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WO1996041173A1 - Nanoparticules empreintes de sites de reconnaissance pour des molecules cibles - Google Patents

Nanoparticules empreintes de sites de reconnaissance pour des molecules cibles Download PDF

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Publication number
WO1996041173A1
WO1996041173A1 PCT/US1996/007936 US9607936W WO9641173A1 WO 1996041173 A1 WO1996041173 A1 WO 1996041173A1 US 9607936 W US9607936 W US 9607936W WO 9641173 A1 WO9641173 A1 WO 9641173A1
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det
particle
recognition sites
particles
polymeric
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PCT/US1996/007936
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English (en)
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Graham D. Darling
Seymour Heisler
Brent R. Stranix
Petra Turkewitsch
Barbara Wandelt
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Martinex R & D Inc.
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Publication of WO1996041173A1 publication Critical patent/WO1996041173A1/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/531—Production of immunochemical test materials
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2600/00—Assays involving molecular imprinted polymers/polymers created around a molecular template

Definitions

  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • racemic mixtures are disclosed to be solely due to the asymmetry of the cavities in the polymer. See also, G. Wulff et al., J. Liq. Chrom.. ⁇ , 2987 (1990).
  • These polymers are prepared by polymerizing a monomer comprising a covalently linked optically active compound (see Example 1).
  • FIG. 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).
  • 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.
  • 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 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 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.
  • 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.
  • 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.
  • 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).
  • 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.
  • Det is also capable of reversibly binding to T, to yield the unit B-Det-T.
  • 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 olef ⁇ nically-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.
  • 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).
  • 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.
  • the bonding is ion-ion, dipole-ion or both.
  • Det is a fluorescent moiety or "fluorophore", and its fluorescence changes when T binds to the recognition site.
  • 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.
  • 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.
  • 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) n are repeating units of A and X is H or B-T;
  • (B) also is bound to detection moiety (Det), so that there is a close association between T and Det during imprinting.
  • 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.
  • 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.
  • 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.
  • this embodiment of the invention provides a method of preparing imprinted polymeric particles comprising:
  • 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.
  • Det can also reversibly bind to T.
  • 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.
  • 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.
  • 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.
  • T preselected template compound
  • VBP free vinylbenzyl piperazine
  • DsCl dansyl chloride
  • 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.
  • 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.
  • 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.
  • a suitable liquid medium such as a high freezing point organic solvent
  • 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.
  • the polymeric matrix of the present 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.
  • A olefinically unsaturated organic monomers
  • 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.
  • 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.
  • 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.
  • 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.
  • a catalyst can be used such as, e.g., in radical polymerization: azobis(isobutyronitrile), IgracureTM (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: TiCl 4 , BF 3 , H 2 SO 4 , alkali metals, butyllithium, sodium or potassium naphthalide; in insertion polymerization: Ziegler-Natta-catalysts, e.g., Al(Et) 3 and TiCl 4 ; or initiation by heat or by ultrasonic waves, UV-,
  • 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.
  • useful crosslinkers include glycerine, cyanuric acid, phenol, melamine, trichlorosilane, hexamethylenetetramine.
  • useful crosslinkers include 2,4,6-tricyanatotoluene, glycerol, sorbitol, and ethylene tetramine.
  • 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.
  • the monomer can be 100% cross-linkable (DVB) or, in some cases, no cross-linking agent is used.
  • the polymerization is effected in the presence of an inert solvent or solvent mixture.
  • G. Wulff et al. U.S. Patent No.4,111,863
  • K. Mosbach U.S. Patent No. 5,110,833
  • 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.
  • the finished particles are 10-100 nm in diameter.
  • Such particles can readily be formulated into solutions or suspensions.
  • Chain Transfer Agent (OTA) 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.
  • Chain-transfer 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.
  • 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)CH 2 CH 2 NH Me 2 ) + RCOO ).
  • T template molecule
  • RCOOH D- or L- amino acid
  • 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.
  • 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.
  • initiator or CTA compound B will also comprise one or more functional groups which can reversibly bind to template compound T.
  • the B-T bond can be due to, or enhanced by, ⁇ - ⁇ interactions, hydrogen bonding, dipol interactions, electrostatic interactions, charge-transfer complexes, chelation and the like.
