A New Light-Harvesting Conjugated Polyelectrolyte Microgel for DNA

pubs.acs.org/Langmuir © 2009 American Chemical Society

A New Light-Harvesting Conjugated Polyelectrolyte Microgel for DNA and Enzyme Detections† Xuli Feng, Qingling Xu, Libing Liu, and Shu Wang* Beijing National Laboratory for Molecular Sciences, Key Laboratory of Organic Solids, Institute of Chemistry, Chinese Academy of Sciences, Beijing 100190, P. R. China Received April 23, 2009. Revised Manuscript Received May 15, 2009 A new fluorescent microgel containing the CP moiety (PFP-NIPAm) was prepared with the size within 100 nm. Covalent linking of the conjugated polyelectrolyte moiety into the microgel prevents leakage of the fluorophore while keeping its fluorescence property. The new fluorescent microgel can be used as an optical platform to detect DNA and enzyme. More importantly, because the electrostatic attraction dominates the interactions between cationic PFPNIPAm microgel and negatively charged target, washing with high ionic strength aqueous solution can block their interactions and displace the target from the PFP-NIPAm microgel. Thus, PFP-NIPAm can be readily reusable for detection. Another unique feature is that the PFP-NIPAm microgel extends the detection media from homogeneous solution to solid phase, which shows great potential for biodetection in the real world using conjugated polymers. Furthermore, the detection can be carried out under UV light, and no expensive detection instrumentation is needed. In principle, such an assay system could be expanded to a high-throughput assay.

1. Introduction In recent years, conjugated polyelectrolytes (CPs) have attracted intense attention as new optical platforms for biosensors. In comparison to small molecule-based assays, the CPs collect the action of a large number of absorbing units, and the transfer of excitation energy along the whole backbone to the chromophore reporter results in the amplification of fluorescence signals, which improves the detection sensitivity significantly.1-6 † Part of the “Langmuir 25th Year: Molecular and macromolecular selfassemblies” special issue. *Corresponding author. E-mail: [email protected].

(1) (a) Swager, T. M. Acc. Chem. Res. 1998, 31, 201. (b) Thomas, S. W.III; Joly, G. D.; Swager, T. M. Chem. Rev. 2007, 107, 1339. (c) McQuade, D. T.; Pullen, A. E.; Swager, T. M. Chem. Rev. 2000, 100, 2537. (2) Bunz, U. H. F. Chem. Rev. 2000, 100, 1605. (3) Liu, B.; Bazan, G. C. Chem. Mater. 2004, 16, 4467. (4) Ho, H. A.; Najari, A.; Leclerc, M. Acc. Chem. Res. 2008, 41, 168. (5) Feng, F.; He, F.; An, L.; Wang, S.; Li, Y.; Zhu, D. Adv. Mater. 2008, 20, 2959. (6) 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. (7) (a) Gaylord, B. S.; Heeger, A. J.; Bazan, G. C. Proc. Natl. Acid. Sci. U.S.A. 2002, 99, 10954. (b) Wang, S.; Gaylord, B. S.; Bazan, G. C. J. Am. Chem. Soc. 2004, 126, 5446. (c) Liu, B.; Bazan, G. C. J. Am. Chem. Soc. 2006, 128, 1188. (d) Gaylord, B. S.; Massie, M. R.; Feinstein, S. C.; Bazan, G. C. Proc. Natl. Acad. Sci. U.S.A. 2005, 102, 34. (8) (a) Wang, D.; Gong, X.; Heeger, P. S.; Rininsland, F.; Bazan, G. C.; Heeger, A. J. Proc. Natl. Acad. Sci. U.S.A. 2002, 99, 49. (b) Fan, C.; Plaxco, K. W.; Heeger, A. J. Trends Biotechnol. 2005, 23, 186. (c) Xu, Q.-H.; Wang, S.; Korystov, D.; Mikhailovsky, A.; Bazan, G. C.; Moses, D.; Heeger, A. J. Proc. Natl. Acad. Sci. U.S.A. 2005, 102, 530. (9) (a) Chen, L. H.; McBranch, D. W.; Wang, H. L.; Helgeouson, R.; Wudl, F.; Whitten, D. G. Proc. Natl. Acad. Sci. U.S. A. 1999, 96, 12287. (b) Kumaraswamy, S.; Bergstedt, T. S.; Shi, X.; Rininsland, F.; Kushon, S. A.; Xia, W.; Ley, K. D.; Achyuthan, K. E.; McBranch, D.; Whitten, D. G. Proc. Natl. Acad. Soc. U.S.A. 2004, 101, 7511. (c) Hemburu, S. C.; Corbitt, T. S.; Ista, L. K.; Ji, E.; Fulghum, J.; Lopez, G. P.; Ogawa, K.; Schanze, K. S.; Whitten, D. G. Langmuir 2008, 24, 11053. (d) Chen, L.; Xu, S.; McBranch, D. W.; Whitten, D. G. J. Am. Chem. Soc. 2000, 122, 9302. (10) (a) Yang, C. Y. J.; Pinto, M. R.; Schanze, K. S.; Tan, W. H. Angew. Chem., Int. Ed. 2005, 44, 2572. (b) Pinto, M. R.; Schanze, K. S. Proc. Natl. Acad. Soc. U.S. A. 2004, 101, 7505. (c) Liu, Y.; Ogawa, K.; Schanze, K. S. Anal. Chem. 2008, 80, 150. (d) Liu, Y.; Schanze, K. S. Anal. Chem. 2009, 81, 231.

