Poly(m-Phenylenediamine) Nucleic Acid DetectionPoly(m-Phe nylen ediam ine) Nano spheres and Nano rods:Selective Synthesis and Their Application for Multiplex Nucleic Acid DetectionYingwei Zhang 1. , Hailong Li1,2 . , Yonglan Luo 1, Xu Shi 3, Jingqi Tian 1,2 , Xuping Sun 1*1 State Key Lab of Electroanalytical Chemistry, Changchun Institute of Applied Chemistry, Changchun, Jilin, People ' Republic of China, 2 Chinese Academy of Sciences, Graduate School of the Chinese Academy of Sciences, Beijing, People ' Republic of China, 3 Institute of Virology and AIDS Research, First Affiliated Hospital, Jilin University, Changchun, Jilin, People ' Republic of ChinaAbstractIn this paper, we demonstrate for the first time that poly(m-phenylenediamine) (PMPD)nanospheres and nanorods can be selectively synthesized via chemical oxidation polymerization of m-phenylenediamine (MPD) monomers using ammonium persulfate (APS)asan oxidant at room temperature. It suggests that the pH value plays a critical role in controlling the the morphology of the nanostructures and fast polymerization rate favors the anisotropic growth of PMPD under homogeneous nucleation condition. We further demonstrate that such PMPD nanostructures can be used as an effective fluorescent sensing platform for multiplex nucleic acid detection. A detection limit as low as 50 pM and a high selectivity down to single-base mismatch could be achieved. The fluorescence quenching is attributed to photoinduced electron transfer from nitrogen atom in PMPD to excited fluorophore. Most importantly, the successful use of this sensing platform in human blood serum system is also demonstrated.Citatio n: Zha ng Y, Li H, Luo Y, Shi X, Tia n J, et al. (2011) Poly(m-Phe nyle nediami ne) Nan ospheres and Nano rods: Selective Sy nthesis and Their Applicatio n forMultiplex Nucleic Acid Detection. PLoSONE6(6): e20569. doi:10.1371/journal.pone.0020569Editor: Meni Wanunu, University of Pennsylvania, United States of AmericaReceived February 14, 2011; Accepted May 4, 2011; Published June 23, 2011Copyright: ? 2011 Zhang et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permitsunrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.Funding: The authors have no support or funding to report.Competing Interests: The authors have declared that no competing interests exist.* E-mail: sunxp@. These authors contributed equally to this work.PLoS ONE | www.plosone.org # June 2011 | Volume 6 | Issue 6 | e20569Poly(m-Phenylenediamine) Nucleic Acid DetectionPLoS ONE | www.plosone.org # June 2011 | Volume 6 | Issue 6 | e20569Poly(m-Phenylenediamine) Nucleic Acid DetectionIn troducti onDuring the past decades, conducting polymers (CPs) have constituted a subject of research for their unique properties and important application potential [1]. Polyaniline is one of the most studied CPs due to its chemical stability and relative high conductivity [2] and, at the same time, polymers based on aniline derivatives have also been widely investigated [3]. Among them, poly(phenylenediamine) (PPD) homopolymer is a highly aromatic polymer containing 2,3-diaminophenazine or quinoraline repeating unit and exhibiting high thermostability and has found important applications in sensor designing, immunospecies detection, and as component of rechargeable cells etc [4 - 13]. PPD is usually prepared by electrochemical [14] and chemical oxidation polymerization [15]. Although we [16] and other researchers[17 - 19have successfullyprepared poly(o-phenylene- diamine) nanobelts and microparticles by chemical oxidation polymerization method, respectively,the selectivesynthesisof PPD with different morphologies has not been addressed so far.On the other hand, it isvitally important to develop rapid, costeffective, sensitiveand specific methods for the detection of nucleic acid due to their various applications in gene expression profiling, clinical disease diagnostics and treatment [20]. The increasing availability of nanostructures has created widespread interest in their usein biotechnological systemfor diagnostic application [21]. Indeed, the useof a variety of nanostructures for this purpose has been well-demonstrated [22]. Recently, there have been many efforts toward developing homogeneous fluorescence assaysbased on fluorescence resonance energy transfer (FRET) or quenching mechanism for nucleic acid detection [23]. The use of nanostructures asa ‘‘ nanoquenchees'remarkable advantage in that the same nanostructure has the ability to quench dyes of different emission frequencies and thus the selection issueof a fluorophore- quencher pair is eliminated from the nanostructure-involved system [23,24]. Up to now, a number of structures have been successfully used by us and other researchers in this assay, including gold nanoparticles, single-walled carbon nanotubes (SWCNTs), multi-walled carbon nanotubes, carbon