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`
`US1X16022748A
`
`[19]
`United States Patent
`6,022,748
`['11] Patent Number:
`Charych et at. Feb. 8, 2000 [45] Date of Patent:
`
`
`
`
`
`[S4] SOL-GEL MATRICES FOR DIRECT
`COLORIMETRIC DETECTION ()Ir
`ANALYTES
`
`[75]
`
`Inventors: Deborah 1-]. Charych, Albany. Calif.;
`Darryl Snsaki, Albuquerque, N.Mex.;
`Stacey Ynmnnnka, Dallas, Tex.
`
`|73| Assignee: Sandie Corporation - New Mexico
`Regents of the University of
`California. Calif.
`
`[21] App}. No: 081920501
`
`[22
`
`Filed:
`
`Aug. 29, 1997
`
`Int. Cl? ..................... ._ (10m 33.1552; GU'IN 33549
`|51|
`I52] U.S. C1.
`.......................... 4361527; 436528; 436.535;
`436.805; 430i811; 436(815; 43619923; 436i829
`Field of Search
`436.628, 829,
`
`
`436527, 535, 811, SIS, 805. 823; 424i1.2'l,
`450, 94.3, 812
`
`[58]
`
`[56]
`
`References Cited
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`5,571,568
`5,591.453
`5,618,735
`5.622.872
`5,017.507
`
`.
`.
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`
`it997 Saul ct al.
`4361518
`4it99‘i Rihi ....................................... .. 43oi518
`.......................... .. 436i16o
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`(List continued on next page.)
`
`Primary: Examiner—Mary E. Ceperlcy
`Attorney, Agent, or Firm—Medlen & Carrol], LLP
`
`|57|
`
`ABSTRACT
`
`The present invention relates to methods and compositions
`for the direct detection of analytes using color changes that
`occur in immobilized hiopolymeric material in response to
`selective binding of analytes to their surface. In particular,
`the present
`invention provides methods and compositions
`related to the encapsulation ot" hiopolymeric material into
`metal oxide glass using the sol-gel method.
`
`6 Claims, 14 Drawing Sheets
`
`0001
`
`US. Patent No. 8,652
`
`Apple
`APLl
`
`
`
`Apple Inc.
`APL1061
`U.S. Patent No. 8,652,040
`
`0001
`
`

