unbinding and separating of step iii) (and any repeated step iii)) comprises unbinding the magnetically supported template molecule from the molecularly imprinted polymer of the magnetic template-bound molecularly imprinted polymer, and thereafter magnetically separating the magnetically supported template molecule from the molecularly imprinted polymer.are the a the a 10 . tois defined by configuration iii) and / or iv).or an to d- is or , or or (or orof silica or functionalised silica.41. The method as defined in paragraph 30 or any of 31 to 40 where dependent on paragraph 30, wherein coupling a template molecule to the functionalised magnetic support comprises forming a reaction mixture in a reaction vessel, the reaction mixture comprising the functionalised magnetic support (suitably a suspension thereof), the template molecule, and a solvent or solvent system; and allowing coupling to take place at a temperature of 25oC + / - 25oC (preferably 25oC + / - 10oC). 42. A molecularly imprinted polymer obtained by the method as defined in any preceding paragraph. 43. A method of manufacturing a functionalised magnetic support, the method comprising: mixing together one or more magnetic core-forming components with a functionalising molecule, and optionally further processing, to form a functionalised magnetic support comprising a magnetic core, derived from the magnetic core-forming components, the magnetic core bearing, at the surface thereof, one or more reactive functional group(s) derived from the functionalising molecule; wherein the magnetic core is free of a silica coating or functionalised silica; and the one or more reactive functional groups are directly attached to the magnetic core; optionally wherein the functionalised magnetic support and / or its method of manufacture is further defined by any of paragraphs 30 to 40. 44. A functionalised magnetic support obtained by the method of paragraph 43. 45. A method of manufacturing a magnetically supported template molecule, the method comprising: providing a functionalised magnetic support by, or obtained by, the method of manufacturing a functionalised magnetic support as defined in paragraph 43, and coupling a template molecule to said functionalised magnetic support via a (preferably direct) chemical reaction between the template molecule and a reactive functional group borne on the surface of the functionalised magnetic support; optionally wherein the template molecule is further defined by any preceding paragraph. 46. A magnetically supported template molecule obtained by the method of paragraph 45. 47. A method of manufacturing a magnetic template-bound molecularly imprinted polymer, which comprises or consists of the (or one or more of the, preferably a plurality of) magnetically supported template molecule(s) bound to a molecularly imprinted polymer via a (or one or more, preferably a plurality of) corresponding complementary imprint(s) within the molecularly imprinted polymer(s), the method comprising: polymerising one or more monomer(s) (preferably at least one backbone-forming monomer and a cross-linking monomer) in the presence of a magnetically supported template molecule obtained by or provided via the method of manufacturing a magnetically supported template molecule as defined in paragraph 45. 48. A method of manufacturing a (free) molecularly imprinted polymer (MIP), the method comprising: i) providing a magnetic template-bound molecularly imprinted polymer by, or obtained by, the method of manufacturing a magnetic template-bound molecularly imprinted polymer as defined in paragraph 47; ii) separating the magnetically supported template molecule from the molecularly imprinted polymer of the magnetic template-bound molecularly imprinted polymer; iii) isolating: a. the molecularly imprinted polymer; and optionally also b. the magnetically supported template molecule as a recycled magnetically supported template molecule; and optionally repeating steps i)-iii) by reusing the recycled magnetically supported template molecule isolated in step iii). 49. A (free) molecularly imprinted polymer (MIP) obtained by the method of paragraph 48. 50. A method of manufacturing a molecularly imprinted polymer-bearing electrode, the method comprising: i) providing a (pre-formed) molecularly imprinted polymer; ii) providing an electrode; iii) forming a coating polymer upon the surface of the electrode by electrochemically initiating and / or propagating polymerisation of one or more coating monomers in the presence of the molecularly imprinted polymer, suitably to trap the molecularly imprinted polymer within the coating polymer as the coating polymer is formed upon the surface of the electrode. 51. The method as defined in paragraph 50, wherein the molecularly imprinted polymer is a (free) molecularly imprinted polymer obtained by the method of manufacturing a (free) molecularly imprinted polymer as defined in any of paragraphs 1 to 41 or paragraph 48. 52. The method as defined in paragraph 50 or 51, wherein the electrode is a screen-printed electrode. 53. The method as defined in any of paragraphs 50 to 52, wherein the electrode is an electrode for cyclic voltammetry (CV) and / or electrochemical impedance spectroscopy (EIS). 54. The method as defined in paragraph 53, wherein the electrode is an electrode device comprising a working electrode, a counter electrode, and a reference electrode. 55. The method as defined in paragraph 54, wherein the method comprises contacting the working electrode, the counter electrode, and the reference electrode of an electrode device with a reaction mixture, which initially comprises the molecularly imprinted polymer (suitably dispersed or suspended) and the one or more coating monomers (suitably dissolvedor dispersed) in a solvent or solvent system, and initiating and / or propagating a polymerisation reaction to cause a polymer or co-polymer comprising (or formed from) the one or more coating monomers to form on the surface of the electrode (or electrode device) producing a polymer coating entrapping or incorporating the molecularly imprinted polymer (which are suitably not covalently bonded to the polymer coating) therein. 56. The method as defined in paragraph 55, wherein the electrochemical initiation and / or propagation (and the polymerisation reaction in general) is caused by cyclic voltammetry (CV), suitably b...

