Counterion Effects on Fluorescence Energy Transfer in Conjugated

Apr 6, 2009 - Cationic poly[9,9′-bis[6′′-(N,N,N-trimethylammonium)hexyl]fluorene-co-alt-phenylene]s with five different counterions (CIs) were ...
0 downloads 0 Views 145KB Size
Download PDF
5788

J. Phys. Chem. B 2009, 113, 5788–5793

Counterion Effects on Fluorescence Energy Transfer in Conjugated Polyelectrolyte-Based DNA Detection Okhil Kumar Nag,† Mijeong Kang,† Sungu Hwang,† Hongsuk Suh,‡ and Han Young Woo*,† Department of Nanofusion Technology (BK21), Pusan National UniVersity, Miryang 627-706, Republic of Korea, and Department of Chemistry and Chemistry Institute for Functional Materials, Pusan National UniVersity, Busan 609-735, Republic of Korea ReceiVed: December 8, 2008; ReVised Manuscript ReceiVed: February 2, 2009

Cationic poly[9,9′-bis[6′′-(N,N,N-trimethylammonium)hexyl]fluorene-co-alt-phenylene]s with five different counterions (CIs) were synthesized and studied as fluorescence resonance energy transfer (FRET) donors (D) to dye-labeled DNA (FRET acceptor, A). The polymers with different CIs show the same π-conjugated electronic structure with similar absorption (λabs ) ∼380 nm) and photoluminescence (λPL ) ∼420 nm) emission spectra in water. The CIs accompanying the polymer chain are expected to affect the D/A complexation and modify the D-A intermolecular separation by acting as a spacer. Polymers with different CIs function differently as FRET excitation donors to fluorescein (Fl)-labeled single-stranded DNA (ssDNAFl). The FRET-induced Fl emission was enhanced significantly by the larger CI-exchanged polymers. The polymers with the CIs of tetrakis(1-imidazolyl)borate (FPQ-IB) and tetraphenylborate (FPQ-PB) showed a 2-4-fold enhancement in the FRET-induced signal compared with the polymer with bromide (FPQ-BR). The delayed FRET signal saturation and low association constants (Ka) with ssDNA-Fl (3.53 × 106 M-1 for FPQ-BR and 1.80 × 106 M-1 for FPQ-PB) were measured for the polymers with larger CIs. The delayed acceptor saturation strengthens the antenna effect and reduces self-quenching of Fl by increasing the polymer concentration near Fl. The weak polymer/ssDNA-Fl association reduces the amount of energy-wasting charge transfer by increasing D-A intermolecular separation. The combined effects lead to increase the overall FRET-induced signal. Introduction In recent years, water-soluble conjugated polyelectrolytes (CPs) have attracted considerable attention as a useful platform for chemical and biological assays owing to their unique optical, optoelectronic, and electrochemical properties.1 CPs are structurally modified conjugated polymers containing ionic substituents (cationic or anionic).2 The π-conjugation along the polymeric backbone provides their useful optoelectronic properties, and the ionic groups endow their electrolytic properties and solubility in highly polar media such as water. A noble DNA detection and signal amplification strategy using cationic CPs (CCPs) was suggested by Bazan. This detection scheme employs fluorescence resonance energy transfer (FRET) from CCPs to fluorophore-labeled peptide nucleic acid (PNA-C*) or DNA-C*, which are complementary to the target DNA (t-DNA).3 Electrostatic complexation between the positive CCPs and the hybridized duplex (negatively charged PNA-C*/t-DNA or DNA-C*/t-DNA) brings them into close proximity and gives rise to an efficient FRET to signal a sequence-specific target DNA. The FRET-based real time DNA detection has been extensively studied for sequencing a DNA strand1b,c and identifying genetic mutation, etc.4,5 This assay benefits from the light-harvesting properties of water-soluble CCPs to achieve sensory signal amplification and improved detection sensitivity. However, the quenching phenomena due * Corresponding author. Telephone: 82-55-350-5300. Fax: 82-55-3505653. E-mail: [email protected]. † Department of Nanofusion Technology. ‡ Department of Chemistry and Chemistry Institute for Functional Materials.

to photoinduced charge transfer (PCT) in the CCPs/DNA-C* complex and self-interaction of C* within the complex reduce the overall level of signal amplification.6,7 Recent efforts to optimize the FRET based-DNA sensors include the structural modification of CCPs for matching the energy levels between the FRET donor (D) and acceptor (A) pairs,7 controlling the intermolecular D-A separation to minimize the energy-wasting PCT quenching,8 altering the working medium (buffer/organic solvent mixture) to tune the optical properties and electrostatic complexation,9 and reducing the charge density on the CCPs by employing anionic surfactants to increase the donor concentration around the acceptor.10 The fine structure of the polymer/DNA complex, the distance between the optically active backbone and the probe dye, and the degree to which FRET or PCT occurs are strongly influenced by the structural attributes of the CCPs. The CCPs are accompanied by counterions (CIs) with an opposite charge for charge compensation. These CIs are expected to influence the electrostatic complexation and perturb the fine structure of the D/A complex, i.e., D-A intermolecular separation, which strongly affects competition between the desirable FRET and energy-wasting PCT. Recently, the influence of CIs on the optical and optoelectronic properties in CP-based electronic devices has been discussed.11 Understanding the D/A fine structure and how to control it on the molecular scale are very important factors for optimizing CPbased FRET biosensors. This paper reports the molecular design and FRET-related photophysical properties (as a FRET donor) of cationic poly(fluorene-alt-phenylene) copolymers with five different counterions: poly[9,9′-bis[6′′-(N,N,N-trimethylammonium)hexyl]fluorene-co-alt-phenylene] with bromide (FPQ-BR), tetrafluoroborate