`

`
`
`6,022,748
`
`Page 2
`
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`
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`chrome c and catalasc in SiO2 sol—gel materials.” J. Nonv—
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`Press, Oxford, pp. 33—104 [1990].
`Okahata and Kunitakc, “Formation of Stable Monolayer
`Membranes and Related Structurs in Dilute Aqueous Solu~
`tion from Two—Headed Ammonium Amphiphilcs." J. Am.
`Chem. Soc. 101: 5231—5234 [1979].
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`Detection of Cholera Toxin by Polfldiacetylene) Liposorrtes
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`with Sialic Acid Bind and Colorimetricaliy Detect Influenza
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`Ligand for the CD4 ('I'4);’I-luman Immunodeficiency Virus
`Receptor," J. Near-oser'encc Res. 18: 102—107 [1987].
`Sheplcy et al.. “Monoclonal antibody identification of a
`liKt—kDa membrane protein in HeI.a cells and human spinal
`cord involved in poliovirus attachment.” Pmc. Natl. Acnd.
`Sci. 35: r743—7747 [I988].
`Spevak et 31.. “Polymerized I..iposornes Containing CwGly-
`cosides of Sialic Acid: Potent Inhibitors of Influenza Virus
`in Vitro Infectivity," .J'. Am. Client. Soc. 115:
`ll46—II47
`[[993].
`Weis et at. "Structure of the influenza virus haemagglutinin
`complexed with its receptor, sialic acid," Narran 333:
`426-431 [1988 ].
`White el al., “Viral Receptors of the Immunoglobulin Super-
`t‘amily," Cell 56:?25—728 [1989].
`Wyrick el 31. “Entry of Genital Chlamydia trndwntnrr's into
`Polarized Iiu man Epithelial Cells,” Infect.
`Innnnn. 57:
`2378—2389 [1 989].
`
`Yamanaka et al., "Nicotinamide Adenine Dinucleotidc Phos-
`phate Fluoresaence and Absorption Monitoring of Enzy-
`matic Activity in Silicate Sol-«Gels for Chemical Sensing
`Applications." J. Am. Chem. Soc. 11.719095-9096 {1995].
`Zhao and Reichert, “Influence of Biotin Lipid Surface
`Density and Accessibility on Avid in Binding to the Tip of an
`Optical Fiber Sensor," Langmuir 8: 2785—2791 [1992].
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`Porous Silicate Glasses Prepared by the Sol—Gel Method,"
`Science 255:1113—1115 (1992).
`Chai et at, "Sol—Gel Derived Hydroxyapatite Coatings for
`Biomedical Applications," Marci: Mann}?
`Processes
`10205-416 (1995).
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`Syntp. Proc. 282:153—161 (1993).
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`Surface of Polymerized Monolayers and Liposornes," PhD.
`Dissertation, University of California at Berkeley (1993).
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`Spectra of Diacetylenic Phospholipid Polymers," Biocliim.
`Biophys. Acm 693:461—465 (1982).
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`caused by pre—annealing in poiydiacetylenc Langmuir—
`Blodgell films," Thin Solid Films 2]ili211:548—550 (1992).
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`I’olydiacety—
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`Lio el 21]., “Atomic force microscope study of chromatic
`transitions in polydiacetylene thin films," J. Vac. Sci. Tech-
`nol. 14(2):l481—l436 (1996).
`Leung et aI.. "Imaging of polydiacetylene on graphite by
`scanning
`tunneling microscopy,"
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`69(4}:2044—2047 (1991).
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`Patterned
`SEW—Assembled Monolayers."
`Langmuir
`10:619—622 (1994}.
`Dagani. “Lipids and Minerals Forrn Novel Composite
`Microstructurcs," Client. & Eng. News, 19-20 (1993).
`Yamanaka et aI., “Solid Phase Immobilization of Optically
`Responsive Liposomes in Sol—gel Materials for Chemical
`and Biological Sensing,” Langmuir 13:5049—5053 (1997).
`Zink el al., “Inorganic Sol—Gel Glasses as Matrices for
`Nonlinear Optical Materials,” ACS Symposium Series, vol.
`455, pp. 541—552 (1991).
`
`0002
`
`
`
`0002
`
`

`

`US. Patent
`
`Feb. 8, 2000
`
`Sheet 1 of 14
`
`6,022,748
`
`FIGURE 1
`
`\\
`
`\\
`
`1
`
`O
`
`O
`
`HO
`
`\\
`
`O
`N.» WQNOJN
`H
`H
`
`HO
`
`HO
`
`“a
`
`Ho
`
`COOH
`
`ACNH
`
`O
`
`HO
`
`0
`
`0003
`
`0003
`
`

`

`US. Patent
`
`Feb. 8, 2000
`
`Sheet 2 0f 14
`
`6,022,748
`
`FIGURE 2
`
`Wavelength (nm)
`
`Absorbance
`
`400
`
`500
`
`600
`
`700
`
`0004
`
`0004
`
`

`

`US. Patent
`
`Feb. 8, 2000
`
`Sheet 3 0f 14
`
`6,022,748
`
`(D
`
`FIGURE 3
`
`Wavelength (nm)
`
`400
`
`500
`
`600
`
`700
`
`0005
`
`OC (
`
`5 e OU
`
`)
`.0
`<3:
`
`0005
`
`

`

`Feb. 8, 2000
`
`Sheet 4 of 14
`
`6,022,748
`
`FIGURE 4
`
`US. Patent
`
`0006
`
`0006
`
`