Claims

CLAIMS 1. A method of manufacturing a (free) molecularly imprinted polymer (MIP), the method comprising: i) providing a magnetically supported template molecule, which comprises or consists of a magnetic core coupled or tethered to one or more (preferably a plurality) of template molecule(s); ii) forming a magnetic template-bound molecularly imprinted polymer, which comprises or consists of the (or one or more of the, preferably a plurality of) magnetically supported template molecule(s) bound to a molecularly imprinted polymer via a (or one or more, preferably a plurality of) corresponding complementary imprint(s) within the molecularly imprinted polymer(s), by a method comprising: polymerising one or more monomer(s) (preferably at least one backbone-forming monomer and a cross-linking monomer) in the presence of the magnetically supported template molecule (or a recycled magnetically supported template molecule, when this step is repeated); iii) unbinding and separating the magnetically supported template molecule from the molecularly imprinted polymer of the magnetic template-bound molecularly imprinted polymer, to yield a (free) molecularly imprinted polymer and a recycled magnetically supported template molecule; iv) repeating, one or more times, steps ii)-iii) using the recycled magnetically supported template molecule of step iii) (or of repeated step iii)) as the magnetically supported template molecule for repeated step ii).

2. The method as claimed in claim 1, wherein the (free) molecularly imprinted polymer is isolated (optionally in a solvent or solvent system) after each step iii) (including repeats of step iii)) in a plurality of batches of the (free) molecularly imprinted polymer, and the plurality of batches are combined.

3. The method as claimed in any preceding claim, wherein step iv) comprises repeating, at least three times, steps ii)-iii) using the recycled magnetically supported template molecule of step iii) (or of repeated step iii)) as the magnetically supported template molecule for repeated step ii).

4. The method as claimed in any preceding claim, wherein the unbinding and separating of step iii) (and any repeated step iii)) comprises unbinding the magnetically supported template molecule from the molecularly imprinted polymer of the magnetic template-bound molecularly imprinted polymer by subjecting the magnetic template-bound molecularly imprinted polymer to unbinding conditions, said unbinding conditions comprising agitating the magnetic template-bound molecularly imprinted polymer in a solvent or solvent system.

5. The method as claimed in claim 4, wherein agitating the magnetic template-bound molecularly imprinted polymer comprises sonicating (i.e. via the application of ultrasound) the magnetic template-bound molecularly imprinted polymer in a solvent or solvent system, suitably at an ultrasound frequency between 10 and 200 kHz and ultrasound power between 50 and 1,000 Watts for a time period between 1 minute and 15 minutes.

6. The method as claimed in any preceding claim, wherein the unbinding and separating of step iii) (and any repeated step iii)) comprises unbinding the magnetically supported template molecule from the molecularly imprinted polymer of the magnetic template-bound molecularly imprinted polymer, and thereafter magnetically separating the magnetically supported template molecule from the molecularly imprinted polymer.

7. The method as claimed in any preceding claim, wherein the unbinding and separating of step iii) are performed at a temperature of 25oC + / - 25oC.