Langmuir 2009, 25(24), 13737–13741

They have been widely employed to detect nucleic acid, protein, enzyme, metal ions, and biological small molecules.7-17 Biosensors based on CPs exhibit outstanding features, such as fast response speed, high sensitivity, convenience, and so on. However, most of these biosensors are confined to detect biomacromolecules in homogeneous solutions, which reduces the possibility of commercialization of such detection methods, because a great part of existing commercialized biosensing devices are based on solid detection manner. Therefore, finding a way to transfer detection using CPs from aqueous solution to the solid phase is needed. In this respect, microspheres modified with CPs (11) (a) Kim, I. B.; Erdogan, B.; Wilson, J. N.; Bunz, U. H. F. Chem.;Eur. J. 2004, 10, 6247. (b) Phillips, R. L.Miranda, O. R.; You, C. C.; Rotello, V. M.; Bunz, U. H. F. Angew. Chem., Int. Ed. 2008, 47, 2590. (c) 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. (12) (a) Wosnick, J. H.; Mello, C. M.; Swager, T. M. J. Am. Chem. Soc. 2005, 127, 3400. (b) Satrijo, A.; Swager, T. M. J. Am. Chem. Soc. 2007, 129, 16020. (c) Kim, J.; McQuade, D. T.; McHugh, S. K.; Swager, T. M. Angew. Chem., Int. Ed. 2000, 39, 3868. (d) Kuroda, K.; Swager, T. M. Macromolecules 2004, 37, 716–724. (13) (a) Ho, H. A.; Boissinot, M.; Bergeron, M. G.; Corbeil, G.; Dor
e, K.; Boudreau, D.; Leclerc, M. Angew. Chem., Int. Ed. 2002, 41, 1548–1551. (b) Dor
e, K.; Dubus, S.; Ho, H. A.; L
evesque, I.; Brunette, M.; Corbeil, G.; Boissinot, M.; Boivin, G.; Bergeron, M. G.; Boudreau, D.; Leclerc, M. J. Am. Chem. Soc. 2004, 126, 4240–4244. (c) Ho, H. A.; B
era-Ab
erem, M.; Leclerc, M. Chem.;Eur. J. 2005, 11, 1718–1724. (d) Ab
erem, M. B.; Najari, A.; Ho, H. A.; Gravel, J.-F.; Nobert, P.; Boudreau, D.; Leclerc, M. Adv. Mater. 2006, 18, 2703. (14) (a) Nilsson, K. P. R.; Ingan€as, O. Nat. Mater. 2003, 2, 419. (b) Nilsson, K. P. R.; Rydberg, J.; Baltzer, L.; Ingan€as, O. Proc. Natl. Acad. Sci. U.S.A. 2003, 100, 10170. (c) Sigurdson, C. J.; Nilsson, K. P. R.; Hornemann, S.; Manco, G.; Polymenidou, M.; Schwarz, P.; Leclerc, M.; Hammarstr€om, P.; W€uthrich, K.; Aguzzi, A. Nat. Methods 2007, 4, 1023. (d) Herland, A.; Nilsson, K. P. R.; Olsson, J. D. M.; Hammarstrom, P.; Konradsson, P.; Ingan€as, O. J. Am. Chem. Soc. 2005, 127, 2317. (15) Li, C.; Numata, M.; Takeuchi, M.; Shinkai, S. Angew. Chem., Int. Ed. 2005, 44, 6371. (16) (a) Lee, K.; Rouillard, J.-M.; Pham, T.; Gulari, E.; Kim, J. Angew. Chem., Int. Ed. 2007, 46, 4667. (b) Lee, K.; Povlich, L. K.; Kim, J. Adv. Funct. Mater. 2007, 17, 2580. (17) (a) He, F.; Tang, Y.; Wang, S.; Li, Y.; Zhu, D. J. Am. Chem. Soc. 2005, 127, 12343. (b) Duan, X.; Li, Z.; He, F.; Wang, S. J. Am. Chem. Soc. 2007, 129, 4154. (c) Feng, F.; Wang, H.; Han, L.; Wang, S. J. Am. Chem. Soc. 2008, 130, 11338. (d) Feng, F.; Tang, Y.; Wang, S.; Li, Y.; Zhu, D. Angew. Chem., Int. Ed. 2007, 46, 7882.

