Conjugated Polyelectrolyte Based Fluorescence Turn-On Assay for

Anal. Chem. 2010, 82, 8604–8610

Conjugated Polyelectrolyte Based Fluorescence Turn-On Assay for Real-Time Monitoring of Protease Activity Yanyan Wang, Yong Zhang, and Bin Liu* Department of Chemical and Biomolecular Engineering, 4 Engineering Drive 4, National University of Singapore, Singapore 117576, Singapore A fluorescence “turn-on” assay for monitoring protease activity is developed on the basis of a water-soluble carboxylated polyfluorene derivative, PFP-CO2Na, and its different fluorescence response toward cytochrome c (cyt c) and its fragments. PFP-CO2Na is synthesized via Suzuki coupling polymerization between 2,7-dibromo-9,9-bis(3′-tert-butyl propanoate)fluorene and 1,4-bis(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzene, followed by treatment with trifluoroacetic acid and Na2CO3. The fluorescence of PFP-CO2Na can be significantly quenched by cyt c due to complexationmediated electron transfer between the polymer and protein. Using the complex of PFP-CO2Na/cyt c as a substrate, a real-time fluorescence turn-on assay for trypsin activity study has been developed. Addition of trypsin to the substrate solution induces gradual recovery of the fluorescence intensity for PFP-CO2Na due to trypsin-catalyzed hydrolysis of cyt c, which dissociates the heme moiety from the polymer vicinity. The time-dependent fluorescence intensity increase of PFP-CO2Na in the presence of trypsin allows us to derive the initial reaction rates and kcat/Km (5350 M-1 s-1) for trypsin-catalyzed hydrolysis. Addition of trypsin inhibitor efficiently inhibits trypsin-catalyzed hydrolysis reaction of cyt c, which leads to a decreased fluorescence turn-on response of PFP-CO2Na. Conjugated polyelectrolytes (CPEs) combine the optoelectronic properties of conjugated polymers with the electrostatic behaviors of polyelectrolytes to provide a unique platform for construction of chemical and biological sensors.1-3 CPEs have shown lightharvesting properties, and their delocalized backbone structures allow for the development of various assays based on both superquenching4,5 and fluorescence resonance energy transfer * Corresponding author. Phone: +65-65168049. Fax: +65-67791936. E-mail: [email protected]. (1) Thomas, S. W.; Joly, G. D.; Swager, T. M. Chem. Rev. 2007, 107, 1339– 1386. (2) Ho, H. A.; Najari, A.; Leclerc, M. Acc. Chem. Res. 2008, 41, 168–178. (3) Liu, B.; Bazan, G. C. Chem. Mater. 2004, 16, 4467–4476. (4) Achyuthan, K. E.; Bergstedt, T. S.; Chen, L.; Jones, R. M.; Kumaraswamy, S.; Kushon, S. A.; Ley, K. D.; Lu, L.; McBranch, D.; Mukundan, H.; Rininsland, F.; Shi, X.; Xia, W.; Whitten, D. G. J. Mater. Chem. 2005, 15, 2648–2656. (5) Chen, L. H.; McBranch, D. W.; Wang, H. L.; Helgeson, R.; Wudl, F.; Whitten, D. G. Proc. Natl. Acad. Sci. U.S.A. 1999, 96, 12287–12292.

