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Publication numberWO1996041173 A1
Publication typeApplication
Application numberPCT/US1996/007936
Publication date19 Dec 1996
Filing date29 May 1996
Priority date7 Jun 1995
Publication numberPCT/1996/7936, PCT/US/1996/007936, PCT/US/1996/07936, PCT/US/96/007936, PCT/US/96/07936, PCT/US1996/007936, PCT/US1996/07936, PCT/US1996007936, PCT/US199607936, PCT/US96/007936, PCT/US96/07936, PCT/US96007936, PCT/US9607936, WO 1996/041173 A1, WO 1996041173 A1, WO 1996041173A1, WO 9641173 A1, WO 9641173A1, WO-A1-1996041173, WO-A1-9641173, WO1996/041173A1, WO1996041173 A1, WO1996041173A1, WO9641173 A1, WO9641173A1
InventorsGraham D. Darling, Seymour Heisler, Brent R. Stranix, Petra Turkewitsch, Barbara Wandelt
ApplicantMartinex R & D Inc.
Export CitationBiBTeX, EndNote, RefMan
External Links: Patentscope, Espacenet
Nanoparticles imprinted with recognition sites for target molecules
WO 1996041173 A1
Abstract
The present invention provides solid imprinted particles comprising recognition sites spaced on the surface thereof which selectively bind a preselected template compound (T), wherein said recognition sites are each defined by a shaped polymeric matrix conforming to the shape and size of T, comprising a unit of the formula -B-Det, wherein B is bound to the polymeric matrix and also comprises a functional group which reversibly binds to T, and Det is a moiety capable of generating a detectable signal which changes following binding of B to template compound T.
Claims  (OCR text may contain errors)
WHAT IS CLAIMED IS:
A solid particle comprising recognition sites spaced on the surface thereof which selectively bind a preselected template compound (T), wherein said recognition sites are each defined by a shaped polymeric matrix conforming to the shape and size of T, comprising a unit of the formula
-B-Det, wherein B is bound to the polymeric matrix and also comprises a functional group which reversibly binds to T, and Det is a moiety capable of generating a delectable signal changes following binding of B to template compound T.
The particle of claim 1 wherein Det is also capable of reversibly binding to T.
The particle of claim 2 wherein Det generates a signal by fluorescence, that changes following binding of B or Det to T.
4. The particle of claim 1 wherein B is grafted onto the polymeric matrix.
5. The particle of claim 1 wherein -B-Det is the residue of an olefinically unsaturated polymerizable monomer that is part of the shaped polymeric matrix.
6. The particle of claim 1 which is spherical.
The particle of claim 6 which is about 10-100 nm in diameter.
8. A method for preparing imprinted polymeric particles, comprising the steps of:
(a) spraying droplets of water enclosing polymerizable organic micelles which comprise a mixture of at least one polymerizable monomer (A), a complex (B-T) between template component and a unsaturated amphiphilic agent B which comprises a functional group which is reversibly bound to a hydrophilic template compound T, and a polymerization initiator, into an organic liquid or onto a metal substrate, held at a temperature so as to freeze said droplets and said micelles; (b) raising the temperature of the organic liquid so that the water remains frozen and the micelles thaw; (c) polymerizing said micelles to yield solid particles comprising units of the formula (A)n-(B-T)p, wherein (A)n are repeating polymerized units of A, B is grafted onto A, and p is a number less than n; (d) thawing the water and recovering the solid particles; and (e) removing T from the surface of the particles to yield imprinted particulate solid particles comprising recognition sites spaced on the surface thereof which selectively bind T; wherein the recognition sites as each defined by a shaped polymeric matrix conforming to the size and shape of T, comprising units of the formula -(A)n-(B)p.
9. The method of claim 8 wherein the metal substrate is a mesh.
10. The method of claim 8 wherein the organic liquid is a hydrocarbon.
11. The method of claim 8 wherein the micelles are photopolymerized.
12. The method of claim 8 wherein the complex B-T further comprises a detectable moiety, Det, bound to B that is capable of generating a detectable signal. 13. The method of claim 12 wherein Det is also reversibly bound to T.
14. The method of claim 12 wherein Det is a fluorophore that changes fluorescence following binding of T to the recognition sites.
15. The method of claim 8 wherein the solid particles are spherical.
16. The method of claim 15 wherein the particles are about 10- 100 nm in diameter.
17. Imprinted polymeric particles prepared by the method of claim 8.
18. A solid particle comprising recognition sites spaced on the surface thereof which selectively bind a preselected template compound (T); wherein said recognition sites are each defined by a shaped polymeric matrix conforming to the shape and size of T, comprising a unit of the formula (-(A)n-B), wherein (A)n are repeating units of a polymerizable monomer A, and B is a nonpolymerizable organic polymerization initiator or chain transfer agent, which comprises a functional group which reversibly binds to template compound (T).
19. The particle of claim 18 wherein the recognition sites comprise a unit of the formula -(A)n-B-Det, wherein Det is a moiety capable of generating a detectable signal following binding of B or Det to template compound T.
20. The particle of claim 19 wherein Det generates a signal by fluorescence, that changes following binding of B or Det to T.
21. The particle of claim 20 wherein Det is an aromatic fluorophore.
22. The particle of claim 18 which is spherical. 23. The particle of claim 22 which is about 10- 100 nm in diameter.
24. The particle of claim 18 wherein B is derived from an allyl amine.
25. The particle of claim 18 wherein A is derived from an olefinically unsaturated monomer.
26. A method for preparing imprinted polymeric particles, comprising the steps of: (a) spraying droplets of a solvent enclosing polymerizable organic micelles which comprise a mixture of at least one polymerizable monomer (A), a complex (B-T) between template component and a nonpolymerizable chain transfer agent B which comprises a functional group reversibly bound to template compound T and a polymerization initiator, into an organic liquid or onto a metal substrate, held at a temperature so as to freeze said droplets and said micelles; (b) raising the temperature of the organic liquid so that the water remains frozen and the micelles thaw; (c) polymerizing said micelles to yield solid particles comprising units of the formula X-(A)n-B-T, wherein (A)n represents repeating polymerized units of A and X is H or B-T;
(d) thawing the water and recovering the solid particles; and
(e) removing T from the surface of the particles to yield imprinted solid particles comprising recognition sites spaced on the surface thereof which selectively bind T; wherein the recognition sites as each defined by a shaped polymeric matrix conforming to the size and shape of T, comprising units of the formula -(A)n-B.
27. The method of claim 26 wherein the droplets of a liquid are water droplets. 28. The method of claim 26 wherein the metal substrate is a mesh.
29. The method of claim 26 wherein the organic liquid is a hydrocarbon.
30. The method of claim 26 wherein the micelles are photopolymerized.
31. The method of claim 26 wherein the complex B-T further comprises a detectable moiety, Det, bound to B, that is capable of generating a detectable signal.
32. The method of claim 31 wherein Det is also reversibly bound to T.
33. The method of claim 32 wherein Det is a flurophore, that changes fluoresence when the recognition sites bind to T.
34. The method of claim 26 wherein the solid particles are spherical.
35. The method of claim 34 wherein the particles are about 10-100 nm in diameter.
36. Imprinted polymeric particles prepared by the method of claim 26.
37. A solid composite particle comprising a polymeric core particle enclosed by a polymeric coating having recognition sites spaced on the surface of said coating, which sites selectively bind to a preselected template compound (T); wherein the recognition sites are each defined by a shaped polymeric matrix conforming to the size and shape of T, wherein said sites are formed by (a) binding moieties of the formula Det to the surface of the polymeric core, wherein Det is a moiety capable of generating a detectable signal that can both bind to the polymeric core and reversibly bind to T; (b) applying the polymeric coating to the core in the presence of T to form Det-T moieties on the surface of the core which are surrounded by said polymeric coating, and (c) removing said T moieties from the surface of said composite particulate solid to yield said spaced recognition sites.
38. The particle of claim 37 wherein Det generates a signal by fluorescence, that changes following of spaced recognition sites to T.
39. The particle of claim 38 wherein Det is an aromatic fluorophone.
40. The particle of claim 37 wherein the solid composite particle is spherical.
41. The particle of claim 40 wherein the solid composite particle is about 10-100 nm in diameter.
42. A solid composite particle comprising a polymeric core particle enclosed by a polymeric coating having recognition sites spaced on the surface of said coating, which sites selectively bind to a preselected template compound (T); wherein the recognition sites are each defined by a shaped polymeric matrix conforming to the size and shape of T, wherein said sites are formed by (a) binding moieties of the formula Det-T to the surface of the polymeric core, wherein Det is a moiety capable of generating a detectable signal that can both bind to the polymeric core reversibly bound to T; (b) applying the polymeric coating to the core to form Det-T moieties on the surface of the core which are surrounded by said polymeric coating, and (c) removing said T moieties from the surface of said composite particulate solid to yield said spaced recognition sites.
43. The particle of claim 42 wherein Det generates a signal by fluorescence, which changes following binding of said spaced recognition sites to T. 44. The particle of claim 43 wherein Det is an aromatic fluorophone.
45. The particle of claim 42 wherein the solid composite particle is spherical.
46. The particle of claim 45 wherein the solid composite particle is about 10-100 nm in diameter.
47. The particle of claim 46 which is less than about 10-50 nm in diameter.
Description  (OCR text may contain errors)

NANOP ARTICLES IMPRINTED WITH RECOGNITION SITES FOR TARGET MOLECULES

Background of the Invention

Molecular recognition and its underlying mechanisms provide the basis for most biological processes, such as antibody-antigen binding, membrane transport or receptor binding, and enzymatic catalysis. In view of the difficulties in preparing and manipulating complex biomolecules, extensive research has been directed toward the development of synthetic recognition systems which can specifically bind to a given molecule in the presence of other structurally related molecules. One approach, termed "molecular imprinting," refers generally to a process for preparing polymers that are selective for a particular compound (the "print molecule" or "template molecule"). As discussed by B. Ekberg et al., TIBTECH. 7, 92 (1989) and in TIBS.19, 9 (January 1994), the technique involves: (1) prearranging the "template" molecule and the monomers and allowing complementary interactions to develop; (2) polymerizing the monomers around the template molecule-monomer complex; and (3) removing the template molecule from the resultant solid polymer by extraction.

Polymerization thus preserves the complementarity to the print molecule and the polymer can potentially selectively adsorb the print molecule, when subsequently contacted therewith, in the presence of structural analogs. The technique has also been referred to as "host-guest" polymerization or "template" polymerization. Thus, the imprinted polymers can be used as synthetic antibodies or catalysts, to accomplish separations, the resolution of enantiomeric mixtures, the promotion of reaction rates and the like.

Two fundamentally different approaches have been employed to develop molecular imprinting systems: (1) the monomers and the print molecule are covalently, but reversibly, bound, or (2) the initial interactions between monomers and the print molecule are non-covalent. Both approaches have used matrices based on styrene, acrylates and silica, and a number of structurally different print molecules and representative approaches are summarized on Table 1.

For example, G. Wulff et al. (U.S. Patent No. 4,111,863) claim a three-dimensional polymeric material formed from an "olefinically unsaturated compound" or a "polycondensation polymer" having a void shaped structurally and functionally to correspond to an "optically active compound," so that the polymeric material will preferentially sorb an optically active compound when a racemate of the compound is contacted with the material. The polymeric material has recurring units of =A-B, wherein A is the polymerizable monomer and B is the residue. For example, B can be a "polyfunctional achiral matrix molecule," that can be removed from the polymer to yield cavities containing functional groups oriented to separate the matrix molecules from complex mixtures. The resolution of racemic mixtures is disclosed to be solely due to the asymmetry of the cavities in the polymer. See also, G. Wulff et al., J. Liq. Chrom.. ϋ, 2987 (1990). These polymers are prepared by polymerizing a monomer comprising a covalently linked optically active compound (see Example 1).

