Springer Series on Fluorescence Methods and Applications Series Editor: O.S Wolfbeis For further volumes: http://www.springer.com/series/4243 Springer Series on Fluorescence Series Editor: O.S Wolfbeis Recently Published and Forthcoming Volumes Advanced Fluorescence Reporters in Chemistry and Biology II Molecular Constructions, Polymers and Nanoparticles Volume Editor: A.P Demchenko Vol 9, 2010 Advanced Fluorescence Reporters in Chemistry and Biology I Fundamentals and Molecular Design Volume Editor: A.P Demchenko Vol 8, 2010 Lanthanide Luminescence Photophysical, Analytical and Biological Aspects Volume Editors: P Haănninen and H Haărmaă Vol Standardization and Quality Assurance in Fluorescence Measurements II Bioanalytical and Biomedical Applications Volume Editor: Resch-Genger, U Vol 6, 2008 Standardization and Quality Assurance in Fluorescence Measurements I Techniques Volume Editor: U Resch-Genger Vol 5, 2008 Fluorescence of Supermolecules, Polymeres, and Nanosystems Volume Editor: M.N Berberan-Santos Vol 4, 2007 Fluorescence Spectroscopy in Biology Volume Editor: M Hof Vol 3, 2004 Fluorescence Spectroscopy, Imaging and Probes Volume Editor: R Kraayenhof Vol 2, 2002 New Trends in Fluorescence Spectroscopy Volume Editor: B Valeur Vol 1, 2001 Advanced Fluorescence Reporters in Chemistry and Biology II Molecular Constructions, Polymers and Nanoparticles Volume Editor: Alexander P Demchenko With contributions by G Bergamini Á S.M Borisov Á P Ceroni Á J Chen Á A.P Demchenko Á A.B Descalzo Á I Dı´ez Á T Fischer Á M Grabolle Á M.A Habeeb Muhammed Á Y Jin Á C.L John Á I Klimant Á O.P Klochko Á S Liang Á B Liu Á M.Yu Losytskyy Á E Marchi Á T Mayr Á G Mistlberger Á T Nann Á R Nilsson Á R Nitschke Á L.D Patsenker Á K Peter Á T Pradeep Á K.-Y Pu Á R.H.A Ras Á U Resch-Genger Á M.A Reppy Á K Rurack Á R.A Simon Á W Tan Á A.L Tatarets Á E.A Terpetschnig Á S Xu Á V.M Yashchuk Á H Yao Á Q Yuan Á J.X Zhao Á S Zhu Volume Editor Prof Dr Alexander P Demchenko Palladin Institute of Biochemistry National Academy of Sciences of Ukraine Kyiv 01601 Ukraine alexdem@ukr.net ISSN 1617-1306 ISBN 978-3-642-04699-5 DOI 10.1007/978-3-642-04701-5 Springer Heidelberg Dordrecht London New York Library of Congress Control Number: 2010934374 # Springer-Verlag Berlin Heidelberg 2010 This work is subject to copyright All rights are reserved, whether the whole or part of the material is concerned, specifically the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting, reproduction on microfilm or in any other way, and storage in data banks Duplication of this publication or parts thereof is permitted only under the provisions of the German Copyright Law of September 9, 1965, in its current version, and permission for use must always be obtained from Springer Violations are liable to prosecution under the German Copyright Law The use of general descriptive names, registered names, trademarks, etc in this publication does not imply, even in the absence of a specific statement, that such names are exempt from the relevant protective laws and regulations and therefore free for general use Cover design: WMXDesign GmbH, Heidelberg, Germany Printed on acid-free paper Springer is part of Springer Science+Business Media (www.springer.com) Series Editor Prof Dr Otto S.Wolfbeis Institute of Analytical Chemistry Chemo- and Biosensors University of Regensburg 93040 Regensburg Germany otto.wolfbeis@chemie.uni-regensburg.de Aims and Scope Fluorescence spectroscopy, fluorescence imaging and fluorescent probes are indispensible tools in numerous fields of modern medicine and science, including molecular biology, biophysics, biochemistry, clinical diagnosis and analytical and environmental chemistry Applications stretch from spectroscopy and sensor technology to microscopy and imaging, to single molecule detection, to the development of novel fluorescent probes, and to proteomics and genomics The Springer Series on Fluorescence aims at publishing state-of-the-art articles that can serve as invaluable tools for both practitioners and researchers being active in this highly interdisciplinary field The carefully edited collection of papers in each volume will give continuous inspiration for new research