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(FRET)6-8 mechanisms. So far, various analytes, such as metal ions,9,10 small molecules,11,12 enzymes,13 proteins,14,15 DNA,16,17 and RNA,18 have been detected in aqueous solution or on solid substrates. Among these analytes, enzyme detection and their activity study are of high importance for the screening of pathologies and for the development of efficient therapeutics, due to their involvement in a variety of biochemical processes.19 Traditional methods for enzyme activity study include capillary isoelectric focusing, gel electrophoresis, high-performance liquid chromatography (HPLC), and enzyme-linked immunosorbent assay (ELISA).20-23 These methods require fluorescent labels, sophisticated instrumentations, and specific synthesis of peptides, which are time-consuming and are of high cost. To address these problems, fluorescence-based enzymatic assays have been developed to monitor enzyme activities by taking advantage of the intrinsic fluorescent signal change of CPEs upon interaction with different substrates and enzyme-digested products.24-34 One (6) Ho, H. A.; Dore, K.; Boissinot, M.; Bergeron, M. G.; Tanguay, R. M.; Boudreau, D.; Leclerc, M. J. Am. Chem. Soc. 2005, 127, 12673–12676. (7) Liu, B.; Bazan, G. C. J. Am. Chem. Soc. 2006, 128, 1188–1196. (8) Pu, K. Y.; Liu, B. Biosens. Bioelectron. 2009, 24, 1067–1073. (9) Kim, I. B.; Bunz, U. H. F. J. Am. Chem. Soc. 2006, 128, 2818–2819. (10) Wang, Y. S.; Liu, B. Macromol. Rapid Commun. 2009, 30, 498–503. (11) Wang, Y. Y.; Liu, B. Analyst 2008, 133, 1593–1598. (12) Zhan, R. Y.; Fang, Z.; Liu, B. Anal. Chem. 2010, 82, 1326–1333. (13) Tang, Y. L.; Feng, F. D.; He, F.; Wang, S.; Li, Y. L.; Zhu, D. B. J. Am. Chem. Soc. 2006, 128, 14972–14976. (14) Aberem, M. B.; Najari, A.; Ho, H. A.; Gravel, J. F.; Nobert, P.; Boudreau, D.; Leclerc, M. Adv. Mater. 2006, 18, 2703–2707. (15) Wang, Y. Y.; Liu, B. Langmuir 2009, 25, 12787–12793. (16) Gaylord, B. S.; Heeger, A. J.; Bazan, G. C. Proc. Natl. Acad. Sci. U.S.A. 2002, 99, 10954–10957. (17) Lee, K.; Povlich, L. K.; Kim, J. Adv. Funct. Mater. 2007, 17, 2580–2587. (18) Liu, B.; Baudrey, S.; Jaeger, L.; Bazan, G. C. J. Am. Chem. Soc. 2004, 126, 4076–4077. (19) Reich, E.; Rifkin, D. B.; Shaw, E. Proteases and Biological Control; Cold Spring Harbor Laboratory Press: New York, 1975. (20) Shimura, K.; Matsumoto, H.; Kasai, K. Electrophoresis 1998, 19, 2296– 2300. (21) Johnston, D.; Hermans, J. M.; Yellowlees, D. Arch. Biochem. Biophys. 1995, 324, 35–40. (22) Vestling, M. M.; Murphy, C. M.; Fenselau, C. Anal. Chem. 1990, 62, 2391– 2394. (23) Borovsky, D.; Powell, C. A.; Carlson, D. A. Arch. Insect Biochem. Physiol. 1992, 21, 13–21. (24) Pinto, M. R.; Schanze, K. S. Proc. Natl. Acad. Sci. U.S.A. 2004, 101, 7505– 7510. (25) Kumaraswamy, S.; Bergstedt, T.; Shi, X. B.; Rininsland, F.; Kushon, S.; Xia, W. S.; Ley, K.; Achyuthan, K.; McBranch, D.; Whitten, D. G. Proc. Natl. Acad. Sci. U.S.A. 2004, 101, 7511–7515. (26) Wosnick, J. H.; Mello, C. M.; Swager, T. M. J. Am. Chem. Soc. 2005, 127, 3400–3405. 10.1021/ac101695x  2010 American Chemical Society Published on Web 09/16/2010

