EP1436599A2 - Detecteur d'anions en solution a base de polymere a empreinte moleculaire - Google Patents

Detecteur d'anions en solution a base de polymere a empreinte moleculaire

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Publication number
EP1436599A2
EP1436599A2 EP02801765A EP02801765A EP1436599A2 EP 1436599 A2 EP1436599 A2 EP 1436599A2 EP 02801765 A EP02801765 A EP 02801765A EP 02801765 A EP02801765 A EP 02801765A EP 1436599 A2 EP1436599 A2 EP 1436599A2
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EP
European Patent Office
Prior art keywords
lanthanide
vinyl
sensor device
analyte
chelated
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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EP02801765A
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German (de)
English (en)
Inventor
George M Murray
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Johns Hopkins University
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Johns Hopkins University
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
    • G01N21/77—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator
    • G01N21/7703—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator using reagent-clad optical fibres or optical waveguides
    • 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
    • G01N33/84—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving inorganic compounds or pH
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
    • G01N21/77—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator
    • G01N21/7703—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator using reagent-clad optical fibres or optical waveguides
    • G01N2021/7706—Reagent provision
    • G01N2021/772—Tip coated light guide
    • 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

  • the present invention relates generally to the use of molecularly imprinted polymers comprising chelated lanthanides in methods and apparatus for detecting the presence of an analyte.
  • Methods and apparatus for the efficient and accurate detection and quantification of analytes, including polyatomic anion analytes, are of particular interest for use in a wide range of applications.
  • such methods and apparatus are useful in the detection, monitoring, and management of environmental pollutants, including organophosphorus-based pesticides.
  • Organophosphorus-based pesticides, including paraoxon, parathion, and diazinon are widely used in the agriculture industry. Because such materials exhibit a relatively high toxicity to many forms of plant and animal life, and also exhibit relatively high solubility in water, organophosphorus-based pesticides pose a clear threat to aquatic life and to our drinking water.
  • nitrate run off from agriculture can cause problems for water quality, especially for children, resulting in the "blue baby" syndrome.
  • Detecting dissolved nutrients, i.e. phosphate and nitrate, is a critical need for evaluating environmental pollution.
  • conventional optical sensors for the detection of aqueous analytes typically rely on small changes in the indices of refraction in response to the presence of an analyte.
  • conventional optical sensors include planar waveguides, optical fibers, metallized prisms, and diffraction gratings. These and other conventional methods typically require extensive analysis procedures that can take up to 24 hours to perform. Although all these techniques have some degree of sensitivity, they lack specificity, rapid detection, real time analysis, easy operation, low cost, and portability.
  • the present invention overcomes the aforementioned disadvantages by providing optical sensors that are capable of detecting a variety of analytes, especially polyatomic anionic analytes, with a relatively high degree of selectivity and sensitivity, and also offer the advantages of real time analysis, easy operation, low cost, and portability.
  • MIPs molecularly imprinted polymers
  • the lanthanide-containing MIPs of the present invention exhibit selective binding characteristics for a wide range of target analytes and thus allow for the detection of such target analytes with a relatively high degree of selectivity and sensitivity, and in less time and with fewer false positives than conventional optical sensors.
  • the chelated lanthanides embedded within the present Mff s can be sensitized to absorb excitation energy provided by a low-cost light and power source, such as a light-emitting diode (LED), and to subsequently luminesce to allow for the detection of analytes. Accordingly, the sensors of the present invention tend to be low-cost, portable, yet highly effective, analyte sensors.
  • the sensor devices of the present invention comprise a molecularly imprinted polymer containing a chelated lanthanide capable of binding the analyte to be detected, and which has operatively associated therewith: a light source for generating excitation energy for the chelated lanthanide of the molecularly imprinted polymer, wherein at least a portion of the excitation energy is absorbed molecularly imprinted polymer; and a detector for detecting luminescent energy generated by the chelated lanthanide upon excitation.
  • a molecularly imprinted polymer comprising: mixing a lanthanide salt with one or more polymerizable/lanthanide-coordinating ligand compounds and a polyatomic anion target analyte under conditions effective to produce a chelated lanthanide-analyte complex; co-polymerizing the lanthanide-analyte complex with one or more cross-linking monomers and one or more matrix monomers to form a polymer structure; and removing the polyatomic anion from the polymer structure to form an MIP.
  • Figure 1 is a schematic drawing of an optical sensor according to one embodiment of the present invention.