10.1021/jp8107733 CCC: $40.75  2009 American Chemical Society Published on Web 04/06/2009

FRET in CP-Based DNA Detection

J. Phys. Chem. B, Vol. 113, No. 17, 2009 5789

SCHEME 1: Molecular Structures of FPQ Polymers with Different Counterions

(FPQ-FB), hexafluorophosphate (FPQ-FP), tetrakis(1-imidazolyl)borate (FPQ-IB), and tetraphenylborate (FPQ-PB) as counterions (Scheme 1). These polymers have the same π-conjugated electronic structure with the main structural difference being the accompanying CIs for charge compensation of the terminal hexyltrimethylammonium groups. A fluorescein (Fl)-labeled single-stranded DNA (ssDNA-Fl, 5′-Fl-TTAA TCGA GTTA CCGC AATC) was used as a FRET acceptor. As described in detail below, cationic polymers with different CIs behave differently as excitation donors to ssDNA-Fl. By simply exchanging the CIs, the FRET-induced Fl signal was successfully enhanced ca. 2-6-fold relative to the direct excitation of ssDNA-Fl. The delayed FRET signal saturation and low association constants with ssDNA-Fl were observed for the polymers with larger CIs. These combined effects cause an increase of the antenna effect of the polymers and decrease the charge transfer quenching in the D/A complex. This simple approach suggests an effective way of controlling the fine structure of the polymer/DNA electrostatic complex and the FRET process on the molecular scale for maximizing the signal amplification in conjugated polymer based FRET biosensors. Experimental Section General. All chemicals were purchased from Aldrich Chemical Co. and used as received, unless otherwise mentioned. HPLC-purified single-stranded DNA labeled with fluorescein at the 5′-position (ssDNA-Fl) with 20 bases with the sequence of 5′-Fl-TTAA TCGA GTTA CCGC AATC was obtained from Sigma-Genosys. The 1H NMR spectra were recorded on a JEOL (JNM-AL300) Fourier transform (FT) NMR system. The X-ray photoelectron spectroscopy (XPS) data were measured at the Korea Basic Science Institute in Pusan National University (ESCALAB 250 XPS spectrometer). The UV/vis absorption spectra were measured using a Jasco (V-630) spectrophotometer. The photoluminescence (PL) spectra were obtained on a Jasco (FP-6500) spectrofluorometer with a xenon lamp excitation source, using 90° angle detection for the solution samples. All FRET PL spectra were measured in 5 mM phosphate buffer, unless otherwise stated. The fluorescence quantum yield was measured relative to freshly prepared aqueous solution of fluorescein at pH 11. Synthesis and Characterization. Scheme 1 shows the molecular structures of the cationic FPQ series with different CIs. The synthetic approach to the neutral precursor, poly[9,9′bis(6′′-bromohexyl)fluorene-co-alt-1,4-phenylene] (FPN), involves the Suzuki copolymerization of 2,7-dibromo-9,9-bis(6′bromohexyl)fluorene and 1,4-phenylenebisboronic ester using Pd(PPh3)4 in toluene/H2O (2:1) at 85 °C for 24 h (yield 81%, Supporting Information).1c,7 The number-average molecular

weight of FPN was measured to be Mn ) 16 700 g/mol (polydispersity index (PDI) ) 2.32) using gel permeation chromatography (in THF) relative to a polystyrene standard. The cationic poly[9,9′-bis[6′′-(N,N,N-trimethylammonium)hexyl]fluorene-co-alt-phenylene] bromide (FPQ-BR) was obtained by treating FPN with a 30% aqueous solution of trimethylamine in THF/methanol for 24 h. The almost quantitative quaternization was confirmed by measuring the integration ratio among the proton signals for -CH2CH2X (X ) Br, N+(CH3)3Br-) and -CH2CH2N+(CH3)3Br- in the 1H NMR spectra. The other polymers (FPQ-FB, -FP, -IB, -PB) were prepared by CI-exchange reactions (repeated two to three times). The 1H NMR and XPS data support the completeness of the CI exchange for all the polymers. In the XPS spectra of the CPs with the exchanged CIs, the Br peak nearly disappeared and the characteristic elemental peaks were recorded for corresponding exchanged ions. The detailed experimental procedures and XPS data for the CI-exchanged polymers are provided in the Supporting Information. Poly[9,9′-bis(6′′-bromohexyl)fluorene-co-alt-1,4-phenylene] (FPN). H NMR (300 MHz, CDCl3): δ (ppm) 7.83-7.58 (br, 10H), 3.31 (br, 4H), 2.1 (br, 4H), 1.7 (br, 4H), 1.23 (br, 8H), 0.83 (br, 4H).

