(12) INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT)
`
`(19) World Intellectual Property Organization
`International Bureau
`
`11111111111111111111111111111111111111111111111111111111111111111111111111111111
`
`(43) International Publication Date
`27 September 2001 (27.09.2001)
`
`PCT
`
`(10) International Publication Number
`WO 01/71317 A1
`
`G01N 21/00,
`(51) International Patent Classification7:
`21/01, 21/17, 31/20, 33/544, 33/538, 33/567, 33/537,
`33/543, 33/53, 33/546, 33/552, C12M 1/00, C12N 1/00,
`1/20, ll/00, C12Q 1/68, C07H 21/04
`
`(21) International Application Number: PCT/US01/08790
`
`(22) International Filing Date: 20 March 2001 (20.03.2001)
`
`(25) Filing Language:
`
`(26) Publication Language:
`
`English
`
`English
`
`(74) Agents: AMERNICK, Burton, A. et al.; Connolly Bove
`Lodge & Hutz, P.O. Box 19088, Washington, DC 20036
`(US).
`
`(81) Designated States (national): AE, AG, AL, AM, AT, AU,
`AZ, BA, BB, BG, BR, BY, BZ, CA, CH, CN, CR, CU, CZ,
`DE, DK, DM, DZ, EE, ES, Fl, GB, GD, GE, GH, GM, HR,
`HU, ID, IL, IN, IS, JP, KE, KG, KP, KR, KZ, LC, LK, LR,
`LS, LT, LU, LV, MA, MD, MG, MK, MN, MW, MX, MZ,
`NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, SL, TJ, TM,
`TR, TT, TZ, UA, UG, US, UZ, VN, YU, ZA, ZW.
`
`(30) Priority Data:
`60/190,091
`
`20 March 2000 (20.03.2000) US
`
`(71) Applicant (for all designated States except US): ANA(cid:173)
`LYTICAL BIOLOGICAL SERVICES, INC. [US/US];
`Cornell Business Park 701-4, Wilmington, DE 19801 (US).
`
`(84) Designated States (regional): ARIPO patent (GH, GM,
`KE, LS, MW, MZ, SD, SL, SZ, TZ, UG, ZW), Eurasian
`patent (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), European
`patent (AT, BE, CH, CY, DE, DK, ES, Fl, FR, GB, GR, IE,
`IT, LU, MC, NL, PT, SE, TR), OAPI patent (BF, BJ, CF,
`CG, CI, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG).
`
`(72) Inventors; and
`(75) Inventors/Applicants (for US only): REPPY, Mary, A.
`[US/US]; 811 N. Franklin Street, Wilmington, DE 19806
`(US). SPORN, Sarah, A. [US/US]; 204 W. Longspur
`Drive, Wilmington, DE 19808 (US). SALLER, Charles,
`F. [US/US]; 11812 Paseo Lucido #2011, San Diego, CA
`92128 (US).
`
`Published:
`-
`with international search report
`
`For two-letter codes and other abbreviations, refer to the "Guid(cid:173)
`ance Notes on Codes and Abbreviations" appearing at the begin(cid:173)
`ning of each regular issue of the PCT Gazette.
`
`iiiiiiii
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`---iiiiiiii
`------
`
`iiiiiiii
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`iiiiiiii ----
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`,..-.! <
`
`t"--(cid:173)
`,..-.1
`~
`,..-.!
`..._
`t'-----------------------------------------------------------------------------
`,..-.! (54) Title: METHOD FOR DETECTING AN ANALYTE BY FLUORESCENCE
`0
`0 (57) Abstract: Two-dimensional and three-dimensional arrays of a polydiacetylene backbone having a substrate incorporated are
`> used in chemical sensing methods to detect the interaction of an analyte with the substrate by monitoring the change in the fluores(cid:173)
`~ cence of the array.
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`METHOD FOR DETECTING AN ANAL YTE BY FLUORESCENCE
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`DESCRIPTION
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`5
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`Technical Field
`
`The present invention relates to a method for detecting the presence of an analyte
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`in a sample, and more particularly, to a method involving the monitoring of the change in
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`fluorescence. According to the present invention, an array incorporating a
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`10
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`polydiacetylene backbone with a substrate incorporated in the array is employed.