  • Figure 2 is a schematic representation of molecular imprinting to obtain a molecularly imprinted polymer according to certain embodiments of the present invention.
  • Figure 3 depicts the structural representation of an exemplary chelated lanthanide-analyte complex according to certain embodiments of the present invention.
  • Figure 4 depicts the structural representation of an exemplary chelated lanthanide-analyte complex according to certain other embodiments of the present invention.
  • Figure 5 is a schematic drawing of an optical sensor according to certain preferred embodiments of the present invention.
  • Figure 6 is laser excited luminescence spectra of Eu(DMMB) 3 (NO 3 ) 3 and Eu(DMMB) 3 PMP(NO 3 ) 2 crystalline solids excited at 465.8 nm.
  • Figure 7 shows the effect of pH on the temporal response of an optic sensor of the present invention.
  • Figure 8 shows the effect of the thickness of the polymer coating on temporal response.
  • Figure 9 shows response of an optical sensor of the present invention to selected interferents (pesticides) excited at 465.8 nm.
  • Figure 10 is LED excited luminescence spectra of Eu(dibenzoylmethane) 3 and NaEu(dibenzoylmethane) 3 H 2 PO 4 crystalline solids excited at 370 nm.
  • the present invention provides optical sensors that employ a molecularly imprinted polymer containing chelated lanthanides, in conjunction with a light source and a detector, to detect a variety of analytes with a relatively high degree of selectivity and sensitivity.
  • MIP molecularly imprinted polymer
  • MIPs refers generally to a polymeric mold-like stracture having one or more pre-organized recognition sites which complement the shape of at least a portion of a target or imprint molecule and which contain interactive moieties that complement the spacing of, and exhibit an affinity for, at least a portion of the binding sites on the target or imprint molecule.
  • MIPs are typically formed by coordinating imprint molecules with one or more functional monomers to form imprint/monomer complexes (wherein the imprint molecule interacts or bonds with a complementary moiety of the functional monomer via covalent, ionic, hydrophobic, hydrogen-bonding, or other interactions).
  • FIG. 1 is a schematic representation of one method of molecular imprinting showing self assembly of an imprint to form a imprint complex (1,2); inco ⁇ oration of the imprint complex into the polymer matrix (3); removal of the imprint molecule; and formation of the imprinted cavity (5).
  • the MIPs of the present invention comprise lanthanide-containing polymeric structures that exhibit selective binding characteristics towards an analyte to be detected by a sensor device of the present invention (a "target analyte").
  • a target analyte an analyte
  • Applicants have recognized that such MIPs can be used advantageously as part of an optical sensor device to selectively capture target analyte molecules, by associating such molecules with the MIP lanthanide binding sites, from an analyte solution for detection of the target analyte by the sensor.
  • the present MIP s act not only to provide a site for selectively rebinding the target analyte, but also, act as a source of luminescence, which can be analyzed to determine the amount of target analyte in an analyte solution.
  • the present chelated lanthanides can be sensitized to absorb light energy, including light in the blue region of electromagnetic spectrum, from a variety of light sources, including low-cost LEDs, and to luminesce with an enhanced, detectable intensity.
  • the intensity of a certain luminescence line will vary with the amount of anion bound to the polymer (wherein the an amount bound in the MIP is in equilibrium with amount in solution). Such characteristic luminescence can be detected and analyzed to determine the amount of target analyte in solution according to the present invention.
  • An MIP in accordance with the principles of the present invention can be prepared via any of a wide range of known methods including those described in U.S. Pat. Nos. 5,110,883; 5,321,102; 5,372,719; 5,310,648; 5,208,155; 5,015,576; 4,935,365; 4,960,762; 4,532,232; 4,415,655; and 4,406,792, the entire disclosures of which are inco ⁇ orated herein by reference.
  • the MIPs of the present invention are formed by: mixing a lanthanide salt with one or more polymerizable ligand compounds and a target analyte under conditions effective to produce a chelated lanthanide-analyte complex; co-polymerizing the lanthanide-analyte complex with one or more cross-linking monomers, and optionally, one or more matrix monomers to form a polymer structure; and removing the imprint molecule from the polymer stracture to form an MIP.
  • chelated lanthanide-analyte complex refers generally to a complex comprising a lanthanide ion having one or more polymerizable ligands associated therewith, wherein the chelated lanthanide is chemically bonded to a target analyte.
  • chemically bonded refers generally to any two moieties that are associated via covalent, ionic, hydrophobic, steric, electrostatic, hydrogen-bonding, or other bonding interactions.