Figure 1 is a schematic depiction of the preparation of molecularly imprinted polymers, which are formed by non-covalent imprinting, as discussed by G. Vlatakis et al., Nature. 361. 645 (1993). In Panel a, functional monomer, methacrylic acid, (MAA) is mixed with print molecule, e.g., theophylline, and optionally, a crosslinking monomer, in a suitable solvent. MAA is selected for its ability to form hydrogen bonds with a variety of chemical functionalities of the print molecule. The polymerization reaction is started by addition of initiator (2,2'-azobis(2-methylpropionitrile), AIBN). A rigid insoluble polymer is formed. 'Imprints', which are complementary to the print molecule in both shape and chemical functionality, are now present within the polymeric network, as shown in Panel b. The print molecule is removed by solvent extraction, to yield a recognition site for the print molecule which comprises functional COOH groups from MAA.

An example of the noncovalent approach is disclosed in Mosbach (U.S. Patent No. 5,110,833), that claims a method of producing "synthetic enzymes or synthetic antibodies" by orienting excess monomers around a "print molecule;" polymerizing the monomers with crosslinking agent and subsequently removing the print molecule, to create a cavity in the polymer corresponding to the print molecule. Example 1 discusses imprinting p- nitrophenyl methylphosphonate, a transition state analog, in poly[4(5)- vinylimidazole] to yield a synthetic enzyme-type polymer which hydrolyses p- nitrophenyl acetate at an increased rate.

Noncovalent molecular imprinting is disclosed to be useful to resolve mixtures of enantiomers by Kempe and Mosbach in J. Chromatography. 664.276 (1994), where the resolution of naproxen is disclosed using poly-4- vinylpyridine crosslinked with EGDA. Fischer, MuUer and Mosbach (published PCT application WO 93/09075) disclose a chiral solid-phase chromatography support, which contains molecular imprints of an optically pure enantiomer. The support is prepared by polymerizing a monomer in the presence of a cross- linking agent and a resolved enantiomer of an aryloxipropanolamine derivative to be separated, which forms a molecular imprint of the optically pure enantiomer in the polymer by non-covalent interactions between the monomer and the optically pure enantiomer.

Vlatakis, Anderson and Muller (published PCT application WO 94/11403) disclose artificial antibodies prepared by polymerizing functionalized monomers with a crosslinker in the presence of a print molecule, followed by removal of the print molecule to yield specific multi-functional binding sites for the print molecule. These materials can potentially be used to assay for the print molecule such as a drug in serum. See Vlatakis et al., Nature. 361. 645 (1993). All of these procedures involve bulk copolymerizations to yield blocks of generally macroporous material that are then ground and washed to yield only relatively coarse particles suitable as chromatographic media. Association constants, Ka, of correspondingly-imprinted polymers for their templates, have been reported at 7.9 x 104 M'1 for 9-ethyladenine in chloroform (K.J. Shea et al., J. Amer. Chem. Soc, 115, 3368 (1993)), and at 106 to 109 M'1 for the nitrogenous compounds theophylline and diazepam (G. Viatakis et al., Nature, 361, 645 (1993). Data from aqueous solutions are less available, but association here may be even higher due to additional hydrophobic effects.

Arnold et al. (U.S. Patent No. 5,310,648) claim an "imprinted matrix" comprising a polymer structure with "chelated metals" at "spaced locations" which bind with functional groups of a template molecule. The "imprinted matrix" can be a "lipid membrane," and the examples disclose the synthesis of a polymerizable metal-chelating amphiphilic lipid monomer and a lipid comonomer for the purpose of preparing protein-imprinted liposomes. In Example XI, the binding sites are oriented to a template protein by equilibration of the liposomes with a solution of the protein, then the liposome-protein complexes are cooled, the lipids are polymerized and the template protein is removed. It is disclosed that the lipid containing the interactive moiety hypothetically can diffuse in the bilayer membrane of the liposome to match the protein template. The position of the complexed metal ions is fixed by polymerization of the lipids, followed by removal of the template matrix to yield a two-dimensional assay of interactive sites. See D.R. Shrek et al., Langmuir. 10, 2382 (1994).

While molecular imprinting that employs non-covalent interactions between the template molecule and the polymeric matrix is relatively simple to carry out, the resultant selectivity that is achieved may be unacceptably slow. On the other hand, while the strong interactions which arise due to covalent bonds between functional groups on the monomers and the template molecules can lead to a good "fit" between the template molecule and the polymeric matrix, the drawback of this system is that complex chemical syntheses are required. Therefore, a continuing need exists for polymeric materials that can be readily prepared in a controlled fashion to comprise specific molecular recognition sites imprinted on the surface thereof.

Summary of the Invention The present invention provides a solid particle comprising recognition sites spaced on the surface thereof which selectively bind a preselected template compound (T); wherein said recognition sites are each defined by a shaped polymeric matrix conforming to the shape and size of T, comprising a unit of the formula -B-Det, wherein B is bound to the polymeric matrix and also comprises a functional group which reversibly binds to T, and Det is a moiety capable of generating a detectable signal which changes following binding of B to template compound T. Preferably, Det is also capable of reversibly binding to T, to yield the unit B-Det-T.

As used herein, with respect to B, B-Det or B-Det-T, the term "bound to the polymeric matrix" encompasses covalent attachment of B to the polymeric matrix, (A)n, by grafting B, B-Det, or B-Det-T at spaced intervals onto the polymeric chain; by copolymerization of olefϊnically-unsaturated B with one or more monomers, A, including cross-linking agents; or by terminating (A)n chains, as occurs when B is selected from a functionalized organic initiator or a chain transfer/chain termination agent, as discussed hereinbelow.

In one embodiment, the particulate solid comprises recognition sites spaced on the surface thereof which selectively bind a preselected template compound (T); wherein said recognition sites are each defined by a shaped polymeric matrix conforming to the size and shape of T, comprising a unit of the formula: (-(A)n-B), wherein (A)n are repeating units of a polymerizable monomer A, and B is a nonpolymerizable chain transfer agent or organic polymerization initiation which comprises a functional group which reversibly binds to template compound (T).

Preferably, the recognition site comprises a unit of the formula -(A)n-B-Det, wherein Det is a moiety capable of generating a detectable signal both before and after binding of B to template compound T, preferably Det does not interfere substantially with the binding of B to T, and most preferably, Det is selected to reversibly bind to T or to otherwise enhance the ability of the recognition site to bind to T.

The bonding of B and/or Det to T can be one or more of ion-ion, dipole-ion, dipole-dipole, Van der Waals, hydrogen bonding, chelation, or covalent bonding, or a mixture thereof. Preferably, the bonding is ion-ion, dipole-ion or both. Preferably, Det is a fluorescent moiety or "fluorophore", and its fluorescence changes when T binds to the recognition site. Preferably, the particles are substantially homogeneous in shape and/or size, with spherical microparticles, most preferably, nanoparticles being preferred, e.g., particles about 10-90 nm in average diameter. Particles of submicron size are advantageous in that their high surface area yields higher effective reaction rates per unit weight of particle. The preferred nanoparticles are <50 nm particles that can yield essentially clear solutions. Such solutions are more readily analyzed, i.e., bound analytes are more readily detected, as by spectrofluorimetry. The nanoparticles can also be targeted to, and introduced into, living cells by techniques such as microinjection, liposome fusion, and the like, more readily than microparticles. Such particles can also be more easily bound to components of optoelectronic apparatuses, i.e, applied to the tips of optical fibers used in biosensors.

In order to control its incorporation into the polymeric matrix, moiety B can be selected from functionalized organic polymerization initiators or from functionalized chain transfer agents or CTAs. CTAs are compounds comprising active hydrogen atoms that are not polymerizable monomers, but which permit polymerization by terminating the polymerization of one (A)n chain and initiating the polymerization of another chain. The moiety B is preferably selected and used in an amount relative to monomer A so that the number of recognition sites on the particulate solid is controlled, both in toto and on the surface. The moiety B is also preferably selected with respect to its polarity, so as to maximize the formation of recognition sites on the surface of the particles, as at an nonpolar/polar phase interface.

The use of nonpolymerizable CTA moieties (B) thus permits the particles of the invention to be imprinted in a reproducible manner, so that each particle will be functionally identical, e.g., recognition of T will be identical in terms of orientation, binding affinity (Ka, Kd) and the like. The ability to form a high number of recognition sites per unit surface area of a particle permits efficient binding of large T molecules, that cannot readily bind to sites positioned in interior channels or pores of the particles. The ability to control the distance between recognition sites permits the reduction or elimination of steric interference when template compounds (T) occupy adjacent recognition sites. The present invention also provides a method for preparing the imprinted polymeric particles, comprising the steps of:

(a) spraying droplets of a liquid enclosing polymerizable organic micelles which comprise a mixture of at least one polymerizable monomer (A), a complex (B-T) between template component and a nonpolymerizable chain transfer agent B which comprises a functional group which is reversibly bound to template compound T, and a polymerization initiator, into an organic liquid or onto a metal substrate held at a temperature so as to freeze said droplets and said micelles;

(b) raising the temperature of the organic liquid so that the water remains frozen and the micelles thaw;

(c) polymerizing, i.e., photopolymerizing, said micelles to yield solid particles comprising units of the formula X-(A)n-B-T, wherein

(A)n are repeating units of A and X is H or B-T;

(d) thawing the water and recovering the solid particles; and

(e) removing T from the surface of the particles to yield imprinted particulate solid particles comprising recognition sites spaced on the surface thereof which selectively bind T; wherein the recognition sites as each defined by a shaped polymeric matrix conforming to the size and shape of T, comprising units of the formula -(A)n-B.

Preferably, (B) also is bound to detection moiety (Det), so that there is a close association between T and Det during imprinting. Most preferably, Det also reversibly binds to T. Det is thus associated with, or close to, the resultant recognition site, which will comprise -(A)n-B-Det. Preferably, Det is a fluorescent group that is selected so that it changes in wavelength as well as in emission intensity when T is present in the recognition site. This permits a detection and quantitative measurement of the amount of T which is subsequently bound to the recognition site, as discussed hereinbelow. In a further embodiment of the invention, agent B can be an unsaturated amphiphilic agent which reversibly binds to T, and can be grafted onto the (A)n chains to yield particles having units of the formula (A)n-(B-T)p, wherein p is a number less than n, if T is selected to be hydrophilic, the complexes B-T will preferably orient within the liquid micelles so that T is at the surface of the micelle, at the organic-aqueous interface. Thus, this embodiment of the invention provides a method of preparing imprinted polymeric particles comprising:

(a) spraying droplets of water enclosing polymerizable organic micelles which comprise a mixture of at least one polymerizable monomer (A), a complex (B-T) between template component and an olefinically unsaturated amphiphilic agent B which comprises a functional group which is reversibly bound to hydrophilic template compound T, and a polymerization initiator, into an organic liquid or onto a metal substrate, held at a temperature so as to freeze said droplets and said micelles;

(b) raising the temperature of the organic liquid so that the water remains frozen and the micelles thaw;

(c) polymerizing said micelles to yield solid particles comprising units of the formula X-(A)n-(B-T)p, wherein (A)n are repeating polymerized units of A, B-T is grafted onto A, and p is a number less than n;

(d) thawing the water and recovering the solid particles; and

(e) removing T from the surface of the particles to yield imprinted particulate solid particles comprising recognition sites spaced on the surface thereof which selectively bind T; wherein the recognition sites as each defined by a shaped polymeric matrix conforming to the size and shape of T, comprising units of the formula -(A)n-(B)p. Preferably, the complexes also comprise a detectable moiety, Det, bound to B in a manner which does not interfere with the ability of B to bind to T. Preferably, Det can also reversibly bind to T. Thus, the recognition sites in the resultant particles will be defined by a shaped polymeric matrix conforming to the size and shape of T, comprising a unit of the formula (A)n-(B)p or (A)n-(B- Det)p, wherein p < n so that B is bound to less than all of the polymerized A units at spaced intervals.

