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Ru(bpy)32+-doped silica nanoparticle DNA probe for the electrogenerated chemiluminescence detection of DNA hybridization
Electrochimica Acta 52 (2006) 575–580
Ru(bpy)32+-doped silica nanoparticle DNA probe for the electrogenerated chemiluminescence detection of DNA hybridization Zhu Chang a,b , Jingming Zhou a , Kun Zhao a , Ningning Zhu c , Pingang He a , Yuzhi Fang a,∗ a
Department of Chemistry, East China Normal University, Shanghai 200062, China b Department of Chemistry, Shangqiu Teachers College, Shangqiu 476000, China c Department of Chemistry, Shanghai Normal University, Shanghai 200234, China
Received 18 January 2006; received in revised form 27 March 2006; accepted 11 May 2006 Available online 5 July 2006
Abstract A sensitive electrogenerated chemiluminescence (ECL) detection of DNA hybridization, based on tris(2,2 -bipyridyl)ruthenium(II)-doped silica nanoparticles (Ru(bpy)3 2+ -doped SNPs) as DNA tags, is described. In this protocol, Ru(bpy)3 2+ -doped SNPs was used for DNA labeling with trimethoxysilylpropydiethylenetriamine(DETA) and glutaraldehyde as linking agents. The Ru(bpy)3 2+ -doped SNPs labeled DNA probe was hybridized with target DNA immobilized on the surface of polypyrrole (PPy) modified Pt electrode. The hybridization events were evaluated by ECL measurements and only the complementary sequence could form a double-stranded DNA (dsDNA) with DNA probe and give strong ECL signals. A three-base mismatch sequence and a non-complementary sequence had almost negligible responses. Due to the large number of Ru(bpy)3 2+ molecules inside SNPs, the assay allows detection at levels as low as 1.0 × 10−13 mol l−1 of the target DNA. The intensity of ECL was linearly related to the concentration of the complementary sequence in the range of 2.0 × 10−13 to 2.0 × 10−9 mol l−1 . © 2006 Elsevier Ltd. All rights reserved. Keywords: Ru(bpy)3 2+ ; Silica nanoparticles; Electrogenerated chemiluminescence; DNA hybridization
1. Introduction Sensitive DNA detection is extremely important in clinical diagnostics, gene therapy, and a variety of biomedical studies. Recently, great efforts have been made to develop new biotechnologies to improve the sensitivity and selectivity for gene analysis [1–3]. Therefore, many detection techniques of DNA sequence have been developed in recent years. These detection techniques include radiochemical [4], enzymatic [5], fluorescent [6], and electrogenerated chemiluminescence (ECL) methods [7]. Surface plasmon resonance spectroscopy [8], electrochemical methods [9] and quartz crystal microbalance [10] are also widely used. Compared with other detection techniques, the ECL has some advantages: no radioisotopes are used, detection limits are extremely low, the dynamic range for quantification extends over six orders of magnitude, the labels are extremely
∗
Corresponding author. Fax: +86 21 62233508. E-mail addresses: [email protected] (P. He), [email protected] (Y. Fang). 0013-4686/$ – see front matter © 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.electacta.2006.05.036
stable compared with those of most other chemiluminescence (CL) systems and the measurement is simple and rapid [11,12]. Ru(bpy)3 2+ has become the most attractive ECL label because of its stability, regenerability, excellent luminescence properties and compatibility with a wide range of analyses [13]. The ECL reaction of Ru(bpy)3 2+ allows the detection of a wide range of analytes, such as C2 O4 2− [14,15], DNA damage [16], electrochemiluminescence immunoassay [17], aliphatic amines [18]. Santra et al. [19] encapsulated ruthenium complexes in thin silica layers as biomarkers for leukemia cell recognition. The emergence of nanotechnology is opening new horizons for the application of nanoparticles in analytical chemistry. Nanoparticles as quantitation tags, such as the optical detection of quantum dots and the electrochemical detection of metallic nanoparticles materials [20,21], offer excellent prospects for chemical and biological sensing because of their unique optical or electrical properties and evidently amplifying signal. The nanoparticles as biomolecules labels in DNA hybridization detection assays and immunoassays for anti-protein are also widely applied. Mirkin and co-workers [22] fabricated ‘biobarcodes’ in which gold nanoparticles are derivatized with distinct
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sequences of DNA, and then taking advantage of the distinct melting temperature of the different DNA sequences, to determine the ‘barcode’ identity. Thanh and Rasenzweig [23] developed an assay in which antibody concentration was determined as a function of aggregation of antigen-coated gold particles. Wu et al. [24] described a Her2 assay for breast cancer with semiconductor quantum dots. Novel classes of optically active nanoparticles are also being developed as next-generation quantitation tags. Our group also developed nanoparticles as DNA labels, such as metallic particle (Au [25], Au@Ag [26]), quantum dots (CdS, PdS, ZnS) [27–29] and core-shell nanoparticles of Co(bpy)3 3+ doped silica nanoparticles (SNPs) [30], to electrochemical detection DNA hybridization. However, electrochemiluminescent detection of DNA hybridization based on the use of metal chelate doped SNPs tags has rarely been reported. Herein, we attempt to combine Ru(bpy)3 2+ -doped SNPs as DNA tags and develop a sensitive electrochemiluminescent method to detection DNA hybridization by amplifying ECL signal through boosting up the amount of Ru(bpy)3 2+ labeled on DNA probe. We succeeded in doping the luminescent reagent of Ru(bpy)3 2+ into SNPs and labeling oligonucleotides onto the nanoparticles surface. We demonstrate that different concentration of Ru(bpy)3 2+ doped SNPs will give different ECL intensity. The performance of DNA biosensors based the Ru(bpy)3 2+ doped SNPs labeled DNA probes with respect to sensitivity, linear range, and the characterization of Ru(bpy)3 2+ -doped SNPs are presented and discussed. 2. Experimental 2.1. Apparatus All ECL measurements were carried out using a laboratory constructed ECL system. A Model 173 potentiostat (Princeton Applied Research, PAR, EG & G) and an XFD-8A waveform generator (Ningbo Radio Factory, Ningbo, China) were used for giving and controlling waveforms and potential. The ECL emission was detected and amplified using a 9901 luminometer (Rongsheng Electronic Equipment Co., Shanghai, China) and the output signal which is directly related to the ECL light inten-
sity was recorded using a Chart Recorder (Dahua Instrument Co, Shanghai, China). The three-electrode system was used. A Pt plate (4 mm × 10 mm) fixed on black plexiglass was used as a working electrode. An Ag/AgCl (saturated KCl) and a platinum wire were used as reference electrode and auxiliary electrode. All ECL reactions were carried out in a 1 ml cell placed directly in the front of photomultiplier tube. A CHI 630 electrochemical analyzer (CHI Instruments Inc., USA), a transmission electron microscope (TEM) (Hitachi, Japan), and a TDL-16B centrifuge (Anting Science Instrument Inc., Shanghai, China) were employed for the experimentation. 2.2. Reagents 24-Base synthetic oligonucleotides (24-base probe sequence: 5 -NH2 -GAG CGG CGC AAC ATT TCA GGT CGA-3 ; its complementary sequence: 5 -TCG ACC TGA AAT GTT GCG CCG CTC-3 ; a non-complementary sequence: 5 -GAG CGG CGC AAC ATT TCA GGT CGA-3 ; a three-base mismatch sequence: 5 -TCG TCC TGA AAC GTT GCG CCT CTC-3 ) were purchased from Shenggong Bioengineering Ltd. Company (Shanghai, China). Tris(2,2 -bipyridyl)ruthenium(II) chloride was purchased from Adlrich (USA). Tetraethylorthosilicate (TEOS) and trimethoxysilylpropydiethylenetriamine (DETA) were purchased from United Chemical Technologies (Bristol, PA). Pyrrole was provided by Shanghai Chemical Factory (Shanghai, China) and was twice distilled before use. Other reagents were commercially available and were all of analytical reagent grade. PBS solution (40 mmol l−1 NaCl + 50 mmol l−1 sodium phosphate buffer, pH 6.8), 0.1 mol l−1 oxalic acid solution and other solutions were prepared with deionized water from an Aquapro system (Yize Co., China). 2.3. The preparation of Ru(bpy)3 2+ -doped SNPs labeled DNA probe and ECL detection The scheme representation of Ru(bpy)3 2+ -doped SNPs labeled DNA probe and the procedure of ECL detection of DNA hybridization based on the probe are illustrated in Fig. 1A and B, respectively.
