CHEMICALLY TAILORABLE NANOPARTICLES REALIZED THROUGH METAL-METALLOLIGAND COORDINATION CHEMISTRY

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Application Serial No. 60/685,786, filed May 31, 2005, and U.S. Provisional Application Serial No. 60/777,638, filed February 28, 2006, each of which is incorporated in its entirety by reference.

STATEMENT OF GOVERNMENTAL INTERESTS [0002] This invention was made with government support under National Science Foundation grant Nos. EEC-0118025/003 and CHE-0447674; Office of Naval Research grant No. 00014-06-1-0078; Air Force Office of Scientific Research grant No. F49620-00-1-0283; and Homeland Security Advanced Research Projects Agency grant No. W8IXWH-05-2- 0036. The government has certain rights in this invention.

BACKGROUND

[0003] Micro- and nano-sized particles play important roles in many different areas, including catalysis, optics, biosensing, and data storage. Recent advances in this field have made it possible to control many of the chemical and physical properties of these solid-state materials through control over their size, shape, and composition. (Peng, et al. Nature 404:59 (2000); Horn et al., Angew. Chem. Int. Ed. 40:4331 (2001); and Chen et al. Angew. Ckem. Int. Ed. 44:2589 (2005)). One important goal in this area is an ability to intentionally interconvert different particle compositions and structures through chemistry that occurs throughout the particle structure, in addition to chemistry on its surface. There are two ways to effect such transformations, either through a thermal or photochemically induced shape change (Jin et al., Science 294:1901 (2001)) or by chemical means that transform the particle from one chemical composition to another. (Son et al., Science 306:1009 (2004)) Even though the latter approach, in principle, can provide a straightforward, economic, and potentially powerful way to access new classes of micro- and nanoparticle materials, very few such approaches have been developed, and those that have been realized are quite limited in scope.

[0004] Typically, chemical transformations in the solid state require a high activation energy for the diffusion of small molecules or ions into the solid. Therefore, the reactions are usually slow and involve high temperature and/or pressure. (Feng et al. Ace. Chem. Res .34:239(2001) and Schaak et al., Chem. Mαter.l4:1455(2002)) One exception is that CdSe can be converted to Ag ₂ Se in the context of nano-sized particles at room temperature.

(Son et al., Science 306:1009 (2004)) In contrast, metal-organic coordination polymers are highly porous materials, that often allow small molecules to move freely within such structures. For this reason, they are typically used for gas storage (Yaghiet al. Nature 423:705 (2003) and Zhao et al. Science 306:1012 (2004)) and catalysis. (Seo et al. Nature 404:982 (2000))

[0005] Thus, there remains a need for particles which can be easily assembled, can be modified to provide desired physical and/or chemical properties, and can be used a multitude of applications.

SUMMARY

[0006] Disclosed herein are colloidal particles comprising metal-organic coordination polymers. The colloidal particles are assembled from a coordination of a metal salt and bis- metallo-tridentate Schiff base (BMSB) building blocks. The colloidal particles of the present invention have a formula (I) or (II):

wherein R ¹ is independently selected from the group consisting of H, hydroxyl, C _1-8 alkyl, OC _1-8 alkyl, Ci _-8 heteroalkyl, OC _1-8 heteroalkyl, P(R ² ) ₂ , aryϊ, heteroaryl; R ² is the same or different and is selected from aryl or heteroaryl; R ³ is independently selected from the group consisting of H, hydroxyl, C _1-8 alkyl, OCi _-8 alkyl, C _1-8 heteroalkyl, OC _1-8 heteroalkyl, P(R ² ) ₂ , aryl, heteroaryl; M and M' are independently selected from the group consisting of Zn, Cu, Mn, Pb, Ni, Cd, Co, and Cr; L is null or a ligand selected from the group consisting of pyridine, methanol, acetate, dimethylsulfoxide, dimethylformamide, acetone, water, chloride, fluoride, iodide, bromide, and hydroxide; x is an integer from 0 to 3; n is an integer from 0 to 5; and m is an integer from about 100 to about 10,000. In some embodiments, M and M' are the same metal ion, while in other embodiments, M and M' are different metal ions.

[0007] Also disclosed herein is a method of synthesizing the colloidal particles, wherein a metal salt solution and BMSB are admixed, then a nonpolar solvent is added to the

admixture. Nonlimiting examples of a nonpolar solvent are pentane, diethyl ether, hexane, benzene, xylenes, cyclohexane, and toluene. In some embodiments^ the nonpolar solvent is added rapidly to the mixture of metal salt and BMSB, and the resulting colloidal particles have a diameter of about 100 to less than 1 μm. In other embodiments, the nonpolar solvent is added slowly, and the resulting colloidal particles have a diameter of about 1 μm to about 5 μm.

[0008] Also disclosed herein are methods of using the colloidal particles in biological or chemical detection assays using a sandwich assay, hi one embodiment, a sample containing or suspected of containing an analyte of interest is mixed with (1) a magnetic microparticle (MMP) which is modified with a tag. on its surface that is specific for the analyte and (2) a colloidal particle of the present invention which is modified with a tag on its surface that is specific for the analyte. A sandwich structure of MMP-analyte-colloidal particle results, and is isolated, e.g., using a magnet. The sandwich structure then is dissolved in pyridine, and a fluorescence is measured. The fluorescence is correlated to the presence or concentration of the analyte of interest. In some embodiments, the colloidal particles comprise Zn.

BRIEF DESCRIPTION OF THE DRAWINGS

[0009] FIG. 1 shows a schematic of the preparation of colloidal particles of the invention from metal salts and bis-metallo-tridentate Schiff base (BMSB) building blocks;

[0010] FIG. 2 shows images, of colloidal particles prepared by the disclosed methods and analyzed by (a) optical microscopy, (b) and (c) fluorescence microscopy, and (d) scanning electron microscopy (SEM) where the inset in (d) is a high-resolution zoom-in image of the colloidal particles;

[0011] FIG. 3 shows SEM and optical microscopy images of example micro- and nanoparticles formed through the methods disclosed herein and the proposed mechanism of formation, where (a)-(c) are SEM images monitoring the growth of Zn-BMSB-Zn colloidal particles, (a) is an early intermediate cluster formed by aggregation of small particles, (b) is an intermediate at a later stage, showing surface annealing, (c).is a fully formed spherical particle, (d) is a dark-field optical microscopy image of cluster aggregates (bottom) and a fully formed particle (top), (e) is a schematic representation of the proposed cluster-fusion growth mechanism, (f) is an image of spherical particles of Zn-BMSB-Zn with an average diameter of 190 ± 60 nm (s.d., n= 50) as determined by SEM and 176 ran as determined by dynamic light scattering (DLS); (g) is an image of spherical particles of Zn-BMSB-Zn with

an average diameter of 780 ± 230 nm (s.d., n= 50) as determined by SEM and 575 nm as determine by DLS;

[0012] FIG. 4 shows emission spectra (excitation wavelength of 420 nm) of Zn-BMSB-Zn particles where the ancillary ligands have been systematically changed to dimethyl sulfoxide (DMSO), pyridine, dimethylformamide, acetone, methanol, or water;

[0013] FIG. 5 shows a schematic of selective cation exchange of disclosed colloidal particles, where mixture of a colloidal particle and a metal salt (M") allows for the exchange of the M' ion of the colloidal particle to the M" ion, while the M ion remains unaffected;

[0014] FIG. 6 shows the chemical transformation of Zn-BMSB-Zn colloidal particles to Cu-BMSB-Zn particles via cation exchange, where (a) shows both the optical microscopy (OM, top) and fluorescence microscopy (FM, bottom) images of the mixture at time 0, (b) is at time 1 minute, (c) time 5 minutes, and (d) at time 60 minutes, (e) is a photograph of Zn- BMSB-Zn (right) and Cu-BMSB-Zn (left), and (f) is an emission spectra obtained by exciting at 420 nm;

[0015] FIG. 7 shows SEM images of (a) Zn-BMSB-Zn and (b) Cu-BMSB-Zn, where the scale bars indicate 5 μm;

[0016] FIG. 8 shows OM and FM (inset) images of (a) Zn-BMSB-Zn, (b) Cu-BMSB-Zn, (c) Mn-BMSB-Zn, and (d) Pb-BMSB-Zn, and (e) and (f) are photographs of, from left to right, Zn-BMSB-Zn, Cu-BMSB-Zn, Mn-BMSB-Zn, and Pb-BMSB-Zn, where (f) in with UV-irradation, indicating that only Zn-BMSB-Zn is fluorescent;

[0017] FIG. 9 shows a schematic of an analyte detection method using the colloidal- particles disclosed herein where a capture DNA-modified magnetic probe is mixed with a target DNA and a fluorescent colloidal particle further modified with a capture DNA to form a "sandwich" of the magnetic probe-target DNA-colloidal particle (step 1) and then isolated using a magnetic field (step 2), and dissolved with pyridine to create an amplified fluorescence signal (step 3); and

[0018] FIG. 10 shows (a) fluorescence spectroscopy of solution of colloidal particles dissolved in pyridine as a function of target DNA concentration in PBS buffer, and (b) shows fluorescence intensity of the particles quantified by integration of the area of the spectra from 580 to 615 nm for target DNA concentrations from 5 aM to 50 fM.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0019] Disclosed herein are colloidal particles of polymerized metal-ligand networks made by coordination-chemistry-induced assembly of metal ions and carboxylate functionalized bis-metallo-tridentate Schiff base (BMSB) building blocks (FIG. 1). The colloidal particles are formed by the polymerization of the BMSB and metal, ions, and the size of the colloidal particles can be controlled by the manner in which the BMSB and metal ions are mixed together.