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`Background of the Invention
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`Polydiacetylenes are conjugated polymers with backbones of alternating double
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`15
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`and triple bonds formed from the 1 ,4-addition polymerization of 1 ,3-diacetylenes
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`(Figure!). Polydiacetylenes generally absorb well in the visible region of the spectrum,
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`and hence are highly colored, ranging from blue to yellow. There has been intense
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`interest in the non-linear optic properties ofpolydiacetylenes and extensive study has
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`been made of both the salvo-chromic properties of solubilized polydiacetylenes and the
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`20
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`thermo-chromic properties of polydiacetylene films and single crystals. It is well known
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`that to form polydiacetylene, the diacetylene monomers must be in an ordered packing to
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`allow the polymerization to occur. It seems to be generally accepted, though the
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`inventors are not bound herein, that disruption of the packing of the side chains can affect
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`the conjugation length of the backbone, and hence the chromic properties.
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`25
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`Diacetylene monomers have been used to form various ordered systems, including
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`crystals, liquid crystals, liposomes and films that were then polymerized to form the
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`polymer. Liposomes have been made from monomers with two diacetylene chains and
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`polar head groups (such as phosphotidylcholines, and its analogues) and from monomers
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`30 with single diacetylene chains. The liposomes can be polymerized with UV light or y(cid:173)
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`radiation. Monomer films have been formed by Langmuir Blodgett methods or cast from
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`solvents and then also polymerized with UV light or y-radiation. The choice of monomer
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`structure, conditions of liposome or film formation, and polymerization conditions all
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`affect the conjugation length of the polydiacetylene backbone, and hence the color of the
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`system. Upon heating, these polymerized systems can undergo a change in the effective
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`conjugation length, from the longer length forms (blue and purple) to the shorter length
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`forms (red and yellow). This change has been attributed to the side-chains moving and
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`5
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`repacking upon being heated. Soluble polydiacetylenes show solvo-chromic behavior
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`and polydiacetylene films often change color upon exposure to solvent vapors.
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`Polydiacetylene films and liposomes formed from diacetylene surfactants also often
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`change color with change in pH. In the case of the packed polymer arrays that form the
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`films and liposomes, it is generally accepted that changes in the environment that affect
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`10
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`the organization and packing of the side chains coming off the conjugated backbone can
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`affect the conjugation length and hence the chromic and electronic properties of the
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`polymer.
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`These polydiacetylene films and liposomes have been suggested for chromogenic
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`15
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`assays that depend upon color change (Charych et al, US Patent 6,001,556; Charych et al,
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`US Patent 6,180,135; Charych et al, US Patent 6,080,423; Charych, US Patent 6,183,772;
`
`Charych et al, US Patent 6,022,748). It has been hypothesized by Charych (Okada S. et
`
`al, Ace. Chem. Res., 1998, 31, 229-239) that binding to a ligand incorporated in a blue
`
`polydiacetylene films or liposomes perturbs the side chains of the polydiacetylene and
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`20
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`hence the conjugation length of the polydiacetylene and changes the color of the film or
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`liposomes to red. The color change is proposed to be measured either by eye or by a
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`UV NIS spectrophotometer and comparison of the absorbance at a wavelength above
`
`600nm and the absorbance of a wavelength below 600nm.
`
`25
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`The phenomenon of fluorescence is distinct from the absorbance properties that
`
`give systems their color. In order to be fluorescent, the system must absorb one
`
`wavelength of light and then emit another. Upon absorbing the light, the system is
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`excited to a higher energy state. It can then return to the ground state by a variety of
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`mechanisms, most of which do not lead to fluorescence. These alternative, non-
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`30
`
`radiative, mechanisms for returning to the ground state lead to many strongly absorbing
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`species to be non-fluorescent, and makes the prediction of which species will be
`
`fluorescent a difficult task and therefore not apparent to those skilled in the art.
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`For instance, while some organic systems with extended conjugation
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`exhibit fluorescence, many more do not.
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`Along these lines, generally species absorb light in the ultraviolet and
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`5
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`visible ranges. The ultraviolet wavelength range is approximately 190nm-380nm; the
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`visible light range runs from approximately 380nm to 800nm. Upon absorption of the
`
`light the species move to a higher energy electronic excited state. What happens then
`
`determines if the species is fluorescent. If a species absorbs light at one wavelength, is
`
`excited to a higher energy state, and then emits light at a different wavelength and returns
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`10
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`to the ground state, it is fluorescent (or phosphorescent). For fluorescence to occur the
`
`excited species must be capable of emitting light; generally for the fluorescence to be
`
`measurable the emitted light must be at a different wavelength than the excitation. The
`
`Stokes shift is the difference between the excitation and emission wavelengths. Most
`
`species that absorb light are not capable of light emission; they return to the ground state
`
`15
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`by a variety of non-radiative mechanisms. Furthermore, fluorescent species often absorb
`
`wavelengths of light that do not cause fluorescence, as well as absorbing wavelengths that
`
`do cause fluorescence. In short, absorbance of light is necessary for fluorescence but
`
`does not guarantee it.
