Application of conjugated polyelectrolyte in biosensor

APPLICATION OF CONJUGATED POLYELECTROLYTE IN BIOSENSOR PU KANYI NATIONAL UNIVERSITY OF SINGAPORE 2010 APPLICATION OF CONJUGATED POLYELECTROLYTE IN BIOSENSOR PU KANYI (M.S., FUDAN UNIV.) A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DEPARTMENT OF CHEMICAL AND BIOMOLECULAR ENGINEERING NATIONAL UNIVERSITY OF SINGAPORE 2010 ACKNOWLEDGEMENTS I would like to express my sincere gratitude to my supervisor, Associate Prof. LIU Bin, for her constructive guidance, continuous inspirations and encouragements throughout my doctoral study. Her enthusiasm and persistence in science carried me forward to many interesting and challenging research topics in conjugated polyelectrolytes. I wish to acknowledge the National University of Singapore and Singapore Ministry of Education for providing the opportunity for me to pursue my Ph. D. degree here. I also would like to thank Chinese government for giving me the award of outstanding self-financed students abroad in 2008. I would like to thank all the people in our group, particularly Mr. LI Kai for his support in the cell culture experiment, Dr. FANG Zhen, Dr. CAI Liping and Mr. WANG Guan for their helps in the NMR experiment. I would love to give my deep and special thanks to my family members including my parents, my wife and my parents in law for their unconditional love, support and understanding through all of these years. i TABLE OF CONTENTS ACKNOWLEDGEMENTS . i TABLE OF CONTENTS ii SUMMARY v LIST OF FIGURES . vii LIST OF SCHEMES xi NOMENCLATURES . xiv CHAPTER 1. INTRODUCTION 1.1. Conjugated Polyelectrolyte Based Biosensors 1.2. Research Objectives 1.3. Thesis Outline CHAPTER 2. LITERATURE REVIEW . 2.1. Conjugated Polyelectrolytes . 2.2. Fluorescence Quenching Sensors . 11 2.3. Fluorescence Turn-on Sensors . 15 2.4. Colorimetric Sensors 17 2.5. FRET Sensors 20 2.5.1. DNA Detection 22 2.5.2. Protein Detection . 25 2.5.3. Small Molecule Detection 29 2.5.4. Influencing Factors for FRET 32 CHAPTER 3. MULTICOLOR CONJUGATED POLYELECTROLYTE WITH ENERGY TRANSFER BACKBONE FOR VISUAL DETECTION OF HEPARIN 36 3.1. Introduction . 36 3.2. Experiment 39 3.2.1. Instruments . 39 3.2.2. Materials 40 3.2.3. Synthesis 40 3.3. Results and Discussion 43 3.3.1. Synthesis and Characterization 43 3.3.2. Optical Properties . 45 3.3.3. Aggregation-Induced FRET . 47 3.3.4. Heparin Quantification . 50 ii 3.4. Conclusion . 52 CHAPTER 4. MULTICOLOR INTERCALATING-DYE-HARNESSED CONJUGATED POLYELECTROLYTE FOR VISUAL DETECTION OF DOUBLE-STRANDED DNA 54 4.1. Introduction . 54 4.2. Experiment 56 4.2.1. Instruments . 56 4.2.2. Materials 56 4.2.3. Synthesis 57 4.3. Results and Discussion 60 4.3.1. Synthesis and Characterization 60 4.3.2. Optical Properties . 62 4.3.3. Fluorescence Response toward DNA 64 4.3.4. Comparison with Free TO/PFP System . 68 4.3.5. Recognition of dsDNA in Serum . 71 4.4. Conclusion . 74 CHAPTER 5. CONJUGATED POLYELECTROLYTE BLEND AS PERTURBABLE ENERGY TRANSFER ASSEMBLY FOR MULTICOLOR FLUORESCENT RESPONSES TOWARD PROTEINS 76 5.1. Introduction . 76 5.2. Experiment 78 5.2.1. Instrument 78 5.2.2. Materials 78 5.2.3. Synthesis 78 5.3. Results and Discussion 81 5.3.1. Sensing Mechanism . 81 5.3.2. Synthesis and Characterization 83 5.3.3. Optical Properties . 85 5.3.4. Fluoresecence Responses toward Proteins . 87 5.3.5. Ferritin Dection in Serum 90 5.4. Conclusion . 91 CHAPTER 6. MANNOSE-SUBSTITUTED CONJUGATED POLYELECTROLYTE AND OLIGOMER AS AN SMART ENERGY TRANSFER PAIR FOR DETECTION OF CONCANAVALIN A . 93 6.1. Introduction . 93 6.2. Experiment 96 6.2.1. Instruments . 96 6.2.2. Materials 96 6.2.3. Synthesis 96 6.3. Results and Discussion 104 iii 6.3.1. Synthesis and Characterization 104 6.3.2. Optical Properties . 109 6.3.3. Protein Sensing 111 6.3.4. Protein Quantification 116 6.4. Conclusion . 117 CHAPTER 7. CONJUGATED OLIGOELECTROLYTE-SUBSTITUTED POSS AS UNIMOLECULAR NANOPARTICULATE ENERGY DONOR FOR FLUORESCENCE AMPLIFICATION IN CELL . 