[0020] Colloidal particles of the present invention have a formula (I) or (II):

wherein R ¹ is independently selected from the group consisting of H, hydroxy!, C _1-8 alkyl, C _1-8 alkoxy, C _1-8 heteroalkyl, OC _1-8 heteroalkyl, P(R ² ) ₂ , aryl, heteroaryl; R ² is the same or different and is selected from aryl or heteroaryl; R ³ is independently selected from the group consisting of H, hydroxyl, C _1-8 alkyl, OC _1-8 alkyl, C _1-8 heteroalkyl, OC _1-8 heteroalkyl, P(R ² ) ₂ , aryl, heteroaryl; M and M' are independently selected from the group consisting of Zn, Cu, Mn, Pb, Ni, Cd, Co, and Cr; L is null or a ligand selected from the group consisting of pyridine, methanol, acetate, dimethylsulfoxide, dimethylformamide, acetone, water, chloride, fluoride, iodide, bromide, and hydroxide; x is an integer from 0 to 3; n is an integer from 0 to 5; and m is an integer from about 100 to about 10,000. In some embodiments, M and M' are the same metal ion, while in other embodiments, M and M' are different metal ions.

[0021} As used herein, the term "alkyl" includes straight chained and branched hydrocarbon groups containing the indicated number of carbon atoms, such as methyl, ethyl, and straight chain and branched propyl and butyl groups. The hydrocarbon group can contain up to 8 carbon atoms. The term "alkyl" also encompasses alkyl groups which are optionally substituted with, e.g., one or more halogen atoms, one or more hydroxyl groups, or one or more thiol groups. Also encompassed by the term "alkyl" are cycloalkyl groups. "Cycloalkyl" is defined as a cyclic C ₃ -C ₈ hydrocarbon group, e.g., cyclopropyl, cyclobutyl,

cyclohexyl, and cyclopentyl. "Heteroalkyl" and "heterocycloalkyl" are defined similar to alkyl and cycloalkyl, except at least one heteroatom is present in the structure. Suitable heteroatoms include N, S, and O.

[0022] The term "halo" or "halogen" is defined herein to include fluorine, bromine, chlorine, and iodine.

[0023] The term "aryl," alone or in combination, is defined herein as a monocyclic or polycyclic aromatic group, preferably a monocyclic or bicyclic aromatic group, e.g., phenyl or naphthyl. Unless otherwise indicated, an "aryl" group can be unsubstituted or substituted, for example, with one or more, and in particular one tα three, halo, alkyl, hydroxy, C(=O)OR, hydroxyalkyl, alkoxy, alkoxyalkyl, halαalkyl, haloalkoxy, cyano, nitro, amino, alkylamino, acylamino, alkylthio, alkylsulfinyl, and alkylsulfonyl. Exemplary aryl groups include phenyl, naphthyl, tetrahydronaphthyl, 2-chlorophenyl, 3-chloropheny.l, 4-chlorophenyl, 2- methylphenyl, 4-methoxyphenyl, 3-trifluoromethylphenyl, 4-nitrophenyl, and the like. The terms "arylC _1-3 alkyl" and "heteroarylC _1-3 alkyl" are defined as an aryl or heteroaryl group having a C _1-3 alkyl substituent.

[0024] The term "heteroaryl" is defined herein as a monocyclic or bicyclic ring system containing one or two aromatic rings and containing at least one nitrogen, oxygen, or sulfur atom in an aromatic ring, and which can be unsubstituted or substituted, for example, with one or more, and in particular one to three, substituents, like halo, alkyl, hydroxy, hydroxyalkyl, alkoxy, alkoxyalkyl, haloalkyl, nitro, amino, alkylamino, acylamino, alkylthio, alkylsulfinyl, and alkylsulfonyl. Examples of heteroaryl groups include thienyl, furyl, pyridyl, oxazolyl, quinolyl, isoquinolyl, indolyl, triazolyl, isothiazolyl, isoxazolyl, imidizolyl, benzothiazolyl, pyrazinyl, pyrimidinyl, thiazolyl, and thiadiazolyl.

[0025] The term "hydroxy" is defined as -OH.

[0026] The term "alkoxy" is defined as -OR, wherein R is alkyl.

[0027] Colloidal particles of the present invention have a diameter of about 100 nm to about 5 μm, which is dependent upon the manner in which the colloidal particles are prepared. When the colloidal particles are formed through the fast addition of a nonpolar solvent (such as, e.g., diethyl ether or pentane), the result is smaller diameter colloidal particles, while the slow addition of the nonpolar solvent results in larger diameter colloidal particles.

[0028] BMSB molecules can be prepared through known chemical techniques. One such technique is outlined in the following scheme for the formation of a specific, nonlimiting BMSB.

BMSB

[0029] Different BMSBs can be prepared by altering the starting di-aldehyde, aromatic amine, and/or metal salt. The BMSB is then used to prepare the colloidal particles of the present invention. The BMSB building blocks are important components of the strategy for making the colloidal particles disclosed herein. They are readily polymerizable through their carboxylate groups and allow manipulation of the chemical and physical properties of the resulting colloidal particles in a systematic manner through choice of BMSB ligand, type of metallation, and ancillary ligands. The BMSB is mixed with a metal ^* salt (such as, e.g., M' (OAc) ₂ ) in a pyridine solution. The metal of the BMSB may be the same or different from the metal salt used. The colloidal particles are prepared by the addition, either fast (to form smaller diameter colloidal particles) or slow (to form larger diameter colloidal particles), of a nonpolar solvent. The nonpolar solvent, typically pentane or diethyl ether, affects the solubility of the forming colloidal particle. The fast addition of the nonpolar solvent causes the forming colloidal particle to precipitate out of solution more quickly, and the resulting colloidal particles are therefore smaller in diameter. Slow addition of the nonpolar solvent, however, allows for the colloidal particles to grow in solution for a longer period of time before precipitating out of solution, and, therefore, have larger diameters than those produced from the fast addition of the nonpolar solvent.

[0030] The nonpolar solvent used also effects the size of the resulting colloidal particles. Diethyl ether typically provides smaller diameter colloidal particles, whereas pentane

provides larger diameter colloidal particles under the same conditions. For example, the fast addition of diethyl ether results in colloidal particles having a diameter of about 190 ± 60 rnn, while fast addition of pentane results in colloidal particles having a diameter of about 780 ± 230 nm. Slow addition of diethyl ether results in colloidal particles having a diameter of about 1.60 ± 0.47 μm, and slow addition of pentane results in colloidal particles having a diameter of about 5 μm. Therefore, colloidal particle diameter can be controlled through the appropriate choice of nonpolar solvent and rate of addition of the nonpolar solvent to the mixture of BMSB and metal salt. The polarity of the solvent affects the solubility of the resulting colloidal particles and, therefore, their average size.

[0031] When pentane is used as an initiation solvent instead of diethyl ether, larger spherical microp articles (5 ± 3 μm) are formed (FIG. 2). The growth of the particles under this set of conditions can be observed by taking aliquots at various stages and characterizing the particles by SEM and OM. This set of experiments also provides some mechanistic insight into the particle growth process. Two kinds of intermediate particles, were observed, i.e., clusters formed by aggregation of small particles and a fused version of these clusters. At an early stage of the reaction, clusters of smaller particles are observable (FIG. 2 A and 2D). These structures slowly anneal into single particles having smoother surfaces (FIG. 2B) and ultimately form uniform spherical particles (FIG. 2C and FIG. 2D). Based upon these observations, the proposed mechanism of the formation of the structures is via a two step cluster-fusion growth process (FIG. 2E). The first step is aggregation of several small particles to form large cluster particles. The second step is intraparticle fusion, resulting in large uniform spherical particles. This can occur because of the reversible nature of the metal coordination chemistry, which allows the system to anneal into a smooth particle. The cluster fusion step can involve a plurality of particles or only a few, depending upon conditions, and the ultimate size of the large spherical particles depends upon the number of smaller particles involved in the fusion process.

