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pH-Responsive and pyrene based electrospun nanofibers for DNA adsorption and detection

A facile and labeling-free DNA adsorption and detection method is developed by pH-responsive and luminescent electrospun (ES) nanofiber mats fabricated using poly((2-(dimethylamino)ethyl methacrylate)-co-(stearyl acrylate)-co-((1-pyrene)methyl 2-methyl-2-propenoate)) (poly(DMAEMA-coSA-co-Py)). FE-SEM images show the ES nanofibers have good wettability and integrity in aqueous solution with smooth and bead-free morphology. A decrease in the excimer-to-monomer fluorescent intensity ratio of pyrene moiety from pH 7 to pH 5 indicates a deswelling–swelling transition behavior due to protonation of DMAEMA segment. However, an obvious increase of excimer-to-monomer ratio is observed in the presence of DNA because of the electrostatic interaction between cationic fibers and negative charged phosphate groups from DNA. The detachment of DNA could be modulated by adjusting the pH value to 7. These results indicate this new ES nanofibrous mat has the potential applications for DNA adsorption, detection and separation.


Introduction
Electrospinning (ES) is a well-known and simple technique that utilizes electrostatic forces to produce various functional and composite nanobrous lms. [1][2][3][4][5] In this process, a high electrical eld is applied to polymer solution or melts to create a polymer jet at spinneret, which is then elongated until reaching the collector to form a continuous and ultrane ber. 1 Unlike conventional solution-and melt-spinning techniques, ES process is capable of generating bers with a diameter ranging from tens of nanometers to a few microns. 4 In addition, ES nanobers with three-dimensional scaffolds have been proved to possess high porosity and large surface area that is approximately 1 to 2 orders of magnitude larger than that of the continuous thin lms. 6 These unique characteristics render ES nanobers feasible for adsorption/separation of various heavy metal ions, 6,7 pollutants, 8 microorganism 9 and biomolecules (e.g. DNA, 10 and large proteins 11,12 ).
Deoxyribonucleic acid (DNA) is a biopolymer containing genetic information that plays an important role in many applications ranging from medical, genetic, diagnostic, environmental, agriculture and food sciences. Immobilized DNA is demanded for development of DNA chips and arrays, DNA sensors and gene therapy. Although surface-modied substrate (e.g. cationic polymer coated surface) or continuous thin lms have been reported, 13,14 low adsorption/binding capacity is still main disadvantages due to their low surface area. ES nanobers with high surface area-to-volume ratio are favorable for DNA detection/immobilization. However, little attention has been devoted to utilizing ES nanobers as solid supports for DNA detection/immobilization. Uyar et al. reported surface modication of ES cellulose acetate nanobers with cationic polymer brush was used for DNA adsorption. 10 They demonstrated the enhanced DNA adsorption capacity and reusability of the nano-bers, which provided a facile method for DNA immobilization. Kong et al. reported a brous membrane of polyethylene glycol-modied polyethylenimine via electrospinning was utilized to immobilize DNA onto it for effective gene delivery. 15 They suggested the combination of non-viral gene therapy and ES nano-brous scaffolds has potential applications in tissue regeneration. Chen et al. utilized a aligned ES uorescent nano-bers via blends of cationic polyuorene derivatives and poly(methyl methacrylate) for DNA detection. 16 They showed micromolar DNA detection range of nanobers. In addition to DNA detection/immobilization, ES nanobers can be used as adsorptive membrane for purication or separation of DNA, which is favorable for gene therapeutic agent production.
Pyrene is a good candidate as a uorescent probe because it shows high sensitivity to the micro-environment 17,18 and has a strong tendency to form excimer via intermolecular p-p stacking, 17,19 which exhibits a broad, structureless and red-shied uorescent emission with respect to that of monomeric pyrene. The unique monomer and excimer emissions at different wavelength are considerably dependent on the relative distance and geometry between pyrene molecules. 20 In the past decades, several pyrene-based DNA sensors have been developed. [21][22][23] For example, we reported the cationic charged amino moiety at the periphery of the dendrons with functional pyrene as uorescent probe for DNA detection. 23 Most of these pyrenebased DNA sensors are still operated in solutions that limit their applications. Recently, pyrene-labeled ES nanobers have been developed via physical doping or covalent attachment of pyrene to nanobers for TNT and metal ions detection. 6,24 However, its applications for DNA detection have not been explored yet.