1

Poly[9,9′-bis[6′′-(N,N,N-trimethylammonium)hexyl]fluoreneco-alt-phenylene] Bromide (FPQ-BR). 1H NMR (300 MHz, DMSO): δ (ppm) 8.10-7.60 (br, 10H), 3.17 (br, 4H), 2.96 (s,18H), 1.6-0.9 (br, 20H). Poly[9,9′-bis[6′′-(N,N,N-trimethylammonium)hexyl]fluoreneco-alt-phenylene] Tetrafluoroborate (FPQ-FB). 1H NMR (300 MHz, DMSO): δ (ppm) 8.10-7.60 (m, 10H), 3.10 (br, 4H), 2.92 (s,18H), 1.6-0.9 (br, 20H). Poly[9,9′-bis[6′′-(N,N,N-trimethylammonium)hexyl]fluoreneco-alt-phenylene] Hexafluorophospate (FPQ-FP). 1H NMR (300 MHz, DMSO): δ (ppm) 8.10-7.60 (m, 10H), 3.10 (br, 4H), 2.92 (s,18H), 1.6-0.9 (br, 20H). Poly[9,9′-bis[6′′-(N,N,N-trimethylammonium)hexyl]fluoreneco-alt-phenylene] Tetrakis(1-imidazolyl)borate (FPQ-IB). 1H NMR (300 MHz, DMSO): δ (ppm) 8.10-7.60 (br, 10H), 7.02 (s, 8H), 6.91 (s, 8H), 6.74 (s, 8H), 3.11 (br, 4H), 2.92 (s,18H), 1.6-0.9 (br, 20H). Poly[9,9′-bis[6′′-(N,N,N-trimethylammonium)hexyl]fluoreneco-alt-phenylene] Tetraphenylborate (FPQ-PB). 1H NMR (300 MHz, DMSO): δ (ppm) 8.10-7.60 (br, 10H), 7.17 (m, 16H), 6.90 (m, 16H), 6.77 (m, 8H), 3.04 (br, 4H), 2.83 (s,18H), 1.6-0.8 (br, 20H).

5790 J. Phys. Chem. B, Vol. 113, No. 17, 2009

Nag et al. TABLE 1: Spectroscopic Properties of FPQ Polymers

SEV (Å3) for CIs in DMSO in water ΦPL λabs (nm) Figure 1. Normalized absorption and PL spectra of FPQ-BR (a), FPQFB (b), FPQ-FP (c), FPQ-IB (d), and FPQ-PB (e) in DMSO (A) and in 5 mM phosphate buffer (B).

Results and Discussion Absorption and Fluorescence Spectroscopic Properties of FPQ Polymers. The effect of the CIs on the optical (and electronic) properties of the polymers was examined in an aqueous buffer and dimethyl sulfoxide (DMSO) by UV/vis and photoluminescence (PL) spectroscopies. Figure 1A shows that the absorption and PL spectra in DMSO (in which all polymers are very soluble) are identical for the five polymers with different CIs. The maxima in the absorption (λabs) and PL (λPL) for all polymers in DMSO were measured at 383 and 418 nm, respectively. All polymers also showed a similar PL quantum efficiency (ΦPL) of ∼70% in DMSO. This suggests that the polymers have the same electronic structures (with the same HOMO-LUMO energy levels) because the CIs do not interact with the π-conjugated main backbone. However, the accompanied CIs are expected to have different ion-pair dissociation properties and alter the average interchain separation in the condensed phases, which would affect the conformation and morphology of the polymers.12 For all polymers, the similar absorption (λabs ) ∼380 nm) and PL (λPL ) ∼420 nm) spectra were also measured with ΦPL ) ∼40% in 5 mM phosphate buffer (Figure 1B).12c The slight difference in the spectra (λabs ) 375 nm and λPL ) 425 nm) for FPQ-PB might be due to the limited solubility (and resulting conformational change or partial aggregation)13 in the buffer as a consequence of the hydrophobic nature of PB. The volume of the structure is expected to increase with increasing number of atoms consisting of the CIs. An estimation of the Connolly solvent-excluded volume (SEV) of the CIs, which is computed by summing the volumes of small cubes contained within the solvent-accessible surface of the molecule,14,15 indicates that size of the CIs increases in the following order: bromide (27.0 Å3) < tetrafluoroborate (51.8 Å3) < hexafluorophosphate (79.2 Å3) < tetrakis(1-imidazolyl)borate (278.1 Å3) < tetraphenylborate (353.9 Å3) in dimethyl sulfoxide (DMSO). A similar trend of SEV for the CIs was observed in water. Properties of the FPQ polymers are summarized in Table 1. Counterion Effects on FRET-Induced PL Spectroscopy. The FRET-induced emission of Fl was investigated in the presence of the polymers with different CIs to study how the accompanying CIs influence the PL energy transfer to fluoresceinlabeled single-stranded DNA (ssDNA-Fl). Fluorescein (λabs ) 490 nm, λPL) 514 nm) was chosen as the FRET acceptor due to its good spectral overlap with the emission of the polymers. The opposite charges on the polymer backbone and DNA allow the formation of an electrostatic complex, CCP/ssDNA-Fl, enabling facile energy transfer from the CCPs to ssDNA-Fl. The FRET PL measurements were carried out in 5 mM phosphate buffer (pH 8.20) at a fixed concentration of [ssDNAFl] ) 20 nM with increasing polymer concentration. Figure 2A shows the typical FRET-induced PL spectra for FPQ-BR/

