Poly(o-phenylenediamine) Colloid-Quenched Fluorescent

Dec 27, 2010 - pH Manipulation: A Facile Method for Lowering Oxidation State and Keeping Good Yield of Poly(m-phenylenediamine) and Its Powerful Ag Ad...
29 downloads 12 Views 2MB Size
Download PDF
pubs.acs.org/Langmuir © 2010 American Chemical Society

Poly(o-phenylenediamine) Colloid-Quenched Fluorescent Oligonucleotide as a Probe for Fluorescence-Enhanced Nucleic Acid Detection Jingqi Tian,†,‡ Hailong Li,†,‡ Yonglan Luo,† Lei Wang,† Yingwei Zhang,† and Xuping Sun*,† †

State Key Lab of Electroanalytical Chemistry, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun 130022, Jilin, China, and ‡Graduate School of the Chinese Academy of Sciences, Beijing 100039, China Received September 22, 2010. Revised Manuscript Received December 20, 2010 In this Letter, we demonstrate that chemical oxidation polymerization of o-phenylenediamine (OPD) by potassium bichromate at room temperature results in the formation of submicrometer-scale poly(o-phenylenediamine) (POPD) colloids. Such colloids can absorb and quench dye-labeled single-stranded DNA (ssDNA) very effectively. In the presence of a target, a hybridization event occurs, which produces a double-stranded DNA (dsDNA) that detaches from the POPD surface, leading to recovery of dye fluorescence. With the use of an oligonucleotide (OND) sequence associated with human immunodeficiency virus (HIV) as a model system, we demonstrate the proof of concept that POPD colloid-quenched fluorescent OND can be used as a probe for fluorescence-enhanced nucleic acid detection with selectivity down to single-base mismatch.

Fluorescent nucleic acid probes are fluorophore-labeled oligonucleotides (ONDs) capable of energy transfer. These probes signal detection of their targets by changing either the intensity or the color of their fluorescence. In recent years, fluorescent probes have multiplied at a high rate and have found numerous applications in fields including drug discovery, gene expression profiling, clinical disease diagnostics, and treatment.1 Among them, Taqman probes, molecular beacons (MBs), and Scorpions are labeled with both a reporter and a quencher dye, and the fluorescence is only released from the reporter when the two dyes are physically separated via hybridization or nuclease activity.1a Although they have been extensively employed in a broad spectrum of applications,1,2 they still have some drawbacks in that they require labeling at both ends of the OND probe with specific dyes that suffer in overall yield and are expensive.3 Single-labeled fluorescent OND probes with only one fluorophore tag have also been developed; however, nanostructures have to be used as a nanoquencher for the fluorophore. It is a demonstrated fact that the same nanostructure is able to quench dyes of different emission frenquencies and hence the selection issue of fluorophorequencher pair is eliminated from the nanostructure-involved, single-labeled OND-based assay system.4 Up to now, only limited nanostructures including gold nanoparticles,4a,5 single-walled *To whom correspondence should be addressed. Telphone/Fax: 0086-43185262065. E-mail: [email protected]. (1) (a) Didenko, V. V., Ed. Fluorescent Energy Transfer Nucleic Acid Probes: Designs and Protocols; Human Press: Totowa, NJ, 2006. (b) Gresham, D.; Ruderfer, D. M.; Pratt, S. C.; Schacherer, J.; Dunham, M. J.; Botstein, D.; Kruglyak, L. Science 2006, 311, 1932. (2) (a) Holland, P. M.; Abramson, R. D.; Watson, R.; Gelfand, D. H. Proc. Natl. Acad. Sci. U.S.A. 1991, 88, 7276. (b) Tyagi, S.; Kramer, F. R. Nat. Biotechnol. 1996, 14, 303. (c) Yang, C. J.; Medley, C. D.; Tan, W. Curr. Pharm. Biotechnol. 2005, 6, 445. (d) Marras, S. A. E. Methods Mol. Biol. 2006, 335, 3. (3) Misra, A.; Kumar, P.; Gupta, K. C. Anal. Biochem. 2007, 364, 86. (4) (a) Ray, P. C.; Darbha, G. K.; Ray, A.; Walker, J.; Hardy, W. Plasmonics 2007, 2, 173. (b) Yang, R.; Tang, Z.; Yan, J.; Kang, H.; Kim, Y.; Zhu, Z.; Tan, W. Anal. Chem. 2008, 80, 7408. (5) (a) Dubertret, B.; Calame, M.; Libchaber, A. J. Nat. Biotechnol. 2001, 19, 365. (b) Maxwell, D. J.; Taylor, J. R.; Nie, S. J. Am. Chem. Soc. 2002, 124, 9606. (c) Li, H.; Rothberg, L. J. Anal. Chem. 2004, 76, 5414. (d) Song, S.; Liang, Z.; Zhang, J.; Wang, L.; Li, G.; Fan, C. Angew. Chem., Int. Ed. 2009, 48, 8670. (e) Li, D.; Song, S.; Fan, C. Acc. Chem. Res. 2010, 43, 631.

