WO2006076430A1

WO2006076430A1 - Nerve agent detection method and device

Info

Publication number: WO2006076430A1
Application number: PCT/US2006/000980
Authority: WO
Inventors: E. Roland Menzel
Original assignee: Texas Tech University; MENZEL, Theresa
Priority date: 2005-01-14
Filing date: 2006-01-12
Publication date: 2006-07-20

Abstract

A method for detecting a nerve agent, comprising the steps of exposing a test zone to a test sample for an adequate time period for any nerve agent or nerve agent components in said test sample to contact said test zone, said test zone comprising a nerve agent detection composition, to form an exposed test zone; viewing said exposed test zone under a light source suitable for allowing observation of any optical alterations; and concluding that said test sample contains a nerve agent or nerve agent component after comparing the optical alterations observed to those occurring in a control test zone. An apparatus for nerve agent detection comprising at least four nerve agent detection compositions.

Description

NERVE AGENT DETECTION METHOD AND DEVICE

FIELD OF THE INVENTION

[0001] The invention relates to the field of compositions, methods and devices for the detection of nerve agents.

BACKGROUND OF THE INVENTION

[0002] Organophosphates are a potent class of nerve agents. Both volatile and non- volatile nerve agents are of concern to public health. Volatile nerve agents have high vapor pressures and are readily deployed and preparable. Examples of volatile nerve agents are Sarin and Soman. Such agents may be inhaled as well as absorbed through the skin. Sarin (O-Isopropyl Methylphosphonofluoridate), has a formula of CH₃P(O)(F)OCH(CH₃)₂. Sarin is classified as a weapon of mass destruction by the United Nations, according to UN Resolution 687, and was employed by terrorists in an attack on the Tokyo subway system in 1995. The compound inhibits the acetylcholinesterase enzyme needed for relaxation of a muscle or organ after stimulation. By binding to the enzyme, it in effect causes nerve impulses to be continuously transmitted because acetylcholine builds up. A dose of 0.01 mg per kilogram of body weight may be lethal in as little as one minute, and sublethal doses can cause permanent damage to the nervous system. Soman (O-Pinacolyl methylphosphonofluoridate), also known as "GD," is also classified as a nerve agent and as a weapon of mass destruction by the United Nations according to UN Resolution 687. Soman is more lethal than Sarin and is volatile, corrosive and (in pure form) a colorless liquid or (in impure form) a yellow to brown color with an odor. There are also non-volatile nerve agents that may be deployed in warfare or terroristic attacks as an aerosol. An example of such an agent is VX (Phosphonothioic acid, methyl-,S-(2- (diisopropylamino)ethyl) O-ethyl ester). A need exists for rapid detection of nerve agents in the field as well as the clinical laboratory.

BRIEF SUMMARY OF SOME OF THE PREFERRED EMBODIMENTS [0003] Disclosed herein is a method for detecting a nerve agent, comprising the steps of exposing a test zone to a test sample for an adequate time period for any nerve agent or nerve agent components in said test sample to contact said test zone, said test zone comprising a nerve agent detection composition, to form an exposed test zone; viewing said exposed test zone under a light source suitable for allowing observation of any optical alterations; and concluding that said test sample contains a nerve agent or nerve agent component after comparing the optical alterations observed to those occurring in a control test zone. [0004] Further disclosed herein is a method of detecting an organophosphate nerve agent comprising the steps of providing a nerve agent detector composition test zone; adding a test sample containing an organophosphate nerve agent to form an exposed test zone; observing such exposed test zone under ultraviolet or other appropriate light; and concluding that said test sample likely contains an organophosphate if said exposed test zone exhibits changes in spectral properties as compared to a control test zone.

[0005] Further disclosed herein is an apparatus for nerve agent detection comprising at least four nerve agent detection compositions.

[0006] Further disclosed herein is a method of detecting an optically reactive target compound comprising placing at least one optical sensor chemical in contact with the target compound; allowing the optical sensor chemical to react with the target compound and; detecting any optical alterations produced when the said optical sensor chemical reacts with said target compound.

[0007] Further disclosed herein is a method of detecting a nerve agent in a sample comprising hydrolyzing said nerve agent; reacting the hydrolysis product of said nerve agent with a lanthanide coordination complex; detecting alterations in the luminescence of the lanthanide coordination complex and; determining the presence or absence of a nerve agent in said sample.

[0008] Further disclosed herein is a method of detecting an organophosphate nerve agent comprising the steps of (a) providing a nerve agent detector composition test zone; (b) adding a test sample containing an organophosphate nerve agent to form an exposed test zone; (c) observing such exposed test zone under ultraviolet or other appropriate light; and (d) concluding that said test sample likely contains an organophosphate if said exposed test zone exhibits changes in spectral properties as compared to a control test zone. [0009] The foregoing has outlined rather broadly the features and technical advantages of the present invention in order that the detailed description of the invention that follows may be better understood. Additional features and advantages of the invention will be described hereinafter that form the subject of the claims of the invention. It should be appreciated by those skilled in the art that the conception and the specific embodiments disclosed may be readily utilized as a basis for modifying or designing other structures for carrying out the same purposes of the present invention. It should also be realized by those skilled in the art that such equivalent constructions do not depart from the spirit and scope of the invention as set forth in the appended claims. BRIEF DESCRJPTION OF THE DRAWINGS

[0010] Figures Ia-Ib are schematics of devices for nerve agent detection. [0011] Figure 2 is a room light photo showing the color of the reaction solutions. [0012] Figures 3-4 are photos of reaction solution fluorescence.

DETAILED DESCRIPTION

[0013] Disclosed herein are compositions for the detection of nerve agents and methods of using same. Compositions for the detection of nerve agents (NADs) may comprise a metal complex, a metalloid compound, an organic chromophore or combinations thereof. Herein a metal complex refers to a compound having at least one metal ion in communication with at least one other atom and a metalloid compound refers to a compound comprising at least one metalloid. A metallod may be defined as an atom having properties intermediate between those of metals and nonmetals. The known metalloids are boron, silicon, germanium, arsenic, antimony, tellurium and polonium. Herein an organic chromohphore refers to any organic compound that absorbs and transmits light energy. NADs may function in the detection of nerve agents such as organophosphate nerve agents or non-phosphate containing nerve agents. In an embodiment, a NAD reacts with a nerve agent to produce an alteration in the optical properties of the NAD, the nerve agent or both. Such optical alterations may include without limitation changes in spectral wavelength, changes in spectral intensity or combinations thereof. For example, in one embodiment volatile nerve agents such as Sarin, Soman and Cyclosarin may be detected. In another embodiment, non-volatile nerve agents are detected, such as, for example, VX.