and will point to exciting new trends Preface A variety of fluorescent and luminescent materials in the form of molecules, their complexes, and nanoparticles are available for implementation as reporting units into sensing technologies Increasing demands from these application areas require development of new fluorescence reporters based on association and aggregation of fluorescence dyes and on their incorporation into different nanostructures Interactions between these dyes and their incorporating matrices lead to new spectroscopic effects that can be actively used for optimizing the sensor design One of these effects is a spectacular formation of J-aggregates with distinct and very sharp excitation and emission bands By incorporation into nanoparticles, organic dyes offer dramatically increased brightness together with improvement of chemical stability and photostability Moreover, certain dyes can form nanoparticles themselves so that their spectroscopic properties are improved Semiconductor quantum dots are the other type of nanoparticles that possess unique and very attractive photophysical and spectroscopic properties Many interesting and not fully understood phenomena are observed in clusters composed of only several atoms of noble metals In conjugated polymers, strong electronic conjugation between elementary chromophoric units results in dramatic effects in quenching and in conformationdependent spectroscopic behavior Possessing such powerful and diverse arsenal of tools, we have to explore them in novel sensing and imaging technologies that combine increased brightness and sensitivity in analyte detection with simplicity and low cost of production The present book overviews the pathways for achieving this goal In line with the discussion on monomeric fluorescence reporters in the accompanying book (Vol of this series), an insightful analysis of photophysical mechanisms behind the fluorescence response of composed and nanostructured materials is made Based on the progress in understanding these mechanisms, their realization in different chemical structures is overviewed vii viii Preface Demonstrating the progress in an interdisciplinary field of research and development, this book is primarily addressed to specialists with different background – physicists, organic and analytical chemists, and photochemists – to those who develop and apply new fluorescence reporters It will also be useful to specialists in bioanalysis and biomedical diagnostics Kyiv, Ukraine June 2010 Alexander P Demchenko Contents Part I General Aspects Nanocrystals and Nanoparticles Versus Molecular Fluorescent Labels as Reporters for Bioanalysis and the Life Sciences: A Critical Comparison Ute Resch-Genger, Markus Grabolle, Roland Nitschke, and Thomas Nann Optimization of the Coupling of Target Recognition and Signal Generation 41 Ana B Descalzo, Shengchao Zhu, Tobias Fischer, and Knut Rurack Collective Effects Influencing Fluorescence Emission 107 Alexander P Demchenko Part II Encapsulated Dyes and Supramolecular Constructions Fluorescent J-Aggregates and Their Biological Applications 135 Mykhaylo Yu Losytskyy and Valeriy M Yashchuk Conjugates, Complexes, and Interlocked Systems Based on Squaraines and Cyanines 159 Leonid D Patsenker, Anatoliy L Tatarets, Oleksii P Klochko, and Ewald A Terpetschnig Part III Dye-Doped Nanoparticles and Dendrimers Dye-Doped Polymeric Particles for Sensing and Imaging 193 Sergey M Borisov, Torsten Mayr, Guănter Mistlberger, and Ingo Klimant ix Fluorescence Reporting 437 mixture as compared to that of buffer [81]; dilution of dye-labeled probe using unlabeled ssDNA or cationic surfactant can relax the compaction of the complex structures, leading to minimized acceptor self-quenching [82]; addition of nonionic surfactant increases the quantum yields of donors and reduces donor self-quenching upon complexation with DNA as a result of the incorporation of CCP molecules into the surfactant micelles [83, 84]; and utilization of silica nanoparticles (NPs) as the sensing venue to immobilize DNA can further reduce acceptor quenching and also allow excess DNA probes on the NP surface to capture CCPs, leading to increased local concentration of