`
`20
`
`On the other hand, color is an absorbance property; the colors we see are
`
`related to the wavelengths oflight that the species is absorbing. For example if the
`
`species absorbs light primarily at 650 nm, we will see it as blue, while if it absorbs
`
`primarily at 550 nm, we will see it as red. Color arises from absorbance of light in the
`
`visible range. Most colored species are not fluorescent. If a colored species is
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`25
`
`fluorescent, it will normally appear one color, but when it is excited with the appropriate
`
`wavelength, it will glow with the color of the emitted light. For example, a fluorophore
`
`may look like an orange powder, but glow green under a UV lamp.
`
`Polydiacetylenes can show fluorescence. However, their ability to fluoresce is
`
`30
`
`dependent on the structural form and organization of the polymers (particularly the
`
`conjugation length and aggregation state), whether in solution, a film, or formed into
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`liposomes or other three-dimensional structures.
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`It is known that polydiacetylene films have an intrinsic fluorescence when
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`produced in the red or yellow form, and are not fluorescent (by conventional
`
`measurements) when the film is made in the blue form (Yasuda A. et al, Chem. Phys.
`
`Lett., 1993, 209(3), 281-286). This fluorescent property of the films has been used for
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`5 microscopic imagining of film domains and defects.
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`Ribi et al have suggested two sensors using polydiacetylene film fluorescence.
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`The first sensor (Saul et al, U.S. Patent 5,415,999 and US Patent 5,618,735) uses a red,
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`fluorescent, polydiacetylene film layered with a fluorescence modulation reagent non-
`
`1 0
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`covalently associated with the film that modulates the measured emission of the film, e.g.
`
`by absorbing the emitted light, in the presence of an analyte. The fluorescent state of the
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`film does not change during the assay; rather the emission is obscured or revealed by the
`
`action of the fluorescence modulation agent. The second suggested sensor (Ribi, U.S.
`
`Patent 5,622,872) uses a film of specific composition for detection of an analyte by
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`15
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`change in the fluorescence of a film of this composition. The films in the detection
`
`method claims comprise a polymerized film, polymerized from diacetylene monomers of
`
`the defined formulation (A)a(D)aCx(C C)zCyLB wherein A is a functional group used to
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`link the film to an underlying substrate, a is 0 or 1, Cis carbon, x andy are 1 or greater
`
`and (x+y) is in the range of 4-32, D and L are bond or linking groups and B is a specific
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`20
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`binding member which binds to a specific analyte, one terminus of each monomer is
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`proximal to the underlying substrate and the other terminus comprising B (i.e. the film is
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`a mono-layer with every polydiacetylene side-chain either terminating in proximity to the
`
`underlying substrate, or in a binding member). Neither Ribi nor others, to our knowledge,
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`have suggested detection of analytes using three-dimensional arrays of polydiacetylenes
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`25
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`(e.g. liposomes or tubules) and measuring the change in fluorescence arising from the
`
`interaction of the analyte and the polydiacetylene three-dimensional array.
`
`Summary of Invention
`
`30
`
`The present invention provides a sensing method that measures fluorescent
`
`changes in polydiacetylene films as they convert from the non-fluorescent form
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`(generally blue or purple) to the fluorescent forms (generally red to yellow). More
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`particularly, the present invention provides for the detection of an analyte in a sample,
`
`which comprises contacting the sample to be tested with a two-dimensional or three(cid:173)
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`dimensional array of a polydiacetylene backbone with substrate (e.g. a ligand or reactive
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`substrate) covalently or non-covalently incorporated. The two-dimensional arrays of the
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`5
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`present invention, i.e. films, comprise polymerized diacetylene two-dimensional arrays
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`wherein no more that 90% of the diacetylenes terminate in groups that will bind
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`specifically to the analyte (i.e. specific binding members). The ligand or reactive substrate
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`has direct affinity for the analyte or can function as a binder to the analyte or can undergo
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`a chemical or biological reaction or process with the analyte. The change in fluorescence
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`10
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`is measured or detected to indicate the presence of the analyte.