[0014] In an embodiment, an NAD comprises a metal complex, alternatively a lanthanide coordination complex (LCC), alternatively a europium coordination complex, alternatively a terbium coordination complex , or combinations thereof. In another embodiment, an NAD comprises a lanthanide ion and one or more ligands. Such ligands will be described in more detail later herein.

[0015] Lanthanides (also known as rare earth elements) are elements with atomic numbers 57-71 in the periodic table of the elements. Lanthanide complexes are known to be highly luminescent wherein luminescence refers to the emission of light after the absorption of a photon. Lanthanide luminescence may result from exposure to ultraviolet (UV) or near UV irradiation in the wavelength range of about 200 ran to 380 nm. Such UV irradiation results in excitation of the lanthanide ion and hence the devices capable of producing the UV irradiation are also termed excitation sources. Any device capable of producing UV irradiation may serve as an excitation source for lanthanide luminescence. Examples of excitation sources include lasers such as tunable diode lasers, excimer lasers, HeCd lasers and nitrogen lasers and halogen lamps such as a xenon lamp. Such excitation sources and UV irradiators may be used with other NADs and embodiments as disclosed herein.

[0016] Luminescence can be of two distinct types. If the emission occurs from an excited state that has the same spin multiplicity as the ground state, then the emission is called fluorescence. Phosphorescence occurs when the excited state is of different spin multiplicity than the ground state. The spin multiplicity may be defined as the number of possible orientations calculated as 2S + 1, of the spin angular momentum corresponding to a given total spin quantum number (S) for the same spatial electronic wavefunction and is directly related to the number of unpaired spins in a molecule. Fluorescence and phosphorescence can also be distinguished based on the time frame for emission of light following irradiation. Fluorescence light emission typically occurs within nanoseconds to milliseconds after irradiation whereas phosphorescence light emission occurs over much longer time periods (i.e. minutes to hours) after initial irradiation. The fundamental equation of luminescence spectroscopy is

0(X) = KI(λ)Φ(λ)F(λ) (1) where O(λ) is the observed luminescence intensity when the sample is being irradiated at wavelength λ with an incident intensity (quanta/sec) of IQC). The luminescence quantum yield, Φ(λ) is the probability that once a molecule has absorbed a photon it will emit a photon. F(X) is the fraction of incident light absorbed by the sample, and K is a proportionality constant. For most pure substances the emission spectrum is independent of the exciting radiation. K is then usually constant as long as the detector wavelength, system gain, and geometry do not change. The fraction of radiation absorbed, F(X), is given by Beer's Law:

where: ε(λ) is the molar absorptivity at wavelength, λ;

C is the concentration in moles/liter; and £ is the optical path length

[0017] The luminescence behavior of lanthanides is well known in the art. For example, it is well known in the art that both europium and terbium, Eu³⁺ and Tb³⁺, luminescence with high quantum efficiency. Without wishing to be limited by theory, the most intense luminescence of the europium ion occurs at about 615 nm (red), arising from the transition from the upper ⁵D₀ to the lower ⁷F₂ state. For terbium, the corresponding states are ⁵D₄ and ⁷F₅, respectively, with transitions resulting in green luminescence at 545 nm. The luminescence may be detected using any optical device for the detection of luminescence or the luminescence may be directly observable. Although the luminescence efficiencies can be high, the luminescence intensities are generally quite low because the lanthanide molar extinction coefficients, which are proportional to the ion's ability to absorb light, are very low. The low molar extinction coefficients are due to the fact that the transitions from the ground state (⁷F₀ for the europium and ⁷F₆ for the terbium ion) violate selection rules governing electronic transitions. Specifically, electronic transitions involving a change in the number of unpaired electrons are forbidden.

[0018] m an embodiment, the LCCs are europium and/or terbium coordination complexes. Said coordination complexes may comprise europium and/or terbium in the +3 oxidation state bonded to at least one ligand. The coordination complexes may comprise the lanthanide and any number of ligands, alternatively the coordination complexes comprise the lanthanide and a sufficient number of ligands to form a nine-fold coordination sphere around the lanthanide. For example, a LCC may be of the form LX₃^oH₂O, where L is the bivalent lanthanide cation (for example Eu³⁺and/or Tb³⁺) and X is a monovalent anion (for e.g. chloride or nitrate), with 6 water molecules completing the coordination. In some embodiments, the LCC comprises the lanthanide ion and at least one monodentate ligand. Alternatively, the LCC comprises the lanthanide ion and at least one multidentate ligand. Alternatively, the LCC comprises the lanthanide ion and a combination of monodentate and multidentate ligands. Herein the term monodentate refers to a ligand with the ability to form one bond with a metal ion whereas multidentate refers to a ligand with the ability to form multiple bonds to a metal ion. For example a bidentate ligand is capable of forming two bonds while a tridentate ligand is capable of forming three bonds.

[0019] In an embodiment, the monodentate or multidentate ligand may be sensitizing, nonsensitizing, or densensitizing. Herein the terms sensitizing, nonsensitizing and densensitizing refer to the ligand's ability to absorb light well and efficiently transfer the absorbed light (excitation energy) to the lanthanide ion via a process known as the Forster energy transfer process. The Forster type fluorescence energy transfer (FRET=Fluorescence Resonance Energy Transfer) process is a probabilistic process based on dipole-dipole interaction between suitable donor and acceptor molecules. Its efficiency depends on the donor (ligand) fluorescence-acceptor (lanthanide ion) absorption spectral overlap as well as proximity between donor and acceptor. A ligand capable of FRET is referred to herein as a sensitizing ligand because it strongly increases the lanthanide luminescence via the energy transfer process. In an embodiment, the LCC comprises a lanthanide ion and at least one sensitizing ligand. In an embodiment, the sensitizing ligand is thenoyltrifluoroacetone (TTFA), 1,10 phenanthroline (OP), bipyridine, terpyridine, triphenylphosphine, or combinations thereof. Alternatively, the sensitizing ligand is thenoyltrifluoroacetone , 1,10 phenanthroline, or combinations thereof. Methods of selecting an appropriate sensitizing ligand are known to one of ordinary skill in the art. For example, one method of selecting an appropriate sensitizing ligand may involve performing luminescence spectroscopy on the proposed ligand-lanthanide coordination complex and observing alterations in lanthanide luminescence. [0020] In an alternative embodiment, the LCC comprises a lanthanide ion and at least one non-sensitizing ligand. Nonsensitizing ligands herein refer to ligands that may occupy the lanthanide binding sites and prevent the coordination of desensitizing ligands. Without limitation, an example of a non-sensitizing ligand is ethylenediaminetetraacetic acid (EDTA) which may form as many as five bonds with the lanthanide ion.