donor units [85, 86] Protein Biosensor Since many diseases not have a specific genetic signature, but rather have a variation in protein expression, biosensors for protein detection are of particular significance in medical diagnostics and pathogen recognition [87] However, proteins are much more complex and sensitive than oligonucleotides, which merit additional severe considerations in the course of assay design [88] Immunoassays are conventional methods based on specific antibody–antigen recognition for protein detection Enzyme immunosorbent assay (ELISA) is the most widely used immunoassay in clinics, which requires antibodies to be immobilized on the substrate to capture antigens and the secondary antibodies [89] The enzymes attached to the secondary antibodies serve as the catalyst to generate detection signals Despite its high sensitivity, ELISA demands tedious protein modification, surface immobilization, blocking and washing and is limited by the availability of commercial antibodies Thus, there is an ever-growing and urgent demand for developing new protein detection strategies that are simple and economical, with high selectivity and sensitivity CPEs have been explored for protein detection based on nonspecific interaction induced perturbation in their photophysical properties Fluorescence quenching of CPEs in the presence of proteins via electron transfer or aggregation mechanisms allowed protein discrimination according to the pattern of Stern–Volmer quenching constants [90–92], and a protein sensor array has been built with six water-soluble poly(p-phenylene ethynylene)s (PPEs) by Bunz’s group [93] These works provide important fundamental information about how proteins interact with CPEs and in turn affect the polymer fluorescence, which form a reference basis to be consulted during the design of CPE-based protein sensor involving FRET protocols 3.1 Antibody–Antigen Based Sensor FRET-based protein biosensors have been developed using CPEs as the lightharvesting donors in conjugation of “lock–key” recognition Streptavidin is a 438 K.-Y Pu and B Liu tetrameric protein that binds up to four molecules of biotin with the dissociation constant estimated to be  10À14 M In 2004, Zheng and Swager reported FRET experiments between a biotin-substituted PPE and three different dye-labeled streptavidins [94] Among these dye-labeled streptavidins, TR-labeled streptavidin showed the highest FRET-induced dye fluorescence upon excitation of the biotinsubstituted PPE, which was followed by rhodamine B-labeled streptavidin and then Fl-labeled donor streptavidin Of significance is the fact that a structurally similar PPE without biotin groups gave no observable FRET-induced dye emission, indicating the importance of specific antibody–antigen interaction in controlling FRET In 2006, Wang et al reported a specific streptavidin assay using 1Br as the lightharvesting donor and F1-labeled biotin (Fl-B) as the acceptor probe [95] The working mechanism of this assay is illustrated in Scheme Addition of analyte proteins into the solution of Fl-B leads to two situations Situation A refers to the target protein, streptavidin The biotin moiety of Fl-B strongly associates with streptavidin and Fl is deeply buried in the adjacent vacant binding sites Thus, after the addition of 1Br into the mixture solution, although there are strong electrostatic interactions between 1Br and the Fl-B/streptavidin complexes, the distance between 1Br and Fl-B does not meet the requirement for FRET As a result, the Fl fluorescence is weak Situation B corresponds to a nonspecific protein, such as BSA Under these conditions, Fl-B remains separated from the protein Therefore, electrostatic attraction between oppositely charged 1Br and F1-B can bring them into close proximity, leading to efficient FRET from 1Br to F1 and thus strong F1 fluorescence The discrepancy in Fl emission intensity thus allows one to distinguish streptavidin from other proteins Recently, we reported a CPE-amplified silica NP-based immunoassay for IgG detection [96] Silica NPs (100 nm) were used as the substrate in view of their small size and high surface-to-volume ratio for maximum protein loading, which could A