Published on Web 06/15/2009

DOI: 10.1021/la901444c

13737

Article

Feng et al.

on their surfaces have been developed for DNA detection.18-21 However, the reusable solid-phase detection system based on CPs still remains a challenge. More and more researchers pay attention to fluorescent colloid spheres that can be used as markers in biological detection.22,23 Various organic dyes have been embedded into monodisperse polymers and silica spheres to generate fluorescent spheres.24 Nevertheless, the undesirable leakage and unstable luminescent properties of these fluorescent spheres restrict their application. Semiconductor nanocrystals (NCs) have also been used for fluorescent detection due to their high photostability.25 The existing major ways to incorporate NCs into colloid spheres are based on either layer-by-layer assembly methods or direct doping of the preformed NCs.26 Due to the penetration of NCs through porous layers, long-term stability of these resulting spheres is problematical. In this article, we present a brand new way to generate inherently fluorescent microgel-containing CP moieties for DNA and enzyme detections. Microgels based on cross-linked poly(N-isopropylacrylamide) (PNIPAM) are well-known;27 herein, we employ N-isopropylacrylamide and vinyl-substituted cationic conjugated polyfluorene to prepare cross-linked fluorescent microgel (PFP-NIPAm) that can overcome the long-term leakage problem while preserving good fluorescence property of polyfluorene. The PFP-NIPAm can be used as optical platform to detect DNA and enzyme. More importantly, the microgel shows good reusability with a simple recovery process.

source. SEM images were taken on Hitachi S-4300 scanning electron microscopy. Preparation of Polymer 2. Excess triethylamine was added to a solution of polymer 1 (12 mg) in anhydrous DMSO followed by stirring at room temperature for 1 h. To the above solution, 15 μL of acryloyl chloride was added dropwise, and the reaction solution was further stirred at room temperature for 24 h. The solvent was removed under reduced pressure to afford the crude product that was dialyzed using a dialysis membrane with a cutoff at M= 3500 g/mL for 2 days to obtain polymer 2.

Preparation of Fluorescent Microgel PFP-NIPAm.