[0021] hi an alternative embodiment, the LCC comprises a lanthanide ion and at least one desensitizing ligand. Desensitizing ligands herein refer to ligands whose coordination to the lanthanide would function to reduce the lanthanide luminescence. An example of a desensitizing ligand includes without limitation water. The bonding of the water to the lanthanide ion through the oxygen end of the water is neither ionic nor of the typical covalent bonding variety of organic molecules. It has some electrostatic characteristics, namely, the attraction between charged object and an electric dipole, reminiscent of hydrogen bonding, and some covalent characteristics as well.

[0022] In an embodiment, the LCC comprises a lanthanide ion and no more than one water ligand. With regard to lanthanide luminescence, the coordination of one water molecule may not substantively affect the luminescence of the metal but a larger number of waters of hydration may result in the loss (quenching) of lanthanide luminescence, the quenching being proportional to the number of hydration waters. The intrinsic preference of lanthanides for ligands which coordinate via an oxygen atom (i.e. water) may increase further when the oxygen acts as if it were a negatively charged entity, as it does in most explosives via the ubiquitous NO₂ functionality. For example, the proclivity of lanthanides for water is such that a number of lanthanide coordination complexes that are highly luminescent are quenched once placed in the presence of water because the water displaces the sensitizing ligands. [0023] It is to be understood that the choice of lanthanide ion, sensitizing ligands, nonsensitizing ligands and desensitizing ligands are independent of each other, thus any combination of these components to form an NAD is contemplated in this disclosure. [0024] In an embodiment, the LCC comprises europium and/or terbium in the +3 oxidation state and a multidentate ligand. In some embodiments, the multidentate ligand is sensitizing, alternatively nonsensitizing, alternatively desensitizing. In an embodiment, the ligand is OP, alternatively the ligand is TTFA. TTFA and OP both sensitize red europium luminescence (under near-UV illumination), but TTFA is preferred. The OP ligand, rather than the TTFA ligand, serves a luminescence sensitization function (under deep-UV illumination) to produce green terbium luminescence. TTFA, however, is spectrally mismatched with terbium, and thus does not function as sensitizing ligand. Thus it may only function as a ligand that excludes water.

[0025] In an embodiment, the LCC comprises Eu³⁺ complexed with four TTFA ligands. Alternatively, the LCC comprises Tb³⁺ complexed with OP ligands, or alternatively Eu3⁺ complexed with TTFA and OP ligands. In an embodiment, the LCC comprises a coordination complex of the type represented by structure I:

TTFA

Structure I

OP

[0026] Without wishing to be limited by theory, LCCs comprising a lanthanide ion and a nonsensitizing ligand may react with the hydrolysis product of a nerve agent such as Sarin or VX. For example, hydrolysis of Sarin liberates fluoride ions, which may then attack the LCC. Once the LCC is attacked by the fluoride anion resulting from the hydrolysis of the fluorophosphate nerve agent, the ligand, TTFA, is displaced. The free LCC coordination sites may then be occupied by any available water, which results in a quenching of the lanthanide luminescence.

[0027] Alternatively, LCCs comprising a lanthanide ion and a sensitizing ligand may react with the hydrolysis product of a nerve agent such as Sarin or VX. In this case, the hydrolysis product (fluoride ion) is unable to displace the sensitizing ligand (e.g. OP) and instead displaces the one water of hydration. Displacement of the water that occupies the ninth binding site of the lanthanide by a hydrolysis product of the nerve agent may result in a luminescence enhancement.

[0028] LCCs of the type disclosed herein may be prepared using any suitable method known in the art. For example, a LCC can be prepared by mixing in a solvent in which it is soluble the lanthanide chloride hexahydrate with the desired ligand, such as TTFA and/or OP. It is preferable to use an alcohol solvent such as methanol or ethanol because these are appropriate for solubility and are quick drying. For a single ligand complex, it is preferable to use excess ligand to ensure fullest coordination with that ligand. A preferred ratio is 1:5 lanthanide compound to ligand. For a mixed ligand complex, excess ligand is employed, and the preferred ratio is 1:3.3. The complex formation occurs rapidly on mixing. [0029] In an embodiment, an NAD comprises a metalloid compound, alternatively a semiconductor nanoparticle. Herein a semiconductor refers to a substance, usually a solid chemical element or compound, that can conduct electricity under some conditions but not others, making it a good medium for the control of electrical current while a nanoparticle refers to a particle with at least one dimension less than lOOnm. Such semiconductor nanoparticles may also be referred to herein as quantum dots. Any semiconductor nanoparticle that reacts with a nerve agent to produce an alteration in the optical properties of the NAD, the nerve agent or both may be suitable for use in this disclosure. In an embodiment, the NAD comprises a semiconductor nanoparticle further comprising a metalloid. Alternatively, the NAD comprises cadmium selenide (CdSe), cadmium sulfide (CdS), cadmium telluride (CdTe), or combinations thereof. Li an embodiment, the NAD comprises a semiconductor nanoparticle that is part of a core/shell structure. Such core shell structures are known in the art and include for example and without limitation a CdSe core with a zinc sulfide (ZnS) shell. Structures of this form have been described in detail in Tomlinson, LD et al. "The Design and Synthesis of Novel Derivatives of the Dopamine Uptake Inhibitors GBR 12909 and GBR 12935: High-affinity opaminergic Ligands for Conjugation with Highly Fluorescent Cadmium Selenide/Zinc Sulfide Core/Shell Nanocrystals" published in 2003 in Tetrahedron Volume 59 Number 40 pages 8035-8047 and Tomlinson, LD. et al. "A Synthesis of 6-(2,5-dimethoxy-4-(2- aminopropyl)phenyl)-hexylthiol:A Ligand for Conjugation with Fluorescent Cadmium Selenide/Zinc Sulfide Core/Shell Nanocrystals and Biological Imaging" published in 2002 in Molecules Volume 7 Number 11 pages 777-790 each of which are incorporated herein by reference in their entirety.