Fig. 1. Schematic representation of preparation Ru(bpy)3 2+ -doped SNPs oligonucleotides probes (A) and the electrogenerated chemiluminescence detection of DNA hybridization based on the Ru(bpy)3 2+ -doped SNPs labeled oligonucleotides probes (B).
Z. Chang et al. / Electrochimica Acta 52 (2006) 575–580
2.3.1. Ru(bpy)3 2+ -doped nanoparticle synthesis and surface modification with oligonucleotides DNA probe 2.3.1.1. Nanoparticle synthesis. SNPs were prepared according to the literature [19]. The water-in-oil (W/O) microemulsion was prepared firstly by mixing 1.77 ml of TritonX-100, 7.5 ml of cyclohexane, 1.8 ml of n-hexanol, 340 l of 0.04 mol l−1 of Ru(bpy)3 2+ solution were mixed and stirred for 0.5 h at room temperature, forming a uniform W/O microemulsion. The watersurfactant molar ratio was kept constant at 10. In the presence of 100 l of TEOS, a polymerization reaction [31] was initiated by adding 60 l of NH3 ·H2 O (28–30 wt.%). The reaction was allowed to continue for 24 h. After the reaction was completed, the Ru(bpy)3 2+ -doped SNPs were isolated by acetone, followed by centrifuging, ultrasonicating and washing with ethanol and water several times to remove any residual surfactant molecules or any physically adsorbed Ru(bpy)3 2+ from the surface of the particles. Finally, the required orange-colored Ru(bpy)3 2+ doped SNPs were obtained. The Ru(bpy)3 2+ -doped SNPs were suspended in 2 ml PBS(pH 6.8) by ultrasonication for the following experiments. 2.3.1.2. Surface modification of Ru(bpy)3 2+ -doped SNPs and covalent conjugation of oligonucleotides onto the nanoparticle surface. Functionalization of nanoparticles was performed according to the literature [30] by immersion in freshly prepared 1% (v/v) solution of DETA and 1.0 × 10−3 mol l−1 acetic acid for 30 min at room temperature (25 ◦ C). The DETA modified SNPs were thoroughly rinsed with deionized water to remove excess DETA. The above amine-functionalized Ru(bpy)3 2+ -doped SNPs were reacted with 5% glutaraldehyde solution in a water bath at 37 ◦ C for 120 min with shaking. After that, the nanoparticles were washed with deionized water several times to remove excess glutaraldehyde. Then 2.0 OD of 5 -amine-capped oligonucleotides diluted in PBS buffer (pH 6.8) was added to the functionalized SNPs solution, and stirring was continued for 120 min in a water bath at 37 ◦ C (final oligonucleotides concentration is 1.27 × 10−5 mol l−1 ). Oligonucleotides probe conjugated nanoparticles were then treated with 10 ml of 30 mmol l−1 glycine solution for 30 min. The final product was washed and centrifuged, resuspended in PBS (pH 6.8) buffer, either used immediately for test or stored at 4 ◦ C for later usage. 2.3.2. Immobilization of target ssDNA on PPy/Pt electrode The immobilization of target ssDNA on a Pt electrode was carried out according to the literature [32]. Briefly, the Pt electrode was polished carefully with alumina powder on a soft polishing cloth. After sonicating in absolute ethanol, then in water for 5 min, the pretreated Pt electrode was immersed into 0.1 mol l−1 KCl solution containing 0.05 mol l−1 pyrrole monomer (pH was adjusted to 3.0 with sodium acetate buffer). Fourteen cyclic voltammetric (CV) scans were performed from 0.0 to +0.7 V (versus Ag/AgCl) at a scan rate of 50 mV s−1 , after the CV scans a thin polypyrrole (PPy) was formed on the Pt electrode surface. After preparing the polymer, the PPy/Pt electrode was rinsed with acetate buffer and immersed into a
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0.1 mol l−1 acetate buffer solution (pH 5.2) containing target oligonucleotide, while applying a constant potential at +0.5 V (versus Ag/AgCl) for 5 min. The electrode was then immersed with PBS and water for 5 min, respectively to remove any nonspecific adsorption of DNA. 2.3.3. Hybridization reaction The electrode immobilized with target ssDNA was immersed into a stirred hybridization solution containing Ru(bpy)3 2+ doped SNPs labeled DNA probe at 40 ◦ C for 50 min. After that, the electrode was thoroughly washed three times with the same buffer and was then employed as working electrode for the ECL measurement. 2.3.4. ECL detection The electrodes were immersed into an ECL cell containing 1.0 ml of PBS (pH 6.6) solution and 25 l of 0.1 mol l−1 H2 C2 O4 . The cell was placed in the dark chamber without stirring. A pulse potential was exerted on the working electrode starting from +0.40 V (versus Ag/AgCl) with pulse amplitude of +0.77 V, pulse period of 20 s and pulse width of 4 s. The recorded height of ECL signal was used for quantification. 3. Results and discussion 3.1. The Characterization of ECL of Ru(bpy)3 2+ -doped SNPs The Ru(bpy)3 2+ -doped SNPs synthesized by reverse microemulsion method were extremely uniform in size, with the average diameter of 90 ± 5 nm as measured 100 nanoparticles by transmission electron microscope (Fig. 2). Ru(bpy)3 2+ doped SNPs had a obvious oxidation current signal at +1.07 V versus Ag/AgCl potential (Fig. 3A) and the ECL intensity of Ru(bpy)3 2+ -doped SNPs was enhanced at present of C2 O4 2− (Fig. 3B), which are similar with the reaction of free Ru(bpy)3 2+ cation [15,33]. Therefore, the ECL mechanism of Ru(bpy)3 2+ -
Fig. 2. TEM image of Ru(bpy)3 2+ -doped SNPs.