874 DOI: 10.1021/la103799e

carbon nanotubes (SWCNTs),4b,6 multiwalled carbon nanotubes,7 carbon nanoparticles,8 mesoporous carbon microparticles,9 graphene oxide (GO),10 poly(p-phenylenediamine) nanobelts,11 and polyaniline nanofibers12 have been successfully used in this assay so far. Therefore, the exploration of new nanostructures suitable as an effective fluorescent sensing platform for nucleic acid detection is highly desirable. In this Letter, we report on the simple synthesis of submicrometerscale conjugation polymer poly(o-phenylenediamine) (POPD) colloids by direct mixing of aqueous OPD and potassium bichromate solutions at room temperature. It is interestingly found that such colloids can absorb and quench dye-labeled singlestranded DNA (ssDNA) very effectively. With the use of an OND sequence associated with human immunodeficiency virus (HIV) as a model system, we demonstrate the proof of concept that POPD colloid-quenched fluorescent OND can be used as a probe for fluorescence-enhanced nucleic acid detection with selectivity down to single-base mismatch. The detection of DNA is accomplished by two steps: (1) POPD absorbs and quenches the fluorescence of dye-labeled ssDNA probe. (2) In the presence of target, a hybridization event occurs, which produces a doublestranded DNA (dsDNA) that detaches from the POPD surface, leading to recovery of dye fluorescence. All chemically synthesized oligonucleotides were purchased from Shanghai Sangon Biotechnology Co. Ltd. (Shanghai, China). DNA concentration was estimated by measuring the absorbance at 260 nm. All the other chemicals were purchased from Aladin Ltd. (Shanghai, China) and used as received without further (6) Yang, R.; Jin, J.; Chen, Y.; Shao, N.; Kang, H.; Xiao, Z.; Tang, Z.; Wu, Y.; Zhu, Z.; Tan, W. J. Am. Chem. Soc. 2008, 130, 83. (7) Li, H.; Tian, J.; Wang, L.; Zhang, Y.; Sun, X. J. Mater. Chem. 2011, 21, 824. (8) Li, H.; Tian, J.; Wang, L.; Zhang, Y.; Sun, X. Chem. Commun. 2011, 47, 961. (9) Liu, S.; Li, H.; Wang, L.; Tian, J.; Sun, X. J. Mater. Chem. 2011, 21, 339. (10) (a) Lu, C.; Yang, H.; Zhu, C.; Chen, X.; Chen, G. Angew. Chem., Int. Ed. 2009, 48, 4785. (b) He, S.; Song, B.; Li, D.; Zhu, C.; Qi, W.; Wen, Y.; Wang, L.; Song, S.; Fang, H.; Fan, C. Adv. Funct. Mater. 2010, 20, 453. (11) Wang, L.; Zhang, Y.; Tian, J.; Li, H.; Sun, X. Nucleic Acids Res. 2010, http://dx.doi.org/10.1093/nar/gkq1294. (12) Liu, S.; Wang, L.; Luo, Y.; Tian, J.; Li, H.; Sun, X. Nanoscale, submitted for publication.