8. The method as claimed in any preceding claim, wherein the forming a magnetic template-bound molecularly imprinted polymer of step ii) comprises co-polymerising one or more backbone-forming monomer(s) and one or more (preferably only one) cross-linking monomer(s) in the presence of the magnetically supported template molecule (or the recycled magnetically supported template molecule, when this step is repeated) and suitably also an initiator or initiator system.

9. The method as claimed in claim 8, wherein the one or more backbone-forming monomers are selected from the group consisting of optionally substituted acrylamide(s), optionally substituted alkacrylamide(s) (e.g. optionally substituted methacrylamides), optionally substituted acrylic acid(s), optionally substituted alkacrylic acid(s) (e.g. optionally substituted methacrylic acids), optionally substituted acrylate ester(s), optionally substituted alkacrylate ester(s) (e.g. optionally substituted methacrylate esters), or any combination thereof (suitably a combination of two or more thereof where plurality of backbone-forming monomers are used); optionally wherein the one or more cross-linking monomers are selected from the group consisting of N,N’methylenebisacrylamide (BIS, MBAm), allyl methacrylate (AM), divinylbenzene, ethyleneglycol dimethacrylate, dipentaerythritol pentaacrylate, pentaerythritol tetraacrylate, and any combination (though preferably only a single cross- linking monomer).

10. The method as claimed in any preceding claim, wherein the forming a magnetic template-bound molecularly imprinted polymer of step ii) is performed at a temperature of 25oC + / - 25oC (more preferably 25oC + / - 10oC).

11. The method as claimed in any preceding claim, wherein the (or each) molecularly imprinted polymer comprises a plurality of complementary imprints, preferably at least 10 complementary imprints.

12. The method as claimed in any preceding claim, wherein the average particle size of the (free) MIP is between 10 and 1000 nm (preferably between 50 and 500 nm), or is between 30 and 500 nm (preferably between 80 and 300 nm).to 1 to 50 an of to d- to ofc. Repeating heating step b) until a predetermined maximum temperature (Tmax) is reached;wherein: the difference between the start temperature (Tmin) and maximum temperature (Tmax) is between 50 and 300oC (preferably between 70 and 250oC); the maximum temperature is between 60 and 350oC (preferably between 150oC and 250oC); the total ramp time (ttotal) (between step a), when heating first begins, and the end of step c), reaching the maximum temperature) is between 2 and 60 mins (preferably between 5 and 20 mins); and the ramp rate is between 5oC / min and 100oC / min (preferably between 8oC / min and 30oC / min).

24. The method as claimed in any of claims 19 to 23, wherein the magnetic core, and indeed the functionalised magnetic support as a whole, is free of silica or functionalised silica; preferably wherein the magnetic core is free of any silica or functionalised silica coating; preferably wherein the magnetically supported template molecule is free of silica or functionalised silica.

25. The method as claimed in claim 19 or any of claims 20 to 24 where dependent on claim 19, wherein coupling a template molecule to the functionalised magnetic support comprises forming a reaction mixture in a reaction vessel, the reaction mixture comprising the functionalised magnetic support (suitably a suspension thereof), the template molecule, and a solvent or solvent system; and allowing coupling to take place at a temperature of 25oC + / - 25oC (preferably 25oC + / - 10oC).

26. The method as claimed in any preceding claim, wherein the (free) molecularly imprinted polymer (MIP) is further processed.

27. The method as claimed in any preceding claim, wherein the method comprises polymerising one or more monomers (or optionally copolymerising two or more monomers, preferably wherein at least one of the two or more monomers is a cross-linking monomer) in the presence of the magnetically supported template molecule; wherein one or more of the monomers comprises a detectable moiety, such as a fluorescent moiety.

28. The method as claimed in any preceding claim, wherein the method comprises polymerising one or more monomers (or optionally copolymerising two or more monomers, preferably wherein at least one of the two or more monomers is a cross-linking monomer) in the presence of the magnetically supported template molecule; wherein one or more of the monomers comprise reactive moieties that are coupled to a detectable moiety after the polymer has been formed (suitably after unbinding and separating).

29. The method as claimed in any preceding claim, wherein the (free) molecularly imprinted polymer (MIP) is labelled.

30. The method as claimed in claim 29, wherein the (free) molecularly imprinted polymer (MIP) is radio-labelled or fluorescent-labelled or redox-labelled.

