Inter Partes Review of U.S. Patent No. RE46,817
`Declaration of Kirk S. Schanze, Ph.D. (TFS1002)
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`UNITED STATES PATENT AND TRADEMARK OFFICE
`
`____________________________
`
`BEFORE THE PATENT TRIAL AND APPEAL BOARD
`
`____________________________
`
`THERMO FISHER SCIENTIFIC INC.
`Petitioners
`
`v.
`
`THE REGENTS OF THE UNIVERSITY OF CALIFORNIA
`Patent Owner.
`____________________________
`
`Case IPR2018-01156
`U.S. Patent No. RE46,817
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`
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`
`
`Declaration of Kirk S. Schanze, Ph.D.
`
`
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`
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`
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`TFS1002
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`
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`
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`UC Ex-2003
`Thermo Fisher v. UC Regents
`IPR2018-01347
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`

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`Inter Partes Review of U.S. Patent No. RE46,817
`Declaration of Kirk S. Schanze, Ph.D. (TFS1002)
`
`
`TABLE OF CONTENTS
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`I.(cid:3)
`
`Overview .......................................................................................................... 4(cid:3)
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`II.(cid:3) My background and qualifications .................................................................. 4(cid:3)
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`III.(cid:3) Basis for my opinion ........................................................................................ 6(cid:3)
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`IV.(cid:3) Person of ordinary skill in the art .................................................................. 10(cid:3)
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`V.(cid:3)
`
`State of the art before August 26, 2002 ......................................................... 11(cid:3)
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`A.(cid:3) Overview of Förster resonance energy transfer (FRET) ............................... 11(cid:3)
`
`B.(cid:3)
`
`Chromophores used in FRET ........................................................................ 14(cid:3)
`
`1.(cid:3)
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`2.(cid:3)
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`3.(cid:3)
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`Fluorescent Dyes used as FRET acceptors .................................................... 14(cid:3)
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`Quenchers used as FRET acceptors .............................................................. 15(cid:3)
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`Conjugated polymers as donors in FRET systems ........................................ 17(cid:3)
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`C.(cid:3) Nucleic acid hybridization using FRET was well known by August 2002 .. 27(cid:3)
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`D.(cid:3)
`
`Signal amplification in FRET sensing systems ............................................. 32(cid:3)
`
`VI.(cid:3) The '817 patent and its claims ....................................................................... 38(cid:3)
`
`A.(cid:3) Independent claim 1 ....................................................................................... 46(cid:3)
`B.(cid:3) Dependent claim 3 ......................................................................................... 47(cid:3)
`VII.(cid:3) Meaning of Claim Terms ............................................................................... 47(cid:3)
`
`A.(cid:3) Multichromophore system: ............................................................................ 47(cid:3)
`B.(cid:3) Other claim terms: ......................................................................................... 48(cid:3)
`VIII.(cid:3) The basis of my analysis with respect to obviousness .................................. 48(cid:3)
`
`IX.(cid:3) The Challenged Claims would have been obvious over Cardullo, McQuade
`and LeClerc .................................................................................................... 49(cid:3)
`
`
`
`2
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`Inter Partes Review of U.S. Patent No. RE46,817
`Declaration of Kirk S. Schanze, Ph.D. (TFS1002)
`
`Claim 1 ........................................................................................................... 50(cid:3)
`
`A POSA would have had a reason to combine Cardullo, McQuade, and
`LeClerc ........................................................................................................... 69(cid:3)
`
`A POSA would have had a reasonable expectation of success at arriving at
`the claimed multichromophore system .......................................................... 77(cid:3)
`
`A.(cid:3)
`
`1.(cid:3)
`
`2.(cid:3)
`
`B.(cid:3) Dependent claim 3 ......................................................................................... 86(cid:3)
`
`X.(cid:3)
`
`A.(cid:3)
`
`1.(cid:3)
`
`2.(cid:3)
`
`The Challenged Claims would have been obvious over Cardullo, LeClerc
`and Harrison ................................................................................................... 87(cid:3)
`
`Claim 1 ........................................................................................................... 88(cid:3)
`
`A POSA would have had a reason to combine Cardullo, LeClerc and
`Harrison ......................................................................................................... 98(cid:3)
`
`A POSA would have had a reasonable expectation of success at arriving at
`the claimed multichromophore system ........................................................108(cid:3)
`
`B.(cid:3) Dependent claim 3 .......................................................................................112(cid:3)
`
`XI.(cid:3)
`
`I am not aware of any objective indicia of nonobviousness that would
`change my opinion regarding the obviousness of the challenged claims ...114(cid:3)
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`3
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`Inter Partes Review of U.S. Patent No. RE46,817
`Declaration of Kirk S. Schanze, Ph.D. (TFS1002)
`
`
`I, Kirk S. Schanze, Ph.D., do hereby declare as follows:
`
`I.