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`The two-dimensional and three-dimensional polydiacetylene arrays of the present
`
`invention either change their fluorescent state upon interaction with the analyte, or change
`
`their fluorescence polarization upon interaction with the analyte. The change from the
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`15
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`non-fluorescent form to the fluorescent forms ofthe polydiacetylene of the array, occurs
`
`due to interaction of the analyte of interest with the substrate incorporated in the array.
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`Measuring the increase in the fluorescence of the array as the array changes from non(cid:173)
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`fluores·cent to fluorescent can monitor these interactions as a new detection method,
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`which is more sensitive than monitoring color change. This increase in sensitivity is
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`20
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`necessary for the detection system to have actual utility as a sensor for many applications.
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`It is also possible to begin with the array partially in the fluorescent form and measure the
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`increase in fluorescence as the interaction with the analyte takes place. The arrays may
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`also contain auxiliary fluorescence species ( fluorophores ), and the fluorescence of these
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`fluorophores may be modulated by the polydiacetylene array and thus change as the
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`25
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`polydiacetylene changes its fluorescent form.
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`The assay method of the present invention makes possible a continuous
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`monitoring of the binding of the analyte. The analyte is added to the array and the
`
`fluorescence can be measured over time. Since no wash steps are required, the method of
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`30
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`the present invention is relatively simple and inexpensive to carry out.
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`Summary of Drawings
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`monomers.
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`Figure 1 illustrates the formation ofpolydiacetylene from diacetylene
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`PCT/USOl/08790
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`Figure 2 illustrates the fluorescence spectrum of the non-fluorescent and
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`5
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`fluorescent form of a polydiacetylene liposome employed in the process of the present
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`invention.
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`Figure 3 illustrates the changes in absorbance and fluorescence of
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`polydiacetylene liposomes as they convert from one form to the other.
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`10
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`Figure 4 illustrates the fluorescence ofpolydiacetylene liposomes with and
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`without fluorophores incorporated.
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`Figure 5 illustrates the fluorescence spectra of a polydiacetylene array with
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`15
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`fluorophores and anti-chlamydia antibodies incorporated, coated onto a nanoporous
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`membrane, before and after exposure to chlamydia elementary bodies.
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`Best and Various Modes for Carrying Out Invention
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`In order to facilitate an understanding of the present invention the
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`20
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`following definitions which are used herein are presented :
`
`• Substrate: A chemical or biological entity
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`• Reactive substrate: a substrate that can undergo a chemical or biological
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`reaction or process
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`25
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`• Ligand: An entity or substrate that can preferentially interact with an
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`analyte by a covalent or non-covalent binding interaction
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`• Analyte: Any entity (physical, chemical or biological) that is to be
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`detected
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`• Non-fluorescent form: Low over-all fluorescence above 500 nm of the
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`30
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`polydiacetylene as compared to its corresponding fluorescent form.
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`The two-dimensional and three-dimensional arrays employed according to
`
`the present invention comprise a polydiacetylene backbone. Incorporated in the
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`polydiacetylene array is a ligand or reactive substrate that has direct affinity for the
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`analyte or which can function as a competitive binder to the analyte or can react with the
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`analyte. The ligand or reactive substrate is lipophilic or contains lipophilic portions or is
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`conjugated to a non-polar or polar species rendering the overall conjugate lipophilic. The
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`5
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`lipophilic or non-polar portions may contain diacetylenes or other polymerizable groups
`
`but it is not necessary. The arrays are prepared by polymerization of precursor
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`diacetylene arrays. The diacetylene precursor two and three-dimensional arrays may also
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`contain non-substrate species that are not diacetylenes. The two-dimensional arrays are
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`formed from polymerization of two-dimensional arrays of diacetylenes wherein the arrays
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`1 0
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`are comprised of not more than 90% of diacetylenes that terminate in functional groups
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`that are specific binding groups for the analyte (i.e. that are members of specific binding
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`pair with the analyte). More typically the two-dimensional precursor diacetylene arrays
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`are comprised of 60% or less of diacetylenes that terminate in specific binding groups for
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`the analytes.