These methods are represented schematically in Figure 2, wherein the template molecule (T) is represented by the large, notched hexagon, the chain transfer agent (B) which complexes with T is represented by the fork-like structure, wherein the tines represent the functional group that binds to T, and the monomers A are represented by the crossed dashed lines. As shown in

Figure 2, at least the functional group on B that binds to T is preferably selected to exhibit an affinity for the aqueous environment of the droplet prior to freezing, thus orienting B-T at the surface of the micelles prior to polymerization. The liquid droplets can be water droplets or droplets of an organic solvent in which said micelles are stable, such as benzene. The organic liquid into which the droplets are sprayed can be a hydrocarbon solvent or a liquified organic gas such as butane or propane. Alternatively, in step (a), the droplets can be sprayed onto a chilled metal substrate, such as a wire or mesh, to freeze said droplets on said metal substrate. The frozen droplets are then warmed and the micelles polymerized in situ.

A further embodiment of the invention provides a solid composite particle comprising a polymeric core particle enclosed by a polymeric coating having recognition sites spaced on the surface of said coating, which sites selectively bind to a preselected template compound (T); wherein the recognition sites are each defined by a shaped polymeric matrix conforming to the size and shape of T, wherein said sites are formed by (a) binding moieties of the formula Det to the surface of the polymeric core, wherein Det is a moiety capable of generating a detectable signal that can both bind to the polymeric core and reversibly bind to T; (b) applying the polymeric coating to the core in the presence of T to form Det-T moieties on the surface of the core which are surrounded by said polymeric coating, and (c) removing said T moieties from the surface of said composite particulate solid to yield said spaced recognition sites. As shown schematically in Figure 3, free vinylbenzyl piperazine (VBP) is produced from the dihydrochloride 2 by reaction with NaOH. Copolymerization of VBP with hexadecyltrimethylammonium bromide, divinylbenzene and ethylvinylbenzene, in aqueous emulsion, initiated by AIBN thermally or photolytically, yields core particles with pendant piperazinyl groups (11). These groups are reacted with dansyl chloride (DsCl) in the presence of triethylamine to yield dansylated nanoparticles, wherein Dansyl is Det.

The particles are then enclosed in a polymeric shell formed by copolymerizing a mixture of HEMA and TMPTMA containing a template molecule, such as a D- or L- amino acid (HOOC-R), to yield composite particles (13) having surface recognition sites which can selectively bind to T.

Alternatively, the recognition sites can be formed by binding moieties of the formula Det-T to the surface of the polymeric core, wherein T is reversibly bonded (covalently or ionically bound) to Det in a previous step. Det is bound to the core via reaction of functional groups on Det which act to graft Det-T onto (A)n chains.

Step (b) can be accomplished by suspending the polymeric core particles in a suitable liquid medium, such as a high freezing point organic solvent, along with the template molecules and the monomeric components of the coating. The medium is then frozen at a temperature at which the monomer/T mixture associated with the surface and/or interior of the core particles remains liquid. This mixture is then polymerized to yield the coated particles, the medium is thawed, and the template is removed from the coating or shell to yield composite imprinted particles.

Detailed Description of the Invention Polymeric Matrix

The polymeric matrix of the present particles, including both the polymeric core particle and the polymeric coating of the composite particles, is preferably formed of a synthetic organic polymer that can be prepared by polymerizing one or more olefinically unsaturated organic monomers (A), preferably in the presence of a minor but effective amount of a cross-linking agent. As discussed below, at least one chain transfer agent can be included in the reaction mixture, which will act to terminate the polymeric chain -(A)n, while initiating polymerization of a new chain. Thus, the recognition site moiety -(A)n-B represents one terminus of one polymeric chain of the polymeric matrix which defines the recognition site. The remainder of the moiety -(A)n-B can be depicted as X-(A)m- wherein n and m are selected so as to define the average molecule weight of the polymer X-(A)m-(A)n-B and X is preferably H or B. Each moiety A in (A)n or (A)m may be identical or the moieties (A)n or (A)m may represent a mixture of monomeric residues, including residues of multifunctionalized monomers or crosslinking agents that can react to cross-link the polymer chains, as discussed hereinbelow.

Useful monomers (A) include olefinically unsaturated compounds of general formula (R')(R )C=C(R3)Z wherein R1, R2, R3, and R4 are individually H or (C,-C4)alkyl and Z is phenyl, pyridyl, imidazolyl, pyrrolidinyl, tolyl, Si(R2)3aminophenyl, CN, CO2R', ethylphenyl, or C(O)R4 wherein R4 is (C,-C4)alkyl, hydroxy(C,-C4)alkoxy, O(C,-C4)alkyl, phenyl, N(R')(R4), OR5 or SR5 where R5 is phenyl or (C,-C4)alkyl, and the like. Such polymers include polyacrylates, polymethacrylates, polystyrenes, polyesters, polyurethanes, polyamides, polyvinylpyridines, polyvinylimidazoles, polyvinylpyrrolidones, polyvinylethers, polyvinylsiloxanes, phenolic polymers, such as phenol formaldehyde and urea polymers such as urea formaldehyde resins, isocyanurates and derivatives thereof, including derivatives containing furfural and furfuryl alcohol. The monomeric units are preferably selected so that the polymeric particles are essentially stable in the media to which they are exposed during use, including physiological fluids.

Generally speaking, n and m will be selected so that the molecular weights of the polymeric matrices will be between 20,000 and the molecular weight of three-dimensional cross-linked polymers, preferably above 500,000. These molecular weights are determined in accordance with the measurement of light-scattering in corresponding polarometers. The polymers will generally have more than 150 repeating units. In such connection, it is important to note that if the monomer contains a plurality of polymerizable or polycondensible groups, the resultant polymer will be, at least to a minor extent, cross-linked.

The polymerization can be carried out in the absence of a catalyst by subjecting the reaction mixture to the appropriate polymerization parameters, such as light, heat, or pressure. If desired, a catalyst can be used such as, e.g., in radical polymerization: azobis(isobutyronitrile), Igracure™ (Ciba-Geigy), 2,2'- azobis(2-amidino propane) (V-50, Wako, Inc.), dibenzoyl peroxide, potassium persulfate; cumylhydroperoxide, ferric ion and potassium persulfate; in ionic polymerization: TiCl4, BF3, H2SO4, alkali metals, butyllithium, sodium or potassium naphthalide; in insertion polymerization: Ziegler-Natta-catalysts, e.g., Al(Et)3 and TiCl4; or initiation by heat or by ultrasonic waves, UV-, X- and γ- rays.

Since organic polymerization initiators become covalently attached at the ends of polymeric chains (A)n, functionalized initiators can be chosen to bind to template molecules T, while still retaining their ability to initiate polymerization. An example of such a molecule is V-50, which can photolytically or thermally bind to anionic T compounds in a radical form that initiates polymerization. It is particularly desirable to include in the reaction mixture a polyfunctional cross-linking agent. Particularly useful cross-linking agents for polymerization include trimethylolpropane trimethacrylate (TMPTMA) divinylbenzene (DVB), butandiol diacrylate, glycol dimethacrylate, glycol divinyl ethers, adipic acid divinylester, allyl-vinyl ethers, and unsaturated polyesters. For polycondensation, useful crosslinkers include glycerine, cyanuric acid, phenol, melamine, trichlorosilane, hexamethylenetetramine. For polyaddition, useful crosslinkers include 2,4,6-tricyanatotoluene, glycerol, sorbitol, and ethylene tetramine.

Generally, the cross-linking agents are employed in an amount between 0.5 and 80 weight percent, preferably between 25 and 40-wt. percent based upon the weight of the monomer or monomers. However, the monomer can be 100% cross-linkable (DVB) or, in some cases, no cross-linking agent is used. Preferably, particularly in the case of polymerization in the presence of a cross-linking agent, the polymerization is effected in the presence of an inert solvent or solvent mixture. Other useful monomers, monomer mixtures, catalysts, and crosslinkers are disclosed in G. Wulff et al. (U.S. Patent No.4,111,863), K. Mosbach (U.S. Patent No. 5,110,833), and Table 1, hereinabove, including the references cited therein.

The polymers can be selected to be biocompatible, in the case of particles intended for administration to mammals, e.g., for human therapy.

Preferably, the finished particles are 10-100 nm in diameter. Such particles can readily be formulated into solutions or suspensions. Chain Transfer Agent (OTA)

Chain transfer agents (B) useful to prepare the present particles include organic compounds containing active hydrogen atoms such as SH, NH, or allylic hydrogens, e.g., thiols, 3,3-dialkyl-l-propenes, allyl amines, ethers, esters, or amides. CTAs do not form high polymers under the conditions employed to form the polymeric matrix. Instead of adding to the C=C of an allyl group to propagate a polymer chain, an attacking radical will tend to abstract one of the CTAs allylic hydrogens to terminate growth of the current chain, and generate a stable allylic radical able to reinitiate a new chain. This process is called chain-transfer and several kinds of chain-transfer agents, particularly thiols, can be added in small amounts to polymerizing monomers to limit the molecular weights of the resulting polymers. See, G. Odion, Principles of Polymerization. Wiley-Interscience, Toronto, Canada (1991). Though such compounds cannot be said to polymerize or copolymerize with monomers, chain- transfer does allow them to become bound at the ends of the resulting polymer chains. The reactions of allylic chain-transfer agents during radical polymerization are shown in Scheme 1, below. Scheme 1.

Termination

Chain- transfer

Re-initiation

An example of the preparation and use of a thiol-containing CTA in the preparation of composite particles is shown schematically in Figure 4. Transformation of polyethylene glycol monomethyl ether ( 10) to the chloride (15), then to the thiol (14) by the method of Bayer et al., Polym. Bull, 8, 585 (1982) gives a chain-transfer agent that can add polyoxyethylene groups to ((OCH2CH2)n) to nanoparticle surfaces at some point during the first polymerization, to give both core and later core/imprinted shell nanoparticles capable of remaining suspended in water without the need to use large amounts of other exogenous surfactants. Copolymerization of monomers divinylbenzene and ethyl(vinyl)benzene with the hydrophilic aminoalkanethiol (7) results in surface template-binding groups necessary for eventual molecular imprinting, as shown for core nanoparticle (16). Core particle (16) can then be labelled with dansyl chloride (DsCl) to yield surface-labelled core particles (17). These particles are coated with a polymeric shell by copolymerization with HEMA and TMPTA in the presence of a template molecule (T) such as a D- or L- amino acid (RCOOH) to yield imprinted composite particles (18), wherein the polymeric coating comprises ethylenoxy groups and B-Det-T moieties, (- N(Ds)CH2CH2NH Me2)+RCOO ).

A template-complexing initiator, though it is not a polymerizable monomer, will in general be covalently attached to a polymer matrix (at one end of each polymer chain), as shown in Figure 5. In Fig. 5, 2,2'-azobis(2- amidinopropane) dihydrochloride (4) ("V-50," Wako Inc.), is reacted with 2 moles of cAMP in step 1, and exposed to heat or light to yield intermediate radical 2; which initiates the copolymerization of HEMA and TMPTMA to yield an imprinted polymeric matrix (shaded area) having a recognition site (defined by the curved line) complexed with cAMP. Removal of the cAMP yields a shaped recognition site comprising a H2N-C=NH2 +C1* functionality. In the practice of the present invention, initiator or CTA compound B will also comprise one or more functional groups which can reversibly bind to template compound T. Such functional groups include CO2H, C=O, SO3H, boronic acid, phosphonic acid, phosphoric acid mono- and diesters, amino, imino, acylamino, nitro, OH, SH, (C,-C4)alkylamino- or ammonium, hydroxylamino or hydrazino, and the like. For example, the B-T bond can be due to, or enhanced by, π-π interactions, hydrogen bonding, dipol interactions, electrostatic interactions, charge-transfer complexes, chelation and the like. For specific examples of such interactions, see G. Wulff et al. (U.S. Patent No. 4,111,863).