Fluorescein Streptavidin FRET Biotin B Nonspecific protein Scheme Schematic illustration of streptavidin assay operation Fluorescence Reporting 439 provide improved assay performance relative to those based on planar microwells or glass substrates [97] In addition, the antibody-immobilized silica NPs allowed for efficient target capture and thorough interference isolation by simple centrifugation washing–redispersing procedures CCP 10 and Cy3-labeled anti-goat IgG were chosen as the donor and acceptor, respectively The assay scheme is shown in Scheme 10 Antigoat IgG–NP is first prepared by conjugating anti-goat IgG onto triazine-functionalized silica NPs The free triazine groups on the NP surface are further blocked by casein to minimize the nonspecific interaction between antigoat IgG–NP conjugates and interference proteins Goat IgG is then captured by antigoat IgG–NP conjugates, which is followed by the formation of a sandwich structure with the signaling antigoat IgG–Cy3 to afford fluorescent NPs CCP 10 is then introduced into the NP suspension Electrostatic attractions between the protein and 10 bring Cy3 and the polymer into close proximity to allow for efficient FRET, consequently yielding amplified Cy3 emission On the contrary, in the presence of nonspecific proteins, the NPs remain nonfluorescent because of the inexistence of the sandwich structure, and thus no FRET occurs upon addition of the polymer Fig shows the PL spectra of antigoat IgG–NP after incubation with goat IgG, thrombin, and BSA in the presence of 10 upon excitation at 370 nm Intense Cy3 fluorescence at 575 nm is observed for the NP suspension incubated with goat IgG, while very weak Cy3 fluorescence is observed for NPs with thrombin and BSA treatment The fluorescence intensities at 575 nm of anti-goat IgG NPs incubated with nonspecific proteins (BSA and thrombin) are less than 6% of that generated by goat IgG, resulting in a high signal-to-noise ratio of $17 The limit of detection for IgG is calculated to be $1.1 ng/mL, which is comparable to other NP-based fluoroimmunoassay containing multiple dye molecules [98] In addition, a sevenfold improvement in the limit of detection is achieved in the presence of 10 as Casein blocking Antibody immobiliation Antigen recognition FR ET hν hν′ Dye labeled antibody binding Signal amplification Anti-goat lgG Casein Goat lgG Anti-goat IgG-Cy3 10 Scheme 10 Schematic illustration of the CCP-amplified NP-based fluoroimmunoassay 440 K.-Y Pu and B Liu 350 Goat IgG BSA Thrombin 300 PL Intensity (a.u.) 250 200 150 100 50 540 560 580 600 Wavelength (nm) 620 640 Fig PL spectra of the NP suspension in the presence of 10 (8 mM) for antigoat IgG–NPs (0.2 mg) incubated with various proteins (1 mg/mL) and subsequent treatment with antigoat IgG–Cy3 (0.18 mg/mL), excitation at 370 nm compared to that upon direct excitation of Cy3 in the absence of 10, as a result of efficient FRET This study highlights that utilization of CPE as the energy donor can impart signal amplification and eliminate cross-talking fluorescence for protein immunoassays, which provides an opportunity to minimize the experimental errors and to improve the detection sensitivity 3.2 Aptamer-Based Sensor The specific aptamer–protein interaction has recently been used for protein detection with high selectivity [99] Aptamers are artificial nucleic acids selected in vitro with high affinity to proteins and other biological compounds In comparison with traditional specific antibody–antigen recognition, the application of aptamer as the probe has several advantages, which include easy preparation, stability, reusability and general availability for almost any protein Recently, we reported a strategy for lysozyme detection that took advantage of specific aptamer/protein interaction and aptamer/protein complexation mediated FRET between an ACP and a dye-labeled aptamer [100] The working mechanism for the assay is displayed in Scheme 11, which involves poly[9,9-bis(40 -sulfonatobutyl)fluorene-co-alt-1,4-phenylene] sodium salt (5) and a 6-carboxylfluorescein (FAM) labeled lysozyme aptamer as the energy donor and acceptor, respectively ACP was chosen as the light-harvesting molecule because of its