  • suitable chelated lanthanide-analyte complexes can be made according to the present inveniton by mixing stoichiometric amounts of a lanthanide metal salt with one or more complexing ligands in an aqueous solution and evaporating to near dryness. Water or alcohol/water mixtures of the lanthanide metal, ligands, and target analyte in stoichiometric ratios, evaporated to dryness, are preferred to obtain near quantitative yields of the desired chelated lanthanide-analyte complex.
  • a lanthanide is chosen as the transducer because the lanthanide ions have excellent spectroscopic properties such as long luminescence lifetimes and narrow bandwidths, usually only a few nanometers. Any of a wide range of lanthanide metal salts capable of dissociating in solution to form a lanthanide ion, and combinations of two or more thereof, are suitable for use in the present invention.
  • lanthanide salts include halides, nitrates, perchlorates, and the like, of lanthanum (La), cerium (Ce), praseodymium (Pr), neodymium (Nd), promethium (Pm), samarium (Sm), europium (Eu), gadolinium (Gd), terbium (Tb), dysprosium (Dy), holmium (Ho), erbium (Er), thulium (Tm), ytterbium (Yb), and lutetium (Lu).
  • La lanthanum
  • Ce cerium
  • Pr praseodymium
  • Nd neodymium
  • promethium Pm
  • Sm samarium
  • Eu europium
  • Gd gadolinium
  • Tb terbium
  • Dy dysprosium
  • Ho holmium
  • Er erbium
  • Tm thulium
  • Yb
  • Preferred lanthanide salts of lanthanide ions that exhibit a narrow-line luminescence including salts of the +3 ions of samarium, europium, dysprosium, terbium, and neodymium.
  • the lanthanide salts are salts of the +3 ions of europium and terbium.
  • Any suitable complexing ligand compounds capable of coordinating with a lanthanide of the present invention and being capable of being polymerized with one or more other polymerizable monomers while chelated to the lanthamde can be used in the present invention.
  • suitable complexing ligand monomers include a wide range of mono- and bi-dentate ligands including nitrogen-, hydroxyl-, acid-, and/or ester- containing organic compounds such as: beta-diketones, including, vinyldibenzoylmethane, divinyldibenzoylmethane, and the like; phenanthrolines, including vinyl-substituted 1,10-phenanthroline, and the like; mono-, di-, and tri- acids and esters, including 4- vinyl benzoic acid, methyl-3,5-divinyl benzoate, and the like; oximes , including 4-vinyl-2-hydroxybenzaldehyde oxime (vinylsalicylaldoxime), and the like; 2-hydroxy-l,2-di-4-vinylphenylethanone (benzoin oxime vinyl derivative), and the like; polyaminopolycarboxylic acids including EDTA, and the like; (poly)pyridines; calix
  • Suitable ligand monomers for use in the present invention include those described in Jenkins, A., et al., "Ultratrace Determination of Selected Lanthanides by Luminescence Enhancement," Anal. Chem., 68(17):2974-2980 (1996)(the entire disclosure of which is inco ⁇ orated herein by reference).
  • Certain preferred complexing ligand compounds include vinyldibenzoylmethane, divinyldibenzoylmethane, vinyl-substituted 1,10- phenanthroline, 4-vinyl benzoic acid, methyl-3,5-divinyl benzoate, 4-vinyl-2 ⁇ hydroxybenzaldehyde oxime, 2-hydroxy-l,2-di-4-vinylphenylethanone (benzoin oxime vinyl derivative), and mixtures of two or more thereof.
  • the particular combination of complexing ligands and relative amounts thereof used for any given application of the present invention may vary depending on a number of factors including, the lanthanide ion to be used, the target analyte (in particular the anionic charge associated therewith, if any) to be bound thereto, and the light source to be used in the sensor device.
  • the chelated lanthanide monomer suitable for any particular application according to the present invention should have a charge which complements the charge (if any) on the target analyte such that the chelated lanthanide is capable of bonding with the target analyte.
  • the lanthanide monomer and target analyte form a bond that will resist dissociation during the polymerization process, but will subsequently release the target analyte to leave behind a suitable set of chelated lanthanide binding sites when the target analyte is removed.
  • the lanthamde ion is europium 3+ and the target analyte is a polyatomic anion, including organophosphorus anions
  • Fig. 3 shows an example of such a chelated lanthanide-analyte complex.
