Technical Field

The present invention relates to a molecularly imprinted polymer (MIP) and a method of making the molecularly imprinted polymer. It also relates to a MIP sensor.

Background

Molecularly imprinting of polymers is widely used in chemical and biochemical sensing, catalysis, compound separation and purification, and removal of hazardous substances. For a molecularly imprinted polymer (MIP) to be used as a sensor, the MIP needs to be attached to a sensing substrate. However, peeling of the MIP from the sensing substrate is a common problem due to inadequate adhesion of the MIP to the sensing substrate and this therefore affects the stability of the MIP sensor.

There is therefore a need for an improved MIP for use in a MIP sensor.

Summary of the invention

The present invention seeks to address these problems, and/or to provide an improved MIP for use in a MIP sensor, as well as a method for making the improved MIP.

According to a first aspect, the present invention provides a method of making a molecularly imprinted polymer (MIP) comprising:

preparing a MIP solution comprising a monomer host, an anchoring group- containing monomer, one or more target molecules and a first solvent;

polymerising the MIP solution to form MIP comprising the anchoring group; and

removing the one or more target molecules from the MIP to form cavities in the MIP comprising the anchoring group,

wherein the anchoring group comprises at least one of: cyclic imide, O-acylisourea, azide, carboxylic acid, amine, hydroxyl, or thiol.

The anchoring group-containing monomer may comprise at least one functional group selected from, but not limited to: cyclic imide, O-acylisourea, azide, carboxyl, amine, hydroxyl, or thiol. The anchoring group-containing monomer may be any suitable anchoring group- containing monomer. For example, the anchoring group-containing monomer may comprise at least one functional group selected from, but not limited to: cyclic imide, O- acylisourea, azide, carboxylic acid, amine, hydroxyl, or thiol. According to a particular aspect, the cyclic imide group may comprise, but is not limited to: succinimide ester or sulfosuccinimide ester. The O-acylisourea group may comprise, but is not limited to: 1-ethyl-3-(3-dimethylaminopropyl) O-acylisourea, dicyclehexyl O-acylisourea, N,N’-diisopropyl O-acylisourea, or 1-cyclohexyl-(2- morpholinoethyl) O-acyliso metho-p-toluene sulfonate (CMCT). According to a particular aspect, the anchoring group-containing monomer may be, but not limited to: an N-hydroxysuccinimide (NHS)-containing monomer, a 1-ethyl-3-(3- dimethylamidopropyl)carbodiimide (EDC)-containing monomer, or a co-monomer thereof.

According to a particular aspect, the anchoring group-containing monomer may be, but not limited to: N-(Allyloxycarbonyloxy)succinimide, methacrylic acid N- hydroxysuccinimide ester, acrylate-polyethylene-NHS, methacrylic acid O-acylisourea, methacrylic azide, methacrylic acid, 3-(2-Amino-3-pyridyl)acrylic acid, 2-hydroxylethyl methacrylate, 10-sulfanyldecyl methacrylate, or a co-monomer thereof.

The monomer host may be any suitable monomer host. For example, the monomer host may comprise, but is not limited to: ionic liquid monomer, acrylate monomer, acrylamide monomer, or co-monomers thereof. In particular, the monomer host may be selected from the group consisting of, but not limited to: 1-allyl-3-methylimidazolium dicyanamide, 1-allyl-3-methylimidazolium dibromide, vinyl 3H-imidazolium bis(trifluoromethanesulfonyl)imide, N,N,N,N-butyldimethylmethacryloyloxyethyl bis(trifluoromethanesulfonyl)imide, 3-ethyl-1-vinylimidazolium bis(trifluoromethanesulfonyl)imide, 1 -allyl-1 -methylpyrrolidinium bis(trifluoromethanesulfonyl)imide, 1 ,4-butanediyl-3,3’-bis-1-vinylimidazolium)di- bis(trifluoromethanesulfonyl)imide, acrylic acid, methacrylic acid, ethyl 2-ethylacrylate, 4-acetoxyphenethyl acrylate, benzyl 2-propylacrylate, 2-carboxyethyl acrylate, 2- hydroxyethyl acrylate, [2-(acryloyloxy)ethyl]trimethylammonium chloride, PEG diacrylate, PEG methyl ether acrylate, acrylamide, alkylacrylamide, 3- (acrylamide)phenylboronic acid, 2-acrylamido-2-methyl-1 -propane sulfonic acid, 2- aminoethyl methacrylamide, N-hydroxyethyl acryamide, (4-hydroxyphenyl)acrylamide, N-phenylacryamide, N-(triphenylmethyl) acrylamide, N-

[tris(hydroxymethyl)methyl]acryamide, or co-monomers thereof.

According to a particular aspect, the one or more target molecule may comprise a biotoxin or a heavy metal ion. The biotoxin may be any suitable biotoxin. The heavy metal ion may be any suitable heavy metal ion.