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Fig. 3. (A) The cyclic voltammograms of Ru(bpy)3 2+ -doped SNPs in PBS solution (pH 6.6). Scan rate: 0.1 V/s, scan range: 0.0–1.25 V (vs. Ag/AgCl). (B) The ECL intensity of Ru(bpy)3 2+ -doped SNPs in absent (a) and present (b) C2 O4 2− PBS solution (pH 6.6).
doped SNPs should still be expressed in accordance with the Ru(bpy)3 2+ [33,34]. A series of very sharp ECL peak could be observed when pulse potentials were applied from +0.40 to +1.17 V pulse potential as shown in Fig. 4. The onset of light emission for each peak occurred simultaneously with the appearance of the pulse potential reaching more positive pulse, which indicates that ECL reaction of Ru(bpy)3 2+ -doped SNPs is fast enough in the oxalate solution. In order to get maximal ECL signals and balance the concentration of the diffusion layer around the electrode, a relative long rest time at +0.40 V should be kept, the results of experiment showed that the pulse potential with pulse amplitude of +0.77 V, pulse period of 20 s and pulse width of 4 s was optimal ECL potential. We also investigated the effects of concentration of C2 O4 2− and the pH of the buffer solution on ECL
Fig. 4. Voltage pulses (A) and corresponding light pulses (B). Conditions: Ru(bpy)3 2+ -doped SNPs (diluted 100-fold with PBS) PBS buffer (pH 6.6) containing 2.5 × 10−3 mol l−1 C2 O4 2− .
Fig. 5. Effect of C2 O4 2− concentration on the response signal. Conditions: pulse amplitude: +0.40 to +1.17 V (vs. Ag/AgCl), pulse period of 20 s with pulses width of 4 s; Ru(bpy)3 2+ -doped SNPs (diluted 50-fold with PBS); PBS buffer (pH 6.6).
intensity of Ru(bpy)3 2+ -doped SNPs. The results demonstrated that maximal ECL intensity was obtained when the concentration of C2 O4 2− was over 2.5 × 10−3 mol l−1 (Fig. 5), and the ECL intensity increased greatly with the increasing of the pH value until it reached a plateau at the pH 6.5–6.9 and declined sharply at the pH >6.9 (Fig. 6) which is similar to the literature [15,34]. Therefore, we emphatically studied the ECL of Ru(bpy)3 2+ -doped SNPs in PBS solution (pH 6.6) containing 2.5 × 10−3 mol l−1 oxalate. For the SNPs preparation, a concentration of 0.04 mol l−1 Ru(bpy)3 2+ was usually used, however, for optimization of the Ru(bpy)3 2+ concentration, experiments were also performed at 0.01, 0.02, 0.03 and 0.05 mol l−1 . As a result, the ECL intensity of SNPs no obviously increased when the concentration of Ru(bpy)3 2+ more than 0.04 mol l−1 . The nanoparticles were kept stable for 2 months at room temperature.
Fig. 6. Effect of pH on the response signal. Conditions: pulse amplitude: +0.40 to +1.17 V (vs. Ag/AgCl), pulse period of 20 s with pulses width of 4 s; Ru(bpy)3 2+ -doped SNPs (diluted 50-fold with PBS); PBS buffer containing 2.5 × 10−3 mol l−1 C2 O4 2− .
Z. Chang et al. / Electrochimica Acta 52 (2006) 575–580
Fig. 7. ECL intensity of different condition on the PPy/Pt electrode. PPy/Pt electrode immersed (1) Ru(bpy)3 2+ -doped SNPs with applying +0.5 V potential, (2) Ru(bpy)3 2+ -doped SNPs labeled DNA probe and (3) Ru(bpy)3 2+ -doped SNPs labeled DNA probe with applying +0.5 V potential for 200 s.
3.2. The Characterization of ECL of Ru(bpy)3 2+ -doped SNPs labeled DNA probe The surface of Ru(bpy)3 2+ -doped SNPs was first silanized with DETA, a silanization reagent attached the primary amine group to the silica surface. Then the 5 -amine-capped oligonucleotides was covalently linked to the amine-functionalized SNPs using glutaraldehyde as linking agent [22]. Three PPy/Pt electrodes were immersed into acetate buffer solution (pH 5.2), the first and the second were immersed the solution containing same concentration of Ru(bpy)3 2+ -doped SNPs and Ru(bpy)3 2+ doped SNPs labeled DNA probe, respectively, while applying a constant potential at +0.5 V (versus Ag/AgCl) for 200 s, the third PPy/Pt electrode was immersed into the same Ru(bpy)3 2+ doped SNPs labeled DNA probe solution for 200 s without applying any potential. After washed by PBS buffer carefully, the electrodes were carried out ECL detection in PBS buffer (pH 6.6) containing C2 O4 2− . The results in Fig. 7 show that the second electrode has a very strong ECL emission, but the first and the third electrodes have hardly ECL emission. The phenomena distinctly evince that the positively charged PPy film on the Pt electrode can specifically adsorb negative DNA strand labeled Ru(bpy)3 2+ -doped SNPs while applying a positive potential [31,35], but the adsorption is not strong enough that it can be easily washed off if without applying +0.5 V potential, while Ru(bpy)3 2+ -doped SNPs nanoparticles are difficult to be adsorbed on the PPy film. Therefore, Ru(bpy)3 2+ -doped SNPs were successfully labeled onto ssDNA and the Ru(bpy)3 2+ doped SNPs labeled probe possess of excellent ECL character.