[0032] The phrase "rapid addition," or interchangeably "fast addition," means herein that the nonpolar solvent is added to the mixture at a rate of at least 1 mL/sec, preferably at least 3 mL/sec, and most preferably at least 5 mL/sec. The phrase "slow addition" means herein that the nonpolar solvent is added to the mixture at a rate of less than 1 mL/sec, preferably less than 1 mL/min, and most preferably, less than 1 mL/hour.

[0033] The term "nonpolar solvent" as used herein means a solvent which is immiscible or only slightly miscible in pyridine. One means of determining whether a solvent is suitable is by its solubility parameter. Solvents with solubility parameters (δ) of less than 19.0 can be

used in the methods disclosed herein, more preferably less than 18.0, and most preferably less than 16.0. Pentane has a solubility parameter of 14.4, diethyl ether has a solubility parameter of 15.4, and toluene a solubility parameter of 18.3. Solubility parameters can be found in any chemistry handbook, such as, e.g., Barton, Handbook of Solubility Parameters, CRC Press (1983). Other examples of nonpolar solvents include benzene, xylenes, hexanes, cyclohexane, amyl acetate, and the like.

[0034] The physical properties of the colloidal particles can be affected both by the manner in which they are synthesized, as. discussed above, but also by the ancillary ligand, L, of the colloidal particle. The ancillary ligand of the colloidal particles can be changed by mixing the colloidal particle with various solvents. For example, a red toluene suspension of colloidal particles 3a (FIG. 1), (M and M' are Zn, L is pyridine, and the BMSB is a compound of formula (I), where R ¹ is null) turns yellow when methanol is added. This color change is attributable to the replacement of the pyridine ligand on the Zn metal centers with methanol. The solvent can be removed from this complex and the resulting powder redispersed in toluene having 10% dimethylsulfoxide (DMSO). The yellow solution turns red due to the formation of a zinc-DMSO adduct. The emission spectra of colloidal particles having Zn metal centers and various ancillary ligands is shown in FIG. 4, where the ancillary ligand is DMSO, DMF, acetone, methanol, pyridine, or water. The ancillary ligand can be easily altered by simple addition of the appropriate small molecule, and is completely reversible, with the exception of pyridine which dissolves the colloidal particle into its BMSB and metal salt components.

[0035] In a typical experiment, a precursor pyridine solution consisting of a mixture of the appropriate metal acetate salt (e.g., M'(OAc) ₂ , where M' = Zn, Cu, or Ni and OAc = acetate) and BMSB 2 is prepared (FIG. 1). Addition of an initiation solvent such as diethyl ether or pentane results in the spontaneous formation of spherical inorganic polymer particles 3 (FIG. 1). These particles form via coordination of the carboxylate groups on the BMSB precursor with the metal ions supplied by the acetate salt, and the polymerization process is completely reversible as evidenced by the formation of the starting materials upon addition of excess pyridine. Spherical microscale colloidal particles are formed, instead of a macroscopic polymeric material of other reported organometallic polymerizations, due to the slow diffusion of a nonpolar solvent in the pyridine solution of the BMSB and metal salt at room temperature. The addition of diethyl ether or pentane to the polar (e.g., pyridine) precursor solution results in precipitation due to the low solubility of the particles in nonpolar media.

The resulting particles are stable in organic solvents (toluene, methanol, DMF and dimethyl sulfoxide (DMSO)), water, and in the dried state.

[0036] Optical microscopy (OM, FIG. IA), fluorescence microscopy (FM, FIG. 1, B and C), and scanning electron microscopy (SEM, FIG. ID) images of example compositions show the spherical particles with an average diameter of 1.6 ± 1.2 μm. The chemical composition of the particles was determined by energy dispersive X-ray spectroscopy (EDX) and elemental analysis. Control experiments with a BMSB which does not contain a free carboxylate group (2d in FIG. 1) show that the coordination polymer and therefore colloidal particles as disclosed herein will not form in the absence of the carboxylate groups. In addition to the aforementioned control experiments, Fourier transform IR spectroscopy, ¹ H and ¹³ C NMR spectroscopy, and electrospray ionization mass spectrometry are all consistent with the proposed mode of polymerization. Featureless powder X-ray diffraction data for these particles show that they are amorphous and not crystalline materials.

[0037] Colloidal particles of the present invention can be prepared with a variety of different metals, including Zn, Cu _^ Ni, Pd, and Mn, by the choice of starting metal salt and starting BMSB. Spherical nanoparticles of Cu-BMSB-Cu 3b (FIG. 1) synthesized by fast addition of pentane into a precursor solution having Cu-BMSB 2b and Cu(OAc) ₂ -(H ₂ O) Ib in pyridine, have diameters similar to Zn-BMSB-Zn particles prepared via analogous procedures. Slow diffusion of pentane into a precursor solution containing 2b and Ib yields particles that are on average significantly larger than the particles formed from the fast addition method. Similarly, the reaction between Ni-BMSB 2c and Ni(OAc) ₂ -4(H2O) Ic gives spherical particles Ni-BMSB-Ni 3c. hi contrast to 3a, particle compositions of 3b and 3c are not fluorescent because they are not made of fluorescent BMSB building blocks.

[0038] The synthetic methods disclosed herein provide dispersible micro- and nano-scale particles having highly tailorable physical and chemical properties, which can be controlled through choice of polymerizing metal ion, such as Zn(II), Cu(II), or Ni(II), BMSB, and ancillary ligands. Because certain BMSB building blocks can be prepared in enantiopure form (such as, e.g., those based upon a bi-naphthyl ring system) and are important compounds in homogeneous catalysis, this new class of material has great potential in asymmetric catalysis and chiral separations.

[0039] One can also transform the particles prepared as described above into new compositions through selective cation exchange reactions without significantly affecting the size or morphology of the particle. The approach utilizes two different types of ions, ones

coordinated to the tridentate pockets of the BMSB ligands and ones that link the BMSB ligands thorough the carboxylate groups to form the polymers. When Zn(II) is used as the metal ion, it can be readily displaced from the BMSB tridentate pockets, but not from the carboxylate sites with a variety of metal ions (FIG. 5).

[0040] In a typical reaction, a mixture of Zn-BMSB-Zn microparticles (3a, FIG. 1) (average diameter ~ 1.6 μm) and Cu(OAc) ₂ -H ₂ O in methanol was prepared under ambient conditions. The mixture contains Cu(II) ions in a slight excess to ensure the replacement of all the Zn(II) ions coordinated to the tridentate Schiff base moieties within the particles. Zn- BMSB building blocks are highly fluorescent, as are Zn-BMSB-Zn particles, however, the product resulting from cation exchange, Cu-BMSB-Zn is not. Therefore, one can easily monitor particle transformation by optical microscopy (OM) and fluorescence microscopy (FM, FIG. 6 A -6D, compare the OM and FM images). Within five minutes, the majority of the fluorescent Zn-BMSB-Zn particles had been transformed into the Cu-BMSB-Zn particles (FIG. 6C), and by 60 minutes the reaction was complete (FIG. 6D). This transformation was followed by fluorescence or by the naked eye as evidenced by a concomitant color change from orange (Zn-BMSB-Zn) to brown (Cu-BMSB-Zn).

[0041] In addition to these optical property changes, energy dispersive X-ray (EDX) spectra, ICP analyses , and emission spectra measured before and after the cation- exchange reaction were consistent with the formation of the Cu-BMSB-Zn product. The emission spectrum of the resulting particles reveals complete disappearance of fluorescence indicating full exchange of two equivalents of Cu(II) for the two Zn(II) ions contained within the Zn- BMSB building blocks. A separate study of the Zn-BMSB monomer shows that one must remove both Zn ions within the Zn-BMSB to quench the fluorescence from this system. In addition, an EDX spectrum of the Cu-BMSB-Zn particles measured after the cation exchange shows the expected Cu signal in addition to Zn. Finally, ICP analysis data are consistent with a change in the number of metal ions within the particles from three to one Zn(II) ions and from zero to two Cu(II) ions per repeating unit.

[0042] Scanning electron microscopy (SEM) images of initial and cation-exchanged particles show that the size and shape of the particles are preserved after the reaction. From these data, it is concluded that the Cu(II) ions exchange only with the Zn(II) coordinated to the tridentate Schiff base (i.e., the M metal of formulae (I) or (H)) and not the Zn(II) ions forming the interconnecting nodes (i.e, the M' metal of formulae (I) or (IT)). When excess Cu(II) ions are used in the exchange reaction, the reaction occurs at a faster rate, but results in the same product, Cu-BMSB-Zn. Under the conditions studied, there is no evidence for the

formation of the Cu-BMSB-Cu product, which is a reflection of the difference in binding strength of the two coordination sites.

[0043] The cation exchange reaction rate is size dependent. Particles of about 200 nm undergo Cu(II) exchange reaction in a few minutes as compared to the hour required to effect it with the micron-scale particles. Again, FM and OM images clearly show the transformation with a minimal change in particle size; the average particle sizes measured before (212 nm) and after the cation exchange reaction (215 nm) by dynamic light scattering (DLS) are nearly identical (FIG. 8A vs. FIG. 8B).