The MIP solution may further comprise one or more of: cross-linkers, polymerisation initiators.

The removing may be by any suitable means. According to a particular aspect, the removing may comprise extracting the one or more target molecules from the MIP using a second solvent, wherein the one or more target molecules may be soluble in the second solvent.

According to a particular aspect, the method may further comprise forming a MIP sensor by covalently immobilizing the MIP comprising the anchoring group on a substrate surface comprising a coupling group. In particular, the substrate surface may be functionalised by the coupling group. The coupling group may be any suitable coupling group. For example, the coupling group may comprise, but is not limited to: an amino group, a carboxyl group or a combination thereof.

According to a particular aspect, the anchoring group of the MIP comprising the anchoring group may covalently bond with the coupling group of the substrate surface comprising the coupling group.

According to a second aspect, there is provided a molecularly imprinted polymer (MIP) sensor made from the method of the first aspect.

There is also provided a molecularly imprinted polymer (MIP) comprising an anchoring group, the anchoring group comprising at least one of: cyclic imide, O-acylisourea, azide, carboxylic acid, amine, hydroxyl, or thiol according to a third aspect of the present invention.

The cyclic imide group and the O-acylisourea group may be as described above. According to a fourth aspect, there is provided a molecularly imprinted polymer (MIP) sensor comprising a MIP according to the third aspect.

Brief Description of the Drawings

In order that the invention may be fully understood and readily put into practical effect there shall now be described by way of non-limitative example only exemplary embodiments, the description being with reference to the accompanying illustrative drawings. In the drawings:

Figure 1 shows anchoring reactions between anchoring groups and coupling groups according to various embodiments of the present invention; Figure 2 shows a schematic representation of the preparation of a MIP comprising an anchoring group and a sensor comprising the MIP according to one embodiment of the present invention;

Figure 3 shows a schematic representation of the preparation of a MIP comprising an anchoring group and a sensor comprising the MIP according to one embodiment of the present invention;

Figure 4 shows a schematic representation of the preparation of an endotoxin MIP comprising anchoring groups;

Figure 5 shows the plotted calibration curve of an endotoxin PA-10 MIP sensor;

Figure 6 shows the high selectivity of Zn ²⁺ imprinted MIP based sensor against various other interfering ions.

Detailed Description

As explained above, there is a need for an improved MIP for use in a MIP sensor, as well as a method for making the improved MIP.

The present invention also provides a method for making a polymer-based sensor. In particular, the sensor made from the method of the present invention is a molecularly imprinted polymer sensor which enables better adhesion of the MIP to the sensor substrate. The sensor shows good response in detecting target molecules which may include biotoxins and heavy metal ions. Further, the sensor made also exhibits a fast response time in detecting target molecules and a low limit of detection. It is also more cost-effective to make the sensor of the present invention.

Molecular imprinting is a technique to produce molecule specific receptors analogous to those receptor binding sites in biochemical systems. A molecularly imprinted polymer (MIP) is a polymer that is formed in the presence of a template or target analyte molecule producing a complementary cavity that is left behind in the MIP when the template is removed. The MIP demonstrates affinity for the original template molecule over other related and analogous molecules.

In general terms, the present invention relates to a method of making MIP comprising anchoring groups which could covalently bond to a substrate surface, to be used as sensors for detecting biotoxins and heavy metal ions. In particular, the anchoring groups enable better adhesion of the MIP to the substrate surface, thereby preventing peeling off of the MIP from the substrate surface and improving the stability of the MIP sensor. According to a first aspect, the present invention provides a method of making a molecularly imprinted polymer (MIP) comprising:

preparing a MIP solution comprising a monomer host, an anchoring group- containing monomer, one or more target molecules and a first solvent;

polymerising the MIP solution to form MIP comprising the anchoring group; and

removing the one or more target molecules from the MIP to form cavities in the MIP comprising the anchoring group,

wherein the anchoring group comprises at least one of: cyclic imide, O-acylisourea, azide, carboxylic acid, amine, hydroxyl, or thiol. The anchoring group-containing monomer may comprise at least one functional group selected from, but not limited to: cyclic imide, O-acylisourea, azide, carboxyl, amine, hydroxyl, or thiol. In particular, the cyclic imide group may comprise a succinimide ester group.

The anchoring group-containing monomer may be any suitable anchoring group- containing monomer. The anchoring group containing monomer may comprise co monomers. For example, the anchoring group-containing monomer may comprise at least one functional group selected from, but not limited to: cyclic imide, O-acylisourea, azide, carboxylic acid, amine, hydroxyl, or thiol. In particular, the cyclic imide group may comprise a succinimide ester group.