`
`Overview
`
`1.
`
`I am over the age of 18 and otherwise competent to make this
`
`declaration. I have been retained as an expert on behalf of Thermo Fisher
`
`Scientific Inc. ("Thermo Fisher"). I understand this declaration is being submitted
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`together with a petition for Inter Partes Review ("IPR") of claims 1 and 3 ("the
`
`challenged claims") of U.S. Patent No. RE46,817 ("the '817 patent") (TFS1001).
`
`2.
`
`I am being compensated for my time in connection with this IPR at
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`my standard legal consultant rate of $375/hr. I have no personal or financial
`
`interest in Thermo Fisher or in the outcome of this proceeding.
`
`3.
`
`In preparing this declaration, I have reviewed the '817 patent
`
`(TFS1001) and considered each of the documents cited therein, in light of the
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`general knowledge in the art before August 26, 2002. I have also relied upon my
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`experience in the relevant art and considered the viewpoint of a person of ordinary
`
`skill in the art ("POSA"; defined in § IV) before August 26, 2002.
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`II. My background and qualifications
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`4.
`
`I am currently the Robert A. Welch Distinguished University Chair in
`
`the Chemistry Department at the University of Texas, San Antonio. A copy of my
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`CV is attached as Exhibit TFS1027.
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`
`
`4
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`Inter Partes Review of U.S. Patent No. RE46,817
`Declaration of Kirk S. Schanze, Ph.D. (TFS1002)
`
`I earned by B.S. in Chemistry from Florida State University in 1979
`
`5.
`
`and my Ph.D. in Organic Chemistry from the University of North Carolina,
`
`Chapel Hill, in 1983. I was a Postdoctoral Research Associate in the Department
`
`of Chemistry at the University of North Carolina, Chapel Hill from 1983-1984. I
`
`was then a Miller Postdoctoral Fellow in the Chemistry Department of the
`
`University of California, Berkley from 1984-1986.
`
`6.
`
`In 1986 I was appointed an Assistant Professor in the Department of
`
`Chemistry of the University of Florida. I became an Associate Professor at Florida
`
`in 1992 and was appointed full Professor in 1997. From 1997 to 2016 I held
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`various Professorship chairs as outlined in my CV. In 2016 I moved to my current
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`position at the University of Texas, San Antonio, where I serve as the Welch
`
`Distinguished Professor.
`
`7.
`
`Throughout my career I have earned various honors and awards,
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`including the Globalization Award from American Chemical Society (ACS) in
`
`2018. I was made a Fellow of the ACS in 2011, and won the Florida Award from
`
`the Florida Section of ACS in 2009. I have also been awarded many Visiting
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`Professor and Lecturer appointments at various universities throughout Asia. A
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`full list of my Honors and Awards is provided in my CV.
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`8.
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`I have also held many professional service positions, such as the Chair
`
`of the Editor Search Committee for the ACS in 2014 and 2015 and I was the Chair
`
`
`
`5
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`Inter Partes Review of U.S. Patent No. RE46,817
`Declaration of Kirk S. Schanze, Ph.D. (TFS1002)
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`of the ACS Publications Editors Conference in San Diego in 2016. I was
`
`Associate Editor of the American Chemical Society (ACS) Journal Langmuir
`
`from 2000 – 2008. Since 2008 I have served as founding Editor in Chief (EIC) of
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`ACS Applied Materials & Interfaces. This is one of the largest technical journals
`
`in the field of materials chemistry and materials science. As EIC, I overview the
`
`review of more than 20,000 papers that are submitted annually for consideration
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`to be published.
`
`9.
`
` My research primarily focuses on work in the fields of conjugated
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`polymers, conjugated polyelectrolytes, luminescence imaging, photochemistry
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`and photophysics and photoresponsive materials. I have been conducting research
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`on conjugated polymers, luminescence imaging and photoresponsive materials
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`since at least 1986. As such, as of August 2002 I met the qualifications for a
`
`person of ordinary skill in the art (POSA) as defined below, and I am qualified to
`
`opine on what the opinion of a POSA would have been at that time.