Either the initiator or chain-transfer type of template-complexing and polymer-binding "non-monomers" (fluorescent or otherwise) have the following advantages over the functionalized monomers which have been employed for molecular imprinting;

(1) Ease and versatility of synthesis: as shown in Figure 6, allyl derivatives capable of chain-transfer, and including fluorescent Det and other functionality, can be made simply by reaction of appropriate nucleophile such as compound (5_), with an allyl halide to yield B-Det compound (6-Br). In Figure 6, 6-Br is then reacted with template cAMP to yield 6-cAMP.

An electrophile is reacted with an allyl amine or allyl alcohol. A thiol group conferring chain-transfer ability can be introduced into B by a convenient and general "mercaptoethylation" reaction, either with episulfide alone under controlled conditions to yield compound 7 (S.J. Lippard et al., J. Am. Chem. Soc. 98. 6951 (1976)) or in the presence of activating chlorotrimethylsilane, to yield compound 8.

(2) Stability during purification and storage. It is sometimes difficult to prevent rapid polymerization of many functional monomers once they are made. Inhibitors may have to be added, that then have to be removed again before polymerization. Concentrated non-monomers do not polymerize, and so store much better.

(3) Stability after binding to polymer: allylic functionalities that have become bound to polymer matrix via carbon, or thiol-containing compounds that bind via sulfide links, do not exhibit the potential hydrolytic or other instability of the ester or amide links of acrylic or methacrylic derivatives, nor of the benzylic links of vinylbenzyl derivatives.

(4) Position and distribution in product: unlike functional monomers, functional initiators and chain-transfer agents would show little or no undesired tendency to bind to each other in preference to a growing polymer matrix. This can avoid the possibility of a hydrophilic template-complexing compound (B or B-Det) forming a soluble polymer in the solvent phase instead of in the polymer phase near an imprinted cavity. The resulting binding sites would also be more evenly distributed over a solid polymer surface, and so better defined and more selective, than sites which can form using the imprinting species that are able to bind via covalent bonds directly with each other. Detectable Moiety

Compound B will further preferably comprise a detectable moiety (Det) which then becomes held in proximity to, and preferably associated with, the recognition site. Preferably, Det also can bind to T. Det moieties can generate detectable signals before and after binding to T. Thus, the Det moiety can contain a fiuorescing component or fluorophore, such as fluorescein, DMASP, rhodamine, dabsyl or dansyl; a magnetic component, such as a magnetic oxide; an electron-dense component such as ferritin; or a radioisotope. For example, see Engelhardt et al. (U.S. Patent No. 5,241,060). Preferred Det moieties alter their detectability when T is bound to B, thus providing a means to determine the extent to which T can occupy the recognition sites.

The use of fluorescence as a means of signal transductions is highly preferred, as it is a highly sensitive detection technique which necessitates only very small quantities of fluorophore. Fluorescence measurements also permit the observations of both excitation and emission wavelengths, in a non destructive manner. The fluorescence emission may be monitored as lifetime measurements, rotation anisotropy, intensity, and intensity ratio.

Fluorescence lifetimes can be measured by depolarization, and wavelengths of excitation or emission bands by scanning the relevant spectra, but emission intensities at chosen fixed wavelengths are easiest to accurately measure. Preferably, the modes of fluorescence of a chemosensor should be distinctly different between the bound and unbound states, the latter acting as an internal standard so that the ratio of the two can provide (after standardization) the concentrate of substrate, in a manner independent of the concentration of chemosensor, thickness or other geometry of the sample, or optics of the particular measuring instrument (such as a fluorescence microscope to study individual cells loaded with chemosensor). Other requirements for an effective fluorescent chemosensor or "molecular probe" include: that the dissociation constant for host-guest interaction be in the range of interest for the substrate concentration; that excitation be generable and emission be measurable with available equipment; that both binding and fluorescent portions of the whole fluorescent chemosensor molecule be chemically stable. The dyes exemplified hereinbelow (dimethylaminostyrylpyridinium and dansyl derivatives) do not shift emission wavelengths or quench with any specific chemical interaction with the templates, but rather, rely on the physical change in their proximity, such as the change in polarity, viscosity or some form of radiative energy transfer. (J.P. Gao, J. Amer. Chem. Soc. 114. 3997 (1992); K.J. Shea et al., Macromol.. 22, 1722 (1981)). These effects have been studied on these systems both as small molecules and polymer bound versions. They also do not vary significantly with small changes in pH, or photobleach easily. These dyes are preferably immobilized onto the polymer matrix by polymerization of pendant monomeric units, a chain transfer mechamsm involving the pendant chain transfer groups B, or simply by nucleophilic substitution after polymerization, as in the case of the composite particles.

Besides choosing the detectable polymer-binding component B- Det to be able to associate with the template during and after the polymerization, it would also help for different portions of B-Det to associate, during the polymerization, either with the very polar protic liquid phase (e.g., ions, OH groups), or with the less polar monomer or gel phase (e.g., aromatic groups). Such amphiphilic or surfactant complexes would thus tend to congregate at the polymer/liquid interface, so that cavities would be formed that are already partially open for exchange of substrate with surrounding liquids.

As an example of an olefinically-unsaturated B-Det compound, that functions as a comonomer with A during polymerization, compound (1) was prepared as shown in Figure 7, by reacting m-vinylbenzyl chloride with 4-(4- dimethylaminostyryl)pyridine. Compound (1) is a fluorescent molecule which can closely associate with negatively-charged aromatic or heterocyclic templates through ionic and aryl-stacking forces. The general chemical structure of N-substituted A-p- dimethylaminostyrylpyridinium compounds (R-DMASP+), such as 1, permits them to be photoexcited to either normal planar (NP) or twisted intramolecular charge transfer (TICT) energy states, enabling dual fluorescence in the same molecule. (See, W. Retig, Angew. Chem. Int. Ed. Engl., 25, 971 (1986).) In either single-excitation/double-emission or double-excitation/single-emission experiments, one of the two modes of such dual fluorescence is much more sensitive than the other to changes in polarity or rigidity of the molecule's microenvironment, such as may accompany binding of T into a nearby attached binding site. This makes possible the use of TICT-capable molecules in 'ratioing' techniques of fluorescence measurement to probe polymer and other environments, K. H. Hamasaki et al., J. Amer. Chem. Soc, 115, 5035 (1993); D.W. Cho et al., J. Phys. Chem., 98, 558 (1994).

The 5-dimethylaminonaphthalenesulfonyl or dansyl group also changes its fluorescence pattern according to the polarity and other characteristics of its microenvironment, typically by a shift in wavelength of its emission band, which is referred to as solvatofluochromism. (See, K.J. Shea et al., Macromolec, 22, 1722 (1981).) Though this molecule does not exhibit dual fluorescence, ratioing techniques can still be used for fluorescent chemosensors employing it, for example, between the signals received from one detector slit adjusted to a higher-wavelength portion of the shifting band, and another to a lower- wavelength. The synthesis via 2 of 1, an olefinically-unsaturated molecule containing dansyl whose protonated form can also complex anionic templates, is shown on Figure 8. Template Molecule (T)

A wide variety of template molecules can be employed to form the recognition sites, so long as they can be oriented to present a given molecular configuration to the surface of the polymer matrix. The template molecule used to impart the desired molecular configuration to the recognition site may consist of the target molecule itself (the end-product), a derivative of that target molecule, a transition state molecule in a catalytic reaction sequence, or a molecular mimic or model of the above.

After the polymerization, the template molecule T is partially or completely removed from the polymer. The removal is performed with known cleaving agents which dissolve the link to the functional groups on B (or A) so that, on the one hand, optically active or achiral T compounds are formed and on the other hand, the polymers develop which contain the functional radicals at the points at which the optically active or achiral compound was extracted, these radicals being bound to the polymer in the steric configuration predetermined by the matrix. The dissolution of the optically active or achiral compound from the polymer matrix can be performed, for example, by hydrolysis with water, by acid hydrolysis, acid alcoholysis, alkaline hydrolysis, hydrogenation, exchange reactions with low-molecular amines, aldehydes, etc., double-bond cleavage, glycol cleavage, reduction, or oxidation. The particular reaction or reactions chosen depend upon the nature of the linkage to be cleaved. Neutral hydrolysis can be performed using water or mixtures of water with Cr to C8- alcohols or mixtures of water with acetone or other water-soluble solvents at room temperature or at elevated temperatures ranging from 30C to 150C. The resultant recognition sites bind selectively to T in the presence of one or more preselected non-T molecules, such as analogs, isomers, derivatives of T or T intermediates. Typical T molecules include bioactive compounds such as a drug, vitamin, metabolite, nucleotide, nucleic acid, carbohydrate (sugars), protein, toxin, steroid, amino acids, prostaglandin, hormone, biocide (pesticide, herbicide, fungicide, etc.) or cytokine.

The resultant imprinted particles can be used as (a) separation materials, to accomplish stereo- and regioselective separations, as to resolve dl mixtures of drugs, or as antibody mimics in assays; (b) enzyme mimics which exhibit substrate selective catalytic behavior, e.g., by preparing recognition sites against transition state analogs; and (c) as sensor components. See K. Mosbach, TIBS. 19. 9 (1994). Methodology to Prepare Imprinted Polymeric Nanoparticles

As shown in Fig. 9, a small quantity of B-Det compound (1) can be combined with excess template (T), such a sodium cAMP, along with monomer A (hydroxyethyl methacrylate (HEMA)), crosslinking monomer trimethylolpropane trimethylacrylate (TMPTMA), solvent, and free-radical initiator azobis(isobutyronitrile) (AIBN). Polymerization can be initiated either with heat or, at room temperature, irradiation with ultraviolet light, or by a combination of the two. It is expected that, under these conditions, the anionic cAMP moiety would preferably associate with the cationic fluorescent dye as shown by the bracketed intermediate complex (B-Det-T) as B becomes part of the surrounding polymer matrix (shaded area). Following removal of cAMP, the resulting porous solid is then ground to finer particles, that are then washed extensively to give imprinted polymer fluorescent chemosensor 4a, which can later be suspended in water containing different concentrations of c AMP or cGMP, while being monitored by spectrofluorimetry. As controls, similar imprinting is conducted with cGMP, or in the absence of nucleotide template altogether.

A preferred method of preparing crosslinked polymer nanoparticles is by thermal- or photochemical-initiated polymerization of a microemulsion of micelles of a di- or poly-functional hydrophilic monomer in water, which micelles are preferably stabilized by a surfactant layer (Turro et al., Macromolecules, 20, 1216 (1987); Antonetti et al., Macromolecules, 24, 6136 (1991); Antonetti et al., Macromolecules, 25, 1139 (1992); Wang et al., Prog. Polym. Sci., 19, 703 (1994)). A complex of template compound (T) with B, Det or B-Det, that can bind to monomer A or to (A)n, and that is strongly amphiphilic (i.e. hydrophobic/hydrophilic), can help to stabilize such micelles. Such a complex can function as an "internal surfactant," to position a hydrophilic template compound (T) at the surface of the micelle so that the recognition site that forms around it will be a cavity in the particle that is open on one side, as shown in Fig. 2. The uniform depth, orientation and distribution expected of the complexed template as it "floats" as part of a surfactant's head group should result in imprinted recognition sites that are similarly uniform with respect to shape, exposure to the liquid phase, relative position to nearby functionalities that are capable of binding, detectability, such as fluorescence and the like. In practice, several factors can complicate this approach. Thermodynamically-stable microemulsions of significant quantities of monomer are in fact difficult to achieve, particularly with relatively small quantities of effective surfactants and as little as possible of others. Dense packing of template-complexing polymer-binding molecules at the monomer/water interface would likely result in less well-defined imprinted cavities. Though thermodynamically unstable microemulsions of monomers with smaller quantities of less-effective surfactants, as prepared by vigorous agitation with a homogenizer or sonicator, can be still kinetically stable for hours or days, attempts to polymerize these often result in rapid coagulation as certain droplets containing free radicals recruit more monomer from outside their boundaries and grow at the expense of other (Odian, Principles of Polymerization, Wiley- Interscience, Toronto, CA (1991)). Such particles can also agglomerate as they pass through a "sticky phase" where a single glancing collision will irreversibly fuse one to another. Surfactants that are polymer-binding can also be covered over during such growth of crosslinked polymer particles.