  • ⁇ - ⁇ interactions hydrogen bonding, dipol interactions, electrostatic interactions, charge-transfer complexes, chelation and the like.
  • 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.
  • Compound B will further preferably comprise a detectable moiety (Det) which then becomes held in proximity to, and preferably associated with, the recognition site.
  • Det also can bind to T.
  • Det moieties can generate detectable signals before and after binding to T.
  • 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.
  • 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
  • radioisotope see Engelhardt et al. (U.S. Patent No. 5,241,060).
  • 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.
  • 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).
  • 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 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.
  • B-Det 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.
  • very polar protic liquid phase e.g., ions, OH groups
  • the less polar monomer or gel phase e.g., aromatic groups
  • compound (1) 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.
  • R-DMASP + N-substituted A-p- dimethylaminostyrylpyridinium compounds
  • NP normal planar
  • TICT twisted intramolecular charge transfer
  • 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.
  • 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.
  • solvatofluochromism See, K.J. Shea et al., Macromolec, 22, 1722 (1981).
  • 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.
  • 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.
  • 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 C r to C 8 - alcohols or mixtures of water with acetone or other water-soluble solvents at room temperature or at elevated temperatures ranging from 30°C to 150°C.
  • 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
  • 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).
  • T excess template
  • monomer A hydroxyethyl methacrylate (HEMA)
  • TMPTMA crosslinking monomer trimethylolpropane trimethylacrylate
  • AIBN free-radical initiator azobis(isobutyronitrile)
  • Polymerization can be initiated either with heat or, at room temperature, irradiation with ultraviolet light, or by a combination of the two.
  • 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).
  • B-Det-T bracketed intermediate complex
  • 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.
  • 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)).
  • 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.
  • detectability such as fluorescence and the like.
  • 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.
  • thermodynamically unstable microemulsions of monomers with smaller quantities of less-effective surfactants 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.
  • 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.
  • the liquid propane can then be replaced with another less volatile hydrophobic liquid (e.g., hexanes), as the temperature is raised (as to -14°C, 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.
  • DMASP 4-(p-Dimethylaminostyryl)pyridine
  • Monomers and initiators were purchased from Aldrich Chemical Co.
  • cAMP and cGMP were used as received from Sigma
  • 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- >-dimethylaminosry ⁇ yIpyridinium chloride (1).
  • DMASP 0.508 g, 2.26 mmol
  • Toluene 25 ml was added to the flask, and the system was flushed with nitrogen, then heated to 60°C.
  • m-Vinylbenzyl chloride (3.03 g, 19.9 mmol) was then added dropwise.
  • Vinylbenzyl chloride (7.69 g, 49.3 mmol) was then added in one portion and the mixture stirred on a cold water bath (10°C) for 1 hr. The solution was cooled to - 13°C for 2hrs and the resulting white precipitate removed. The filtrate was acidified with dry HCl and cooled to -10°C for 30 min..
  • 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.
  • Example 4 Bulk Fluorescent Chemosensor for cAMP (4-cAMP). Sodium cyclic adenosine 3',5'-monophosphate (Na + " cAMP, 41.7 mg, 118.7 ⁇ mol) was weighed into a glass specimen bottle, followed by N-(/w-vinylbenzyl)-4-p- dimethylaminostyrylpyridinium chloride 1 (31.5 mg, 83.6 ⁇ mol) and 30 mL methanol.
  • TMPTMA trimethylolpropane trimethacrylate
  • HOM 2-hydroxyethyl acrylate
  • AIBN 2,2'- azobis(isobutyronitrile)
  • 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 (25.0 ⁇ 0.1°C) 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.
  • PTI Photon Technology International
  • 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 I 0 . 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/H 2 O) was added. After incubating in a shaker bath at 25°C 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.
  • sample solution such as Na + " cAMP/H 2 O
  • 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
  • [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
  • 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 ⁇ mol/g polymer.
  • 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 10 4 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.
  • 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.
  • cGMP quenching values are the results of a single experiment. Error is standard error in the case of cAMP values.
