SE544779C2 - Template-imprinted polymer particles - Google Patents

Abstract

A template-imprinted polymer particle of a size of about 10 to 1000 nm prepared from a nucleated composition, formed spontaneously and instantaneously in absence of surfactant, stabilizer, or dispersant and without extended agitation, and comprising a monomer of formula (I), a process for preparation of the template-imprinted polymer particle, and a method for application of the template-imprinted polymer particle as a molecular recognition element are provided:(I)wherein;(a) X is -CH2-, -0-CH2-CH2-, -0-CH2-CH(CH3)-, or -0-CH(CH3)-CH2-;(b) Y is -0-C(0)-CH=CH2, -0-C(0)-C(CH3)=CH2, -NH-C(0)-CH=CH2,-NH-C(0)-C(CH3)=CH2,-0-CH=CH2, -0-CH2-CH=CH2, -CH=CH2,-CH2-CH=CH2, -0-C(0)-CH2-CH2-SH, -NH-C(0)-CH2-CH2-SH, or SH; (c) Z is -CH2-XP-Y, a hydrogen, a functional group, an alkyl group, or a functionalized alkyl group; and(d) each of 1, m, n, and p is independently a number in the range 0-10.

Description

Molecular imprinting, also referred to as template polymerization, is a method that tailors a polymer to recognize and bind a desired molecule or class of molecules. Recognition sites of pre- deterrnined selectivity are produced in the polymer network during the polymerization of the polymer°s building blocks (monomers) [Kempe, H; Kempe, M. Molecularly imprinted polymers. In “The Power of Functional Resins in Organic Synthesis”, Femando Albericio and Judit Tulla-Puche, Eds. Wiley- VCH, Weinheim, Germany, 2008: l5-44]. A typical molecular imprinting procedure starts with the preparation of a pre-polymerization mixture, comprising a template, functional and cross-linking monomers, and a free-radical initiator, all dissolved in an organic solvent. The functional monomers interact with the template, forrning self-assembled complexes. The main role of the cross-linker is to rigidify the polymer. Functionalized cross-linkers providing crucial interactions with the template are rare but have been reported [Sibrian-Vazquez, M.; Spivak, D. A. J. Am. Chem. Soc. 2004, 126, 7827- 7833] Co-polymerization of monomers and cross-linkers provides a polymer network with precise imprints of the template. After polymerization, the template is extracted from the polymer network, leaving recognition sites capable of selective binding.

Molecularly imprinted polymers (MIPs) have traditionally been prepared as monolithic polymers, which are transforrned into particles by crushing and grinding. The procedure is tedious and typically provides irregularly shaped particles of broad size distributions; fractionation gives particles of the desired size range in relatively low yields. An advantage of the approach is, however, that the interactions between the template and the polymer building blocks, which are essential for the molecular recognition capacity of the final polymer, are maintained and not compromised by the introduction of potentially interfering agents. Such agents, including surfactants, stabilizers, dispersants, and additional liquid phases and co-solvents are commonly used in many classical methods for the production of spherical beads, making the procedures sub-optimal for the preparation of MIPs. In suspension polymerization, water-insoluble monomers are suspended in a continuous water phase by vigorous mixing using stabilizers, for example, polymers or detergents, to stabilize and prevent coalescence of the droplets formed during the agitation. Mixing is continued throughout the full polymerization time period. A Wide range of particle sizes, from sub-micrometer to several millimeters in diameter, can be produced by suspension polymerization. In emulsion polymerization, Water-insoluble monomers and surfactants are dispersed in a continuous Water phase by vigorous stirring, resulting in the forrnation of monomer-containing small micelles and larger droplets of monomers. Polymerization of the monomers in the micelles, using a Water-soluble initiator, provides polymer particles, Which continuously grow by diffusion of monomers from the larger monomer droplets to the micelles until the monomers are depleted in the droplets. Typical sizes of the resulting particles are around 100 nm. In mini-emulsion polymerization, a co-stabilizer is used in addition to the surfactant and high-sheer mixing by, for example, ultrasound is applied. Polymerization using a Water-soluble initiator provides polymer particles in the size range 50-500 nm. In micro-emulsion polymerization, a high concentration of surfactant is used to produce a therrnodynamically stable micro-emulsion of monomer-filled micelles, Which are polymerized after addition of a Water-soluble initiator. The typical resulting particles size in this case is in the range 10-50 nm.

Due to the difficulties in producing MIP particles by the classical methods, several altemative routes to spherical MIP beads have been explored during the past decades. Spherical micro-sized MIP beads have been produced by modified suspension polymerization procedures using either perflurocarbon liquid or mineral oil as the continuous medium, thereby avoiding the use of Water [Mayes and Mosbach, Anal. Chem. 1996, 68, 3769-3774; Kempe and Kempe, Macromol. Rapid Commun. 2004, 25, 3l5-320]. Precipitation polymerization, a method Where particles precipitate as they polymerize from a homogenous highly diluted solution of monomer(s) and cross-linker(s) dissolved in an organic solvent, has been explored for the production of both nano- and micro-sized MIP particles [Wang et al. AngeW. Chem. Int. Ed. 2003, 42, 5336-5338] Key to precipitation polymerization is that the solvent should be a good solvent for the polymer building blocks but should be a poor solvent for the polymer particles to promote precipitation during the polymerization. Acetonitrile is often used as the solvent and the polymerization typically takes place at 60 °C for 24 h. Modified procedures that reduce the reaction time by carrying out the polymerization at the boiling temperature, either under pure reflux conditions or by distilling off some of the solvent, have been reported [Shen et al. RSC Adv., 2016, 6, 81491-81499; Yang et al. Macromolecules 2009, 42, 8739- 8746]. Although the above described methods for the preparation of MIP particles are useful for many applications, there is a need for more facile, less labor-intense, more environmentally friendly, and more energy-efficient methods.

MIPs typically shoW optimal recognition and rebinding in organic solvents. Selective recognition and rebinding in Water has, hoWever, proven to be more difficult. This limitation is problematic since many potential applications of MIPs require that the polymers are functional in aqueous media. Such applications are found, for example, in the medical, biomedical, pharrnaceutical, environmental, and food sectors. There is therefore a large need of methods that provide MIP particles capable of molecular recognition in aqueous media.

A broad range of molecules present in aqueous media are potential targets to be addressed by the molecular imprinting technology. The steroids is one group of molecules that have attracted interest in the scientific community. Molecules classified as steroids are based on a carbon core structure of seventeen carbon atoms arranged in four interconnected rings (three six-membered rings and one five- membered ring). Several steroids contain the intact steroid core, While others contain cleaved, contracted, and/or expanded rings. The steroid carbon core structure is typical for the steroid horrnones. In humans, the steroid horrnones can be divided into corticosteroids (adrenocortical horrnones) and androgens (sex horrnones). The former class comprises the glucocorticoids, exemplified by cortisol and cortisone, and the mineralcorticoids, exemplified by aldosterone. Synthetic glucocorticoid analogs of important pharrnacological roles include prednisone, methylprednisone, and dexamethasone. The androgens include progesterone, testosterone, and estradiol, among others. The human steroid horrnones are either synthesized from cholesterol, a steroid provided by ingested food, or synthesized de nova in the body from acetyl CoA via the mevalonate pathWay. The open-ring steroids include the different vitamin D forms (e. g., ergocalciferol, cholecalciferol, and calcitriol). A number of synthetic compounds, collectively named endocrine disruptors, mimic the horrnones and interfere With the horrnonal systems. Examples of endocrine disruptors With estrogenic effects include alkyl phenols, bisphenol A (BPA), polychlorinated biphenyls (PCB), dichlorodiphenyltrichloroethane (DDT), polybrominated diphenyl ethers, and phthalates.

The steroid horrnones have important physiological roles in the body. For example, the main roles of cortisol are to increase plasma glucose levels by activating gluconeogenesis and to exert immunosuppressive and anti-inflammatory activities. Cortisol is secreted folloWing a circadian rhythm, With high levels in the moming and low levels at midnight. In addition, the hormone is released in response to stress and is therefore a popular stress level biomarker. Abnormal levels of cortisol are indicators of adrenal disorders such as Cushing°s syndrome and Addison°s disease. The monitoring of cortisol, as Well as other steroid horrnones, steroid hormone analogs used as drugs, and endocrine disruptors is of clinical relevance. To this end, selective recognition elements capable of recognizing and binding the substances are highly desirable for the construction of assays and sensors. Such recognition elements may also be useful as carriers for controlled and/or sustained drug administration.

As discussed above, MIPs typically show optimal recognition and rebinding in organic solvents. Previously reported MIPs targeted for steroids have in fact required the recognition and rebinding steps to be carried out in organic solvents. Solvents and solvents mixtures reported in the scientific literature for the rebinding steps include tetrahydrofuran-heptane-acetic acid [Ramström et al. Chemistry & Biology 1996, 3, 47l-477], acetonitrile-Water (337) [Fitzhenry et al. Microchim Acta 2013, 180, 1421- 1431], methanol-ethyl acetate (9:1) [Liu et al., J. Colloid Interface Science 2017, 504, 124-133], chloroforrn-hexane (431) [Murase et al., J. Mater. Chem. B 2016, 4, 1770-1777; Murase et al., Macromol. Chem. Phys. 2015, 1396-1404], chloroforrn-acetic acid (199:1) [Baggiani et al., Talanta 2000, 51, 71-75], acetonitrile [Liu et al., J. Colloid Interface Science 2017, 504, 124-133; Baggiani et al., J. Am. Chem. Soc. 2012, 134, 1513-1518; Sreenivasan & Sivakumar, J. Appl. Polym. Science, 1999, 71, 1823-1826; Sreenivasan, J. Polym. Research 2001, 8, 197-200], and dichloromethane [Sreenivasan, J. Appl. Polym. Science 2001, 82, 889-893].