119 7.1. Introduction . 119 7.2. Experiment 121 7.2.1. Instruments . 121 7.2.2. Materials 121 7.2.3. Cell cultures . 122 7.2.4. Confocal Imaging . 122 7.2.5. Cytotoxicity Test 123 7.2.6. Synthesis 123 7.3. Results and Discussion 126 7.3.1. Synthesis and Characterization 126 7.3.2. Optical Properties . 129 7.3.3. FRET in Solution . 130 7.3.4. Cell Imaging . 132 7.3.5. Cytotoxicity 135 7.4. Conclusion . 136 CHAPTER 8. CONCLUSIONS AND RECOMMENDATIONS 138 8.1. Conclusions 138 8.2. Recommendations . 143 REFERENCES . 147 LIST OF PUBLICATIONS 157 iv SUMMARY Reliable technologies for the detection of chemical and biological substances are of great scientific importance and economic interest because of their vital applications in clinical diagnosis, environmental monitoring, forensic analysis and antiterrorism. In this regard, conjugated polyelectrolytes (CPEs) with electron-delocalized fluorescent backbones and water-soluble ionic side chains have provided a unique platform for the construction of biosensors. However, fast, simple and label-free visual sensing strategies remain lacking in CPE-based assays. In this thesis, a series of new CPEs are designed and synthesized to constitute effective förster resonance energy transfer (FRET) probes for label-free visual detection of physiologically important biomolecules such as heparin, double-stranded DNA (dsDNA), and proteins. Two kinds of FRET probes are developed, which include the CPEs with intramolecular energy donor-acceptor architecture (single-component systems) and the CPE blends with energy donor-acceptor pair (bicomponent system). In general, these CPE-based probes vary the fluorescent colors upon interacting with the targets of interest due to enhanced FRET, consequently making visual sensing feasible. As nonspecific interactions between CPEs and biomolecules are inevitably in existence and likely to disturb fluorescent signals, two molecular engineering methods are created to increase the detection selectivity. Incorporation of fluorescent dyes with biorecognition capability as the energy acceptor to the CPEs significantly enhances the detection selectivity, allowing for visual detection of dsDNA even in mixed samples; whereas, attachment of biorecognition groups to both the donor and v acceptor of the CPE-based biocomponent probes is proven to be effective in highly selective visual detection of a specific protein. In addition to label-free visual detection in solution, efficient FRET in cell is observed for the CPEbased probes, which enables to light up and visualize the cellular structure using the commercial dyes with low brightness. Such a primary application in cell not only illustrates the importance of three-dimensional nanoparticle architecture of CPE in achieving whole-cell permeability, but also offers the opportunities of CPE-based probes in cellular sensing and imaging applications. The label-free visual assays developed herein together with the underlying mechanisms unraveled thereof should also provide useful guidelines for the further advance of CPEs in biological applications. vi LIST OF FIGURES Figure 2.1 (A) Absorption [(a) green and (c) orange] and emission [(b) blue and (d) red] spectra of CCP 1I and single-stranded PNA1Fl, respectively. Fluorescence was measured by exciting at 380 and 480 nm, for 1I and PNA1-Fl, respectively. (B) PL spectra of PNA-C* in the presence of complementary [(a) red] and noncomplementary [(b) black] DNA by excitation of CCP 1I. Conditions are in water at pH = 5.5. The spectra are normalized with respect to the emission of CCP 1I.63 Copyright 2002 National Academy of Sciences U S A. Reproduced with permission from Ref 63. Figure 3.1 Normalized absorption (a) and PL spectra (b) of PFOBT at [RU] = μM in water (excitation at 365 nm). Figure 3.2 Normalized PL spectra of PFOBT and PFBT5% at [RU] = μM (a) and [RU] = 60 μM (b) in mM PBS buffer at pH = 7.4 (excitation at 365 nm). Figure 3.3 (a) PL spectra of PFOBT at [RU] = 60 μM in mM