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Fig. 8. Comparison of hybridization event of different sequence 24-base oligonucleotide with Ru(bpy)3 2+ -doped SNPs labeled DNA probe. (1) Blank measurement, (2) non-complementary sequence, (3) three-base mismatch sequence, (4) complementary sequence (the concentration of different sequence oligonucleotide: 2.0 × 10−11 mol l−1 ; hybridization solution: PBS (pH 6.8) containing 10−8 mol l−1 Ru(bpy)3 2+ -doped SNPs labeled DNA probe; hybridization time: 50 min; hybridization temperature: 40 ◦ C.
at 40 ◦ C for 50 min. The electrode was washed thoroughly to remove the nonspecific adsorption probe and then used as working electrode for the ECL measurement. As shown in Fig. 8, only complementary sequence gave significant ECL emission (Fig. 8(4)). The non-complementary sequence had negligible responses (Fig. 8(2)), which were equivalent to that of the blank measurement. Although three-base mismatch sequence had slight emission (Fig. 8(3)), it still could be identified with complementary sequence. This proved that the prepared Ru(bpy)3 2+ -doped SNPs labeled DNA probe in this work was highly selective to the target DNA sequence and could be used to identified the DNA sequence with three-base mismatch. Fig. 9 shows that the ECL intensity has a linear response with the concentration of complementary
3.3. The recognition of target DNA and ECL detection of DNA hybridization Different oligonucleotide sequences (complementary sequence, non-complementary sequence and three-base mismatch sequence) were immobilized on the PPy/Pt electrode and hybridized with Ru(bpy)3 2+ -doped SNPs labeled DNA probe
Fig. 9. ECL intensity for different target concentration: (a) 2.0 × 10−13 mol l−1 ; (b) 2.0 × 10−12 mol l−1 ; (c) 2.0 × 10−11 mol l−1 ; (d) 2.0 × 10−10 mol l−1 ; (e) 2.0 × 10−9 mol l−1 . Inset: the logarithmic standard plot.
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sequence in the range of 2.0 × 10−13 to 2.0 × 10−9 mol l−1 . The regression equation is IECL = 0.275 lg CDNA + 5.38 (CDNA is the concentration of complementary sequence, M), and the regression coefficient (R) of the linear curve is 0.9966. The detection limit is 1.0 × 10−13 mol l−1 (s/n = 3). Reproducibility of the measured light intensities was determined by performing the ECL experiments on different days or on the same day using fresh solutions. The light intensities were obtained by recording an emission transient during a 20-s long potential step, the integrated light intensities did not vary significantly with the method employed. 4. Conclusion In summary, we have developed a sensitive ECL detection of DNA hybridization with a 1.0 × 10−13 mol l−1 detection limit, based on Ru(bpy)3 2+ -doped SNPs as DNA tags. The Ru(bpy)3 2+ -doped SNPs synthesized using a modified reverse microemulsion exhibit an excellent signal enhance ability in the presence of a trace amount of DNA targets. With an effective surface modification, nonspecific binding and nanoparticles aggregation are all minimized. It also promotes the application of the technique of ECL nanoparticles tag in biochemical analysis and biomolecular interaction studies. Acknowledgement The authors are grateful for the financial support received from the National Nature Science Foundation of China (no. 29975010). References [1] L. He, M.D. Musick, S.R. Nicewarner, F.G. Salinas, S.J. Benkovic, M.J. Natan, C.D. Keating, J. Am. Chem. Soc. 122 (2000) 9071. [2] J. Wang, D.K. Xu, R. Polsky, J. Am. Chem. Soc. 124 (2002) 4208. [3] G.M. Makrigiorgos, S. Chakrabarti, Y.Z. Zhang, M. Kaur, B.D. Price, Nat. Biotechnol. 20 (2002) 936.
[4] G. Sutherland, J. Mulley, in: R.H. Symons (Ed.), Nucleic Acid Probes, CRC Press, Boca Raton, FL, 1989, p. 159. [5] M.A. Augustin, W. Ankenbauer, B. Angerer, J. Biotechnol. (2001) 289. [6] Z. F¨oldes-Papp, B. Angerer, W. Ankenbauer, R. Rigler, J. Biotechnol. 86 (2001) 237. [7] X.H. Xu, A.J. Bard, J. Am. Chem. Soc. 117 (1995) 2627. [8] K.A. Peterlinz, R.M. Georgiadis, T.M. Herne, M.J. Tarlov, J. Am. Chem. Soc. 119 (1997) 3401. [9] A.B. Steel, T.M. Herne, M.J. Tarlov, Anal. Chem. 70 (1998) 4670. [10] Y. Okahatz, M. Kawase, K. Niikura, F. Ohtake, H. Furasawa, Y. Ebara, Anal. Chem. 70 (1998) 1288. [11] G.F. Blackburn, H.P. Shah, J.H. Kenten, J. Leland, R.A. Kamin, J. Link, J. Peterman, M.J. Powell, A. Shah, D.B. Talley, S.K. Tyagi, E. Wilkins, T.G. Wu, R.J. Massey, Clin. Chem. 37 (1991) 1534. [12] D.R. Deaver, Nature 377 (1995) 758. [13] D.M. Hercules, F.E. Lytle, J. Am. Chem. Soc. 88 (1966) 4745. [14] J. Rublnstein, C. Martin, A.J. Bard, Anal. Chem. 55 (1983) 1580. [15] F. Li, X.Q. Lin, H. Cui, Electroanal. Chem. 534 (2002) 91. [16] W.C.W. Chan, S.M. Nie, Science 281 (5385) (1998) 2013. [17] X. Peng, L. Manna, W. Yang, J. Wickham, E. Scher, A. Kadavanich, A.P. Alivisatos, Nature 404 (6773) (2000) 59. [18] D.J. Maxwell, J.R. Taylor, S. Nie, J. Am. Chem. Soc. 124 (2002) 9606. [19] S. Santra, P. Zhang, K.M. Wang, R. Tapec, W. Tan, Anal. Chem. 73 (2001) 4988. [20] Y.P. Ho, M.C. Kung, S. Yang, T.H. Wang, Nano Lett. 5 (2005) 1693. [21] J. Wang, Anal. Chim. Acta 500 (2003) 247. [22] J. Nam, S. Park, C.A. Mirkin, J. Am. Chem. Soc. 124 (2002) 3820. [23] N.T.K. Thanh, Z. Rosenzweig, Anal. Chem. 74 (2002) 1624. [24] X. Wu, H. Liu, K.N. Haley, J.A. Treadway, P.J. Larson, N. Ge, F. Peale, M.P. Bruchez, Nat. Biotechnol. 21 (2003) 41. [25] H. Cai, Y. Xu, P.G. He, Y.Z. Fang, J. Electroanal. Chem. 510 (2001) 78. [26] H. Cai, Y.Q. Wang, P.G. He, Y.Z. Fang, Anal. Chim. Acta 469 (2002) 165. [27] N.N. Zhu, A.P. Zhang, P.G. He, Y.Z. Fang, Analyst 128 (2003) 260. [28] N.N. Zhu, A.P. Zhang, P.G. He, Y.Z. Fang, Electroanalysis 16 (2004) 577. [29] J. Wang, L. Guodong, R. Gustavo, Anal. Chem. 75 (2003) 4667. [30] N.N. Zhu, H. Cai, P.G. He, Y.Z. Fang, Analyst 481 (2003) 181. [31] W. Stober, A. Fink, J. Colloid Interface Sci. 26 (1968) 62. [32] M.L. Yang, C.Z. Liu, K.J. Qian, Y.Z. Fang, P.G. He, Analyst 127 (2002) 1267. [33] I. Rubinstein, A.J. Bard, J. Am. Chem. Soc. 103 (1981) 512. [34] D. Ege, G.W. Becker, J.A. Bard, Anal. Chem. 56 (1984) 2413. [35] Liz A. Thompson, K. Janusz, J. Mira, J. Jiri, J. Am. Chem. Soc. 125 (2003) 324.
