Synthesis and fluorescence study of water-soluble conjugated polymers for efficient FRET-based DNA detection

Current Applied Physics 9 (2009) 636–642

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Synthesis and fluorescence study of water-soluble conjugated polymers for efficient FRET-based DNA detection Rati Ranjan Nayak a, Okhil Kumar Nag a, Han Young Woo a,*, Sungu Hwang a, Doojin Vak b, Dmitry Korystov b, Youngeup Jin c, Hongsuk Suh c a

Department of Nano Fusion Technology (BK21), Pusan National University, Miryang 627-706, Republic of Korea Institute for Polymers and Organic Solids, University of California, Santa Barbara, CA 93106, USA c Department of Chemistry, Pusan National University, Busan 609-735, Republic of Korea b

a r t i c l e

i n f o

Article history: Received 23 April 2008 Accepted 23 April 2008 Available online 7 June 2008 PACS: 36.20.-r Keywords: Water-soluble conjugated polymers FRET Energy transfer Fluorescence Conjugated polyelectrolytes

a b s t r a c t Two cationic conjugated polyelectrolytes (CPs, P1i and P2i) were synthesized and examined as a fluorescence resonance energy transfer (FRET) donor to fluorescein (Fl)-labeled single-stranded DNA (ssDNA–Fl) using steady-state and time-resolved photoluminescence (PL) spectroscopy. The two polymers have the same p-conjugation with the main structural difference being the presence of the spiro-anthracenyl substituents orthogonal to the polymer backbone of P2i. These spiro-substituents can function as a molecular spacer that increases the intermolecular separation in the electrostatic complex with ssDNA–Fl. We measured almost complete PL quenching of the excited Fl* after electrostatic complexation with P1i (PL lifetime 4 ns ? 78 ps) and relatively moderate quenching with P2i (PL lifetime 4 ns ? 552 ps). A quenching efficiency (UeT) of 98% and 86% was obtained for P1i/ssDNA–Fl and for P2i/ssDNA–Fl, respectively. Both systems have same thermodynamic driving force for quenching as a result of them having the same electronic structures. This discrepancy can be explained in terms of the reduced quenching (via electron transfer, eT) by the increased D–A distance due to the existence of spiro-attached molecular spacers in P2i. It shows that thermodynamically favorable eT quenching can be controlled kinetically by modulating the D–A intermolecular distance using molecular spacers, which suggests an important molecular design guideline for efficient CPs-based DNA detection. Ó 2008 Elsevier B.V. All rights reserved.

1. Introduction There has been increasing interest in water-soluble conjugated polyelectrolytes (CPs) as optical platforms in fluorescence-based sensor applications [1–7]. The great potential of CPs in these applications can be attributed to the unique light-harvesting and superquenching of the emitted fluorescence. Recent papers have reviewed the synthesis and sensing-related properties of various conjugated polyelectrolytes, including polythiophene, poly(p-phenylene), poly(phenylenevinylene), poly(phenyleneethynylene), and oligonucleotide-functionalized polymers, etc. [8– 12]. Homogeneous DNA hybridization assays based on a fluorescence resonance energy transfer (FRET) or photo-induced charge transfer (PCT) mechanism are attractive and constitute an important detection scheme [13–19]. Bazan demonstrated successful DNA detection based on the FRET method, which involves the use of cationic CPs and hybridization between the fluorophore-labeled peptide nucleic acids (PNA) and complementary (or non-

* Corresponding author. Tel.: +82 55 350 5300; fax: +82 55 350 5653. E-mail address: [email protected] (H.Y. Woo). 1567-1739/$ - see front matter Ó 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.cap.2008.04.014

complementary) DNA [20,21]. Electrostatic attractions between positive CPs and negatively charged DNA can be coordinated to produce efficient FRET and design sequence-specific DNA assays. This scheme benefits from using the light-harvesting (or antenna-like) properties of water-soluble CPs to achieve the sensory signal amplification in the presence of suitable energy or electron acceptors. The interaction between cationic CPs and negatively charged DNA results in the formation of electrostatic complexes, the structure of which is poorly understood, and needs to be controlled in order to optimize the FRET process. The molecular structure of the CPs must play an important role in determining the overall aggregate size, the distance between the optically active backbone and the acceptor dye, and the degree to which FRET or photo-induced charge transfer (PCT) takes place [22–24]. Understanding how these variations work together in complicated biological mixtures is essential for a rational design of the fully optimized FRETbased DNA assays. This paper reports the synthesis and photoluminescence spectroscopic studies of two water-soluble polymeric structures, poly(9,90 -bis(6-N,N,N-trimethylammoniumhexyl)fluorene-alt-1,4(2,5-bis(6-N,N,N-trimethylammoniumhexyloxy))phenylene) tetra-