[0030] As is known to one of ordinary skill in the art, the particle size of the semiconductor nanoparticle may be designed so as to provide the optical and mechanical properties desired by the user. Any method and condition for the preparation of semiconductor nanoparticles of a desired size and having the desired optical properties may be used.

[0031] In an embodiment, the semiconductor nanoparticles of this disclosure may be prepared by any means known to one of ordinary skill in the art. In an embodiment, a semiconductor nanoparticle is prepared using a simple aqueous precipitation process. For example, a sulfide salt, such as ammonium sulfide may be contacted in aqueous media with a cadmium salt such as cadmium sulfate to form cadmium sulfide nanoparticles. Alternatively, a cadmium sulfide nanoparticle may be prepared using a process involving templates. Any material containing regular nano-sized pores or voids may be used as a template to form the semiconductor nanoparticles. Examples of such templates include without limitation porous alumina, zeolites, di-block co-polymers, dendrimers, proteins and other molecules. In an embodiment, the semiconductor nanoparticles are cadmium sulfide nanoparticles formed using a dendrimer as a template. Herein a dendrimer refers to a tree-like highly branched polymer molecule. Methods and conditions for the synthesis of semiconductor nanoparticles using a dendrimer template are known to one of ordinary skill in the art.

[0032] In an embodiment, the NAD comprises a semiconductor nanoparticle which when reacted with a nerve agent or component of a nerve agent results in a detectable alteration in the optical properties of the NAD, the nerve agent or both. Alternatively, the NAD comprises a semiconductor nanoparticle which when reacted with a nerve agent or component of a nerve agent displays a detectable alteration in the fluorescence spectroscopy of the NAD. Such alterations may be changes in the fluorescence intensity, fluorescence emission energy or combinations thereof.

[0033] In an embodiment, the NAD comprises a semiconductor nanoparticle immobilized on suitable substrates. Suitable substrates will be described later herein. Alternatively, the NAD comprises a semiconductor nanoparticle in a liquid media. Methods for the use of an NAD either immobilized on a substrate or in a liquid media will be described later herein. [0034] In an embodiment, an NAD comprises an organic chromophore, alternatively a fluorescent dye, fluorescent dye derivative or combinations thereof. The fluorescent dye may be chosen to have emissions in any energy range desired by the user. As would be understood by one of ordinary skill in the art, the fluorescent dye may also be chosen to have an excitation energy in a range desired by the user. Many such dyes are readily available from numerous commercial sources.

[0035] hi an embodiment, the fluorescent dye is of the type represented by Structures II and in:

Structure II

Structure III

wherein the fluorescent dye may be coumarin, fluorescein, rhodamine, derivatives thereof or combinations thereof. In an embodiment, the fluorescent dye is coumarin 6 and is represented by Structure IV:

Structure IV

[0036] In an embodiment, the NAD is any fluorescent dye which when reacted with a nerve agent or component of a nerve agent results in detectable alterations in the optical properties of the NAD, the nerve agent or both. Alternatively, the NAD is a fluorescent dye which when reacted with a nerve agent or component of a nerve agent displays a detectable alteration in the fluorescence spectroscopy of the NAD. Such alterations may be changes in fluorescence intensity, fluorescence emission energy or combinations thereof. In an embodiment, the NAD comprises a fluorescent dye, which exhibits differing optical alterations dependent upon the nature of the nerve agent that it is contacted with. Such dyes may provide the additional advantage of being able to discriminate between nerve agents. In an embodiment, the optical alterations produced by the contacting of a nerve agent with a fluorescent dye are empirically determined. These determinations may be made for any number of nerve agents and any number of dyes of the type disclosed herein. Such information may be used to both detect and identify a nerve agent present in a sample. Such methods of determining and cataloging the optical alterations produced by contacting a nerve agent with a fluorescent dye are known to one of ordinary skill in the art and may be conducted manually or may be automated.

[0037] In an embodiment, the NAD comprises a fluorescent dye immobilized on a suitable substrate. Suitable substrates will be described later herein. Alternatively, the NAD comprises a fluorescent dye in a liquid media.

[0038] In an embodiment, the NADs disclosed herein react with a nerve agent and produce a detectable optical alteration in less than about 1 minute, alternatively in less than about 30 seconds, alternatively upon contacting of the nerve agent with the NAD. [0039] In an embodiment, a sample containing a nerve agent designated X, may react with the NAD and result in an alteration in the emission energy of the NAD for example from an emission energy of about 475 nm to an emission energy of about 510 nm. Such an alteration would be observed as a blue to green transition that may serve as an indicator of the presence of a nerve agent in said sample. Another sample of said NAD may be reacted with a sample potentially containing a nerve agent designated Z and result in an optical alteration, such as a change in the emission energy from about 475 nm to about 570 nm. Such an alteration would be observed as a blue to yellow transition that may serve as an indicator of the presence of a nerve agent in said sample. In an embodiment, the optical alteration observed may comprise a method of identification of the nerve agent in the sample. For example, the extent of change in the emission energy of the NAD, i.e from blue to green for nerve agent X or from blue to yellow for nerve agent Z may be indicative of the identity of the nerve agent in the sample. As will be understood by one of ordinary skill in the art, the reaction of the NAD with the nerve agent may produce multiple optical alterations such as both changes in emission energy and changes in emission intensity. These changes may be cataloged and comprise a methodology for identification of the particular nerve agent.

[0040] In an embodiment, the NAD comprises an LCC, a semiconductor nanoparticle, a fluorescent dye or combinations thereof. In an embodiment, the NAD comprises at least one LCC and at least one semiconductor nanoparticle. In an embodiment, the NAD comprises at least one LCC and at least one fluorescent dye. In another embodiment, the NAD comprises at least one semiconductor nanoparticle and at least one fluorescent dye. In yet another embodiment, the NAD comprises at least one LCC, at least one semiconductor nanoparticle and at least one fluorescent dye. Said NADs may be used as a component of a solution or in the solid phase (i.e. immobilized on a substrate) as will be described herein. [0041] In an embodiment, a testing substrate is impregnated with one or more of the NADs disclosed herein to form an immbolized NAD. Any substrate that can be impregnated with, or bound to, the NAD can be used, such as, for example filter papers, cloth, non-woven fabric, and other surfaces commonly used in the laboratory or field test kits. The NAD may also be deposited on, or chemically linked to, a non-absorberit surface. One appropriate testing substrate is filter paper, and it has been found that Whatman 1 filter paper (Whatman PIc, Middlesex, U.K.) is one appropriate testing substrate.