Materials and Measurements. Polymer 128 and enzymatic substrate DA+17d were synthesized according to the procedures in the literature. DNAP-Fl and its complementary strand were purchased from Sunbiotech Co. Ltd. (Beijing, China). Their concentrations were determined by measuring the absorbance at 260 nm in 250 μL quartz cuvettes. ssDNA-F1 was annealed at 80 °C for 20 min and slowly cooled to room temperature to get the hairpin structure. Water was purified using a Millipore filtration system. Fluorescence emission spectra were recorded at room temperature with a Hitachi F-4500 fluorescence spectrophotometer with a xenon lamp as excitation source. Phase-contrast bright-field and fluorescence images were taken with fluorescence microscopy (Olympus 1
71) with a mercury lamp (100 W) as light

N-Isopropylacrylamide (50 mg), polymer 2 (6 mg), potassium persulfate (5 mg), and N,N0 -methylene-bis(acrylamide) (6 mg) were dissolved in 10 mL of water. The polymerization was conducted at 70 °C for 4 h under N2. The as-prepared colloid spheres were purified by centrifugation at 2000 rpm for 10 min and redispersed in water for later use. DNA Detection. To a solution containing fluorescent microgel was added DNAP-Fl ([DNAP-Fl] = 5.0
10-6 M). After incubation for about 10 min, microgel was washed with pure water three times; then, the complementary strands ([ssDNAC]= 5.0
10-6 M) and EB ([EB]=5.0
10-6 M) were added. Upon annealing for 10 min at room temperature, microgel was washed with pure water to get rid of nonspecific absorption of ssDNAC and then was dotted on a glass slide with an Eppendorf pipet. The images were taken with a Canon IXUS 750 digital camera under a UV lamp with an excitation wavelength of 365 nm. Recycling Procedure for Microgel PFP-NIPAm. To a solution containing fluorescent microgel was added DNAP-Fl ([DNAP-Fl]=5.0
10-6 M). After incubation for about 10 min, microgel was washed with pure water three times followed by taking a photo under UV light (λmax=365 nm). The microgel was then washed with 800 mM NaCl twice to remove the DNA and then redispersed in pure water for reuse, followed by taking a photo under UV light (λmax = 365 nm). The same process was repeated three times. Enzyme Detection. Acetylcholinesterase (AchE) was added to a solution containing microgel PFP-NIPAm and enzymatic substrate DA+ in phosphate buffer (25 mM, pH = 8.5). The resulting solution was incubated at 37 °C for a certain period of time (from 0.5 to 3 h) followed by centrifugation at 1000 rpm for 0.5 min, and then the microgel was dotted on a glass slide with an Eppendorf pipet and the images were taken with a Canon IXUS 750 digital camera under UV lamp with an excitation wavelength of 365 nm.

(18) (a) Xu, H.; Wu, H.; Huang, F.; Song, S.; Li, W.; Cao, Y.; Fan, C. Nucleic Acid Res. 2005, 33, e83. (b) Ren, X. S.; Xu, Q. H. Langmuir 2009, 25, 43. (c) Tian, N.; Xu, Q. H. Adv. Mater. 2007, 19, 1988. (d) Tian, N.; Tang, Y.; Xu, Q. H.; Wang, S. Macromol. Rapid Commun. 2007, 28, 729. (19) (a) Kushon, S. A.; Ley, K. D.; Bradford, K.; Jones, R. M.; McBranch, D.; Whitten, D. Langmuir 2002, 18, 7245. (b) Xia, W.; Rininsland, F.; Wittenburg, S. K.; Shi, X.; Achyuthan, K. E.; McBranch, D. W.; Whitten, D. G. Assay Drug. Dev. Technol. 2004, 2, 183. (20) Lee, K.; Maisel, K.; Rouillard, J.-M.; Gulari, E.; Kim, J. Chem. Mater. 2008, 20, 2848. (21) Raymond, F. R.; Ho, H.-A.; Peytavi, R.; Bissonnette, L.; Boissinot, M.; Picard, F. J.; Leclerc, M.; Bergeron, M. G. BMC Biotechnol. 2005, 5, 10. (22) Kuang, M.; Wang, D.-Y; Bao, H.-B; Gao, M.-Y; Mohwald, H.; Jiang, M. Adv. Mater. 2005, 17, 267. (23) (a) Battersby, B. J.; Lawrie, G. A.; Johnston, A. P. R.; Trau, M. Chem. Commun. 2002, 1435. (b) Trau, M.; Battersby, B. J. Adv. Mater. 2001, 13, 975. (24) (a) Fulton, R. J.; McDade, P. L.; Smith, P. L.; Kineker, L. J.; Kettman, J. R. Jr. Clin. Chem. 1997, 43, 1749. (b) Battersby, B. J.; Bryant, D.; Meutermans, W.; Matthews, D.; Smythe, M. L.; Trau, M. J. Am. Chem. Soc. 2000, 122, 2138. (c) Steemers, F. J.; Ferguson, J. A.; Walt, D. R. Nat. Biotechnol. 2000, 18, 94. (25) (a) Chan, W. C. W.; Maxwell, D. J.; Gao, X.; Bailey, R. E.; Han, M.; Nie, S. Curr. Opin. Biotechnol. 2002, 13, 40. (b) Bruchez, M.; Moronne, M.; Gin, P.; Wiess, S.; Alivisatos, A. P. Science 1998, 281, 2013. (c) Chan, W. C.; Nie, S. Science 1998, 281, 2016. (26) (a) Marrinez-Rubio, M. I.; Ireland, T. G.; Fern, G. R.; Silver, J.; Snowden, M. J. Langmuir 2001, 17, 7145. (b) Rogach, A. L.; Nagesha, D.; Ostrander, J. W.; Giersig, M.; Kotov, N. A. Chem. Mater. 2000, 12, 2676. (27) Pelton, R. Adv. Colloid Interface Sci. 2000, 85, 1. (28) He, F.; Feng, F.; Wang, S.; Li, Y.; Zhu, D. J. Mater. Chem. 2007, 17, 3702.