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`15
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`The polydiacetylene backbones employed according to the present invention are
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`known and need not be described herein in any detail and can range from being
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`oligiomeric (from the reaction of three or more monomers) to polymeric. For example
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`see U.S. Patent 6,001,556 to Charych et al, disclosures of which are incorporated herein
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`20
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`by reference. The selection of a desirable polydiacetylene for a particular application can
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`be determined by persons skilled in the art and aware of the disclosure in this application
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`without undue experimentation.
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`In one embodiment of the invention the ligand or reactive substrate is
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`25
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`bound to the polydiacetylene backbone, preferably via a linear structural linker. The
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`linear structure linkers typically have two terminal ends, wherein the linkers are attached
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`to their first terminal ends to the ligand or reactive substrate moieties, a portion of the
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`middle of the linker is incorporated in the polydiacetylene backbone, and the remainder
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`including the second terminal end becomes a side chain of the polydiacetylene. It is also
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`30
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`possible for the second terminal end to be incorporated in the polydiacetylene backbone.
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`In this embodiment the polydiacetylene is formed from polymerizing a three-dimensional
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`or two-dimensional array of diacetylenes comprising a mixture of diacetylenes and of
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`diacetylenes with the ligand or reactive substrate moieties attached covalently to the
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`polymerizable diacetylene group. The array may also contain non-diacetylene species.
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`In another embodiment of the present invention the ligand or reactive
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`5
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`substrates are incorporated in the polydiacetylene array without a covalent attachment
`
`between the polydiactylene backbone and the ligand or reactive substrate. The array may
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`also contain other non-diacetylene species.
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`Also, side chains with ordering head groups are typically bound to the
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`10
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`polydiacetylene backbone. The head groups are typically polar.
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`The polydiacetylene backbone is preferably in its non-fluorescent form.
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`The arrays are formed by polymerizing arrays of diacetylene monomers.
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`15
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`The typical monomers are single or multi-tailed diacetylene surfactants with polar head
`
`groups. More typically used are single or his-tailed diacetylene surfactants with polar
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`head groups. The invention is not dependant on use of any specific diacetylene
`
`surfactant, tail structure, or polar head group, but can be used with any diacetylene
`
`monomer that can be polymerized to give polydiacetylene in its non-fluorescent form or
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`20
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`polydiacetylene in a fluorescent form that is converted to a fluorescent form having a
`
`different emission and preferably a higher emission upon interaction with the analyte.
`
`Materials typically used as head groups in the present invention include, but are
`
`not limited to: carboxylic acids, carboxylate salts, amides, ethanol amide, amines,
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`25
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`ammoniums, imines, imides, alcohols, carbamates, carbonates, thio-carbamates,
`
`hydrazides, hydrazones, phosphates, phosphonates, phosphoniums, thiols, sulfates,
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`sulfonates, sulfonic acids, sulfonic amines, sulfonamides, amino acids, peptides, nitro(cid:173)
`
`functionalized moieties, carbohydrates, choline, ethylene glycol, oligiomeric ethylene
`
`glycol, poly( ethylene glycol), propylene glycol, oligiomeric propylene glycol, and
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`30
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`poly(propylene glycol), and combinations thereof.
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`The ligands and reactive substrates employed in the present invention can be of a
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`wide variety of materials and are defined herein as species incorporated in the
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`polydiacetylene arrays that interact with analytes. The main criterion is that the ligand or
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`reactive substrate has an affinity for the analyte of choice. The ligand or reactive
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`substrate may be of a broad range, such as when a class of materials is to be assayed.
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`Appropriate ligands include, but are not limited to: peptides, proteins, antibodies,
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`5
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`antibody fragments, antigens and the epitopes thereof, enzymes, carbohydrates, nucleic
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`acids, amino acids, crown ethers, cage compounds, small molecules, organo-metallic
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`compounds, salts, or any biological or organic compound or species which bind to
`
`analytes. Appropriate reactive substrates include phospholipids, peptides, proteins,
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`protease substrates, kinase substrates, carbohydrates, nucleic acids, amino acids, or any
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`10
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`biological, organic or organo-metallic compound or species that can undergo a chemical
`
`or biological reaction or process. Analytes include, but are not limited to: membranes,
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`receptors, cells, bacteria, viruses, toxins, proteins, enzymes, proteases, kinases, antigens,
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`antibodies, nucleic acids, small molecules, and any biological, organic or organo-metallic
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`species that can interact with a ligand or reactive substrate.