high quantum yield in water ($0.9 in water) and Fluorescence Reporting 441 C Lysozyme Nontarget Protein ET FR C C Scheme 11 The lysozyme detection mechanism (left) and the chemical structure (right) of poly [9,9-bis(40 -sulfonatobutyl)fluorene-co-alt-1,4-phenylene] sodium salt (5) good emission spectral overlap with the absorption of FAM The detection starts with a solution composed of and FAM-labeled lysozyme aptamer Since both the polymer and the aptamer are negatively charged, they separate from each other because of electrostatic repulsion and FRET does not occur in solution upon excitation of at 370 nm Thus, as shown in Fig 10, there is no FAM emission initially In the presence of lysozyme, the aptamer specifically binds to lysozyme, forming a complex with net positive charge As a result, electrostatic attraction between and the aptamer/lysozyme complex occurs to bring and dye into close proximity for FRET to light up the FAM emission (Fig 10) In the presence of nonspecific proteins, there is no recognition between the lysozyme aptamer and proteins, and the aptamer surface charge remains negative The distance between the lysozyme aptamer and the polymer remains too far for 442 K.-Y Pu and B Liu Norm FL Intensity (a.u.) 1.0 Lysozyme Thrombin 0.8 Trypsin BSA 0.6 0.4 0.2 0.0 400 450 500 550 Wavelength (nm) 600 650 Fig 10 Normalized PL spectra of alone and with FAM-labeled lysozyme aptamer preincubated with lysozyme, thrombin, trypsin and BSA, respectively: [FAM-labeled lysozyme-aptamer] ¼ 4.5  10À8 M, [lysozyme] ¼ 2.4 mg/mL in mM Tris buffer; [5] ¼ 4.6  10À7 M in mM Tris buffer; Excitation at 370 nm FRET to occur, and thus no Fl emission is observed The specificity of this assay was also examined for mixed samples The mixed lysozyme samples were prepared in fetal bovine serum (FBS), human saliva and human urine It was found that FAM emission was still visible upon addition of each mixed sample, implying that this assay has a great potential for the detection of real biological samples This study illuminates that introduction of specific aptamer/protein interaction as the recognition event, and utilization of FRET as the signal transduction channel, is an effective way to develop CPE-based protein sensors with good specificity We also utilized CCP and aptamer-functionalized silica NPs to develop a sandwich assay for optical detection of thrombin in biological media with high sensitivity and selectivity [101] CCP 10 and Fl were chosen as the donor and the acceptor, respectively The assay scheme is shown in Scheme 12 The primary thrombin-binding aptamer (TBA) is covalently attached to triazine-functionalized silica NPs, which is followed by BSA blocking on the free sites of NP surface During the sensing process, TBA forms an intramolecular G-quartet (known as the guanine tetrad that consists of four guanine bases in a square planar array arranged in a cyclic hydrogen-bonding pattern, where each guanine is both the donor and acceptor of two hydrogen bonds) and recognizes the active site of thrombin, affording thrombin-bound TBA–NPs The Fl-labeled secondary TBA is then added to associate with the remaining thrombin binding sites, resulting in the dye-containing NPs Finally, addition of CCP 10 into the NP suspension induces FRET, leading to amplified Fl emission In contrast, in the presence of nonspecific proteins, the primary TBA-immobilized NPs cannot capture the Fl-labeled secondary TBA Fluorescence Reporting 443 Block Primary TBA Thrombin NP (100 nm) hn CCP Secondary TBA thrombin BSA hn ′ 10 Primary TBA: 5'-amino-T8 AGTCCGTGGTAGGGCAGGTTGGGGTGACT-3' or 5'-amino-T8 GGTTGGTGTGGTTGG-3' Secondary TBA: 5'-FAM-GGTTGGTGTGGTTGG- 3' Scheme 12 Working principle of CCP-amplified NP-based thrombin detection and no polymer-sensitized Fl emission is observed after addition of 10 Therefore, thrombin detection can be easily realized by monitoring the amplified Fl emission of NPs On the basis of this assay, a thrombin detection limit of 1.06 nM is obtained Moreover, the NP-based assay can be conducted in blood serum, which benefits from its solid-state detection platform that allows eliminating nonspecific adsorption through centrifugation–washing–redispersing circles