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`
`
`III. Basis for my opinion
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`10.
`
`In formulating my opinion, I have considered all documents cited
`
`herein, including the following:
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`
`
`6
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`

`

`Thermo
`Fisher
`Exhibit #
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`1001
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`1002
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`1003
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`1004
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`1005
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`1006
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`1007
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`1013
`
`1014
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`1015
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`Inter Partes Review of U.S. Patent No. RE46,817
`Declaration of Kirk S. Schanze, Ph.D. (TFS1002)
`
`
`Description
`
`Bazan et al,. "Methods and Compositions For Detection And
`Analysis of Polynucleotides Using Light Harvesting
`Multichromophores," U.S. Patent No. RE46,817, (filed March 29,
`2017; issued May 1, 2018)
`Declaration of Kirk S. Schanze, Ph.D.
`Cardullo, R., et al., "Detection of nucleic acid hybridization by
`nonradiative fluorescence resonance energy transfer," PNAS 85:
`8790-8794 (1988)
`LeClerc et al., "Detection of Negatively Charged Polymers Using
`Water-Soluble, Cationic, Polythiophene Derivatives," International
`Publication No. WO 02/081735, (filed April 5, 2002; published
`October 17, 2002)
`McQuade, D., et al., "Signal Amplification of a "Turn-On" Sensor:
`Harvesting the Light Captured by a Conjugated Polymer," J. Am.
`Chem. Soc. 122: 12389-12390 (2000)
`Harrison, B., et al., "Amplified Fluorescence Quenching in a
`Poly(p-phenylene)-Based Cationic Polyelectrolyte," J. Am. Chem.
`Soc. 122: 8561-8562 (2000)
`Bazan et al., "Methods and Compositions For Detection and
`Analysis of Polynucleotides Using Light Harvesting
`Multichromophores," U.S. Patent No. 9,085,799 (filed August 14,
`2014; issued July 21, 2015)
`Burgess et al., "Through Bond Energy Transfer In Fluorescent Dyes
`For Labelling Biological Molecules," U.S. Patent No. 6,340,750
`(filed December 14, 1999; issued January 22, 2002)
`Reppy et al., "Method For Detecting An Analyte By Fluorescence,"
`International Publication No. WO 01/71317 (filed March 20, 2001;
`published September 27, 2001)
`Kool, "Fluorescent Nucleoside Analogs and Combinatorial
`Fluorophore Arrays Comprising Same," International Publication
`No. WO 01/044220 (filed December 13, 2000; published June 21,
`2001)
`
`
`
`7
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`

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`Inter Partes Review of U.S. Patent No. RE46,817
`Declaration of Kirk S. Schanze, Ph.D. (TFS1002)
`
`
`Description
`
`Kang, T., et al., "Photoluminescence properties of various
`polythiophene derivatives," Synthetic Metals 69: 377-378 (1995)
`Glazer et al., "Dyes Designed For High Sensitivity
`Detection of Double-Stranded DNA," U.S. Patent No. 5,783,687
`(filed September 5, 1996; July 21, 1998)
`De Angelis, D., "Why FRET over genomics?" Physiological
`Genomics 1: 93-99 (1999)
`Didenko, V., "DNA Probes Using Fluorescence Resonance Energy
`Transfer (FRET): Designs and Applications," Biotechniques 31:
`1106–1121 (2001)
`LeClerc, M., "Optical and Electrochemical Transducers
`Based on Functionalized Conjugated Polymers," Adv. Mater. 11:
`1491 – 1498 (1999)
`Wang, J., et al., "Photoluminescence of Water-Soluble Conjugated
`Polymers: Origin of Enhanced Quenching by Charge Transfer,"
`Macromolecules 33: 5153-5158 (2000)
`Liu, B., et al., "Synthesis of a novel cationic water-soluble efficient
`blue photoluminescent conjugated polymer," Chem. Comm. 551-
`552 (2000)
`Gaylord, B., et al., "DNA detection using water-soluble conjugated
`polymers and peptide nucleic acid probes," PNAS 99: 10954-10957
`(2002)
`The Nobel Prize in Chemistry 2000, Conductive Polymers, The
`Royal Swedish Academy of Sciences
`Chen, L., et al., "Highly sensitive biological and chemical sensors
`based on reversible fluorescence quenching in a conjugated
`polymer," PNAS 96: 12287-12292 (1999)
`Wang, S., et al., "Fluorescein Provides a Resonance Gate for FRET
`from Conjugated Polymers to DNA Intercalated Dyes," J. Am.