  • the quenching values for the 2nd template extraction are the results of a single experiment. Error is standard error for 1.00 x 10 _3 and 1.00 x 10' 6 M 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 CH 2 C1 2 , 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 K 2 CO 3 /H 2 O, and the organic phase dried over MgSO 4 , and evaporated to dryness, yielding hard glassy material 5 (0.91 g, 91%).
  • 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.
  • 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 75°C 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 70-76°C; 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 CHC1 3 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 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 -155°C during the subsequent spraying process. The spraying was done using an air brush connected to a nitrogen outlet set at 30psi.
  • 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 lOO ⁇ l of the solution.
  • dialysates were found to contain 1.70% solid for the Cl and 1.74% for the cAMP.
  • 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/H 2 O (0.1 mL, 10 ⁇ mol), Na + cAMP (0.0250 g, 71 ⁇ mol), 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.
  • 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 ⁇ mol) 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.
  • 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 ⁇ mol) 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.
  • 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/H 2 O (A) causes more change in fluorescence than as much as 10 "4 M of cGMP/H 2 O (B), indicating specificity.
  • Figure 14 depicts the fluorescence emission spectra of nanoparticles 10-cAMP, excited at 365 nm, at 25°C, in water (A) and in the presence of 2.5 x 10 "4 M of cAMP/H 2 O (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.
  • 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/H 2 O.
  • 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.
  • Example 17 Fluorescent labelling hydrophobic nanoparticle cores (12).
  • Amino-functionalized nanoparticle cores 11 (1.22 g, 0.23 mmol R 2 NH) 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 60°C. 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.
  • TMPTMA (0.9002 g, 2.7 mmol), HEM (0.0987 g, 0.76 mmol) and Irgacure 184 (0.0062 g) were mixed together.
  • fluorescent nanoparticle cores 12 0.0126 g, 2.2 ⁇ mol R 3 N ⁇ NDs
  • N-dansyl-L-phenylalanine 0.0051 g, 13 ⁇ mol
  • 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 (-170°C).
  • 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 -14°C for 16 hrs, before being placed in an ice-salt bath at -14.1 to -12°C for irradiation in a Rayonet Photochemical Reactor for 2 hours.
  • Figure 15 depicts a plot of F/F 0 ("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 25°C, in chloroform.
  • F 0 is the emission intensity prior to addition of substrate.
  • Example 19 Nanoparticle Fluorescent Chemosensor for N-Dansyl-D- Phenylalanine Via Imprinting of Hydrophobic Fluorescent Cores (13-DDP).
  • 13-DLP 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).
  • 13-DLP and 13- DDP 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 H 2 O to give a slightly iridescent solution that could be filtered (0.45 ⁇ m) to clear yet bright fluorescent green liquid.
  • Example 21 ⁇ -Chloro- ⁇ -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 29-31°C.
  • Example 22 ⁇ -Mercapto- ⁇ -methoxy-polyethylene glycol (15).(Bayer, Zheng et al. 1982) ⁇ -Chloro- ⁇ -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/H 2 O, then the solution was filtered and the ethanol evaporated.
  • 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/H 2 O.
  • 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 ⁇ 45°C 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 ⁇ -mercapto- ⁇ - 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).
  • 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 80°C 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 R 2 NH) was taken up in CHC1 3 , 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.
  • 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 ⁇ mol R 3 N ⁇ NDs). N-dansyl-L-phenylalanine (4.0080 mg, 10.1 ⁇ mol), the methacrylate monomer mixture used in 13-DLP (5.7 mg), 1.0 mL of degassed deionized water.
  • the pouch was then opened and 20 mL of 5% NEt 3 /CH 3 OH was added, producing a white precipitate that was separated by centrifugation at 0°C.
  • the pellet was resuspended in 5 mL warm water, then 5% NEt 3 /CH 3 OH 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 60°C overnight, yielding a faint yellow glass with baby blue fluorescence.
  • Example 26 Nanoparticle Fluorescent Chemosensor for N-Dansyl-D- Phenylalanine Via Imprinting of Hydrophilic Fluorescent Cores (18-DDP).
  • Example 27 Nanoparticle Fluorescent Chemosensor for R-2- Phenylpropanoic Acid Via Imprinting of Hydrophilic Fluorescent Cores (18-RP).