A review of the patent literature shows a number of patents mentioning molecular imprinting of cortisol. None of the identified patents describe, however, a MIP that can rebind cortisol in water. The first example, US 6,833,274 B2 (“Cortisol sensor”), describes a cortisol sensor based on a cortisol- imprinted polymer. The patent does not detail a procedure for the preparation of said cortisol-imprinted polymer, but tells that the MIP can be prepared “by adapting for use herein any of a wide range of known methods including those described in U.S. Pat. Nos. 5,110,883; 5,321,102; 5,372,719; 5,310,648; 5,208,155; 5,015,576; 4,935,365; 4,960,762; 4,532,232; 4,415,655; and 4,406,792, as well as, U.S.patent application Ser.No.09/300,867, “Imprinted Polymers as Protecting Groups for Regioselective Modification of Polyfunctional Substrates C. Alexander et al., J. Amer:Chem.Soc., 1999, 121, 6640-6651, and “Chromatographic characterization of a molecular imprinted polymer binding cortisol” Baggiani et al., Talanta, 2000, 51, 71-75.” A critical review of the cited references shows that none of them provide a MIP capable of binding cortisol in water or provide methods that can be adapted for its preparation: US 5,110,883 (“Process for the production of high molecular weight copolymers of diallyl ammonium monomers and acrylamide monomers in solution”) does not describe any MIP or suitable methods for its preparation. US 5,321,102 (“Molecular engineering of porous silica using aryl templates”) uses a series of organic templates, none of which are cortisol, for the purpose of manipulating the porosity of silica. US 5,372,719 (“Molecular imaging”) provides surfaces selective for macromolecules (i.e., proteins and peptides); cortisol is not included. US 5,310,648 (“Composition of matter comprising an imprinted matrix exhibiting selective binding interactions through chelated metals”) provides an imprinted matrix which exhibits selective binding interactions through metal chelates with templates exemplified by bis-imidazoles and proteins; cortisol does not contain any functional group suitable for metal chelating and is not mentioned in the patent. US 5,208,155 (“D- amino acid oxidase and method for isolation thereof”) describes an enzyme and its isolation; no MIP is described. US 5,015,576 (“Macroporous particles for cell cultivation or chromatography”) and US 4,935,365 (“Macroporous particles for cell cultivation or chromatography”) describe particles for cell cultivation and chromatography; none of the particles are molecularly imprinted. US 4,960,762 (“Chiral two-phase system and method for resolution of racemic mixtures and separation of diastereomers”) describes a system comprising two immiscible liquid phases and one or more enantioselectively binding chiral component, the latter exemplified by cyclodextrin, cellulose, and chiral amino acids; no MIPs are described in the patent. US 4,532,232 (“Lectin-containing separation agent”) describes a separation agent composed of a Water insoluble solid substance With a covalently bound lectin; no MIP or methods for its preparation are described. US 4,415,655 (“Electrophoretic separation of isoenzymes utilizing a stable polyacrylamide system”) describes a method for separating isoenzymes by polyacrylamide gel electrophoresis; no MIP or methods for its preparation are described. US 4,406,792 (“Separation agent”) describes a chromatographic separation agent based on silica/silicate With covalently bound dihydroxyboryl groups; no MIP is described in the patent. The paper “Imprinted Polymers as Protecting Groups for Regioselective Modification of Polyfunctional Substrates. C. Alexander et al., J. Amer:Chem.Soc. 1999, 121, 6640-6651” describes covalent molecular imprinting of androst-5-ene- 3beta,17beta-diol and structural analogs using polymerizable and cleavable templates. Rebinding Was demonstrated in chloroforrn, tetrahydrofuran, and ethyl acetate; no binding in aqueous media Was demonstrated. The paper “Chromatographic characterization of a molecular imprinted polymer binding cortisol. Baggiani et al.,Talanta 2000, 51, 71-75” provides a MIP imprinted With cortisol. Rebinding Was carried out in chloroforrn-acetic acid (199: 1); no recognition in Water Was demonstrated.

The second example, patent application WO2013046826A1 (“Molecular template and method for producing same”), describes cortisol-imprinted monolithic polymers made from itaconic acid, styrene, and divinylbenzene, Wherein cortisol is provided as a template either in its free form or in a polymerizable form. Rebinding Was carried out in acetonitrile and chloroforrn-hexane (431), respectively. The third example, patent application WO2017046836A1 (“Chemical analysis device”), provides core-shell magnetic MIP particles imprinted With cortisol that require rebinding in 50% of acetonitrile. The fourth example, patent US8241575B2 (”Molecularly Imprinted Polymer Sensor Device”), describes cortisol-binding MIPs prepared by RAFT polymerization. Analysis of cortisol in biological fluids is claimed but no examples of binding in a biological fluid is included; the only provided example of rebinding Was carried out in methanol. The f1fth example, SE509863 (“Material för selektering av substanser ur kombinatoriska bibliotek”), describes MIPs imprinted With 11-alpha- progesterone and corticosterone, respectively, applied as HPLC stationary phases for the separation of a mixture of steroids, Wherein cortisol 21-acetate Was included. The separation used dichloromethane- acetic acid (99.9:0.1) and dichloromethane-acetic acid (99.5:0.5), respectively, as mobile phases. The sixth example, WO1997038015A1 (”Artif1cial antibodies to corticosteroids prepared by molecular imprinting”), describes MIPs imprinted With cortisol. Optimal binding performance Was achieved in mixtures of tetrahydrofuran and n-heptane; binding in aqueous media is not covered in the patent.

Summarizing, previous patents and papers on cortisol-imprinted polymers require rebinding in organic solvents. Since cortisol presents itself in biological samples and aqueous matrices, previous MIPs are of limited practical application for direct binding of the molecule. The situation is similar for other steroids as Well as many other substances. Hence, novel MIPs capable of molecular recognition and selective rebinding in aqueous media are needed. This invention addresses this need.

SUMMARY OF THE INVENTION The invention provides in one embodiment synthetic polymer particles With a molecular recognition capacity for a target molecule or class of molecules. Said molecular recognition capacity is created by molecular imprinting, Which means that the polymer particles are molecularly imprinted With a template, thereby making them, after removal of said template, capable of recognizing and binding said target molecule or class of target molecules. The template used in the invention is a molecule that is more hydrophobic than Water. In one embodiment, the template is identical With the target molecule. In another embodiment, the template is a derivative of the target molecule. In one embodiment, the template is derivatized With a polymerizable group. In another embodiment, the template is an analog of the target molecule. The template-imprinted polymer particle of the invention is comprised of a polymerized monomer of the following forrnula (I) and is prepared from a nucleated composition comprising said monomer: (1) å. --------- --------- ~ Säg ------------- -- Y l MX» ------- *f Wherein; (a) X is -CH2-, -O-CH2-CH2-, -O-CH2-CH(CH3)-, or -O-CH(CH3)-CH2-; (b) Y is -O-C(O)-CH=CH2, -O-C(O)-C(CH3)=CH2, -NH-C(O)-CH=CHz, -NH-C(O)-C(CH3)=CHz, -O-CH=CHz, -O-CH2-CH=CH2, -CH=CHz, -CHz-CH=CHz, -O-C(O)-CHz-CHz-SH, -NH-C(O)-CHz-CHz-SH, or SH; (c) Z is -CHg-Xp-Y, a hydrogen, a functional group, an alkyl group, or a functionalized alkyl group; and (d) each of l, m, n, and p is independently a number in the range 0- In one embodiment, the polymer particle is further comprised of a co-polymerized monomer.

In another embodiment, the invention provides a process for preparing said template-imprinted polymer particle comprising the steps: (a) selecting a template capable of providing a molecular recognition of a target molecule; (b) dissolving the monomers(s) in a Water-miscible solvent, forrning a solution comprising said monomer(s); (c) interfacing said solution and a Water phase to form a mixture in Which spontaneous and instantaneous formation of nucleated droplets comprising said monomer(s) take place; (d) providing the template either in said solution or in said Water phase; and (e) polymerizing said monomer(s) by a polymerization reaction.

The invention provides, spontaneously and instantaneously, droplets of monomers through an energy- efficient and environmentally friendly procedure. The droplets nucleate When a preferred proportion of a solution, comprising monomers dissolved in a Water-miscible solvent, and Water are interfaced. The preferred proportions are estimated from experimentally deterrnined phase diagrams. Ethanol is particularly suitable to be used as the Water-miscible solvent. Only a brief initial mixing is required to interface the tWo liquids for the formation of the droplets. No extended mixing/agitation and no stabilizer is needed for the formation of the droplets When the composition is located in the nucleation region of the phase diagram. The droplets remain stable in the mixture during time periods that are suff1ciently long to allow for a polymerization of the monomer(s) to form solid polymer particles. The recognition sites of the polymer particles are created by template molecules being present during the polymerization. The polymer particles are suitable to be used as molecular recognition elements or as synthetic antibodies in applications Where selective or specific binding is required.

BRIEF DESCRIPTION OF THE DRAWINGS Figure l. (a) Right triangle phase diagram of the temary system PETRA-ethanol-Water before polymerization. The region providing nucleated droplets, appropriate for polymerization to particles, is located beloW the dotted line. (b) Right triangle phase diagram after polymerization.

Figure 2. Influence of interfacing method on the particle size distribution of poly(PETRA). Interfacing Was carried out by (a) manual shaking (20 inversions); (b) stirring using an overhead stirrer equipped With a radial flow impeller (700 rpm, 3 min); (c) homogenization using a homogenizer equipped With a dispersing element (8 000 rpm, l min); and (d) homogenization using an ultrasonic homogenizer equipped With an ultrasonic probe (6 >< 10 s, 20 W). DLS analysis (intensity based, n=30) Was carried out on particles dissolved in Water.

Figure 3. Influence of polymerization conditions on the particle size distribution of poly(PETRA). Polymerization Was carried out by subjecting the samples to (a) heat (60 °C for 6 h); or (b) UV light (350 nm for 2 h). DLS analysis (intensity based, n=l0) Was carried out on particles dissolved in Water.

Figure 4. Particle size distribution of (a) cortisol-imprinted polymer particles no. l2 (MIP l2) and (b) non-imprinted polymer particles no. l2 (NIP l2). DLS analysis (intensity based, n=l0) Was carried out in Water.

Figure 5. One-site Langmuir binding isotherrns showing the binding of cortisol to cortisol- imprinted polymer particles no. 12 (MIP 12; filled Squares, solid line) and corresponding non-imprinted polymer particles no. 12 (NIP 12; open circles, dotted line). The insert shows the Scatchard plot for the binding of cortisol to MIP l DETAILED DESCRIPTION Several embodiments of the present invention will be described in more detail below with reference to the accompanying drawings in order for those skilled in the art to be able to carry out the invention. The invention may, however, be embodied in many different forrns and should not be construed as limited to the embodiments set forth herein. Furthermore, the terrninology used in the detailed description of the particular embodiments illustrated in the accompanying drawings is not intended to be limiting of the invention.

The invention provides in one embodiment molecularly imprinted polymer particles with recognition sites for a chosen target molecule. The target molecule of the invention can be selected from a wide range of molecules. Examples of target molecules include, but are not limited to, amino acids, peptides, peptide mimetics, proteins, enzymes, antibodies, carbohydrates, nucleotides, nucleosides, oligonucleotides, peptide nucleic acids, lipids, triglycerides, fatty acids, polycyclic aromatic hydrocarbons, macrocyclic organic compounds, fats, waxes, steroids, flavonoids, alkaloids, prostaglandins, vitamins, cofactors, pesticides, herbicides, antimicrobials, metabolites, nutrients, xenobiotics, horrnones, toxins, neurotransmitters, signal molecules, cytokines, adhesion molecules, growth factors, biomarkers, drugs, and analogs, residues, or derivatives thereof.