λPL (nm)

FPQBR

FPQFB

FPQFP

FPQIB

FPQPB

27.0 27.0 70.5a (43.8)b 383 (382) 418 (419)

51.8 50.8 72.8 (45.2) 383 (382) 418 (420)

79.2 75.1 75.5 (44.0) 383 (381) 418 (421)

278.1 260.4 76.3 (43.0) 383 (381) 418 (421)

353.9 314.0 75.8 (-)c 383 (375) 418 (425)

a Values in DMSO. b Values in parentheses are in 5 mM phosphate buffer. c The accurate measurement of ΦPL was not possible due to limited solubility in buffer.

Figure 2. FRET-induced PL spectra by exciting at 380 nm (A) and Fl emission spectra of FPQ-BR/ssDNA-Fl by directly exciting Fl at 490 nm with increasing [FPQ-BR] (B). [ssDNA-Fl] ) 20 nM.

ssDNA-Fl by exciting the polymer at 380 nm. Each polymer addition corresponds to 200 nM (based on repeat units) and gives rise to an increase in charge ratio ([+] in FPQ-BR/[-] in ssDNA-Fl) by 1. Note that molecular weight of ssDNA-Fl is 6615 g/mol and it contains 20 bases and 20 negative phosphate linkers in the strand. The negative charge concentration in ssDNA-Fl can be calculated in the following way: [-] ) 20[ssDNA-Fl] ) 20 × 20 nM ) 400 nM. The FPQ polymers contain two positive charges per repeat unit, and the positive charge concentration in the polymer is calculated in a similar way: [+] ) 2[FPQ] ) 2 × 200 nM ) 400 nM. In the absence of ssDNA-Fl, the PL emission of the polymer is increased with increasing [FPQ-BR], the intensity of which is linearly proportional to the polymer concentration in our experimental range (Supporting Information). However, the polymer emission was quenched and the Fl emission signal began to increase via FRET upon the addition of FPQ-BR to the solution containing ssDNAFl. This suggests that energy of the excited polymer is transferred efficiently to Fl via FRET and the excited Fl* finally decays radiatively. The FRET Fl signal increased with increasing [FPQ-BR] due to the antenna effect of the polymer and finally became saturated at [FPQ-BR] ) 600 nM with a charge ratio of 3:1 ([+] in FPQ-BR:[-] in ssDNA-Fl). Acceptor saturation with a similar charge ratio was observed with a slight decrease in the FRET-dye emission after saturation.1c,3,7 The main driving force to form the FPQ-BR/ssDNA-Fl complex is electrostatic attraction.16 Therefore, the charge ratio should be around 1:1 at the saturation point. However, the higher charge ratio to achieve saturation indicates that not all the alkylammonium groups on the polymer side chain contribute to the formation of the electrostatic D/A complex. The detailed complexation process and fine structure of the CCP/ssDNA-Fl complex are not completely understood at present. There are several possibilities for the higher charge ratio at the acceptor saturation. One possibility is that charge dissociation of the alkylammonium group and bromide does not

FRET in CP-Based DNA Detection

Figure 3. FRET-induced Fl signal intensity (by exciting at 380 nm) with increasing polymer concentration. Emission intensity was taken at the Fl emission maximum. [ssDNA-Fl] ) 20 nM.