  • 18-DLP employing R-enantiomer 2-phenylpropanoic acid (3.9873 mg, 24.3 ⁇ mol), and slightly different quantities of hydrophilic fluorescent nanoparticle cores 17 (4.8976 mg, 2.5 ⁇ mol R 3 N ⁇ 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).
  • 18-DLP As for 18-DLP, but employing S-enantiomer 2-phenylpropanoic acid (3.9866 mg, 24.43 ⁇ mol), and slightly different quantities of hydrophilic fluorescent nanoparticle cores 17 (4.8977 mg, 2.5 ⁇ mol R 3 N ⁇ 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).
  • 18-DLP employing no template, and slightly different quantities of hydrophilic fluorescent nanoparticle cores 17 (4.6808 mg, 2.3 ⁇ mol R 3 N ⁇ 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.
  • NanP + Dans Piperazine blue-green - 520 increase in intensity
  • These particles may further be functionalized with chain tranfer agents (such as HS-polyethyleneglycol-OMe) capable of solubilizing the nanoparticles in water.
  • chain tranfer agents such as HS-polyethyleneglycol-OMe
  • 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.
  • FIG. 16 depicts a plot of F 0 /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.
  • F 0 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").

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Abstract

L'invention concerne des particules empreintes solides comprenant des sites de reconnaissance espacés sur la surface desdites particules et se fixant sélectivement à un composé gabarit présélectionné (T), lesdits sites de reconnaissance étant définis chacun par une matrice polymère façonnée se conformant à la forme et à la dimension de T, comprenant une unité de formule -B-Det, dans laquelle B est fixé à la matrice polymère et comporte également un groupe fonctionnel qui se fixe à T de façon réversible et où Det représente une fraction capable de générer une modification détectable de signal à la suite de la fixation de B au composé gabarit T.
PCT/US1996/007936 1995-06-07 1996-05-29 Nanoparticules empreintes de sites de reconnaissance pour des molecules cibles WO1996041173A1 (fr)

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WO2000009480A1 (fr) * 1998-08-11 2000-02-24 Daiichi Pharmaceutical Co., Ltd. Nouveaux derives sulfonyle
EP1039291A1 (fr) * 1999-03-26 2000-09-27 Sony International (Europe) GmbH Capteur optochimique et procédé pour sa fabrication
WO2000066790A1 (fr) * 1999-05-05 2000-11-09 The Regents Of The University Of California Procede de detection d'agents biologiques
WO2001090228A1 (fr) * 2000-05-22 2001-11-29 Klaus Mosbach Empreinte moleculaire
WO2007110538A3 (fr) * 2006-03-24 2008-10-09 Oreal Composition de teinture comprenant un colorant fluorescent thiol masque a groupe dimethylamino, a charge cationique interne, procede d'eclaircissement des matieres keratiniques a partir de ce colorant et de colorant disufure
FR2921376A1 (fr) * 2007-09-21 2009-03-27 Oreal Compose styryl tetrahydroquinolinium thiol/disulfure, procede d'eclaircissement des matieres keratiniques a partir de ce colorant
US7717964B2 (en) 2007-09-21 2010-05-18 L'oreal S.A. Styryl thiol/disulfide compound with a hydroxy(cyclo)alkylamino unit, process for lightening keratin materials using same
US7727287B2 (en) 2007-09-21 2010-06-01 L'oreal S.A. Hemicyanin styryl thiol/disulfide dye, composition comprising hemicyanin styryl thoil/disulfide dye, and method for lightening keratin materials using hemicyanin styryl thiol/disulfide dye
US7744658B2 (en) 2007-09-21 2010-06-29 L'oreal S.A. Indole-derived styryl dye comprising an alkylene linker, a dye composition comprising this dye, and a process for lightening keratin materials using this dye
US7780743B2 (en) 2006-03-24 2010-08-24 L'oreal S.A. Fluorescent entity, dyeing composition containing at least one fluorescent entity, and method for lightening keratin materials using said at least one fluorescent entity