`

`U. S. Patent
`
`Feb. 8, 2000
`
`Sheet 5 0f 14
`
`6,022,748
`
`FIGURE 5
`
`Wavelength (nm)
`
`o c m eoU
`
`a)
`
`)
`.0
`<3:
`
`400
`
`500
`
`600
`
`700
`
`0007
`
`0007
`
`

`

`US. Patent
`
`Feb. 8, 2000
`
`Sheet 6 of 14
`
`6,022,748
`
`Wavelength (nm)
`
`Absorbance
`
`FIGURE 6
`
`400
`
`500
`
`600
`
`700
`
`0008
`
`0008
`
`

`

`FIGURE 7
`
`Small
`Molecules
`
`CI
`
`x
`
`US. Patent
`
`Feb. 8, 2000
`
`Sheet 1' of 14
`
`6,022,748
`
`\
`
` xx \
`
`0009
`
`0009
`
`

`

`Feb. 8, 2000
`
`Sheet 8 0f 14
`
`6,022,748
`
`
`
`US. Patent
`
`
`
`ABSORBANCB(AU)
`
`FIGURE 8
`
`0010
`
`0010
`
`

`

`US. Patent
`
`Feb. 8,2000
`
`Sheet 9 0f 14
`
`6,022,748
`
`FIGURE 9
`
`
`
`1-butanol
`
`1-hexanol
`
`l-octanol
`
`CH2C12
`
`CCM
`
`CHC13
`
`toluene
`
`benzene
`
`cyclohexane
`
`:1ieLhylether
`
` Colometnc
`mS8'5‘8E8388
`
`Re5ponse(‘79)
`
`
`
`Solvent
`
`Saturation
`concentration in
`water (191%)
`
`Sa! vent
`
`Saturation
`commune“
`in water (wr%)
`
`0011
`
`0011
`
`

`

`U.S. Patent
`
`Feb. 8, 2000
`
`Sheet 10 of 14
`
`96 Colodni‘etfic
`
`Concentratioa (wt %) of 1-banol in water
`
`FIGURE 10
`
`35
`
`30
`
`z;
`
`20
`
`15
`
`-1
`
`0
`
`I
`
`2
`
`3
`
`i
`
`5
`
`6
`
`7
`
`3
`
`0012
`
`0012
`
`

`

`US. Patent
`
`Feb. 8, 2000
`
`Sheet 11 of 14
`
`6,022,748
`
`FIGURE 11
`
`I>
`
`700
`
`800
`
`
`
`[au]
`absorbance
`[au]
`absorbance
`
`400
`
`500
`
`500
`
`03
`
`wavelength [nm]
`
`400
`
`500
`
`500
`
`700
`
`800
`
`wavelength [nm]
`
`0013
`
`0013
`
`

`

`US. Patent
`
`Feb.8,2000
`
`Sheet 12 of 14
`
`6,022,748
`
`FIGURE 12
`
`A
`
`Wavelength (m)
`
`(AU)
`
`400
`
`5m
`
`m
`
`m
`
`800
`
`Wavelengthfnm)
`
`5‘
`s.
`
`Ee9
`
`..[J
`<
`
`Absurhance
`
`0014
`
`0014
`
`

`

`
`
`US. Patent
`
`Feb. 8, 2000
`
`Sheet 13 0f 14
`
`6,022,748
`
`FIGURE 13
`
`Add‘cfthupc
`
`NacrdibdGupz
`
`finicfladGup:
`
`it’ll."ll
`0
`
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`
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`
`2.4 (PDA)
`
`25 (GUI-FDA)
`
`2.5 (EA-FDA)
`
`2.7
`
`2.8 (EDA-FDA]
`
`2.9 (PEG-FDA)
`
`2.10
`
`1.1!
`
`2.12
`
`2.13
`
`0015
`
`
`
`0015
`
`

`

`US. Patent
`
`Feb. 8, 2000
`
`Sheet 14 0f 14
`
`6,022,748
`
`FIGURE 14
`
`u
`
`1.1
`
`1 . 1 c
`
`éI:rINiaV’\/\‘K‘k\/\u’\l‘vfi\/\J'
`
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`
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`
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`
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`
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`
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`
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`
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`tea-x
`
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`
`0016
`
`0016
`
`