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Scheme 1. Fluorescence resonance energy transfer from CPs to ssDNA–Fl via electrostatic complexation.

bromide (P1i) and poly((10,100 -bis(6-N,N,N-trimethylammoniumhexyl)-10H-spiro(anthracene-9,90 -fluorene))-alt-1,4-(2,5-bis(6-N,N,Ntrimethylammoniumhexyloxy))phenylene) tetrabromide (P2i). The two polymers have the same p-conjugated electronic structures with the main structural difference being the presence of the spiro-attached anthracenyl substituents on the polymer backbone of P2i. Fluorescence energy transfer from the two polymers to fluorescein-labeled single-stranded DNA (ssDNA–Fl) was investigated using steady-state and time-resolved PL spectroscopy (Scheme 1). The results showed almost complete PL quenching of the excited Fl* in P1i/ssDNA–Fl and relatively moderate quenching in P2i/ssDNA–Fl. As detailed below, intermolecular distance modulation using spiro-attached molecular spacers in P2i can allow the fine-control of PCT induced quenching of the Fl in the complex, which suggests an important molecular design guideline for efficient CPs-based biosensors.

2. Experimental part 2.1. General details All chemicals were purchased from Aldrich Co. and used without further purification. The oligonucleotides were purchased from Genscript Corp. and DNA concentrations were determined by measuring the absorbance at 260 nm in a 200 lL quartz cuvette. The ssDNA–Fl is a 20 base single-stranded DNA with a sequence of 50 -fluorescein-ATCTT GACTA TGTGG GTGCT-30 . FRET experiments were performed by successive addition of polymers to a solution of ssDNA–Fl (108 M) in water at pH 8 at room temperature. 1 H and 13C NMR spectra were collected on a Varian Unity 200 MHz (or JEOL JNM-AL 300 MHz) spectrometer. The UV/vis absorption spectra were recorded on a Shimadzu UV-2401 PC diode array spectrometer. The photoluminescence spectra were obtained on a PTI Quantum Master fluorometer equipped with a Xenon lamp excitation source. The fluorescence quantum yields were measured relative to fluorescein and 9,10-diphenylanthracene. Mass spectrometry was performed by UC Santa Barbara and Korea Advanced Institute of Science and Technology Mass Spectrometry Lab. PL lifetime measurements were performed using a time-correlated single-photon counting technique (TCSPC). The PL was generated by laser pulses with a duration of nearly 100 fs, which are produced via second harmonic generation process from the output of mode-locked Ti:sapphire laser (Spectrophysics Tsunami). The repetition rate of the laser pulses was reduced using a home-made acousto-optical pulse picker. The luminescence was dispersed in a spectrometer and detected by a microchannel-plate photomultiplier tube (MCP PMT; Hamamatsu R3809U-51).