US8070830B2 (en) 2006-03-24 2011-12-06 L'oreal S.A. Fluorescent entity, dyeing composition containing at least one fluorescent entity comprising at least one heterocycle, with at least one internal cationic charge, and method for lightening keratin materials using said at least one fluorescent entity
US8192762B2 (en) 2005-06-01 2012-06-05 Cranfield University Preparation of soluble and colloidal molecularly imprinted polymers by living polymerization
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CN104897846A (zh) * 2015-06-23 2015-09-09 江南大学 一种基于原位形成光活性纳米材料模拟酶的碱性磷酸酯酶活性检测方法
CN107880227A (zh) * 2017-10-26 2018-04-06 江南大学 一种基于大分子自组装制备糖蛋白印迹荧光纳米粒子的方法
CN111760561A (zh) * 2020-07-23 2020-10-13 桂林理工大学 一种基于MCM-41分子筛表面的As(Ⅲ)离子印迹材料的制备方法
CN114755401A (zh) * 2022-03-24 2022-07-15 北京理工大学 一种识别未知或模糊目标物的分子印迹材料以及基于分子印迹的夹心式传感器检测方法

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WO2000009480A1 (fr) * 1998-08-11 2000-02-24 Daiichi Pharmaceutical Co., Ltd. Nouveaux derives sulfonyle
EP1039291A1 (fr) * 1999-03-26 2000-09-27 Sony International (Europe) GmbH Capteur optochimique et procédé pour sa fabrication
US7223367B1 (en) 1999-03-26 2007-05-29 Sony International (Europe) Gmbh Chemical sensor arrangement
WO2000066790A1 (fr) * 1999-05-05 2000-11-09 The Regents Of The University Of California Procede de detection d'agents biologiques
US6589731B1 (en) * 1999-05-05 2003-07-08 The Regents Of The University Of California Method for detecting biological agents
WO2001090228A1 (fr) * 2000-05-22 2001-11-29 Klaus Mosbach Empreinte moleculaire
US8192762B2 (en) 2005-06-01 2012-06-05 Cranfield University Preparation of soluble and colloidal molecularly imprinted polymers by living polymerization
US7780743B2 (en) 2006-03-24 2010-08-24 L'oreal S.A. Fluorescent entity, dyeing composition containing at least one fluorescent entity, and method for lightening keratin materials using said at least one fluorescent entity
WO2007110538A3 (fr) * 2006-03-24 2008-10-09 Oreal Composition de teinture comprenant un colorant fluorescent thiol masque a groupe dimethylamino, a charge cationique interne, procede d'eclaircissement des matieres keratiniques a partir de ce colorant et de colorant disufure
US8070830B2 (en) 2006-03-24 2011-12-06 L'oreal S.A. Fluorescent entity, dyeing composition containing at least one fluorescent entity comprising at least one heterocycle, with at least one internal cationic charge, and method for lightening keratin materials using said at least one fluorescent entity
US7727287B2 (en) 2007-09-21 2010-06-01 L'oreal S.A. Hemicyanin styryl thiol/disulfide dye, composition comprising hemicyanin styryl thoil/disulfide dye, and method for lightening keratin materials using hemicyanin styryl thiol/disulfide dye
US7744658B2 (en) 2007-09-21 2010-06-29 L'oreal S.A. Indole-derived styryl dye comprising an alkylene linker, a dye composition comprising this dye, and a process for lightening keratin materials using this dye
US7744657B2 (en) 2007-09-21 2010-06-29 L'oreal S.A. Styrl tetrahydroquinolinium thiol/disulfide dye compound and method for lightening keratin materials using the same
FR2921376A1 (fr) * 2007-09-21 2009-03-27 Oreal Compose styryl tetrahydroquinolinium thiol/disulfure, procede d'eclaircissement des matieres keratiniques a partir de ce colorant
US7717964B2 (en) 2007-09-21 2010-05-18 L'oreal S.A. Styryl thiol/disulfide compound with a hydroxy(cyclo)alkylamino unit, process for lightening keratin materials using same
EP2075289A1 (fr) * 2007-09-21 2009-07-01 L'Oréal Composé styryl tetrahydroquinolinium thiol/disulfure, procédé d'éclaircissement des matières kératiniques à partir de ce colorant
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