`Chem. Soc., 126:5446-5451 (2004)
`Curriculum Vitae for Kirk S. Schanze, Ph.D.
`
`Thermo
`Fisher
`Exhibit #
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`1016
`
`1017
`
`1018
`
`1019
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`1020
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`1021
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`1022
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`1023
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`1024
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`1025
`
`1026
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`1027
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`
`
`8
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`

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`Inter Partes Review of U.S. Patent No. RE46,817
`Declaration of Kirk S. Schanze, Ph.D. (TFS1002)
`
`
`Description
`
`Swager, T. "Fluorescence Studies of Poly(p-
`phenyleneethyny1ene)s: The Effect of Anthracene Substitution," J.
`Phys. Chem. 99, 4886-4893 (1995)
`Chen et al., "Method For Detecting Biologlcal Agents," U.S. Patent
`Application Publication No. 2004/0023272 (filed April 10, 2003;
`February 5, 2004)
`Swager, T. "The Molecular Wire Approach to Sensory Signal
`Amplification," Ace. Chem. Res. 31:201-207 (1998)
`Gruber, H., et al., "Biotin-Fluorophore Conjugates with
`Poly(ethylene glycol) Spacers Retain Intense Fluorescence after
`Binding to Avidin and Streptavidin," Bioconjugate Chem. 8: 552-
`559 (1997)
`Haugland, R., "Section 6.4 – Phycobiliproteins" in Handbook of
`Fluorescent Probes and Research Products, 9th Ed., Molecular
`Probes, Inc. (2002)
`Haugland, R., "Section 6.4 – Phycobiliproteins" in Handbook of
`Fluorescent Probes and Research Products, 8th Ed., Molecular
`Probes, Inc. (2001)
`Pinto, M. and Schanze, K., "Conjugated Polyelectrolytes: Synthesis
`and Applications," Synthesis 9: 1293 - 1309 (2002)
`Alberts, Molecular Biology of the Cell, 2nd ed., Garland Publishing,
`Inc. (1989)
`Swager et al., "Emissive Polymers And Devices Incorporating
`These Polymers," International Publication No. WO 99/57222 (filed
`May 5, 1998; November 11, 1999)
`Hancock et al., "Luminescent Polymer Particles," U.S. Patent
`Application Publication No. 2003/0134959 (filed November 30,
`2001; July 17, 2003)
`
`Thermo
`Fisher
`Exhibit #
`
`1028
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`1029
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`1030
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`1031
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`1032
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`1033
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`1034
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`1035
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`1036
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`1037
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`
`
`
`
`9
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`

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`Inter Partes Review of U.S. Patent No. RE46,817
`Declaration of Kirk S. Schanze, Ph.D. (TFS1002)
`
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`IV. Person of ordinary skill in the art
`
`11.
`
`It is my understanding that a person of ordinary skill in the art
`
`("POSA") is a hypothetical person who is presumed to be aware of all pertinent
`
`art, thinks along conventional wisdom in the art, and is a person of ordinary
`
`creativity. A POSA in the technical field of the '817 patent (conjugated polymer
`
`energy transfer detection systems) would have had knowledge of the scientific
`
`literature concerning methods of synthesizing fluorescent conjugated polymers
`
`and signaling chromophores and using these polymers and chromophores for
`
`detecting binding events.
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`12. Here, a POSA would typically have had (i) a Ph.D. in Chemistry, or in
`
`a related field in the chemical sciences, and have at least about two years of
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`experience in chemical synthesis and application of fluorescent conjugated
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`polymers; or (ii) a Master's degree in the same fields with at least about five years
`
`of the same experience. Also, a POSA may have worked as part of a
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`multidisciplinary team and drawn upon not only his or her own skills, but of
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`others on the team, e.g., to solve a given problem. For example, a biochemist,
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`molecular biologist, and a clinician specializing in detection of biological
`
`molecules may have been part of a team.
`
`
`
`10
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`

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`Inter Partes Review of U.S. Patent No. RE46,817
`Declaration of Kirk S. Schanze, Ph.D. (TFS1002)
`
`
`State of the art before August 26, 2002
`
`V.