[0042] A method for detection of an immobilized NAD comprises immersion of a substrate into a NAD solution and drying of the immersed substrate. The substrate may be dried for equal to or greater than about fifteen minutes at conditions of about 21 °C and 30% humidity or until reasonably dry. Preferably, the paper should be dried for one hour or more. Equivalent aided drying may also be used. Methods of aided drying, for example the use of drying ovens, are well known to one of ordinary skill in the art.

[0043] hi an embodiment, a method for detection of a volatile nerve agent comprises exposure of an immbolized NAD to the volatile nerve agent that has been collected or by placing the substrate comprising an immobilized NAD in the vicinity of the volatile nerve agent and allowing the NAD and nerve agent to react as described herein, hi a preferred embodiment for detection of fluorophosphate vapor and liquid (e.g. Sarin), filter paper (Whatman 1) may be immersed in a NAD solution. The filter paper may then be dried to form an immobilized NAD and subsequently exposed to the volatile nerve agent.

[0044] hi an embodiment, the immobilized NAD comprises a LCC further comprising a sensitizing ligand, a nonsensitizing ligand, a desensitizing ligand or combinations thereof. Such an NAD when exposed to a fluorophosphates nerve agent (e.g., Sarin), may have a quenched luminescence, hi the VX-type instance, one can either quench the LCC chemical luminescence or one can increase it upon exposure to the nerve agent. As is known to one of ordinary skill in the art, the degree to which the luminescence of the LCC is altered (quenched or enhanced) is dependent on the concentration of both the LCC and nerve agent. [0045] hi an embodiment, the nerve agent is nonvolatile. Alternatively, the nerve agent is non-fluorine containing. Non-fluorine containing nerve agents such as VX are not volatile, and thus would be deployed in a terrorist attack in airborne drops or droplets (aerosols). In an embodiment, a nonvolatile, non-fluorine containing nerve agent may be detected by collecting the droplets of said agent and applying a sample of said nerve agent to a substrate comprising at least one immobilized NAD. [0046] In an embodiment, the NADs disclosed herein are provided as solutions. The NAD solution concentration may be any concentration effective for the detection of a nerve agent in a period of time of less than about 24 hours. Alternatively, the solution concentration is from about 10^'4 M to about 10^"2 M. As is understood by one of ordinary skill in the art, the lower the concentration used, the stronger light source needed for fluorescence detection. In an embodiment, the volatile nerve agent may be collected and bubbled through a solution containing an NAD.

[0047] In an embodiment, the nerve agent is nonvolatile. Alternatively, the nerve agent is non-fluorine containing. Non-fluorine containing nerve agents such as VX are not volatile, and thus would be deployed in a terrorist attack in airborne drops or droplets (aerosols). In an embodiment, a nonvolatile, non-fluorine containing nerve agent may be detected by collecting the droplets of said agent in a solution comprising an NAD. In such an embodiment, the NAD may comprise coordination complexes such as Eu-OP and Eu-TTFA. For example, air- containing droplets can be collected with a suction pump and bubbled through a solution of NAD and solvent. In another embodiment, the aerosol droplets may be collected as they fall via gravity into the test solution. In an embodiment the solvents may be polar or nonpolar. Examples of solvents suitable for use in this disclosure are methanol, ethanol, heptane, water or combinations thereof.

[0048] In an alternative embodiment, an NAD may be prepared in spray form and sprayed on clothing or a surface suspected to have been exposed to a nerve-agent. A contaminant on a suspected contaminated material may also be collected by swab, for example, and the collector sprayed with or immersed in a sensor chemical comprising an NAD.

[0049] In some embodiments, an apparatus for detection of a nerve agent may comprise a plurality of compartments containing the NADs of this disclosure in a form ready to be contacted with a sample potentially containing a nerve agent. In some embodiments, the apparatus comprising a plurality of compartments makes use of a plurality of NADs. Said NADs may be present in the apparatus the form of a compound immobilized on a suitable substrate, a component in a solution or combinations thereof. Such an apparatus having a plurality of compartments and employing a plurality of NADs may result in the accurate detection of the presence of a nerve agent with a reduced incidence of false positives or false negatives.

[0050] In one embodiment, an apparatus for detection of a nerve agent may comprise three compartments, one compartment having a control compound and two additional compartments each containing at least one of the NADs disclosed herein. For example, the apparatus may have one compartment, compartment A, comprising an NAD further comprising an LCC with a sensitizing ligand, a second compartment, compartment B, comprising an NAD further comprising an LCC with a desensitizing ligand and a third compartment, compartment C, containing a control sample. For purposes of this example the control sample may comprise an LCC with a sensitizing ligand. Application of a portion of a sample containing a nerve agent to compartments A and B may result in luminescence quenching in compartment A and luminescence enhancement in compartment B indicating the presence of a nerve agent. The extent of the change in luminescence may be compared to that observed in compartment C containing an unreacted sample of the LCC.

[0051] In another embodiment, referring to Figure Ia, an apparatus for the detection of nerve agents, 200, may comprise for example eight compartments. The apparatus 200 may be constructed of any chemically resistant materials compatible with the components of the disclosed methodology. For example, the apparatus 200 may largely be of polymeric composition; alternatively the apparatus may be constructed of stainless steel. In an embodiment, the apparatus 200 may have one compartment 15 containing a lanthanide coordination complex with a sensitizing ligand such as Eu-OP 20, a second compartment 25 containing a lanthanide coordination complex with a nonsensitizing ligand such as Eu-TTFA 30, a third compartment 35 containing a semiconductor nanoparticle such as CdS 40, a fourth compartment 45 containing a fluorescent dye 50 and control compartments 55 containing samples of the disclosed NADs 60. These control samples 60 may be reacted with a positive control, for example a sample of a nerve agent model compound or a nerve agent or may be reacted with a negative control and compared to the NAD reacted with sample. The area wherein the NAD is located and able to contact a sample or at least a portion of a sample is referred to herein as the test zone.

[0052] hi an alternative embodiment, an apparatus for the detection of nerve agents may be of the form given in Figure Ib. In this instance, the apparatus 300, may comprise a substrate such as those described herein with different NADs immobilized in different locations on the substrate.