Ru(bpy)32+-doped silica nanoparticle DNA probe for the electrogenerated chemiluminescence detection of DNA hybridization Zhu Chang a,b , Jingming Zhou a , Kun Zhao a , Ningning Zhu c , Pingang He a , Yuzhi Fang a,∗ a
Department of Chemistry, East China Normal University, Shanghai 200062, China b Department of Chemistry, Shangqiu Teachers College, Shangqiu 476000, China c Department of Chemistry, Shanghai Normal University, Shanghai 200234, China
Received 18 January 2006; received in revised form 27 March 2006; accepted 11 May 2006 Available online 5 July 2006
Abstract A sensitive electrogenerated chemiluminescence (ECL) detection of DNA hybridization, based on tris(2,2 -bipyridyl)ruthenium(II)-doped silica nanoparticles (Ru(bpy)3 2+ -doped SNPs) as DNA tags, is described. In this protocol, Ru(bpy)3 2+ -doped SNPs was used for DNA labeling with trimethoxysilylpropydiethylenetriamine(DETA) and glutaraldehyde as linking agents. The Ru(bpy)3 2+ -doped SNPs labeled DNA probe was hybridized with target DNA immobilized on the surface of polypyrrole (PPy) modified Pt electrode. The hybridization events were evaluated by ECL measurements and only the complementary sequence could form a double-stranded DNA (dsDNA) with DNA probe and give strong ECL signals. A three-base mismatch sequence and a non-complementary sequence had almost negligible responses. Due to the large number of Ru(bpy)3 2+ molecules inside SNPs, the assay allows detection at levels as low as 1.0 × 10−13 mol l−1 of the target DNA. The intensity of ECL was linearly related to the concentration of the complementary sequence in the range of 2.0 × 10−13 to 2.0 × 10−9 mol l−1 . © 2006 Elsevier Ltd. All rights reserved. Keywords: Ru(bpy)3 2+ ; Silica nanoparticles; Electrogenerated chemiluminescence; DNA hybridization
1. Introduction Sensitive DNA detection is extremely important in clinical diagnostics, gene therapy, and a variety of biomedical studies. Recently, great efforts have been made to develop new biotechnologies to improve the sensitivity and selectivity for gene analysis [1–3]. Therefore, many detection techniques of DNA sequence have been developed in recent years. These detection techniques include radiochemical [4], enzymatic [5], fluorescent [6], and electrogenerated chemiluminescence (ECL) methods [7]. Surface plasmon resonance spectroscopy [8], electrochemical methods [9] and quartz crystal microbalance [10] are also widely used. Compared with other detection techniques, the ECL has some advantages: no radioisotopes are used, detection limits are extremely low, the dynamic range for quantification extends over six orders of magnitude, the labels are extremely
∗
Corresponding author. Fax: +86 21 62233508. E-mail addresses: [email protected] (P. He), [email protected] (Y. Fang). 0013-4686/$ – see front matter © 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.electacta.2006.05.036
stable compared with those of most other chemiluminescence (CL) systems and the measurement is simple and rapid [11,12]. Ru(bpy)3 2+ has become the most attractive ECL label because of its stability, regenerability, excellent luminescence properties and compatibility with a wide range of analyses [13]. The ECL reaction of Ru(bpy)3 2+ allows the detection of a wide range of analytes, such as C2 O4 2− [14,15], DNA damage [16], electrochemiluminescence immunoassay [17], aliphatic amines [18]. Santra et al. [19] encapsulated ruthenium complexes in thin silica layers as biomarkers for leukemia cell recognition. The emergence of nanotechnology is opening new horizons for the application of nanoparticles in analytical chemistry. Nanoparticles as quantitation tags, such as the optical detection of quantum dots and the electrochemical detection of metallic nanoparticles materials [20,21], offer excellent prospects for chemical and biological sensing because of their unique optical or electrical properties and evidently amplifying signal. The nanoparticles as biomolecules labels in DNA hybridization detection assays and immunoassays for anti-protein are also widely applied. Mirkin and co-workers [22] fabricated ‘biobarcodes’ in which gold nanoparticles are derivatized with distinct
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sequences of DNA, and then taking advantage of the distinct melting temperature of the different DNA sequences, to determine the ‘barcode’ identity. Thanh and Rasenzweig [23] developed an assay in which antibody concentration was determined as a function of aggregation of antigen-coated gold particles. Wu et al. [24] described a Her2 assay for breast cancer with semiconductor quantum dots. Novel classes of optically active nanoparticles are also being developed as next-generation quantitation tags. Our group also developed nanoparticles as DNA labels, such as metallic particle (Au [25], Au@Ag [26]), quantum dots (CdS, PdS, ZnS) [27–29] and core-shell nanoparticles of Co(bpy)3 3+ doped silica nanoparticles (SNPs) [30], to electrochemical detection DNA hybridization. However, electrochemiluminescent detection of DNA hybridization based on the use of metal chelate doped SNPs tags has rarely been reported. Herein, we attempt to combine Ru(bpy)3 2+ -doped SNPs as DNA tags and develop a sensitive electrochemiluminescent method to detection DNA hybridization by amplifying ECL signal through boosting up the amount of Ru(bpy)3 2+ labeled on DNA probe. We succeeded in doping the luminescent reagent of Ru(bpy)3 2+ into SNPs and labeling oligonucleotides onto the nanoparticles surface. We demonstrate that different concentration of Ru(bpy)3 2+ doped SNPs will give different ECL intensity. The performance of DNA biosensors based the Ru(bpy)3 2+ doped SNPs labeled DNA probes with respect to sensitivity, linear range, and the characterization of Ru(bpy)3 2+ -doped SNPs are presented and discussed. 2. Experimental 2.1. Apparatus All ECL measurements were carried out using a laboratory constructed ECL system. A Model 173 potentiostat (Princeton Applied Research, PAR, EG & G) and an XFD-8A waveform generator (Ningbo Radio Factory, Ningbo, China) were used for giving and controlling waveforms and potential. The ECL emission was detected and amplified using a 9901 luminometer (Rongsheng Electronic Equipment Co., Shanghai, China) and the output signal which is directly related to the ECL light inten-
sity was recorded using a Chart Recorder (Dahua Instrument Co, Shanghai, China). The three-electrode system was used. A Pt plate (4 mm × 10 mm) fixed on black plexiglass was used as a working electrode. An Ag/AgCl (saturated KCl) and a platinum wire were used as reference electrode and auxiliary electrode. All ECL reactions were carried out in a 1 ml cell placed directly in the front of photomultiplier tube. A CHI 630 electrochemical analyzer (CHI Instruments Inc., USA), a transmission electron microscope (TEM) (Hitachi, Japan), and a TDL-16B centrifuge (Anting Science Instrument Inc., Shanghai, China) were employed for the experimentation. 2.2. Reagents 24-Base synthetic oligonucleotides (24-base probe sequence: 5 -NH2 -GAG CGG CGC AAC ATT TCA GGT CGA-3 ; its complementary sequence: 5 -TCG ACC TGA AAT GTT GCG CCG CTC-3 ; a non-complementary sequence: 5 -GAG CGG CGC AAC ATT TCA GGT CGA-3 ; a three-base mismatch sequence: 5 -TCG TCC TGA AAC GTT GCG CCT CTC-3 ) were purchased from Shenggong Bioengineering Ltd. Company (Shanghai, China). Tris(2,2 -bipyridyl)ruthenium(II) chloride was purchased from Adlrich (USA). Tetraethylorthosilicate (TEOS) and trimethoxysilylpropydiethylenetriamine (DETA) were purchased from United Chemical Technologies (Bristol, PA). Pyrrole was provided by Shanghai Chemical Factory (Shanghai, China) and was twice distilled before use. Other reagents were commercially available and were all of analytical reagent grade. PBS solution (40 mmol l−1 NaCl + 50 mmol l−1 sodium phosphate buffer, pH 6.8), 0.1 mol l−1 oxalic acid solution and other solutions were prepared with deionized water from an Aquapro system (Yize Co., China). 2.3. The preparation of Ru(bpy)3 2+ -doped SNPs labeled DNA probe and ECL detection The scheme representation of Ru(bpy)3 2+ -doped SNPs labeled DNA probe and the procedure of ECL detection of DNA hybridization based on the probe are illustrated in Fig. 1A and B, respectively.