`

`6,022,?48
`
`2
`that occur in immobiliyed hiopolymeric material in response
`to selective binding of analytes to their surface.
`The present
`invention provides various methods and
`compositions useful for the detection of analytes.
`In one embodiment, the present inventionprovides meth-
`ods for immobilizing biopolymeric material: providing a
`metal oxide, biopolymeric material, an acid, a buffer, and a
`sonicating means; sonicating the metal oxide and the acid
`using the sonicating means to produce a souicated solution;
`adding the buffer to the sonicated solution to produce a
`buffered solution; and adding the biopolyrneric material to
`the buffered solution to produce an organiciinorganic solu-
`tion.
`
`In alternative embodiments of the methods, the present
`invention further comprises the steps of applying the
`organici'inorganic solution to a formation support to produce
`a formed organiciinorganic solution; and gelling the formed
`organicfirtorganic solution to produce an organicfinorganic
`device.
`
`
`
`1
`SOL-GEL MATRICES FOR DIRECT
`COLORIMICTRIC DETECTION OF
`ANALYTES
`
`FIELD OF THE INVENTION
`
`The present invention relates to methods and composi-
`tions for the direct detection of analytcs using color changes
`that occur in immobilized hiopolymeric material in response
`to selective binding of analytes to their surface.
`
`BACKGROUND OF THE INVENTION
`
`A major goal of analer detection research is to develop
`inexpensive,
`fast,
`reliable, and sensitive detectors.
`Unfortunately, the technologies developed to date have only
`met some of these goals, and no single device has sulfi—
`ciently attained a majority of them.
`Classical detection methods such as liquid chromatogra-
`phy (LC), gas chromatography (GET), and supercritical fluid
`chromatography (SFC).
`in combination with mass
`spectrometry, are widely uned and provide accurate identi-
`fication of analytes and quantitative data. However. these
`techniques are time consuming, extremely expensive,
`require sample preconeentration, and are difficult or impos—
`sible to adapt to field use.
`Biosensors (i.e., devices containing biological material
`linked to a transducing apparatus} have been developed to
`overcome some of the shortcomings of the classical analyte
`detection techniques. Many currently used biosensors are
`associated with transducer devices that use photometry,
`fluorimetry, and chemiluminescence; fiber optics and direct
`optical sensing (cg, grating coupler); surface plasmon
`resonance; potentiometric and ampcromctric electrodes;
`field effect
`transistors; picrneleclt’ic sensing; and surface
`acoustic wave (Kramer, J. AOAC Intern. 79: 1245 [1996]).
`However.
`there are major drawbacks to these devices.
`including their dependence on a transducing device, which
`prevents miniaturization and requires a power source. 'I'hese
`disadvantages make such devices too complex, expensive,
`or unmanageable for many routine analer detection appli-
`cations such as field work or home use. Additionally, many
`of these devices are limited by the lack of stability and
`availability of the biological materials (e.g., proteins,
`antibodies, cells, and organelles).
`Immunoassay methods are also used for detecting certain
`types of analytes. In these methods, antibodies are devel—
`oped to specifically bind to a target of interest (eg, an
`analyte). By labeling the antibody (cg, with dye or fluo~
`resccnt or radioactive material), binding of the antibody to
`an analyte can be detected. However, immunoassay methods