appear to be completed in 5 mM phosphate buffer at pH 8.20. The ionic dissociation constant is closely related to the molecular structure of the ion pairs.17 The environment around the ion pairs, such as ionic strength and pH, also influences the ion dissociation.18 Another possibility is partial aggregation of the polymers due to the limited solubility caused by the hydrophobic nature of the conjugated backbone. Previous light-scattering experiments demonstrated that a water-soluble phenylenevinylene oligomer and polyfluorene copolymer had effective diameters of ∼190 and ∼350 nm in water due to their aggregation, the structures of which are similar to that of FPQ-BR.12a,19 The aggregation can prevent the ammonium ionic groups from reaching the vicinity of the negative backbone of DNA. For example, it is difficult for the positive groups inside the aggregated polymer to reach the DNA relative to those on the surface. Consequently at a charge ratio of 1:1, the concentration of available free ammonium groups is deficient for complete complexation with DNA. Figure 2B shows the decrease of the Fl emission intensity upon direct excitation at 490 nm with increasing polymer concentration. This implies that there is a serious PL quenching of Fl upon complexation with the polymers. The acceptor quenching after complexation will be discussed in the next section. Figure 3 shows the FRET-induced Fl signal intensity (at the Fl emission maximum, λPL) 530 nm) for the complex, FPQ/ ssDNA-Fl, as a function of the polymer concentration (full spectra in Supporting Information). The FRET emission intensity increases with increasing [FPQ]. Polymers with larger CIs (IB or PB) show more enhanced FRET-induced Fl signal relative to those with smaller CIs (FPQ-BR, -FB, -FP). The FRET process and resulting signal amplification show a clear dependence on the existing CIs in the cationic FPQ polymers. Interestingly, a different charge ratio ([+] in FPQ:[-] in ssDNAFl ) 3:1 for FPQ-BR, 4:1 for FPQ-FB, 5:1 for FPQ-FP, 7:1 for FPQ-IB, and 9:1 for FPQ-PB) was measured at Fl signal saturation for each polymer with different counterions. The highest intensity of the FRET signal for each polymer (in Figure 3) indicates the saturation point of the FRET-induced Fl signal. The acceptor saturation was delayed in the following order: BR < FB < FP < IB < PB. This might be related to the different ionic properties and/or hydrophobic nature (with different ionic dissociation property) of each ion pair on the polymer chain. Ion dissociation in water should decrease with increasing hydrophobic nature of the ion pairs. As mentioned above, polymer aggregation in an aqueous medium also influences the charge dissociation and electrostatic complexation with DNA. Figure 4 shows the FRET-induced PL spectra of ssDNA-Fl in the presence of FPQs with different CIs at the Fl signal saturation. The FRET-induced Fl signals for FPQ-FP/ssDNAFl, FPQ-IB/ssDNA-Fl, and FPQ-PB/ssDNA-Fl were enhanced

J. Phys. Chem. B, Vol. 113, No. 17, 2009 5791

Figure 4. FRET-induced PL spectra at the Fl signal saturation for FPQ-BR/ssDNA-Fl at [FPQ-BR] ) 600 nM (a), FPQ-FB/ssDNA-Fl at [FPQ-FB] ) 800 nM (b), FPQ-FP/ssDNA-Fl at [FPQ-FP] ) 1000 nM (c), FPQ-IB/ssDNA-Fl at [FPQ-IB] ) 1400 nM (d), and FPQ-PB/ ssDNA-Fl at [FPQ-PB] ) 1800 nM (e). All spectra were obtained upon excitation at 380 nm. For comparison, the Fl emission is presented by directly exciting at 490 nm in the absence of the polymers (f). [ssDNAFl] ) 20 nM.

1.3-, 2.0-, and 4.0-fold, respectively, relative to that of FPQBR/ssDNA-Fl. Similar FRET Fl signals were observed for both FPQ-FB/ssDNA-Fl and FPQ-BR/ssDNA-Fl. The FRET signal is clearly dependent on the CIs on the polymeric chain, which highlights the potential for controlling or modulating the FRET signal amplification and antenna effects by exchanging the CIs on the CPs. Effects of Counterions on Acceptor Quenching. Additional important information concerning the effects of CIs was obtained from the Fl emission measurements by directly exciting Fl at 490 nm in the absence and presence of excess polymers. As shown in Figure 5A, the Fl emission spectra are compared after complexation for each polymer at a charge ratio of 3:1, where the acceptor saturation was measured for FPQ-BR/ssDNA-Fl. The absence of uncomplexed free ssDNA-Fl was confirmed by the same emission maximum at λPL ) 530 nm for both the FRET-induced and direct Fl excitation PL spectra of FPQ-BR/ ssDNA-Fl. Free ssDNA-Fl shows the PL maximum at λPL ) 518 nm in the absence of the polymers. The Fl emission was significantly quenched in the presence of excess FPQ-BR. Similar data were obtained for the polymers with smaller CIs (FB, FP). This is mainly due to electron (or charge) transfer from the polymer to the excited Fl*.7,8a,19 The concentration selfquenching of Fl in the complex CCP/ssDNA-Fl was also suggested as a possible mechanism.9 However, relatively moderate quenching was observed for the larger IB- and PBcontaining polymers. In particular, FPQ-PB induces a ∼50% decrease in Fl emission relative to free ssDNA-Fl. The PL maximum wavelength (λPL ) 520 nm) was measured between those of free ssDNA-Fl and the completely complexed one. This means that the complexed and uncomplexed ssDNA-Fl coexist at a charge ratio of 3:1 for FPQ-PB/ssDNA-Fl. This is well consistent with the delayed acceptor saturation for the polymers with larger CIs (see Figure 3). Figure 5B shows the comparison of the Fl emission spectra of the complexes upon direct excitation at their respective saturation of the FRET signal (additional spectra in Supporting Information). In all cases, the same emission maximum at λPL ) 530 nm (which is identical to that of the FRET Fl signal) indicates that all the ssDNA-Fl are complexed with the polymers at the saturation point. The acceptor quenching was clearly mitigated with the polymers having larger CIs (Supporting Information). By combining the above PL data (by exciting the polymer and Fl), it appears that the larger CIs induce a delay in the acceptor saturation and reduce the acceptor quenching, which increases the donor concentration around the acceptor and amplifies the light-

5792 J. Phys. Chem. B, Vol. 113, No. 17, 2009

Nag et al.