Fig. 1. Schematic representation of preparation Ru(bpy)3 2+ -doped SNPs oligonucleotides probes (A) and the electrogenerated chemiluminescence detection of DNA hybridization based on the Ru(bpy)3 2+ -doped SNPs labeled oligonucleotides probes (B).
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2.3.1. Ru(bpy)3 2+ -doped nanoparticle synthesis and surface modification with oligonucleotides DNA probe 2.3.1.1. Nanoparticle synthesis. SNPs were prepared according to the literature [19]. The water-in-oil (W/O) microemulsion was prepared firstly by mixing 1.77 ml of TritonX-100, 7.5 ml of cyclohexane, 1.8 ml of n-hexanol, 340 l of 0.04 mol l−1 of Ru(bpy)3 2+ solution were mixed and stirred for 0.5 h at room temperature, forming a uniform W/O microemulsion. The watersurfactant molar ratio was kept constant at 10. In the presence of 100 l of TEOS, a polymerization reaction [31] was initiated by adding 60 l of NH3 ·H2 O (28–30 wt.%). The reaction was allowed to continue for 24 h. After the reaction was completed, the Ru(bpy)3 2+ -doped SNPs were isolated by acetone, followed by centrifuging, ultrasonicating and washing with ethanol and water several times to remove any residual surfactant molecules or any physically adsorbed Ru(bpy)3 2+ from the surface of the particles. Finally, the required orange-colored Ru(bpy)3 2+ doped SNPs were obtained. The Ru(bpy)3 2+ -doped SNPs were suspended in 2 ml PBS(pH 6.8) by ultrasonication for the following experiments. 2.3.1.2. Surface modification of Ru(bpy)3 2+ -doped SNPs and covalent conjugation of oligonucleotides onto the nanoparticle surface. Functionalization of nanoparticles was performed according to the literature [30] by immersion in freshly prepared 1% (v/v) solution of DETA and 1.0 × 10−3 mol l−1 acetic acid for 30 min at room temperature (25 ◦ C). The DETA modified SNPs were thoroughly rinsed with deionized water to remove excess DETA. The above amine-functionalized Ru(bpy)3 2+ -doped SNPs were reacted with 5% glutaraldehyde solution in a water bath at 37 ◦ C for 120 min with shaking. After that, the nanoparticles were washed with deionized water several times to remove excess glutaraldehyde. Then 2.0 OD of 5 -amine-capped oligonucleotides diluted in PBS buffer (pH 6.8) was added to the functionalized SNPs solution, and stirring was continued for 120 min in a water bath at 37 ◦ C (final oligonucleotides concentration is 1.27 × 10−5 mol l−1 ). Oligonucleotides probe conjugated nanoparticles were then treated with 10 ml of 30 mmol l−1 glycine solution for 30 min. The final product was washed and centrifuged, resuspended in PBS (pH 6.8) buffer, either used immediately for test or stored at 4 ◦ C for later usage. 2.3.2. Immobilization of target ssDNA on PPy/Pt electrode The immobilization of target ssDNA on a Pt electrode was carried out according to the literature [32]. Briefly, the Pt electrode was polished carefully with alumina powder on a soft polishing cloth. After sonicating in absolute ethanol, then in water for 5 min, the pretreated Pt electrode was immersed into 0.1 mol l−1 KCl solution containing 0.05 mol l−1 pyrrole monomer (pH was adjusted to 3.0 with sodium acetate buffer). Fourteen cyclic voltammetric (CV) scans were performed from 0.0 to +0.7 V (versus Ag/AgCl) at a scan rate of 50 mV s−1 , after the CV scans a thin polypyrrole (PPy) was formed on the Pt electrode surface. After preparing the polymer, the PPy/Pt electrode was rinsed with acetate buffer and immersed into a
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0.1 mol l−1 acetate buffer solution (pH 5.2) containing target oligonucleotide, while applying a constant potential at +0.5 V (versus Ag/AgCl) for 5 min. The electrode was then immersed with PBS and water for 5 min, respectively to remove any nonspecific adsorption of DNA. 2.3.3. Hybridization reaction The electrode immobilized with target ssDNA was immersed into a stirred hybridization solution containing Ru(bpy)3 2+ doped SNPs labeled DNA probe at 40 ◦ C for 50 min. After that, the electrode was thoroughly washed three times with the same buffer and was then employed as working electrode for the ECL measurement. 2.3.4. ECL detection The electrodes were immersed into an ECL cell containing 1.0 ml of PBS (pH 6.6) solution and 25 l of 0.1 mol l−1 H2 C2 O4 . The cell was placed in the dark chamber without stirring. A pulse potential was exerted on the working electrode starting from +0.40 V (versus Ag/AgCl) with pulse amplitude of +0.77 V, pulse period of 20 s and pulse width of 4 s. The recorded height of ECL signal was used for quantification. 3. Results and discussion 3.1. The Characterization of ECL of Ru(bpy)3 2+ -doped SNPs The Ru(bpy)3 2+ -doped SNPs synthesized by reverse microemulsion method were extremely uniform in size, with the average diameter of 90 ± 5 nm as measured 100 nanoparticles by transmission electron microscope (Fig. 2). Ru(bpy)3 2+ doped SNPs had a obvious oxidation current signal at +1.07 V versus Ag/AgCl potential (Fig. 3A) and the ECL intensity of Ru(bpy)3 2+ -doped SNPs was enhanced at present of C2 O4 2− (Fig. 3B), which are similar with the reaction of free Ru(bpy)3 2+ cation [15,33]. Therefore, the ECL mechanism of Ru(bpy)3 2+ -
Fig. 2. TEM image of Ru(bpy)3 2+ -doped SNPs.