Alternatively, a surfactant complex, existing in equilibrium between the droplet interface and the aqueous phase, may find itself excluded as polymerization proceeds in the intervals of its absence, so that whether or not the polymer-binding component ultimately becomes polymer-bound, an imprinted cavity has not been defined by the template in the polymer matrix.

Several of these problems can be circumvented at once if the continuous (i.e. aqueous) phase of the microemulsion is solidified, most simply by rapid freezing. This prevents exchange of monomer, to micelles that are polymerizing, from micelles still awaiting initiation. Surfactants also cannot diffuse away from the monomer droplet, because they would still be held at the surface through hydrogen-bonding or other adhesion to the ice. The low temperatures required to solidify the continuous phase generally preclude thermal initiation of the polymerization. However, since ice is reasonably UV- transparent, photochemical initiation is still possible, so long as the photoinitiator has been included in the monomer mixture.

Since the freezing process itself can cause coagulation of suspended material as the pure solvent crystallizes, freezing the continuous phase as rapidly as possible is desirable to prevent such crystallization. A number of fast-freezing techniques have been developed in the field of microscopy, for 'freeze-fracture' preparation of samples that can prevent distortion and agglomeration of subcellular components and other microscopic entities (Roberts et al., Freeze Fracture Images of Cells and Tissues, Oxford V. Press, N.Y. (1991). One of these is to spray, e.g., with an airbrush, a fine aerosol of the suspension into a liquid organic phase such as liquid propane that has been cooled to liquid nitrogen temperature (-190C). Liquid nitrogen itself flashes into an insulating vapor as it starts to cool anything immersed in it.

After spraying, the liquid propane can then be replaced with another less volatile hydrophobic liquid (e.g., hexanes), as the temperature is raised (as to -14C, in an ice-salt bath) so that the frozen droplets remain solid, but the monomer mixture in the enclosed micelles liquefies prior to photochemical polymerization. Afterwards, simply evaporating the hexanes and melting the ice gives a suspension of nanoparticles in water. The template can then be removed by procedures such as dialysis or ultrafiltration.

The invention will be described by reference to the following detailed examples, wherein dynamic light-scattering was obtained using a Brookhaven BI-2030AT dynamic light-scattering photon correlation spectrometer. !H NMR spectra were obtained on a JEOL 270 MHz spectrometer. FT-IR spectroscopy was performed on a Bruker IFS-48. Spraying of the monomer solutions was done using a Badger model 100 air brush. All solvents used were reagent grade or better, and were used without further purification unless specified. Fluorescence emission was recorded on a Spectro SPEX 1680, 0.22m double spectrometer, SPEX industries (Edison N.J.). Spectrograde solvents were used for analysis only. 4-(p-Dimethylaminostyryl)pyridine (DMASP) was prepared by a published procedure (A.N. Kost et al., J. Gen. Chem. USSR, 84, 4106 (1964)). Monomers and initiators were purchased from Aldrich Chemical Co. cAMP and cGMP were used as received from Sigma

Chemical Co. Aqueous solutions were prepared using double distilled deionized water (Millipore) with a pH at 5.9. Inhibitor was removed from all monomers by passing them through a column of activated aluminum oxide (Aldrich).

Example 1. N-(m-Vinylbenzyl)-4- >-dimethylaminosryιyIpyridinium chloride (1). DMASP (0.508 g, 2.26 mmol) was weighed into a 50 mL round flask equipped with a magneic stirrer, reflux condenser with nitrogen inlet, and heating mantle. Toluene (25 ml) was added to the flask, and the system was flushed with nitrogen, then heated to 60C. m-Vinylbenzyl chloride (3.03 g, 19.9 mmol) was then added dropwise. After overnight (-18 hrs) heating under nitrogen, thin-layer chromatorgraphy (ethanol :toluene 3 :7/SiO2) of the red reaction mixture indicated no DMASP starting material remained. The mixture was cooled to room temperature, and the red-orange precipitate that had formed was collected by filtration, washed several times with hot toluene, then dried under vacuum at 50C, to afford red-orange crystals of 1 (0.198 g, 23% yield) that were stored in a dessicator in the dark: mp 225-227C; 'H-NMR (CDC13) δ_9.0-6.5 (m, 15H, ArH & Ar-CH=CH2), 5.9 (s, 2H, ArCH2Pyr+), 5.5 (dd, 2H, R'-CH=CH-R2), 3.05 (s, 6H, NCH3)2).

Example 2. N-(m-VinylbenzyI)piperazine dihydrochloride (2). Dry piperazine (6.45 g, 75 mmol) was dissolved in 25 mL of dry ethanol. m-

Vinylbenzyl chloride (7.69 g, 49.3 mmol) was then added in one portion and the mixture stirred on a cold water bath (10C) for 1 hr. The solution was cooled to - 13C for 2hrs and the resulting white precipitate removed.The filtrate was acidified with dry HCl and cooled to -10C for 30 min.. The resulting white plates were filtered off and dried in vacuuo, to give 2 (12.89 g, 94%): mp 250- 253 C (dec); 'H-NMR (CDC13) δ 7.53-7.26 (m, 4H, ArH), 6.75-6.62 (m, IH, =CH), 5.83-5.73 (d, IH, =CH), 5.3-5.2 (d, IH, =CH), 4.33 (d, 2H, ArCH2N), 3.48-3.46 (d, 8H, CH2).

Example 3. N-Dansyl-N'-(w-vinylbenzyl)piperazine (3). N-(/w-

Vinylbenzyl)piperazine dihydrochloride 2 (1.19 g, 4.3 mmol) was dissolved in 10 ml distilled water, then 15 ml acetone was added with stirring, then IN NaOH to pH 12 (about 10 ml). Dansyl chloride (0.99g, 3.6 mmol) in 20 ml acetone was then added slowly. After stirring the mixture 15 min at room temperature, the acetone was removed by rotoevaporation, and the resulting precipitate collected by filtration and dried in vaccuo, to give 3 (1.50 g, 95%): one spot by TLC (EtOH), mp 116-120C (dec), 'H-NMR (CDC13) δ 8.65 (d, IH, ArH), 8.45 (d, IH, ArH), 8.15 (d, IH, ArH), 7.73-7.26 (m, 7H, ArH), 6.75-6.62 (m, IH, =CH), 5.83-5.73 (d, IH, =CH), 5.3-5.2 (d, IH, =CH), 4.03 (d, 2H, ArCH2), 3.45-3.3 (m, 4H, CH2NRSO2), 2.96 (d, 6H, ArNCH3), 2.52 ppm (dd, 4 H, CH2NR2).

Example 4. Bulk Fluorescent Chemosensor for cAMP (4-cAMP). Sodium cyclic adenosine 3',5'-monophosphate (Na+ "cAMP, 41.7 mg, 118.7 μmol) was weighed into a glass specimen bottle, followed by N-(/w-vinylbenzyl)-4-p- dimethylaminostyrylpyridinium chloride 1 (31.5 mg, 83.6 μmol) and 30 mL methanol. The mixture was shaken and sonicated until all solids were dissolved, then were added trimethylolpropane trimethacrylate (TMPTMA, 29.0 g, 86 mmol), 2-hydroxyethyl acrylate (HEM, 0.675 g, 5.19 mmol), and finally 2,2'- azobis(isobutyronitrile) (AIBN, 0.665 g, 4.05 mmol). The bottle was purged with nitrogen for 10 minutes, then placed in a Rayonet Photochemical Reactor for 1.5 h, then into an oil bath at 60C for 24 h. The solid polymer mass, free of smell of TMPTMA or HEM, was released by breaking the bottle, wiped free of broken glass, then ground with a mortar and pestle, and wet-sieved with water to collect 45-106 μm particles. These were placed in a soxhlet extractor for washing by 300 mL refluxing H2O:CH3OH 7:3 v:v for more than 24 hours, then 300 mL pure methanol for more than 24 hours, then collected by filtration, and dried under 20 mm Hg vacuum at 35C overnight, and stored in air-tight vials in the dark, giving 4a.

Example 5. Bulk Fluorescent Chemosensor Control (4-CI). As for 4_a above, but omitting Na+ "cAMP or other nucleotide as template.

Example 6. Fluorescence Measurements of Bulk Fluorescent Chemosensors. Fluorescence of 4-cAMP and 4-CI were evaluated on 150 mg samples that had been shaken in 4 mL double-distilled water overnight, seperated by centrifugation, then resuspended in 1.250 mL distilled water in a Hellma 1-cm triangular cuvette. After brief shaking, this was immediately placed in the temperature-controlled (25.00.1C) cuvette holder of a Photon Technology International (PTI) Deltascan 4000 spectrofluorimeter, adapted for front-faced illumination of opaque samples, with excitation slits at 3 nm and emission slits at 2 nm, with a polarizer in the emission beam to reduce scatter. A fluorescence emission spectrum for excitation at 469 nm was acquired in triplicate each 10 minutes (shaking each time) until a stable reading was attained, whose average maximum intensity near 600 nm was taken to be I0. Afterwards the cuvette contents were transferred back to the vial, and the solid contents spun down and the liquid decanted, and 1.250 mL of sample solution (such as Na+ " cAMP/H2O) was added. After incubating in a shaker bath at 25C for 1.5 hours, the contents of the vial were poured into the cuvette, and fluorescence emission spectra acquired in triplicate each 15 minutes for 45 minutes, and the (by then stable) average taken to be I.

For each sample, fluorescence measurements with an exciting wavelength of 469 nm were performed in water and in the presence of aqueous solutions of substrate, to give the maximum emission intensity (590 nm) in the absence (lo), and in the presence (I) of substrate. In order to estimate the association constant for the binding of c AMP to the nonimprinted and imprinted polymers, plots were constructed using the static quenching relationship [J.R. Lakowicz, Principles in Fluorescence Spectroscopy:, Plenum: NY (1983)]:

Io/I = 1 + Ka[Q]

where, lo = the maximum emission intensity in the absence of substrate, I = the maximum emission intensity in the presence of substrate, Ka = the association constant of complex formation, and [Q] = the concentration of quencher.

Figure 10 shows a representative example of the polymer binding time course for the nonimprinted and imprinted polymers. The template, cAMP, was added at time 0 min, and equilibration for both was attained between 60 and 90 min. Thus, in further binding experiments, measurements were performed at

15 min. intervals from 90 to 135 min. A difference in the behavior of the polymers is visible as a greater quenching of the imprinted polymer compared with the nonimprinted polymer.