strategy relies on fluorescence quenching of CPEs upon complexation or covalent binding with quencher-labeled peptide and the subsequent fluorescence increase upon enzymatic hydrolysis of the peptide.24-26 In addition, the fluorescence quenching of CPE by small molecules (e.g., Cu2+) has also been utilized to monitor the alkaline phosphatase and protease activity.27,28 The other strategy involves enzyme-digestion-triggered changes in FRET between CPEs and organic dyes.30-34 Recently, multiplex assays for both nuclease and protease activity study using a synthetic peptide/DNA complex or DNA logic circuits as the substrate have also been demonstrated.30,31 The currently available CPE-based enzymatic assays generally involve either modification of the substrate with fluorescent dye/quencher or premodification of CPE structures, which require multiple-step operations, and the substrate modification may also lead to different kinetic factors and binding constants as compared to those for the natural substrate.35,36 So far, very few CPE-based enzymatic assays involve natural substrates. On the other hand, CPEs have also been reported to interact with proteins to form CPE/protein complexes driven by Coulomb and/or hydrophobic interactions.16,37-39 Previous studies have shown that various proteins can quench the fluorescence of CPEs to different degrees depending on both the charge states and electron transfer character of the proteins.40 The quenching efficiency of CPEs in the presence of different proteins is reflected by the difference in Stern-Volmer quenching constants (Ksv). In general, a higher Ksv value indicates a more efficient fluorescence quenching process. The quenching mechanism could be due to energy transfer or charge transfer between the CPE and proteins or complexation-induced CPE aggregation or the combination of these effects.41 The fluorescence change of CPE upon protein binding has been widely used for protein detection and quantification.37,41-43 Cytochrome c (Cyt c) is a well-known electron transfer protein due to its heme moiety that significantly quenches the fluorescence of many CPEs.40,44 Cyt c has also been reported to be digested into fragments by trypsin, and the metal-containing heme peptide fragment has an isoelectric point (pI) of 7.0, which is (27) Liu, Y.; Schanze, K. S. Anal. Chem. 2008, 80, 8605–8612. (28) Liu, Y.; Schanze, K. S. Anal. Chem. 2009, 81, 231–239. (29) Zhang, T.; Fan, H. L.; Liu, G. L.; Jiang, J.; Zhou, J. G.; Jin, Q. H. Chem. Commun. 2008, 5414–5416. (30) Feng, X. L.; Duan, X. R.; Liu, L. B.; Feng, F. D.; Wang, S.; Li, Y. L.; Zhu, D. B. Angew. Chem., Int. Ed. 2009, 48, 5316–5321. (31) Zhang, Y.; Wang, Y. Y.; Liu, B. Anal. Chem. 2009, 81, 3731–3737. (32) Feng, X. L.; Liu, L. B.; Wang, S.; Zhu, D. B. Chem. Soc. Rev. 2010, 39, 2411–2419. (33) Chemburu, S.; Ji, E.; Casana, Y.; Wu, Y.; Buranda, T.; Schanze, K. S.; Lopez, G. P.; Whitten, D. G. J. Phys. Chem. B 2008, 112, 14492–14499. (34) An, L. L.; Tang, Y. L.; Feng, F. D.; He, F.; Wang, S. J. Mater. Chem. 2007, 17, 4147–4152. (35) Hergenrother, P. J.; Spaller, M. R.; Haas, M. K.; Martin, S. F. Anal. Biochem. 1995, 229, 313–316. (36) Kurioka, S.; Matsuda, M. Anal. Biochem. 1976, 75, 281–289. (37) Zhang, Y.; Liu, B.; Cao, Y. Chem.sAsian J. 2008, 3, 739–745. (38) Ambade, A. V.; Sandanaraj, B. S.; Klaikherd, A.; Thayumanavan, S. Polym. Int. 2007, 56, 474–481. (39) Herland, A.; Inganas, O. Macromol. Rapid Commun. 2007, 28, 1703–1713. (40) Fan, C. H.; Plaxco, K. W.; Heeger, A. J. J. Am. Chem. Soc. 2002, 124, 5642–5643. (41) Kim, I. B.; Dunkhorst, A.; Bunz, U. H. F. Langmuir 2005, 21, 7985–7989. (42) You, C. C.; Miranda, O. R.; Gider, B.; Ghosh, P. S.; Kim, I. B.; Erdogan, B.; Krovi, S. A.; Bunz, U. H. F.; Rotello, V. M. Nat. Nanotechnol. 2007, 2, 318–323. (43) Pu, K. Y.; Liu, B. Chem. Commun. 2010, 46, 1470–2472.

significantly lower than that for cyt c (pI 10.2),45,46 These properties offer us an opportunity to develop a fluorescence turnon assay for trypsin activity study. Trypsin is the most important digestive enzyme produced by the pancreas, which promotes other pancreatic proenzymes to their active forms and controls pancreatic exocrine function.47 A deficiency in the trypsin level or a mutated trypsin can cause pancreatic diseases such as meconium ileus and hereditary pancreatitis.48,49 In addition, the foot-andmouth disease virus has been found to be greatly reduced by trypsin treatment.50 Therefore, the ability to quantitatively monitor trypsin catalytic activity and its inhibitor is important for therapeutics of both pancreatic diseases and epidemic diseases. This paper is organized as follows. We first report the synthesis of a carboxylated poly(fluorene-co-phenylene) (PFP-CO2Na). This is followed by the study of polymer’s optical properties and its fluorescence response to cyt c. A continuous and sensitive “turn-on” enzymatic assay using the complex of PFP-CO2Na and cyt c as the substrate is subsequently developed. The concept of the turn-on assay is based on the quenching and dequenching of PFP-CO2Na by cyt c in the absence and presence of trypsin, respectively. At last, the effect of inhibitor on trypsin activities is studied, and the initial reaction rates and kinetic parameters are derived. Our method has several important features. First, no label is required on the substrate. This would not only reduce the cost, but also provide assay accuracy since labeling on the substrate may lead to low catalytic turnover of the enzymes with reduced reaction rates.35,36 Second, a natural substrate cyt c is employed, which has specific responses to enzymes. Third, this method provides real-time readout for the enzyme activity study. At last, the fluorescence turn-on assay reduces the likelihood of a false positive signal but also enhances the detection sensitivity as compared to that for turn-off assays. EXPERIMENTAL SECTION Materials. 2,7-Dibromo-9,9-bis(3′-tert-butyl propanoate)fluorene (1) and 1,4-bis(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzene (2) were synthesized as described in our previous publications.16,37 Pd(PPh3)4 and trifluoroacetic acid were purchased from Aldrich. Cyt c, trypsin, thrombin, and lysozyme were ordered from Sigma. Alkaline phosphatase (ALP) was ordered from Promega. PBS buffer (10×, pH 7.4, first BASE) was a commercial product and was used as received without further purification. Instrumentation. The 1H and 13C NMR spectra were taken on a Bruker 300 MHz spectrometer using a probe at 300 MHz for 1H NMR and 75 MHz for 13C NMR. UV-vis absorption (44) Liu, M.; Kaur, P.; Waldeck, D. H.; Xue, C. H.; Liu, H. Y. Langmuir 2005, 21, 1687–1690. (45) Busnel, J. M.; Descroix, S.; Le Saux, T.; Terabe, S.; Hennion, M. C.; Peltre, G. Electrophoresis 2006, 27, 1481–1488. (46) http://www . innovagen . se / custom - peptide - synthesis / peptide - property calculator/peptide-property-calculator.asp (accessed May 2010). (47) Rawlings, N. D.; Barrett, A. J. Families of Serine Peptidases. Proteolytic Enzymes: Serine and Cysteine Peptidases; Academic Press: London, 1994; Vol. 244, pp 19-61. (48) Noone, P. G.; Zhou, Z. Q.; Silverman, L. M.; Jowell, P. S.; Knowles, M. R.; Cohn, J. A. Gastroenterology 2001, 121, 1310–1319. (49) Whitcomb, D. C. Gut 1999, 45, 317–322. (50) Hernandez, J.; Valero, M. L.; Andreu, D.; Domingo, E.; Mateu, M. G. J. Gen. Virol. 1996, 77, 257–264.