According to a particular aspect, the cyclic imide group may comprise, but is not limited to: succinimide ester or sulfosuccinimide ester. The O-acylisourea group may comprise, but is not limited to: 1-ethyl-3-(3-dimethylaminopropyl) O-acylisourea, dicyclehexyl O-acylisourea, N,N’-diisopropyl O-acylisourea, or 1-cyclohexyl-(2- morpholinoethyl) O-acyliso metho-p-toluene sulfonate (CMCT).

According to a particular aspect, the anchoring group-containing monomer may be, but not limited to: an N-hydroxysuccinimide (NHS)-containing monomer, a 1-ethyl-3-(3- dimethylamidopropyl)carbodiimide (EDC)-containing monomer, or a co-monomer thereof.

According to a particular aspect, the anchoring group-containing monomer may be, but not limited to: N-(Allyloxycarbonyloxy)succinimide, methacrylic acid N- hydroxysuccinimide ester, acrylate-polyethyleneglycol-NHS, methacrylic acid O- acylisourea, methacrylic azide, methacrylic acid, 3-(2-Amino-3-pyridyl)acrylic acid, 2- hydroxylethyl methacrylate, 10-sulfanyldecyl methacrylate, or a co-monomer thereof.

The monomer host may be any suitable monomer host. The monomer host may comprise co-monomers. The monomer host may be a combination of a monomer unit and a co-monomer unit. For example, the monomer host may comprise one or more monomers, a co-monomer, or a combination of a monomer with other co-monomers.

According to a particular aspect, the monomer host may comprise, but is not limited to: ionic liquid monomer, acrylate monomer, acrylamide monomer, or co-monomers thereof. For example, the monomer host may be selected from the group consisting of, but not limited to: 1-allyl-3-methylimidazolium dicyanamide, 1-allyl-3-methylimidazolium dibromide, vinyl 3H-imidazolium bis(trifluoromethanesulfonyl)imide, N,N,N,N- butyldimethylmethacryloyloxyethyl bis(trifluoromethanesulfonyl)imide, 3-ethyl-1- vinylimidazolium bis(trifluoromethanesulfonyl)imide, 1-allyl-1-methylpyrrolidinium bis(trifluoromethanesulfonyl)imide, 1 ,4-butanediyl-3,3’-bis-1-vinylimidazolium)di- bis(trifluoromethanesulfonyl)imide, acrylic acid, methacrylic acid, ethyl 2-ethylacrylate,

4-acetoxyphenethyl acrylate, benzyl 2-propylacrylate, 2-carboxyethyl acrylate, 2- hydroxyethyl acrylate, [2-(acryloyloxy)ethyl]trimethylammonium chloride, PEG diacrylate, PEG methyl ether acrylate, acrylamide, alkylacrylamide, 3- (acrylamide)phenylboronic acid, 2-acrylamido-2-methyl-1 -propane sulfonic acid, 2- aminoethyl methacrylamide, N-hydroxyethyl acryamide, (4-hydroxyphenyl)acrylamide, N-phenylacryamide, N-(triphenylmethyl) acrylamide, N-

[tris(hydroxymethyl)methyl]acryamide, or co-monomers thereof.

According to a particular aspect, the one or more target molecule may be any suitable target molecule. The target molecule may also comprise homologous molecules, homologs, of the target molecule which the MIP sensor comprising the MIP made from the method of the present invention is going to be used for detecting and/or quantifying. Homologs of the target molecules may include molecules that are similar to the target molecule in various attributes such as, but not limited to, size, electrostatic potentials, electronegativity, charge density, chemical bonding potential, and molecules that have similar shapes to the target molecule. Homologs may include isomers and stereoisomers of the target molecule. In particular, the one or more target molecule may comprise a biotoxin, a heavy metal ion or a combination thereof.

The biotoxin may be any suitable biotoxin. For the purposes of the present invention, a biotoxin may be defined as toxic substance with biological origin. Biotoxins may be poisonous substances produced within living cells or organisms. They come in many forms and are produced in most living organisms such as mycotoxins made by fungi, exotoxin or endotoxin made by bacterial, zootoxins made by animals, and phytotoxins are made by plants. Biotoxins may be in the form of small molecules, peptides, or proteins and may be capable of causing diseases on contact with or absorption by body tissues interacting with biological macromolecules such as enzymes or cellular receptors. For example, the biotoxin may comprise, but is not limited to: endotoxins, ochratoxins, aflatoxins, cytotoxins, neurotoxins, or a combination thereof. In particular, biotoxins may comprise: mycotoxins, originating from fungus, including food-contaminating poisons, for example, ochratoxins found in beverages such as beer and wine; aflatoxins found in peanuts, peanut oil, and maize; citrinin found in infected wheat, rice, corn, barley, oats, and rye; ergot alkaloids found in infected cereals and bread made from them; patulin found in mouldy fruits and vegetables, in particular rotting apples and figs; and fusarium toxins found in infected cereals, including fumonisins, trichothecenes, zearalenone, beauvercin, enniatins, butenolide, equisetin, and fusarins; cytotoxins, which are toxic at the level of individual cells, including Ricin from castor beans, apitoxin from honey bees, T-2 mycotoxin from certain toxic mushrooms, and peanut agglutinin from peanuts; neurotoxins, which affect the nervous systems of animals, including tetrodotoxin - sodium channel inhibitor; chlorotoxin - chloride channel inhibitor; conotoxin - calcium channel inhibitor, botulinum toxin - inhibitor of synaptic vesicle release; bungarotoxin - receptor inhibitor; and 1-pentyl-3-