[0044] The utility of this method was also assessed for producing additional particle compositions with other metal ions such as Mn(II) and Pd(II). A mixture of Zn-BMSB-Zn nanoparticles and Mn(OAc) ₂ or Pd(OAc) ₂ in methanol was prepared under ambient conditions. Ih contrast to the process involving Cu(II) exchange, excess Mn(II) or Pd(II) (e.g., greater than 20 equivalents) was necessary in order to observe significant conversion as measured by fluorescence. The transformations can be monitored by florescence or with the naked eye. In addition, the resulting nanoparticles, like their Cu(II) counterparts, were characterized by EDX, DLS, and emission spectroscopy. Other cation exchanges are contemplated. Any metal salt can be used to exchange with the metal M of formulae (I) or (II). Mn(II), Pb(II), Ni(II), Co(II) and Cu(II) are examples of such suitable metal ions, but metal salts of other metals, or of the same metals but alternative oxidation states (such as, e.g., Mn(IV)) are also contemplated. The associated anion of the metal salt is preferably one which is not strongly coordinated to the metal center and can be replaced by alternate coordinating groups, such as, e.g., Schiff bases of the BMSB ligand moieties. Such anions include acetate, trifluoroacetate, halide, nitrate, perchlorate, phosphate, sulfate, triflate, and the like.

[0045] Selective cation exchange within the context of nano- and microparticles.made from infinite coordination polymers can be used to transform one particle composition into several new ones in a very efficient process. The fast rates of these processes, which derive from the porosity of the structures and the labile nature of the Zn-coordination interactions is unexpected, especially when compared to their solid-state counterparts, but extremely useful because it provides a way of creating one precursor set of particles that can be deliberately and controllably transformed into a new stet of compositions with properties that will derive from the metallated BMSB building blocks.

[0046] The colloidal particles disclosed herein can be used as catalysts or as phase transfer agents. The BMSB-metal moiety is a well-known catalytic center, and the colloidal particles can be used as catalysts in the same manner as a typical Schiff base-metal complex can be used. See, for instance, Desimoni et al, Chem. Rev., 103(8): 3119 (2003) and McManus, et al., Chem. i?ev.,104(9): 4151 (2004), each of which is incorporated herein in its entirety.

[0047] The colloidal particles disclosed herein can be used in biological and chemical detection of analytes. Because the colloidal particles are composed of hundred or thousands of fluorescent monomers (such as, e.g., colloidal particles of Zn-BMSB-Zn), minute quantities of an analyte of interest can be detected. The fluorescence of the colloidal particle is amplified by the dissolution of the colloidal particle into its monomelic components. Concentrations as low as about 50 aM (10 ^"18 M) of analyte are detectable using the colloidal particles disclosed herein. (FIG. 10)

[0048] The colloidal particles disclosed herein can be used in "sandwich" type assays. Such assays include, but are not limited to, ELISA, immunoassays using antibody-antigen- antibody interactions, magnetic microparticle (MMP) sandwich assays, and the like, wherein one component of the sandwich assay is modified to incorporate a colloidal particle as disclosed herein.

[0049] For use in the detection of analytes a probe can be attached to the colloidal particle. In certain embodiments, exemplary probes are antibodies, antigens, polynucleotides, oligonucleotides, receptors, ligands, and the like. The term polynucleotide is used broadly herein to mean a sequence of deoxyribonucleotides or ribonucleotides that are linked together by a phosphodiester bond. For convenience, the term oligonucleotide is used herein to refer to a polynucleotide that is used as a primer or a probe. Generally, an oligonucleotide useful as a probe or primer that selectively hybridizes to a selected nucleotide sequence is at least about 10 nucleotides in length, usually at least about 15 nucleotides in length, and for example between about 15 and about 50 nucleotides in length. Polynucleotide probes are particularly useful for detecting complementary polynucleotides in a biological sample.

[0050] A polynucleotide can be RNA or can be DNA, which can be a gene or a portion thereof, a cDNA, a synthetic polydeoxyribonucleic acid sequence, or the like, and can be single stranded or double stranded, as well as a DNA/RNA hybrid. In various embodiments, a polynucleotide, including an oligonucleotide (e.g., a probe or a primer) can contain nucleoside or nucleotide analogs, or a backbone bond other than a phosphodiester bond. In general, the nucleotides comprising a polynucleotide are naturally occurring

deoxyribonucleotides, such as adenine, cytosine, guanine or thymine linked to T- deoxyribose, or ribonucleotides such as adenine, cytosine, guanine or uracil linked to ribose. However, a polynucleotide or oligonucleotide also can contain nucleotide analogs, including non-naturally occurring synthetic nucleotides or modified naturally occurring nucleotides.

[0051] The covalent bond linking the nucleotides of a polynucleotide generally is a phosphodiester bond. However, the covalent bond also can be any of numerous other bonds, including a thiodiester bond, a phosphorothioate bond, a peptide-like amide bond or any other bond known to those in the art as useful for linking nucleotides to produce synthetic polynucleotides. The incorporation of non-naturally occurring nucleotide analogs or bonds linking the nucleotides or analogs can be particularly useful where the polynucleotide is to be exposed to an environment that can contain a nucleolytic activity, including, for example, a tissue culture medium or upon administration to a living subject, since the modified polynucleotides can be less susceptible to degradation.

[0052] As used herein, the term selective hybridization or selectively hybridize, refers to hybridization under moderately stringent or highly stringent conditions such that a nucleotide sequence preferentially associates with a selected nucleotide sequence over unrelated nucleotide sequences to a large enough extent to be useful in identifying the selected nucleotide sequence. It will be recognized that some amount of non-specific hybridization can occur, but is acceptable provided that hybridization to a target nucleotide sequence is sufficiently selective such that it can be distinguished over the non-specific cross- hybridization, for example, at least about 2-fold more selective, generally at least about 3.-fold more selective, usually at least about 5-fold more selective, and particularly at least about 10- fold more selective, as determined, for example, by an amount of labeled oligonucleotide that binds to target nucleic acid molecule as compared to a nucleic acid molecule other than the target molecule, particularly a substantially similar or homologous nucleic acid molecule other than the target nucleic acid molecule. Conditions that allow for selective hybridization can be determined empirically, or can be estimated based, for example, on the relative GC: AT content of the hybridizing oligonucleotide and the sequence to which it is to hybridize, the length of the hybridizing oligonucleotide, and the number, if any, of mismatches between the oligonucleotide and sequence to which it is to hybridize.

[0053] An example of progressively higher stringency conditions is as follows: 2 x SSC / 0.1% SDS at about room temperature (hybridization conditions); 0.2 x SSC / 0.1% SDS at about room temperature (low stringency conditions); 0.2 x SSC / 0.1% SDS at about 42°C (moderate stringency conditions); and 0.1 x SSC at about 68°C. (high stringency conditions).

Washing can be carried out using only one of these conditions, for example, high stringency conditions, or each of the conditions can be used, for example, for 10-15 minutes each, in the order listed above, repeating any or all of the steps listed. However, as mentioned above, optimal conditions will vary, depending on the particular hybridization reaction involved, and can be determined empirically.

[0054] In some embodiments, the colloidal particles can include an antibody probe. As used herein, the term antibody is used in its broadest sense to include polyclonal and monoclonal antibodies, as well as antigen binding fragments of such antibodies. An antibody useful in a method of the invention, or an antigen binding fragment thereof, is characterized, for example, by having specific binding activity for an epitope of an analyte.

[0055] An antibody can be detected using these antibody probe-modified colloidal particles. The antibodies which can be detected include naturally occurring antibodies as well as non-naturally occurring antibodies, including, for example, single chain antibodies, chimeric, bifunctional and humanized antibodies, as well as antigen-binding fragments thereof. Such non-naturally occurring antibodies can be constructed using solid phase peptide synthesis, can be produced recombinantly or can be obtained, for example, by screening combinatorial libraries consisting of variable heavy chains and variable light chains. These and other methods of making, for example, chimeric, humanized, CDR- grafted, single chain, and bifunctional antibodies are well known to those skilled in the art.

[0056] The term binds specifically or specific binding activity, when used in reference to an antibody means that an interaction of the antibody and a particular epitope has a dissociation constant of at least about 1 x 10 ^~6 , generally at least about 1 x 1(J ⁷ , usually at least about 1 x 10 ^"8 , and particularly at least about 1 x 10 ^"9 or 1 x lO ^"10 or less. As such, Fab, F(ab')2, Fd and Fv fragments of an antibody that retain specific binding activity for an epitope of an antigen, are included within the definition of an antibody.