31. The method as claimed in claim 29, wherein the (free) molecularly imprinted polymer (MIP) is enzyme-labelled.

32. The method as claimed in any preceding claim, wherein the (free) molecularly imprinted polymer (MIP) is linked (suitably via conjugation) to an enzyme, suitably by chemically coupling (or conjugating, be it directly or indirectly) the enzyme to the MIP, suitably after the MIP has been formed (and suitably after unbinding and separating).

33. A molecularly imprinted polymer obtained by the method as claimed in any preceding claim.

34. A method of manufacturing a functionalised magnetic support, the method comprising: mixing together one or more magnetic core-forming components with a functionalising molecule, and optionally further processing, to form a functionalised magnetic support comprising a magnetic core, derived from the magnetic core-forming components, the magnetic core bearing, at the surface thereof, one or more reactive functional group(s) derived from the functionalising molecule; wherein the magnetic core is free of a silica coating or functionalised silica; and the one or more reactive functional groups are directly attached to the magnetic core; optionally wherein the functionalised magnetic support and / or its method of manufacture is further defined by any preceding claim.

35. A functionalised magnetic support obtained by the method of claim 34.

36. A method of manufacturing a magnetically supported template molecule, the method comprising: providing a functionalised magnetic support by, or obtained by, the method of manufacturing a functionalised magnetic support as claimed in claim 27, and coupling a template molecule to said functionalised magnetic support via a (preferably direct) chemical reaction between the template molecule and a reactive functional group borne on the surface of the functionalised magnetic support; optionally wherein the template molecule is further defined by any preceding claim.

37. A magnetically supported template molecule obtained by the method of claim 36.

38. A method of manufacturing a magnetic template-bound molecularly imprinted polymer, which comprises or consists of the (or one or more of the, preferably a plurality of) magnetically supported template molecule(s) bound to a molecularly imprinted polymer via a (or one or more, preferably a plurality of) corresponding complementary imprint(s) within the molecularly imprinted polymer(s), the method comprising: polymerising one or more monomer(s) (preferably at least one backbone-forming monomer and a cross-linking monomer) in the presence of a magnetically supported template molecule obtained by or provided via the method of manufacturing a magnetically supported template molecule as claimed in claim 36.

39. A method of manufacturing a (free) molecularly polymer (MIP), the method comprising: i) providing a magnetic template-bound molecularly imprinted polymer by, or obtained by, the method of manufacturing a magnetic template-bound molecularly imprinted polymer as claimed in claim 38; ii) separating the magnetically supported template molecule from the molecularly imprinted polymer of the magnetic template-bound molecularly imprinted polymer; iii) isolating: a. the molecularly imprinted polymer; and optionally also b. the magnetically supported template molecule as a recycled magnetically supported template molecule; and optionally repeating steps i)-iii) by reusing the recycled magnetically supported template molecule isolated in step iii).

40. A (free) molecularly imprinted polymer (MIP) obtained by the method of claim 39.

41. A method of manufacturing a molecularly imprinted polymer-bearing electrode, the method comprising: i) providing a (pre-formed) molecularly imprinted polymer; ii) providing an electrode; iii) forming a coating polymer upon the surface of the electrode by electrochemically initiating and / or propagating polymerisation of one or more coating monomers in the presence of the molecularly imprinted polymer, suitably to trap the molecularly imprinted polymer within the coating polymer as the coating polymer is formed upon the surface of the electrode.

42. A molecularly imprinted polymer-bearing electrode obtained by the method as claimed in claim 41.

43. Use of a molecularly imprinted polymer-bearing electrode in the detection, characterisation, and / or quantification of a target molecule (preferably dissolved, dispersed, or suspended in a solvent or solvent system), wherein the molecularly imprinted polymer-bearing electrode is obtained by the method of manufacturing a molecularly imprinted polymer-bearing electrode as claimed in claim 41.

44. A detection device comprising a molecularly imprinted polymer-bearing electrode operable to detect, characterise, and / or quantify a target molecule, wherein the molecularly imprinted polymer-bearing electrode is obtained by the method of manufacturing a molecularly imprinted polymer-bearing electrode as claimed in claim 41.

45. Use of a (free) molecularly imprinted polymer as claimed in claim 33 in an assay, suitably in an immunoassay (e.g. an ELISA, especially where the molecularly imprinted polymer is linked to an enzyme as per claim 32).

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