nanoparticles, carbon nanospheres, nano-C60, mesoporous carbon microparticles, graphene oxide (GO), polyaniline nanofibres, poly(o-phenyl- enediamine) colloids, poly(2,3-diaminonaphthalene) microspheres, coordination polymer colloids and nanobelts, Ag@poly(m-phenyl- enediamine) core-shell nanoparticles, tetracyanoquinodimethane nanoparticles, and supramolecular microparticles [23 - 46For the SWCNT or GO system, it has drawbacks: (1) several hours ' sonication is required to disperse SWCNT in an organic solvent like N,N-dimethylformamide (DMF) [30]; (2)the GO preparation by the Humme' s method is time-consuming and labor-intensive[47] . We have also found that conjugation polymer poly(p- phenylenediamine) nanobelts (PNs) can serve as an effective fluorescent sensing platform for multiplex nucleic acid detection[48] ; however, this system still has two serious drawbacks which limit its practical use: (1)the nanobelts are tens of micrometers in length and thus tend to sink in aqueous solution due to the gravity, Figure 1. Instrumental analysis of the precipitate thus formed. Low water and (C) NMPD as reacti on solve nt, (B) and (D) corresp onding to the doi:10.1371/journal.po ne.0020569.g001causing the problem of stability in detection; (2) it has poor discrimination ability in that it gives only 8.8% difference of detection signal between single-basemismatched and complementary sequences[48]. Accordingly, developing new nanostructurebased fluorescent sensing platform to overcome all these drawbacks is highly desired.In this paper, we report on the selective synthesis of poly(m- phenylenediamine) (PMPD) nanospheresand nanorods by chemical oxidation polymerization of MPD monomers using ammonium persulfate (APS) asan oxidant at room temperature for the first time. It is found that the pH value is key to controlling the morphology of the nanostructures and fast polymerization rate favors the anisotropic growth of PMPD under homogeneous nucleation condition. We further demonstrate that such PMPD nanostructures can serve as an effective fluorescent sensing platform for multiplex nucleic acid detection. A detection limit as low as 50 pM and a high selectivity down to single-base mismatch could be achieved. The fluorescence quenching is attributed to photoinduced electron transfer from nitrogen atom in PMPD to excited fluorophore. Most importantly, the successful use of this sensing platform in human blood serum systemis also demonstrated.Results and DiscussionFigure 1A shows low magnification SEM image of the products thus formed in water (sample1, seeMaterials and Methods for preparation details), indicating that they consist exclusively of a large amount of nanoparticles. A close view of such nanoparticles further revealsthat they are nearly spherical in shapeand have size ranging from 300 to 600 nm, as shown in Figure 1B. The chemical magn ificati on SEM images of the PMPD nano structures formed using (A) high magnification SEM position of the nanospheres was determined by the energy- dispersedspectrum (EDS), as shown in Figure S1. The peaksof C and N elements are observed, indicating the nanospheres are formed from MPD. The presence of the peaks of S and O elements can be attributed to the fact that the polymerization of MPD by APS yields cationic polymer PMPD due to the proton22doping effect, the SO4 (the reduced product of APS) andexcessiveS2O822 as counter-ions, however, will diffuse into the PMPD nanostructures for charge compensation [49,50]. Very interestingly, it is found that the PMPD morphology can be changed by simply varying the reaction solvent used. Figure 1C and 1D shows typical SEM images of the products obtained with the use of N-methylpyrrolidone (NMPD) asreaction solvent, under otherwise identical conditions used for preparing sample 1. It is clearly seenthat a large quantity of nanorods are produced as the main products. It was found that the use of N,N-dimethylforma- mide (DMF) and ethanol as the reaction solvent lead to nanorods (Figure S2A) and nanoshperes (Figure S2B), respectively. The possible mechanism of the effect of solvent in controlling the PMPD morphology is proposed as follows: The polymerization of MPD monomers by APS leads to a decreaseof pH value of the system.Given the weakbasic nature of NMPD and DMF, they are expected to neutralize the protons generated and thus the rate of polymerization of MPD is accelerated, which may favor the anisotropic growth of PMPD under homogeneous nucleation condition [51,52]. It was found that polymerization of MPD monomers using water as reaction solvent but at basic condition also produced rod-like products. (Figure S2C). All these observations indicate that the pH value has played a critical role in controlling the morphology of the nanostructures. It is important to mention that these PMPD nanospheres and nanorods have smaller sizes and higher zeta