[0053] In an embodiment, a methodology for use of the apparatus described herein comprises contacting a nerve agent in a test zone, allowing the nerve agent to react with the NAD in said test zone and detecting the results of the NAD, nerve agent reaction. The test zone may comprise in one instance the individual compartments of an apparatus such as described previously and shown as apparatus 200 or 300 in Figures Ia and Ib or the entire apparatus. A sample potentially containing a nerve agent may be applied to the apparatus 200 or 300 in any convenient manner and allowed to contact the particular NAD in the test zone. Contacting of the sample with the NAD in the test zone may result in a detectable optical alteration such as changes in fluorescence intensity or emission energy. Such changes may be detected using any means known for the detection of optical alterations such as a fluorimeter or a hand-held light source. In an embodiment, the detection of a nerve agent with each NAD constitutes a "mode" of detection. The multimodal detection approach described herein may result in a significant reduction of the instances of false positives or false negatives associated with nerve agent detection. For example, an apparatus as schematized in Figures Ia or Ib and described previously may be adhered to a surface, such as clothing and placed in an area suspected of being contaminated with a nerve agent. Reaction of any nerve agent present in the area with the NADs may result in multiple optical alterations occurring in the apparatus such as luminescence quenching, luminescence enhancement, color changes and/or changes in intensity that could be directly observed. Alternatively, a method for nerve agent detection may involve deploying an apparatus comprising NADs into an area suspected of containing a nerve agent. Said apparatus when reacted with the nerve agent may produce multiple optical alterations that may be readily detectable through visual observation. Alternatively, such an apparatus may comprise additional devices for sensing, recording and reporting any optical alterations occurring upon exposure of the apparatus to a nerve agent. Such an apparatus may have the additional advantage of allowing for the remote testing and detection of nerve agents.

EXAMPLES

[0054] The invention having been generally described, the following examples are given as particular embodiments of the invention and to demonstrate the practice and advantages thereof. It is understood that the examples are given by way of illustration and are not intended to limit the specification of the claims in any manner.

EXAMPLE 1

[0055] Eu-TTFA and Eu-OP complexes were prepared by simply mixing in methanol EuCl₃»6H₂O with TTFA or OP. A 1:4 molar ratio of Euiligand and a 10^"2 M complex concentration was used, but other ratios of excess ligand may also be used. It is preferable for this embodiment to have the complex dissolved in a nonpolar solvent such as heptane. To achieve this, heptane is added to the methanol solution, in volume equal to the methanol. The mixture is then stirred and sonicated for 5-10 minutes. Eu-TTFA readily migrates into the heptane fraction. On standing, the mixture separates, with the heptane traction on top of the methanol fraction. Illumination of the sample with near-ultraviolet light from a hand-held ultraviolet lamp (Model UVGL-58, Minerality, UVP₅ Upland, CA) reveals the characteristic red europium luminescence in both solvent fractions. On the basis of the luminescence intensities, the relative complex concentrations of Eu-TTFA were estimated to be 3:2 in the methanohheptane fractions, i.e., 6 X 10^~3 M ::4 x 10^"3 M. Eu-OP does not easily migrate from methanol to heptane. The relative concentrations in our preparation are roughly 10:1 in methanol:heptane. The methanol or heptane fraction thus prepared may serve as a liquid sensor for nerve agents. Detection of nerve agents via photoluminescence implies the need for a light source that excites the photoluminescence.

EXAMPLE 2

[0056] Treated filter papers were prepared by immersing a Whatman 1 filter paper in a ICT² M solution of Eu-TTFA or Tb-TTFA/OP and drying for 30 minutes at room temperature. The papers were tested by exposing each treated filter paper to diisopropylfluorophosphate, a substance used to mimic the chemical properties of Sarin. The treated paper was placed over the opening of a bottle containing the ligand diisopropylfluorophosphate (D 12,600-4 Sigma- Aldrich, Milwaukee, WI). The filter paper was exposed to the supernatant vapor in the bottle containing the liquid by simply placing the filter paper over the bottle opening. The minimum exposure time that produced an unambiguous result was 2 seconds. Upon removal of the paper from the bottle opening, it was visually inspected under a UV lamp (Model UVGL-58, Miiieralite®, UVP, Upland CA). It was found that the intense luminescence from the impregnated paper was quenched in the area of exposure to the diisopropylfluorophosphate. A dark spot of the dimensions of the bottle opening was seen under the UV lamp, and no optical filter was needed for observation.

EXAMPLE 3

[0057] For the sensing of the paraoxon model compound, which mimics nerve agents such as VX, Eu-TTFA and Eu-OP complexes were prepared by simply mixing in methanol EuCl₃«6H₂0 with TTFA or OP. A 1:4 molar ratio of Eu:ligand and a 10^"2 M complex concentration was used. Once the complex was formed, heptane was added to the methanol solution, in volume equal to the methanol. The mixture was then stirred and sonicated for 5-10 minutes. The agitation formed a temporary emulsion. Eu-TTFA readily migrates into the heptane fraction because it has non-polar functional groups and thus can migrate into the non- polar heptane. On standing, the mixture separated, with the heptane fraction on top of the methanol fraction. The top fraction was decanted. Illumination of the sample with near- ultraviolet light from a hand-held ultraviolet lamp (Model UVGL-58, Mineralite, UVP, Upland, CA) revealed the characteristic red europium luminescence in both solvent fractions. On the basis of the luminescence intensities, the relative complex concentrations were estimated to be 3:2 in the methanol: heptane fractions, i.e., 6 X 10^"3 M::4 x 10^"3 M. Eu-OP does not easily migrate from methanol to heptane. The relative concentrations in our preparation were roughly 10:1 in methanol:heptane. The nerve agent sensor was either the methanol or heptane fraction thus prepared.

[0058] For the sensing of the paraoxon model compound, the sensor solution was in a 50- drop volume (about 1 ml) of either methanol or heptane.

[0059] In some experiments, the effect of hydrolysis was assessed. In those instances, two drops of water was added to each of the 50 drop methanol and heptane solutions. One (undiluted) drop of the model compound was then added to each solution. The solution was then agitated (shaken) for a few seconds. All reagents, including the paraoxon, were obtained from Sigma- Aldrich (Milwaukee, WI) and were used as received. The purpose of the water addition was two-fold: to ensure the occupation by water of the 9th lanthanide binding site and to facilitate organophosphate hydrolysis.