(l-naphthoyl)indole (JWH-018) - receptor agonist; cyanotoxins, produced by bacteria called cyanobacteria (also known as blue algae), such as microcystins, nodularins, anatoxin-a, cylindrospermopsins, Lyngbyatoxin-a, saxitoxin, endotoxins (lipopolysacchrides), aplysiatoxins, and b-methylamino-L-alanine (BMAA); dinotoxins, produced by dinoflagellates, such as saxitoxins, gonyautoxins, and yessotoxins; and myotoxins, small, basic peptides found in snake venoms and lizard venoms, such as crotamine. The heavy metal ion may be any suitable heavy metal ion. Some heavy metals such as lead, cadmium, mercury, and the metalloid arsenic, have serious biotoxic effects, which may damage the central nervous system, cardiovascular system, gastrointestinal system, lungs, kidneys, liver, endocrine glands and bones of living organisms. According to a particular aspect, the heavy metal ion may comprise at least one of, but not limited to, ions of: Zn, Cu, Pb, Hg, As, Cd, Cr, Fe, Co, Ni, Mn, Au, Ag, Mg, Ca, Al, Na, K.

The first solvent may be any suitable solvent for the purposes of the present invention. According to a particular aspect, the first solvent may be water or a non-aqueous solvent. The first solvent may be an organic solvent. Examples of the first solvent include, but is not limited to, water, acetonitrile, or a combination thereof. In particular, the first solvent may be acetonitrile.

The MIP solution may further comprise cross-linkers, polymerisation initiators or a combination thereof. The cross-linker may be any suitable cross-linker. For example, the cross-linker may comprise, but is not limited to, ethyleneglycoldimethacrylate (EGDMA), trimethylolpropane tri methacrylate (TRIM), divinylbenzene (DVB), pentaerythritol triacrylate (PETRA), or a combination thereof. In particular, the cross linker may be TRIM.

The polymerisation initiator may be any suitable initiator. For example, the polymerisation initiator may comprise, but is not limited to, azobisisobutyronitrile, 1 ,T~ aåobis(cyclohexanecarbonitriie), benzoyl peroxide, dicumyl peroxide, persulfates, or a combination thereof. In particular, the initiator may be azobisisobutyronitrile.

The MIP solution may further comprise a porogen. The porogen may be any suitable porogen. For example, the porogen may be, but not limited to, methanol. The preparing may comprise mixing the monomer host, the anchoring group-containing monomer, the one or more target molecules and the first solvent. According to a particular aspect, various orders of addition and mixing of the monomer host, anchoring group-containing monomer, target molecule and first solvent may be used. The amount of monomer host, anchoring group-containing monomer, target molecule and first solvent added to the MIP solution may depend on the monomer host, anchoring group- containing monomer, target molecule and first solvent being used.

According to a particular aspect, the method may further comprise binding the one or more target molecules on a support prior to the preparing. For example, a support may be functionalised such that the target molecule non-covalently or covalently binds to the functionalised support. In particular, a support may be functionalised by immobilising silanes on the surface of the support. Accordingly, the target molecule may bind to the silane functional group.

The polymerising may be under suitable conditions. For example, the conditions may comprise polymerising the MIP solution under ultraviolet (UV) light for a pre-determined period of time. The pre-determined period of time may be 1-60 minutes. In particular, the pre-determined period of time may be 3-45 minutes, 5-40 minutes, 10-30 minutes. Even more in particular, the pre-determined period of time may be 3-10 minutes.

According to a particular aspect, the polymerising may comprise heating at a pre determined temperature. The pre-determined temperature of the heating may be 50- 70°C. In particular, the pre-determined temperature may be 60-70°C. Even more in particular, the pre-determined temperature may be about 70°C. The heating may be for a suitable period of time. The period of time may be 60-600 s, 90-540 s, 120-480 s, 150-450 s, 180-420 s, 210-390 s, 240-360 s, 270-330 s. In particular, the period of time may be 120-180 s. The removing may be by any suitable means and under suitable conditions. When the target molecule is removed, it may leave behind a MIP with imprinted molecular cavities complementary in shape and functionality to the target molecule, which can rebind, in the cavities, a target identical to the original target molecule. The imprinted molecular cavities may be on the surface of the MIP and/or inside of the MIP. According to a particular aspect, the removing may comprise extracting the one or more target molecules from the MIP using a second solvent, wherein the one or more target molecules may be soluble in the second solvent. For example, the extracting may comprise soaking the MIP in the second solvent for a pre-determined period of time. Alternatively, the target molecule may be evaporated from the MIP film if the second solvent has a lower boiling point than the target molecule.