The molecular recognition sites arise from template molecules being present during the polymerization. In one embodiment, the template is identical to the target molecule. To be useful as a template in the invention, the molecule should be less soluble in water than water itself. In one embodiment of the invention, the template is a molecule with an interrnediate solubility in water. In a preferred embodiment of the invention, the template is a molecule with a low solubility in water. Water solubility is deterrnined by the degree of polarity and hydrophilicity of a molecule. Polar and hydrophilic molecules have, in general, a high solubility in water whereas nonpolar and hydrophobic molecules have a low solubility. Hydrophobic molecules can be defined as molecules being more hydrophobic than water on a hydrophobicity-hydrophilicity scale. The degree of hydrophobicity of a molecule can be estimated by the partition coeff1cient (P) or log P in an octanol-water mixture. In a preferred embodiment of the invention, the template is a molecule that is more hydrophobic than water. In another preferred embodiment, a template-monomer conjugate that is more hydrophobic than water is used. In another preferred embodiment, the template is a target molecule derivatized with a hydrophobic molecule, making the adduct more hydrophobic than water.

In one preferred embodiment of the invention the target molecule is a steroid. Steroids are, in general, sufficiently hydrophobic to be used directly as the template. In one preferred embodiment, cortisol (also named hydrocortisone) is the target molecule. With a log P of 1.76 [DörWald, Lead Optimization for Medicinal Chemists: Pharmacokinetic Properties of Functional Groups and Organic Compounds. John Wiley & Sons, 2012], no derivatization is required to engineer the molecule to become sufficiently hydrophobic; cortisol can be used directly as the template. In another preferred embodiment, the template is beta-estradiol (log P = 3.29). In another preferred embodiment, the template is cholesterol (log P = 7.02). Other examples of steroids suited to be used as the template in the invention include, but are not limited to, cortisone, cortisone acetate, hydrocortisone buteprate, hydrocortisone butyrate, hydrocortisone Valerate, pregnenolone, l7-alpha-hydroxypregnenolone, androstenedione, dehydroepiandrosterone, androstenediol, progesterone, l7-alpha-hydroxy- progesterone, testosterone, ll-deoxy-corticosterone, ll-deoxycortisol, corticosterone, aldosterone, estrone, estriol, prednisolone, prednisone, methylprednisolone, levonorgestrel, gestodene, desogestrel, etonogestrel, norethindrone, cloprednol, cyproterone acetate, deflazacort, elcometrine, medrogestone, spironolactone, alphaxalone, dutasteride, ganaxolone, tibolone, norgestimate, forrnestane, istaroxime, f1nasteride, abiraterone, triamincinolone, triamincinolone acetonide, dexamethasone, betamethasone, betamethasone dipropionate, betamethasone Valerate, deoxycortone, fludrocortisone, amcinonide, budesonide, desonide, fluocinolone acetonide, fluocinonide, halcinonide, triamcinolone acetonide, beclomethasone, fluocortolone, halometasone, mometasone, mometasone furoate, alclometasone dipropionate, clobetasol propionate, clobetasone butyrate, fluprednidene acetate, ciclesonide, des- ciclesonide, prednicarbate, tixocortol piValate, flunisolide, fluticasone furoate, fluticasone propionate, beta-sitosterol, campesterol, stigmasterol, stigmastanol, campestanol, brassicasterol, ergosterol, lupeol, cycloartenol, calcitriol, ergocalciferol, cholecalciferol, doxecalciferol, paricalcitol, calcipotriol, alfacalcidol, mifepristone, tirilazad, ulipristal acetate, asoprisnil, and Vecuronium.

In one embodiment, the template is an endocrine disruptor, exemplified by, but not limited to, bisphenol A, polychlorinated biphenyls (PCB), and dichlorodiphenyltrichloroethane (DDT).

In one embodiment, the template is a [non-steroidal] hydrophobic drug. Examples of hydrophobic drugs include, but are not limited to, ciprofloxacin, telmisartan, repaglinide, aspirin, imatinib, fenof1brate, clopidogrel, rosuvastatin, paclitaxel, docetaxel, tacrolimus, teniposide, dihydroergotamine, doxycyclin, methocarbamil, lorazepam, chlordiazepoxide, propanidid, cefuroxime axetil, zafirlukast, nelfinavir, itraconazole, nabilone, nimodipine, etraVirine, carbamazepine, modaf1nil, piroxicam, caffeine, cisplatin, epirubicin, doxorubicin, rapamycin, sirolimus, aprepitant, megestrol, morphine, naloxone, naltrexone, dexmethyl-phenidate, methyl phenidate, tizanidine, camptothecin, diazepam, cyclosporine A, flurbiprofen, prostaglandin-El, thyroxin, propofol, clofazimine, dutasteride, ritonavir, saquinavir, tritionoin, tipranavir, Vincristine, nystatin, bupiVacaine, and ropiVacaine.

In one en1bodin1ent of the inVention the template is a pesticide. Exaniples of pesticides include, but are not lin1ited to, chlorpyriphos, cypeniethrin, diazinon, dicofol, din1ethoate, disulfoton, endosulfan, ethion, fenitrothion, iprodione, nietalaxyl, parathion, perrnethrin, phenthoate, phorate, phosalone, pirin1icarb, and pentachloronitrobenzene.

The polynier particles of the invention are coniposed of polynier networks, forrned by polynierization of n1onon1ers, used as polynier building blocks. The group of n1onon1ers used in the inVention can be divided into cross-linking n1onon1ers and non-cross-linking n1onon1ers. In one en1bodin1ent of the inVention, the polynier particles are con1prised of a polynierized cross-linking n1onon1er of the following forrnula (1): (1) l Ä ------ ~ il -------- ~ --------- ~ X ----------- ~ Y l "mmšßlfñ MMMM! Wherein; (a) X is -CH2-, -O-CHz-CHr, -O-CH2~CH(CH3)-, or -O-CH(CH3)-CH2-; (b) Y is -O-C(O)-CH=CH2, -O-C(O)-C(CH3)=CH2, -NH-C(O)-CH=CHz, -NH-C(O)-C(CH3)=CH2, -O-CH=CH2, -O-CH2-CH=CH2, -CH=CHz, -CH2-CH=CH2, -O-C(O)-CH2-CH2-SH, -NH-C(O)-CH2-CH2~SH, or SH; (c) Z is -CHg-Xp-Y, a hydrogen, a functional group, an alkyl group, or a functionalized alkyl group; and (d) each of l, ni, n, and p is independently a nuniber in the range 0- In one en1bodin1ent, the polynier particles are further con1prised of a co-polynierized n1onon1er.