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Scheme 1. Synthetic Route to Polymers PFP-CO2Bu and PFP-CO2Na

spectra were collected with a Shimadzu UV-2401 recording spectrophotometer. Fluorescence was measured with thermocontrol using a Perkin-Elmer LS-55 equipped with a xenon lamp excitation source and a Hamamatsu (Japan) 928 PMT, using 90° angle detection for solution samples. Fluorescence quantum yields were measured with quinine sulfate in 0.1 M H2SO4 solutions as a reference. The buffer pH was measured using a pH meter (Sartorius PB-10) with a glass/reference electrode, calibrated with buffer solutions of pH 4, 7, and 10. Matrixassisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) measurements were obtained using a Bruker Daltonics Autoflex TOF/TOF instrument. Polymer Synthesis. Poly[9,9-Bis(3′-tert-butyl propanoate)fluorene-co-1,4-phenylene] (PFP-CO2Bu). Monomers 1 (291 mg, 0.5 mmol) and 2 (165 mg, 0.5 mmol), K2CO3 (360 mg), Pd(PPh3)4 (10 mg), toluene (10 mL), and water (3 mL) were mixed in a 50 mL two-neck flask. After degassing, the mixture was heated at 90 °C with vigorous stirring for 48 h. After the mixture was cooled to room temperature, it was poured into methanol. The precipitate was collected by filtration to yield PFP-CO2Bu (210 mg, 84% yield) as fibers. 1H NMR (300 MHz, CDCl3): δ 7.94-7.40 (m, 10H), 2.51 (s, 4H), 1.64 (s, 4H), 1.31 (s, 18H). Sodium Poly[9,9-bis(3′-propanoate)fluorene-co-phenylene] (PFPCO2Na). In a 50 mL flask, PFP-CO2Bu (100 mg) was dissolved in dichloromethane (20 mL). After addition of trifluoroacetic acid (3 mL), the mixture was stirred overnight at room temperature. After removal of the solvent, the yellow-green residue was treated with Na2CO3 aqueous solution (0.05 M, 20 mL) at room temperature for 4 h. The polymer was purified through dialysis against distilled water for 3 days. The solution was freeze-dried to give PFP-CO2Na (65 mg, 76% yield) as a powder. 1H NMR (300 MHz, CD3OD): δ 7.88-7.31 (m, 10H), 2.56 (s, 4H), 1.60 (s, 4H). 13C NMR (75 MHz, CD3OD): δ 150.56, 140.54, 127.48, 126.79, 126.32, 121.68, 120.20, 54.90, 36.84, 32.44. Fluorescence Quenching. The quenching experiments were performed upon successive addition of cyt c to a solution of PFPCO2Na (1 µM based on repeat units) in 20 mM PBS buffer at pH 8.9 at 37 °C. The fluorescence spectra were recorded at various cyt c concentrations under 380 nm excitation. PL0/PL was used for the Ksv plot, where PL0 is the initial fluorescence intensity of PFP-CO2Na and PL is the quenched fluorescence intensity of PFP-CO2Na upon addition of cyt c. 8606