2.2. Materials 2.2.1. Synthesis of 2,7-bis(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2yl)-9,9-bis(60 -bromohexyl)fluorene (2) To a solution containing 2,7-dibromo-9,9-bis(6-bromohexyl)9H-fluorene (3.0 g, 4.6 mmol) in dry THF (70 mL), t-butyllithium (13 mL, 1.7 M in pentane) was added at 78 °C. The solution was stirred at 78 °C for 2 h. To the above solution, 2-isopropoxy4,4,5,5-tetramethyl-1,3,2-dioxaborolane (5.6 mL, 28 mmol) was injected and the resulting solution was stirred at 78 °C for a further 2 h. A small amount of water was added to quench the reaction. The solution was warmed to room temperature, extracted with chloroform and washed several times with water. The organic layer was dried over magnesium sulfate and evaporated under reduced pressure. The product was purified by silica gel column chromatography (ethylacetate/hexane, 1/19) to produce 1.85 g (53.8%) of a white powder. 1 H NMR (300 MHz, CDCl3, d, ppm): 7.82 (m, 6H), 3.28 (t, 4H), 2.03 (m, 4H), 1.67 (m, 4H), 1.37 (s, 24H), 1.08 (m, 8H), 0.55 (m, 4H). 13C NMR (100 MHz, CDCl3, d, ppm): 150.07, 143.89, 133.79, 128.77, 119.46, 83.77, 55.04, 40.0, 33.92, 32.65, 28.94, 27.72, 24.94, 23.37. HRMS (m/z): calcd for C37H54B2Br2O4, 742.26; found, [M]+ = 742.2592. 2.2.2. Synthesis of (6-chlorohexyloxy)(tert-butyl)dimethylsilane (4) Triethylamine (4.62 mL, 33 mmol) was injected into a two-neck flask containing tert-butylchlorodimethylsilane (4.98 g, 33 mmol) and chlorohexanol (4.08 g, 30 mmol) in chloroform (50 mL) under argon. The solution was refluxed for 1 h. An excess of HCl was poured into the reaction mixture and extracted with dichloromethane. The organic layer was washed with water and dried over magnesium sulfate. The product was purified by silica gel column chromatography (dichloromethane/hexane, 1/9) to produce 7.0 g (93%) of a colorless liquid. 1 H NMR (200 MHz, CDCl3, d, ppm): 3.50 (m, 4H), 1.70 (m, 2H), 1.40 (m, 6H), 0.84 (s, 9H), 0.01 (s, 6H). 13C NMR (100 MHz, CDCl3, d, ppm): 63.21, 45.25, 32.84, 26.90, 26.17, 25.35, 18.56, 5.08. HRMS (HREI, m/z): calcd for C12H27ClOSi, 250.15; found [M– C4H9]+ = 193.0824. 2.2.3. Synthesis of 10,10-bis(6-tert-butyldimethylsilyloxyhexyl)-10Hspiro(anthracene-9,90 -(20 ,70 -dibromofluorene)) To a two-neck flask containing compound 3 (0.98 g, 2.0 mmol), compound 4 (1.5 g, 6 mmol) and 18-crown-6 (0.1 g, 0.4 mmol) in THF (40 mL), excess KH in mineral oil was added under an argon flow. The solution was stirred overnight and methanol was added dropwise to the reaction mixture to deactivate the remaining KH. The solution was extracted with dichloromethane and washed several times with water. The organic layer was dried over magnesium sulfate and concentrated under reduced pressure. The product was

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purified by silica gel column chromatography (dichloromethane/ hexane, 1/4) to produce 1.37 g (75%) of a white powder. 1 H NMR (200 MHz, CDCl3, d, ppm): 7.68 (d, 2H, J = 6.6 Hz), 7.47 (m, 4H), 7.23 (m, 2H), 7.05 (s, 2H), 6.90 (t, 2H, J = 8.0 Hz), 6.28 (d, 2H, J = 8.0 Hz), 3.52 (t, 4H, J = 6.6 Hz), 2.12 (m, 4H), 1.41 (m, 4H), 1.21 (br, 8H) 0.87 (m, 22H), 0.00 (s, 12H). 13C NMR (100 MHz, CDCl3, d, ppm): 159.81, 139.04, 138.10, 136.19, 130.59, 129.01, 128.54, 127.50, 126.25, 125.87, 122.30, 121.22, 63.24, 57.81, 46.72, 45.56, 32.74, 29.83, 25.99, 25.79, 25.58, 18.36, 5.27. HRMS (HREI, m/z): calcd for C50H68Br2O2Si2, 914.31; found [M– C4H9]+ = 857.2406.

was extracted with dichloromethane, dried over magnesium sulfate, and concentrated by evaporation. The concentrated solution was distillated under vacuum to remove excess 1,6-dibromohexane. The remaining crude product was purified by silica gel column chromatography (dichloromethane/hexane, 1/4) to produce 5.85 g (65%) of a white powder. 1 H NMR (300 MHz, CDCl3, d, ppm): 7.09 (s, 2H), 3.94 (t, 4H), 3.42 (t, 4H), 1.85 (m, 8H), 1.54 (m, 8H). 13C NMR (100 MHz, CDCl3, d, ppm): 150.06, 118.51, 111.17, 70.03, 33.74, 32.64, 28.91, 27.81, 25.18. HRMS (HREI, m/z): calcd for C18H26Br4O2, 589.87; found [M]+ = 589.8745.