The association constants (Ka) for the binding of cAMP to the corresponding polymers were estimated from the cAMP quenching plots shown in Figure 11. (Note that the x-axis is represented in a log scale for the sake of illustration, however, a linear scale was used for all calculations.) In both cases, nonlinear plots were obtained indicative of a heterogeneous population of binding for the template molecule. This is consistent with previously reported data [G. Viatakis et al., Nature, 361, 645 (1993)]. In each case, the plots were linear over the low concentration range of 10"8 - 5 x 10'6 M cAMP, which is consistent with the print molecule loading of 3.96 μmol/g polymer. Above 5 x 10"6 M cAMP, saturation of the binding sites appears to occur. Association constants, and y-intercepts for the low concentration sites were 4.15 x 103 M"' and 0.99; and 1.52 x 104 M'1 and 1.03 for the nonimprinted and imprinted polymers, respectively. To eliminate the contributions of nonspecific interactions, the nonimprinted value was substracted from the imprinted value to obtain an association constant for the specific binding of cAMP to the imprinted sites of 1.11 x 104 M"' [Shea, J. Amer. Chem. Soc, 115, 3368 (1993); Sellergren, Macromol Chem., 190, 2703 (1989); Sherrington et al., Polymer, v34, 1, 77-84 (1993)].

This data may also be represented by a quenching value for the specific binding of cAMP to the cAMP imprinted cavities obtained by substracting the individual nonimprinted from the imprinted cAMP quenching values at each concentration of cAMP. This quenching value representing the specific binding of cAMP may then be plotted (Figure 12), yielding an identical association constant to that previously calculated (1.11 x 104 M'1). The bars on the plots of Figs. 11-12 indicate standard deviation. This association constant is an order of magnitude higher than that reported by Rebek, J. Amer. Chem. Soc, 116, 3269 (1994) for a water soluble Kemp's triacid imide receptor for cAMP (Ka=6006% M"' in 1 OmM cacodylate buffer, 1.0 mM Aerosol O.T. at an ionic strength of 5 ImM NaCl), and by Conn, J. Amer. Chem. Soc, 115, 3548 (1993) for aqueous receptors for 9-ethyladenine (Ka=351 M*' in methanol). However, it is comparable to polymer complements for 9-ethyladenine binding in chloroform (Ka=7.9 x 104 M-') [Shea, ibid. 1993], despite the fact that the binding here occurred in aqueous media. Molecularly imprinted polymers prepared for the drugs, theophylline and diazepam were reported to have association constants in the range of 109 to 106 M"' for binding in organic solvent mixtures [Viatakis, ibid. 1993].

The selectivity of the cAMP imprinted polymer was assessed by investigating the binding of cGMP, since its phosphate/sugar moiety is similar to that of cAMP, but its base differs. The polymers demonstrated selectivity for cAMP over cGMP in the substrate concentration range 1.00 x 10"' to 1.00 x 10"5 M, as seen by the quenching values for the specific binding in Table 2 below. cGMP produced almost no response compared to cAMP.

Table 2. Quenching (Io/ISB) values for the specific binding of cAMP and cGMP to a cAMP imprinted polymer at various concentrations of substrate3.

[substrate] (M) Io/ISB cAMP Io/ISB cGMP

1.00 10"' 0.054

1.00 x lO"3 0.2321.98 x l0"2 (n=6) 0.039

1.00 x lO"5 0.2235.81 x lO"2 (n=3) -0.01

aThe cGMP quenching values are the results of a single experiment. Error is standard error in the case of cAMP values.

Finally, an interesting feature of these fluorescent sensor polymers is their ability to be recycled or reused. It was possible to reextract cAMP from the particles used in a binding experiment, and obtain similar binding results when the particles were reused, as demonstrated in Table 3 below. Table 3. Quenching (Io/ISB) values for the specific binding of cAMP to a cAMP imprinted polymer after 1st and 2nd template extractions at various concentrations of cAMP\

[cAMP] (M) 1st template extraction 2nd template extraction

1.00 x lO"3 0.2321.98 x 10' (n=6) 0.268 1.00 x lO"6 0.065.69 x l0-3 (n=3) 0.058 1.00 x lO'8 0.0180.001 (n=2) 0.019

aThe quenching values for the 2nd template extraction are the results of a single experiment. Error is standard error for 1.00 x 10_3and 1.00 x 10'6M cAMP, and standard deviation for 1.00 x 10"8 M cAMP.

Example 7. N-Dansyl-N',N'-dimethylethylenediamine (5). Dansyl chloride (0.8450 g, 3.13 mmol) was dissolved in 50ml CH2C12, and N,N- dimethylethylenediamine (0.28 g, 3.18 mmol) was added all at once. The yellow solution was refluxed 15 min, whereupon it became green. After cooling, the solution was extracted with saturated K2CO3/H2O, and the organic phase dried over MgSO4, and evaporated to dryness, yielding hard glassy material 5 (0.91 g, 91%).

Example 8. N-AHyl-(N'-aIlyI-N'-dansylaminoethyl)-N,N- dimethylam onium bromide (6-Br). N-Dansyl-N',N'- dimethylethylenediamine 5 (0.85 g, 2.6 mmol) was dissolved in dry tetrahydrofuran under nitrogen, then sodium hydride (0.10 g, 4 mmol) was added in portions under nitrogen. After stirring for 30 min, allyl bromide (0.5 mL, 6 mmol) was added and the mixture refluxed overnight. After cooling, the solution was filtered to remove white precipitate, and the filtrate evaporated, giving a yellow-green glass 6-Br (1.21 g, 97%): TLC 1 spot (EtOH); 'H-NMR (CDC13) δ 8.5 (d, IH, ArH), 8.3-8.1 (m, 2H, ArH), 7.7-7.5 (m, 2H, ArH), 7.2 (d, IH, ArH), 6.0-5.7 (m, 4H, =CH2), 5.5 (m, 2H, -CH=), 4.5 (m, 2H, CH2N+), 4.1 (d, 2H, CH2NSO2), 4.0-3.8 (m, 4H, CH2-C=), 3.4 (s, 6H, CH3N+), 2.9 (s, 6H, ArNCH3). Example 9. N-Allyl-(N'-allyl-N'-dansyIaminoethyl)-N,N- dimethylammonium cyclic adenosine 3',5'-monophosphate (6-cAMP). N-

Allyl-(N'-allyl-N'-dansylaminoethyl)-N,N-dimethylammonium bromide 6-Br (1.1 Og, 2.28 mmol) was dissolved in 22.25 mL of 90% EtOH/H2O, and 11 mL was then taken from this (1.13 mmol) and added to silver acetate (0.1890 g, 7.12 mmol). After decanting from the resulting precipitate, the supernatant was mixed with cAMP acid (0.3911 g, 1.19 mmol). This solution was then evaporated, and dried under vacuum for three days, giving golden glass 6-cAMP (0.9012 g, 109%): 'H-NMR (D2O) δ 8.5 (d, IH), 8.3-8.1 (m, 4H), 7.7-7.5 (m, 2H), 7.2 (d, IH), 6.18 (s, IH), 6.0-5.7 (m, 4H), 5.5 (m, 2H), 4.9 (m, IH), 4.7 (d, IH), 4.5 (m, 2H), 4,4-4.2 (m, 4H), 4.1 (d, 2H), 4.0-3.8 (m, 4H), 3.4 (s, 6H), 2.9 (s, 6H). This was then diluted with 11.50ml D20. 0.100M solution.

Example 10. N-(2-Mercaptoethyl)-N' ,N'-dimethy .ethylenediamine hydrochloride (7). N,N-dimethylethylenediamine (1.00 g, 11.3 mmol) was dissolved in warm benzene, and filtered ethylene sulfide (1.00 g, 16.6 mmol) was then added in one portion and the solution stirred at 75C for 2 hrs, then at room temperature for 3 hrs. 25 mL of distilled water was then added, and the benzene layer was then separated and dried over MgSO4, filtered, then chlorotrimethylsilane (1.5 mL, 12 mmol) and hot ethanol (5 mL, 90 mmol) were added. Cooling of the solution 1 hr, then filtration, gave white solid 7 (1.6 g, 67%): mp 70-76C; positive lead acetate test for free thiol.

Example 11. N-Mercaptoethyl-4- ?-dimethylaminostyrylpyridinium chloride (8). DMASP (0.23 g, 1 mmol) was dissolved in 25 mL CHC13 in a 50 mL round flask under nitrogen, and chlorotrimethylsilane (0.11 g, 1 mmol) was added, then filtered episulfide (0.10 mL, 1.7 mmol) was added and the red mixture refluxed gently overnight. The bright red solution was then poured into 100 mL CC14, and the resulting bright red precipitate collected and dried under vacuum at 70C, giving 8 red solid (0.16 g, 50%): mp 185-190C; 1 spot by TLC (CHCl3:tol l :l). Example 12. 6-cAMP Polymer (9)

As summarized in Table 4, two samples were prepared in parallel, one containing the imprinting template, cAMP, and one containing chloride ion.

Table 4. Control Mixture Template Mixture

6-C1 in water (lO l 0.100M) 6,-cAMP in water (10μl 0.100M)

1.0031g DVBa 1.0099g DVB

0.079 lg lRGACURE 0.081 lg lRGACURE aDivinyl Benzene

The two mixtures were each emulsified in 10 ml degassed water using the ultrasonicator for 30 min. (The Polytron homogeneizer did not change the consistency of the solution). The resulting emulsions had a pink fluorescence for the 6-cAMP, and a green fluorescence for the 6.-C1. 25ml of propane passed through a tube full of KOH was condensed in a 100ml round- bottomed flask with a side arm, bathed in a Dewar flask of liquid nitrogen. The temperature was carefully monitored and remained at -170 to -155C during the subsequent spraying process. The spraying was done using an air brush connected to a nitrogen outlet set at 30psi.

Short bursts of droplets were sprayed in at a steady rate while mintaining the temperature at the appropriate range. About 5ml of the 6-cAMP emulsion was sprayed in and about 8ml for the 6-C1. The propane was evaporated off placing the propane flask in a ice salt bath at -20C for 5 min and the ice was resuspended in cold hexanes. The flasks were then purged with nitrogen and placed in a ice-salt bath at -21.7C (cAMP pink, Cl Green Fluorescence) and irradiated for 2.5 hrs., until the temperature reached -6.1C. The ice was allowed to thaw and the aqueous phase pipetted out (cAMP bluegreen fluorescence, Cl yellow-green), yielding 2.4ml of liquid for cAMP and 8.8ml of liquid for Cl. Both liquids were at pH 4.4 and both had a slight organic smell. 2.3 ml of the liquid retentate were diluted to 5 ml with water and 3 drops of saturated NaCl and dialysed overnight in 2L of distilled water.

The water had a slight fluorescence (blue) after the dialysis but the dialysate was clearly much more fluorescent. (9-cAMP blue, 9-C1 yellow). The volume was adjusted to 5 ml and % solids was determined by evaporating off lOOμl of the solution.

The dialysates were found to contain 1.70% solid for the Cl and 1.74% for the cAMP.

FT-IR (KBr) (cm''): 3500-3300 (NH), 3100-2800 (CH), 1630 (C=C), 1510, 1486, 1147, 990 (C=C), 903, 836, 797, 706; Particle size: Cl; 240nm very polydispered; cAMP; 60nm very polydispersed.

Example 13. Nanoparticle Fluorescent Chemosensor for cAMP: One-Step Method From Mixture With Template Salt (10-cAMP). 55:45 Divinylbenzene:ethylstyrene (0.5510 g, 2.3:1.9 mmol), 0.100 M N-allyl-(N'- allyl-N'-dansylaminoethyl)-N,N-dimethylammonium bromide 6-Br/H2O (0.1 mL, 10 μmol), Na+ cAMP (0.0250 g, 71 μmol), and Irgacure 184 (0.0511 g) were suspended in 24.50 mL double-distilled water, sonicating for 30 minutes to give a slightly turbid yellow-green fluorescent suspension. This was sprayed into 30-50 mL liquid propane as for 9, which was then replaced by 20 mL chilled hexanes in a -13C ice-salt bath for 6-hour irradiation to give baby-blue fluorescent ice. After thawing and stirring for 30 minutes, the aqueous phase was drawn off and dialyzed (cutoff 12000 g/mol) against 12 L of distilled water for 3 days to give a turbid suspension with some coagulate. This was filtered (0.45 μm) to give about 15 mL slightly turbid solution, of which 10 mL was further dialyzed for 7 days against 12 L H2O, changed every 48 hours, then diluted to 20 mL of 10-cAMP (0.1% solids, 5% yield): FTIR (KBr; of the coagulate portion): 3500-3300 (NH), 3100-2800 (CH), 1630 (C=C), 1510, 1486, 1147, 990 (C=C), 903, 836, 797, 706 cm'1; light scattering showed 260 nm average diameter particles with broad polydispersity.