[0057] The analyte detection methods disclosed herein can be performed, for example, by traditional sandwich assays known in the art. Such assays are described, for instance, in U.S. Patent 5,637,508; 5,639,626; 5,710,006; 6,096,563; 6,544,776; 6,686,208; and 6,670,115, each of which is incorporated herein by reference in its entirety. The detection of the analyte in a sample can be performed by contacting a sample containing an analyte with a colloidal particle of the present invention which is modified to include a probe, wherein the probe binds to the analyte; and further contacting the sample with another probe which also binds to the analyte, wherein this second probe allows for the removal of the bound analyte from the

sample, through, e.g., immobilization, magnetic separation, and the like. The bound colloidal particle, then, can be redissolved in pyridine and the fluorescence signal measured, wherein the fluorescence signal is indicative of the presence and/or concentration of the analyte in the sample. This amplification of fluorescence is such that very low levels of analyte can be detected.

[0058] The term "analyte " means any molecule or compound. An analyte can be in the solid, liquid, gaseous or vapor phase. By gaseous or vapor phase analyte is meant a molecule or compound that is present, for example, in the headspace of a liquid, in ambient air, in a breath sample, in a gas, or as a contaminant in any of the foregoing. It will be recognized that the physical state of the gas or vapor phase can be changed by pressure, temperature as well as by affecting surface tension of a liquid by, for example, the presence of or addition of salts.

[0059] As indicated above, methods of the present invention, in certain aspects, detect binding of an analyte to a probe. The analyte can be comprised of a member of a specific binding pair (sbp) and maybe a ligand, which is monovalent (monoepitopic) or polyvalent (polyepitopic), usually antigenic or haptenic, and is a single compound or plurality of compounds which share at least one common epitopic or determinant site. The analyte can be a part of a cell such as bacteria or a cell bearing a blood group antigen such as A, B, D, etc., or an HLA antigen or a microorganism, e.g., bacterium, fungus, protozoan, or virus. In some cases, the analyte is charged.

[0060] A member of a specific binding pair (sbp member) is one of two different molecules, having an area on the surface or in a cavity which specifically binds to and is thereby defined as complementary with a particular spatial and polar organization of the other molecule. The members of the specific binding pair are referred to as ligand and receptor (antiligand) or analyte and probe. Therefore, a probe is a molecule that specifically binds an analyte. These will usually be members of an immunological pair such as antigen-antibody, although other specific binding pairs such as biotin-avidin, protein-lectin, hormones-hormone receptors, nucleic acid duplexes, IgG-protein A, polynucleotide pairs such as DNA-DNA, DNA-RNA, and the like are not immunological pairs but are included in the invention and the definition of sbp member.

[0061] Specific binding is the specific recognition of one of two different molecules for the other compared to substantially less recognition of other molecules. Generally, the molecules have areas on their surfaces or in cavities giving rise to specific recognition between the two molecules. Exemplary of specific binding are antibody-antigen interactions, enzyme-

substrate interactions, polynucleotide hybridization interactions, and so forth. Non-specific binding is non-covalent binding between molecules that is relatively independent of specific surface structures. Non-specific binding may result from several factors including hydrophobic interactions between molecules.

[0062] The colloidal particles of the present invention can be used to detect the presence of a particular target analyte, for example, a nucleic acid, oligonucleotide, protein, enzyme, antibody, or antigen. The colloidal particles can also be used to screen bioactive agents, such as, for example, drug candidates, for binding, to a particular target or to detect agents like pollutants. As discussed above, any analyte for which a probe moiety, such as a peptide, protein, oligonucleotide or aptamer, may be designed can be used in combination with the disclosed colloidal particles.

[0063] In particular, analytes include poly(amino acids), such as for example, polypeptides and proteins, polysaccharides, hormones, nucleic acids, and combinations thereof. Such combinations include components of bacteria, viruses, prions, cells, chromosomes, genes, mitochondria, nuclei, cell membranes and the like. Additional possible analytes include drugs, metabolites, pesticides, pollutants, and the like. Included among drugs of interest are the alkaloids. Among the alkaloids are morphine alkaloids, which includes morphine, codeine, heroin, dextromethorphan, their derivatives and metabolites; cocaine alkaloids, which include cocaine and benzyl ecgonine, their derivatives and metabolites; ergot alkaloids, which include the diethylamide of lysergic acid; steroid alkaloids; iminazoyl alkaloids; quinazoline alkaloids; isoquinoline alkaloids; quinoline alkaloids, which include quinine and quinidine; diterpene alkaloids, their derivatives and metabolites.

[0064] The term analyte further includes polynucleotide analytes such as those polynucleotides defined below. These include, for example, m-RNA, r-RNA, t-RNA, DNA, and DNA-RNA duplexes. The term analyte also includes receptors that are polynucleotide binding agents, such as, for example, peptide nucleic acids (PNA), restriction enzymes, activators, repressors, nucleases, polymerases, histones, repair enzymes, chemotherapeutic agents, and the like.

[0065] The analyte may be a molecule found directly in a sample such as a body fluid from a host. The sample can be examined directly or may be pretreated to render the analyte more readily detectible. Furthermore, the analyte of interest may be determined by detecting an agent probative of the analyte of interest such as a specific binding pair member complementary to the analyte of interest, whose presence will be detected only when the

analyte of interest is present in a sample. Thus, the agent probative of the analyte becomes the analyte that is detected in an assay. The body fluid can be, for example, urine, blood, plasma, serum, saliva, semen, stool, sputum, cerebral spinal fluid, tears, mucus, and the like.

[0066] In general, probes can be attached to colloidal particles through attachment via surface carboxylate moieties of the colloidal particle. Alternatively, colloidal particles may be coupled with probes through biotin-avidin linkages. For example, avidin or streptavidin (or an analog thereof) can be covalently attached to the surface of the colloidal particles through one or more available carboxylate moieties a and a biotin-modified probe contacted with the avidin or streptavidin-niodified surface forming a biotin-avidin (or biotin- streptavidin) linkage. Optionally, avidin or streptavidin may be attached in combination with another protein, such as BSA, and optionally be crosslinked. Probes having an amine, hydroxyl, or carboxylic acid functional group can be attached through water-soluble carbodiimide coupling reagents, such as EDC (l-ethyl-3 -(3 -dimethyl aminopropyl)carbodiimide), or other coupling agents well known in the art, which couples carboxylic acid functional groups with amine groups, hydroxyl groups, and/or other carboxylic acid functional groups.

[0067] Nucleotides attached to a variety of functional groups may be commercially obtained (for example, from Molecular Probes, Eugene, Oreg.; Quiagen (Operon), Valencia, Calif.; and IDT (Integrated DNA Technologies), Coralville, Iowa) and incorporated into oligonucleotides or polynucleotides. Biotin-modified nucleotides are commercially available (for example, from Pierce Biotechnology, Rockford, 111., or Panomics, Inc. Redwood City, Calif.) and modified nucleotides can be incorporated into nucleic acids during conventional amplification techniques. Oligonucleotides may be prepared using commercially available oligonucleotide synthesizers (for example, Applied Biosystems, Foster City, Calif). Additionally, modified nucleotides may be synthesized using known reactions, such as for example, those disclosed in, Nelson, P., Sherman-Gold, R, and Leon, R, "A New and Versatile Reagent for Incorporating Multiple Primary Aliphatic Amines into Synthetic Oligonucleotides," Nucleic Acids Res., 17:7179-7186 (1989) and Connolly, B., Rider, P. "Chemical Synthesis of Oligonucleotides Containing a Free Sulfhydryl Group and Subsequent Attachment of Thiol Specific Probes," Nucleic Acids Res., 13:4485-4502 (1985). Alternatively, nucleotide precursors may be purchased containing various reactive groups, - such as biotin, hydroxyl, sulfhydryl, amino, or carboxyl groups. After oligonucleotide synthesis, colloidal particles may be attached using standard chemistries. Oligonucleotides of any desired sequence, with or without reactive groups for colloidal particle attachment, may

also be purchased from a wide variety of sources (for example, Midland Certified Reagents, Midland, Tex.).

[0068] Probes, such as polysaccharides, may also be attached to colloidal particles, for example, through methods disclosed in Aslam, M. and Dent, A., Bioconjugation: Protein Coupling Techniques for the Biomedical Sciences, Grove's Dictionaries, Inc., 229, 254 (1998). Such methods include, but are not limited to, periodate oxidation coupling reactions and bis-succinimide ester coupling reactions.

[0069] The following paragraphs include further details regarding exemplary applications of colloidal particle-probes species. It will be understood that numerous additional specific examples of applications that utilize colloidal particle-probes species can be identified using the teachings of the present specification. One of skill in the art will recognize that many interactions between polypeptides and their target molecules can be detected using colloidal particle-labeled polypeptides. In one group of exemplary applications, colloidal particle- labeled antibodies (i.e., antibodies bound to a colloidal particle) are used to detect interaction of the colloidal particle-labeled antibodies with antigens. It will be understood that such immunoassays can be performed using known methods such as, for example, ELISA assays, Western blotting, or protein arrays, utilizing the colloidal particle-labeled antibody or colloidal particle-labeled secondary antibody, in place of a primary or secondary antibody labeled with an enzyme or a radioactive compound.