PBS at pH = 7.4 in the presence of heparin with concentrations ranging from to 50 μM at intervals of μM (excitation at 365 nm); (b) Changes in the fluorescent color of the corresponding solution at intervals of μM under a hand-held UV-lamp with λmax = 365 nm. Figure 3.4 Normalized PL spectra of PFOBT at [RU] = 60 μM in the presence of [heparin] or [HA] = 44 μM in mM PBS at pH = 7.4 (excitation at 365 nm). The inset shows the corresponding fluorescent color under a hand-held UV-lamp with λmax = 365 nm. Figure 3.5 φ as a function of [heparin] and its linear trendline at [RU] = 60 μM in mM PBS at pH = 7.4. The data are based on the average of three independent experiments. Figure 3.6 φ as a function of [heparin] and its linear trendline at [RU] = μM in mM PBS at pH = 7.4. The inset shows the corresponding PL spectra of PFOBT at [RU] = μM in mM PBS at pH = 7.4 upon addition of heparin with concentrations ranging from to 180 nM at intervals of 30 nM. The data are based on the average of three independent experiments. Figure 4.1 H NMR spectra of and 3. vii Figure 4.2 (a) UV absorption spectra of PFPTO and PFP at [RU] = μM, and [1] = 0.3 μM; (b) Normalized PL spectra of PFPTO and PFP upon excitation at 370 nm. Figure 4.3 PL spectra of PFPTO at [RU] = μM in the presence of (a) dsDNA with [DNA] varying from to 8.4 nM at intervals of 1.2 nM, and (b) dsDNA or ssDNA with [DNA] = 8.4 nM in 1×PBS at pH = 7.4, excitation at 490 nm. Figure 4.4 PL spectra of PFPTO at [RU] = μM in the presence of (a) dsDNA with [dsDNA] varying from to 8.4 nM at intervals of 1.2 nM, and (b) dsDNA or ssDNA with [DNA] = 8.4 nM in 1×PBS at pH = 7.4, excitation at 370 nm. Figure 4.5 ΔI as a function of [DNA] upon excitation of PFPTO at 370 (squares) or 490 nm (circles). [RU] = μM in 1×PBS at pH = 7.4. Figure 4.6 PL spectra for solutions of TO/PFP at [RU] = μM and [TO] = 0.06 μM in the absence and presence of dsDNA or ssDNA with [DNA] = 8.4 nM in 1×PBS at pH = 7.4. Excitation at 490 nm (a) and 370 nm (b). Figure 4.7 PL spectra for solutions of TO/PFP at [RU] = μM and [TO] = 0.06 μM (a), and PFPTO at [RU] = μM (b) in the absence (solid line) and presence (dashed line) of dsDNA at [DNA] = 8.4 nM in 2×PBS at pH = 7.4 upon excitation at 370 nm. Figure 4.8 PL spectra of PFPTO (a) in the presence of dsDNA with [DNA] ranging from to 7.2 nM at intervals of 1.2 nM, and (b) in the absence of DNA, and in the presence of dsDNA or ssDNA at [DNA] = 7.2 nM. [RU] = μM in 1× PBS containing 10 vol% serum. (c) Photographs of fluorescence for PFPTO solutions at [RU] = μM in the presence of ssDNA with [DNA] = 7.2 nM, and in the presence of dsDNA with [DNA] ranging from to 6.0 nM at intervals of 1.2 nM in 1× PBS containing 10 vol% serum under a hand-held UV lamp with λmax = 365 nm. Figure 5.1 H NMR spectrum of PFVBT in CD3OD. Asterisk indicates the solvent peak. The spectrum is broken to eliminate the strong peak of water at 4.87 ppm. Figure 5.2 Normalized UV-vis absorption and PL spectra of PFVP and PFVBT in water. Figure 5.3 PL spectra of PFVP/PFVBT mixtures in 25 mM PBS with the ratio ranging from to 0.6. [PFVP] = μM, excitation at 430 nm or 515 nm. viii Chapter For visual nucleic acid sensing in Chapter 4, utilization of a greenemissive CPE backbone (e.g. PFV) as the energy donor instead of the blueemissive PF backbone in Chapter to create the FRET probe may facilitate the visual detection as human eyes are most sensitive to green color. Furthermore, other intercalating dyes can be tethered to a proper CPE structure to achieve a signaling emission at different wavelengths. However, the optoelectronic properties of the intercalating dye and the CPE should be simultaneously optimized in terms of energy-level alignment as described in Chapter in order to achieve efficient FRET. For visual protein sensing in Chapters and 6, increasing the watersolubility while decreasing the charge density of CPEs are crucial for reducing the nonspecific interactions and in turn improving the