2.2.4. Synthesis of 10,10-bis(6-bromohexyl)-10H-spiro(anthracene9,90 -(20 ,70 -dibromofluorene)) (5) A 0.33 M Br2PPh3 solution was prepared by dissolving triphenylphosphine (5.2 g, 20 mmol) in dichloromethane (60 mL), followed by the slow addition of bromine (1.0 mL, 20 mmol) under argon. To a two-neck flask containing 10,10-bis(6-tert-butyldimethylsilyloxyhexyl)-10H-spiro(anthracene-9,90 -(20 ,70 -dibromofluorene)) (1.37 g, 1.5 mmol) in dichloromethane (30 mL), the above Br2PPh3 solution (10 mL) was slowly injected at room temperature. The solution was stirred at room temperature for 30 min. The solution was extracted with dichloromethane and washed with water several times. The organic layer was dried over magnesium sulfate and concentrated by evaporation. The product was purified by silica gel column chromatography (dichloromethane/hexane, 1/4) to produce 0.84 g (66%) of a white powder. 1 H NMR (200 MHz, CDCl3, d, ppm): 7.69 (d, 2 H, J = 8.0 Hz), 7.48 (m, 4H), 7.25 (m, 2H), 7.04 (s, 2H), 6.92 (t, 2H, J = 6.8 Hz), 6.29 (d, 2H, J = 8.0 Hz), 3.33 (t, 4H, J = 6.4 Hz), 2.20 (m, 4H), 1.75 (m, 4H), 1.28 (m, 8H), 0.96 (m, 4H). 13C NMR (100 MHz, CDCl3, d, ppm): 159.72, 138.81, 138.10, 136.21, 130.63, 128.88, 128.60, 127.58, 126.35, 125.78, 122.29, 121.30, 57.77, 46.50, 45.58, 33.99, 32.58, 29.11, 28.02, 25.69. HRMS (HREI, m/z): calcd for C38H38Br4, 809.97; found [M]+ = 809.9697.

2.3. General procedure for polymerization

2.2.5. Synthesis of 10,10-bis(6-bromohexyl)-10H-spiro(anthracene9,90 -(20 ,70 -bis(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)fluorene)) (6) To a two-neck flask containing compound 5 (2.04 g, 2.5 mmol) in THF (70 mL), t-butyllithium (3.1 mL, 1.7 M in pentane) was slowly added at 78 °C. The solution was stirred at 78 °C for 30 min. 2-Isopropoxy-4,4,5,5,-tetramethyl-1,3,2-dioxaborolane (1.4 mL, 7 mmol) was injected and stirred for 30 min at 78 °C. A small amount of water was added to quench the remaining t-butyllithium, and the solution was allowed to warm to room temperature. The solution was extracted with dichloromethane and washed several times with water. The organic layer was dried over magnesium sulfate and concentrated under reduced pressure. The product was purified by silica gel column chromatography (ethylacetate/hexane, 1/19) to produce 1.67 g (73%) of a white powder. 1 H NMR (200 MHz, CDCl3, d, ppm): 7.82 (m, 4H), 7.48 (d, 2H, J = 7.6 Hz), 7.34 (s, 2H), 7.17 (t, 2H, J = 6.8 Hz), 6.83 (t, 2H, J = 6.8 Hz), 6.27 (d, 2H, J = 8.0 Hz), 3.33 (t, 4H, J = 7.0 Hz), 2.21 (m, 4H), 1.75 (m, 4H), 1.5–1.0 (m, 36H) .13C NMR (100 MHz, CDCl3, d, ppm): 157.97, 142.79, 138.84, 137.81, 133.93, 131.70, 129.55, 128.98, 126.70, 125.89, 125.52, 119.40, 83.45, 57.66, 46.84, 45.34, 33.54, 32.86, 29.06, 27.15, 25.07, 24.88. HRMS (HREI, m/z): calcd for C50H62B2Br2O4, 906.32; found [M]+ = 906.3260. 2.2.6. Synthesis of 1,4-bis(6-bromohexyloxy)-2,5-dibromobenzene (7) 1,4-dibromohydroquinone (4.0 g, 15 mmol), tetrabutylammonium bromide (0.35 g) and 1,6-dibromohexane (23 mL, 86.7 mmol) were added to 50 mL of 45 wt.% aqueous KOH solution. The resulting mixture was stirred at 80 °C overnight. The solution