  • the complexing ligands are selected to enhance the luminescence intensity of the lanthanide. It is particularly desirable, in certain preferred embodiments, to sensitize the lanthanide such that, when the chelated lanthanide is inco ⁇ orated in an MIP, a low-cost light source, such as an LED, and be used to cause the lanthanide to luminesce, and in turn, provide a means of target analyte analysis with low limits of detection. As will be recognized, it is desirable for the selected ligands to overlap the triplet state of the lanthanide.
  • the lanthanide ion is europium 3+ and the target analyte is a polyatomic anion, including organophosphates, nitrate, perchlorate, and the like
  • Fig. 4 shows an example of such a preferred chelated lanthanide-analyte complex.
  • the polymerization step comprises co-polymerizing a chelated lanthanide-analyte complex with one or more cross-linking monomers, and optionally, one or more additional matrix monomers to form a polymer stracture.
  • crosslinking monomers Any of a wide range of crosslinking monomers can be used according to the present invention.
  • Suitable crosslinking monomers/agents that lend rigidity to the MIP include di-, tri- and tetrafunctional acrylates or methacrylates, divinylbenzene (DNB), alkylene glycol and polyalkylene glycol diacrylates and methacrylates, including ethylene glycol dimethacrylate (EGDMA) and ethylene glycol diacrylate, vinyl or allyl acrylates or methacrylates, divinylbenzene, diallyldiglycol dicarbonate, diallyl maleate, diallyl fumarate, diallyl itaconate, vinyl esters such as divinyl oxalate, divinyl malohate, diallyl succinate, triallyl isocyanurate, the dimethacrylates or diacrylates of bis-phenol A or ethoxylated bis-phenol A, methylene or polymethylene bisacrylamide or bismethacrylamide, including hexamethylene bisacrylamide or hexamethylene bisme
  • Any suitable monomer that provides an accurate imprint of the imprint molecule upon polymerization may be optionally used in addition to the crosslinking monomers and chelated lanthanide-analyte complexes to synthesize a MIP in accordance with the principles of the present invention.
  • suitable monomers include any of the complexing ligand monomers described above for forming a chelated lanthanide- analyte complex.
  • Suitable non-limiting examples of monomers that can be used for preparing a MIP of the present invention include: methylmethacrylate, other alkyl methacrylates, alkylacrylates, allyl or aryl acrylates and methacrylates, cyanoacrylate, styrene, alpha-methyl styrene, vinyl esters, including vinyl acetate, vinyl chloride, methyl vinyl ketone, vinylidene chloride, acrylamide, methacrylamide, acrylonitrile, methacrylonitrile, 2-acetamido acrylic acid; 2-(acetoxyacetoxy)ethyl methacrylate 1- acetoxy- 1,3 -butadiene; 2-acetoxy-3-butenenitrile; 4-acetoxystyrene; acrolein; acrolein diethyl acetal; acrolein dimethyl acetal; acrylamide; 2-acrylamidoglycolic acid; 2- acrylamido-2-methyl propane
  • Acrylate-terminated or otherwise unsaturated urethanes, carbonates, and epoxies can also be used in the MIP.
  • An example of an unsaturated carbonate is allyl diglycol carbonate (CR-39).
  • Unsaturated epoxies include, but are not limited to, glycidyl acrylate, glycidyl methacrylate, allyl glycidyl ether, and 1,2-epoxy- 3-allyl propane.
  • Preferred examples of matrix monomers include styrene, or styrene derivatives, especially those that can act as an optical antenna.
  • Any ratio of simple monomers to crosslinking monomers that provides a polymeric stracture of appropriate integrity can be used to produce an MIP according to the present invention.
  • those skilled in the art will be readily able to select suitable ratios of monomers to provide the desired structural integrity and produce MIPs according to the present invention, without undue experimentation.
  • any suitable conditions effective to polymerize the monomers of the present invention to produce an MIP without dissociating the chelated lanthanide-analyte complex may be used.
  • the monomers of the present invention may be polymerized via cationic polymerization, anionic polymerization, free radical polymerization, and the like. In preferred embodiments, free radical polymerization is used.
  • UN and thermal free radical initiators include benzoyl peroxide, acetyl peroxide, lauryl peroxide, azobisisobutyronitrile (AIB ⁇ ), t-butyl peracetate, cumyl peroxide, t-butyl peroxide; t- butyl hydroperoxide, bis(isopropyl)peroxy-dicarbonate, benzoin methyl ether, 2,2'- azobis(2,4-dimethylvaleronitrile), tertiarybutyl peroctoate, phthalic peroxide, diethoxyacetophenone, and tertiarybutyl peroxypivalate, diethoxyacetophenone, I- hydroxycyclohexyl phenyl ketone, 2,2-dimethyoxy-2-phenylacetophenone, and phenothiazine, and
  • the crosslinked polymer may be washed, cryogenically ground to a uniformly fine powder, and/or extensively eluted with nonpolar solvents to remove any unreacted lanthanide-analyte complex.