Herein, the aim of this study is to fabricate a uorescentlabeled ES nanober mat that not only provides uorescent signal to detect DNA, but adsorbs/detaches DNA for purication or separation. A series of pH-responsive and luminescent random copolymers poly((2-(dimethylamino)ethyl methacrylate)-co-(stearyl acrylate)-co-((1-pyrene)methyl 2-methyl-2propenoate)) (P(DMAEMA-co-SA-co-Py), Pm1-Pm3) with different molar ratios of SA has been synthesized via free radical polymerization. The pH-responsive DMAEMA segment with a pK a around 7.3-7.5 25,26 is utilized to form cationic amino groups for DNA adsorption where the degree of ionization could be manipulated by pH ranging from 7 to 5. In addition, the physical cross-linkable SA segment plays an important role to maintain the integrity of nanobers as immersed in aqueous environment. The chemical structures and properties of all copolymers were characterized by 1 H-NMR, gel permeation chromatography (GPC), differential scanning calorimetry (DSC) and thermogravimetric analysis (TGA). The morphology of ES nanobers with different SA content was investigated by eldemission scanning electron microscope (FE-SEM) and photoluminescence spectroscopy (PL) at pH ranging from 7 to 5, showing deswelling-swelling characteristics and change of pyrene excimer emission due to the different degree of ionization. DNA adsorption/detection on cationic nanobers has been characterized in terms of FE-SEM, PL, and laser confocal microscope.
Synthesis of poly(DMAEMA-co-SA-co-Py) copolymers Poly(DMAEMA-co-SA-co-Py) (Pm1-Pm3) was synthesized by free radical polymerization with different monomer ratios of DMAEMA, SA and Py monomers as listed in Table 1. The concentration of AIBN used as the initiator was 0.003 M and the monomer to initiator ratio was xed at 450/1. Typically for synthesis of Pm3, DMAEMA (3.99 g, 25.4 mmol), SA (2.21 g, 6.81 mmol), Py (573 mg, 1.91 mmol) and AIBN (12.4 mg) were added to Schlenk tube and dissolved in 24 mL benzene. Nitrogen was bubbled through the mixture for 30 min, and then degassed by three freeze-pump-thaw cycles. The reaction was stirred under positive nitrogen pressure and placed into an oil bath at 65 C for 18 h. Then, the reaction solution was precipitated into hexane and ltered to remove residual monomers. The obtained copolymer was dried in vacuum oven at 40 C to yield white powder 4.9 g with yield of 73%.

Preparation of electrospun nanobers
Poly(DMAEMA-co-SA-co-Py) copolymers were dissolved in chloroform with polymer concentration of 250 mg mL À1 and 5 wt% benzyl triethylammonium chloride (BTEAC, corresponding to polymer) was added to enhance the conductivity of electrospun solution. The solution conductivity was 9.5 ms cm À1 measured by Clean CON500 conductivity meter. The mixture was stirred for one day to give a clear homogeneous solution before preparing the ES nanobers. The ES nanobers were prepared using a single-capillary spinneret. First, the polymer solution was fed into the syringe pump (Chemyx Fusion 100, USA) connected to a 22 gauge metallic needle, with feed rate of 0.3 mL h À1 . The metallic needle was connected to a high voltage power supply (You-Shang Technical Corp., Taiwan) with voltage set at 11 kV, and a piece of aluminum foil or glass was placed 15 cm below the tip of the needle (working distance) to collect the ES nanobers. All experiments were carried out at room temperature (27 C) and around 43% relative humidity. The prepared nanobers were sealed with nitrogen and stored under 0 C in dark.

Preparation of dip-coating lms
For comparison with the properties of the ES nanobers, the corresponding polymer lms were prepared on glass substrate with the same concentration of copolymer (250 mg mL À1 ) mixture by dip-coating method. The glass substrate was dipped in the polymer solution for 3 min and then the substrate was drawn at the rate of 3 cm min À1 by the syringe pump. The prepared lms were dried in an airow hood and the thickness was 3.3 mm measured by FE-SEM (Hitachi S-4800). The prepared lms were sealed with nitrogen and stored under 0 C in dark.