Exaniples of other cross-linking n1onon1ers that can be applied as a polynier building block in the inVention include, but are not lin1ited to, divinylbenzene [H2C=CH-C6H4-CH=CH2], glycerol diglycidyl ether [C9H16O5], glycerol diniethacrylate [H2C=C(CH3)CO2CH2CH[OR(H)]CH2OH(R); R = H or COC(CH3)=CH2], diVinyl sulfone [(H2C=CH)2SO2], Vinyl acrylate [HzC=CH-C(O)-O- CH=CH2], Vinyl niethacrylate [HgC=C(CH3)-C(O)-O-CH=CH2], ethylene glycol diacrylate [H2C=CH-C(O)-O-CH2-CH2-O-C(O)-CH=CH2], ethylene glycol diniethacrylate [H2C=C(CHs)- C(O)-O-CHz-CHz-O-C(O)-C(CH3)=CH2], di(ethylene glycol)din1ethacrylate [[H2C=C(CH3)~C(O)- O-CH2CH2]2O], di(ethylene glycol) diacrylate [[H2C=CH-C(O)-O-CH2-CHz]2O], l,4-butanediol [H2C=CH-C(O)-O-(CHz)4-O-C(O)-CH=CH2), l ,4-butanediol diniethacrylate [H2C=C(CH3)-C(O)-O-(CH2)4~O-C(O)-C(CH3)=CHz), glycerol propoxylate triglycidyl ether, diacrylate tri(propy1ene glycoßdiacrylate [H2C=CH-C(O)-(O(CH2)3)3-O-C(O)-CH=CH2], tri(propy1ene glycoßdirnethacrylate [H2C=C(CH3)-C(O)-(O(CH2)3)3-O-C(O)-C(CH3)=CH2], p01y(ethy1ene glycol- 400)-diacry1ate [H2C=CH-C(O)-(OCHzCHz)9-O-C(O)-CH=CHz], p01y(ethy1ene g1yc01-400)-dirneth- [H2C=C(CH3)-C(O)-(OCHzCHz)9~O-C(O)-C(CH3)=CH2], N,N”-methylenediacrylamide [H2C=CH-C(O)-NH-CH2-NH-C(O)-CH=CH2], N,N°-methylenedimethacrylamide [H2C=C(CH3)- C(O)-NH-CH2-NH-C(O)- C(CH3)=CH2], N,Nïphenylenediacrylamide [H2C=CH-C(O)-NH-C6H4- NH-C(O)- CH=CH2], N,N°-phenylenedimethacrylamide [H2C=C(CH3)-C(O)-NH-C6H4-NH-C(O)- C(CH3)=CH2], 3,5-bis(acry1oy1arnid0)benzoic acid [H2C=CH-C(O)-NH-C6H3(COzH)-NH-C(O)- CH=CH2], 3,5-bis(rnethacryloylarnido)benzoic acid [H2C=C(CH3)~C(O)-NH-C6H3(CO2H)-NH- C(O)-C(CH3)=CH2], N,O-bisacryloyl-L-phenylalaninol [H2C=CH-C(O)-NH-CH(CH2-C6Hs)~CHz- O-C(O)-CH=CH2], N,O-bismethacryloyl-L-phenylalaninol [HgC=C(CH3)-C(O)-NH-CH(CH2- C6H5)-CH2-O-C(O)-C(CH3)=CH2], Vinyl acrylate [H2C=CH-C(O)-O-CH=CH2], Vinyl rnethacrylate [H2C=C(CH3)-C(O)-O-CH=CH2], diallyl succinate [(CH2CO2CH2CH=CH2)2], trimethylolpropane trivinyl ether [(H2C=CH-O-CH2)3C(C2H5)], 2,4,6-tria11y10xy-1,3,5-triazine [C12H15N3O3], 1,3,5- triallyl- 1 ,3 ,5 -triazine-2,4,6(1H,3H,5H)-tri0ne, tris [2-(acryloyloxy)ethyl] -isocyanuratq difirirnethylol- propane) [((H2C=CH-C(O)-O-CH2)2C(C2H5)-CH2)2O], tetramethacrylate [((H2C=C(CH3)-C(O)-O-CH2)zC(C2H5)- CH2)2O], pentaerythritol tetraacrylate [C(CH2-O-C(O)-CH=CH2)4], pentaerythritol tetramethacrylate [C(CH2-O-C(O)-C(CH3)=CH2)4], pentaerythritol (PETRA) [HO-CHz-C(CH2-O-C(O)-CH=CH2)3], pentaerythritol trimethacrylate [HO-CH2-C(CH2-O-C(O)-C(CH3)=CH2)3], trirnethylolpropane triacrylate [CHg- CHg-C(CH2-O-C(O)-CH=CH2)3], trimethylolpropane trimethacrylate (TRIM) [CH3-CH2-C(CH2-O- C(O)-C(CH3)=CH2)3], trimethylolpropane benzoate diacrylate [(H2C=CH-C(O)-O-CH2)2C(C2H5)- CH2-O-C(O)-C6H5], dirnethacrylate [(H2C=C(CH3)-C(O)-O- CH2)2C(C2H5)-CH2-O-C(O)-C6H5], trimethylolpropane allyl ether [H2C=CH-CH2-O-CH2- C(C2Hs)(CH2OH)2, diallyl [CzHsC(CH2OCH2CH=CHz)zCH2OH], trimethylolpropane ethoxylate (lEO/OH) rnethyl ether diacrylate [H2C=CH-C(O)-O-CHz-CHz-O- CH2)2C(C2H5)-CH2-O-CHg-CHg-O-CH;], pentaerythritol ethoxylate (3/4 EO/OH) tetraacrylate [C[CH2(OCH2CH2)HO-C(O)-CH=CH2]4, pentaerythritol ethoxylate (3/4 EO/OH) tetramethacrylate [C[CH2(OCH2CH2)HO-C(O)-C(CH3)=CH2]4, pentaerythritol propoxylate (5/4 PO/OH) tetraacrylate [C[CHg[OCH2CH(CH3)]11O-CH=CH2]4, pentaerythritol propoxylate (5/4 PO/OH) tetramethacrylate [C[CHg[OCH;CH(CH3)]nO-C(CH3)=CH2]4, pentaerythritol ethoxylate (15/4 EO/OH) tetraacrylate [C[CH2(OCH2CH2)HO-C(O)-CH=CH2]4, pentaerythritol ethoxylate (15/4 EO/OH) tetramethacrylate [C[CH2(OCH2CH2)HO-C(O)-C(CH3)=CH2]4, trimethylolpropane ethoxylate (lEO/OH) rnethyl ether [H2C=C(CH3)-C(O)-O-CHz-CHz-O-CHz)zC(CzHs)~CHz-O-CHz-CHz-O-CH;], trimethylol-propane ethoxylate (1/3 EO/OH) triacrylate [(H2C=CH-C(O)-O-(CHz-CHz-O)nCHz)sC- acrylate tetraacrylate difirirnethylolpropane) triacrylate trimethylolpropane benzoate trimethylolpropane ether dirnethacrylate C2H5], triniethylolpropane ethoxylate (1/3 EO/OH) triniethacrylate [(H2C=C(CH3)-C(O)-O-(CH2- CH2-O)nCH2)3C-C2H5], triniethylolpropane ethoxylate (7/3 EO/OH) triacrylate [(H2C=CH-C(O)-O- (CH2-CH2-O)11CH2)3C-C2H5], (7/3 EO/OH) triniethacrylate [(H2C=C(CH3)-C(O)-O-(CH2-CH2-O)11CH2)3C-C2H5], triniethylolpropane ethoxylate (14/3 EO/OH) triacrylate [(H2C=CH-C(O)-O-(CH2-CH2-O)HCHz)3C-CzHs], triniethylolpropane ethoxylate (14/3 EO/OH) triniethacrylate [(H2C=C(CH3)-C(O)-O-(CH2-CH2-O)nCH2)3C-C2H5], triniethylolpropane propoxylate (1/PO/OH) triacrylate [(H2C=CH-C(O)-O-(C3H6O)n-CHz)3C-C2Hs], triniethylolpropane propoxylate (1/PO/OH) triniethacrylate [(H2C=C(CH3)-C(O)-O-(C3H6O)n-CH2)3C-C2H5], triniethylolpropane propoxylate (2PO/OH) triacrylate [(H2C=CH-C(O)-O-(C3H6O)n-CH2)3C-C2H5], triniethylolpropane propoxylate (2PO/OH) triniethacrylate [(H2C=C(CH3)-C(O)-O-(C3H6O)n- CH2)3C-C2H5], glycerol propoxylate (lPO/OH) triacrylate [[H2C=CH-C(O)-O-[CH(CH3)-CH2-O]1- CHz] [H2C=CH-C(O)-O-[CH(CH3)~CH2 O]m] [H2C=CH -C(O)-O-[CH(CH3)~CHz-O]n-CH2] [~CH], glycerol ethoxylate-co-propoxylate triacrylate [CH2=CH-C(O)-O-(C3H6O)X-(CH2CH2O)y-CH[CHg- (OCHzCHfly-(OCaHøk-O-C(O)-CH=CHz]2], [CHs-(CH2)7~CH=CH-(CH2)7~C(O)-O- CH(CH2-O-C(O)-(CH2)7-CH=CH-(CH2)7-CH3)2], triniethylolpropane triglycidyl ether [C15H26O6], 2,2-bis[4-(2-hydr0xy-3-niethacryloyloxypropoxy)phenyl]-propane, bisphenol-A diniethacrylate, pentaerythritol tetrakis(3-n1ercapt0pr0pi0nate) [(HS-CHz-CHz-C(O)-O-CHz)4C], [(HS-CH2-CH2-C(O)-O-CHz)3C-C2Hs], 2- hydroxymethy1-2-n1ethy1-1ß-propanediol tris-(3-n1ercapt0pr0pi0nate) [(HS-CH2-CH2-C(O)-O- CHz)3C-CH3] , 2,2-bis(su1fany1n1ethy1)-1 ,3 -propanedithiol [(HS-CH2)4C] , di-3 - niercaptopropionate, p01y(ethy1ene glycol) dithiol [HS-CHg-CHg-(O-CHg-CHfln-SH], 1,3,4- thiadiazole-2,5-dithiol, [C2H2N2S3], t01uene-3,4-dithi01 [CH3C6H3(SH)2], benzene-1,4-dithiol, benzene-1,2-dithiol, 1,3-benzenedithi01 [C6H4(SH)2], biphenyl-4,4'-dithiol [HS-C6H4-C6H4-SH], p- terpheny1-4,4”-dithi01 [HS-C6H4-C6H4-C6H4-SHL hexa(ethy1ene glycol) dithiol [HS-(CHg-CHQ- O)5-CH2-CH2-SH], tetra(ethy1ene glycol) dithiol [HS-(CHg-CHg-Ofi-CHg-CHg-SH], 2,2'- (ethylenedioxy)diethanethiol [HS-CHz-CHz-O-CHz-CH2~O-CH2-CH2~SH], 1,4-benzenedin1ethane- thiol [C6H4(CH2SH)2], 1,16-hexadecanedithi01 [HS-(CH2)16-SH], dithiothreitol [HS-CH2-CH(OH)- CH(OH)-CH2-SH], 2-n1ercapt0ethy1 ether [O(CH2CH2SH)2], LS-propanedithiol [HS-(CH2)3-SH], 1,4-butanedithiol [HS-(CH2)4-SH], 1,5-pentanedithiol [HS-(CH2)5-SH], 1,6-hexane dithiol [HS- (CH2)6-SH], 4arrn-PEG-SH [C(CHg-O-(CH2CH2O)nCH2CH2SH)4], Sarrn-PEG-SH, and analogs and triniethylolpropane ethoxylate triolein ethoxylated triniethylolprop ane tris(3 -n1ercaptopr0pi0nate) glycol derivatives of these.

Exaniples of non-crosslinking niononiers that can be used in the invention include, but are not limited to, acid-containing niononiers such as acrylic acid, niethacrylic acid (MAA), trifluoroniethacrylic acid, itaconic acid, Vinylacetic acid, 4-Vinylbenzoic acid (4-VBA), 4- Vinylphenylboronic acid, Vinylsulfonic acid, and Vinylphosphonic acid; ester-containing niononiers such as Vinyl acetate, Vinyl propionate, Vinyl pivalate, allyl acetate, n1ethyl acrylate, n1ethyl niethacrylate, ethyl acrylate, ethyl niethacrylate, propyl acrylate, propyl niethacrylate, butyl acrylate, butyl niethacrylate, pentyl acrylate, pentyl niethacrylate, cyclohexyl acrylate, cyclohexyl niethacrylate, benzyl acrylate, benzyl niethacrylate, isobornyl acrylate, isobornyl niethacrylate, hydroxybutyl acrylate, hydroxybutyl niethacrylate, Vinyl decanoate, Vinyl 4-tert-butylbenzoate, glycidyl acrylate, and glycidyl niethacrylate; an1ide-containing n1onon1ers such as acrylaniide, n1ethacrylan1ide, and N- Vinylacetaniide; an1ino-containing niononiers such as allyl an1ine, 2-an1inoethyl niethacrylate, 2- (diethylan1ino)ethyl niethacrylate, (Vinylbenzyl)trin1ethylan1n1oniun1 chloride, and 4-an1inostyrene; heteroaroniatic n1onon1ers such as l-Vinyliniidazole, 4-Vinylpyridine, 2-Vinylpyridine, l-Vinyl-2- pyrrolidinone, N-Vinylcaprolactani, and N-Vinylphtaliniide; hydroxyl-containing n1onon1ers such as 4- hydroxystyrene, alpha-Vinylbenzyl alcohol, 2-hydroxyethyl acrylate, 2-hydroxyethyl niethacrylate (HEMA), S-hydroxypropyl acrylate, 3-hydroxypropyl niethacrylate, 4-hydroxypropyl acrylate, 4- hydroxypropyl niethacrylate, acrylate, niethacrylate, 2,3- -hydroxypentyl 5-hydroxypentyl dihydroxypropyl acrylate, 2,3-dihydroxypropyl niethacrylate, N-hydroxyniethylacrylaniide, N- hydroxyniethylniethacrylaniide, allyl alcohol, hydroxyethyl Vinyl ether, and allyl-2-hydroxy-2-phenyl ether; Vinyl acid halides such as acryloyl chloride, n1ethacryloyl chloride; halide-containing n1onon1ers such as Vinyl broniide and Vinyl chloride; thiol-containing n1onon1ers such as 2-propene-l-thiol; silane- containing n1onon1ers such as Vinyltriniethylsilane, Vinyltriniethoxysilane, and 3- glycidoxypropyltriniethoxysilane; aron1atic n1onon1ers such as styrene, 2-Vinyl anthracene, 9-Vinyl anthracene, l-Vinylnaphtalene, 2-Vinylnaphtalene, l-Vinylphenanthrene, 9-Vinylphenanthrene, 4- Vinylbiphenyl, 4-Vinyl-o-terphenyl, 4-Vinylpyrene, 5-Vinylpyrene, 2-Vinyltetracene; and analogs and derivatives of these.