The second solvent may be any suitable solvent. The second solvent may be selected such that the monomer host is insoluble in the second solvent and the target molecules are soluble in the second solvent, thereby facilitating the removal of the target molecules. For example, the second solvent may comprise an alcohol, a liquid organic acid, a surfactant, Dl water, pH buffer, phosphate-buffered saline, or the like. In particular, the second solvent may comprise a solvent selected from the group consisting of, but not limited to: methanol, dilute aqueous acetic acid, sodium dodecyl sulphate, Dl water, pH buffer, phosphate-buffered saline, and a combination thereof.

Once the target molecules have been removed from the MIP, the MIP comprising the anchoring group is formed. The MIP comprising the anchoring group may be rinsed and dried. The rinsing may be by using any suitable solvent. For example, the rinsing may be by using deionised water. The rinsing is to remove any impurities and unpolymerised monomers.

The drying following the rinsing may be performed under any suitable conditions. For example, the drying may comprise air-drying or nitrogen-drying. The drying may be at a suitable temperature, such as £ 60°C. In particular, the drying may be at room temperature.

According to a particular aspect, the method may further comprise forming a MIP sensor by covalently immobilizing the MIP comprising the anchoring group on a sensing substrate surface comprising a coupling group. In particular, the sensing substrate surface may be functionalised by the coupling group. The coupling group may be any suitable coupling group. For example, the coupling group may comprise, but is not limited to: an amino group, a carboxyl group, or a combination thereof.

In particular, the anchoring group of the MIP comprising the anchoring group may covalently bond with the coupling group of the sensing substrate surface comprising the coupling group. The anchoring group of the MIP comprising the anchoring group provide binding sites for the coupling group of the sensing substrate surface comprising the coupling group, thereby enabling the strong bonds to be formed between the MIP and the substrate surface. In this way, there is strong adhesion of the MIP to the sensing substrate, thereby minimising the risk of the MIP peeling off the surface of the sensing substrate and improving the stability of the MIP sensor.

Figure 1 provides some examples of how anchoring groups of the MIP comprising the anchoring group may covalently bond with the coupling group of the sensing substrate surface comprising the coupling group. In particular, the bonding between the anchoring group and the coupling group may require addition of further reagents. As can be seen from Figure 1 , the succinimide ester group of the anchoring group reacts with the -NH ₂ of the coupling group to form an amide bond, the O-acylisourea group of the anchoring group reacts with the -OH of the coupling group to form an ester bond, the azide group of the anchoring group reacts with the alkyne of the coupling group to have a [3+2] cycloaddition, the azide of the anchoring group reacts with acetyl chloride of the coupling group, carboxylic acid of the anchoring group reacts with -NH ₂ of the coupling group through addition of EDC-NHS to form an amide bond, and thiol group of the anchoring group reacts with carboxylic acid of the coupling group through addition of EDC-NHS to form a thiol ester bond.

The sensing substrate may be any suitable sensing substrate for the purposes of the present invention. In particular, the sensing substrate may be selected such that the sensing substrate is suitable for being able to indicate the change in a measureable property of the Ml P during the detection or target molecules. For example, the sensing substrate may be, but not limited to: a coated or uncoated quartz crystal substrate, a metal-based substrate, a glass substrate, a plastic substrate, a silicon substrate, and the like. In particular, the sensing substrate may be, but not limited to, a gold-coated quartz crystal substrate, silver coated quartz crystal, an oxide-based conductive glass substrate.

According to a particular aspect, the sensing substrate may be a quartz crystal substrate. The quartz crystal substrate may be coated, for example with silver, gold, or the like. The quartz crystal may be suitable for quartz crystal microbalance. Alternatively, the sensing substrate may be a metal-based substrate. The metal-based substrate may be suitable for surface plasmonic resonance (SPR). Another example of a suitable sensing substrate may be indium tin oxide (ITO) or fluoride-doped tin oxide (FTO) conductive glass substrate suitable for electrochemical sensing systems.

According to a particular aspect, the sensing substrate surface onto which the MIP comprising the anchoring group is provided may be cleaned prior to the forming of the sensor. The cleaning of the sensing substrate surface may be by any suitable method using any suitable solvent. For example, the solvent may be methanol.