The diboronic ester monomer (2 or 6, 0.5 mmol), compound 7 (0.5 mmol) and Pd(PPh3)4 (0.02 mmol) were dissolved in THF (60 mL). The degassed 2 M aqueous K2CO3 solution (15 mL) was added to the above solution. The mixture was degassed, purged with argon and refluxed for 24 h in an argon atmosphere. The resulting solution was added dropwise to the 300 mL of water/ methanol (1:2) mixture. The precipitate was filtered, dissolved in chloroform and re-precipitated into methanol. The precipitate was filtered and dried under vacuum. The neutral precursor polymers (P1n, P2n) were dissolved in THF (30 mL) and cooled to 78 °C with a dry ice/acetone bath. A large excess of condensed trimethylamine (2 mL) was added to the solution. The solution was allowed to warm to room temperature for 12 h, and methanol (20 mL) was added to dissolve the precipitated compounds. Another portion of condensed trimethylamine (2 mL) was added at 78 °C and the mixture was stirred for a further 12 h to complete quaternization. After removing the solvents and excess trimethylamine under vacuum, the residue was dissolved in methanol and filtered. The evaporation of methanol afforded the ionic polymers (P1i and P2i), which were washed in acetone and dried under vacuum. 2.4. P1n 1 H NMR (200 MHz, CDCl3, d, ppm): 7.85–7.50 (br m, 6H), 7.20 (s, 2H), 4.02 (m, 4H), 3.35 (m, 8H), 2.04–0.81 (br, 36H). Mn = 19,910 (Mw/Mn = 1.42).

2.5. P1i 1 H NMR (200 MHz, DMSO, d, ppm): 7.97–7.60 (br m, 6H), 7.20 (s, 2H), 4.11 (br, 4H), 3.40 (m, 8H), 3.10 (br, 36H), 1.72–1.0 (br, 36H).

2.6. P2n 1

H NMR (200 MHz, CDCl3, d, ppm): 7.83 (d, 2H), 7.39 (br, 4H), 7.12 (br, 4H), 6.78 (m, 4H), 6.41 (d, 2H), 3.49 (br, 4H), 3.34 (t, 4H), 3.03 (t, 4H), 2.17 (br, 4H), 1.80–1.41 (br m, 10H), 1.30–0.80 (br, 22H). Mn = 29,600 (Mw/Mn = 1.69). 2.7. P2i 1 H NMR (200 MHz, DMSO, d, ppm): 8.09 (br, 2H), 7.60–7.10 (br m, 8H), 6.91 (br, 4H), 6.22 (br, 2H), 3.56 (br, 12H), 3.05 (br, 36H), 2.18 (br, 4H), 1.80–0.7 (br m, 32H).

3. Results and discussion The synthetic routes for preparing the monomers and the final cationic polyelectrolytes are shown in Schemes 2 and 3. Compound

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Scheme 2. Reagents and conditions for synthesis of the monomers: (i) 1,6-dibromohexane, tetrabutylammonium bromide, 80 °C , 45% aqueous KOH solution; (ii) t-BuLi, 2isopropoxy-4,4,5,5-tetramethyl-1,3,2-dioxaborolane, 78 °C, THF; (iii) 4, KH, 18-crown-6, THF; (iv) 0.33 M Br2PPh3 in CH2Cl2.

Scheme 3. Reagents and conditions for synthesis P1i and P2i: (i) Pd(PPh3)4, aqueous 2 M K2CO3 solution, reflux in THF; (ii) N(CH3)3, THF/H2O, room temperature, 24 h.