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`15
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`Some specific ligands include GM 1-ganglioside, sialic acid, serotonin,
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`diactadecyl glycerylether-j3-gluocoside, anti-Salmonella antibodies, anti-Listeria
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`antibodies, anti-Campylobacter antibodies, anti-Chlamydia antibodies, anti(cid:173)
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`Cryptosporidium antibodies, anti Escherica coli antibodies, HIV protease substrate, MAP
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`20
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`kinase substrates, MEK kinase substrates, and hexokinase. Some specific reactive
`
`substrates include dimyristoyl phosphotidylcholine, dipalmitolyl phosphotidylcholine,
`
`peptides containing myelin basic protein residue sequences, peptides containing tyrosine
`
`hydroxylase residue sequences, peptides containing MAP kinase residue sequences, and
`
`phosphatidylinositol-4,5-biphosphonate substrate for Phosphoinositide 3-kinase. Some
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`25
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`specific analytes include influenza virus, cholera toxin, phospholipases such as
`
`phospholipase A2, HIV protease, MAP kinases, MEK kinases, Phosphoinositide 3-
`
`kinases, Salmonella, Listeria, Eschericia coli, Chlamydia, Cryptosporidium, and
`
`Campylobacter.
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`30
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`When the arrays of the present invention are to be secured or anchored to a
`
`support surface, the tails of the lipids can be selected to provide this function.
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`The two-dimensional and three-dimensional arrays of the present invention
`
`can be produced in any number of forms. One of the suitable three-dimensional array
`
`forms that can be produced are liposomes. The liposomes can be formed in a number of
`
`different sizes and types. For instance, it is possible to form the liposomes as simple bi-
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`5
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`layer structures. Additionally, they can be multi-layered in an onion type structure. Their
`
`size can also be varied. A suitable two-dimensional array form that can be produced is a
`
`film. The film can be mono-layered, hi-layered, or multi-layered.
`
`Numerous other shapes can also be produced. Lamellae (Rhodes et al,
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`10
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`Langmuir, 1994, 10, 267-275), hollow tubules and braids (Frankel et al, JAm. Chem.
`
`Soc., 1994, 116, 10057-10069.), crystals, lyotropic and thermotropic liquid crystalline
`
`phases, gels and amorphous structures are among the other shapes that can be formed.
`
`When these assemblies are immobilized they can collectively form even larger constructs.
`
`15
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`Diacetylene liposomes can be converted to tubules before polymerization by
`
`controlled cooling, concentration changes, or addition of ethanol. The tubules can be
`
`photopolymerized to give the non-fluorescing form ofpolydiacetylene, and then used in
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`assays with fluorescence monitoring. Polydiacetylene can also be formed as blue or red
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`gels with a net-work structure of aggregated fibers. Polydiacetylenes have been used in
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`the formation of composite materials, including layering with inorganic clays.
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`Two or three-dimensional arrays of diacetylene long chain monomers are prepared
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`with ligands or substrates incorporated for use in chemical reactions. These ligands or
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`substrates can be functionalized diacetylenes, or .simply be sufficiently lipophilic to mix
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`25 with the diacetylene monomers. The arrays are usually in the form of mono-layer or
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`multi-layer films, or liposomes in solution. Film arrays of diacetylenes or
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`polydiacetylenes may be used in the free form, or supported on glass, ceramic, polymer,
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`paper, metal, or other surfaces. The supports may be porous, including, but not limited
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`to, nano and micro porous membranes. Diacetylene coatings may also be cast onto glass,
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`ceramic polymer, paper,metal or other surfaces and photopolymerized to give the
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`polydiacetylene array described above.
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`Diacetylene and polydiacetylene liposomes may be attached to, supported on, or
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`absorbed in, solids, including, but not limited to: polymers such as polystyrene,
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`polycarbonate, polyethylene, polypropylene and polyfluorocarbons such as Teflon®;
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`silicon chips; beads; filters and membranes; glass; gold; silica; sephadex; sepharose;
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`porous or swelling solids such as polyacrylates and polyacetonitrile; and sol-gels. In the
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`case of diacetylene liposomes and film arrays, they are polymerized after incorporation
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`with or attachment to the solid support.
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`A preferred embodiment of solid supported polydiacetylenes is as an array on
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`nano-porous membranes. A more preferred embodiment is as an array on polycarbonate
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`nano-porous membranes.