  • the steps of grinding and/or freezing in liquid nitrogen may be used to maximize surface area and allow for access by the various reagents and samples. Freezing allows the polymer to become brittle enough to be ground and prevents distortions of the polymer by the heat of friction.
  • Polymers used in the construction of optical sensors may be prepared in situ on the distal end of an optical fiber whose surface is prepared by binding a polymerizable agent on the surface.
  • the imprint molecule may be removed in a mam er that does not adversely affect the imprinted cavity.
  • any appropriate method can be used to cleave the covalent bond, although the covalent bond formed should preferably be cleaved under conditions suitable to release the imprint molecule after the MIP is formed, without adversely affecting the selective binding characteristics of the MJJP.
  • acetone or other suitable organic solvent may be used to swell the resultant polymers, allowing greater access to the coordinated metal ions because imprinted resins have a relatively low amount of functionalization and are primarily nonionic matrices.
  • the covalent bond that is cleaved to release the imprint molecule can optionally provide an additional polar or ionic site for design and imprinting of the imprint molecule, hi preferred embodiments wherein the target analyte is associated with the lanthanide in a non-covalent manner, the non-covalently bound analyte is simply leached or washed out after polymerization.
  • a 1 N aqueous acidic solution may be mixed into the acetone washes, with increasing aqueous acidic phase in each sequential wash, to remove the imprint molecule from the cavities.
  • an acidic solvent having a pH of about 4.5 or less is used.
  • resin mass action is used to replace a target anion with an easily exchangeable anion by immersing the polymer in a solution containing the easily exhangable anion at a suitable pH.
  • the MIP of the present invention is used in conjunction with a light source and a detector to form an optical sensor device for detecting a target analyte.
  • the term "light” refers to optical radiation, whether ultraviolet, visible or infrared. Suitable non-limiting examples of light sources include an argon laser, blue laser, tunable laser, light emitting diode (LED), combinations of two or more thereof, and the like.
  • any of a wide range of suitable detectors can be used according to the present invention.
  • suitable detectors include a spectrophotometer, spectrometer (gas or mass), photomultiplier tube, monochromator equipped with a CCD camera, filters, the naked eye, combinations of two or more thereof, and the like.
  • a sensor device of the present invention is produced by operatively associating at least one light source and at least one detector with an MIP.
  • two objects are considered to be "operatively associated" when connected or arranged in a manner such that excitation or luminescent energy produced by one of the objects is capable of being absorbed or detected by the other object.
  • the light source, detector and MIP of the present invention may be operatively associated in any manner such that excitation energy from the light source is transmitted to the MIP and absorbed by the chelated lanthanide, and the luminescent energy produced by the excited lanthanide is transmitted to, and detected by, the detector, h addition, the components of the present sensor devices may be connected or arranged with or in any suitable medium through which excitation or luminescent energy can be transmitted.
  • suitable media include air, optical devices, such as films or fibers, and combinations of two or more thereof.
  • the light source, MIP and detector are associated through optical fibers to provide a fiber optic sensor device.
  • the fiber optic sensor device for detecting the presence of at least one analyte in a sample, such as an organophosphorus compound comprises: at least one optical fiber having a proximal end and a distal end for transmitting light energy, the proximal end being disposed within a probe housing, a molecularly imprinted polymer containing a lanthanide-complex disposed on, or bonded to, the distal end of the optical fiber means, wherein the lanthanide-complex is capable of chemically binding with said analyte, a light source for generating excitation energy, said light source being operatively associated with said optical fiber such that said excitation energy passes through said optical fiber means to said MIP, and detection means operatively associated with said optical fiber means, for detecting luminescent energy generated by said lanthanide complex.
  • the device may employ a modulated light emitting diode (LED) for excitation and a small photosensor module for detection, with the output going to a microprocessor controlled grated integrator.
  • LED light emitting diode
  • an optical multiplex switch may be inco ⁇ orated into the design so that many sensors can be coupled to one control system, which will allow monitoring of a large area such as found in a building, subway station, shopping mall, ai ⁇ ort, etc.