Figure 5. PL spectra of FPQ/ssDNA-Fl by directly exciting Fl at 490 nm when the charge ratio is 3:1 ([+] in FPQ:[-] in ssDNA-Fl) (A) and when the FRET Fl signal is saturated for each system (B). [ssDNA-Fl] ) 20 nM.

Figure 6. Stern-Volmer plot of ssDNA-Fl quenched by FPQ polymers (A) and the linear region of the Stern-Volmer plot (B).

harvesting antenna effect of the polymers. In addition, there is another important point to consider in the FRET-induced Fl signal enhancement for FPQ-PB/ssDNA-Fl. As shown in Figure 1, FPQ-PB shows a clear red shift in the UV/vis spectrum, which causes an increase of the overlap between the emission of FPQPB and the Fl absorption. This in turn facilitates the FRET process because the FRET rate is also proportional to the spectral overlap of donor emission and acceptor absorption. All the FPQ polymers show the same π-conjugated electronic structures, so they should have same thermodynamic driving forces for either FRET or charge transfer.8a However, the CIs accompanying the polymer chain may perturb the complexation and modify the fine structure of the D/A electrostatic complex, i.e., D-A intermolecular separation, which kinetically influences the competition between FRET and fluorescence quenching. The acceptor (Fl) quenching by the polymers with the different CIs was examined by quantifying the overall quenching effects using the Stern-Volmer (SV) equation.20

I0 /I ) 1 + KSV[Q] where I0 and I are the fluorescence intensity of the acceptor (Fl) in the absence and presence of the quencher (Q, FPQ polymers), respectively. KSV is the Stern-Volmer quenching constant. The KSV can be used to quantify the quenching efficiency of the polymers.21 Figure 6 shows the SV plot of ssDNA-Fl quenched by the FPQ polymers with different CIs. The concentrations of the polymers range from 0 to 300 nM. At a low polymer concentration ([RU] ) 0-75 nM), a linear plot with different KSV values was obtained for each FPQ polymer. The KSV values calculated from the linear region of the SV plots are 3.53 × 106 M-1 for FPQ-BR, 3.24 × 106 M-1 for FPQ-FB, 3.23 × 106 M-1 for FPQ-FP, 2.53 × 106 M-1 for FPQ-IB, and 1.80 × 106 M-1 for FPQ-PB, respectively. The measured KSV constants clearly indicate that Fl fluorescence quenching is reduced for the polymers with larger CIs. With the fluorescence lifetime (τ ) ∼4 ns) of Fl in water,22 the

Figure 7. Maximum FRET-induced signal amplification (at each acceptor saturation) relative to ssDNA-Fl emission by direct excitation at λex ) 490 nm. [ssDNA-Fl] ) 20 nM.

estimated quenching rate (KSV/τ) of Fl in the presence of FPQBR, FPQ-FB, FPQ-FP, FPQ-IB, and FPQ-PB was 8.82 × 1014, 8.10 × 1014, 8.07 × 1014, 6.32 × 1014, and 4.5 × 1014 M-1 s-1, respectively. These values are far above the upper limit of diffusion-controlled dynamic quenching.20,21 Therefore, the static quenching mechanism is expected to be favorable at low polymer concentrations. In addition, within the limits where static quenching dominates, the KSV value can be treated as an association constant (Ka) for the formation of a ground-state complex between D and A.21 The lower Ka for FPQ-IB and FPQ-PB (compared with FPQ-BR) indicates that the CIs act as a spacer between the polymers and ssDNA-Fl and loosen the compaction of D and A in the complex. The weak D-A association produces a less tight complex (with ssDNA-Fl) with longer D-A intermolecular separation compared with the polymers with smaller CIs, which decreases the PL quenching process. More importantly, the longer D-A separation should cause a more substantial decrease in the PCT relative to FRET. The PCT process is more sensitive to the D-A intermolecular distance (rD-A). The PCT rate (kPCT) shows an exponential decrease with increasing rD-A, while the FRET rate (kFRET) is proportional to rD-A-6.20 Furthermore, the SV plots curve upward at higher polymer concentrations. This might be due to several processes that contribute concurrently to the overall quenching process, which include mixed static and dynamic quenching, variations of the association constant with increasing polymer concentration, and different aggregation tendencies induced by the CIs of the polymers in solution. Figure 7 shows the maximum FRET signal amplification compared to ssDNA-Fl emission by direct excitation, at each acceptor saturation point. Polymers with larger CIs show more pronounced signal amplification than the polymers with smaller CIs. Combining the PL (by exciting the polymer and Fl) and quenching spectroscopic data, it appears that the larger CIs induce the delayed acceptor saturation with increasing polymer concentration around Fl and mitigate PCT quenching with increased D-A separation. Increased polymer concentration around Fl strengthens