[0060] With Eu-OP, it was found that strong europium luminescence quenching was produced. When water was added, a temporary emulsion was formed on agitation after the organophosphate addition, which later separated into a fraction below the heptane solvent. With Eu-TTFA, it was found that a heptane/water solution enhanced europium luminescence. It may be efficaceous to have three sensitizing ligands and three waters of hydration (to be displaced) bound to the lanthanide ion, rather than four sensitizing ligands and one water of hydration, in the instance of luminescence enhancement. It is concluded that detection of nonvolatile organophosphates can be accomplished by either measuring the quenching of Eu-OP or the enhancement of Eu-TTFA in heptane/water. In this enhancement case, the sensitivity limit was found to be 1/100 drop of the paraoxon (one drop of paraoxon diluted by a factor of 100 with heptane solvent).

EXAMPLE 4

[0061] In order to determine if a nerve gas of the Sarin type has been released in a given area, treated filter paper can be placed in the area and viewed under a UV light. In a clinical setting, one would swab the victim with an impregnated filter paper to note luminescence quenching or one would swab with a cotton swab or other absorbent collection device and stir it in a solution system to note either luminescence quenching or luminescence enhancement. This would be a presumptive test to be followed by more accurate laboratory analytical chemistry.

EXAMPLE 5

[0062] Clothing or sensors for persons at risk for exposure to nerve agents can be made and used as follows: the clothing can be impregnated with the sensor chemical. Clothing may be useful for military personnel in war zones so that it can be quickly determined if exposure to a nerve gas has occurred so that an antidote can be administered as soon as possible. A sensor serving the same function can be attached to a helmet or other article. Equally importantly, a soldier wearing bio/chemical safety gear needs to be examined to determine whether decontamination procedures have to be implemented before that safety gear is removed. The invention can also be used to assess the need for decontamination of equipment.

EXAMPLE 6

[0063] One can envision dropping sensors ahead of troops and illuminating them by laser (the lasers have collimated beams of very long range to illuminate small areas). The laser illumination would produce a luminescence response (quenching or enhancement) detected by observation through a telescope. Alternatively, the sensor system would be placed into an unmanned drone that files over the area of concern to detect clouds of nerve agent.

EXAMPLE 7

[0064] The preparation of the CdS nanoparticles is facile and has been described before (E. R. Menzel, M. Takatsu, R. H. Murdock, K. Bouldin, K. H. Cheng, Photoluminescent CdS/dendrimer nanocomposites for fingerprint detection, J. Forensic Sci. 45, 758 (2000) (and references therein), herein incorporated by reference. It involves dendrimers that are either functionalized with amino (PAMAM Generation 4) or carboxylate (PAMAM Generation 3.5) terminal groups, 64 of them in the dendrimers used. The CdS nanoparticles form intercalated within the dendrimers.

[0065] Diisopropylfluorophosphate is a model compound for Sarin and similar nerve agents. It thoroughly quenches the fluorescence of CdS in both Generation 3.5 and 4 methanol solution (no organophosphate hydrolysis) formulations. In methanol/water solution formulations (hydrolysis occurs), the quenching is only mild, and its extent depends on the methanol/water mix. There clearly is a connection here with the (rapid) hydrolysis of the fluorine-phosphorus bond. There is a time dependence, which eventually leads to thorough quenching as well (likely connected with the phosphate hydrolysis product, which acts more slowly than the fluoride hydrolysis product), but this is not overly interesting in that rapid detection only is of concern. There is a distinct difference in the methanol/water systems between what happens with Generation 3.5 and 4, namely the relative luminescence intensities (less quenching in Gen 4 than 3.5 for low water content and more quenching in Gen 4 than 3.5 for high water content), which thus may serve the practical sensing function. There are spectral color changes (apart from those related to the solvent mixes in terms of the sizes of the nanoparticles) which result from the interaction of the organophosphate with the nanoparticle. These may serve the sensing function in addition to the intensity changes. [0066] Paraoxon immediately quenches all CdS systems thoroughly. This is perhaps not entirely surprising in that its hydrolysis is slower than that of the fluorophosphate. This is in keeping with the fact that the real counterpart, VX is a persistent nerve agent, whereas Sarin, the real counterpart to diisopropylfluorophosphate, degrades fairly rapidly. [0067] In a preferred embodiment a two-compartment sensor may be employed with the "control" sample in one and the "sensor" sample in the other. A preferred embodiment would be a three-compartment sensor. One compartment would have a reference sample, one a sensor sample and the third a second sensor sample that responds differently than the first. The objective is to minimize false positives or negatives. The same applies to the lanthanide modalities. There, one sensor compartment would produce a quenching and the other an enhancement.

[0068] A combined sensor system that involves the above nanoparticle approach together with the lanthanide approach described previously will help in connection with false positives and negatives. In both approaches, the same photoluminescence excitation light source (low power, near-UV) is pertinent and the result is seen by mere visual observation (or ordinary TV camera). The sensor involving the nanoparticles (and also that for the paraoxon sensing by europium complexes) is either liquid-based as set forth in this example, or can be a dry sensor, as exemplified by the filter paper embodiment described herein for sensing diisopropylfluorophosphate vapor. Regardless, the sensing in all cases is quick, as required in a field scenario. It involves dendrimers that are either functionalized with amino (e.g., PAMAM Generation 4) or carboxylate (e.g., PAMAM Generation 3.5) terminal groups, 64 of them in the dendrimers used. Other nanoparticle systems, such as involving CdSe, with or without dendrimers, functionalized in various ways, can also be employed. EXAMPLE 8

[0069] Coυmarin 6 was reacted with diisopropylfluorophosphate and the resulting solution evaluated for optical alterations. Figure 2 is a room light photo showing the color of the dye solution (left) is modified (middle) upon addition of one drop of paraxon and again modified (right) upon addition instead of one drop of diisopropylfuorophosphate. Figure 3 shows the fluorescence of these solutions under near UV illumination and Figure 4 shows the fluorescence under 532 nm excitation using a Nd-YAG laser. The results demonstrate the ability of the fluorescent dyes to produce detectable spectrophotometric alterations when reacted with the described nerve agents. Furthermore, the system has the added advantage of being able to discriminate between nerve agents based on the nature of the spectrophotometric alteration.