`are limited in that
`they require production of antibodies
`against each analyte of interest. Antibodies cannot be gen-
`erated against some types of analytes and their generation
`can be time consuming and expensive.
`The art remains in need of analytc detectors that provide
`the specificity of biosensors but also provide the cost-
`cfliciency, stability, accuracy, reliability, reproducibility, and
`robustness that is lacking from available technologies. In
`particular, development of devices that can be miniaturized
`with controlled shapes and that do not rely on an energy
`source would also be very beneficial, particularly for routine
`field work and home use.
`
`SUMMARY OF THE INVEN'I‘ION
`
`The present invention relates to methods and composi-
`tions for the direct detection of analytcs using color changes
`
`10
`
`15
`
`-
`
`3t)
`
`35
`
`40
`
`45
`
`5f]
`
`55
`
`6(1
`
`()5
`
`0017
`
`inorganic device produced according to any and all of the
`
`the metal oxide comprises
`In preferred embodiments,
`tetrlmethylorthosilieate, although it is contemplated that arty
`material
`that can be used to produce substantially
`transparent, porous glass will he used in the methods of the
`present invention.
`is
`the biopolymeric material
`In some embodiments,
`selected from the group consisting of liposomes,
`films,
`multilayers, braided, lamellar, helical, tubular, and fiber-like
`shapes, solvated rods, solvated coils, and combinations
`thereof.
`
`the biopolymeric material corn-
`In other embodiments,
`prises a plurality of self-assembling monomers selected
`from the group consisting of diacetylcnes, acetylenes,
`alkenes, thiophenes, polythiophenes, imides, acrylamides,
`methacrylates, vinylether, malic anhydride, urethanes,
`allylamines, siloxanes anilines, pyrroles, vinylpyridinium,
`and combinations thereof, although any selfaassemhling
`monomer that will form biopolymer‘ic material is contem-
`plated hy the present invention. In preferred embodiments,
`the diacetylenes are selected from a group consisting of
`5,7-docosadiynoic acid, 10,12-pentacosadiynoic acid, 5,7-
`pentacosadiynoic acid, and combinations thereof.
`In yet
`other embodiments, the sell-assembling monomers contain
`head groups selected from the group consisting of carboxy—
`lic acid, hydroxyl groups, amine groups, amino acid
`derivatives, and hydrophobic groups, although any head
`group that exists or can be synthesized on self-assembling
`monomers is contemplated by the presently claimed inven-
`tion.
`
`In some preferred embodiments, the biopolymeric mate-
`rial t'urther comprises a ligand. In some embodiments, the
`ligand is selected from the group consisting of peptides,
`carbohydrates, nucleic acids, biotin, drugs, chromophorcs,
`antigens, chelau'ng compounds, molecular
`recognition
`complexes,
`ionic groups, polymerizable groups,
`linker
`groups, electron donors, electron acceptor groups, hydro-
`phobic groups, hydrophilic groups, receptor binding groups,
`antibodies, and combinations thereof, although any ligand
`that can be linked to, or associated with, hiopolymcric
`material is contemplated by the present invention.
`In some preferred embodiments,
`the acid comprises
`hydrochloric acid, while in other preferred embodiments, the
`buffer comprises 3—[ N—Morpholino]propanesulfonic acid, In
`other embodiments, the sonicating is conducted at a tem-
`perature from 0° C. to 20° C.
`The present
`invention further provides an organic}
`
`0017
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`