FRET in CP-Based DNA Detection the antenna effect and reduces self-quenching of Fl in the complex CCP/ssDNA-Fl. These processes contribute simultaneously to the overall FRET signal amplification. Conclusion In summary, cationic poly(fluorene-alt-phenylene) copolymers (FPQ) with five different counterions (CIs) were synthesized and characterized as FRET donors to a fluorescein-labeled oligonucleotide (ssDNA-Fl). All FPQ polymers have the same π-conjugated electronic structure with the main structural difference being the accompanying CIs for charge compensation. In the FRET-induced PL spectra, polymers with larger counterions showed delayed acceptor saturation and reduced acceptor quenching when forming an electrostatic complex with ssDNAFl. This strengthens the antenna effect by increasing the polymer concentration near Fl and decreasing energy-wasting charge transfer. Consequently, the FRET-induced signal was amplified 2-6-fold relative to direct ssDNA-Fl emission. Although the precise structure of the CCP/ssDNA-Fl complex is unclear at present, these findings suggest a simple approach for controlling the fine structure of the D/A complex on the molecular scale and increasing the antenna effects for signal amplification in CP-based FRET DNA detection. Acknowledgment. This work was supported for 2 years by Pusan National University Research Grant. The authors greatly thank Dr. Rati Ranjan Nayak for helpful discussions. Supporting Information Available: Synthetic details, XPS data, and additional PL and FRET-induced PL spectra for FPQ polymers. This material is available free of charge via the Internet at http://pubs.acs.org. References and Notes (1) (a) Ho, H. A.; Boissinot, M.; Bergeron, M. G.; Corbeil, G.; Dore, K.; Boudreau, D.; Leclerc, M. Angew. Chem., Int. Ed. 2002, 41, 1548. (b) Liu, B.; Wang, S.; Bazan, G. C.; Mikhailovsky, A. J. Am. Chem. Soc. 2003, 125, 13306. (c) Liu, B.; Bazan, G. C. J. Am. Chem. Soc. 2004, 126, 1942. (d) Wang, S.; Gaylord, B. S.; Bazan, G. C. AdV. Mater. 2004, 16, 2127. (e) Gaylord, B. S.; Massie, M. R.; Feinstein, S. C. Proc. Natl. Acad. Sci. U.S.A. 2005, 102, 34. (f) Leclerc, M. AdV. Mater. 1999, 11, 1491. (g) Ambade, A. V.; Sandanaraj, B. S.; Klaikherd, A.; Thayumanavan, S. Polym. Int. 2007, 56, 474. (h) Thomas, S. W., III; Joly, G. D.; Swager, T. M. Chem. ReV. 2007, 107, 1339. (i) Feng, F.; Tang, Y.; He, F.; Yu, M.; Duan, X.; Wang, S.; Li, Y.; Zhu, D. AdV. Mater. 2007, 19, 3490. (2) (a) McQuade, D. T.; Pullen, A. E.; Swager, T. M. Chem. ReV. 2000, 100, 2537. (b) Nilsson, K. P. R.; Ingana¨s, O. Nat. Mater. 2003, 2, 419. (c) Pinto, M. R.; Schanze, K. S. Proc. Natl. Acad. Sci. U.S.A. 2004, 101, 7505. (d) Kumaraswamy, S.; Bergstedt, T.; Shi, X.; Rininsland, F.; Kushon, S.; Xia, W.; Ley, K.; Achyuthan, K.; McBranch, D.; Whitten, D. Proc. Natl. Acad. Sci. U.S.A. 2004, 101, 7511. (e) Liu, B.; Bazan, G. C. Chem. Mater.