[0070] While preferred embodiments of the invention have been shown and described, modifications thereof can be made by one skilled in the art without departing from the spirit and teachings of the invention. The embodiments described herein are exemplary only, and are not intended to be limiting. Many variations and modifications of the invention disclosed herein are possible and are within the scope of the invention. Where numerical ranges or limitations are expressly stated, such express ranges or limitations should be understood to include iterative ranges or limitations of like magnitude falling within the expressly stated ranges or limitations (e.g., from about 1 to about 10 includes, 2, 3, 4, etc.; greater than 0.10 includes 0.11, 0.12, 0.13, etc.). Use of the term "optionally" with respect to any element of a claim is intended to mean that the subject element is required, or alternatively, is not required. Both alternatives are intended to be within the scope of the claim. Use of broader terms such as comprises, includes, having, etc. should be understood to provide support for narrower terms such as consisting of, consisting essentially of, comprised substantially of, etc. [0071] Accordingly, the scope of protection is not limited by the description set out above but is only limited by the claims which follow, that scope including all equivalents of the subject matter of the claims. Each and every claim is incorporated into the specification as an embodiment of the present invention. Thus, the claims are a further description and are an addition to the preferred embodiments of the present invention. The discussion of a reference herein is not an admission that it is prior art to the present invention, especially any reference that may have a publication date after the priority date of this application. The disclosures of all patents, patent applications, and publications cited herein are hereby incorporated by reference, to the extent that they provide exemplary, procedural or other details supplementary to those set forth herein.

Claims

CLAIMSWhat is claimed is:

1. A method for detecting a nerve agent, comprising the steps of:

(a) exposing a test zone to a test sample for an adequate time period for any nerve agent or nerve agent components in said test sample to contact said test zone, said test zone comprising a nerve agent detection composition, to form an exposed test zone;

(b) viewing said exposed test zone under a light source suitable for allowing observation of any optical alterations; and

(c) concluding that said test sample contains a nerve agent or nerve agent component after comparing the optical alterations observed to those occurring in a control test zone.

2. The method of claim 1, wherein said test zone is a solid surface impregnated with said nerve agent detection composition, a liquid composition comprising a nerve agent detection composition or combinations thereof.

3. The method of claim 1 wherein the optical alterations are changes in spectral intensity, changes in spectral emission energy, changes in spectral absorption energy or combinations thereof.

4. The method of claim 1, wherein said nerve agent detection composition comprises a lanthanide coordination complex, a semiconductor nanoparticle, a fluorescent dye or combinations thereof.

5. The method of claim 4 wherein the lanthanide coordination complex comprises europium, terbium, sensitizing ligands, nonsensitizing ligands, desensitizing ligands or combinations thereof.

6. The method of Claim 4, wherein said lanthanide coordination complex is Eu- TTFA or Tb-TTFA/OP.

7. The method of claim 1 wherein nerve agent detection composition comprises a lanthanide-TTFA complex and the optical alteration observed is luminescence quenching.

8. The method of claim 1 wherein said nerve agent detection composition comprises a lanthanide-OP complex and the optical alteration observed is luminescence enhancement.

9. The method of claim 4 wherein the semiconductor nanoparticle comprises cadmium selenide, cadmium sulfide, cadmium telluride or combinations thereof.

10. The method of claim 4 wherein the fluorescent dye comprises fluorescein, rhodamine, coumarins, derivatives thereof or combinations thereof.

11. The method of claim 1 wherein said light source is ultraviolet light.

12. A method of detecting an organophosphate nerve agent comprising the steps of:

(a) providing a nerve agent detector composition test zone;

(b) adding a test sample containing an organophosphate nerve agent to form an exposed test zone;

(c) observing such exposed test zone under ultraviolet or other appropriate light; and

(d) concluding that said test sample likely contains an organophosphate if said exposed test zone exhibits changes in spectral properties as compared to a control test zone.

13. The method of claim 12 wherein said nerve agent detector composition test zone is a solid surface with an immobilized nerve agent detection composition, a solution comprising a nerve agent detection composition, an aerosol comprising a nerve agent detection composition or combinations thereof.

14. The method of claim 12 wherein said nerve agent detection composition is a lanthanide coordination complex, a fluorescent dye, a semiconductor nanoparticle or combinations thereof.

15. The method of claim 14 wherein the semiconductor nanoparticle is prepared using a dendrimer in a methanol/water solution.

16. The method of claim 14 wherein the semiconductor nanoparticle is cadmium selenide, cadmium sulfide, cadmium telluride or combinations thereof.

17. The method of claim 14 wherein the fluorescent dye is fluorescein, rhodamine, coumarin, derivatives thereof or combinations thereof.

18. An apparatus for nerve agent detection comprising at least four nerve agent detection compositions.

19. The apparatus of claim 18 wherein the nerve agent detection compositions are lanthanide coordination complexes, semiconductor nanoparticles and fluorescent dyes.

20. The apparatus of claim 18 wherein the nerve agent detection compositions are immobilized on a solid substrate, are components of a solution, are aerosols or combinations thereof.

21. A method of detecting an optically reactive target compound comprising: (a) placing at least one optical sensor chemical in contact with the target compound;

(b) allowing the optical sensor chemical to react with the target compound; and

(c) detecting any optical alterations produced when the said optical sensor chemical reacts with said target compound.

22. The method of claim 21 wherein the optical sensor compound comprises a nerve agent detection composition.

23. The method of claim 21 wherein the target compound is a nerve agent.

24. The method of claim 21 wherein the optical sensor chemical is remotely placed in contact with the target compound.

25. The method of claim 21 wherein the optical alterations produced are detectable using visual observation, a spectrophotometric device or combinations thereof.

26. The method of claim 25 wherein the spectrophotometric device comprises a UV illuminator, a fluorimeter, an infrared detector or combinations thereof.

27. The method of claim 25 wherein the visual observation occurs with the aid of a remote viewing device.

28. The method of claim 26 wherein the remote viewing device comprises a camera, a television, a telescope or combinations thereof.

29. A method of detecting a nerve agent in a sample comprising:

(a) hydrolyzing said nerve agent;

(b) reacting the hydrolysis product of said nerve agent with a lanthanide coordination complex;

(c) detecting alterations in the luminescence of the lanthanide coordination complex; and

(d) determining the presence or absence of a nerve agent in said sample.

30. The method of claim 29 wherein the lanthanide coordination complex comprises europium, terbium, sensitizing ligands, nonsensitizing ligands, desensitizing ligands or combinations thereof.

31. The method of claim 29 wherein the alteration in luminescence comprises luminescence enhancement.

32. The method of claim 29 wherein the alteration in luminescence comprises luminescence quenching.