2 was synthesized by a reaction of 2,7-dibromo-9,90 -bis(600 -bromohexyl)fluorene and 2-isopropoxy-4,4,5,5,-tetramethyl-1,3,2dioxaborolane with t-BuLi at 78 °C in THF. The spiro-substituted dibromofluorene 3 was synthesized according to the procedures reported elsewhere [25]. The protons at the 10 position of the spiro-anthracenyl group were removed by excess KH, and further reacted with (6-chlorohexyloxy)-tert-butyldimethylsilane 4. The terminal tert-butyldimethylsilyloxy group was successfully trans-

formed to bromide to give compound 5 using dibromotriphenyl phosphine (Br2PPh3) in dichloromethane at room temperature. The spiro-substituted fluorene diboronic ester 6 was obtained by treating 5 with t-BuLi and 2-isopropoxy-4,4,5,5-tetramethyl1,3,2-dioxaborolane in THF at 78 °C. The comonomer 7, 1,4bis(6-bromohexyloxy)-2,5-dibromobenzene was synthesized by the simple alkylation of 2,5-dibromobenzene-1,4-diol with 1,6-dibromohexane in 45% aqueous KOH solution. All the

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intermediates and monomer structures were confirmed by 1H and 13 C NMR spectroscopy. Further evidence of the identity of compounds 1–7 was obtained by high resolution mass spectrometry. Suzuki coupling of the dialkylated fluorene monomer 2 or its spiro-substituted counterpart 6 with 7 using Pd(PPh3)4 in THF/ H2O (2:1) gave the neutral precursor polymers, P1n and P2n in 40–60% yields. The degree of polymerization was determined to be Mn = 19,000 (PDI = 1.42) for P1n and Mn = 29,000 (PDI = 1.69) for P2n using GPC (solvent: chloroform), respectively. The watersoluble quaternized polymers, P1i and P2i were obtained by treating of P1n and P2n with condensed trimethylamine in THF/H2O for 24 h. Table 1 summarizes the linear absorption and photoluminescence (PL) spectroscopic data for P1i and P2i in water. Due to the break-up of electronic communication between the fluorene and spiro-attached anthracene units in P2i, the two polymers should have the same p-conjugated structures. The spiro-anthracenyl substituents increase the intermolecular distances in the aggregated phases without perturbation on the p-conjugation of the main polymer chain. They can function as molecular spacers or molecular bumpers. As presented in Table 1, the absorption and PL maxima in water show little difference for both structures. The same electrochemical oxidation potentials (Eox = 0.8 V) were also measured by cyclic voltammetry (CV), which confirms P1i and P2i to have identical highest occupied molecular orbital (HOMO) and lowest unoccupied molecular orbital (LUMO) structures with the combined UV/vis measurements. The polymer PL quenching and fluorescence energy transfer from P1i and P2i to ssDNA–Fl were examined by steady-state and time-resolved PL spectroscopy. A single-stranded ssDNA–Fl corresponding to the sequence, 50 -fluorescein-ATCTT GACTA TGTGG GTGCT-30 was purchased from Genscript Corp. Fig. 1a shows the PL (upon excitation of the polymer at 380 nm) spectra of P1i in the presence of ssDNA–Fl as a function of [P1i] in water

Table 1 Spectroscopy summary Polymer

kabs (nm)

kPL (nm)

UPLa

Eox (onset,V)b

HOMO (eV)/LUMO (eV)c

P1i P2i ssDNA–Fl

362 362 495

411 411 513

0.43 0.39 0.80

0.80 0.83 –

5.6/2.5 5.6/2.5 5.8/3.4

a

PL quantum yield was measured relative to fluorescein at pH 11. Oxidation potential was measured relative to ferrocene by cyclic voltammetry (CV). CV was carried out using polymer film on Pt working electrode in 0.1 M nBu4NPF6 in CH3CN with a scan rate of 10 mV/s. c The HOMO and LUMO energy levels were estimated by the electrochemical CV measurements and the corresponding bandgap from absorption measurements. b

Table 2 Summary of time-resolved photoluminescence data (kex = 380 nm) PL lifetime, sm (ps) P1i P2i P1i + ssDNA–Fl P2i + ssDNA–Fl a