The method may further comprise rinsing and drying the MIP sensor formed. The rinsing and drying may be under any suitable conditions. The rinsing may be with a suitable solvent. For example, the solvent may be water. The drying may be at a suitable temperature. For example, the drying may be at a temperature of £ 60°C. In particular, the drying may be at 15-60°C, 20-55°C, 25-50°C, 30-45°C, 35-40°C. Even more in particular, the drying may be at 20-25°C. According to a particular embodiment, the drying may be by air drying or nitrogen drying. Figure 2 shows a schematic representation of the method of the present invention. The method as shown in Figure 2 begins with the preparation of the target molecules, which in the case of Figure 2 is a biotoxin. In particular, functional silanes are immobilised onto a solid support. Biotoxin target molecules are then bound, either covalently, non- covalently or a combination of both, to the silane functional groups on the support to form the biotoxin target molecules for use in the method of the invention. Ionic liquid monomers, acrylates with appropriate functional groups, NHS- and/or EDC-containing monomers, cross-linkers, polymerisation initiators, and optionally porogens, are added to the biotoxin target molecules and mixed in a first solvent to form a mixture and the mixture is sufficiently agitated. The mixture is then allowed to polymerise to form a MIP.

The MIP formed is then purified by washing the MIP to remove any unpolymerised monomers to prevent further polymerisation which may affect the properties of the MIP. Following the purification, the MIP comprising the anchoring group is eluted from the support. The support may still carry the immobilized functional silane with the target molecule bonded. The substrate may be re-used in subsequent preparations of the MIP. Figure 2 also shows the preparation of a biotoxin sensor comprising the obtained MIP comprising the anchoring group. A sensing substrate surface is functionalised with a coupling group. The coupling group may comprise functional amine and/or carboxyl groups. A dispersion of MIP comprising the anchoring group is provided on the sensing substrate surface and the dispersion is allowed to react. In particular, the anchoring group of the MIP comprising the anchoring group and the coupling group of the sensing substrate surface react to covalently immobilise the MIP comprising the anchoring group on the sensing substrate surface to form a MIP sensor.

Another example of a preparation of a endotoxin sensor is as shown in the schematic representation of Figure 3. First, dodecyl and methoxy silane are functionalised onto a solid support. The solid support may be S1O2 particles. The functionalised S1O2 particles then non-covalently or covalently bind to endotoxin as target molecules. In particular, the silane functional groups on the support bind to the endotoxin through interactions such as hydrophobic, electrostatic, hydrogen bonding, or covalent bonding interaction. A polymerizable aqueous or non-aqueous mixture solution comprising ionic liquid monomers such as 1-allyl-3-methylimidazolium dicyanamide, NHS-containing or EDC- containing monomers such as acrylate-PEG2K-NHS, acrylate or acrylamide monomers with appropriate functional groups such as 2-hydroxyethyl acrylate, cross-linkers such as trimethylolpropane trimethacrylate, polymerisation initiators such as azobisisobutyronitrile, solvents such as water and acetonitrile and porogens such as further solvents, or soluble non-polymerizable substances are added to the bound target molecules. The mixture is sufficiently agitated and allowed to polymerize to form MIP on the support.

Subsequently, the MIP is washed to purify the MIP. This step is to remove monomer residues, low-affinity oligomers, and others. The washing may be using a suitable solvent such as acetonitrile and is carried out at low temperature such as 0°C. The MIP is then eluted with solvents such as acetonitrile at elevated temperature of about 50-60°C from the support, which still carries the immobilized functional silane with bonded endotoxin. The support may be re-used. The solution comprising the MIP may be kept in the solvent or be concentrated by evaporation, or dried to get a dry sample. A substrate surface is prepared by functionalising with functional amine such as propylamine or carboxyl groups. The MIP nanoparticle dispersion obtained is then made to react with the amino- or carboxyl-functionalized substrate surface to immobilize the MIP particles on the substrate surface. The by-product is NHS. In particular, the MIP particles are MIP comprising an anchoring group in which the anchoring group covalently bonds with the functional group of the functionalised substrate surface.

According to a second aspect, there is provided a molecularly imprinted polymer (MIP) sensor comprising a MIP made from the method of the first aspect. In particular, the MIP sensor may be any suitable molecularly imprinted polymer sensor suitable for detecting and/or quantifying the presence of target molecules such as, but not limited to, biotoxins and heavy metal ions.

The MIP sensor may be a device that simultaneously monitors one or more target molecules. According to a particular aspect, the MIP sensor may be read visually. According to another particular aspect, the MIP sensor may be coupled to electronics that read the MIP on the sensing substrate and report wirelessly to a central facility. Alternatively, the MIP sensor may be incorporated into a portable and/or handheld device for measurement and processing onsite. For example, the MIP sensor may comprise one or more MIP, wherein each MIP within the MIP sensor, such as a test strip, may be specific to a single target molecule.

According to a third aspect, there is provided a molecularly imprinted polymer (MIP) comprising an anchoring group, the anchoring group comprising at least one of: cyclic imide, O-acylisourea, azide, carboxylic acid, amine, hydroxyl, or thiol.