Trypsin Activity Assay. Trypsin (0, 5, 10, 20, 30, and 40 nM) was first added into the cuvettes containing PFP-CO2Na (1 µM) and cyt c (200 nM) in 20 mM PBS buffer at pH 8.9. The mixtures were then incubated at 37 °C for 30 min. The fluorescence spectra of the mixtures were measured at 60 s intervals under excitation at 380 nm. The emission spectra and the fluorescence intensity at 415 nm were recorded. Trypsin Inhibition Experiment. Trypsin (30 nM) and benzamidine hydrochloride with varying concentrations (0-100 nM) were preincubated at 25 °C for 30 min. The mixtures were added into the solution of PFP-CO2Na (1 µM) and cyt c (200 nM) in 20 mM PBS buffer at pH 8.9. After incubation at 37 °C, the solution fluorescence spectra were measured at 60 s intervals under 380 nm excitation. The emission spectra and the fluorescence intensity at 415 nm were recorded. RESULTS AND DISCUSSION Synthesis and Characterization. The synthetic route to the polymers is shown in Scheme 1. 1 was prepared in 51% yield by direct alkylation of 2,7-dibromofluorene with tert-butyl acrylate in a toluene/aqueous KOH mixture, which was followed by purification using silica column chromatography. 2 was synthesized under Miyaura reaction conditions in the presence of bis(pinacolato)diborane, Pd(dppf)2Cl2, and KOAc using dry DMF as the solvent.51 The NMR spectroscopy, MS, and elemental analysis results show that the monomers were obtained in the correct structures with high purity. Polymerization between 1 equiv of 1 and 2 was conducted in a mixture of toluene and 2 M K2CO3 under an argon atmosphere for 24 h to yield PFP-CO2Bu in 84% yield. The 1H NMR spectrum of PFP-CO2Bu in CDCl3 has shown a chemical shift of 1.31 ppm, which corresponds to the protons for the -C(CH3)3 group, indicating the existence of carboxylic ester groups in the polymer side chains. PFP-CO2Bu is soluble in organic solvents, such as toluene, dichloromethane, chloroform, and THF. PFP-CO2Na was prepared by hydrolysis of PFPCO2Bu in the presence of CF3COOH and dichloromethane (v/v ) 1/1), which was followed by reaction with 0.1 M Na2CO3 overnight. PFP-CO2Na was obtained in 76% yield after purification through dialysis (cutoff molecular weight 3500) against (51) Ishiyama, T.; Murata, M.; Miyaura, N. J. Org. Chem. 1995, 60, 7508–7510.

Figure 2. PL quenching curve of PFP-CO2Na (1 µM) by cyt c in 20 mM PBS with excitation at 380 nm in PBS buffer (pH 8.9). Inset: Stern-Volmer curve of PFP-CO2Na quenched by cyt c with the error bar indicated. PL0 is the initial fluorescence intensity of PFP-CO2Na, and PL is the quenched fluorescence intensity of PFP-CO2Na with a certain cyt c concentration.

Figure 1. (a) Normalized absorption and photoluminescence spectra of PFP-CO2Na in water (pH 6) and in buffer solution (20 mM PBS, pH 8.9). (b) Photoluminescence spectra of PFP-CO2Na at different pH values.

DI water for 3 days to remove salt and small molecular weight fractions. In the 1H NMR spectrum of PFP-CO2Na, the chemical shift at 1.31 ppm disappeared, which indicated the complete conversion of -COOC(CH3)3 to -COONa. In contrast to PFPCO2Bu, PFP-CO2Na is soluble in water, DMSO, and methanol. The water solubility of PFP-CO2Na is >5 mg/mL in deionized water (pH 6) at 25 °C. Optical Properties. The UV-vis and photoluminescence spectra of PFP-CO2Na in water (pH 6.5) and in PBS buffer (20 mM, pH 8.9) are shown in Figure 1a. The PL quantum yields of PFP-CO2Na in water (pH 6.5) and in 20 mM PBS buffer (pH ) 8.9) are 0.21 and 0.59, respectively, measured using quinine sulfate in 0.1 M H2SO4 (quantum yield 0.55) as a reference. The polymer has an absorption maximum of 380 nm in water and 376 nm in buffer. In water, PFP-CO2Na emits blue fluorescence with a main peak at 434 nm and a shoulder at 455 nm. Increasing the pH to 8.9 leads to a blue shift in the emission maximum to 414 nm, which is similar to that of poly(fluorene-co-1,4-phenylene) in organic solvents.52 This clearly indicates the good solubility of PFP-CO2Na in buffer. The PL spectra of PFP-CO2Na at various pH values are shown in Figure 1b. The PL intensity of PFP-CO2Na changes with pH and increases from pH 4 to pH 9. The highest fluorescence intensity was observed at pH 9 in the tested pH range. The (52) Donat-Bouillud, A.; Levesque, I.; Tao, Y.; D’Iorio, M.; Beaupre, S.; Blondin, P.; Ranger, M.; Bouchard, J.; Leclerc, M. Chem. Mater. 2000, 12, 1931– 1936.