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Fig. 3. (A) The cyclic voltammograms of Ru(bpy)3 2+ -doped SNPs in PBS solution (pH 6.6). Scan rate: 0.1 V/s, scan range: 0.0–1.25 V (vs. Ag/AgCl). (B) The ECL intensity of Ru(bpy)3 2+ -doped SNPs in absent (a) and present (b) C2 O4 2− PBS solution (pH 6.6).
doped SNPs should still be expressed in accordance with the Ru(bpy)3 2+ [33,34]. A series of very sharp ECL peak could be observed when pulse potentials were applied from +0.40 to +1.17 V pulse potential as shown in Fig. 4. The onset of light emission for each peak occurred simultaneously with the appearance of the pulse potential reaching more positive pulse, which indicates that ECL reaction of Ru(bpy)3 2+ -doped SNPs is fast enough in the oxalate solution. In order to get maximal ECL signals and balance the concentration of the diffusion layer around the electrode, a relative long rest time at +0.40 V should be kept, the results of experiment showed that the pulse potential with pulse amplitude of +0.77 V, pulse period of 20 s and pulse width of 4 s was optimal ECL potential. We also investigated the effects of concentration of C2 O4 2− and the pH of the buffer solution on ECL
Fig. 4. Voltage pulses (A) and corresponding light pulses (B). Conditions: Ru(bpy)3 2+ -doped SNPs (diluted 100-fold with PBS) PBS buffer (pH 6.6) containing 2.5 × 10−3 mol l−1 C2 O4 2− .
Fig. 5. Effect of C2 O4 2− concentration on the response signal. Conditions: pulse amplitude: +0.40 to +1.17 V (vs. Ag/AgCl), pulse period of 20 s with pulses width of 4 s; Ru(bpy)3 2+ -doped SNPs (diluted 50-fold with PBS); PBS buffer (pH 6.6).
intensity of Ru(bpy)3 2+ -doped SNPs. The results demonstrated that maximal ECL intensity was obtained when the concentration of C2 O4 2− was over 2.5 × 10−3 mol l−1 (Fig. 5), and the ECL intensity increased greatly with the increasing of the pH value until it reached a plateau at the pH 6.5–6.9 and declined sharply at the pH >6.9 (Fig. 6) which is similar to the literature [15,34]. Therefore, we emphatically studied the ECL of Ru(bpy)3 2+ -doped SNPs in PBS solution (pH 6.6) containing 2.5 × 10−3 mol l−1 oxalate. For the SNPs preparation, a concentration of 0.04 mol l−1 Ru(bpy)3 2+ was usually used, however, for optimization of the Ru(bpy)3 2+ concentration, experiments were also performed at 0.01, 0.02, 0.03 and 0.05 mol l−1 . As a result, the ECL intensity of SNPs no obviously increased when the concentration of Ru(bpy)3 2+ more than 0.04 mol l−1 . The nanoparticles were kept stable for 2 months at room temperature.
Fig. 6. Effect of pH on the response signal. Conditions: pulse amplitude: +0.40 to +1.17 V (vs. Ag/AgCl), pulse period of 20 s with pulses width of 4 s; Ru(bpy)3 2+ -doped SNPs (diluted 50-fold with PBS); PBS buffer containing 2.5 × 10−3 mol l−1 C2 O4 2− .
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Fig. 7. ECL intensity of different condition on the PPy/Pt electrode. PPy/Pt electrode immersed (1) Ru(bpy)3 2+ -doped SNPs with applying +0.5 V potential, (2) Ru(bpy)3 2+ -doped SNPs labeled DNA probe and (3) Ru(bpy)3 2+ -doped SNPs labeled DNA probe with applying +0.5 V potential for 200 s.
3.2. The Characterization of ECL of Ru(bpy)3 2+ -doped SNPs labeled DNA probe The surface of Ru(bpy)3 2+ -doped SNPs was first silanized with DETA, a silanization reagent attached the primary amine group to the silica surface. Then the 5 -amine-capped oligonucleotides was covalently linked to the amine-functionalized SNPs using glutaraldehyde as linking agent [22]. Three PPy/Pt electrodes were immersed into acetate buffer solution (pH 5.2), the first and the second were immersed the solution containing same concentration of Ru(bpy)3 2+ -doped SNPs and Ru(bpy)3 2+ doped SNPs labeled DNA probe, respectively, while applying a constant potential at +0.5 V (versus Ag/AgCl) for 200 s, the third PPy/Pt electrode was immersed into the same Ru(bpy)3 2+ doped SNPs labeled DNA probe solution for 200 s without applying any potential. After washed by PBS buffer carefully, the electrodes were carried out ECL detection in PBS buffer (pH 6.6) containing C2 O4 2− . The results in Fig. 7 show that the second electrode has a very strong ECL emission, but the first and the third electrodes have hardly ECL emission. The phenomena distinctly evince that the positively charged PPy film on the Pt electrode can specifically adsorb negative DNA strand labeled Ru(bpy)3 2+ -doped SNPs while applying a positive potential [31,35], but the adsorption is not strong enough that it can be easily washed off if without applying +0.5 V potential, while Ru(bpy)3 2+ -doped SNPs nanoparticles are difficult to be adsorbed on the PPy film. Therefore, Ru(bpy)3 2+ -doped SNPs were successfully labeled onto ssDNA and the Ru(bpy)3 2+ doped SNPs labeled probe possess of excellent ECL character.
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Fig. 8. Comparison of hybridization event of different sequence 24-base oligonucleotide with Ru(bpy)3 2+ -doped SNPs labeled DNA probe. (1) Blank measurement, (2) non-complementary sequence, (3) three-base mismatch sequence, (4) complementary sequence (the concentration of different sequence oligonucleotide: 2.0 × 10−11 mol l−1 ; hybridization solution: PBS (pH 6.8) containing 10−8 mol l−1 Ru(bpy)3 2+ -doped SNPs labeled DNA probe; hybridization time: 50 min; hybridization temperature: 40 ◦ C.
at 40 ◦ C for 50 min. The electrode was washed thoroughly to remove the nonspecific adsorption probe and then used as working electrode for the ECL measurement. As shown in Fig. 8, only complementary sequence gave significant ECL emission (Fig. 8(4)). The non-complementary sequence had negligible responses (Fig. 8(2)), which were equivalent to that of the blank measurement. Although three-base mismatch sequence had slight emission (Fig. 8(3)), it still could be identified with complementary sequence. This proved that the prepared Ru(bpy)3 2+ -doped SNPs labeled DNA probe in this work was highly selective to the target DNA sequence and could be used to identified the DNA sequence with three-base mismatch. Fig. 9 shows that the ECL intensity has a linear response with the concentration of complementary
3.3. The recognition of target DNA and ECL detection of DNA hybridization Different oligonucleotide sequences (complementary sequence, non-complementary sequence and three-base mismatch sequence) were immobilized on the PPy/Pt electrode and hybridized with Ru(bpy)3 2+ -doped SNPs labeled DNA probe
Fig. 9. ECL intensity for different target concentration: (a) 2.0 × 10−13 mol l−1 ; (b) 2.0 × 10−12 mol l−1 ; (c) 2.0 × 10−11 mol l−1 ; (d) 2.0 × 10−10 mol l−1 ; (e) 2.0 × 10−9 mol l−1 . Inset: the logarithmic standard plot.