Example 14. Nanoparticle Fluorescent Chemosensor for cGMP: One-Step Method From Mixture With Template Salt (10-cGMP). As for 10-cAMP, but using Na+ 'cGMP (0.025 g, 68 μmol) and slightly different quantities of 55:45 divinylbenzene:styrene (0.5423 g, 2.3:1.9 mmol) and Irgacure 184 (0.0534 g). Final dilution was made to 15 mL of 10-cGMP in water (0.06% solids, 2% yield): light scattering showed 300 nm average diameter particles with broad polydispersity.

Example 15. Nanoparticle Fluorescent Chemosensor Control: One-Step Method From Mixture With Template Salt (10-Cl). As for 10-cAMP and 10- cGMP, but using NaCl (0.01 g, 170 μmol) and slightly different quantities of 55:45 divinylbenzene:styrene (0.5110 g, 2.2:1.8 mmol) and Irgacure 184 (0.0521 g). Final dilution was made to 21 mL of 10-Cl in water (0.1% solids, 6% yield): light scattering showed 190 nm average diameter particles with broad polydispersity.

The main difficulties surrounding this type of emulsification- polymerization resides in the lack of control of droplet size, uncertainty of the extent of chain transfer, particle stability in water (triphasic system) and the small quantities generated. Several factors influence this such as temperature, monomer concentration, solution viscosity and time of mechanical disruption. The resulting emulsion is also subject to shear forces during the spraying step into the liquid propane medium. Spraying on a non-freezing surface resulted in rapid phase separation of the droplets. The resulting icy solid from the spraying in liquid propane is not completely transparent, this being a problem for photoinitiation of the polymerization due to the decrease in intensity of incident light.

The resulting polymers from Examples 12-15 using compound 6 and cAMP reflect several characteristics due to these problems, including large polydispersity of the particle sizes and significant coagulation of the resulting solids. However, the persistent presence of the fluorophore after washing (some portions were washed in swelling organic solvents) indicated the chain-transfer addition of the fluorophore in fact occured. Also, since a fraction of the dispersed polymer did not coagulate, the samples were used for analysis.

Fluorescence emission spectra of nanoparticles 9-cAMP, excited at 365 nm, at 25 C are shown in Fig. 13. The "ref. sp." peak originates from the crosslinked polystyrene matrix on excitation at 254 nm, is relatively constant vs [cAMP] or other solutes, and can thus be used as an internal standard for a "ratioing" technique that would be independent of chemosensor concentration or sample thickness. Note that as little as 10'6 M of cAMP/H2O (A) causes more change in fluorescence than as much as 10"4 M of cGMP/H2O (B), indicating specificity. Neither cAMP nor cGMP has much effect on similar control "nonimprinted" nanoparticles 9-C1. Thus, the dispersion of nanoparticles, 9_ cAMP. generated using 6-cAMP in Example 12 showed definite selectivity of binding when the fluorescence was measured in the presence of the template (cAMP) and a similar small molecule cGMP. These results are summarized on Table 5.

Table 5. Effect of cAMP or cGMP on Nanoparticle Fluorescence8

Concentration

3.4 x lO"6 1.0 x lO"5 3.0 x lO"5 2.3 x lO"4 2.3 x lO"4 (24h, 48h) impr + cAMP +6.6% +12.7% +3.0% +0.5% +26.4% non + cAMP -2.0% -3.5% -9.0% -7.0 -3.2% imp + cGMP -1.9% -4.0% -6.6 -10.0% +4.3% non + cGMP -0.5% - - - -

Volume: 1.5 3 6 9 12 12 mL + #ml

-Measured at 25.6C. Solvent was distilled water.

Figure 14 depicts the fluorescence emission spectra of nanoparticles 10-cAMP, excited at 365 nm, at 25C, in water (A) and in the presence of 2.5 x 10"4 M of cAMP/H2O (B). Note that the presence of c AMP causes significant change in the shape of the emission peak, as highlighted by deconvolution of each experimental band (curve through points) into two underlying Gaussian curves (or other curves), probably due to changing relative contributions of emissions from fluorophores near empty or cAMP-filled binding sites. Comparison of intensities at higher vs lower wavelengths would give a "ratioing" technique for measuring [cAMP] that would be independent of chemosensor concentration or sample thickness.

Example 16. Amino-functionalized hydrophobic nanoparticle cores (11).

Hexadecyltrimethylammonium bromide (10.00 g, 27 mmol) was dissolved in 90 mL double-distilled degassed water in a 250 mL 2-neck round flask, and its pH adjusted to 13.5 with 1-2 mL of 30% NaOH/H2O. Divinylbenzene: ethylstyrene 55:45 (7.50 g, 32:26 mmol), 2.50 mL toluene, N-(m-vinylbenzyl)piperazine dihydrochloride 2 (0.50 g, 1.8 mmol) and AIBN (0.05 g, 0.30 mmol) were mixed together and slowly added to the aqueous mixture with vigorous manual stirring over 5 min, followed by mild magnetic stirring for 16 hrs at room temperature to form a water-clear microemulsion. Nitrogen gas was then passed through for 2.5 hrs, then the mixture placed in a Rayonet Photochemical Reactor and irradiated for 6 hrs at room temperature. After this, no monomer smell was evident, and 50 mL of the mixture was poured into 275 mL of hot methanol. The coagulate was separated by centrifugation, re-dispersed in THF and re-coagulated, three times before the final pellet was dried at 60C under vacuum overnight to a clear solid pellet of 11 (4.38 g, 120%): FTIR (KBr) 3500-3300 (NH), 3100-2800 (CH), 1630 (C=C), 1510, 1486, 1147, 990 (C=C), 903, 836, 797, 706 cm"', for 23% residual vinyl groups (Bartholin et al., Makromol. Chem., 182, 2075 (1981)).

Example 17. Fluorescent labelling hydrophobic nanoparticle cores (12).

Amino-functionalized nanoparticle cores 11 (1.22 g, 0.23 mmol R2NH) were dissolved in 25 mL THF, then triethylamine (0.1 mL, 0.72 mmol) and dansyl chloride (0.1001 g, 0.37 mmol) were added, and the yellow-orange solution was stirred at 60C. After a few minutes, the fluorescent green solution was poured into 150 mL methanol with more triethylamine (0.1 mL, 0.72 mmol), and the resulting precipitate was separated by centrifugation, resuspended in THF, and the process was repeated 7X until no fluorescence was visible in the supernatant. The resulting pellet was dried at 60C under vacuum to give 12: FT-IR (KBr) 3100-2800 (CH), 1630 (C=C), 1510, 1486, 1289, 1147, 990 (C=C), 903, 836, 797, 706; for 23% residual vinyl groups (Bartholin et al., cited above (1981)).

Examle 18. Nanoparticle Fluorescent Chemosensor for N-Dansyl-L- Phenylalanine Via Imprinting of Hydrophobic Fluorescent Cores (13-DLP).

TMPTMA (0.9002 g, 2.7 mmol), HEM (0.0987 g, 0.76 mmol) and Irgacure 184 (0.0062 g) were mixed together. To a clean dry 5 mL vial were added fluorescent nanoparticle cores 12 (0.0126 g, 2.2 μmol R3N~NDs), N-dansyl-L-phenylalanine (0.0051 g, 13 μmol)(whose addition blue-shifted the solution's colour), 0.60 mL dry benzene, then the TMPTMA:HEM:Irgacure 184 mixture (0.0327 g)(shifting the colour to green). The solution was mixed on a rotary rotary mixer for 30 min in the dark, then the vial purged with nitrogen, and the contents slowly dripped into a 50 mL aluminum cup immersed in liquid nitrogen (-170C). The resulting clear glassy pellet (alternatively, dropping on similarly 250 mm cooled copper mesh gave an easily-handled clear frozen disk) was then tranferred to a 50 ml vial precooled and given an inert atmosphere by prior addition of a few drops of liquid nitrogen, then left in a refrigerator at -14C for 16 hrs, before being placed in an ice-salt bath at -14.1 to -12C for irradiation in a Rayonet Photochemical Reactor for 2 hours. After warming to room temperature, 10 mL of methanol with 5% triethyl amine was then added to the vial to give a white precipitate. After cooling in ice 15 min, the precipitate was isolated by centrifugation, 1.5 mL of warm benzene was added and the mixture shaken for 30 min, before repetition of MeOH/TEA precipitation. This was repeated 5X until no fluorescence was detected in the supernatant. The pellet was dried in a vacuum oven at 60C overnight, yielding faint yellow glassy pellet 13-DLP that fluoresced baby blue in the solid or blue in benzene solution (0.0099 g, 22%): FT-IR (KBr) 3400-3300 (OH), 3100-2800 (CH), 1730 (C=O), 1630 (C=C), 1510, 1486, 1 170 (C-O), 1 147, 990 (C=C), 903, 836, 797, 706, for 15% residual vinyl groups. Figure 15 depicts a plot of F/F0 ("relative response," linear scale) vs either [N-dansyl-L-phenylalanine] or [N-dansyl-D-phenylalanine] (log scale), for nanoparticles 13-DLP, excited at 365 nm, emission measured at 460 nm, at 25C, in chloroform. F0 is the emission intensity prior to addition of substrate. Note the significantly greater fluorescent response, beginning at 2 x 10'6 M, of 13-DLP towards increasing quantities of the enantiomer towards which it had been originally imprinted ("L"), than towards the other enantiomer of the same molecule ("D"). Bars show standard error of multiple measurements.

Example 19. Nanoparticle Fluorescent Chemosensor for N-Dansyl-D- Phenylalanine Via Imprinting of Hydrophobic Fluorescent Cores (13-DDP). As for 13-DLP, but employing slightly different quantities of fluorescent nanoparticle cores 12 (0.0123 g), D-enantiomer N-dansylated amino acid (0.0057 g), and monomer mixture (0.0337 g), and resulting in 13-DDP product (0.0121 g, 26%) of similar fluorescent appearance.

Example 20. Nanoparticle Fluorescent Chemosensor Via Imprinting of Hydrophobic Fluorescent Cores, Control (13-DDP). As for 13-DLP and 13- DDP, but employing no N-dansylated amino acid, and slightly different quantities of fluorescent nanoparticle cores 12 (0.0144 g), and monomer mixture (0.0337 g), and resulting in 13-X product (0.0100 g, 21%) of similar final fluorescent appearance. This product was insoluble in pure water, but 50 mg of 13-X and 92 mg of Triton X-100 in 1 mL of ethanol could be slowly diluted in 10 mL H2O to give a slightly iridescent solution that could be filtered (0.45 μm) to clear yet bright fluorescent green liquid.

Example 21. α-Chloro-γ-methoxy-polyethylene glycol (14).(Bayer, Zheng et al. 1982) Polyethylene glycol) monomethyl ether 750 g/mol (26.16 g, 34.8 mmol) was added to fresh thionyl chloride (48 ml , 480 mmol) under nitrogen, and the mixture refluxed overnight. Distillation gave a light yellow crystalline solid residue 14 (25.43 g, 95%): mp 29-31C.