General characterization
1 H NMR spectra were measured in CDCl 3 by Bruker-DPX-400 instrument spectrometer. GPC analysis was performed with a Waters 1420 pump and a Waters 2410 refractive index detector, in reference with a series of PS standards and THF as the eluent at the ow rate of 1 mL min À1 at 35 C. Thermogravimetric analysis was carried out by Dupont TA instrument TGA 2050 analyzer with heating range from 100 to 800 C at a heating rate of 10 C min À1 under dry nitrogen rate of 90 mL min À1 . Differential scanning calorimetry was performed on a TA instruments-Modulated DSC 2910. DSC curve of each sample was obtained from the second heating run at a rate of 5 C min À1 from À35 to 160 C under dry nitrogen rate of 50 mL min À1 . Lower critical solution temperature (LCST) and upper critical solution temperature (UCST) of prepared polymer solution was determined by monitoring the transmittance at 500 nm with a UV-vis spectrometer (JASCO V-650) equipped with an EHCS-760 Thermostat. The concentration of polymer in water was 4 mg mL À1 . The temperature was raised from 15 to 60 C in every 2.5 C increment and all samples were equilibrated for 5 min before measurement. LCSTs and UCSTs of polymer solutions were determined at the temperature showing 50% optical transmittance of polymer solution.

Morphological characterization
The morphologies of the ES nanobers were characterized by eld-emission scanning electron microscopy (FE-SEM, Hitachi S-4800). The images were taken using a microscope operated at an accelerating voltage of 15 kV. Before imaging, the samples were sputtered with Pt. The average diameter of nanobers was estimated over y bers in SEM images from each sample. For sample preparation, the ES nanobers on aluminum foil were immersed in different pH or DNA solution for certain minutes and then quickly put into a ask containing liquid nitrogen. The frozen samples were lyophilized to maintain their original morphology. Fluorescent microscope images of the ES nano-bers were taken using Olympus FV1000 Confocal Microscope. A 405 nm laser diode was used to excite pyrene moiety.

Photophysical properties
Photoluminescence spectra (PL) were recorded on a Hitachi F-4500 spectrophotometer at excitation wavelength of 341 nm. The ES nanobers on glass substrate or dip-coating lms were inserted diagonally in the cuvettes and xed to ensure the detection at the same position during every measurement. 3 mL of aqueous solution with different pH value or DNA solution with different concentration was lled the cuvette and the bers or lms were incubated for 3 min before measurement.

Results and discussion
Characterization of poly(DMAEMA-co-SA-co-Py) copolymers A series of pH-responsive and pyrene-based copolymers, poly((2-(dimethylamino)ethyl methacrylate)-co-(stearyl acrylate)-co-((1-pyrene)methyl 2-methyl-2-propenoate)) (poly(DMAEMA-co-SAco-Py), Pm1-Pm3), was synthesized via free radical polymerization with feeding ratio of monomer to initiator xed at 450/1. In addition, the feeding ratio of DMAEMA to Py was xed at 40/3 in order to keep the copolymers to have the same ratio of uorescent probes and pH-responsive properties, while varied with different SA content, which showed physical cross-linking function to prevent the dissolution of the ES nanobers and regulate the volume change in different pH value. The obtained copolymers were fully characterized by gel permeation chromatography (GPC) and 1 H-NMR spectra. Fig. 1 shows 1 H-NMR spectra of the copolymers in CDCl 3 . The chemical compositions of the copolymers were determined by comparing the proton signal of aromatic ring on pyrene units observed at approximately 7.99-8.34 ppm (peak a), the methylene protons of DMAEMA at 2.54 ppm (peak