  • a target analyte if present, binds to the lanthanide in the molecularly imprinted polymer causing it to luminesce differently under appropriate excitation.
  • Light from the light source means travels along the optical fiber to its distal end where it undergoes a change caused by interaction with the lanthanide-complex.
  • the modified light returns along the same or another fiber to the detection means which inte ⁇ rets the returned light signal. Detection is based on the change that occurs in the lanthanide's luminescence spectrum when an analyte binds to the lanthanide-complex.
  • Fig. 1 illustrates an exemplary fiber optic portable sensor device according to certain preferred embodiments of the present invention.
  • the sensor device 10 in Fig. 1 comprises an optical fiber 11 having a proximal end disposed within a sensor housing 12 and a distal end having a molecularly imprinted polymer 13 disposed on (bonded to) the distal end of optical fiber 11.
  • Light source 14 is a blue LED diode from which light in the blue range of the spectrum is emitted. The light is emitted through a dichroic mirror 15 to the proximal end of fiber 11 wherein the light energy is transmitted to the chelated lanthanides in the MJJP 13. Any luminescene generated by the lanthanides travels back through fiber 11 and is reflected off the dichroic mirror 15 to detector 16 which comprises a filter 17, a photadiode 18, and a readout 19.
  • exemplary device shown in Fig. 1 comprises a single housing for the detector and light source only, any suitable combination of one or more of the light source, detector, and/or MIP can be housed within one or more device housings according to the present invention.
  • the distal end (working end) of the sensor maybe enclosed within a semi-permeable membrane to separate the analyte-containing media being analyzed from the probe.
  • a semi-permeable membrane to separate, as far as possible, the analyte (i.e., those components in a sample that can bind to the probe) from interferents (i.e., compounds which may be present but are undesirable because they either interfere with the progress of the desired determination reactions or take part in reactions of their own which compete with those of the component sought and distort or overwhelm the signals that are to be measured).
  • the semi-permeable membrane may be impregnated with an alkaline solution or coated with a nonvolatile alkaline oil, to catalyze the hydrolysis of the nerve agents soman and sarin to their respective hydrolysis products.
  • Fig. 5 illustrates an exemplary sensor according to an embodiment.
  • the optical sensor devices of the present invention can be used to detect any of a wide variety of analytes.
  • the present sensors can be used to great advantage in the selective and accurate detection of polyatomic anion analytes.
  • exemplary polyatomic anions detectable by the present sensors include: organic polyatomic anions, such as, organophosphates including sarin, soman, tabun, NX, malation, parathion, paraoxon, diazinon, adenizine triphosphate (ATP), hydrolysis products thereof, and the like; nitrate, sulfate, sulfite, selenate, other oxyanions including pertechnitate, molybdate, perchlorate, periodate, hypochlorite, and the like.
  • the present invention will be further illustrated in the following, non- limiting Examples.
  • the Examples which exemplify a sensor device for detecting benzoates and the hydrolysis products of the nerve gases soman and sarin, are illustrative only and do not limit the claimed invention regarding the materials, conditions, process parameters and the like recited herein.
  • reagent materials were obtained from commercial suppliers and used without further purification. Analytical reagent grade chemicals were used along with deionized water to prepare solutions. PMP and sodium phosphate were obtained from Aldrich (Aldrich, Milwaukee, WI 53233). Neat liquid standards of Phosdrin and Dichlorvos as well as solid standards of Methyl Parathion and Dimethoate were obtained from Supelco (Supelco Chromatography Products, Bellefonte, PA 16823).
  • Spectra were also obtained with an Ocean Optics 52000 Miniature Fiber Optic Spectrometer (Ocean Optics, Dunedin, FL 34698) equipped with a 1200 line holographic grating, permanently installed 100 micron slits and a 440 nm cutoff filter.
  • EXAMPLE 2 Compound Preparation [0058] Lanthanide complex compounds were synthesized using a stoichiometric ratio of one mole of europium to one mole of benzoate anion analyte, namely triethylammonium benzoate (TEAB) or methyl ammonium benzoate (MAB), and about 3 moles of beta-diketone ligating molecules.
  • the benzoate anions were formed by reaction of benzoic acid with a primary and/or a tertiary amine.
  • TEAB and MAB are soluble in organic solvents, which eases the reaction with a hydrophobic tris(beta-diketone) europium complex.
  • MAB and TEAB were reacted with tris(vinyl-benzoylacetonate) europium (TBAE) and tris(l,3- diphenylpropandionate) europium (DPPE) to give four new anionic complexes.