potential (5 mV) and thus their dispersion exhibits better stability than PNs [48]. Indeed, we have found that such PMPD nanostructures are well-dispersed in water or buffer solutions. The resultant dispersions are very stable and no precipitation is observed within a couple of days.To test the feasibility of using PMPD nanostructures as an effectivefluorescent sensingplatform for nucleic acid detection, we chose PMPD nanorods and an oligonucleotide sequence associated with human immunodeficiency virus (HIV) as a model system.Figure 2A showsthe fluorescenceemission spectra of Phiv , the FAM-labeled probe, at different conditions. In the absenceof PMPD, the fluorescence spectrum of Phiv exhibits strong fluorescence emission due to the presence of the fluorescein-based dye (curve a). However, the presenceof PMPD results in about 96% quenching of the fluorescence emission (curve c), revealing that PMPD can strongly adsorb ssDNA and quench the fluorescent dye very effectively. However, the Phiv - PMPD complex exhibits significant fluorescence enhancement upon its incubation with complementary target T1 over a 1-h period, leading to a 77% fluorescence recovery (curve d). Note that the fluorescenceof the free Phiv was, however, scarcely influenced by the addition of T1 in the absenceof PMPD (curve b). It should be mentioned that the PMPD sample exhibits weak fluorescence emission (curve e) which contributes a little to the whole fluorescence intensity of each sample measured. Hence, a background fluorescence subtraction is performed for all PMPD- involved measurements. Figure 2A inset illustrates the fluorescence intensity changes(F/ Fo - 1Qf Phiv - PMPDcomplex upon addition of different concentrations of T1, where F0 and F are FAM fluorescence intensities at 522 nm in the absence and presence of T1, respectively. In the DNA concentration range of 5 - 3001M, a dramatic increase of FAM fluorescence intensity was observed, which suggests that the nanorod/DNA assembly approach is effective in probing biomolecular interactions due to the excellent signalling process .It is worthwhile mentioning that optimal signal- to-noise ratio of 3.8:1 and thus low detection limit can be achieved by decreasing the amount of PMPD and Phiv used. A detection limit as low as50 pM can be achieved when 1-mL PMPD sample and 500 pM Phiv are used in this system(Figure 2B).Because PMPD is a p-rich polymer, it can strongly and effectively adsorb single-stranded DNA (ssDNA) on its surface via p-p stacking between unpaired DNA basesand PMPD [53]. The zeta potential of the nanorods was measured to be about 5 mV in water, indicating that the nanorod has a low positively charged surface. However, the electrostatic attractive interactions between nanorod and negatively charged backbone of ssDNA contribute little to the adsorption of ssDNA on nanorod in the presenceof a large amount of salt in buffer [44]. In contrast, the PMPD nanorod should have weak or even no binding with doublestranded DNA (dsDNA) due to the absence of unpaired DNA basesand the rigid conformation of dsDNA. Figure 3A presentsa schematic to illustrate the fluorescence-enhanced nucleic acid detection using PMPD nanorod as a sensing platform. The DNA detection is accomplished by the following two steps: (1) PMPD binds dye-labeled ssDNA and quenchesthe fluorescenceof the dye when they are brought into close proximity. (2) The subsequent hybridization of the probe with its target produces dsDNA which detaches from PMPD, leading to fluorescence recovery. The releaseof the resultant dsDNA from PMPD can be supported by the following experimental observation that there is no obvious fluorescence change observed after the removal of the PMPD nanorods from the hybridized solution by centrifugation, as shown in Figure S3. The observed fluorescencequenching in our present study could be attributed to photoinduced electron transfer (PET) from nitrogen atom in PMPD to excited fluorophore FAM when they are brought into close proximity [54]. Figure 3B illustrates the quenching mechanism involved. When the fluorophore FAM is excited, an electron from the highest occupied molecular orbital (HOMO) is promoted to the lowest unoccupied molecular orbital (LUMO), leaving an electronic vacancy in the fluorophore HOMO, which is filled by transfer of an electron from the higher energy HOMO of the nitrogen atom in PMPD serving asa donor. The overall effect of PET processis that the excited state life time is shortened and little fluorescenceoccurs, leading to fluorescence quenching. Upon protonation of the donor, however, the redox potential of the protonated donor is raised and its HOMO becomes lower in energy than that of the fluorophore. Consequently, electron transfer is hindered and fluorescence quenching is suppressed. This PET-based fluorescence quenching is confirmed by the experimental observation