The cyclic imide group and the O-acylisourea group may be as described above.

According to a particular aspect, the MIP comprising the anchoring group may be used comprised in a MIP sensor for sensing and detecting a particular target molecule. The target molecule may be, but not limited to, a biotoxin and/or a heavy metal ion. The MIP comprising the anchoring group may be formed from the method of the first aspect described above.

According to a fourth aspect, there is provided a molecularly imprinted polymer (MIP) sensor comprising a MIP according to the third aspect. The sensor may have a low limit of detection, and therefore high sensitivity.

The MIP sensor may be any suitable molecularly imprinted polymer sensor suitable for detecting and/or quantifying the presence of target molecules such as, but not limited to, biotoxins and heavy metal ions.

The MIP sensor may be a device that simultaneously monitors one or more target molecules. According to a particular aspect, the MIP sensor may be read visually. According to another particular aspect, the MIP sensor may be coupled to electronics that read the MIP on the sensing substrate and report wirelessly to a central facility. Alternatively, the MIP sensor may be incorporated into a portable and/or handheld device for measurement and processing onsite. For example, the MIP sensor may comprise one or more MIP, wherein each MIP within the MIP sensor, such as a test strip, may be specific to a single target molecule.

The MIP sensor according to any aspect of the present invention may be stable over a wide range of temperature. In particular, the sensor may be stable for storage from -80- 50°C without the MIP peeling off the substrate surface. According to another aspect of the present invention, there is provided a method for detecting and/or quantifying a target molecule using the molecularly imprinted polymer sensor described above. The method comprises: exposing the MIP sensor to a sample of fluid containing or thought to contain the target molecule, thereby allowing the target molecule, if present, to be received within cavities of the sensor; and

detecting the presence of and/or quantifying the amount of the target molecule bound to the cavities of the sensor using electrochemical, acoustical, spectroscopic, optical or indirect chromatographic techniques, wherein the target molecule is, a biotoxin, a heavy metal ion, or a combination thereof.

According to a particular aspect, the detecting using electrochemical, acoustical, spectroscopic or optical techniques may comprise measuring a change of a measurable property of the MIP, wherein a change comes about when the target molecule is detected in the MIP. The change of the measureable property may be a change in capacitance, resistance, colour, mass, resonance frequency, surface plasmon resonance shift or the like.

The present invention will be exemplified by the following non-limiting examples.

Example 1 : Preparation of Endotoxin Molecularly Imprinted Polymer (MIP) An endotoxin MIP was prepared as follows. A schematic representation of the process for preparing the endotoxin MIP is as shown in Figure 4.

Preparation of substrate

Firstly, 10 g of acid washed glass beads measuring 425-600 pm were activated with 50 ml_ of 2M of sodium hydroxide (NaOH) for 15 minutes and thereafter, rinsed with deionized water to remove any excess NaOH. The glass beads were dried at 80°C for 3 hours.

The dried beads were then incubated in 4 ml_ of 2 vol. % 3- aminopropyltrimethoxysilane dry toluene solution for 24 h for silanization, to obtain amine (-NH2-) bearing beads. Thereafter, the glass beads are decanted into a Buchner funnel with suitable filter paper disc and rinsed with deionized water, followed by drying under vacuum.

The glass beads were then incubated with 10 ml_ of 100 ppm of PA-10 endotoxin template dissolved in 10 mM phosphate-buffered saline (PBS) at pH 7.2 for more than 5 h to obtain template-immobilised glass beads. The template-immobilised glass beads were filtered and rinsed with water, then rinsed with acetone, dried under vacuum, and transferred into a 200 ml_ flat bottomed glass vessel.

Preparation of pre-polymerization mixture

Pre-polymerization mixture was prepared by mixing 20 wt % acrylic acid, 10 wt % 1- allyl-3-methyltrimidazolium bromide, 2 wt % acrylic acid N-hydroxysuccinimide ester, 2 wt % azobisisobutyronitrile (AIBN) and 66 wt % ethylene glycol dimethacrylate (EGDMA) into acetonitrile (ACN), in a solute:solvent ratio of 5:95.

The pre-polymerization mixture was purged with nitrogen gas for at least 20 minutes to remove oxygen via a plastic pipette positioned in the bulk of the solution. Polymerization of MIP on substrate

10 mL of the pre-polymerization mixture was poured onto the glass beads in the flat bottomed glass vessel, and the vessel placed under UV light for 1-60 minutes under a continuous stream of N2.