3. Results and Discussion Preparation of microgel PFP-NIPAm. The procedures for the preparation of PFP-NIPAm microgel are shown in Scheme 1. Polymer 1 bearing amino group was synthesized by a palladiumcatalyzed Suzuki cross-coupling reaction with weight-average molecular weight of about 25 000 and a polydispersity index of 2.4 that was reported in our previous work.28 Reaction of acryloyl chloride with polymer 1 in anhydrous DMSO in the presence of excess triethylamine afforded the crude product followed by dialysis using a membrane with cutoff at M = 3500 g/mL to obtain vinyl-substituted polymer 2. The cross-linked light-harvesting microgel PFP-NIPAm was prepared by reacting NIPAm and polymer 2 in the presence of potassium persulfate with N,N0 methylene-bis(acrylamide) (MBAm) as a cross-linking agent. After the surfactant-free emulsion polymerization, the PFPNIPAm was purified by centrifugation and washing with deionized water. Morphology and Optical Property of Microgel PFPNIPAm. The morphology of PFP-NIPAm was studied by optical microscope (Figure 1a), where an intense hydrogel structure was observed. To get more insight into the morphology of the

2. Experimental Section

13738 DOI: 10.1021/la901444c

Langmuir 2009, 25(24), 13737–13741

Feng et al.

Article Scheme 1. Synthesis of Fluorescent Microgel PFP-NIPAm.a

a

The red and green segmented blocks are respectively polymerized from 2 and MBAm, and the blue rod stands for conjugated polyelectrolyte moiety.

Figure 1. (a) Phase contrast bright-field image of PFP-NIP Am microgel. Lower magnification (b) and higher magnification (c) SEM images of PFP-NIPAm microgel. (d) Size distribution of PFP-NIPAm microgel.

PFP-NIPAm hydrogel, its microstructural characterization was performed using scanning electron microscopy (SEM). As shown in Figure 1b, the microgel structure was formed for PFP-NIPAm (also shown in Scheme 1). Higher-magnification SEM image (Figure 1c) indicated that the size of the PFP-NIPAm microgel was about 60 nm (Figure 1d). The fluorescence property of PFP-NIPAm microgel was studied by fluorescent microscope and emission spectra. Fluorescent microscope image (Figure 2a) showed the hydrogel morphology of PFP-NIPAm and confirmed its fluorescence characteristic. As shown in Figure 2b, the emission spectra of PFP-NIPAm exhibited a maximum peak at 420 nm with a shoulder peak at 442 nm, which is characteristic of polyfluorenes upon excitation of the polyfluorene unit at 380 nm.29 These results show that the fluorescence property of polyfluorene is retained in the microgel. It is noted that strong scattering was observed below 400 nm in the emission spectra of PFP-NIPAm, (29) Scherf, U.; List, E. J. W. Adv. Mater. 2002, 14, 477.