[0070] Another group of exemplary methods, uses colloidal particle probes to detect a target nucleic acid. Such a method is useful, for example, for detection of infectious agents within a clinical sample, detection of an amplification product derived from genomic DNA or RNA or message RNA, or detection of a gene (cDNA) insert within a clone. For certain methods aimed at detection of a target polynucleotide, an oligonucleotide probe is synthesized using methods known in the art. The oligonucleotide probe then is used to functionalize a colloidal particle to produce a colloidal particle-labeled oligonucleotide probe. The colloidal particle-labeled oligonucleotide probe is used in a hybridization reaction to detect specific binding of the colloidal particle-labeled oligonucleotide probe to a target polynucleotide. For example, the colloidal particle-labeled oligonucleotide probe can be used in a Northern blot or a Southern blot reaction. Alternatively, the colloidal particle-labeled oligonucleotide probe can be applied to a reaction mixture that includes the target polynucleotide associated with a solid support, to capture the colloidal particle-labeled oligonucleotide probe. The captured colloidal particle-labeled oligonucleotide probe can then be detected using fluorescence spectroscopy, with or without first being released from the

solid-support. Detection of the captured colloidal particle-labeled oligonucleotide probe can be amplified by first dissolving the isolated hybridized entity, wherein the monomelic BMSB are released into solution. Because the fluorescence particles used in this method are composed of a huge number of fluorescence molecules (e.g., Zn-BMSB), the approach allows for detection of small amounts of target analyte, down to about 5 aM (10 ^"18 M).

[0071] In the methods of the invention, a sample includes a wide variety of analytes that can be analyzed using the colloidal particles described herein. For example, a sample can be an environmental sample and includes atmospheric air, ambient air, water, sludge, soil, and the like. In addition, a sample can be a biological sample, including, for example, a subject's breath, saliva, blood, urine, feces, various tissues, and the like.

[0072] Commercial applications for the invention methods employing the colloidal particles described herein include environmental toxicology and remediation, biomedicine, materials quality control, monitoring of food and agricultural products for the presence of ■ pathogens, anesthetic detection, automobile oil or radiator fluid monitoring, breath alcohol analyzers, hazardous spill identification, explosives detection, fugitive emission identification, medical diagnostics, fish freshness, detection and classification of bacteria and microorganisms both in vitro and in vivo for biomedical uses and medical diagnostic uses, monitoring heavy industrial manufacturing, ambient air monitoring, worker protection, emissions control, product quality testing, leak detection and identification, oil/gas petrochemical applications, combustible gas detection, H ₂ S monitoring, hazardous leak detection and identification, emergency response and law enforcement applications, illegal substance detection and identification, arson investigation, enclosed space surveillance, utility and power applications, emissions monitoring, transformer fault detection, food/beverage/agriculture applications, freshness detection, fruit ripening control, fermentation process monitoring and control applications, flavor composition and identification, product quality and identification, refrigerant and fumigant detection, cosmetic/perfume/fragrance formulation, product quality testing, personal identification, chemical/plastics/pharmaceutical applications, leak detection, solvent recovery effectiveness, perimeter monitoring, product quality testing, hazardous waste site applications, fugitive emission detection and identification, leak detection and identification, perimeter monitoring, transportation, hazardous spill monitoring, refueling operations, shipping container inspection, diesel/gasoline/aviation fuel identification, building/residential natural gas detection, formaldehyde detection, smoke detection, fire detection, automatic ventilation control applications (cooking, smoking, etc.), air intake monitoring, hospital/medical

anesthesia and sterilization gas detection, infectious disease detection and breath applications, body fluids analysis, pharmaceutical applications, drug discovery, telesurgery, and the like.

[0073] Another application for the sensor-based fluid detection device in engine fluids is an oil/antifreeze monitor, engine diagnostics for air/fuel optimization, diesel fuel quality, volatile organic carbon measurement (VOC), fugitive gases in refineries,, food quality, halitosis, soil and water contaminants, air quality monitoring, fire safety, chemical weapons identification, use by hazardous material teams, explosive detection, breathalyzers, ethylene oxide or anesthetics detectors.

EXAMPLES

[0074] Solvents and all other chemicals were obtained from commercial sources and used as received unless otherwise noted. AU deuterated solvents were purchased and used as received from Cambridge Isotopes Laboratories. ¹ H and ¹³ C NMR spectra were obtained using a Varian Mercury 300 MHz or a Varian INOVA 400 MHz FT-NMR spectrometers. Infrared spectra of solid samples were obtained on a Thermo Nicolet Nexus 670 FT-IR spectrometer as KBr pellet. Diffuse reflectance spectra were obtained on a Varian Gary 5000 UV-Vis-NIR spectrophotometer. Emission spectra were obtained on a Jobin Yvon SPEX Fluorolog fiuorometer using quartz cells (10 x 4 mm light path). Electrospray ionization mass spectrometric (ESI-MS) spectra were obtained on a Micromass Quatro II Triple Quadrupole mass spectrometer, and all peaks are cosnsitent with a natural abundance isotopic distribution patterns. Elemental analyses were obtained from Quantitative Technologies Inc., Whitehouse, NJ. All scanning electron microscopy (SEM) images and energy dispersive X- ray (EDX) spectra were obtained using a Hitachi S-4500 cFEG SEM (Electron Probe Instruments Center (EPIC), NUANCE, Northwestern University) equipped with an Oxford Instruments EDS system. All optical and fluorescence microscopy images were obtained using a Zeiss Axiovert IOOA inverted optical/fluorescence microscope (Thomwood, NY) equipped with a Penguin 600CL digital camera (HQ FITC/Boρidy/Fluo3/Di o/EGFP and HQ Texas Red filter sets were used for green and red emission, respectively). Particle size, size distribution, and ζ-potential measurements in solution were performed with a Zetasizer Nano- ZS.

SYNTHESIS OF BIS-METALLIC SCHIFF BASE (BMSB) COMPOUNDS

(Compound SIa)

[0075] (S) 3,3'-difoϊmyl-2,2'-dihydroxy-l,l '-binaphthyl (200-mg, 0.585 mmol), which was synthesized according to the literature procedures (Zhang et al., J. Org. Chem. 66:481 (2001)), and 3-amino-4-hydroxy-benzoic acid (198 mg, 1.29 mmol) were mixed in ethanol (15 mL). The resulting solution was refluxed for 1 h. During this time, a yellow product precipitated. The precipitate product (BSB SIa) was filtered and washed with hot ethanol (85% yield). ¹ H NMR (300 MHz, DMSO-^, 25 °C): δ 13.40 (s, 2H, OH), 12.70 (br, 2H, CO ₂ H), 10.65 (s, 2η, OH), 9.37 (s, 2η, CH=N), 8.51 (s, 2H), 8.04 (s, 2H), 8.02 (d, J- 8.2 Hz, 2H), 7.76 (d, J= 8.4 Hz, 2H), 7.36 (m, 4H), 7.07 (d,J= 8.1 Hz, 2H), 7.04 (d, J= 8.4 Hz, 2H). ¹ H NMR (300 MHz, pyridine-^, 25 ⁰ C): δ 9.36 (s, 2H, CH=N), 8.62 (d, J= 1.2 Hz, 2H), 8.36 (dd, J= 8.7 Hz, J= 1.2 Hz, 2H), 8.26 (s, 2H), 8.09 (m, 2H), 7.64 (m, 2H), 7.38 (m, 4H) ₅ 7.31 (d, J= 8.7 Hz, 2H). ¹³ C( ¹ H) NMR (100.7 MHz, DMSO-^ ₅ 25 °C): δ.167.03 (CO ₂ H) ₅ 163.21 (C=N) ₅ 155.76 (C-OH), 154.47 (C-OH) ₅ 135.16, 134.88, 134.62, 130.02, 129.23, 128.64, 127.24, 124.28, 123.41, 122.14, 121.45, 120.85, 116.37, 116.26. ¹³ C( ¹ H) NMR (100.7 MHz, pyridine-dj, 25 °C): δ 169.35 (CO ₂ H), 163.47 (C=N), 157.72 (C-OH), 156.45 (C-OH), 137.05, 136.61, 135.50, 131.52, 130.68, 130.22, 129.49, 128.83, 126.04, 124.51, 123.05, 122.99, 118.29, 117.67. Anal. Calcd for C ₃₆ H ₂₄ N ₂ O ₈ : C, 70.58; H, 3.95; N, 4.57. Found: C, 70.38; H, 4.02; N, 4.34. ESI-MS (m/z, DMSO/CH ₂ C1 ₂ ): calcd for [SIa + H ⁺ ] ⁺ ₅ 613.2; found, 613.1. IR (KBr pellet, cm ^"1 ): 1684s, 1615s, 1596s, 1506m, 1438m, 1412w, 1384w, 1363w, 134Ow, 1293m, 1270m, 1213w, 1185w, 1152m, 1119m, 1043w, 98Ow, 952m, 905w, 889w, 832w, 776m, 757m, 63Ow.