In another en1bodin1ent, the inVention provides a process for preparing said ten1plate-in1printed polynier particles, con1prising the steps: (a) selecting a ten1plate capable of providing a n1olecular recognition of a target n1olecule: (b) dissolVing the n1onon1er(s) in a Water-n1iscible solVent, forrning a solution con1prising said n1onon1er(s); (c) interfacing said solution and a Water phase to forrn a niixture in Which spontaneous and instantaneous forrnation of nucleated droplets con1prising said n1onon1er takes place; (d) providing the ten1plate either in said solution or in said Water phase; and (e) polynierizing said n1onon1er(s) by a polynierization reaction.

The process of the inVention is particularly adVantageous since it is energy-efficient and environnientally friendly. The first step is the preparation of a solution of n1onon1er(s) dissolVed in a Water-niiscible solVent. The resulting solution is hereafter referred to as the solution. Exaniples of suitable solVents for the preparation of the solution include, but are not lin1ited to, Water-niiscible alcohols, acetonitrile, N,N-dimethylforrnamide (also named DMF), dimethyl sulfoxide (also named DMSO), acetone, acetic acid, acetaldehyde, hexamethylphosphoric triamide (also named HMPT), dimethoxyethane (also named glyme, monoglyme, dimethyl glycol, ethylene glycol dimethyl ether, dimethyl cellosolve, and DME), 1,4-dioxane, N-methyl-2-pyrrolidone (also named NMP), pyridine, tetrahydrofuran (also named THF), or combinations thereof. Examples of alcohols include, but are not limited to, methanol, ethanol, l,2-ethanediol (also named ethylene glycol), l-propanol, 2-propanol, l,2- propanediol (also named propylene glycol), l,3-propanediol, l-butanol, 2-butanol, l,2-butanediol, 1,3- butanediol, l,4-butanediol, l-pentanol, 2-pentanol, 3-pentanol, l,2-pentanediol, l,3-pentanediol, 2,3- pentanediol, 2,4-pentanediol, 1,4-pentanediol, 1,5-pentanediol, glycerol (also named glycerine), erythritol (also named butane-l,2,3,4-tetrol), pentaerythritol (also named 2,2- bis(hydroxymethyl)propane-l,3-diol), furfuryl alcohol, diethylene glycol (also named DEG), triethylene glycol (also named triglycol, TEG, and TREG), tetraethylene glycol, and pentaethylene glycol. In one embodiment of the invention, ethanol is particularly suitable to be used as the Water- miscible solVent.

The next step comprises interfacing the solution With Water by simply adding the solution to Water or by, in addition, including a brief mixing/blending procedure, thereby forrning a mixture of monomer(s), Water-miscible solvent, and Water. In some cases, the momentum of the solution being added to the Water phase is enough to cause sufficient turbulence to mix the two liquids. Upon mixing/blending the three (or more) components, the Water-miscible solVent partitions into the Water phase and nucleation (droplet formation) of the monomer(s) occur. Key to successful nucleation is to provide preferred fractions of the three (or more) components in the mixture. The preferred fractions are estimated from an experimentally deterrnined phase diagram. As an example, a temary phase diagram of pentaerythritol triacrylate (PETRA), ethanol, and Water is provided in the format of a right triangle phase diagram in Figure la. Within the nucleation region, located beloW the dotted line in Figure la, nucleation occurs instantaneously and spontaneously When the components are interfaced/mixed. In the most preferred embodiments, the composition is comprised of at the most about 20 mass percent (i.e., at the most about 0.2 mass fractions) of a Water miscible solVent and at the most about 1 mass percent (i.e., at the most about 0.01 mass fractions) of monomer(s). Only an interfacing or initial brief mixing/blending is required; no continued mixing or agitation is required. The droplets remain stable in the mixture during time periods that are sufficiently long to allow for a polymerization of the monomer(s) to form solid polymer particles. The size of the droplets is indicated in Figure la. Mixtures of compositions located outside of the nucleation region form either (i) a single phase or (ii) unstable, aggregated droplets. Upon polymerization, mixtures from the single-phase region may undergo precipitation polymerization and mixtures from the unstable region may form aggregated polymer particles of large size distributions and/or monolithic gels. Hence, polymer particles of narrow size distribution are formed only from mixtures located in the nucleation region. Figure lb shoWs the resulting particle sizes after subjecting the temary mixtures of PETRA, ethanol, and Water in Figure la to polymerization conditions.

As mentioned above, no extensive mixing or agitation is needed for nucleation to take place When the composition of the components is located Within the nucleation region of the phase diagram. The methods for interfacing the solution and the Water phase include, but are not limited to, batch-Wise methods and flow system methods. The batch-Wise methods include, but are not limited to, mixing by turbulent addition, mixing by manual inversion, mixing by automated inversion, mixing by manual shaking, mixing by automated shaking, mixing using a vortexer, mixing by ultrasonic treatment, mixing using a magnetic stirrer, mixing using an overhead stirrer, mixing using a blender, mixing using a disperser device, and mixing using a homogenizer. The flow system methods include, but are not limited to, mixing using a static mixer, mixing using a microfluidic mixer, mixing using a micromixer, continuous-flow capillary mixing, microfluidic mixing using Y junctions, microfluidic mixing using T junctions, microfluidic mixing using cross-junctions, and mixing using various three-Way intersections or connectors. Mixing devices produced by microfabrication or 3D-printing are useful. Industrial scale mixing devices include, but are not limited to, impellers, turbines, anchors, helical ribbons, high-shear dispersers, ribbon blenders, paddle mixers, double cone blenders, static mixers, liquid Whistles, dispersion mixers, mixing paddles, and continuous-flow mixers.

Figure 2 shoWs that there is no significant influence of the interfacing method on the resulting particle size distribution.

Although no extended mixing/agitation is needed for the formation of the droplets When the composition is located in the nucleation region of the phase diagram, more extensive mixing procedures as Well as prolonged mixing periods may be applied in some cases for the purpose of affecting the final particle size. Although no stabilizer is needed for the formation of droplets When the composition is located in the nucleation region of the phase diagram, one or more stabilizers may be added for the purpose of affecting the mean particle size, the polydispersity, and the particle size distribution of the resulting polymerized particles or for the purpose of extending the time period that the droplets are stable in the mixture. The term stabilizers are here used to denote suspension stabilizers, suspending agents, suspension agents, emulsifiers, emulsion agents, emulsifying agents, dispersants, dispersing agents, and other agents used for similar purpose as the ones listed. The terms are used interchangeably herein. Stabilizers may be minimally Water-soluble inorganic salts such as those selected from the group consisting of tribasic calcium phosphate, calcium carbonate, calcium sulfate, barium sulfate, barium phosphate, magnesium carbonate, and mixtures thereof Water soluble organic stabilizers, such as, but not limited to, those selected from the group consisting of polyvinyl alcohol, poly-N vinylpyrrolidone, polyacrylic acid, polyacrylamide, and hydroxyalkyl cellulose, can also be used. Surfactants can also be used, including anionic surfactants, cationic surfactants, or nonionic surfactants. Anionic surfactants include, but are not limited to, the group consisting of alcohol sulfates, alkyl aryl sulfonates, ethoxylated alkyl phenol sulfates, ethoxylated alkyl phenol sulfonates, and mixtures thereof More specific examples of anionic surfactants include, but are not limited to, sodium lauryl sulfate, ammonium laureate, and sodium dioctyl sulfosuccinate. Cationic surfactants include, but are not limited to, the group consisting of quatemary ammonium salts. Nonionic surfactants useful in the present inVention include, but are not limited to, the group consisting of ethoxylated alkyl phenols, ethoxylated fatty acids, and ethoxylated fatty alcohols and mixtures thereof. A combination of more than one stabilizer can also be useful in the inVention.

In the final step, the polymer networks are formed by assembling the monomers through a polymerization reaction, including, but not limited to, a free-radical polymerization, a thiol-ene polymerization, an anionic polymerization, a cationic polymerization, a redox-initiated polymerization, a chain-growth polymerization, a step-growth polymerization, a condensation polymerization, a living polymerization, a reVersible-deactivation radical polymerization, and a reVersible addition- fragmentation chain transfer (RAFT) polymerization. Free-radical initiators useful in the present inVention include those norrnally suitable for free-radical initiation. These species include, but are not limited to, azo compounds, organic peroxides, benzoine ethers, benzyl ketals, alpha-dialkoxy acetophenones, alpha-hydroxy alkylphenones, alpha-amino alkylphenones, acyl phosphine oxides, benzophenones, and thio-xanthones. Examples of azo compounds include low molecular Weight azo initiators and macro azo initiators. LoW molecular Weight azo initiators include, but are not limited to, 2,2 °-azobis(2-methylbutyronitrile), 2,2 °-azobis(2-methylpropionitrile), l , l ”-azobis(cyclohexanecarbo- nitrile), phenyl-azo-triphenylmethane, and 4,4”-azobis(4-cyanovaleric acid). Commercial products of this type include, but are not limited to, VAZO 52, VAZO 64, VAZO 67, and VAZO 88 initiators from DuPont. Macro azo initiators include, but are not limited to, compounds comprising polydimethylsiloxane units, such as the VPS series from Wako, and compounds comprising polyethyleneglycol units, such as the VPE series from Wako. Examples of peroxides include, but are not limited to, tert-butyl peroxide, cumyl peroxide, acetyl peroxide, benzoyl peroxide, lauroyl peroxide, tert-butyl hydroxyperoxide, and tert-butyl perbenzoate. The rate of the decomposition of peroxides may be increased by the addition of tertiary amines, such as, but not limited to, N,N-dimethylaniline. Anionic initiators useful in the present inVention include those norrnally suitable for anionic initiation. Examples of anionic initiators include, but are not limited to, metal alkyls such as n-butyllithium. Cationic initiators useful in the present inVention include those norrnally suitable for cationic initiation. Examples of cationic initiators include, but are not limited to, sulfuric acid, tin(IV)chloride, boron trifluoride, and iodine. Redox-initiated polymerizations use a redox pair for initiation, exemplified by, but not limited to, organic peroxides and tertiary amines, such as the benzoyl peroxide and N,N-dimethyl-p-toluidine redox pair. The polymerization may be manipulated by the addition of quenchers or inhibitors. In the case of thiol-ene polymerizations, examples of quenchers include, but are not limited to, 3- mercaptopropionic acid and other thiols. Initiators, quenchers, and/or inhibitors are added either to the solution or to the Water phase.

To promote the polymerization, the monomers can be exposed to a source of energy, such as, but not limited to, ultraviolet (UV) radiation, gamma radiation, and/or a heat source providing an elevated temperature for a time period sufficiently long to promote the polymerization. Preferably, the reaction is carried out at a temperature of about -30-100 °C, more preferably at a temperature of 10-90 °C, and most preferably at a temperature of 30-70 °C. For polymerizations promoted by UV light, radiation in a Wavelength of about 200-400 nm is preferred. Combinations of elevated temperatures and UV light can also be applied. Figure 3 shows the resulting particle size distributions When the polymerizations Were carried out at an elevated temperature and under the influence of UV light, respectively.