`

`
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`6.022348
`
`3
`methods described above. In addition, the present invention
`provides biopolyTneric material encapsulated in sol-gel
`glass. In some embodiments. the glass comprises tetram-
`ethylorthosilicate.
`In some preferred embodiments, the biopolymeric mate-
`rial encapsulated in sol~gel glass is selected from the group
`consisting of liposomes,
`films, multilayers, braided,
`lamellar, helical,
`tubular, and fiber-like shapes, solvated
`rods, solvated coils, and combinations thereof. In particu-
`larly preferred embodiments,
`the biopolyrneric material
`comprises Self-assembling monomer selected from the
`group consisting of diacetylertes. acetylenes, alkenes,
`thiophenes, polythiophenes,
`imides, acrylamides,
`methacrylates, vinylether, maiic anhydride. urethanes.
`allylamines, siloxanes anilines, pyrroles, vinylpyridinium,
`and combinations thereof, although any self-assembling
`monomer that will form biopolymeric material is contem-
`plated by the present invention. In preferred embodiments,
`the diacetytenes are selected from a group consisting of
`5,7-Clooosadiyttoic acid. 10,12-pentacosadiynoic acid, 5,7— '
`pentacosadiynoic acid. and combinations thereof.
`In yet
`other embodiments, the self-assembling monomers contain
`head groups selected from the group consisting of carboxy-
`lic acid. hydroxyl groups. amine groups, amino acid
`derivatives. and hydrophobic groups, although any head —
`group that exists or can be synthesized on self-assembling
`monomers is contemplated by the presently claimed inven-
`tion.
`
`It]
`
`15
`
`In some preferred embodiments, the biopolymeric mate-
`rial encapsulated in sol-gel glass further comprises a ligand.
`In some embodiments, the ligand is selected from the group
`consisting of peptides, carbohydrates, nucleic acids, biotin,
`drugs, chromophores, antigens, chelating compounds,
`molecular recognition complexes, ionic groups, polymeriz-
`able groups, linker groups, electron donors. electron accep—
`tor groups, hydrophobic groups, bydrophilic groups, recep-
`tor binding groups. antibodies, and combinations thereof,
`although any ligand that can be linked to, or associated with,
`biopotymeric material is contemplated by the present inven-
`tion.
`
`invention further provides methods for
`The present
`detecting analytes: providing biopolymeric material encap-
`sulated in sol—gel glass, a detection means, and one or more
`analyte; exposing the hiopolymeric material encapsulated in
`sol-gel glass to the analer to produce a response; and
`detecting said response using the detection means.
`In preferred embodiments,
`the glass comprises
`tetramethylorthosilicate, although it is contemplated that any
`material
`that can be used to produce substantially
`transparent. porous glass will be used in the methods of the
`present invention.
`is
`the biopolymerie material
`In some embodiments,
`selected from the group consisting of liposomes,
`films,
`multilayers, braided, lamellar, helical, tubular, and fiber-like
`shapes, solvated rods, solvatcd coils, and combinations
`thereof.
`
`In other embodiments, the biopolymeric material corn-
`prises a plurality of self-assemblng monomers selected
`from the group consisting of diacetylenes. acetylenes,
`alkenes,
`thiophcnes, polythiophenes, imidcs, acrylamides,
`methacrylates, vinylether, malic anhydride, urethanes,
`allylamines, siloxanes anilines, pyrroles, vinylpyridinium,
`and combinations thereof, although any self-assembling
`monomer that will form biopolymeric material is contem-
`plated by the present invention. In preferred embodiments,
`the diacetylenes are selected from a group consisting of
`
`3t)