620a 633 (70%), 65 (30%)a 30 (kdet = 412 nm)b 78 (kdet = 526 nm)c 50 (kdet = 412 nm)b 800 (60%), 180 (40%) (kdet = 526 nm)c

keT (s1)

UeT (%)

1.26  1010 9

1.56  10

98 86

PL signal was detected at kdet = 412 nm. PL decay of the polymers in the presence of ssDNA–Fl was measured at kdet = 412 nm. [P1i or P2i] = 1.5  107 M ([+] = 6  107 M), [ssDNA–Fl] = 3  108 M ([] = 6  107 M). The value is beyond of our instrumental resolution range. c PL decay of fluorescein in the electrostatic complex [P1i or P2i]/ssDNA–Fl, was measured at kdet = 526 nm. [P1i or P2i] = 4.5  107 M ([+] = 18  107 M), [ssDNA– Fl] = 1.5  108 M ([] = 3  107 M). b

at pH 8. Fig. 1b shows the PL spectra with P2i. In both cases, polymer emission is quenched with ssDNA–Fl. There was an increase in FRET induced Fl emission with increasing [P2i] but no Fl emission was measured from the P1i/ssDNA–Fl solution. Despite the identical electronic structures for both P1i and P2i, they function differently as a FRET excitation donor to fluorescein-labeled DNA. It is believed that this difference is related to the fine structures of the electrostatic complexes of P1i/ssDNA–Fl and P2i/ssDNA–Fl. The time-resolved PL decays of P1i and P2i were measured in the presence or absence of ssDNA–Fl using a time-correlated single-photon counting (TCSPC) technique (Table 2) [26]. The donor (P1i and P2i) excited state showed significantly faster decay when the acceptor (ssDNA–Fl) was present, which is consistent with the steady-state data (Fig. 2). P1i in the absence of ssDNA–Fl showed single exponential PL decay with sm = 620 ps in water at pH 8. After adding ssDNA–Fl ([P1i] = 1.5  107 M ([+] = 6  107 M), [ssDNA– Fl] = 3  108 M ([] = 6  107 M), there was a significant decrease in the measured PL lifetime (sm), which suggests that the quenching process through either FRET or PCT is very rapid (within 50 ps), beyond the temporal resolution of the instrument used in this study. The same trend was measured with P2i and ssDNA–Fl. The quenching processes for P1i/ssDNA–Fl and P2i/ssDNA–Fl could not be distinguished in the polymer PL decay measurements. However, electron transfer (eT) quenching of the excited Fl* in the electrostatic complexes, P1i/ssDNA–Fl and P2i/ssDNA–Fl, could be estimated successfully. The free ssDNA–Fl in water at pH 8 showed sm = 4 ns in the absence of the polymers, which is consistent with previously reported data [27]. After the formation of an electrostatic complex with P1i, the lifetime of the excited Fl* showed a substantial decrease to ca. 78 ps. Relatively moderate Fl quenching was observed in the case of P2i/ssDNA–Fl; the PL decay could be fitted approximately with the double exponential time constants of 800 ps (60%) and 180 ps (40%) with an average lifetime of 552 ps (Fig. 3). It was not possible to tune the laser wavelength to around 500 nm (absorption maximum of Fl) in order to directly excite Fl. A wavelength of 380 nm was chosen for excitation, in which excited Fl* can be generated through fluorescence energy transfer from the polymers. Excess polymers were added ([P1i or P2i] = 4.5  107 M [ssDNA–Fl] = 1.5  108 M) to make sure complete complexation of ssDNA–Fl. The significant decrease in the excited lifetime of Fl in the presence of the polymers was closely related to PL quenching through electron transfer from the neighboring polymers (P1i or P2i in the ground state) to the excited Fl* [22]. Electron transfer to Fl* is thermodynamically favorable due to the arrangement of the HOMO and LUMO energy levels of the polymers and Fl (Table 1). The quenching rate and quenching efficiency by electron transfer were also estimated using the time-resolved PL data. The calculated eT rate (keT) for P1i/ssDNA–Fl and P2i/ ssDNA–Fl was 1.26  1010 s1 and 1.56  109 s1, respectively. A quenching efficiency (UeT) was obtained to be 98% for P1i/ ssDNA–Fl and 86% for P2i/ssDNA–Fl, respectively [28]. These results are in good agreement with the steady-state measurements within experimental error. The steady-state PL quantum efficiency was measured to be 80% for ssDNA–Fl in the absence of polymers. After electrostatic complexation, the UPL value of Fl was determined to be 1% for P1i/ssDNA–Fl and 27% for P2i/ssDNA–Fl, respectively [23]. Electron transfer is sensitive to the intermolecular distance between the donor and acceptor. PL quenching via electron transfer is well understood and has been suggested as a possible mechanism for PL quenching in a fluorescence-based biosensor [5,22]. Electron transfer is basically a contact process with an exponential D–A distance dependence [29,30]. Previously, Götz et al. examined PL quenching and time-resolved absorption measurements of the protein-bound fluorescein at the femtosecond region [27]. They reported that Anticalin FluA complexes fluorescein as a ligand with