On the other hand, the successful detection of lysozyme in a similar manner also highlights the generality of this protein assay scheme Protein biochip for thrombin detection was also reported by Leclerc’s group using the complex of a cationic polythiophene (20) and dye-labeled ssDNA aptamer as the FRET-based probe as illustrated in Scheme 13 [102] The complex probe was prepared by stoichiometrically mixing CCP 20 with 30 -Cy3-labeled ssDNA aptamer Two ssDNA sequences were used, one was the specific thrombin aptamer, ssDNA9-Cy3 (50 -NH2-C6-GGTTGGTGTGGTTGG-Cy3-30 ), and the other was the random ssDNA, ssDNA10–Cy3 (50 -NH2-C6-GGTGGTGGTTGTGGT-Cy3-30 ) The presence of amine group at the 50 -end of the ssDNA aptamer allows the complex of 20/ssDNA to covalently binding onto treated glass slides to form the solid-state array In the presence of thrombin, a significant fluorescence intensity increase is observed for the spots with 20/ssDNA9–Cy3 complexes (binding sequence) In the presence of two nonspecific proteins, BSA and immunoglobulin E (IgE), the fluorescence intensities of the spots remain low, revealing an excellent specificity of the detection with respect to the target On the other hand, the spots with 20/ssDNA10–Cy3 complexes (nonbinding sequence) not exhibit fluorescence enhancement for human thrombin, confirming the specificity of the detection in terms of the complex probe Correlation of the fluorescence intensities as a function of protein concentration using the spots of 20/ssDNA9–Cy3 reveals a limit of detection of $1.5  107 molecules for human thrombin (i.e., 6.2  10À11 M in 0.4 mL) together with a very good specificity This work highlights that biochips can 444 K.-Y Pu and B Liu Scheme 13 Illustration of the specific detection of target proteins by using the complex of a cationic polythiophene(21)/dye-labeled ssDNA aptamer on glass slides Reproduced with permission from [102] be established by taking advantage of CPEs in association with dye-labeled aptamers, which opens new feasibilities for simple and rapid multiplex analysis in proteomics 3.3 CPE Complex Based Sensor In addition to protein detection using specific antibody/antigen and aptamer/protein interactions, array detection was demonstrated based on nonspecific interactions between the CPE/dye-labeled ssDNA complexes and proteins [103] The design concept is motivated by the fact that external agents can effectively perturb the electron coupling of optical units within the complex of CPE/ssDNA-C* and in turn vary the FRET-induced fluorescence of both CPE (donor) and C* (acceptor) Owing to discrepancies in local hydrophobic and charged domains of different Fluorescence Reporting 445 proteins, it is suggested that the concerted action of both the higher order aggregate structure and the ssDNA sequence/length would lead to differential responses to protein structures A series complexes of a cationic conjugated oligomer (21, chemical structure in the inset of Fig 11) and ssDNA–FAM with different sequences were used to form complex-based FRET probes to generate an array fluorescence response toward proteins For detection, 21/ssDNA–FAM solutions is prepared by mixing ssDNA–FAM (5.7  10À6 M, based on nucleotide bases) with 21 (1.0  10À6 M) in potassium phosphate/sodium hydroxide buffer solution (50 mM, pH ¼ 7.4) The charge ratio between 21 and ssDNA is kept at 0.7 in order to avoid precipitation As shown in Fig 11, addition of proteins into the solution of 21/ssDNA results in fluorescence intensity decreases in both the emission of 21 at 400 nm and the FRET sensitized FAM emission at 525 nm More importantly, the FRET-induced fluorescence is sensitive to ssDNA sequence, allowing for cross-check of the detection reliability The fluorescence response of 21/ssDNA was also examined upon addition of other proteins, including myoglobin, lysozyme, hemoglobin, BSA, PS–BSA, PT–BSA, PY–BSA, streptavidin, proteinase K, T4 PNK, T4 DNA, DNA polymerase, and Taq polymerase The resulting proteindependent fluorescence changes were patterned and subjected to principle component analysis (PCA) PCA is a mathematical transformation used to extract variance between entries in a data matrix, by reducing the redundancy in the dimensionality Fig 11 Chemical structure of 21, the sequences of ssDNA11 and ssDNA12, and PL spectra of 22 (1.0  10À6 M) before (black), after (red) the addition of (a) ssDNA11–FAM and (b) ssDNA12– FAM (5.7  10À6 M, nucleicbase), and after addition of 10 ng/mL thrombin (blue) Measurements were done in PBS (pH ¼ 7.4) by excitation at 338 nm Reproduced with permission from [103] 446 K.