i) to the protons of alkyl side chain from SA moiety at 1.25 ppm (peak f). The calculated copolymer compositions from the proton peak integration are consistent with the initial feed ratios, indicating the successful synthesis of the copolymers. The calculated molar fractions of SA moiety to Pm1-Pm3 copolymers are 0 mol%, 5.3 mol% and 14 mol%, respectively. The number-average molecular weights (M n ) of the Pm1-Pm3 copolymers evaluated by GPC are 18 800, 14 900, and 35 500 g mol À1 , with the corresponding PDI of 2.54, 2.01, and 1.89, respectively. The chemical compositions, molecular weight, and thermal properties are summarized in Table 1. The thermal properties of the copolymers were  investigated by TGA and DSC ( Fig. S1 and S2 in the ESI †). The thermal decomposition temperature (T d ) of the copolymers increased from 256 to 286 C with increasing SA content. On the other hand, the glass transition temperature (T g ) of Pm1 and Pm2 copolymers was 24.8 and 12.1 C, respectively. Pm1 has a higher T g than reported PDMAEMA homopolymer (14 C) 28 because of the incorporation of rigid pyrene moiety. However, a broad endothermic peak was appeared at Pm3 copolymer, which had 14 mol% of SA content and the long alkyl chain caused formation of a crystalline aggregation structure. No obvious T g was observed and thus we suggested its T g was below 15 C. The temperature-responsive behaviors of Pm1-Pm3 copolymers in pH 7 and pH 5 aqueous solutions were evaluated by temperature-controlled UV-visible spectroscopy. The LCSTs (lower critical solution temperatures) and UCSTs (upper critical solution temperatures) were determined by observing the 50% optical transmittance of copolymer solutions at 500 nm as a function of temperature (Fig. 2). It was found that the change in solution pH affected the phase transition of the copolymers. At pH 7, the LCSTs were decreased from 40.2, 33.6, to 25.8 C with increasing SA content due to its hydrophobic characteristic of long alkyl side chain that facilitated chain aggregation in aqueous solution and reduced the LCST. At pH 5, no LCST of Pm1 copolymer was observed in the studied temperature range from 15 to 60 C, indicating their strong hydrophilicity due to the protonation of PDMAEMA segments. Interestingly, Pm2 and Pm3 exhibited UCST characteristics, which were found insoluble at low temperature and became soluble as temperature above 23.0 and 32.2 C, respectively. This might be attributed to the physically crosslinking via hydrophobic interaction of the stearyl side chains, 28,29 resulting in the aggregation of the copolymers in water at low temperature. However, when the temperature was raised, the intermolecular hydrophobic interactions no longer existed and hydrophilicity of protonated DMAEMA became dominant, leading the copolymers to rehydrate in aqueous solution.
Morphology of poly(DMAEMA-co-SA-co-Py) Fig. 3 shows the FE-SEM images of poly(DMAEMA-co-SA-co-Py) nanobers fabricated by electrospinning with BTEAC salt added to increase the conductivity of the polymer solution in methanol. Pm1 and Pm2 nanobers have average diameters of 723 AE 153 and 526 AE 136 nm in a dry state, respectively. The average diameter of nanobers was estimated over y bers from each sample. The inset SEM images show a smooth and nonporous surface of as-prepared Pm1 and Pm2 nanobers. However, unclear and collapsed brous structures were observed on Pm3, because it has much lower T g than room temperature. Nevertheless, stearyl acrylate has been reported to exhibit the shape memory effect and the formation of crystalline aggregates  among the long alkyl side chains would locks its shape. 30 Thus, we believe the bers could maintain their cylindrical shape even at room temperature.