As described above, the recognition sites of the polymer particles of the invention are created by template molecules being present during the polymerization. The template molecules are added to either the solution or to the Water phase. In one understanding, the template molecules act as molds to produce cavities or imprints that are complementary to the templates in size, shape, and positioning of functional groups. The template molecules can be covalently coupled to one or more of the monomers or can interact via non-covalent interactions With one or more of the monomers. In both cases, the templates are removed from the polymer after the polymerization, leaving recognition sites capable of molecular recognition and binding. The former type of molecular imprinting, often referred to as covalent molecular imprinting, uses templates derivatized With a polymerizable group and requires removal of the template by cleavage after polymerization. In the latter type of molecular imprinting, often referred to as non-covalent molecular imprinting, removal of the template is carried out by extraction. The template can also be left purposely in the polymer network for later extraction. This approach is particularly suitable for applications involving sustained release and template delivery.

The target molecule can be used directly as a template. Altematively, a derivative of the target molecule can be used as a template. In some cases, it is more preferred to use an analog of the target molecule as a template. Derivatized target molecules or analogs of the target molecules can be used to solve problems of solubility or stability in cases of target molecules With limited solubility or stability. For example, the solubility of a target molecule in the solution can be increased by derivatization With a hydrophobic molecule. Typical hydrophobic structural elements that can be used to increase the hydrophobicity are long-chain hydrocarbons, aromatic hydrocarbons, and long-chain polyethylene glycols. Examples of hydrophobic molecules include, but are not limited to, fatty acids, fats, triglycerides, Waxes, alkanes, long-chain polyethylene glycols, aromatic hydrocarbons, steroids, fat- soluble vitamins, fat-soluble horrnones, hydrophobic drugs, and hydrophobic pesticides. In some cases, an analog is used as the template because it is more stable, cheaper, or less toxic than the target molecule itself. In covalent molecular imprinting, the target molecule is derivatized With a polymerizable group. Examples of polymerizable groups include, but are not limited to, the acrylate group, the methacrylate group, the Vinyl group, the allyl group, and the thiol group.

In one embodiment of the invention, a polymer particle comprising molecular recognition sites for a steroid is provided. Polymer networks comprised of polymerized pentaerythritol triacrylate (PETRA) (forrnula I, Wherein Z is -CHg-OH; each of l, m, and n is 0; and Y is -O-C(O)-CH=CH2) are particularly suitable for recognizing and binding steroids.

In one embodiment of the invention, a process is provided that is particularly suitable for preparing a steroid-imprinted polymer particle, for example, but not limited to, a cortisol-imprinted polymer particle, a beta-estradiol-imprinted polymer particle, and a cholesterol-imprinted polymer particle. The process comprises the steps: (a) dissolving pentaerythritol triacrylate (PETRA) (forrnula I, Wherein Z is -CHz-OH; each of l, m, and n is 0; and Y is -O-C(O)-CH=CH2) in ethanol, forrning an ethanolic solution comprising PETRA; (b) interfacing said solution and Water in the proportions Which, according to Figure la, induce spontaneous and instantaneous formation of nucleated droplets comprising PETRA; (c) providing the template either in said solution or in said Water phase; (d) providing a free-radical initiator either in said solution or in said Water phase; and (e) polymerizing PETRA by free-radical polymerization.

As seen in the saturation binding isotherrns in Figure 5, the binding capacity of the cortisol- imprinted polymer particles (MIP 12) is signif1cantly higher than the binding capacity of the non- imprinted polymer particles (NIP l2), prepared as the imprinted ones but in absence of a template. The figure clearly demonstrates the usefulness of the invention°s polymer particles.

The polymer particles of the present invention can be used as molecular recognition elements or as synthetic antibodies in applications Where selective or specific binding is required. Applications include, but are not limited to, the use as recognition elements in sensors and assays, as stationary phases for chromatography and solid-phase extractions, as carriers in drug delivery systems, as sorption materials for removal of toxins, metabolites, nutrients, or other compounds, and as carriers in delivery of pesticides, flavors, and fragrances among other substances.

EXAMPLES Example l Identification of Compositions Providing Nucleated Droplets and Polymerized Particles Firstly, a temary PETRA-ethanol-Water phase diagram Was constructed for identification of the nucleation region. Water and ethanol Were purged With nitrogen gas prior to use. Temary mixtures (10 mL) of PETRA, ethanol, and Water Were prepared by interfacing volumes (l-50 vol%) of a solution of PETRA (2.5 mM-150 mM) in ethanol With volumes (5 0-99 vol%) of Water. The solution Was added to the Water phase and the tWo liquids Were manually interfaced by inverting (shaking) the sample approximately 20 times. The resulting mixtures Were inspected and subj ected to dynamic light scattering (DLS) analysis using a Nanotrac Ultra Particle Size Analyzer from Mictrotrac (Montgomeryville, PA, USA) to determine the size distribution of the droplets. The mean droplet size Was calculated based on intensity using the Rayleigh-Debye theory (n = 10). The mass fractions of ethanol and PETRA, respectively, in each of the temary mixtures Were calculated. The calculated data Were combined With the DLS data and plotted in a phase diagram (Figure la). Secondly, mixtures for polymerization testing in small scale Were prepared following the same procedure, except that the Water phases Were preheated to 60 °C and the ethanolic solutions included AIBN (40 mM) used as a free-radical initiator. Polymerizations Were carried out for 6 h in a Water bath set at 60 °C and the samples Were cooled to room temperature before characterization. The resulting mixtures Were inspected and analyzed by DLS.

The data Were plotted in a phase diagram (Figure lb).

Example 2 Synthesis of Poly(PE T RA ) Particles; Demonstration of the Applicabilitj/ of Various Interfacing Methods A solution containing PETRA (0.04 M) and AIBN (008 M) in ethanol Was prepared. The ethanol used had previously been purged With nitrogen gas for 10 min. The solution (l volume part) Was then added to preheated (60 °C) Water that had previously been purged With nitrogen gas for l h (39 volume parts). The solution and the Water phase Were interfaced by applying either (i) manual shaking (20 inversions); (ii) an IKA Labortechnik Eurostar digital overhead stirrer (IKA-Werke Gmbh & Co., Staufen, Germany) equipped With a Heidolph TR 20 radial flow impeller operating at 700 rpm for 3 min; (iii) a model Dl25 basic dispersing device equipped With an S25N-25F dispersing element (IKA-Werke Gmbh & Co., Staufen, Germany) operating at 8000 rpm for l min; or (iv) a Bandelin Sonoplus ultrasonic homogenizer equipped With a VS70T titanium alloy probe (Bandelin Gmbh, Berlin, Germany) operating at 20 W for 6 x 10 s. The total volume of each mixture Was 500 mL.

Therrnolytically initiated polymerizations Were carried out in a 60 °C Water bath for 6 h. The polymerized mixtures Were centrifuged (9 500 rpm, 2 h). The supematants Were discarded and the particles Were retained. The particles Were incubated repeatedly first With ethanol, then With Water, and finally again With ethanol. After each incubation, the samples Were centrifuged (9 500 rpm, 2 h) and decanted. The absorbance of the supematants Was measured at 210 nm. Extractions Were repeated until the absorbance of the supematants Was < 0.05 absorbance units. Finally, the particles Were dried in vacuo oVemight. The dried particles Were dissolved in Water for DLS analysis (Table l, Figures 2a-d). The mean particle size Was calculated based on intensity using the Rayleigh-Debye theory. The polydispersity index Was calculated as dv/dN, Where dv and dN are the mean particle sizes based on Volume and numbers, respectively, calculated using the Lorenz-Mie theory.

Table 1. Influence of Interfacing Method on Size and Polydispersity of Po1y(PETRA) Particles (analysis by DLS , n=3 0) Interfacing Method Mean Particle Size i SD (nm) Mean Polydispersity Index i SD Manual shaking (20 inversions) 262 i 21 1.27 i 0.11 Overhead stirrer (700 rpm, 3 min) 248 i 25 1.36 i 0.18 Disperser/homogenizer (8 000 rpm, 1 min) 258 i 16 1.29 i 0.16 Ultrasonic homogenizer (6 X 10 s, 20 W) 258 i 18 1.33 i 0.14 ExampleSynthesis of Poly(PE T RA ) Particles; Demonstration of the Applicabililj/ of Dzflerent F :fee-Radical Generation Methods A solution Was prepared by dissolving PETRA (0.04 M) and AIBN (0.04 M) in ethanol that had previously been purged With nitrogen gas for 10 min. The solution (l Volume part) Was then added to Water that had previously been purged With nitrogen gas for l h (9 Volume parts). Preheated (60 °C) Water Was used for therrnolytically initiated polymerizations and Water of room temperature Was used for photolytically initiated polymerizations. The solution and the Water phase Were interfaced by inverting the sample manually (shaking) approximately 20 times. The total Volume of the mixture Was 250 mL. Therrnolytically initiated polymerizations Were carried out in a 60 °C Water bath for 6 h. Photolytically initiated polymerizations Were carried out under a DYMAX UV (350 nm) curing flood lamp model PC-2000 (Torrington, CT) for 2 h. The polymerized mixtures Were centrifuged (9 500 rpm, 2 h) and the supematants Were discarded. The particles Were incubated repeatedly first With ethanol, then With Water, and finally again With ethanol. After each incubation, the samples Were centrifuged (9 500 rpm, 2 h) and decanted. The absorbance of the supematants Was measured at 210 nm. Extractions Were repeated until the absorbance of the supematant Was < 0.05 absorbance units. Finally, the particles Were dried in vacuo ovemight. The dried particles Were dissolved in Water for DLS analysis (Table 2, Figures 3a,b).