`
`A. Overview of Förster resonance energy transfer (FRET)
`
`13. The theory of Förster resonance energy transfer or fluorescence
`
`resonance energy transfer (FRET) was originally developed by Theodor Förster in
`
`the 1940s. TFS1019, 3. FRET occurs between two light-sensitive molecules
`
`(chromophores): a donor chromophore and an acceptor chromophore.
`
`14. The donor is excited by light radiation of a specific wavelength. Once
`
`excited, the donor then transfers energy to the acceptor via a dipole-dipole
`
`interaction. When the donor transfers its energy, the donor's fluorescence
`
`decreases, i.e., is quenched. Once the energy is transferred to the acceptor, it loses
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`this energy either through a fluorescence emission of light or as heat (also known
`
`as non-radiative emission). TFS1019, 3. The figure below illustrates the energy
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`transfer:
`
`Donor
`
`Acceptor
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`15. The donor and acceptor chromophores each have absorption and
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`emission spectra. The absorption spectrum is the range of wavelengths at which a
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`11
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`Inter Partes Review of U.S. Patent No. RE46,817
`Declaration of Kirk S. Schanze, Ph.D. (TFS1002)
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`chromophore absorbs radiation. The emission spectrum is the range of
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`wavelengths at which a chromophore emits radiation when excited. The peak at
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`which the highest amount of radiation is absorbed is called the absorbance
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`maximum, while the peak at which the highest amount of radiation is emitted is
`
`called the emission maximum. The emission maximum always occurs at a longer
`
`wavelength (i.e., less energy) than the absorption maximum. See TFS1018,
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`5:Figure 1A.
`
`16.
`
`In a FRET system, the donor and acceptor have different absorption
`
`and emission spectra. For FRET to occur, the emission spectrum of the donor
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`must overlap with
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`the absorption spectrum of
`
`the acceptor, as shown
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`schematically in the figure below.
`
`
`
`12
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`Inter Partes Review of U.S. Patent No. RE46,817
`Declaration of Kirk S. Schanze, Ph.D. (TFS1002)
`
`17. The overlap between the donor emission spectrum and the acceptor
`
`absorption spectrum allows emission from the donor to excite the acceptor. The
`
`acceptor then emits radiation in the wavelengths of the acceptor emission
`
`spectrum. TFS1018, 4:1; TFS1019, 3. Thus, in a FRET system, an excitation of
`
`the donor leads to a detected signal from the acceptor.
`
`18. The strength of emission of the acceptor in a FRET system is
`
`dependent on the distance between the acceptor and the donor. TFS1019, 3-4.
`
`FRET will occur only when the donor and acceptor are brought into close enough
`
`proximity to allow for energy transfer between the two. Because of this property,
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`FRET systems can be used as "molecular rulers," measuring distances of 0.5-10
`
`nm with high precision. TFS1019, 4. Measurements of distances in this range are
`
`very useful for measuring binding in biological systems. Indeed, one of the
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`earliest uses of FRET in analyzing biological systems was for studying protein-
`
`protein interactions in the 1970s. TFS1019, 3. But other uses for FRET in
`
`biosensing systems began to develop throughout the 1980s and 1990s, particularly
`
`FRET systems for detecting nucleic acid hybridization, as described below.
`
`TFS1019, 3.
`
`
`
`13
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`Inter Partes Review of U.S. Patent No. RE46,817
`Declaration of Kirk S. Schanze, Ph.D. (TFS1002)
`
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`B. Chromophores used in FRET
`
`1.
`
`Fluorescent Dyes used as FRET acceptors
`
`19. One standard type of chromophores used in FRET systems as either
`
`the donor, acceptor, or both, are fluorescent dyes. Many such dyes were
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`commercially available in August 2002. TFS1033; TFS1032; TFS1019, 4-9.
`
`20. Researchers had used fluorescent dyes in FRET systems by August
`
`2002. For example, Burgess describes a system for detecting nucleic acid
`
`hybridization by detecting FRET between two fluorescent dyes. Burgess used the
`
`dyes fluorescein, rhodamine and BODIPY. TFS1013, 9:36-45. Additional
`
`research groups had also developed FRET systems that used commercially
`
`available fluorescent dyes as the acceptor. For example, McQuade described a
`
`FRET system with fluorescein as the acceptor. TFS1005, 3:2:1. Reppy also
`
`described a FRET system where various fluorescent dyes are used as the acceptor,
`
`including BODIPY derivatives. TFS1014, Tables 1 and 2. And Kool described a
`
`system where conjugated nucleic acid base substitutes transfer energy via FRET
`
`to rhodamine to detect nucleic acid binding. TFS1015, 55, Example 8.