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Fig. 1. PL spectra of P1i (a) and P2i (b) in the presence of ssDNA–Fl in water at pH 8. All spectra were obtained by exciting at 380 nm. [ssDNA–Fl] = 1.5  108 M [P1i] or [P2i] = 5  108 M  3  107 M.

Fig. 2. Time-resolved PL spectra of P1i in the absence of ssDNA–Fl (a) and in the presence of ssDNA–Fl (b). (kex = 380 nm and kdet = 412 nm).

almost complete quenching of its fluorescence, and the mechanism of PL quenching of fluorescein through ultrafast electron transfer (within 400 fs) from the neighboring protein (tryptophan, tyrosine) to the bound fluorescein. This can similarly be applied to the present FRET-based DNA detection system. After the excited Fl* is formed in the complex (P1i or P2i)/ssDNA–Fl, some portions are deactivated radiationless though electron transfer quenching from the neighboring polymer donors. The faster eT rate and almost complete Fl PL quenching for P1i/ssDNA–Fl can be explained by shorter D–A distance due to the absence of molecular spacers in P1i/ssDNA–Fl. Other factors such as the thermodynamic driving force for quenching should be the same for both systems. Similar studies have been reported in non-conjugated and/or conjugated dendrimer systems, in which energy transfer from the periphery donors to the acceptor core of the dendrimer is followed by electron transfer quenching of the core excited state through electron transfer from one of the donors to the excited core [31–35]. This study suggests an important molecular design tip for efficient FRET-based DNA detection systems. Energy wasting eT quenching

Fig. 3. Time-resolved PL spectra of (a) ssDNA–Fl, (b) P2i/ssDNA–Fl and (c) P1i/ ssDNA–Fl. [P1i or P2i] = 4.5  107 M [ssDNA–Fl] = 1.5  108 M (kex = 380 nm and kdet = 526 nm).

could be controlled kinetically through intermolecular D–A distance modulation using spiro-attached molecular spacers. 4. Conclusions In summary, this paper reports the steady-state and time-resolved PL studies of fluorescence energy transfer from cationic conjugated polymers (two polymers with same p-conjugation) to the DNA-bound fluorescein. The almost complete PL quenching of the excited Fl* in P1i/ssDNA–Fl and relatively moderate quenching in P2i/ssDNA–Fl could be explained by the decreased eT quenching as a result of the increased intermolecular distance due to the existence of spiro-substituted molecular bumpers in P2i. This shows that the thermodynamically favorable eT quenching can be controlled kinetically by modulating the D–A intermolecular distance using molecular bumpers. For efficient FRET-based biosensors, molecular design strategies will be needed to prevent quenching of the excited acceptor in the D–A complex and improve the FRET

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efficiency. A combined kinetic and thermodynamic control by modulation of the intermolecular D–A distance as well as adjusting the HOMO/LUMO levels to match the D–A energy levels for favorable energy transfer is necessary for the optimized CPs-based DNA detection. Acknowledgement This work was supported by the Korea Research Foundation Grant funded by the Korean Government (MOEHRD, KRF-2006312-C00584). This study was also financially supported by Pusan National University in program, Post-Doc. 2007 (Dr. R.R. Nayak). References [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13]

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