pH-dependent optical behavior of PFP-CO2Na is believed to be associated with protonation and deprotonation of the -CO2Na group in the polymer side chain. The pKa value of PFP-CO2Na was estimated to be around 4-6 (pKa values for heptane diacid are pKa1 ) 4.48 and pKa2 ) 5.42) due to the similarity between heptane diacid and the side chains of PFP-CO2Na.53 In water, some terminal groups of polymer side chains could exist as -COOH.37 In PBS buffer solution (pH 8.9), negatively charged polymer side chains became dominant in polymer solution. The repulsion between these negatively charged side chains led to minimum polymer aggregation and a high quantum yield of 0.59. Fluorescence Quenching. The fluorescence quenching of PFP-CO2Na was studied in PBS buffer (pH 8.9). This pH is selected to ensure cyt c is positively charged while the peptide fragment containing the heme moiety is negatively charged (pI 7.0). As shown in Figure 2, addition of cyt c (0-200 nM) to 1 µM PFP-CO2Na in PBS buffer quenches the polymer fluorescence, and the extent of quenching increases with increased cyt c concentration. The corresponding Stern-Volmer plot (PL0/PL vs [cyt c]) is shown in the inset of Figure 2. The plot is almost linear in the low cyt c concentration range, and the Stern-Volmer constant (Ksv) derived from the linear region is calculated to be ∼1.32 × 107 M-1. Overview of the Fluorescence Turn-On assay. The illustration of the trypsin turn-on assay is shown in Scheme 2. PFP-CO2Na emits strong blue fluorescence in solution at pH 8.9. Upon addition of cyt c, PFP-CO2Na/cyt c complexes formed simultaneously due to electrostatic attraction. This leads to quenched fluorescence of PFP-CO2Na. Introduction of trypsin to the polymer/protein complex induces hydrolysis of cyt c. Trypsin catalyzes the hydrolysis of peptide bonds by the C side of lysine or arginine in proteins to give more than 15 fragments.45 The digestion converts positively charged cyt c to a negatively charged heme-containing fragment at pH 8.9, which disturbs the complexation of PFP-CO2Na/heme center due to electrostatic repulsion between the fragments and the polymer. As a (53) Nachod, F. C.; Zuckerman, J. J. Determination of Organic Structures by Physical Methods; Academic Press: New York, 1972.


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Scheme 2. Schematic Illustration of the Fluorescence “Turn-On” Assay for Trypsin Screening

consequence, after cleavage of cyt c by trypsin, the prequenched PFP-CO2Na/cyt c could regain blue fluorescence. A fluorescence turn-on trypsin assay could thus be realized by the naked eye using the PFP-CO2Na/cyt c complex as the substrate. By measuring the change of the PFP-CO2Na emission intensity, kinetic investigation and inhibition study of trypsin are also allowed. Assay for Trypsin Activity Study. The trypsin-catalyzed hydrolysis of cyt c was monitored in the presence of PFP-CO2Na. The full sequence of cyt c, the possible hydrolysis point by trypsin, and the corresponding pI of the digested fragments are provided in Figure S1 and Table S1 in the Supporting Information.45,54 After full cleavage, the fragment with the heme complex should be left in the form of Cys-Ala-Gln-Cys-His-ThrVal-Glu-Lys-heme, which has net negative charges at pH 8.9. We thus expect much less efficient electron transfer after cleavage since both the polymer and the heme-containing fragment are negatively charged. Figure 3 shows the typical change in fluorescence spectra for a trypsin digestion assay carried out at 37 °C. Upon addition of 200 nM cyt c to 1 µM PFP-CO2Na, the solution fluorescence (1 µM) is significantly quenched to 10% of the original intensity. After further addition of 30 nM trypsin to this solution followed by 15 min of incubation, the prequenched polymer fluorescence is recovered up to 60% of the original intensity (Figure 3a). Meanwhile, a series of experiments were performed under the same conditions using control enzymes such as lysozyme, ALP, and thrombin. As shown in Figure 3b, none of these enzymes can recover the quenched fluorescence of PFP-CO2Na. These observations confirm that the fluorescence recovery of PFP-CO2Na is generated from trypsincatalyzed hydrolysis of cyt c, which exhibits high specificity of trypsin turn-on assay with the PFP-CO2Na/cyt c complex as the substrate. To further understand the cleavage of cyt c by trypsin, MALDITOF-MS was used to identify the peptide fragments. Figure 4 highlights the MALDI mass spectra (mass range from 800 to 1800 Da) of cyt c (1 × 10-3 M) after digestion by trypsin in 20 mM PBS buffer with pH 8.9. The full MALDI mass spectrum (mass range from 280 to 1800 Da) is shown in Figure S2 (Supporting Information). As shown in Figure 4, the heme/peptide complex was detected with a molecular mass of 1634.95 Da, which is in good agreement with the expected molecular mass (1635.1 Da) of the heme/peptide complex. The peak at 1168.97 Da is ascribed to the fragment of TGPNLHGLFGR with pI 11.0. As shown in Figure S2, the major fragments have molecular mass between 280 and 400 Da, which could only slightly affect the fluorescence of (54) Bushey, M. M.; Jorgenson, J. W. J. Microcolumn Sep. 1990, 2, 293–299.