Published on Web 12/27/2010

Langmuir 2011, 27(3), 874–877

Tian et al.

Letter

Figure 1. (A) Low and (B) high magnification SEM images of the products thus formed.

purification. The water used throughout all experiments was purified through a Millipore system. POPD colloids were prepared as follows: In brief, 250 μL of 0.1 M OPD aqueous solution was diluted with water to 3 mL, followed by the introduction of 190 μL of 0.1 M K2Cr2O7 aqueous solution under stirring. The resulting mixture was kept at room temperature overnight. The solution was washed with water by centrifugation twice, and the resulting precipitate was then redispersed in water and stored at 4 °C for characterization and further use. Scanning electron microscopy (SEM) measurements were made on a XL30 ESEM FEG scanning electron microscope at an accelerating voltage of 20 kV. Fluorescent emission spectra were recorded on a PerkinElmer LS55 luminescence spectrometer (PerkinElmer Instruments, U.K.). Zeta potential measurements were performed on a Nano-ZS Zetasizer ZEN3600 Instrument (Malvern Instruments Ltd., U.K.). Oligonucleotide sequences used in the present study are listed as follows: PHIV (FAM dye-labeled ssDNA): 50 -FAM-AGT CAG TGT GGA AAA TCT CTAGC-30 T1 (complementary target): 50 -GCT AGA GAT TTT CCA CAC TGA CT-30 T2 (single-base mismatched target): 50 -GCT AGA GAT TGT CCA CAC TGA CT-30 (mismatch underlined) T3 (noncomplementary target): 50 -TTT TTT TTT TTT TTT TTT TTT TT-30 Figure 1A shows a low magnification SEM image of the products thus formed, indicating the formation of a large amount of spherical colloids ranging from 450 to 550 nm. A close view of such colloids shown in Figure 1B further reveals that they have smooth surfaces. Li et al. have reported that the chemical oxidation of OPD monomers by ammonium persulfate leads to POPD microparticles.13 Similarly, in our present study, the formation of spherical colloids can also be attributed to the chemical oxidation polymerization of OPD by Cr2O72-. The chemical composition of these colloids was determined by the energy-dispersed spectrum (EDS) shown in Figure S1 in the Supporting Information. The peaks of C, N, and Cr elements are observed, indicating that the particles are formed from OPD and Cr2O72-. The observation of the peak of Cr be attributed to that the polymerization of OPD produces cationic polymers, but at the same time Cr2O72- will serve as counterions and diffuse into the POPD colloid for charge compensation.14 Note that the other peaks can be assigned to the (13) Li, X.; Ma, X.; Sun, J.; Huang, M. Langmuir 2009, 25, 1675. (14) (a) Wan, M.; Yang, J. J. Appl. Chem. Sci. 1995, 55, 399. (b) Trueba, M.; Montero, A. L.; Rieumont, J. Electrochim. Acta 2004, 49, 4341.

Langmuir 2011, 27(3), 874–877

Figure 2. Fluorescence emission spectra of PHIV (50 nM) at different conditions: (a) PHIV; (b) PHIV þ 300 nM T1; (c) PHIV þ POPD; (d) PHIV þ POPD þ 300 nM T1. Curve e is the emission spectrum of the POPD sample. Inset: fluorescence intensity ratio of PHIV-POPD complex with F/F0 - 1 (where F0 and F are the fluorescence intensity without and with the presence of T1, respectively) plotted against the logarithm of the concentration of T1. Excitation was at 480 nm, and the emission was monitored at 526 nm. All measurements were done in Tris-HCl buffer in the presence of 5 mM Mg2þ (pH: 7.4) at room temperature.