J. Phys. Chem. B, Vol. 113, No. 17, 2009 5793 2004, 16, 4467. (f) Lee, K.; Povlich, L. K.; Kim, J. AdV. Funct. Mater. 2007, 17, 2580. (g) Haskins-Glusac, K.; Pinto, M. R.; Tan, C.; Schanze, K. S. J. Am. Chem. Soc. 2004, 126, 14964. (3) (a) Gaylord, B. S.; Heeger, A. J.; Bazan, G. C. Proc. Natl. Acad. Sci. U.S.A. 2002, 99, 10954. (b) Gaylord, B. S.; Heeger, A. J.; Bazan, G. C. J. Am. Chem. Soc. 2003, 125, 896. (4) (a) Duan, X.; Li, Z.; He, F.; Wang, S. J. Am. Chem. Soc. 2007, 129, 4154. (b) He, F.; Tang, Y.; Yu, M.; Feng, F.; An, L.; Sun, H.; Wang, S.; Li, Y.; Zhu, D.; Bazan, G. C. J. Am. Chem. Soc. 2006, 128, 6764. (5) (a) Ho, H.; Leclerc, M. J. Am. Chem. Soc. 2004, 126, 1384. (b) Haskins-Glusac, K.; Pinto, M. R.; Tan, C.; Schanze, K. S. J. Am. Chem. Soc. 2004, 126, 14964. (c) Li, C.; Numata, M.; Takeuchi, M.; Shinkai, S. Angew.Chem., Int. Ed. 2005, 44, 6371. (d) Wosnick, J. H.; Mello, C. M.; Swager, T. M. J. Am. Chem. Soc. 2005, 127, 3400. (e) Tang, Y.; Feng, F.; He, F.; Wang, S.; Li, Y.; Zhu, D. J. Am. Chem. Soc. 2006, 128, 14972. (f) Miranda, O. R.; You, C. C.; Phillips, R.; Kim, I. B.; Ghosh, P. S.; Bunz, U. H. F.; Rotello, V. M. J. Am. Chem. Soc. 2007, 129, 9856. (g) Ho, H.; Dore´, K.; Boissinot, M.; Bergeron, M. G.; Tanguay, R. M.; Boudreau, D.; Leclerc, M. J. Am. Chem. Soc. 2005, 127, 12673. (6) Bazan, G. C. J. Org. Chem. 2007, 72, 8615. (7) Liu, B.; Bazan, G. C. J. Am. Chem. Soc. 2006, 128, 1188. (8) (a) Woo, H. Y.; Vak, D.; Korystov, D.; Mikhailovsky, A.; Bazan, G. C.; Kim, D.-Y. AdV. Funct. Mater. 2007, 17, 290. (b) Liu, B.; Gaylord, B. S.; Wang, S.; Bazan, G. C. J. Am. Chem. Soc. 2003, 125, 6705. (c) Heeger, P. S.; Heeger, A. J. Proc. Natl. Acad. Sci. U.S.A. 1999, 96, 12219. (d) Xu, Q.-H.; Gaylord, B. S.; Wang, S.; Bazan, G. C.; Moses, D.; Heeger, A. J. Proc. Natl. Acad. Sci. U.S.A. 2004, 10, 11634. (9) (a) Wang, S.; Bazan, G. C. Chem. Commun. 2004, 2508. (b) Liu, B.; Bazan, G. C. Chem. Asian J. 2007, 2, 499. (10) Pu, K.-Y.; Pan, S. Y.-H.; Liu, B. J. Phys. Chem. B 2008, 112, 9295. (11) Yang, R.; Wu, H.; Cao, Y.; Bazan, G. C. J. Am. Chem. Soc. 2006, 128, 14422. (12) (a) Yang, R.; Garcia, A.; Korystov, D.; Mikhailovsky, A.; Bazan, G. C.; Nguyen, T.-Q. J. Am. Chem. Soc. 2006, 128, 16532. (b) McCullough, R. D.; Ewbank, P. C.; Loewe, R. S. J. Am. Chem. Soc. 1997, 119, 633. (c) Due to the limited solubility in water, we prepared a stock (10-2-10-3 M) solution of each polymer in dimethyl sulfoxide and the stock solution was diluted ∼1000 times in water for FRET PL experiments. (13) Nguyen, T.-Q.; Doan, V.; Schwartz, B. J. J. Chem. Phys. 1999, 110, 4068. (14) Connolly, M. L. J. Am. Chem. Soc. 1985, 107, 1118. (15) Richmond, T. J. J. Mol. Biol. 1984, 178, 63. (16) Wolfert, M. A.; Dash, P. R.; Navarova, O.; Oupicky, D.; Seymour, L. W.; Smart, S.; Strohalm, J.; Ulbrich, K. M. A. Bioconjugate Chem. 1999, 10, 993. (17) Wu, H. S.; Fang, T.-R.; Meng, S.-S.; Hu, K.-H. J. Mol. Catal. A: Chem. 1998, 136, 135. (18) Ganachaud, F.; Elaı¨ssari, A.; Pichot, C.; Laayoun, A.; Cros, P. Langmuir 1997, 13, 701. (19) Hong, J. W.; Benmansour, H.; Bazan, G. C. Chem.sEur. J. 2003, 9, 3186. (20) Lakowicz, J. R. Principles of Fluorescence Spectroscopy; Kluwer Academic/Plenum: New York, 1999. (21) Tan, C. Y.; Atas, E.; Mu¨ller, J. G.; Pinto, M. R.; Kleiman, V. D.; Schanzec, K. S. J. Am. Chem. Soc. 2004, 126, 13685. (22) (a) Go¨tz, M.; Hess, S.; Beste, G.; Skerra, A.; Michel-Beyerle, M. E. Biochemistry 2002, 41, 4156. (b) Nayak, R. R.; Nag, O. K.; Woo, H. Y.; Hwang, S.; Vak, D.; Korystov, D.; Jin, Y.; Suh, H. Curr. Appl. Phys. 2009, 9, 636.

JP8107733