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PFP-CO2Na through electrostatic-interaction-mediated polymer aggregation. The peaks in the MALDI mass spectrum of cyt c after digestion strongly support the observed fluorescence recovery (turn-on) of the PFP-CO2Na/cyt c complex substrate as discussed above. Determination of Trypsin-Catalyzed Cyt c Hydrolysis Kinetic Parameters. To demonstrate the feasibility of using PFPCO2Na/cyt c substrate for real-time trypsin activity study, the trypsin-catalyzed hydrolysis of cyt c as a function of time upon incubation of PFP-CO2Na/cyt c with several different trypsin concentrations (0-40 nM) was investigated. These experiments were conducted in 20 mM PBS buffer (pH 8.9) at 37 °C

Figure 3. (a) PL spectra of 1 µM PFP-CO2Na (blue, 1), upon addition of 200 nM cyt c (red, 2), and after 15 min of incubation with 30 nM trypsin (green, 3). Inset: Photographs were taken upon UV lamp excitation at 365 nm. (b) Changes in fluorescence intensity at 415 nm after 15 min of incubation of PFP-CO2Na/cyt c with 40 nM lysozyme, 40 nM thrombin, or 1 unit/mL ALP. Conditions: 20 mM PBS buffer (pH 8.9) at 37 °C, λex ) 380 nm.

Figure 4. MALDI mass spectra of tryptic peptides originated from cyt c digested with trypsin under 37 °C in 20 mM PBS buffer with pH 8.9.

Figure 5. Fluorescence intensity changes of the PFP-CO2Na (1 µM) and cyt c (200 nM) complex at 415 nm in 20 mM PBS buffer (pH 8.9) at 37 °C against time after addition of trypsin (0, 5, 10, 20, 30, and 40 nM), λex ) 380 nm.

containing 1 µM PFP-CO2Na and 200 nM cyt c. Figure 5 illustrates the increase of PFP-CO2Na fluorescence intensity at 415 nm with increased incubation time at each trypsin concentration, where the fluorescence intensity was measured every 60 s. It is observed that the fluorescence recovers more quickly and more completely in the presence of high trypsin concentrations, which is further demonstrated by the initial rate of trypsin-catalyzed reaction (v0). The Stern-Volmer plot shown in the inset of Figure 2 affords the calibration curve for calculating the substrate concentration, [cyt c], remaining in solution at any specific digestion time by measuring PL0/PL. The plot of [cyt c] vs time is shown in Figure S3 in the Supporting Information. The slopes of the plots at early time were calculated to afford v0 for the six different concentrations of trypsin investigated, which was then plotted against [trypsin] (Figure 6). The linear relationship between enzyme concentration and initial rate indicates that trypsin-catalyzed hydrolysis reaction is kinetically controlled using the PFP-CO2Na/cyt c complex as the substrate. The limit of detection (LOD) of trypsin is ∼1.7 nM, which is calculated from the calibration curve in Figure 6 (based on 3σ from five independent measurements).

Figure 6. Relationship between the initial rate of trypsin-catalyzed hydrolysis reaction (v0) and the trypsin concentration with the error bar indicated. The PFP-CO2Na (1 µM) and cyt c (200 nM) complex was incubated with various trypsin concentrations in 20 mM PBS buffer (pH 8.9) at 37 °C.

Figure 7. Kinetic parameters (Vmax/Km) as a function of the trypsin concentration with the error bar indicated. The PFP-CO2Na (1 µM) and cyt c (200 nM) complex was incubated with various trypsin concentrations in 20 mM PBS buffer (pH 8.9) at 37 °C.