indium tin oxide (ITO) glass slide used as substrate for SEM and EDS measurements. To demonstrate the proof of concept that POPD colloidquenched fluorescent OND can be used as a probe for fluorescence-enhanced nucleic acid detection, we chose an OND sequence associated with HIV as a model system. Figure 2 shows the fluorescence emission spectra of PHIV, the FAM-labeled probe, at different conditions. In the absence of POPD, PHIV in Tris-HCl buffer exhibits strong fluorescence emission due to the presence of the fluorescein-based dye (curve a). However, in the presence of POPD, about 63% fluorescence quenching occurs (curve c), indicating that POPD can adsorb ssDNA and quench the fluorescent dye very effectively. The PHIV-POPD complex exhibits dramatic fluorescence enhancement upon the addition of complementary target T1, leading to a 56% fluorescence recovery (curve d). When there is no POPD existing in the system, we should note that the addition of T1 only has small influence on the fluorescence of the free PHIV (curve b). Furthermore, it is also worthwhile mentioning that POPD itself shows weak fluorescence emission (curve e) which also contributes to the whole fluorescence intensity of all POPD-involved samples measured. Therefore, a background subtraction is performed for all POPDinvolved measurements. Figure 2 inset shows the fluorescence intensity changes (F/F0 - 1) of the PHIV-POPD complex upon addition of different concentrations of T1, where F0 and F are FAM fluorescence intensities at 526 nm in the absence and the presence of T1, respectively. A notable increase in the FAM fluorescence intensity was observed as T1 concentration increased from 3.0 to 400 nM, indicating that the POPD/ DNA assembly approach is effective in probing biomolecular interactions. The zeta potential of the POPD colloids was measured to be about -12.7 mV, meaning there is electrostatic repulsion interactions between the negatively charged POPD colloids and negatively charged backbone of ssDNA. Considering that POPD is π-rich in nature, there should be strong π-π stacking of the DNA nucleobases and nucleosides on the colloid surface.15 On DOI: 10.1021/la103799e

875

Letter

Tian et al.

Scheme 1. Schematic (Not to Scale) Illustrating the Fluorescence-Enhanced Nucleic Acid Detection Using POPD Colloid-Quenched Fluorescent ONDs as a Probe

Figure 3. Fluorescence emission spectra of PHIV (50 nM) under different conditions: (a) PHIV-POPD complex; (b) PHIV-POPD complex þ 300 nM T1; (c) PHIV-POPD complex þ 300 nM T2; (d) PHIV-POPD complex þ 300 nM T3. Inset: fluorescence intensity histogram with error bar. Excitation was at 480 nm, and the emission was monitored at 526 nm. All measurements were done in Tris-HCl buffer in the presence of 5 mM Mg2þ (pH: 7.4) at room temperature.

the other hand, however, POPD should have weak or even no association with dsDNA due to its negatively charged surface nature and the unavailability of free nucleobases and nucleosides for dsDNA. Scheme 1 presents a schematic illustrating the fluorescence-enhanced nucleic acid detection using POPD colloid-quenched fluorescent ONDs as a probe. The detection is accomplished by two steps: (1) Owing to the strong π-π interactions between DNA nucleobases and nucleosides and POPD, the colloid adsorbs dye-labeled ssDNA, which leads to fluorescence quenching of FAM due to their close approximation. (2) In the presence of target, a hybridization event occurs, which produces a dsDNA that detaches from POPD surface, leading to recovery of dye fluorescence. Supporting Information Figure S2 shows the fluorescence spectra of (a) PHIV-POPD complex þ T1 and (b) the supernatant of (a) after removing POPD colloids by filtration and centrifugation. It is clearly seen that only a small fluorescence intensity change was observed after the separation of POPD (15) Varghese, N.; Mogera, U.; Govindaraj, A.; Das, A.; Maiti, P. K.; Sood, A. K.; Rao, C. N. R. ChemPhysChem 2009, 10, 206.