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sequence in the range of 2.0 × 10−13 to 2.0 × 10−9 mol l−1 . The regression equation is IECL = 0.275 lg CDNA + 5.38 (CDNA is the concentration of complementary sequence, M), and the regression coefficient (R) of the linear curve is 0.9966. The detection limit is 1.0 × 10−13 mol l−1 (s/n = 3). Reproducibility of the measured light intensities was determined by performing the ECL experiments on different days or on the same day using fresh solutions. The light intensities were obtained by recording an emission transient during a 20-s long potential step, the integrated light intensities did not vary significantly with the method employed. 4. Conclusion In summary, we have developed a sensitive ECL detection of DNA hybridization with a 1.0 × 10−13 mol l−1 detection limit, based on Ru(bpy)3 2+ -doped SNPs as DNA tags. The Ru(bpy)3 2+ -doped SNPs synthesized using a modified reverse microemulsion exhibit an excellent signal enhance ability in the presence of a trace amount of DNA targets. With an effective surface modification, nonspecific binding and nanoparticles aggregation are all minimized. It also promotes the application of the technique of ECL nanoparticles tag in biochemical analysis and biomolecular interaction studies. Acknowledgement The authors are grateful for the financial support received from the National Nature Science Foundation of China (no. 29975010). References [1] L. He, M.D. Musick, S.R. Nicewarner, F.G. Salinas, S.J. Benkovic, M.J. Natan, C.D. Keating, J. Am. Chem. Soc. 122 (2000) 9071. [2] J. Wang, D.K. Xu, R. Polsky, J. Am. Chem. Soc. 124 (2002) 4208. [3] G.M. Makrigiorgos, S. Chakrabarti, Y.Z. Zhang, M. Kaur, B.D. Price, Nat. Biotechnol. 20 (2002) 936.
[4] G. Sutherland, J. Mulley, in: R.H. Symons (Ed.), Nucleic Acid Probes, CRC Press, Boca Raton, FL, 1989, p. 159. [5] M.A. Augustin, W. Ankenbauer, B. Angerer, J. Biotechnol. (2001) 289. [6] Z. F¨oldes-Papp, B. Angerer, W. Ankenbauer, R. Rigler, J. Biotechnol. 86 (2001) 237. [7] X.H. Xu, A.J. Bard, J. Am. Chem. Soc. 117 (1995) 2627. [8] K.A. Peterlinz, R.M. Georgiadis, T.M. Herne, M.J. Tarlov, J. Am. Chem. Soc. 119 (1997) 3401. [9] A.B. Steel, T.M. Herne, M.J. Tarlov, Anal. Chem. 70 (1998) 4670. [10] Y. Okahatz, M. Kawase, K. Niikura, F. Ohtake, H. Furasawa, Y. Ebara, Anal. Chem. 70 (1998) 1288. [11] G.F. Blackburn, H.P. Shah, J.H. Kenten, J. Leland, R.A. Kamin, J. Link, J. Peterman, M.J. Powell, A. Shah, D.B. Talley, S.K. Tyagi, E. Wilkins, T.G. Wu, R.J. Massey, Clin. Chem. 37 (1991) 1534. [12] D.R. Deaver, Nature 377 (1995) 758. [13] D.M. Hercules, F.E. Lytle, J. Am. Chem. Soc. 88 (1966) 4745. [14] J. Rublnstein, C. Martin, A.J. Bard, Anal. Chem. 55 (1983) 1580. [15] F. Li, X.Q. Lin, H. Cui, Electroanal. Chem. 534 (2002) 91. [16] W.C.W. Chan, S.M. Nie, Science 281 (5385) (1998) 2013. [17] X. Peng, L. Manna, W. Yang, J. Wickham, E. Scher, A. Kadavanich, A.P. Alivisatos, Nature 404 (6773) (2000) 59. [18] D.J. Maxwell, J.R. Taylor, S. Nie, J. Am. Chem. Soc. 124 (2002) 9606. [19] S. Santra, P. Zhang, K.M. Wang, R. Tapec, W. Tan, Anal. Chem. 73 (2001) 4988. [20] Y.P. Ho, M.C. Kung, S. Yang, T.H. Wang, Nano Lett. 5 (2005) 1693. [21] J. Wang, Anal. Chim. Acta 500 (2003) 247. [22] J. Nam, S. Park, C.A. Mirkin, J. Am. Chem. Soc. 124 (2002) 3820. [23] N.T.K. Thanh, Z. Rosenzweig, Anal. Chem. 74 (2002) 1624. [24] X. Wu, H. Liu, K.N. Haley, J.A. Treadway, P.J. Larson, N. Ge, F. Peale, M.P. Bruchez, Nat. Biotechnol. 21 (2003) 41. [25] H. Cai, Y. Xu, P.G. He, Y.Z. Fang, J. Electroanal. Chem. 510 (2001) 78. [26] H. Cai, Y.Q. Wang, P.G. He, Y.Z. Fang, Anal. Chim. Acta 469 (2002) 165. [27] N.N. Zhu, A.P. Zhang, P.G. He, Y.Z. Fang, Analyst 128 (2003) 260. [28] N.N. Zhu, A.P. Zhang, P.G. He, Y.Z. Fang, Electroanalysis 16 (2004) 577. [29] J. Wang, L. Guodong, R. Gustavo, Anal. Chem. 75 (2003) 4667. [30] N.N. Zhu, H. Cai, P.G. He, Y.Z. Fang, Analyst 481 (2003) 181. [31] W. Stober, A. Fink, J. Colloid Interface Sci. 26 (1968) 62. [32] M.L. Yang, C.Z. Liu, K.J. Qian, Y.Z. Fang, P.G. He, Analyst 127 (2002) 1267. [33] I. Rubinstein, A.J. Bard, J. Am. Chem. Soc. 103 (1981) 512. [34] D. Ege, G.W. Becker, J.A. Bard, Anal. Chem. 56 (1984) 2413. [35] Liz A. Thompson, K. Janusz, J. Mira, J. Jiri, J. Am. Chem. Soc. 125 (2003) 324.