Langmuir 2009, 25(24), 13737–13741

which results from the gel environment around the polyfluorene unit. DNA Detection Using Microgel PFP-NIPAm. Our assay strategy for DNA detection using PFP-NIPAm microgel is illustrated in Scheme 2. The molecular beacon DNA with a five-base-pair double-stranded stem labeled with fluorescein at its 50 -terminus (DNAP-Fl) acts as the probe. To a solution containing PFP-NIPAm microgel was added DNAP-Fl ([DNAP-Fl] = 5.0
10-6 M) and ethidium bromide ([EB] = 5.0
10-6 M) where EB cannot intercalate into the groove of DNAP-Fl. The electrostatic interactions between negatively charged DNAP-Fl and positively charged fluorescent PFP-NIPAm microgel keep them in close proximity, facilitating the energy transfer from PFP-NIPAm to fluorescein and inducing the microgel to turn a green fluorescence color under UV light with excitation at 365 nm (Figure 3). Upon addition of ssDNAC to the assay solution at room temperature, double-stranded DNAPFl/DNAC was formed. In this case, excitation of the PFP-NIPAm microgel led to a two-step fluorescence resonance energy transfer (FRET), from PFP-NIPAm to fluorescein (FRET-1) followed by FRET from fluorescein to EB (FRET-2); thus, we can clearly see the color of the PFP-NIPAm microgel changing from green to salmon pink under UV light with excitation at 365 nm. In addition, PFP-NIPAm microgel can easily be recycled following a simple washing step with 800 mM NaCl solution that emits a blue color the same as that of original PFP-NIPAm (Figure 3). To evaluate the detection specificity of PFP-NIPAm microgel, noncomplementary ssDNANC and two-base mismatched ssDNA2NC were added into PFP-NIPAm/DNAP-Fl/EB, respectively. The two-base mismatch could be clearly distinguished with different emission colors (Figure 3). As reported in our previous work,30 when a DNAP-Fl probe containing a longer double-stranded stem was used, the fluorescein or EB emission signal was hardly distinguishable before and after hybridization of DNAP-Fl with target DNA, because the EB could intercalate into the doublestranded grooves in both cases. Obviously, we can use our naked eye to clearly detect the target DNA. Because of the transfer detection using CPs from aqueous solution to solid phase, we (30) Feng, X.; Duan, X.; Liu, L.; An, L.; Feng, F.; Wang, S. Langmuir 2008, 24, 12138.

DOI: 10.1021/la901444c

13739

Article

Feng et al.

Figure 2. (a) Fluorescence microscope image of PFP-NIPAm microgel with the excitation wavelength of 380/30 nm and the emitting wavelength of 460/50 nm. (b) Fluorescence emission spectra of PFP-NIPAm microgel with the excitation wavelength of 380 nm. Scheme 2. Schematic Representation of DNA Detection Using PFP-NIPAm Microgel and Chemical Structures of Fluorescein, EB, and the DNAsa

a DNAP-Fl is the probe and ssDNAC is complementary to DNAP-Fl, ssDNA2NC is complementary to DNAP-Fl with two-base mismatches (highlighted in purple), and ssDNANC is fully noncomplementary to DNAP-Fl.

Figure 3. DNA detection results using PFP-NIPAm microgel. 1, PFP-NIPAm microgel (blank); 2, PFP-NIPAm microgel after adding DNAP-F1 and EB; 3, PFP-NIPAm microgel after adding DNAP-F1, ssDNAC and EB; 4, recycled PFP-NIPAm microgel; 5, PFP-NIPAm microgel after adding DNAP-F1, ssDNANC, and EB; 6, PFP-NIPAm microgel after adding DNAP-F1, two-base mismatched DNA2NC, and EB. [DNAP-Fl] =5.0
10-6 M, [EB]=5.0
10-6 M. The images were obtained from a glass slide that was dropped with samples under UV light with excitation at 365 nm.