The feasibility of polymer-based trypsin turn-on assay is then investigated via Michaelis-Menten analysis.55 The natural logarithm of [cyt c] as a function of the incubation time is shown in Figure S4 (Supporting Information) by converting the y axis of Figure S3 to the logarithm. Similarly, the slopes of the plots at early time are calculated for each trypsin concentration, which is defined as the kinetic parameter (Vmax/Km)56 on the basis of the method reported by Schanze.27 Figure 7 exhibits a linear relationship between the kinetic parameter and trypsin concentration, and the slope is defined as the specificity constant (kcat/ Km),57 which is calculated to be 5350 M-1 s-1. This constant is reasonable since it is within the range of 3500-62000 M-1 s-1 reported for tryptic hydrolysis of various peptides.58-60 These results demonstrate that it is possible to monitor trypsin in real (55) Lehninger, A. L.; Nelson, D. L.; Cox, M. M. Principles of Biochemistry; Worth Publishers: New York, 1993. (56) Vmax is the maximum rate of the enzyme-catalyzed reaction at the saturation substrate concentration by a fixed enzyme concentration; Km is the Michaelis-Menten constant, which is the substrate concentration that gives 1/2Vmax. (57) kcat is the catalytic constant or turnover number. (58) Huang, G. J.; Ho, Y. L.; Chen, H. J.; Chang, Y. S.; Huang, S. S.; Hung, H. J.; Lin, Y. H. Bot. Stud. 2008, 49, 101–108. (59) Kang, K.; Kan, C. Y.; Yeung, A.; Liu, D. S. Macromol. Biosci. 2005, 5, 344– 351. (60) Caprioli, R. M.; Smith, L. Anal. Chem. 1986, 58, 1080–1083.


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rate of trypsin-catalyzed hydrolysis reaction in the presence of different amounts of inhibitor (v0′) was calculated using the same method applied for calculation of v0 in the absence of inhibitor. Figure 8b shows that the higher inhibitor concentrations lead to lower v0′ values. These results indicate that the digestion of cyt c by trypsin is effectively inhibited by benzamidine hydrochloride. In addition, this study also demonstrates that the PFP-CO2Na and cyt c complex could be used as a sensitive probe to study trypsin activity, as well as to screen potential drugs on the basis of the inhibition of the cleavage reactions catalyzed by trypsin.

Figure 8. (a) Fluorescence intensity changes of the PFP-CO2Na/ cyt c complex at 415 nm as a function of the trypsin digestion time with different amounts of inhibitor. (b) Initial rate of reaction (v0) versus inhibitor concentration with the error bar indicated. Experimental conditions: [PFP-CO2Na] ) 1 µM, [cyt c] ) 200 nM, and [trypsin] ) 30 nM in 20 mM PBS buffer (pH 8.9) at 37 °C, λex ) 380 nm.

time and calculate enzyme kinetic parameters on the basis of the fluorescence change of PFP-CO2Na using the PFP-CO2Na/cyt c complex as the substrate. Inhibition of Trypsin Catalysis. Benzamidine hydrochloride is one of the trypsin activity inhibitors.26,34 It is thus expected that it is able to inhibit the cleavage of cyt c by trypsin, leading to less efficient protein digestion. The inhibition capacity was tested in assays conducted under the same conditions used for the kinetic studies described above. Figure 8a shows the real-time fluorescence intensity change of the PFP-CO2Na (1 µM)/cyt c (200 nM) complex in the presence of 30 nM trypsin and different amounts of benzamidine hydrochloride. The fluorescence recovery (turn-on) of PFP-CO2Na decreased with increased inhibitor amount, indicating that the overall inhibition is more effective at high inhibitor concentrations. The initial

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CONCLUSION In summary, we report the synthesis of a water-soluble carboxylated polyfluorene derivative, PFP-CO2Na. The polymer fluorescence can be quenched by cyt c with a Stern-Volmer constant (Ksv) of ∼1.32 × 107 M-1 at pH 8.9. Using the PFPCO2Na/cyt c complex as the substrate, a continuous fluorescence turn-on trypsin assay has been developed. The fluorescence of the prequenched PFP-CO2Na/cyt c complex recovers due to trypsin-catalyzed cyt c digestion. Monitoring of trypsin activity has been realized by measuring the recovered fluorescence intensity of PFP-CO2Na in the presence of trypsin. The initial reaction rate increases with increased trypsin concentrations. The kcat/Km of trypsin-catalyzed hydrolysis reaction is calculated to be 5350 M-1 s-1, according to the Stern-Volmer plots. Addition of trypsin inhibitor induces a decreased fluorescence turn-on response of PFP-CO2Na and a decreased initial reaction rate. This fluorescence turn-on strategy could also be extended to other CPEs and protein substrates for various enzyme activity studies, provided that the protein is able to quench the fluorescence of the CPE. ACKNOWLEDGMENT We are grateful to the National University of Singapore (NUS Grants ARF R-279-000-197-112/133, R-279-000-234-123, and OLS R-279-000-255-112) for financial support. Y.W. thanks the National University of Singapore for support via a research scholarship. The first two authors contributed equally to this work. SUPPORTING INFORMATION AVAILABLE Synthesis and characterization of the monomers and polymers and related fluorescence quenching curves. This material is available free of charge via the Internet at http://pubs.acs.org. Received for review June 28, 2010. Accepted September 2, 2010. AC101695X

Conjugated Polyelectrolyte Based Fluorescence Turn-On Assay for

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