`
`21. Along with the variety of commercially available dyes, researchers
`
`were also busy developing new biosensing fluorescent dyes and new biosensing
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`systems using fluorescent dyes. For example, Glazer developed new fluorescent
`
`dyes having increased binding to double stranded DNA for detection of nucleic
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`
`
`14
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`Inter Partes Review of U.S. Patent No. RE46,817
`Declaration of Kirk S. Schanze, Ph.D. (TFS1002)
`
`acids. TFS1017. And Gruber developed a system for detecting binding of biotin to
`
`avidin by attaching the known fluorescent dyes fluorescein, rhodamine, Cy3 and
`
`Cy5 to biotin. TFS1031.
`
`22. Thus, a POSA in August 2002 would have known that various
`
`fluorescent dyes could be used as acceptors in FRET systems, as long as the dye
`
`could be brought into close enough proximity with the donor and as long as the
`
`donor emission spectrum overlapped with the acceptor absorption spectrum, as
`
`discussed above.
`
`2.
`
`Quenchers used as FRET acceptors
`
`23. At the same time, other research groups had developed quenching
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`systems that operate according to the principles of FRET. These quenching
`
`systems use a quencher instead of a fluorescent dye. In a quenching system, a
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`fluorescent donor is associated with a quencher molecule, instead of a fluorescent
`
`dye. As discussed above, during FRET, the donor's emission is quenched by
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`absorption by the acceptor which then subsequently emits radiation. A quenching
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`system works in a similar manner.
`
`24. The excited donor chromophore transfers its energy to the acceptor
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`quencher molecule, which results in a quenching of emission from the donor. The
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`quencher molecule, however, does not emit radiation as a fluorescent light signal.
`
`Instead, the quencher dissipates the energy transferred to it as heat. TFS1019, 3.
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`Inter Partes Review of U.S. Patent No. RE46,817
`Declaration of Kirk S. Schanze, Ph.D. (TFS1002)
`
`One example of use of a quencher are systems where the quencher is removed
`
`from its donor. When the quencher is removed, the distance between the donor
`
`fluorophore and the acceptor quencher becomes too large for the energy transfer
`
`between them to continue, so the acceptor quencher will no longer quench the
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`donor fluorophore, which will, in turn, fluoresce. See, e.g., TFS1018, Figure 1.
`
`25. The similarities between fluorescent dyes and quenchers were well
`
`known as of August 2002. In fact, researchers sometimes used the terms
`
`fluorescent dye and quencher interchangeably. For example, in describing a
`
`fluorescence acceptor (A) in a FRET system, De Angelis notes that "[i]f A is
`
`nonfluorescent, it can be referred to as a quencher (Q); in this case, FRET
`
`emission between D and Q results in a net decrease in photo emission from D."
`
`TFS1018, 4:1. As another example, my publication Harrison, describes use of a
`
`Ru(phen')3
`
`4 "quencher," and reports measuring the emission of Ru(phen')3
`
`4,
`
`meaning that a fluorescent signal was emitted from the Ru(phen')3
`
`4. TFS1006,
`
`3:1:2, Figure 1.
`
`26.
`
`In August 2002, and still today, a POSA would have considered
`
`fluorescent dyes and quenchers more generically as fluorescent signaling
`
`molecules. And a POSA would have understood that one could pick and choose
`
`between the different types of fluorescent dyes and quenchers to use as acceptors
`
`depending on the desired outcome of the fluorescent signaling system. If it was
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`Inter Partes Review of U.S. Patent No. RE46,817
`Declaration of Kirk S. Schanze, Ph.D. (TFS1002)
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`more desired to have an increase in fluorescence upon detection of binding in a
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`system, a POSA would have considered bringing a fluorescent dye into proximity
`
`with the molecules being detected or removing a quencher from the proximity of
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`the molecules being detected. Or if it was more desired to have a decrease in
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`fluorescence upon detection of binding, a POSA would have considered removing
`
`a fluorescent dye from the proximity of the molecules being detected or bringing a
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`quencher into proximity with the molecules being detected. See, e.g., TFS1018,
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`4:1, Figure 1B and 1C.