specificity. As well known, poly(ethylene glycol) (PEG) is one of the most effective hydrophilic building blocks that has been widely integrated into biomaterials to suppress nonspecific adsorption of biological substances.187 Therefore, attachment of PEG to CPEs as the side chain should be one of the effective ways to achieve this goal. However, this method requires the precise control and careful selection of PEG length, as it has the possibility to compromise the sensing sensitivity. In a similar way as shown in Chapter 6, other proteins could also be visually detected by attachment of other affinity groups apart from mannose (e.g. biotin for streptavidin). In Chapter 7, other fluorescent biorecognition elements in addition to intercalating dyes can be used as the energy acceptor to form the FRET intracellular probe with the POSS molecule. For instance, utilization of chromophores that show fluorescence quenching or turn-on responses toward 144 Chapter certain metal ions may lead to multicolor intracellular sensing of metal ions. However, good cell-permeability is required for the used chromophores. Moreover, from the materials viewpoint, the emission wavelength, charge nature and diameter of POSS-based fluorescent nanoparticles can be easily adjusted through chemical modification of fluorescent arms so as to fulfill the different requirements of specific applications. In view of their aggregationinhabited nanostructures and environment-resistant fluorescence, CPE-based POSS nanomaterials are also appropriate for signal amplification in various solid-state biological assays, such as DNA and protein microarrays. In-vitro and in-vivo sensing and imaging applications will become another important direction for CPEs-based materials. This is motived by the outstanding fluorescent properties of CPEs and their purely organic ingredients that make them more biocompatible as compared to other inorganic fluorescent nanomaterials (e.g. semiconductor quantum dots).188 Ease of attaching reactive groups (e.g. -NH2 and -COOH) to CPEs as the side chains pre- and post-polymerization is another advantage, which consequently allows for bioconjugation with various recognition elements. In contrast to the traditional linear CPEs as shown Chapters 2-6, CPEs with intrinsic three-dimensional architectures (e.g. hyperbranched and POSSbased polymers) or self-assembled nanostructures (e.g. amphiphilic rod-coil and graft polymers) should be more desired for bioimaging applications, due to their more efficient cellular uptake. Covalent or non-covalent binding of these specifically-designed CPEs with other biorecognition biomolecules, such as antibodies, peptides and aptamers, having high affinity toward certain cells (e.g. Herceptin for cancer cells) could lead to targeted cellular imaging. In 145 Chapter addition, development of CPE-based nanoparticles having suitable size and high stability in bloodstream could lead to targeted in-vivo fluorescent imaging. CPEs are indeed a unique category of optical materials with many utilities that cannot be accomplished by their counterpart organosoluble CPs and also small-molecular chromophores. The application of CPEs in biological sensing and imaging is still its infancy, and there is much work yet to be carried out not only to further understand their interaction and organization in physiological conditions, but also to fully exploit their potential structural diversity that could potentially embody new functions. 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Liu, “Optimization of interactions between a cationic conjugated polymer and chromophore-labeled DNA for optical amplification of fluorescent sensors”, J. Phys. Chem. B 2008, 112, 92959300. 4. K. Y. Pu, B. Liu, “Intercalating dye harnessed cationic conjugated polymer for real-time naked-eye recognition of double-stranded DNA in serum”, Adv. Funct. Mater. 2009, 19, 1371-1378. [Cover] Chapter 5. K. Y. Pu, B. Liu, “Conjugated polyelectrolytes as light-up macromolecular probes for heparin sensing”, Adv. Funct. Mater. 