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`35
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`5f]
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`55
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`6t!
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`()5
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`0018
`
`4
`5,7-docosadiynoic acid, 10,12-pentacosadiynoic acid, 5,7-
`pentacosadiynoic acid, and combinations thereof.
`In yet
`other embodiments, the self-assembling monomers contain
`head groups selected from the group consisting of carboxy-
`lic acid, hydroxyl groups, amine groups, amino acid
`derivatives, and hydrophobic groups, although any head
`group that exists or can be synthesized on self~assembling
`monomers is contemplated by the presently claimed inven-
`tion.
`
`In some preferred embodiments, the biopolymer'ic mate-
`rial further comprises a ligand. In some embodiments, the
`ligand is Selected from the group consisting of peptides,
`carbohydrates. nucleic acids. biotin. drugs. chromophores,
`antigens, chelaling compounds, molecular
`recognition
`complexes,
`ionic groups, polymerizablc groups,
`linker
`groups, electron donors, electron acceptor groups, hydro-
`phobic groups, hydmphilic groups, receptor binding groups,
`antibodies, and combinations thereof, although any ligand
`that can be linked to, or associated with, biopolymeric
`material is contemplated by the present invention.
`In some embodiments, the analytc is selected from the
`group consisting of small molecules. pathogens, bacteria.
`membrane receptors, membrane fragments, enzymes, drugs,
`antibodies. and combinations thereof, although any analytc
`that can be detected through its interaction with a ligand or
`the biopolymeric material is contemplated by the present
`invention.
`
`the biopolymerie material
`In yet other embodiments,
`encapsulated in sol-gel glass comprises a badge.
`In preferred embodiments, the detection means is selected
`from the group consisting of visual inspection. spectrometry,
`optical
`fiber, quartz oscillators, electrode surfaces, and
`Scintillation, although any detection means that provides
`analysis of the presence of an analer is contemplated by the
`present invention.
`In some embodiments. the response is used as a competi-
`tive binding measurement to quantitate and characterize the
`presence of natural binding sites. In other embodiments, the
`biopolymeric material encapsulated in sol-gel glass corn-
`pt'ises an array.
`DESCRIPTION OF THE FIGURES
`
`FIG. I shows a diagram of a receptor-linker—ligand com-
`plex where compound 1 shows a sialic acid group attached
`to 10,12-pentacosadiynoie acid (compound 2} through a
`linker group.
`FIG. 2 shows a visible absorption spectrum of “blue
`phase” DCDA Iiposomes entrapped in sol-gel glass.
`FIG. 3 shows a visible absorption spectrum of "red phase"
`DCDA liposomes entrapped in sol-gel glass
`FIG. 4 shows a diagram of PDA with alterations in the
`position of the diacetylene group from 10,12 to 5,7—
`pentacosadiynoic acid.
`FIG. 5 shows a visible absorption spectrum of “blue
`phase," sialic acid-linked DCDA lipasomes entrapped in
`sol-gel glass.
`FIG. 6 shows a visible absorption spectrum of "red
`phase." sialic acid-linked DCDA liposomes entrapped in
`sol-gel glass.
`FIG. 7 shows a representation of the porous structure of
`sol-gel-prepared material acting as a size selective barrier.
`FIG. 3 shows a visible absorption spectrum ofdiacetylene
`material exposed to l-octanol.
`FIG. 9 shows a bar graph indicating calorimetric
`responses of FDA material
`to various VOCs and a table
`showing the concentration of the VOCs.
`
`
`
`0018
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`