876 DOI: 10.1021/la103799e

Figure 4. (a) Fluorescence quenching of PHIV (50 nM) by POPD and (b) fluorescence recovery of PHIV-POPD by T1 (300 nM) as a function of time. Excitation was at 480 nm, and the emission was monitored at 526 nm. All measurements were done in Tris-HCl buffer in the presence of 5 mM Mg2þ (pH: 7.4) at room temperature.

colloids, indicating that dsDNA indeed detaches from POPD colloid. We should point out that the present PHIV-POPD complex shows ability to discriminate the perfect target and the singlebased mismatched one. Figure 3 shows the fluorescence responses of PHIV-POPD complex toward complementary target T1 and the single-base mismatched target T2. It is found that the F/F0 value obtained upon addition of 300 nM of T2 is about 85% of the value obtained upon addition of 300 nM of T1 into the corresponding PHIV-POPD complex (where F0 and F are FAM fluorescence intensities at 526 nm in the absence and the presence of target, respectively). However, only a very small fluorescence intensity change was observed for the PHIV-POPD complex upon addition of 300 nM noncomplementary target T3, indicating that the observed fluorescence enhancement in our present system is indeed due to the base pairing between probe and its target. Figure 3 inset shows the corresponding fluorescence intensity histogram with error bar. All the above observations clearly indicate the present nucleic acid detection system has selectivity down to single-base mismatch and the results obtained therein exhibit good reproducibility. The kinetic behaviors of PHIV and POPD, as well as of the PHIV-POPD with T1, were monitored by collecting the Langmuir 2011, 27(3), 874–877

Tian et al.

time-dependent fluorescence spectra. Figure 4a shows the fluorescence quenching of PHIV by POPD as a function of incubation time. In the absence of the target, the curve exhibits a rapid decrease and reaches equilibrium within 1 min, indicating the adsorption of ssDNA on POPD is a very fast process. Figure 4b shows the fluorescence recovery of PHIV-POPD by T1 in Tris-HCl buffer as a function of time. In the presence of the target T1, the curve shows a fast increase in the first 5 min, followed by a slow process over a 35 min incubation period. The best fluorescence response was obtained over a 40 min incubation period. We also studied the influence of the amount of POPD used on the fluorescence quenching and the subsequent fluorescence recovery of seven samples with the use of different amounts of POPD colloids. It is found that the quenching efficiency increases but the recovery efficiency decreases with the volume of POPD increasing from 0 to 70 μL. Such an observation can be reasonably explained as follows: POPD has a strong affinity for ssDNA. It is obvious that an increase of POPD in amount leads to more efficient absorption of ssDNA on it and thus a higher quenching efficiency. However, during the subsequent recovery process, the adsorption of target on excessive POPD colloids occurs, which

Langmuir 2011, 27(3), 874–877

Letter

will compete with the hybridization of target with ssDNA absorbed on POPD, leading to decreased hybridization and recovery efficiency. In summary, we successfully synthesize submicrometer-scale POPD colloids via chemical oxidation polymerization of OPD monomers by K2Cr2O7 at room temperature for the first time. We further demonstrate that POPD colloid-quenched fluorescent OND can be used as a probe for fluorescence-enhanced nucleic acid detection with selectivity down to single-base mismatch. We believe that it will provide us a universal fluorescent sensing platform for fluorescence-enhanced detection that is sensitive and selective to the target molecule studied. Acknowledgment. This work was supported by National Basic Research Program of China (No. 2011CB935800). Supporting Information Available: EDS of the POPD colloids; fluorescence spectra of PHIV-POPD complex þ T1 before and after removing POPD colloids. This material is available free of charge via the Internet at http://pubs.acs.org.

DOI: 10.1021/la103799e

877