Table 2. Influence of Polymerization Conditions on Size and Polydispersity ofPa1tic1es Deterrnined by DLS (n=10) Polymerization Conditions Mean Particle Size i SD (nm) Mean Polydispersity Index i SD 60 °C (Water bath), 6 h 415 i 32 1.23 i 0.16 350 nm (UV flood lamp), 2 h 427 i 40 1.23 i 0.18 ExampleSynthesis of Cortisol-Imprintecl Polymer Particles of Various Compositions (MIPs) and Corresponcling Non-Imprintecl Polymer Particles (NIPs); Demonstration of the Particles ' Molecular Recognition Capacity Solutions Were prepared by dissolving the appropriate monomer(s) and AIBN in ethanol that had previously been purged With nitrogen gas for 10 min. Cortisol Was added as a template. All amounts of the components are indicated in Table 3. Each solution Was added to Water that had previously been purged With nitrogen gas for 1 h. Preheated (60 °C) Water Was used for therrnolytically initiated polymerizations and Water of room temperature Was used for photolytically initiated polymerizations. The solutions and the Water phases Were interfaced by inverting the samples manually (shaking) approximately 20 times. The scale of each Synthesis Was in the range 250-1000 mL. Photolytically initiated polymerizations Were earned out under a DYMAX UV (350 nm) curing flood lamp model PC- 2000 (Torrington, CT) for 2 h. Therrnolytically initiated polymerizations Were carried out in a 60 °C Water bath for 6 h. The polymerized mixtures Were centrifuged (9 500 rpm, 2 h) and the supematants Were discarded. The particles Were incubated repeatedly first With ethanol, then With Water, and finally again With ethanol. After each incubation, the samples Were centrifuged (9 500 rpm, 2 h) and decanted. The absorbance of the supematant Was measured at 210 nm. Extractions Were repeated until the absorbance of the supematant Was < 0.05 absorbance units. Finally, the particles Were dried in vacuo ovemight. The binding of cortisol to the particles Was screened in a batch-Wise radioligand binding assay. Quadruplicate samples containing particles (1 mg) and [1,2,6,7-3H]-cortisol (1 pmol), dissolved in Water (1 mL), Were incubated in micro centrifuge tubes for 20 h on a shaking table. The samples Were thereafter centrifuged (2 h, 13 000 rpm). After centrifugation, 0.7 mL of the supematant Was WithdraWn and added to 1.5 mL of Ultima Gold XR scintillation liquid. Radioactivity Was measured using a Perkin Elmer Wallac Guardian 1414 Liquid Scintillation Counter to determine the free cortisol in the supematant. Bound cortisol Was calculated. The partition coefficients (KMIP and KN1P, respectively), showing the partition of cortisol between the polymer particles and the Water phase, and the relative partition coefficients (KMIP/Iímp), sometimes called the imprinting factor, Were calculated from the binding data (Table 3).

Table 3. Composition and Binding Capacity of Cortisol-Imprinted Polymer Particles (MIPs) and Non-Imprinted Polymer Particles (NIPs) Cortisol Molar Ratio Volume AIBN . . MIP Decom- Cross- Functional Com eri- Cornsol* Ram) or position Línker Monomera tratron 1n Cross-Linker~ Solution* KMIP KMIP/KNIP NIP Method Solution Functronal Water (mM) Monomer (V/V) 1 Photolysis PETRA ~ 10 1:4:0 1:9 0.21 3.2 2 Photolysis PETRA ~ 20 1:2:0 1:9 0.29 4.4 3 Photolysis PETRA ~ 40 1: 1:0 1:9 0.3 1 4.7 4 Photolysis PETRA MAA 10 1:4:4 1:9 0 .07 1.0 5 Photolysis PETRA MAA 10 1:4: 8 1:9 0.09 1.2 6 Photolysis PETRA MAA 10 1:4: 32 1:9 0.07 1.7 7 Photolysis PETRA 4-VBA 10 1:4:4 1:9 0 .21 1.0 8 Photolysis PETRA 4-VBA 40 1: 1:1 1:9 0 .3 0 1.5 9 Photolysis TRIM ~ 8 1:4: 0 1:9 0 .07 1.0 10 Photolysis TRIM HEMA 10 1:4:4 1:9 0.03 1.3 11 Thermolysis PETRA ~ 40 1: 1:0 1:9 0.61 6.6 12 Thermolysis PETRA ~ 40 1:1:0 1:39 0.99 8.3 13 Thermolysis PETRA ~ 5 1:1:0 1:1 0.89 1.3 MAA, methacrylic acid; 4-VBA, 4-Vinylbenzoic acid; HEMA, Z-hydroxyethyl methacrylate Example 4 demonstrates that the invention provides (i) template-imprinted polymer particles of Various compositions; (ii) template-imprinted polymer particles With molecular recognition capacity for the template; and (iii) a process for the preparation of said template-imprinted polymer particles.

Example 5 DLS Analysis of Cortisol-Imprinted P0ly(PE T RA ) Particles The dried MIP 12 and NIP 12 particles from Example 4 (Table 3) Were dissolved in Water for DLS analysis (Figure 4a,b). The mean diameter of the particles Was 264 i 12 nm (MIP 12) and 265 i 13 nm (NIP 12).

Example 6 Characterization of the Binding of Cortisol t0 Cortisol-Imprinted P0ly(PE T RA ) Particles Saturation binding isotherrns Were measured by incubating quadruplicate samples of 1 mL of Water solutions containing increasing concentrations of cortisol (0-4 uM), spiked With [l,2,6,7-3H]- cortisol (0.05 nM), and particles of either MIP 12 or NIP 12 (0.5 mg/mL) for 2 days on a shaking table. After centrifugation, samples Were Withdrawn and subj ected to liquid scintillation counting as described in Example 4. Saturation binding data Were frtted to a one-site Langmuir isotherrn and the dissociation constant (KD=4.0 i 0.7 uM) and the number of binding sites (BmaX=2.2 i 0.3 umol/ g) Were calculated using GraphPad Prism (Figure 5). Competitive binding assays Were carried out by incubating quadruplicate 1-mL Water samples containing MIP 12 (0.2 mg/mL), [l,2,6,7-3H]-cortisol (0.05 nM), and increasing concentrations (0-0.1 mM) of either cortisol or a competitor (i.e., cortisone, progesterone, estradiol, propranolol, tetracycline, or caffeine) on a shaking table for 24 h. After centrifugation, samples were withdrawn and subjected to liquid scintillation counting. Binding data were analyzed with GraphPad Prism using a sigmoidal dose-response (Variable slope) model to deterrnine the EC50 values. The cross-reactivity was calculated as the ratio of the ECso of cortisol to the ECso of the competitor (Table 4).

Table 4. ECso and Cross-Reactivity of Cortisol Competitors Competitor ECso (uM) Cross-Reactivity (%) Cortisol (analyte) 2.23 100 Cortisone 1 1.7 19 Progesterone 1.74 128 Estradiol 20.8 1 1 Propranolol 129 100 <0.002 Tetracycline No competition No competition Caffeine No competition No competition Example 7 Synthesis of Cortisol-Imprinted P0ly(PE T RA) Particles Applying Cortisol in the Water Phase The MIP particles were prepared as MIP 12 in Example 4, except that cortisol was added to the water phase instead of to the ethanolic solution. The resultant particles showed similar particle size distributions and binding capacities as MIP Examples 4 and 7 demonstrates that the template can be added either to the ethanolic solution or to the water phase during the preparation of the particles, both cases resulting in particles of similar characteristics.

Example 8 Synthesis of beta-Estradiol-Imprinted P0ly(PE T RA ) Particles A solution was prepared by dissolving PETRA (0.04 M), beta-estradiol (0.04 M), and AIBN (0.08 M) in ethanol (previously purged with nitrogen gas for 10 min). One Volume part of the solution was added to 39 volume parts of 60 °C-pre-heated water (previously purged with nitrogen gas for 1 h). The scale of the synthesis was 1000 mL. The solution and the water phase were interfaced by manual shaking (20 inVersions). A therrnolytically initiated polymerization was carried out in a 60 °C water bath for 6 h. Corresponding non-imprinted poly(PETRA) particles were prepared following the same procedure but omitting the addition of beta-estradiol to the ethanolic solution. DLS analysis of the reaction mixtures after polymerization showed a mean MIP particle size of 239 i 7 nm and a mean NIP particle size of 236 i 10 nm.

Example 9 Synthesis of Cholesterol-Imprinted Poly(PE T RA ) Particles A solution Was prepared by dissolving PETRA (0.04 M), cholesterol (0.04 M), and AIBN (0.08 M) in ethanol (previously purged With nitrogen gas for 10 min). One volume part of the solution Was added to 39 volume parts of pre-heated Water (previously purged With nitrogen gas for l h). The scale of the synthesis Was 500 mL. The solution and the Water phase Were interfaced by manual shaking (20 inversions). Therrnolytically initiated polymerization Was carried out in a 60 °C Water bath for 7 h. Corresponding non-imprinted poly(PETRA) particles Were prepared following the same procedure but omitting the addition of cholesterol to the ethanolic solution. DLS analysis of the reaction mixtures after polymerization showed a mean MIP particle size of 238 i 6 nm and a mean NIP particle size of 209 i 6 nm.

Example 10 Validation of Polj/(PE T RA) MIP Particles as the Recognition Element in a Cortisol Saliva Assay Freshly collected saliva Was stored at -20 °C until used. The sample Was then thaWed and centrifuged (9 500 rpm, 30 min). The supematant Was treated With active carbon for 17 h at 7 °C to deplete the sample from endogenous cortisol. The sample Was again centrifuged and the active carbon Was discarded. l-mL samples containing cortisol-depleted saliva (0.l mL), MIP 12 particles (0.2 mg), cortisol (0.l-l0 ng; corresponding to concentrations of l-l00 ng/mL saliva), and [l,2,6,7-3H]-cortisol (0.3 nM) Were prepared using Water as the diluent. Triplicate samples Were prepared for each cortisol standard concentration. The samples Were incubated on a shaking table for 20 h and then centrifuged (13 000 rpm, 2 h). After centrifugation, particle-free samples Were WithdraWn and subjected to liquid scintillation counting as described in Example 4. Linear regression of binding data provided a standard curve (rz = 0.9986). The accuracy (or trueness) of the measured standard concentrations Was calculated as (i) the mean of the relative errors (in %) of each replicate, Where the relative error of each replicate Was calculated as 100* /Cm-C, //C, (With Cm being the measured concentration and C, the true concentration) and (ii) the mean of the recovered concentration (in %) of each replicate, Where each replicate Was calculated as I 00 *Cm/Cf. The intra-assay precision (repeatability) Was calculated as the relative standard deviation (RSD) in % (i.e., as I 00 *SD/mean Cm). The limit of detection Was calculated as 3*SD0/slope, Where SDo Was the standard deviation of samples containing 0.3 nM [l,2,6,7-3H]- cortisol and slope Was the slope of the standard curve. The parameters are summarized in Table Table 5. Validation of a Cortisol Radioligand Binding Assay in Saliva Standard Concentration Accuracy (Trueness) Precision (Repeatability) ng/mL nmol/L Re1ative Error Ratio of Measured to True Value (Recovery) RSD (%) (%) (%) 100 276 0.2 100.0 0.3 50 138 2.0 100.8 2.4 25 69 11.8 105.8 13.2 10 2.76 3.2 103.2 0.4 5 13.8 7.8 107.8 3.1 1 2.76 6.5 96.1 10.Examples 4-10 demonstrate that the methods of the invention are useful for the preparation of a range of MIP particles. The examples also demonstrate that the MIP particles of the invention possess molecular recognition properties in Water and biological fluids, making the particles suitable as recognition elements in medical, biomedical and pharrnaceutical applications.