2009, 19, 277-284. [Highlighted by ACS as Noteworthy Chemistry, March 2, 2009] 6. K. Y. Pu, K Li, J. B. Shi, B. Liu, “Fluorescent single-molecular core-shell nanospheres of hyperbranched conjugated polyelectrolyte for live-cell imaging”, Chem. Mater. 2009, 21, 3816-3822. 7. K. Y. Pu, L. P. Cai, B. Liu, “Design and synthesis of charge-transferbased conjugated polyelectrolytes as multicolor light-up probes”, Macromolecules 2009, 42, 5933-5940. [Highlighted in the homepage] 8. K. Y. Pu, B. Liu, “Optimizing the cationic conjugated polymer-sensitized fluorescent signal of dye labeled oligonucleotide for biosensor applications”, Biosens. Bioelectron. 2009, 24, 1067-1073. [Review] Chapter 9. K. Y. Pu, R. Y. Zhan, B. Liu, “Surfactant effect on energy transfer between cationic conjugated polymer and dye-attached oligonucleotide”, Macromol. Symp. 2009, 279, 48-51. 10. K. Y. Pu, B. Liu, “Chapter 7.3. Fluorescence reporting based on FRET 157 between conjugated polyelectrolyte and organic dye”, Advanced Fluorescence Reporters in Chemistry and Biology, Springer, 2010, 9, 417454. [Book Chapter] Chapter 11. K. Y. Pu, K. Li, X. H. Zhang, B. Liu, “Conjugated oligoelectrolyte harnessed POSS as light-up hybrid unimolecular nanodots for two-photon fluorescence imaging of cellular nucleus”, Adv. Mater. 2010, 22, 41864189. [Inside Cover] 12. K. Y. Pu, K. Li, B. Liu, “Cationic oligofluorene substituted POSS as light- harvesting unimolecular nanoparticle for fluorescence amplification in cellular imaging”, Adv. Mater. 2010, 22, 643-646. Chapter 13. K. Y. Pu, K. Li, B. Liu, “A molecular brush approach to enhance quantum yield and suppress nonspecific interactions of conjugated polyelectrolyte for far-red/near-infrared fluorescence targeted cell imaging”, Adv. Funct. Mater. 2010, 20, 2770-2777. [Frontispiece] 14. K. Y. Pu, R. Y. Zhan, B. Liu, “Conjugated polyelectrolyte blend as perturbable energy donor-acceptor assembly for multicolor fluorescence responses to proteins”, Chem. Commun. 2010, 46, 1470-1472. Chapter 15. K. Y. Pu, B. Liu, “Fluorescence turn-on responses of anionic and cationic conjugated polymers toward proteins: effect of electrostatic and hydrophobic interactions”, J. Phys. Chem. B 2010, 114, 3077-3084. [Cover] 16. K. Y. Pu, J. B. Shi, L. H. Wang, L. P. Cai, G. Wang, B. Liu, “Mannose- substituted conjugated polyelectrolyte and oligomer as an intelligent energy transfer pair for Visual Detection of Concanavalin A”, Macromolecules 2010, 43, 9690-9697. Chapter 17. K. Y. Pu, K. Li, B. Liu, “Multicolor conjugate polyelectrolyte/peptide complexes as self-assembled nanoparticles for receptor-targeted cellular imaging”, Chem. Mater. 2010, 22, 6736-6741. 18. X. Y. Qi, K. Y. Pu, H. Li, X. Z. Zhou, S. X. Wu, Q. L. Fan, B. Liu, W. Huang, H. Zhang, “Amphiphilic graphene composites”, Angew. Chem. Int. Edit. 2010, 49, 9426-9429. [Co-first author] 19. A. Duarte, K. Y. Pu, B. Liu, G. C. Bazan, “Recent advance in conjugated polyelectrolytes for emerging optoelectronic applications”, Chem. Mater. 2011, 23, 501-515. [Co-first author, Review] 158 20. K. Y. Pu, R. Y. Zhan, J. Liang, B. Liu, “Conjugated polyelectrolyte for label-free visual detection of heparin”, Sci. China Ser. B–Chem. 2011, 54, 567-574. [Feature Article] 159 [...]... pattern allowed the identification of 17 different proteins with a high accuracy of 97% Whitten’s group in 2004 reported the indirect detection of proteins using CPE-based assay, which involves specific metal/protein interactions.40 Kinases control the phosphorylation/dephosphorylation of serine, threonine, and tyrosine residues, affecting and regulating a variety of cellular processes.41 Quaternary... concentration ranging from 0 to 4.5 nM at intervals of 1.5 nM [P1 RU] = 0.1 µM and [6] = 0.05 µM Excitation at 370 nm Figure 7.1 High resolution TEM image of OFP Figure 7.2 Normalized UV-vis absorption spectra of the arm 4, OFP and EB (dashed line), and PL