`

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`
`6,022,?48
`
`5
`FIG. ll] shows a graph comparing colorimetric responses
`of l-bulanol to the concentration of l-butanol.
`
`FIG. Lt shovvs a visible absorption spectrum of sialic
`acid-linked PDA before (solid line) and after (dashed line)
`exposure to influenza virus for: A) bluetpink form; and B)
`purpler‘orange form material.
`FIG. 12 shows a visible absorption spectrum of sialic
`acid—linked PDA; A) before; and B) after exposure to
`cholera toxin.
`FIG. 13 shows derivations of FDA for use in detection
`arrays.
`
`FIG. 14 shows the organic synthesis of compound 2. [0.
`GENERAL DESCRIP'I'ION OF THE. 1N VEN'l'ION
`
`ll]
`
`15
`
`The present invention relates to methods and composi—
`tions for the direct detection of analytes using color changes
`in immobilized biopolymeric material that occur in response
`to selective binding of analytcs to their surface. In particular,
`the present invention provides methods and compositions .
`related to polymerized biological materials immobilized in
`porous glass that undergo conformational changes when
`exposed to analytes. producing a detectable color change.
`although other immobilization means are also contemplated.
`The present invention provides for the direct detection of the _
`presence of a wide range of analytes by changes in color,
`including, but not limited to, small molecules, pathogens,
`bacteria, membrane receptors, membrane fragments, vola-
`tile organic compounds (VOCs), enzymes, drugs,
`antibodies, and other relevant materials. Results can be
`interpreted by an untrained observer, and the methods can be
`conducted under ambient conditions, making them ame-
`nable to numerous uses including, but not limited to, home
`testing diagnostics, detection of air-borne or water-borne
`pathogens for military applications, doctor’s office or point
`of care testing, and many other applications. The present
`invention provides analer detecting technology that does
`not require an energy source and is cost-efficient, stable,
`accurate, reliable, consistent, and robust and can be pro-
`duced in a variety of shapes and sizes. These enhanced
`qualities provide an idea] basis for use in conjunction with
`fiber optic methods for remote sensing, screening new
`compound libraries (e.g., drug screens), drug testing, water
`supply testing, and any area where a quick and accurate
`colorimetric screen is desired.
`
`3t)
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`
`liposomes and other
`research has found that
`Recent
`lipid-based materials can perform as sensitive optical sen-
`sors for the detection of viruses (see e.g., Reichcrt et al., .I.
`Am. Chem. Soc. 117: 829 [1995]; Spevak et al., J. Am.
`Chem. Soc. 115: 1146 [1993]; and Charych et al., Science
`261: 585 [1993]). These materials exhibit rapid response
`times, selectivity, and optical signals that are easily moni-
`tored. As free floating aggregates in solution, these lipid-
`based detectors show promise as simple assay systems. The
`present
`invention provides embodiments in which these
`materials are used immobilized in sol-gel glass, olIering the
`advantages of further chemical and physical stabilization of
`the material, allowing facile handling, and the opportunity of
`recovery and reuse. To date, this efi‘ort has been frustrated by
`the difficulty in immobilizing lipid assemblies to surfaces. A
`few methods have been developed that overcome some of
`the dillicultics by employing polysaccharides and biocom-
`patible acrylate gels to encapsulate liposomes. However,
`until the development of the present invention, low liposome
`entrapment volume. the inability to immobilize pre-formed
`liposomes, and material instability at elevated temperatures
`were shortcomings yet to be resolved.
`
`5f]
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`6
`The present invention provides a means of immobilizing
`pre-formed liposomes and other biopolymeric material at
`low (cg, 4° C.) to ambient temperature, with high material
`entrapment volume,
`in a porous, robust metal oxide gel
`matrix using the solagel method (See generally, Brinker and
`Scherer, Sol-Gel Science, Academic Press, San Diego
`[1995]). Prior to the present invention, lipid entrapment in
`sol-gel material has not previously been reported. The
`unique properties of sol-gel materials of the present inven-
`tion such as optical transparency, durability, and tailorable
`properties (e.g.. porosity, surface functionalization,
`thin
`films, and bulk materials) provide an ideal material for
`sensor applications.
`Sol-gel encapsulation not only provides an excellent way
`to immobilize liposomes and other biopolymeric material,
`but the optical clarity of the metal oxide gel also makes it
`ideal for optical sensor applications. This unique composite
`can easily be applied to surfaces and cast into any shape
`desired, allowing configuration to most any sensor platform.
`The robust nature of the sol-gel material converts the
`biopolymeric material based assays into sensor materials
`that alford good portability, handling, durability, and
`improved storage life (Les shelf life) while maintaining
`sensitivity. In addition. the metal oxide gel’s porous struc-
`ture and ionic surface can be tailored to provide a primary
`screening mechanism and preconcentrator for selective rec-
`ognition and sensing of target analytcs. The biopolymcrici
`sol-gel material is a unique class of organic-inorganic com-
`posite that offers high matrix stability against microbial
`attack, temperature changes, and physical stress as opposed
`to polysaccharide and acrylate gels. The ambient tempera-
`ture at which gel formation takes place and the biologically
`inert metal oxide matrix allows a broad range of biopoly~
`meric material and protein-entrained-biopolymerie material
`to be immobilized.
`
`Thus. the present invention provides methods and com-
`positions that
`fulfill many of the goals of the analytc
`detection field and overcomes many of the disadvantages of
`currently available technologies (e.g., classical methods,
`biosensors, and immunassays).
`The present
`invention provides significant advantages
`over previously Used bios-tensors, as the embodiments of the
`preSent invention are not dependent upon transducing tech-
`nologies. Many proposed biosensors cannot be used because
`of difficulties in transducirtg the molecule recognition event
`into a measurable signal. Additionally, the transducers of
`currently developed devices add cost, create a requirement
`for a power source, are more dillicult to use by untrained
`personnel. and are limited in terms of miniaturization and
`portability. Also, many biosensors do not display the long
`term stability and robustness of the presently claimed inven-