that the quenching is suppressedwith the decreaseof pH value and thus the increaseof protonation degree of donor [18], as shown in Figure 4.It should be noted that the amount of PMPD nanorods usedin this systemhas profound effect on the efficiency of the fluorescence quenching and the subsequent recovery. Figure S4 shows the fluorescence intensity histograms of five sampleswith the use of 0, 5, 10, 15, and 20-mL PMPD nanorods sample, respectively. It suggestedthat the useof increasedamount of nanorods leadsto an increase in quenching efficiency but a decrease in recovery efficiency. The above observations can be reasoned as follows: When the ssDNA probe molecules are mixed with nanorods, they will adsorb on the nanorod surface. Obviously, the use of more nanorods leads to more efficient adsorption of ssDNA and thus higher quenching efficiency. But at the sametime, the possibility of direct surface adsorption of target molecules on those excess nanorods increasesduring the following hybridization process.As a result, decreased hybridization efficiency and thus lower recovery efficiency is observed. Based on these observations, an optimal volume of 10 mL was chosen in our present study if not specified. Figure S5 shows a Stern - Volmeiquenching curve describing F0/ F as a function of MPD concentration, where F0 and F are FAM fluorescence intensities at 522 nm in the absence and the presence of PMPD nanorods, respectively. The plot is linear in the concentration range of 0 to 5 mM and the Stern - Volmer quenching constant (Ksv) is calculated to be 3.7686 105 M2 1 [55].The kinetic behaviors of Phiv and PMPD, as well as of the Phiv - PMPDcomplex incubated with T1, were also studied by collecting the time-dependent fluorescence emission spectra. Curve a in Figure 5A showsthe fluorescence quenching of Phiv in the presenceof PMPD asa function of incubation time at room temperature of 25uC. In the absence of the target, the curve exhibits a rapid reduction in the first 20 min and a slow decrease over a period of 40 min. Curve b in Figure 5A shows the subsequent fluorescence recovery of Phiv - PMPDby T1 in Tris- HCl buffer asa function of incubation time. In the presenceof the target T1, the curve shows a fast increase in the first 10 min, followed by a slow process over a period of 50 min. The best fluorescence response was obtained after 1 h of incubation time. All above observations indicate that both ssDNA- PMPD association and dsDNA- PMPD dissociation occur faster than SWCNT but slower than GO system [24,30,36,37]. These resultsare quite similar to those obtained from PN system [48]. We also investigated the influence of temperature on the kinetic behaviors of thesetwo processes.Figure 5B showsthe corresponding results obtained at 50uC, indicating that the time required to reach equilibrium is greatly shortened for both the quenching and the subsequent recovery process. It should be noted the decreaseofPLoS ONE | www.plosone.org 3 June 2011 | Volume 6 | Issue 6 | e20569Poly(m-Phenylenediamine) Nucleic Acid DetectionPLoS ONE | www.plosone.org 4 June 2011 | Volume 6 | Issue 6 | e20569Poly(m-Phenylenediamine) Nucleic Acid Detection10C / nMPLoS ONE | www.plosone.org # June 2011 | Volume 6 | Issue 6 | e20569Poly(m-Phenylenediamine) Nucleic Acid DetectionPLoS ONE | www.plosone.org # June 2011 | Volume 6 | Issue 6 | e20569Poly(m-Phenylenediamine) Nucleic Acid Detectionno543210PLoS ONE | www.plosone.org # June 2011 | Volume 6 | Issue 6 | e20569Poly(m-Phenylenediamine) Nucleic Acid Detection520540 560 580 600620 640PLoS ONE | www.plosone.org # June 2011 | Volume 6 | Issue 6 | e20569Poly(m-Phenylenediamine) Nucleic Acid DetectionPLoS ONE | www.plosone.org # June 2011 | Volume 6 | Issue 6 | e20569Poly(m-Phenylenediamine) Nucleic Acid DetectionFigure 2. Performa nee of target DNA detect ion and determ in atio n of detecti on limit. (A) Fluoresce nee emissi on spectra of Phiv (50 nM) atdiffere nt con diti on s: (a) PHIV; (b) PHIV + 300 nM T1; (c) PHIV +PMPD nan orods; (d) PHIV + PMPD nano rods +300 nM T1. Curve e is the emissio n spectra ofPMPD nano rods. In set: fluoresce nee inten sity cha nge (F/Fo - 1)of Phiv - PMPDha no rods complex (where Fo a nd Fare the fluoresce nee inten sity without and with the prese nee of T,, respectively) plotted aga inst the logarithm of the concen tratio n of T1. (B) (a) Fluoresce nee emissi on spectra of PHIV (500 pM), (b) fluoresce nee que nchi ng of Phiv (500 pM) by 1-nL PMPD nan orods, and (c) fluoresce nee recovery of (b) by Ti (50 pM). I nset in Figure 2B: the corresp onding fluoresce nee inten sity histograms with error bar. Excitati on was at 480 nm, and the emissi on was mon itored at 522 nm. All measureme nts were done in Tris-HCl buffer in the prese nee of 5 mM Mg2+ (pH: 7.4). 10-nL PMPD nano rods were used in each measureme nt. doi:10.1371/journal.po ne.0020569.g002PLoS ONE 。