Example 22. α-Mercapto-γ-methoxy-polyethylene glycol (15).(Bayer, Zheng et al. 1982) α-Chloro-γ-methoxy-polyethylene glycol 14 (24.13 g, 30.5 mmol) was dissolved in 150 ml dry ethanol, and thiourea (13.0 g, 171 mmol) was added. The mixture was refluxed under nitrogen overnight, then KOH (10 g, 178 mmol) in 25 mL of water was added, and reflux resumed for a further 16 hrs. The pH was then lowered to 4 with concentrated HCl/H2O, then the solution was filtered and the ethanol evaporated. The resulting light brown oil was taken up in CHC13, filtered, and the filtrate dried over MgSO4, again filtered and the solvent evaporated to give light brown oil 15 (21.18 g, 79%): fusion of a drop with 0.1 g benzoin turned lead acetate paper black.

Example 23. Amino-functionalized hydrophilic nanoparticle cores (16). Hexadecyltrimethylammonium bromide (2.00 g, 5.5 mmol) was dissolved in 18 mL double-distilled degassed water in a 25 mL 2-neck round flask, and its pH adjusted to 5 with 1 N HCl/H2O. Divinylbenzene: ethylstyrene 55:45 (1.5 g, 6.3 :5.2 mmol), AIBN (0.05 g, 0.3 mmol) and 0.50 mL toluene were slowly added with vigorous manual stirring over 5 min, and the mixture warmed to ~45C with -120 rpm mild magnetic stirring to form the water-clear microemulsion. Nitrogen gas was then bubbled in to the stoppered flask for 0.5 hrs, then the mixture heated at 75 C for 2 hrs before adding α-mercapto-γ- methoxy-polyethylene glycol 15 (0.70 g, 0.9 mmol) and N-(2-mercaptoethyl)- N',N'-dimethylethylene-diamine hydrochloride 7 (0.2 g, 1.2 mmol). After further stirring at 75C overnight, the hot solution was added to 250 ml hot MeOH containing 5 ml triethylamine, and the resulting precipitate was then spun down on a benchtop centrifuge, isolated and resuspended in THF. This process was repeated twice, then the pellet dried under vacuum at 80C overnight to yield an opaque glass 16 (1.54 g, 68%).

Example 24. Fluorescent hydrophilic nanoparticle cores (17). A portion of the amino-functionalized hydrophilic nanoparticle cores 16 (1.00 g, 0.5 mmol R2NH) was taken up in CHC13, and dansyl chloride (0.4 g, 1.5 mmol) was then added with fresh triethylamine (1 mL, 7.2 mmol), and the mixture warmed with a heat gun for 20 min. After standing at room temperature a further 20 min, the solvent was evaporated and the glassy residue washed 3X with 50 mL hot MeOH, isolating each time by centrifugation, and finally dried in a stream of nitrogen while mildly heating, to give 17 as a fluorescent solid (0.52 g, 47%): light scattering showed an average diameter of 16 nm with moderately wide polydispersity.

Example 25. Nanoparticle Fluorescent Chemosensor for N-Dansyl-L- Phenylalanine Via Imprinting of Hydrophilic Fluorescent Cores (18-DLP). To a clean dry 5 mL vial were added fluorescent hydrophilic nanoparticle cores 17 (4.9896 mg, 2.5 μmol R3N~NDs). N-dansyl-L-phenylalanine (4.0080 mg, 10.1 μmol), the methacrylate monomer mixture used in 13-DLP (5.7 mg), 1.0 mL of degassed deionized water. After shaking the vial in a rotary mixer for 30 min, it was purged with nitrogen, then its greenish contents were slowly dripped onto a 3 cm-diameter disk of 250 μm-space copper mesh kept at - 170C by dipping in liquid nitrogen, forming a clear glassy coating. The disk was then transferred into a Kadpak heat-sealable plastic pouch that had been pre-cooled and purged by adding a few drops of liquid nitrogen. Then the pouch was heat- sealed and stored in the refrigerator at -14C for 30 min, placed in an ice salt bath and irradiated in the Rayonet Photochemical Reactor at -14.1 to -12C for 2 hours. The pouch was then opened and 20 mL of 5% NEt3/CH3OH was added, producing a white precipitate that was separated by centrifugation at 0C. The pellet was resuspended in 5 mL warm water, then 5% NEt3/CH3OH was again added to reprecipitate, and the process repeated 5X until no fluorescence was apparent in the supernatant. About one quarter (-1 mg) was removed from the last precipitate, and dried under vacuum at 60C overnight, yielding a faint yellow glass with baby blue fluorescence. The remainder of the moist precipitate (-3 mg) was resuspended in 250 mL deionized water to give a slightly iridescent solution, which was filtered through a 0.2 μm Supor Acrodisc Gelman filter to give a clear solution that showed intense blue fluorescence. Example 26. Nanoparticle Fluorescent Chemosensor for N-Dansyl-D- Phenylalanine Via Imprinting of Hydrophilic Fluorescent Cores (18-DDP). As for 18-DLP, but employing D-enantiomer N-dansylated amino acid (4.005 mg, 10.1 μmol), and slightly different quantities of hydrophilic fluorescent nanoparticle cores 17 (4.9437 mg, 2.5 μmol R3N~NDs) and methacrylate monomer mixture (5.7 mg), and resulting in solid and solution 18-DDP of similar appearances.

Example 27. Nanoparticle Fluorescent Chemosensor for R-2- Phenylpropanoic Acid Via Imprinting of Hydrophilic Fluorescent Cores (18-RP). As for 18-DLP, but employing R-enantiomer 2-phenylpropanoic acid (3.9873 mg, 24.3 μmol), and slightly different quantities of hydrophilic fluorescent nanoparticle cores 17 (4.8976 mg, 2.5 μmol R3N~NDs) and methacrylate monomer mixture (6.0 mg), and resulting in solid and solution 18- RP of similar appearance.

Example 28. Nanoparticle Fluorescent Chemosensor for S-2- Phenylpropanoic Acid Via Imprinting of Hydrophilic Fluorescent Cores (18-SP). As for 18-DLP, but employing S-enantiomer 2-phenylpropanoic acid (3.9866 mg, 24.43 μmol), and slightly different quantities of hydrophilic fluorescent nanoparticle cores 17 (4.8977 mg, 2.5 μmol R3N~NDs) and methacrylate monomer mixture (5.7 mg), and resulting in solid and solution 18- RP of similar appearance.

Example 29. Nanoparticle Fluorescent Chemosensor Via Imprinting of Hydrophilic Fluorescent Cores, Control (18-X). As for 18-DLP, but employing no template, and slightly different quantities of hydrophilic fluorescent nanoparticle cores 17 (4.6808 mg, 2.3 μmol R3N~NDs), and methacrylate monomer mixture (6.1 mg), and resulting in solid and solution 18- X of similar appearance: light-scattering of the solution showed 47 nm average particle diameter with wide polydispersity. Since the particles of Examples 25-29 formed mostly clear solutions in the solvents employed, no special techniques or apparatus were necessary beyond a SPEX Spectro 1680 spectrofluorimeter in standard configuration, with the samples in a 1 cm cuvette thermostatted to 22C, emission detected perpendicular to excitation at 365 nm, and both excitation and emission slit widths at 2.5 nm. Measurements were made 2.5 hrs after the preparations of solutions, when by then fluorescence response had generally been observed to have stabilized.

Several of the limitations of the one-step process of Example 4 above, such as particle size, fluorophore accesibility and reactivity, can be circumvented by using this multi-step approach. Also use of enantiomeric templates should dispel any doubt as to whether selectivity is due to molecular imprinting. A completely clear solution of nanoparticles containing pendant secondary and tertiary amines along with pendant vinyl groups is easily generated, isolated and quantitated. These particles have a swell ratio of 1.2 in cooled benzene. This swelling behaviour proves that there are in fact micropores present. Further addition of benzene and warming completely dissolves the particles.

Reaction of the secondary amines with dansyl chloride leads to a fluorophore labeled nanoparticle (17) which can be imprinted using further monomers and any of a wide variety of templates. The FT-IR analysis of these imprinted nanoparticles and parent nanoparticles showed residual vinyl groups were still present. Solutions of the particles gave different reactions with different types of substrates, as shown in Table 6. Table 6. Qualitative fluorescence observations of nanoparticles

Sample observed Colour of emmision Notes (Ex 365nm)

NanP in benzene (17) blue ~ 480 nm slightly greenish hue very intense

Dan-L-Phe in benzene yellow green -550 nm very intense 0.01 mg /ml

NanP + Dans-L-phe indigo ~ 450 nm seems quenched, very definite shift , needs- 2min to stabilize

NanP + acetic acid blue ~ 480 no change after 0.5 hrs

NanP + tosic acid blue -480 no change after 0.5 hrs.

NanP + triethyl amine blue - 480 no change after 0.5 hrs

NanP + Dans Piperazine blue-green - 520 increase in intensity

Dans 1 Phe + Dans Pip yellow-green - 550 no visible change

NanP + EtOH blue- green - 520 definite shift

Note all 1.5 ml volume; 15mg NanP in 15 ml dry benzene

These particles may further be functionalized with chain tranfer agents (such as HS-polyethyleneglycol-OMe) capable of solubilizing the nanoparticles in water. In fact, these particles may also be solubilized in water simply by adding the surfactant Triton X-100, giving completely clear aqueous solutions. Since each step in the synthesis provides possible alternative routes, several different nanoparticle systems may be generated. The water soluble PEG grafted particles prepared in Example 23 were imprinted in water with both fluorescent and non- fluorescent enantiomeric substrates.

The nanoparticles generated by these routes gave completely clear solutions and were analysed with a conventional spectrofluorimeter. The imprinted polymers of Examples 25-28 showed promising results. As shown in Table 7, D-Imprinted polymers in chloroform have a greater increase in fluorescence in the presence of D- substrate than the corresponding L- substrate, and the converse showed the same relationship. The near equity of the control vs. the two substrates indicates some degree of certainty as to the reliability of the data. These experiments also showed the fluorescence is unchanged from 1- 24 hrs. indicating stable binding. The experiments were also reproducible with 10-20% error.

Table 7. Rebinding studies

a) 80μg particles/ml chloroform. (8.0 μM binding sites), 25.4C.

The difference in the fluorescence emission of the polymer bound fluorophore and the soluble one in chloroform was not significant (4 nm) thus internal calibration was not possible in chloroform due to several parallel processes occuring in the system.

The water soluble polymer gave an emission characteristic of fluorophore in an organic solvent. This emission is very different from the water soluble substrates and thus a ratioing technique may be used to quantitate. Figure 16 depicts a plot of F0/F ("relative response," linear scale) vs either [R-2- phenylpropanoic acid] or [S-2-phenylpropanoic acid] (log scale), for nanoparticles 18-SP fluorescent chemosensor, excited at 365 nm, emission measured at 460 nm, at 25 C, in water. F0 is the emission intensity prior to addition of substrate. Note the significantly great fluorescent response, beginning at less than 1 x 10"6 M, of 18-SP towards increasing quantities of the enantiomer towards which it had been originally imprinted ("S"), than towards the other enantiomer of the same molecule ("R").

Molecular imprinting, based on electrostatic interactions and hydrogen bonding, is coupled with fluorescence spectroscopy to prepare fluorescent crosslinked polymethacrylate polymers that demonstrated an affinity in aqueous solution comparable to polymer complements designed for nucleotide base binding in chloroform as reported by Shea, ibid., (1993). The results demonstrate the formation of template selective cavities in the polymers, as well as the capability of being recycled. The present routes employ polymerization and chain transfer mechanisms in order to produce imprints. These studies show these imprinted particles have the ability to distinguish between different molecules and enantiomers of the same molecules in organic media. The invention has been described with reference to various specific and preferred embodiments and techniques. However, it should be understood that many variations and modifications may be made while remaining within the invention.

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Classifications
International ClassificationG01N33/531
Cooperative ClassificationG01N33/531, G01N2600/00
European ClassificationG01N33/531
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