(Compound SIb)

[0076] BSB SIb was synthesized using the same method as for SIa, except 2-amino- phenol was used instead of 3-amino-4-hydroxy-benzoic acid (90% yield). ¹ H NMR (300 MHz, DMSO-rf _tf , 25 ⁰ C): δ 13.59 (s, 2H, OH), 9.78 (s, 2H, OH), 9.29 (s, 2H, CH=N), 8.43 (s, 2H) ₅ 8.04 (m, 2H), 7.47 (d, J= 7.5 Hz, 2H), 7.35 (m, 4H), 7.15 (t, J= 7.5 Hz ₃ 2H), 7.06 (m, 2H), 6.97 (d, J= 7.6 Hz, 2H), 6.91 (t, J= 7.5 Hz, 2H). ¹ H NMR (300 MHz, pyridine-^, 25 ⁰ C): δ 14.27 (s, 2H), 12.04 (br, 2H), 9.32 (s, 2H, CH=K), 8.28 (s, 2H), 8.02 (m, 2H>, 7.60 (m, 2H), 7.50 (d, J= 7.8 Hz, 2H), 7.36 (m, 4H), 7.22 (m, 4H>, 7.01 (m, 2H). ¹³ C( ¹ H) NMR (100.7 MHz, DMS(M _? , 25 ⁰ C): δ 162.05 (ON), 154.54 (C-OH), 151.57 (C-OH), 135.01, 134.64, 134.26, 129.12, 128.53, 128.45, 127.22, 124.25, 123.31, 121.53, 119.76, 119.65, 116.68, 116.29. Anal. Calcd for C ₃₄ H ₂₄ N ₂ O ₄ : C, 77.85; H, 4.61; N, 5.34. Found: C, 77.71; H, 4.58; N, 5.33. ESI-MS (m/z, DMSO/CH ₂ C1 ₂ ): calcd for [SIb + H ⁺ ] ⁺ , 525.2; found, 525.4.

[0077] Zn-BMSB 2a: BSB SIa, synthesized as decribed above, (5 mg, 0.008 mmol) and Zn(OAc) ₂ (3 mg, 0.016 mmol) were mixed in DMF (3 mL). The color of the solution changed immediately from yellow to red. Diethyl ether was added to precipitate the product as an orange powder (84% yield). The precipitate product was separated from supernatant and washed with diethyl ether (84% yield). ¹ H NMR (300 MHz, pyridine-Jj, 25 ⁰ C): δ 9.60 (s, 2H, CH=N), 9.11 (d, J= 1.8 Hz, 2H), 8.58 (dd, J= 8.4 Hz, J= 1.8 Hz, 2H), 8.20 (s, 2H), 8.15 (d, J= 7.8 Hz, 2H), 7.79 (d, J= 8.4 Hz, 2H), 7.49 (d, J= 8.4 Hz, 2H), 7.35 (t, J = 7.6 Hz, 2H), 7.28 (t, J= 7.6 Hz, 2H). ¹³ C( ¹ H) NMR (100.7 MHz, pyridine-^, 25 ⁰ C): δ 171.22 (CO ₂ H), 169.81 (C-O), 166.21 (C-O), 159.45 (C=N), 139.02, 136.79, 135.08, 132.98, 131.02, 130.27, 128.12, 126.13, 125.20, 124.74, 120.96, 120.12, 117.97, 116.65. ESI-MS (m/z, pyridine/CH ₂ Cl ₂ ): calcd for [SIa - 4H ⁺ + 2Zn ²⁺ + 2 pyridine + H ⁺ J ⁺ , 895.1; found, 894.9; calcd for [SIa - 4H ⁺ + 2Zn ²⁺ + pyridine + H ⁺ J ⁺ , 816.0; found, 816.0. IR (KBr pellet, cm ⁴ ): 1659s, 1609s, 1588s, 1539w, 1506w, 1491m, 1425m, 1383s, 1339m, 1298m, 1223w, 1198w, 1173w, 1151w, 1121m, 1104m, 1061w, 1044w, 1024w, 958m, 898w, 843m, 788m, 748m, 663m, 478w.

[0078] Cu-BMSB 2b: 2b was prepared using the same method as for 2a, except Cu(OAb) ₂ (H ₂ O) was used instead OfZn(OAc) ₂ . A brown powder was obtained in 86% yield. ESI-MS {ink, pyridine/CH ₂ Cl ₂ ): calcd for [SIa - 4H ⁺ + 2Cu ²⁺ + 2 pyridine + H ⁺ J ⁺ , 893.1; found, 892.9. IR (KBr pellet, cm ^"1 ): 1653s, 1604s, 1585s, 1539w, 1522w, 1491m, 1427m, 1386s, 1369s, 1340m, 13Q6m, 126Ow, 1225w, 1194w, 1173w, 115Ow, 1123m, 1102m, 106 Iw, 1044w, 1023w, 956m, 898w, 846m, 784m, 748m, 666m, 506w, 481 w.

[0079] X-ray crystal structure determination of 2b: A dark tabular crystal was mounted using oil on a glass fiber. Diffraction intensity data were collected with a Bruker SMART- 1000 CCD diffractometer equipped with a graphite-monochromated Mo Ka radiation source. The data collected were processed to produce conventional intensity data by the program SAINT-NT (Bruker). The intensity data were corrected for Lorentz and polarization effects. Absorption corrections were applied using the SADABS empirical method. The structures were solved by direct methods, completed by subsequent difference Fourier syntheses and refined by full matrix least-squares procedures, on F ² . The disordered methanol was refined with a group anisotropic displacement parameter _^ and occupancies adding to fully occupied. The remaining non-hydrogen atoms were refined anisotropically. Hydrogen atoms were included in idealized positions, except those on the disordered and partially occupied methanol, and the hydrogen atom on 05 (methanol), but not refined. The DMF was fixed to ¹ A occupied. All software and sources of scattering factors are contained in the SHELXTL program package (version 5.10, G. Sheldrick, Bruker- AXS, Madison, WI).

[0080] Ni-BMSB 2c: 2c was prepared using the same method as for 2a, except Ni(OAc) ₂ -4(H ₂ O) was used instead OfZn(OAc) ₂ . A red powder was obtained in 82% yield. ESI-MS (m/z, pyridine/CH ₂ Cl ₂ ): calcd for [SIa - 4H ⁺ + 2Ni ²⁺ + 2 pyridine + H ⁺ J ⁺ , 883.1; found, 882.8. IR (KBr pellet, cm ^"1 ): 1655s, 1608s, 1584s, 1545w, 1491m, 1419m, 1387s, 1339m, 1304m, 1263w, 1226w, 1193w, 1173w, 1151w, 1119m, 1104m, 1061w, 1048w, 1026w, 960m, 901 w, 841m, 786m, 749m, 668m, 517w, 479w.

[0081] 2d: 2d was. prepared using the same method as for 2a, except BSB SIb was used instead of BSB SIa. An orange powder was obtained in 81% yield. ¹ HNMR (300 MHz, pyridine-;/?, 25 °C): δ 9.54 (s, 2H, CH=N), 8.31 (s, 2η), 8.07 (d, J= 8.4 Hz, 2H), 7.94 (d, J= 8.1 Hz, 2H), 7.80 (d,J= 8.1 Hz, 2H), 7.48 (d, J- 8.1 Hz, 2H), 7.40 (t, J= 7.4 Hz, 2H), 7.33 (t, J= 7.5 Hz, 2H), 7.24 (t, J= 7.5 Hz, 2H), 7.79 (t, J= 7.5 Hz, 2H). ¹³ C( ¹ H) NMR (100.7 MHz, 25 °C): δ 166.23 (C-O), 165.52 (C-O), 158.53 (ON), 138.86, 136.41, 135.70, 131.19, 130.69, 130.31, 128.01, 126.16, 126.07, 125.64, 120.90, 120.77, 115.69, 114.18. ESI-MS (m/z, pyridine/CH ₂ Cl ₂ ): calcd for [SIb - 4H ⁺ + 2Zn ²⁺ + 2 pyridine + H ⁺ ] ⁺ , 807.1; found, 806.9, calcd for [SIb - 4H ⁺ + 2Zn ²⁺ + pyridine + H ⁺ f, 728.1; found, 728.1.

COLLOIDAL PARTICLE FORMATION

[0082] Slow diffusion method: A precursor solution was prepared by mixing BMSB 2a-c (0.008 mmol) and M(OAc) ₂ (0.008 mmol, M = Zn, Cu, Ni) in pyridine (200 ~ 400 μL) (as an alternative way, by mixing BSB SIa (0.008 mmol) and M(OAc) ₂ (0.024 mmol, M = Zn, Cu, Ni)). Diethyl ether or pentane was allowed to slowly diffuse into the precursor solution. After 1 to 2 hours, micro-size spherical particles 3 form. Particle products were isolated and subsequently washed with toluene via centrifugation-redispersion cycles. Each successive supernatant was decanted and replaced with fresh toluene (74 to 86% yield).