  • Styrenic block copolymers were prepared and the optimal mole percent complex for the preparation of the polymer coating determined.
  • Polymers were prepared by dissolving 1 to 5 mole percent complex compound in 94-98 mole percent styrene. Approximately 1 mole percent of azobisisobutylnitrile (ATBN) was added as an initiator to the mixture described in Example 1.
  • Crosslinked polymers were also prepared using 3 mole percent compound with 1-5 mole percent of a crosslinking agent divinyl benzene (DVB), styrene and ATBN. The resulting solutions were placed in glass vials, purged with nitrogen, and sealed using parafilm and screw on tops.
  • the resulting translucent polymers displayed a slight yellow tint and upon excitation with a uv lamp, displayed the characteristic red-orange luminescence of europium. The best results were obtained from the 3 n i overall, had a diminished analyte peak. Polymers with greater than 5 mole percent complex were not used since they tend to become opaque, reducing optical transduction.
  • the partially polymerized material was placed in an oven at 60°C and allowed to cure overnight.
  • the resulting block copolymers were ground to expose a larger surface area of the polymer and facilitate the removal of the imprinting ion.
  • the imprint ion is removed in two steps (Id.): (1) swelling in water and gradually increasing amounts of methanol (Helferich, F., Ion Exchange; McGraw-Hill: New York, 511(1962)) to remove unreacted monomer and expand the polymer pores, (this produces accessible sites and facilitates the removal of the imprinting ion, and (2) removal of the imprinting ion by acid washing.
  • Acid washing pH of about 4.5
  • the optimal conditions for swelling the polymer include a series methanol/water washes, followed by washing with a weak nitric acid solution.
  • the spectrum of the washed polymer shows the 610 nn peak was no longer visible, demonstrating that PMP was effectively removed.
  • a small residual peak at 610 nm was viewed in some of the polymers resulting from some hydrolysis product trapped in the deeper levels of the polymer.
  • the overall intensity of the polymer's luminescence also decreases upon washing since the nitrate is only weakly coordinated, possibly allowing water to enter the coordination sphere of the lanthanide.
  • the washed polymer was tested for its ability to rebind PMT by exposing it to a 150 ppm PMP solution in aqueous 1M NaOH and obtaining its luminescence spectra. The 610 nm peak was observed in the spectra.
  • a fiber optic sensor comprising a 400 micron optical fiber (Thor Labs, Newton, NJ, 07860) with the polymeric sensing element chemically bound on its distal end was constructed.
  • the fibers were prepared by terminating one end with an SMA connector and removing the cladding from and polishing the distal end using the procedures outlined in the "Thor Labs Guide to Connectorization and Polishing of Optical Fibers".
  • the tips were dipped into the chemically initiated viscous copolymer described in Example 2 leaving a uniform layer on the fiber.
  • the polymer finished curing under a small UN lamp, overnight. Coated fibers were conditioned in a manner similar to the ground polymers as outlined above.
  • Luminescence was excited using the argon laser and the active end of the sensor was placed in a quartz cuvette containing one of the sample dilutions. Two argon ion excitation wavelengths 465.8 and 488 nm, were used with the polymer.
  • the spectrum of the sensor excited with the 465.8 nm laser line displayed better spectral resolution of the 610 nm analyte peak from the 615 rim luminescence peak of the parent europium.
  • the luminescence of the compound excited at 465.8 nm was also more intense. This increase indicates that excitation using the 465.8 nm line results in a near resonant excitation transition from the ground 7Fo level to the SD2 level.
  • Standard 1000 ppm solutions were prepared by the dissolution and/or dilution of the samples in deionized water when possible.
  • the pesticides with limited solubility in water were prepared using a 50:50 water/methanol mixture.
  • the pH of each of the solutions was adjusted to 12 using 1 M sodium hydroxide.
  • Spectra from the fiber for each analyte were taken at regular intervals for 60 minutes. The resulting spectra were then compared with the response from the sensor in 100 ppm PMP.
  • the sensor was cleaned using 1 M nitric acid and rinsed with deionized water between each analysis.
  • Eu(DMMB) 3 PMP(NO " ) 2 demonstrated a relatively easily discernible spectral difference. See Figure 8.
  • the luminescence intensity of this compound was not as large as some of the other candidates, however, the clarity of the spectral difference between the compound with and without the hydrolysis product made detection based on the spectrum a relatively simple process.
  • Eu(PMP) 3 was prepared and its luminescence spectrum generated.