Since the ionic interaction of the ES nanobers are required for DNA adsorption, the cationic amino group of DMAEMA segments should be established under acidic environment. Although physical cross-linked SA moiety was introduced to improve the stability of those ES bers, both thermo-and pHresponsive properties of DMAEMA segments might affect the ber morphology at different pH solution. In this study, the effect of pH on ber morphology was evaluated at pH 7 and pH 5, which were below the pK a of DMAEMA (around 7.3-7.5 in water). 25,26 As shown in Fig. 4a, Pm1 without physical crosslinking moiety was dissolved in water and could not maintain the brous shape at pH 5 or 7 due to protonation of DMAEMA units that increased the hydrophilicity of the nanobers. Moreover, the LCSTs of Pm1 at pH 5 or 7 were higher than room temperature, also resulting in hydrophilic characteristic of Pm1 bers. Fig. 4b shows Pm2 nanobers were also dissolved at pH 7, but a partial dissolution of bers was observed at pH 5. The reasons that cause this phenomenon are still not clear, but we suggest three interactions, including (i) hydrogen-bonding interaction between DMAEMA and water molecules that inuences the LCST, (ii) electrostatic repulsion from the protonation of DMAEMA, and (iii) hydrophobic interaction among the long alkyl side chain of SA, might compete to affect the hydrophilic/ hydrophobic balance of the nanobers. In pH 7 solution at room temperature, less protonation of DMAEMA segments rendered the hydrogen-bonding interaction between DMAEMA and water molecules dominant that caused the nanobers soluble when temperature was below the LCST (33.6 C). On the contrary, at pH 5, although more DMAEMA units were protonated (no LCST), the thermo-responsive behaviors discussed above proved the physically crosslinking via hydrophobic interaction of the stearyl side chains was signicant at pH 5. Therefore, Pm2 became less soluble at pH 5 and exhibited fragmented ber morphology. Fig. 4c shows brous morphology of Pm3 at pH 7 and pH 5 solutions due to its high content of the cross-linkable SA moiety and proper thermal behavior. At room temperature (27 C) which was higher than its LCST (25.8 C) and lower than its UCST (32.2 C), Pm3 became hydrophobic that stabilized the ber structures in aqueous solution. The average diameters of nanobers at pH 7 and pH 5 are 789 AE 240 and 858 AE 156 nm, respectively. Compared to Fig. 3c, the appearance of clear ber structures in wet state relative to those in dry state might be due to penetration of water molecules into bers as well as the electrostatic repulsion between cationic DMAEMA segments that swell the diameter of nanobers. To test the morphological stability of nanobers at pH 5 solution, Pm3 ES nanobers were immersed in solution at different time. The results showed Pm3 nanobers still maintained their well-dened brous structures, indicating they have excellent stability at pH 5 and are suitable for DNA detection (Fig. S3 †). Pm3 nanobers with good stability in different pH solution were chosen for DNA adsorption.
Photoluminescence of poly(DMAEMA-co-SA-co-Py) ES nanobers Fig. 5a shows the photoluminescence (PL) spectra of Pm3 ES nanobers at different pH solution. The emission bands at 378, 386, and 396 nm are corresponded to the monomer emission of pyrene, while the broad emission band centered at 471 nm is ascribed to emission of pyrene excimer. The monomer emission of all PL spectra at 378 nm is normalized in order to distinguish the change of excimer emission. The emerged excimer emission in nanobers indicates the pyrene molecules are in close proximity within nanobers due to strong p-p interactions. 31 As pH decreased from 7 to 5, a signicant decrease in excimer emission was attributed to the separation of neighboring pyrenes from each other. The ratios of excimer to monomer (I 471 / I 378 ) were substantial decreased from 2.24 (pH 7), 1.34 (pH 6) to 0.77 (pH 5). This result conrmed that the quaternized amine groups of DMAEMA under acidic environment caused intensive electrostatic repulsion between DMAEMA segments and swelling of the nanobers (Scheme 1). Moreover, there was no pyrene emission detected in aqueous solution when nanobers were immersed at pH 5, indicating the nanobers were stable (Fig. S4 †). Compared to dip-coating lms (Fig. 5b), the I 471 /I 378 ratios were slightly decreased from 1.14 (pH 7), 1.02 (pH 6) to 0.76 (pH 5). It indicates Pm3 ES nanobers have a better pHresponsive performance and swelling-deswelling transition due to their high surface-to-volume ratio.
The reversibility of pH induced swelling-deswelling transitions of Pm3 nanobers, which correlated with protonation/ deprotonation of DMAEMA segments, was evaluated by monitoring the I 471 /I 378 ratios of pyrene during alternatively changing the pH of solution. Four successive cycles were performed (Fig. 5c). Pm3 nanobers exhibited a notable change of excimer to monomer ratio when immersed in different pH solution. However, the excimer to monomer ratio at pH 7 slightly deviated from its initial value aer three circles, suggesting the nanobers could not completely shrink back to original state within experimental time and reduce p-p interaction between pyrene moieties.