Claims (24)

1. A template-imprinted polymer particle of a size of about 10 to 1000 nm prepared from a nucleated composition, formed spontaneously and instantaneously in absence of surfactant, stabilizer, or dispersant and Without extended agitation, and comprising a monomer of the following forrnula (1): (1) eeeeeeeeeeee ~ X eeeeeeeeeeee ~ Y 2 Ü 'f šššx Kn WWW' i? Wherein; (a) X is -CH2-, -O-CHz-CHr, -O-CH2~CH(CH3)-, or -O-CH(CH3)-CH2-; (b) Y is -O-C(O)-CH=CH2, -O-C(O)-C(CH3)=CH2, -NH-C(O)-CH=CHz, -NH-C(O)-C(CH3)=CHz, -O-CH=CHz, -O-CH2-CH=CH2, -CH=CHz, -CHz-CH=CHz, -O-C(O)-CHz-CHz-SH, -NH-C(O)-CHz-CHz-SH, or SH; (c) Z is -CHg-Xp-Y, a hydrogen, a functional group, an alkyl group, or a functionalized alkyl group; and (d) each of l, m, n, and p is independently a number in the range 0-

2. The template-imprinted polymer particle of claim 1 Wherein the nucleated composition further comprises a monomer.

3. The template-imprinted polymer particle of claim 2 Wherein said monomer contains a functional group selected from the group consisting of the amino group, the hydroxyl group, the carbonyl group, the aldehyde group, the haloforrnyl group, the carboxyl group, the carboxylate group, the ester group, the carbonate ester group, the amide group, the imine group, the imide group, the azide group, the azo group, the hydrocarbyl group, the aromate group, the halo group, the cyanate group, the nitrate group, the nitrile group, the nitrite group, the nitro group, the nitroso group, the pyridyl group, the oxime group, the thiol group, the sulfide group, the disulfide group, the sulfinyl group, the sulfonyl group, the sulf1no group, the sulfo group, the thiocyanate group, the thioketone group, the thio ester group, the phosphino group, the phosphono group, the phosphate group, the borono group, the boronate group, the borino group, and the borinate group.

4. The template-imprinted polymer particle of any of the previous claims Wherein the nucleated composition is formed by interfacing a Water phase and a solution comprising said monomer(s) dissolved in a Water-miscible solvent.

5. The template-imprinted polymer particle of any of the previous claims Wherein the particle is capable of interacting specifically With a target molecule through a molecular recognition effect originating from a template being present during the polymerization.

6. The template-imprinted polymer particle of claim 5 Wherein the target molecule is selected from the group consisting of amino acids, peptides, peptide mimetics, proteins, enzymes, antibodies, carbohydrates, nucleotides, nucleosides, oligonucleotides, peptide nucleic acids, lipids, triglycerides, fatty acids, polycyclic aromatic hydrocarbons, macrocyclic organic compounds, fats, Waxes, steroids, flavonoids, alkaloids, prostaglandins, vitamins, cofactors, pesticides, herbicides, antimicrobials, metabolites, nutrients, xenobiotics, horrnones, toxins, neurotransmitters, signal molecules, cytokines, adhesion molecules, growth factors, biomarkers, drugs, and analogs, residues, or derivatives thereof.

7. The template-imprinted polymer particle of claim 5 Wherein the template is identical With the target molecule.

8. The template-imprinted polymer particle of claim 5 Wherein the template is a derivative or analog of the target molecule.

9. The template-imprinted polymer particle of claim 5 Wherein the template is a hydrophobic molecule.

10. The template-imprinted polymer particle of claim 5 Wherein the template is selected from the group consisting of steroids.

11. ll. A process for preparing a template-imprinted polymer particle of a size of about 10 to 1000 nm, comprising the steps: (a) selecting a template capable of providing a molecular recognition of a target molecule; (b) providing a monomer of the following forrnula (1): (1) WWWWWWWWWWW V» x »»»»»»»»»»»»» ~ v z xw “f íííííí ~ x. ???????????? ~ a' Wherein; (i) X is -CH2-, -O-CHz-CHr, -O-CHz-CH(CHs)-, or -O-CH(CH3)~CHz-; (ii) Y is -O-C(O)-CH=CH2, -O-C(O)-C(CH3)=CHz, -NH-C(O)-CH=CHz, -NH-C(O)-C(CH3)=CH2, -O-CH=CH2, -O-CHz-CH=CH2, -CH=CHz, -CHz-CH=CHz, -O-C(O)-CHz-CHz-SH, -NH-C(O)-CHz-CH2~SH, or SH; (iii) Z is -CHg-Xp-Y, a hydrogen, a functional group, an alkyl group, or a functionalized alkyl group; and (iv) each of l, m, n, and p is independently a number in the range 0- (c) dissolving the monomer in a Water-miscible solVent, forrning a solution comprising said monomer; (d) interfacing said solution and a Water phase, in absence of surfactant, stabilizer, or dispersant and Without extended agitation, to forrn a mixture in Which spontaneous and instantaneous forrnation of nucleated droplets comprising said monomer takes place; (e) providing the template either in said solution or in said Water phase; and (f) polymerizing said monomer by a polymerization reaction.

12. The process of claim ll Wherein said solution further comprises a monomer.

13. The process of claim 12 Wherein said monomer contains a functional group selected from the group consisting of the amino group, the hydroxyl group, the carbonyl group, the aldehyde group, the haloforrnyl group, the carboxyl group, the carboxylate group, the ester group, the carbonate ester group, the amide group, the imine group, the imide group, the azide group, the azo group, the hydrocarbyl group, the aromate group, the halo group, the cyanate group, the nitrate group, the nitrile group, the nitrite group, the nitro group, the nitroso group, the pyridyl group, the oxime group, the thiol group, the sulfide group, the disulfide group, the sulfinyl group, the sulfonyl group, the sulfino group, the sulfo group, the thiocyanate group, the thioketone group, the thio ester group, the phosphino group, the phosphono group, the phosphate group, the borono group, the boronate group, the borino group, and the borinate group.

14. The process of claim ll Wherein said Water-miscible solvent is selected from a group of solvents consisting of methanol, ethanol, 1,2-ethanediol, 1-propanol, 2-propanol, 1,2-propanediol, 1,3- propanediol, 1-butanol, 2-butanol, 1,2-butanediol, 1,3-butanediol, 1,4-butanediol, 1-pentanol, 2- pentanol, 3-pentanol, 1,2-pentanediol, 1,3-pentanediol, 2,3-pentanediol, 2,4-pentanediol, 1,4- pentanediol, 1,5-pentanediol, glycerol, erythritol, pentaerythritol, furfuryl alcohol, diethylene glycol, triethylene glycol, tetraethylene glycol, pentaethylene glycol, acetonitrile, N,N-dimethylforrnamide, dimethyl sulfoxide, acetone, acetic acid, acetaldehyde, hexamethylphosphoric triamide, dimethoxyethane, 1,4-dioxane, N-methyl-2-pyrrolidone, pyridine, tetrahydrofuran, or combinations thereof.

15. The process of claim ll Wherein said interfacing step is augmented by a mixing procedure selected from the group of mixing procedures comprising batch-Wise mixing methods including mixing by turbulent addition, mixing by manual inversion, mixing by automated inversion, mixing by manual shaking, mixing by automated shaking, mixing using a vortexer, mixing by ultrasonic treatment, mixing using a magnetic stirrer, mixing using an overhead stirrer, mixing using a blender, mixing using a disperser device, and mixing using a homogenizer, flow system mixing methods including mixing using a static mixer, mixing using a microfluidic mixer, mixing using a micromixer, continuous-flow capillary mixing, microfluidic mixing using Y junctions, microfluidic mixing using T junctions, and mixing using various three- or four-Way intersections or connectors, mixing by devices produced by microfabrication or 3D-printing, and industrial scale mixing devices including impellers, turbines, anchors, helical ribbons, high-shear dispersers, ribbon blenders, paddle mixers, double cone blenders, static mixers, liquid Whistles, dispersion mixers, mixing paddles, and continuous-flow mixers.

16. The process of claim 11 Wherein said polymerization reaction is a free-radical polymerization.

17. The process of claim 11 Wherein said polymerization reaction is a thiol-ene polymerization.

18. The process of claim 11 Wherein said mixture is comprised of less than about 20 mass percent of a Water miscible solvent and less than about 1 mass percent of monomer.

19. A method for analyzing a target molecule by the steps: (A) providing a template-imprinted polymer particle of a size of about 10 to 1000 nm by: (a) selecting a template capable of providing a molecular recognition of a target molecule; (b) providing a monomer of the following forrnula (1): (1) Wherein; (i) X is -CH2-, -O-CHz-CHr, -O-CH2-CH(CH3)-, or -O-CH(CH3)~CHz-; (ii) Y is -O-C(O)-CH=CH2, -O-C(O)-C(CH3)=CH2, -NH-C(O)-CH=CHz, -NH-C(O)-C(CH3)=CHz, -O-CH=CH2, -O-CHz-CH=CH2, -CH=CHz, -CHz-CH=CHz, -O-C(O)-CH2-CH2-SH, -NH-C(O)-CH2-CHz~SH, or SH; (iii) Z is -CHg-Xp-Y, a hydrogen, a functional group, an alkyl group, or a functionalized alkyl group; and (iv) each of l, m, n, and p is independently a number in the range 0- (c) dissolving the monomer in a Water-miscible solVent, forrning a solution comprising said monomer; (d) interfacing said solution and a Water phase, in absence of surfactant, stabilizer, or dispersant and Without extended agitation, to forrn a mixture in Which spontaneous and instantaneous forrnation of nucleated droplets comprising said monomer takes place; (e) providing the template either in said solution or in said Water phase; (f) polymerizing said monomer by a polymerization reaction; (g) removing said template by extraction With solVent; and (B) subjecting the template-imprinted polymer particle to a sample comprising said target molecule and subsequently quantifying the binding of the target molecule to the polymer particle.

20. The method of claim 19 Wherein the target molecule is selected from the group consisting of amino acids, peptides, peptide mimetics, proteins, enzymes, carbohydrates, nucleotides, nucleosides, oligonucleotides, peptide nucleic acids, lipids, triglycerides, fatty acids, polycyclic aromatic hydrocarbons, macrocyclic organic compounds, fats, Waxes, steroids, flavonoids, alkaloids, prostaglandins, Vitamins, cofactors, pesticides, herbicides, antimicrobials, metabolites, nutrients, xenobiotics, horrnones, toxins, neurotransmitters, signal molecules, cytokines, adhesion molecules, growth factors, biomarkers, drugs, and analogs, residues, or derivatives thereof.

21. 2l. The method of claim 19 Wherein the template is identical With the target molecule.

22. The method of claim 19 Wherein the template is a deriVatiVe or analog of the target molecule.

23. The method of claim 19 Wherein the template is a hydrophobic molecule.

24. The method of claim 19 Wherein the template is selected from the group consisting of steroids.