`
`3.
`
`Conjugated polymers as donors in FRET systems
`
`27. Conjugated polymers were already in use as donors in FRET systems
`
`before August 2002. Conjugated polymers have a system of alternative single and
`
`double (or sometimes triple) bonds. TFS1024, 1-2; TFS1037, [0112]-[0114]. As
`
`shown in the figure below, each bond of the conjugated polymer contains a
`
`sigma((cid:305))-bond between overlapping atomic s-orbitals of the atoms bonded
`
`together. TFS1024, 1-2; TFS1037, [0112]-[0114]. An atomic orbital represents the
`
`possible locations of electrons in the molecule. Each double bond also contains a
`
`pi((cid:652))-bond which is made up over overlapping p-orbitals of the atoms bonded
`
`together. TFS1024, 1-2; TFS1037, [0112]-[0114].
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`Inter Partes Review of U.S. Patent No. RE46,817
`Declaration of Kirk S. Schanze, Ph.D. (TFS1002)
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`28. The alternating double bonds with their overlapping p-orbitals give
`
`conjugated polymers a system of connected p-orbitals. An electron is able to move
`
`throughout this system of connected p-orbitals, covering a great distance along the
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`conjugated polymer. TFS1024, 2; TFS1037, [0112]-[0114]. As the electron is not
`
`limited to a specific bond location in the conjugated polymer and can move
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`around the polymer, the conjugated polymer is considered to have a delocalized
`
`electronic structure. TFS1024, 2; TFS1037, [0112]-[0114]; TFS1023, 3:1:2.
`
`29.
`
` The delocalized electronic structure allows the conjugated polymers
`
`to absorb and emit energy. See, e.g., TFS1037, [0113]-[0114]; TFS1024, 2;
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`TFS1036, 9:30-10:15. Parts of the conjugated system may act as a chromophore,
`
`absorbing energy from light shone on the conjugated polymer and transmitting the
`
`absorbed energy
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`through
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`the conjugated polymer. TFS1036, 9:30-10:15;
`
`TFS1037, [0109]-[0112]. This absorbed energy may be emitted or transferred to
`
`another chromophore through a mechanism such as FRET if the other
`
`chromophore is in close enough proximity. See, e.g., TFS1037, [0113]-[0114];
`
`TFS1018, 4:1, Figure 1B and 1C.
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`Inter Partes Review of U.S. Patent No. RE46,817
`Declaration of Kirk S. Schanze, Ph.D. (TFS1002)
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`30. Conjugated polymers were known to produce an "antenna effect" or
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`"molecular wire effect" whereby the polymer collects light energy it is exposed to
`
`and transmits this energy along the length of the polymer to an acceptor. In this
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`way, the conjugated polymer acts as an antenna or a wire for receiving and
`
`transmitting light energy much in the same way a radio antenna collects a radio
`
`signal and transmits it to a speaker or the way a wire transmits electrical energy.
`
`For example, in discussing anthracene containing conjugated polymers, Swager
`
`notes that "[w]hen anthracene is incorporated into the backbones, the polymers act
`
`as antennae which harvest the optical energy and transfer it to the anthracene
`
`groups, resulting in emission from these chromophores." TFS1028, 8:1:1. Swager
`
`notes that this antenna effect has been demonstrated in a variety of polymeric
`
`systems. TFS1028, 12:1:4.
`
`31. Another paper by Swager's group also discusses how "molecular wires
`
`(conjugated polymers) can be used to amplify molecular chemosensors."
`
`TFS1030, 2-3. And Harrison also notes that "[f]luoresence sensing is amplified by
`
`conjugated polymers because of the 'molecular wire effect'." TFS1006, 3:1:1.
`
`32. Harrison compared the P-NEt3
`
`+ polymer to a non-polymer donor, M-
`
`NEt3
`
`+. A side by side comparison of the two is shown below:
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`Inter Partes Review of U.S. Patent No. RE46,817
`Declaration of Kirk S. Schanze, Ph.D. (TFS1002)
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`+ is more efficiently quenched by an acceptor (quencher)
`
`33.