Figure 4. The fluorescence emission color of PFP-NIPAm upon cycling the sample and washing processes. The photographs were taken under UV light with excitation at 365 nm.

made a stride in DNA detection with cationic conjugated polyelectrolytes that were used to confine the detection in homogeneous solution. Because the electrostatic attraction dominates the interaction between cationic PFP-NIPAm microgel and negatively charged DNA, washing the microgel with high ionic strength aqueous

solution can block the intermolecular interactions and displace the DNA from the PFP-NIPAm microgel. Thus, the PFP-NIPAm microgel can be readily reusable for the DNA detection. As shown in Figure 4, the PFP-NIPAm itself exhibited blue emission under UV light with excitation at 365 nm. Upon addition of DNAP-Fl, the PFP-NIPAm microgel changed emission color from blue to

13740 DOI: 10.1021/la901444c

Langmuir 2009, 25(24), 13737–13741

Feng et al.

Article

Figure 5. (a) The principle of AChE detection using PFP-NIPAm microgel. (b) Images under UV light (λmax=365 nm). 1, PFP-NIPAm itself

(the blank); 2, adding DA+ (12.5 μM) at 37 °C for 3 h (control); 3, adding DA+ (1.25 μM) and AChE (0.038 U) at 37 °C for 0.5 h; 4, adding DA+ (12.5 μM) and AChE (0.057 U) at 37 °C for 3 h.

green. After washing with 800 mM NaCl aqueous solution and pure water, the fluorescence of PFP-NIPAm was recovered to a blue color. This sample and washing processes can be repeated at least three times. Enzyme Detection Using Microgel PFP-NIPAm. The PFP-NIPAm microgel can also be used for enzyme detection. The detection mechanism for acetylcholinesterase (AChE) relative to Alzheimer’s disease is illustrated in Figure 5a. The cationic ACh labeling with a widely used quencher Dabcyl (AD +) is used as AChE substrate.17d As shown in Figure 5b1, the PFP-NIPAm microgel itself exhibited blue emission under UV light with excitation at 365 nm. Addition of ACh-Dabcyl to cationic PFP-NIPAm microgel did not lead to the complex formation due to electrostatic repulsion between them; therefore, the fluorescence of PFP-NIPAm was not quenched by the Dabcyl (Figure 5b2). Upon addition of AChE, ACh-Dabcyl was catalyzed to hydrolysis producing choline and a negatively charged residue containing Dabcyl moiety (AD-). Owing to the electrostatic attraction, PFP-NIPAm/AD- complex formed and the Dabcyl moiety resided closely to the PFP-NIPAm; therefore, the fluorescence of PFP-NIPAm was efficiently quenched (Figure 5b3,4). In light of the quenched fluorescence intensity of PFP-NIPAm, the AchE can be detected in a continuous and real-time manner. The increase of the AChE amount accelerated

Langmuir 2009, 25(24), 13737–13741

the cleavage reaction rate and resulted in a high level of fluorescence quenching.

Conclusion In summary, a new fluorescent microgel containing CP moiety (PFP-NIPAm) was prepared and characterized. Covalent linking of conjugated polyelectrolyte moiety into the microgel prevents the leakage of the fluorophore while keeping its fluorescence property. The PFP-NIPAm microgel can be used for DNA and enzyme detections. The PFP-NIPAm microgel offers several significant features. First, the microgel can be recycled following simple washing steps. Second, the microgel extends the detection media from homogeneous solution to gel phase, which shows the great potential for biodetection in the real world using conjugated polymers. Third, the detection can be carried out under UV light, and no expensive detection instrumentation is needed. Furthermore, such an assay system could be expanded to fluorescencebased high-throughput assays for biomacromolecules. Acknowledgment. The authors are grateful for the financial supports from the National Natural Science Foundation of China (No. 20725308 and 20721061), the National Basic Research Program of China (No. 2006CB806200) and the Major Research Plan of China (No. 2006CB932100).

DOI: 10.1021/la901444c

13741