Detection of DNA via poly(DMAEMA-co-SA-co-Py) ES nanobers
We have demonstrated PL characteristics were correlated with protonation/deprotonation of DMAEMA segments and could be controlled by pH. Because DNA molecules prefer to adsorb on cationic surface via electrostatic interaction, protonated DMAEMA segments of nanobers under acidic pH would facilitate complexation with DNA that might change the morphology of nanobers and further affect the excimer to monomer ratio of pyrene. Fig. 6a shows PL spectra of cationic Pm3 ES nanobers under different concentration of DNA. A gradual increase in I 471 /I 378 ratio from 0.77 (pH 5) to 1.04 upon addition of 10 nM DNA indicated the DNA adsorbed on the nanobers, resulting in more pyrene moieties in close proximity and enhancing the excimer emission. However, the I 471 /I 378 ratio of DNA complexed bers was less than that of its original state at pH 7 (I 471 /I 378 ¼ 2.24). We suggested pyrene moieties might be intercalated into the DNA double helix that prevented Scheme 1 Schematic drawing of swelling-deswelling transition of nanofiber, which correlates with formation of pyrene excimer and DNA detection using the quaternized nanofibers. the excimer formation. 21 In addition, a photoinduced electron transfer process might occur between excited pyrene and nucleobases, resulting in uorescent quenching of pyrene excimer 32,33 (Scheme 1). DNA detachment could be carried out by adjusting the pH values from pH 5 to pH 7. The enhanced excimer emission indicated DNA released from nanobers ( Fig. 6b). At pH 7, the reduced charge density on DMAEMA segments signicantly diminished the interaction between DNA and polymer chains, resulting in increase of excimer emission. Fig. 7 shows FE-SEM images of cationic Pm3 ES nanobers (at pH 5) complexed with DNA molecules. As shown in the gure, the brous structures changed to laments or akes. This is mainly ascribed to the electrostatic interaction between cationic nanobers and negatively charged DNA. In addition, p-p interaction between pyrene and heterocyclic base of DNA as well as hydrogen bonding resulting from the carbonyl groups of copolymers and amino groups of DNA might also cause to change the ber shapes. Although the brous structures were changed, PL spectra demonstrated no dissociated polymer chains were detected in solution (Fig. S5 †). Fig. 8 shows confocal microscope images of Pm3 ES nanobers at different pH and complexed with DNA. The blue emission collected at a range of 430 to 530 nm represents the excimer formation. The intense blue uorescent bers were found at pH 7, while signicantly uorescent quenching was observed at pH 5. This conrmed the change in charge density of DMAEMA caused the nanobers to exhibit swelling-deswelling transitions and affected excimer formation. On the other hands, when nano-bers were complexed with DNA, the increase of blue emission was found due to effectively adsorption of DNA onto the nanobers. These results are consistent with PL measurement.

Conclusions
In summary, we developed a new luminescent and pHresponsive poly(DMAEMA-co-SA-co-Py) nanober mat via free radical polymerization and electrospinning technique for adsorption and detection of DNA. Through the delicate adjustment of SA content, the prepared Pm3 ES nanobers showed good wettability and integrity in different pH solution with smooth and bead-free morphology. The rapid swellingdeswelling transition of the nanobers at different pH solution could be monitored by the excimer-to-monomer ratio (I 471 /I 378 ) of pyrene. The decrease in excimer-to-monomer ratio under acidic environment conrms that the quaternized amine groups of DMAEMA cause intensive electrostatic repulsion between DMAEMA segments and swelling of the nanobers that separate the pyrene excimer. The DNA detection was performed under pH 5 to protonate DMAEMA of the nanobers. The increase in excimer to monomer ratios upon addition of DNA indicate adsorption of DNA on the nanobers through electrostatic interaction causes more pyrene moieties in close   proximity. From all the present results, it can be concluded that the poly(DMAEMA-co-SA-co-Py) nanober mat not only provides uorescent signal to detect and adsorb DNA without dyelabeling, but open up a eld of applications to detect other targets, such as ATP, ADP or protein etc. via electrostatic interaction for purication or bioseparation. Further effort will focus on systematic exploration on uorescent and morphology characterizations of the attachment and detachment of DNA or other targets on the brous mats.