`
` P-NEt3
`
`compared
`
`to M-NEt3
`
`+,
`
`i.e.,
`
`the polymer
`
`is more efficient at energy
`
`transfer.TFS1006, 3:2:1. This increased efficiency in energy transfer in the
`
`polymer is due to the "molecular wire effect" or "antenna affect." TFS1006, 3:1:1;
`
`TFS1030, 2:2:2; TFS1028, 12:1:4. A POSA in August 2002 would have
`
`understood that the "antenna effect" in conjugated polymers was due to the
`
`concentration of multiple chromophore monomers together into a polymer.
`
`34. Thus a POSA would have known in August 2002 that conjugated
`
`polymers have the ability to harvest light energy and transfer it to a donor
`
`chromophore.
`
`35. Cationic, anionic, and uncharged conjugated polymers were all known
`
`as of August 2002. For example, Chen developed an anionically-charged
`
`poly(phenylenevinylene) fluorescent conjugated polymer. TFS1025, 3:1. Both
`
`Harrison and Liu developed cationically-charged poly(phenylene) fluorescent
`
`conjugated polymers. TFS1006, 3:1:2; TFS1022, 3. Swager described neutral
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`Inter Partes Review of U.S. Patent No. RE46,817
`Declaration of Kirk S. Schanze, Ph.D. (TFS1002)
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`poly(p-phenyleneethynylene) fluorescent conjugated polymers. TFS1028, 8:1-2.
`
`And Hancock described anthracene-based fluorescent conjugated polymers that
`
`could be either uncharged or charged. TFS1037, Figure 5, [0129].
`
`36. By August 2002, researchers had already determined that water-
`
`soluble conjugated polymers were especially useful for detection of molecules in
`
`solutions containing biological samples. These water-soluble polymers were
`
`capable of being used in solution in aqueous systems. For example, in discussing
`
`the application of chemical and biological sensors, Harrison developed a water-
`
`soluble, cationic poly(p-phenylene) based polymer and noted that sensors that
`
`operate in aqueous environments would be more useful for detection of analytes in
`
`a liquid or vapor phase TFS1006, 3:1:1. In discussing work before August 2002,
`
`Gaylord noted that "[w]ater-soluble CPs show exceptional fluorescence quenching
`
`efficiencies in the presence of oppositely charged acceptors and are of particular
`
`interest for transduction of biological recognition events." TFS1023, 3:1:2. And
`
`Liu developed a water-soluble, cationic polyphenylene conjugated polymer, and
`
`noted that "[p]otential applications of water-soluble conjugated polymers
`
`include… [use] as highly sensitive fluorescent sensory materials in living bodies."
`
`TFS1022, 3.
`
`37.
`
` The potential applications of water-soluble conjugated polymers in
`
`solution-based and solid state systems for biosensing and for the detection of
`
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`Inter Partes Review of U.S. Patent No. RE46,817
`Declaration of Kirk S. Schanze, Ph.D. (TFS1002)
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`chemical residues led to rapid advancement in the field from 1999 to 2002. As
`
`discussed in more detail below, several groups were successful in synthesizing
`
`such polymers. See, e.g., TFS1004; TFS1015; TFS1025; TFS1014; TFS1021;
`
`TFS1006; TFS1022; and TFS1034. By August 2002 researchers, including
`
`myself, had found that adding hydrophilic side chains (e.g., side chains having
`
`positive or negative charges) was very effective in making conjugated polymers
`
`water soluble. TFS1034, 2; TFS1037, [0126].
`
`38. One way to obtain a water soluble conjugated polymer that was well-
`
`known by August 2002 was to make a charged conjugated polymer. For example,
`
`LeClerc made positively-charged "water-soluble polythiophene derivatives" for
`
`detecting oligonucleotide hybridization that provide an "optical (colormetric,
`
`fluorescent or luminescent) or electrical signal." TFS1004, 4:24-27. And Chen
`
`used the same strategy but made his polymers negatively charged. Chen described
`
`"water-soluble polyanionic" charged poly(phenylene vinylene) polymers
`
`providing a fluorescence signal for use in biosensing assays. TFS1025, 3:1:2;
`
`Figure 2.
`
`39. A POSA would have also known of other strategies for making water-
`
`soluble conjugated polymers in August 2002. For example, Reppy described the
`
`use of polydiacetylene fluorescent conjugated polymers that formed bi-layer
`
`liposome systems that disperse in solution. TFS1014, 11:1-26. And Kool
`
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`Inter Partes Review of U.S. Patent No. RE46,817
`Declaration o