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`We have discovered that diacetylene liposomes can be forced in and onto
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`membranes including 100, 200 and 400 nm membranes and photopolymerized to create
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`non-fluorescent polydiacetylene. These membranes are stable at room temperature, in air,
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`and exposed to light, for at least 12 months. The polydiacetylene array exhibits some
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`resistance to abrasion. It seems possible, though the inventors are not bound thereby, that
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`the diacetylene liposomes partially pass through larger pores at the membrane surface
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`before being trapped by smaller pores within. The polydiacetylene arrays can be
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`converted from the non-fluorescent to the fluorescent form when exposed to appropriate
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`reagents.
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`Nanoporous membranes are available in many materials, including:
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`alumina, polyfluorocarbons such as Teflon®, nylon, polycarbonate, glass and
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`polyvinylene difluoride (PVDF), and also in a variety of pore sizes. We envision using
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`any of these membrane types with pore sizes up to about 600nm for preparing solid
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`supported polydiacetylenes. The polydiacetylene-coated membranes can then be
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`incorporated into filter and flow cells, used as swabs or test strips, or adhered to any solid
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`support.
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`One embodiment of the invention uses a filter or flow cell containing a non(cid:173)
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`fluorescent form polydiacetylene array with a substrate incorporated, inion a membrane.
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`A solution of an analyte is passed through the filter or flow cell and then the fluorescence
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`of the membrane is read. The array may contain fluorophores that does not interact
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`optically or electronically with the polydiacetylene. The fluorescence emission of these
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`fluorophores may be monitored separately as an internal calibration standard. The array
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`may also contain fluorophores that do interact optically or electronically or through
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`resonance coupling with the polydiacetylene.
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`The diacetylene 2-dimensional and 3-dimensional structures are
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`photopolymerized with UV light, or y-radiation, to give organized polydiacetylenes with
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`the longer conjugation lengths characterized by absorption maximum in the range of 500-
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`800nm, preferably in the range 600-750nm, and a blue to purple color. The
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`photopolymerization results in creating mainly the non-fluorescing form and therefore
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`exhibiting low overall fluorescence relative to the background. The term "non(cid:173)
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`fluorescent form" as used herein also refers to these polymers which have low overall
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`fluorescence exhibiting a fluorescent signal above 500 nm that is only about 1-3 times
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`that of the background and less than that of the corresponding fluorescent form. Typically
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`the "non-fluorescent form" exhibits a fluorescent emission above 500 nm that is at least
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`about 10% lower and more typically at least about 50% lower than that of the
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`corresponding fluorescent form. Some diacetylene 2-dimensional and 3-dimensional
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`arrays give polydiacetylene in the fluorescent forms upon photopolymerization; these
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`may still be used in assays if interaction with the analyte converts the arrays to a
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`fluorescing form having a different measurable emission and preferably from a lower(cid:173)
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`fluorescing form to a higher-fluorescing form.
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`The fluorescence of the polymerized arrays is measured, they are exposed to the
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`analyte of interest for a sufficient length of time so that either binding or
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`chemical/biological reaction can occur, and then the fluorescence is measured again. The
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`binding, or chemical/biological reaction, causes a conversion of the array from the non(cid:173)
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`fluorescing form to a fluorescing form, characterized by absorption maxima below
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`590nm, and a red, orange, green or yellow color. The polydiacetylene fluorescence of the
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`fluorescent form may be excited by light with wavelengths between 300 and 600nm, and
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`consists of a broad fluorescence above 500nm with one or two maxima though the
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`invention is not bound by these specifics. A rise in the polydiacetylene fluorescence after,
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`or during, exposure to the analytes or species of interest, indicates binding or reaction.
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`The fluorescence can also be measured periodically during the exposure to follow the
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`course of the interaction and elucidate the kinetics.
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`This sensing method can also be used to measure the inhibition of binding to the
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`ligands, or reaction with the substrate, by active compounds. In the case of inhibition
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`measurements the inhibitor, or test compound, is added to the polydiacetylene arrays.
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`The active species, which can either bind to a ligand or react with a reactive substrate, is
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`also added. If the samples show increase in fluorescence similar to the increase in
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`fluorescence shown by control samples without the test compound added this indicates
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`that the potential inhibitor is not suppressing the activity of the active species. If the
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`samples maintain a low level of fluorescence, relative to the control samples, or the
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`fluorescence increases only a fraction of the rise seen in the control samples, this
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`indicates inhibition of the activity of the active species by the test material.
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`The conditions that cause conversion to the fluorescent form also may cause
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`chromic changes th