  • the peak at 610 nm in the 7 F 2 — 5 D 0 manifold of Eu3+ for the compound was clearly not in the' spectrum of Eu(PMP) 3 .
  • the Eu(PMP) 3 displayed weak luminescence and poor resolution.
  • the Eu(PMP) 3 spectra strongly suggests that the peak at 610 nm was due to the addition of the hydrolysis product to the compound and not an impurity.
  • the performance of the fiber optic sensor with the 1/4 meter monochromator was evaluated.
  • the sensor used to determine the limit of detection consisted of a 400 pm optical fiber with a tapered end. A 50-75 gm layer of the 3 mole percent polymer was directly deposited on to the end. The fiber was cleaned using the method previously described. Using lmW of 465.8 nm for excitation, 200 ⁇ m slits with the monochromator, and an exposure time of 5 seconds, the luminescence spectrum of the sensor in a series of PMP solutions at pH 13, was obtained. The response of the sensor to increasing concentrations of PMT exhibits an increase in the luminescence intensity of the primary europium band as well as an increase in the intensity of the analyte peak.
  • EXAMPLE 6 Response Time and pH Dependence The response time of the sensor is the most crucial characteristic of detectors and sensors for real-time monitoring. Yang, Y.C., et al., Chemical Reviews, 92: 1729-1743(1992). A study was performed using a sensor with a 200 micron coat to determine the effect of pH on the response time and on solutions of PMP prepared with pH values ranging from about 4.5 to 13 over a period of 24 hours. Figure 9 shows the response of the sensor over the initial 30 minute time period. Additional readings were obtained for each pH value at 1 hour and at 24 hours.
  • the sensors show a positive response to the presence of PMP after 3 minutes for all pH values from 6 to 12, and a positive response after 1 minute for the solution with a pH of 13.
  • the response of the sensor is indicative of the removal of PMP from the sensor.
  • This demonstrates the washing process that occurs under acidic conditions.
  • Neutral and slightly basic values (pH from 6-11) provide a response that is consistent over the entire pH range.
  • the full response time for this sensor is 30 minutes. (Response times are typically reported as the time it takes the sensor to reach 80% of maximum.) Report "Assessment of Chemical and Biological Sensor Technologies," National Research Council (1984).
  • the fiber with a 200 micron coat reaches a maximum response within 14 minutes.
  • the 80% response time of the 100 micron coated fiber is decreased to 8 minutes.
  • the compounds that are most chemically analogous to nerve agents are organophosphorus pesticides and herbicides. Many of these compounds exist as liquids,oils or solids at ambient temperatures. Several common pesticides, along with those most chemically similar to the agents sarin and soman were tested using the sensor in order to determine the degree of interference from each pesticide. The concentration used for screening 1000 ppm, is much higher than typically found in water systems even with runoff from nearby agriculture. The pesticide dichlorvos, commonly found in flea collars, was also screened as a possible interference.
  • EXAMPLE 8 Miniaturization
  • the device based on an Ocean Optics spectrometer exhibited favorable sensitivity and selectivity in detecting the agents on a smaller scale.
  • the entire instrument fits on a board 3.5' x 2.5'.
  • the limit of detection for this device was determined using the same procedure used to determine the limit of detection for the larger system.
  • This system provides a limit of detection of 7 parts per trillion using approximately, 1 mW of 465.8 nm laser power and an integration time of 500 microseconds.
  • the linear dynamic range of the device is from 7 ppt to lppm using a 75 p.m coating of fiber.

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Abstract

Cette invention a trait à des dispositifs permettant de détecter et de mesurer une large gamme d'analysats, notamment des anions polyatomiques, tels que des pesticides organophosphorés et des agents neurotoxiques. Ces dispositifs agissent par fixation sélective d'un analysat à un copolymère à empreinte à fonctionnalité luminescente. Ces copolymères renferment un ion lanthanide luminescent à liaison solide, tel que Eu3+, dans un complexe de coordination et ayant fait l'objet d'une empreinte afin de fixer la fonctionnalité chimique. L'invention porte également sur des procédés de production de ces polymères à empreinte moléculaire contenant un lanthanide.
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US8021893B2 (en) 2004-05-07 2011-09-20 Japan Science And Technology Agency Molecular recognition polymer enabling reconstruction of recognition field for target molecule and method of producing the same
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US7520158B2 (en) 2005-05-24 2009-04-21 Baker Hughes Incorporated Method and apparatus for reservoir characterization using photoacoustic spectroscopy
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