Washing After polymerization, the entire contents of the vessel were transferred into a solid phase extraction (SPE) cartridge fitted with a 20-pm porosity frit, and the outlet closed. The SPE cartridge was placed in an ice bath (0°C) for 10 minutes and the supernatant drained by piston or vacuum. The non-polymerized monomers and low-affinity nano sized MIPs were removed by 10 repeated washings with toluene at 0°C. Elution

The SPE cartridge was placed into a water bath at 60°C and fresh toluene (pre-warmed to 60°C) added, and the mixture incubated for 15 minutes. The high affinity nano-sized MIPs were eluted by using a piston or a pump. Fresh acetonitrile (pre-warmed to about 60°C) was added and the SPE cartridge placed into the water bath at 60°C for 2 minutes. The high affinity nano-sized MIPs were collected. This was repeated until about 100 ml_ of the nano-sized MIPs solution had been collected.

The nano-sized MIP solution was cooled. 5 pl_ of the prepared MIP solution was added onto a 3-aminopropyltrimethoxysilane pre-coated quartz crystal with 10 mm-diameter gold (Au) surface, and dried completely before evaluation.

Evaluation A calibration curve of an endotoxin PA-10 MIP was plotted as shown in Figure 5. The limit of detection (LOD) was calculated to be 0.01 Endotoxin Unit (EU), and the lowest tested concentration was 0.005 EU. Thus, in view of the low limit of detection, the MIP sensor comprising the MIP prepared has a high sensitivity.

Example 2: Preparation of Zn(ll) ion (Zn ²⁺ ) Molecularly Imprinted Polymer A Zn(ll) ion (Zn ²⁺ ) MIP was prepared as follows.

Preparation of substrate

Firstly, 10 g of acid washed glass beads measuring 425-600 pm were activated with 50 ml_ of 2M of NaOH for 15 minutes and thereafter, rinsed with deionized water to remove any excess NaOH. The glass beads were dried at 80°C for 3 hours. The dried beads were then incubated in 4 ml_ of 2 vol. % methacrylotrimethoxysilane dry toluene solution for 24 hours for silanization, to obtain -NH2- bearing beads.

Thereafter, the glass beads are decanted into a Buchner funnel with suitable filter paper disc and rinsed with deionized water, followed by drying under vacuum.

Polymerization of MIP on substrate The glass beads were then added to 10 mL of ACN/methanol solution in a 3:1 ratio, with 2 wt. % 2-acryamido-2-methyl-1-propanesulfonic acid, 5 wt. % acrylic acid, 1 wt. % AIBN, and 2 wt. % zinc acetate. This allows immobilization of the coordinating agent onto the beads, thus allowing zinc ions to be bound through coordination.

The solution was placed under UV light and under N ₂ atmosphere for 1-60 minutes.

Subsequently, 0.052 g of methacrylic acid, 0.039 g of 2-hydroxyethyl methacrylate, 0.02 g of acrylic acid N-hydroxysuccinimide ester, 0.595 g of ethylene glycol dimethacrylate, and 0.14 g of AIBN were added and mixed under N ₂ atmosphere.

Thereafter, 30 ml_ of ACN was added under N ₂ atmosphere, stirred for 30 minutes, and the solution heated and maintained in a water bath at 70°C for 3 hours.

Washing After polymerization, the entire contents of the vessel beaker were transferred into a SPE cartridge fitted with a 20-pm porosity frit, and the outlet closed. The SPE cartridge was placed in an ice bath (0°C) for 10 minutes and the supernatant drained by piston or vacuum. The non-polymerized monomers and low-affinity nano-sized MIPs were removed by 10 repeated washings with toluene at 0°C. Elution

The SPE cartridge was placed into a water bath at 60°C and fresh toluene (pre warmed to 60°C) added, and the mixture incubated for 15 minutes.

The high affinity nano-sized MIPs were eluted by using a piston or a pump. Fresh acetonitrile (pre-warmed to about 60°C) was added and the SPE cartridge placed into the water bath at 60°C for 2 minutes. The high affinity nano-MIPs were then collected. This was repeated until about 100 ml_ of the nano-sized MIP solution had been collected.

The nano-sized MIP solution was cooled. 5 pl_ of the prepared MIP solution was added onto a 3-aminopropyltrimethoxysilane pre-coated quartz crystal with 10 mm-diameter Au surface, and dried completely before evaluation.

Evaluation Selectivity of the Zn ²⁺ MIP sensor was tested. High selectivity was exhibited towards Zn ²⁺ ions against Cu ²⁺ , Fe ³⁺ , Co ²⁺ , Ni ²⁺ , Ca ²⁺ , Mg ²⁺ , Al ³⁺ and Na ⁺ ions, as shown in Figure 6. LOD of the Zn ²⁺ sensor was determined to be 0.1 parts per billion (ppb).

As shown in the examples above, the prepared MIP-based endotoxin and ion sensors exhibit strong response to the specific analyte endotoxins or ions.

Whilst the foregoing description has described exemplary embodiments, it will be understood by those skilled in the technology concerned that many variations may be made without departing from the present invention.