spectra of 4 and OFP (solid line) in water Figure 7.3 (a) PL intensity of EB at 610 nm as a function of [OFP] for EB/ssDNA/OFP and EB/dsDNA/OFP mixtures... KSV = 5.6 × 106, while its fluorescence was insignificantly affected by bovine serum albumin (BSA) and jacalin, a galactose-binding protein The selective quenching was ascribed to the aggregation-induced self-quenching of 10 in the presence of Con A Scheme 2.4 Schematic illustration of (a) the displacement of a quenched fluorescent PPE by protein analyte (in blue) from gold NPs to recover the fluorescence,... spectra of 6/P1 blend in PBS (15 mM, pH = 7.2) containing CaCl2 (0.1 mM) and MnCl2 (0.1 mM) in the absence and presence of proteins [P1 RU] = 1 µM, [6] = 0.5 µM and [protein] = 150 nM Excitation at 370 nm (b) The intensity ratio of the yellow emission of 6 at 550 nm to the blue emission of P1 at 422 nm (I550/I422) as a function of proteins The data H NMR spectrum of 5 in CDCl3 Asterisk and hex indicate... spectra of 6/P1 blend in 15 mM PBS (pH = 7.4) containing CaCl2 (0.1 mM) and MnCl2 (0.1 mM) with the molar ratio ranging from 0 to 0.6 µM at intervals of 0.1 µM [[P1 RU] =1 µM Excitation at 370 nm Figure 6.5 PL spectra of 6/P1 blend in PBS (15 mM, pH = 7.2) containing CaCl2 (0.1 mM) and MnCl2 (0.1 mM) in the absence and presence of Con A with the concentration ranging from 0 to 150 nM at intervals of 30... are of vast scientific and economic importance because of their wide applications in clinical diagnosis, environmental monitoring, forensic analysis and antiterrorism.1 In particular, the fast-growing research fields of genomics and proteomics have stimulated the extensive investigations in the development of novel biosensors for the efficient, convenient and specific detection of biomolecules of interest... design and synthesis of a cationic CPE with an energy transfer backbone for label-free visual detection and quantification of heparin The effect of electrostatic interactions in energy transfer properties of the CPE is investigated and revealed, which provides guidelines for polymer design in the following chapters In Chapter 4, a CPE tethered with an intercalating dye as the side chains is developed for... visual detection of dsDNA, which has good selectivity even in serum-containing medium Chapters 5-7 are regarding bicomponent FRET systems Chapter 5 shows the label-free visual detection of proteins based on the nonspecific-interactionperturbed FRET within a bicomponent CPE blend In Chapter 6, the selectivity for visual detection of proteins is further optimized through molecular engineering of CPE blend... responses, making them superior in the transduction of optical signals.7 Water solubility, a prerequisite for fluorescent materials to interrogate biomolecules of interest in physiological environment, necessitates the development of water-soluble CPs CPEs are a kind of CPs with water-soluble side chains.8 These polymers combine the optoelectronic properties of CPs with the charge-mediated behaviors of polyelectrolytes,9... of an analyte, known as fluorescence quenching.30 The first example of fluorescence quenching sensing was demonstrated using organosoluble CPs by Swager’s group in 1995.31 The significant finding of this report is that fluorescence quenching of CPs upon binding to strong electron-withdrawing quenchers (such as explosives) is much more effective than that of their small molecule counterparts This phenomenon . APPLICATION OF CONJUGATED POLYELECTROLYTE IN BIOSENSOR PU KANYI NATIONAL UNIVERSITY OF SINGAPORE 2010 APPLICATION OF CONJUGATED POLYELECTROLYTE IN. spectra of the arm 4, OFP and EB (dashed line), and PL spectra of 4 and OFP (solid line) in water. Figure 7.3 (a) PL intensity of EB at 610 nm as a function of [OFP] for EB/ssDNA/OFP and. 7.4 in the presence of heparin with concentrations ranging from 0 to 50 μM at intervals of 2 μM (excitation at 365 nm); (b) Changes in the fluorescent color of the corresponding solution at intervals