[0083] Fast addition method: A precursor solution was prepared as described above. Diethyl ether or pentane (8 mL) was rapidly added into a precursor solution to form nano-size spherical particles 3. Products were isolated and washed as described above (85 ~ 92% yield).

[0084] IR spectra taken before and after formation of the particles show that the carboxylate groups are coordinating to metal ions as evidenced by a shift in CO stretching frequency from 1653 - 1659 cm ^"1 for the monomeric unbound forms (2a-c) to 1597 - 1605 cm ^"1 for the polymer particles (3a-c). ¹ H and ¹³ C NMR spectra of diamagnetic 3a indicate coordination of the carboxylic acid groups to the metal center. The electrospray ionization mass spectra of 3b and 3c exhibit intense peaks associated with the metal-metalloligand repeat units, [2b - 2H ⁺ + Cu ²⁺ + (pyridine),, + H ⁺ J ⁺ and [2c - 2H ⁺ + Ni ²⁺ + (pyridine),, + H ⁺ ] ⁺ . The NMR spectra of 3b and 3c were not informative because of the paramagnetic nature of these complexes, and the mass spectrum of 3a did not yield a monomer ion. The measurement of Zeta potential of these particles reveals that particles are negatively charged (-12 mV), which is a result of the deprotonated carboxylate groups located on their surfaces.

[0085] Ancillary Iigands exchange reactions: The ¹ H NMR spectra of the methanol-^ and OMSO-dβ suspension of spherical particles 3a with ancillary Iigands L of pyridine and methanol respectively, show the peaks for free pyridine and methanol molecules, which are released from the metal upon replacement with methanol-^ and OMSO-dg, respectively. The elemental analysis measured before and after the exchange of ancillary Iigands L from pyridine to methanol, show a decreasing nitrogen content from 6.33 to 3.35%.

(Zn-BMSB-Zn, compound 3a)

[0086] Zn-BMSB-Zn 3a: ¹ H NMR (300 MHz, pyridine-dj, 25 ⁰ C): δ 9.52 (s, 2H, CH=N), 9.28 (d, J= 1.5 Hz, 2H), 8.77 (dd, J= 8.4 Hz, J= 1.5 Hz, 2H), 8.10 (d, J= 8.1 Hz, 2H), 8.05 (s, 2H), 7.76 (d, J= 8.4 Hz, 2H), 7.50 (d, J= 8.4 Hz, 2H), 7.31 (t, J= 7.6 Hz, 2H), 7.24 (t, J= 7.6 Hz, 2H). ¹³ C( ¹ H) NMR (100.7 MHz, pyridine-^, 25 ⁰ C): δ 175.95 (CO ₂ ), 168.90 (C-O), 166.14 (C-O), 158.75 (C=N), 138.87, 134.65, 133.51, 131.01, 130.16, 127.91, 126.06,, 125.29, 124.67, 124.43, 120.81, 120.50, 119.72, 118.26. Anal. Calcd for Zn ₃ (SIa - 6H)(ρyridine) ₃ : C, 58.90; H, 3.20; N, 6.73. Found: C, 57.83; H, 3.10; N, 6.33. IR (KBr pellet, cm ^"1 ): 1605s, 1591sh, 1539m, 1506w, 1489w, 1465w, 1448m, 1425w, 1375s, 1341m, 1327m,

1299m, 1217m, 1194w, 1171w, 1148m, 1121m, 1069m, 1042m, 1015w, 955w, 894w, 842m, 787m, 749m, 698m, 663m, 636m, 478w, 419w.

(Cu-BMSB-Cu, compound 3b)

[0087] Cu-BMSB-Cu 3b: ESI-MS (m/z, pyridme/CH ₂ Cl ₂ ): calcd for [SIa - 6H ⁺ + 3Cu ²⁺ + 3 pyridine + H ⁺ J ⁺ , 1033.0; found, 1032.7, calcd for [SIa - 6H ⁺ + 3Cu ²⁺ + 2 pyridine + H ⁺ ] ⁺ , 954.0; found, 954.0. Anal. Calcd for Cu ₃ (SIa - 6H)(H ₂ O) ₂ (pyridine) ₃ : C, 57.22; H, 3.48; N, 6.54. Found: C, 57.69; H, 2.96; N, 6.47. IR (KBr pellet, cm ^"1 ): 1602s, 1589sh, 1539m, 1507w, 149Ow, 1466w, 1449m, 1425w, 1369s, 1342m, 1325m, 1262w, 1217m, 119Ow, 1172w, 1148m, 1124m, 1069m, 1043m, 1017w, 955w, 897w, 846m, 783m, 748m, 696m, 671m, 639m, 504w, 479w, 419w.

(Ni-BMSB-Ni, compound 3c)

[0088] Ni-BMSB-Ni 3c: ESI-MS (m/z, pyridine/CH ₂ Cl ₂ ): calcd for [SIa - 6H ⁺ + 3Ni ²⁺ + 4 pyridine + H ⁺ ] ⁺ , 1097.1; found, 1097.0. Anal. Calcd for Ni ₃ (SIa - 6H)(H ₂ O) ₂ (pyridine) ₃ : C, 58.01; H, 3.53; N, 6.63. Found: C, 58.08; H, 3.28; N, 6.57. IR (KBr pellet, cm ^'1 ): 1597s, 1589sh, 1542m, 1507w, 1497w, 1465w, 1448m, 1429w, 1385s, 1369m, 1343m, 1324m, 1269w, 1217m, 1192w, 1172w, 1149m, 1125m, 1070m, 1042m, 1017w, 951w, 897w, 856m, 786m, 757m, 693m, 639w, 516w, 494w, 419w. There are inherent difficulties in formulating

the exact number of pyridine and water in particle 3a-c due to the possibility of exchange with other molecules.

Detection Assay Using Colloidal Particles

[0089] Preparation of the Probes: For the magnetic probe preparation (MMP), 5 '- amino-functionalized DNA (5'-NH ₂ -AAA AAA AAA ATC CTT ATC AAT-3') (SEQ. ID NO: 1) was directly linked to tosyl-activated MMPs using the procedure recommended by Dynal Biotech Inc. For the fluorescence probe preparation, 3 '-amino-functionalized DNA (5'-ATT TAA CAA TAA TCC AAA AAA AAA A-NH ₂ -3') (SEQ. ID NO: 2) was directly attached to the fluorescence particles, which have many carboxylic groups at the surface, by using N-ethyl-N'-(3-dimethylaminoproρyl)carbodiimide (EDC) as a linker. FIG. 9 shows a schematic of the structures of both the MMP and the colloidal particles. The colloidal particles that were used had an average diameter of 1.60 ± 0.47 μm (s.d., n=100).as determined by SEM.

[0090] Fluorescence Amplification Based Bioassay for DNA: The fluorescence amplification based bioassay method was tested with oligonucleotide associated with the anthrax lethal factor (5'-GGA TTA TTG TTA AAT ATT GAT AAG GAT-3') (SEQ. ID. NO: 3) in 0.3 M PBS buffer containing 0.025% Tween 20 as a target over a range of 5 aM (10 ^"18 ) to 50 fM (10 ^"15 M). In a typical assay, a sample solution (50 μL) with a set of target DNA concentration was added to a 50 μL of MMP probes functionalized with a single-strand DNA (SEQ. ID NO: 1) to capture the target DNA, and the solution was shaken on an orbital shaker at 50° C for 15 minutes and at 20°C for 45 minutes. The fluorescence particles functionalized with a single-strand DNA (SEQ. ID NO: 2) to form a sandwich structure with the MMP probes that have captured the target DNA (e.g., SEQ. ID NO: 3) were then added to the solution. This solution was vigorously stirred at 20° C for 2 hours. The magnetic separator was used to concentrate both unreacted magnetic particles and magenetic particles which captured the target DNA and fluorescence colloidal particles. These particles were vigorously washed six times with 0.3 M PBS buffer containing 0.025% Tween 20 to remove any unreacted fluorescence colloidal particles. After the copious washing step, the reacted fluorescence colloidal particles were dissolved in pyridine (100 μL). The solution of particles dissolved with pyridine was studied by fluorescence spectroscopy. The solution was excited at 420 nm and fluorescence emission was measured at 600 nm. In each experiment, a negative control (no target DNA in the reaction mixture) was run for comparison. The area of the spectra from 580 to 615 nm was integrated. AU target concentrations over the 5 aM to 50 fM concentration range was differentiated from negative control (FIG. 10 A). The assay

exhibits a linear relationship between target concentration and fluorescence intensity over a four order of magnitude concentration range (FIG. 10 B).

[0091] The foregoing description of the present invention has been presented for purposes of illustration and description. The description is not intended to limit the invention to the embodiment disclosed herein. Variations and modifications commensurate with the above teachings, and the skill or knowledge of the relevant art, are within the scope of the present invention.