-Y Pu and B Liu Scheme 14 Schematic representations of the assay for nucleases and protease detection based on the complex of ACP, DNA-TR and peptide-FI of the data The fluorescence pattern and the PCA score plots enabled to effectively distinguish 18 proteins from each other Although this array cannot be used for protein quantification and is less suitable for mixed samples, it has good reliability and versatility for the detection of purified proteins Recently, we reported a complex-based FRET assay capable of detecting protease and nuclease in one solution, which relied on a peptide-mediated combinatorial FRET between an ACP and TR-labeled ssDNA [104] The working mechanism of the assay is shown in Scheme 14 A solution of 5, peptide-1 (Arg-Arg-Arg-ArgArg-Arg-Arg-Arg-Arg-Arg), peptide-2 (Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-ArgArg-Fl), and ssDNA8-TR is prepared, within which FRET among 20, Fl, and TR occurs because of the formation of multicomponent complexes Before enzyme digestion, excitation of at 380 nm leads to dominant TR emission (red) in the solution fluorescence as shown in Fig 12 In the presence of trypsin, the peptides are digested into fragments, giving rise to relatively weak electrostatic attraction between peptide fragments and Under these conditions, the complexes become Fluorescence Reporting 447 120 5+peptide-1+TR-ssDNA 5+peptide-1/2 PL intensity (a.u.) 100 5+peptide-1/2 +TR-ssDNA 80 (x 1/4) 60 40 20 400 450 500 550 600 Wavelength (nm) 650 700 Fig 12 PL spectra of 5, 5/peptide-1,2, 5/peptide-1/TR-ssDNA and 5/peptide-1,2/TR-ssDNA upon excitation at 370 nm [5] ¼  10À7 M based on RU, [peptide-1] ¼ 4.5  10À8 M, [peptide-2] ¼  10À9 M, [ssDNA8-TR] ¼  10À9 M loose, and thus TR emission gradually decreases, and finally the polymer emission dominates the solution fluorescence (blue) On the other hand, in the presence of S1 nuclease, ssDNA8-TR is digested, which cause the release of TR from the complexes In this case, the TR emission intensity progressively decreases while the emission intensity for both Fl and increases, ultimately leading to green fluorescence of the solution Accordingly, the cleavage of peptide or DNA by trypsin or S1 nuclease can disturb the FRET within the complex of ACP/peptide-FI/DNA-TR, which allows the detection and monitoring of both protease and nuclease activity in the same solution Summary In light of the sensory systems elucidated herein, it is clear that CPEs have formed an intriguing basis for the construction of advanced FRET-based biosensors capable of detecting a variety of chemical and biological substances Facile modification of the molecular structures of CPEs provides opportunities to fine-tune their optoelectronic properties and charge natures to match specific sensory applications Within these sensors, CPEs not only participate in the signal transudation to diagnostic fluorophores but also serve as powerful light-harvesting donors to amplify signaling fluorescence, ultimately leading to greatly enhanced sensitivity relative to small-molecular fluorophore assays Both electrostatic and hydrophobic interactions 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Published and Forthcoming Volumes Advanced Fluorescence Reporters in Chemistry and Biology II Molecular Constructions, Polymers and Nanoparticles Volume Editor: A.P Demchenko Vol 9, 2010 Advanced Fluorescence. .. 1, 2001 Advanced Fluorescence Reporters in Chemistry and Biology II Molecular Constructions, Polymers and Nanoparticles Volume Editor: Alexander P Demchenko With contributions by G Bergamini Á... otto.wolfbeis@chemie.uni-regensburg.de Aims and Scope Fluorescence spectroscopy, fluorescence imaging and fluorescent probes are indispensible tools in numerous fields of modern medicine and science, including molecular biology,