Dendrimer-encapsulated copper as a novel oligonucleotides label for sensitive electrochemical stripping detection of DNA hybridization

Biosensors and Bioelectronics 48 (2013) 210–215

Contents lists available at SciVerse ScienceDirect

Biosensors and Bioelectronics journal homepage: www.elsevier.com/locate/bios

Dendrimer-encapsulated copper as a novel oligonucleotides label for sensitive electrochemical stripping detection of DNA hybridization Huan Gao, Xue Jiang, Yang-Jun Dong, Wan-Xin Tang, Cong Hou, Ning-Ning Zhu (n) Department of Chemistry, College of Life and Environment Sciences, Shanghai Normal University, Guilin Road 100, Shanghai 200234, China

art ic l e i nf o

a b s t r a c t

Article history: Received 21 January 2013 Received in revised form 17 April 2013 Accepted 17 April 2013 Available online 28 April 2013

This paper describes the synthesis and characterization of a novel electrochemical label for sensitive electrochemical stripping detection of DNA hybridization based on dendrimer-encapsulated copper. The generation 4.5 (G 4.5) carboxyl-terminated poly(amidoamine) dendrimer with a trimesyl core was used as a template for synthesis of Cu2+/dendrimer nanocomposites (Cu-DNCs). Ratios of Cu2+/dendrimer were optimized in order to obtain stable nanocomposites with maximal copper loading in the interior of a polymeric shell. Cu-DNCs labeled DNA probe was employed for determining a target ssDNA immobilized on multi-walled carbon nanotubes-modified glassy carbon electrode (GCE) based on a specific hybridization reaction. The hybridization events were monitored by electrochemical detection of Cu anchored on the hybrids after the release in a diluted nitric acid by anodic stripping differential pulse voltammetry (ASDPV). The results showed that only a complementary sequence could form a dsDNA with the Cu-DNCs DNA probe and give an obvious electrochemical signal. The non-complementary sequence exhibited negligible signal change compared with the blank measurement (means: the electrode containing no target DNA incubating in hybridization buffer solution containing Cu-DNCs DNA probe for a certain time). The use of Cu encapsulated-dendrimer as tags and ASDPV for the detection of the released Cu ions could enhance the hybridization signal, and result in the increase of the sensitivity for the target DNA. Under the conditions employed here, the detection limit for measuring the full complementary sequence is down to pM level. & 2013 Elsevier B.V. All rights reserved.

Keywords: Dendrimer-encapsulated copper Electrochemical DNA hybridization Anodic stripping analysis

1. Introduction The detection of specific sequences of DNA is of use for the sensing or diagnosis of viruses, pathogenic microorganisms and human genetic diseases (Shuber et al., 1997; Saiki et al., 1985). High-sensitivity and high-selectivity detection is necessary for early disease diagnosis and treatment (Golub et al., 1999). Electrochemical methods are of interest because they have the potential for providing sensors of high sensitivity and low cost, suitable for on-site, decentralized testing (Bonanni and Pumera, 2011; Shiddiky et al., 2010; Xia et al., 2010). There have been two main approaches to the electrochemical transduction of DNA hybridization, which can be broadly referred to as labeled methods and label-free methods. Labeled methods are significantly more popular than label-free approaches because there are much more ways in which transduction can be configured with high sensitivity and selectivity (Odenthal and Gooding, 2007). Most available methods involve labeled-based electrochemical assays, in which the target DNA is hybridized with a specific base-sequence probe

(n )

Corresponding author. Tel.: +86 156 183 83006. E-mail address: [email protected] (N.-N. Zhu).

0956-5663/$ - see front matter & 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.bios.2013.04.021

labeled with redox active molecules (Radhakrishnan et al., 2013; Yu et al., 2010), enzymes (Liu et al., 2013; Bakker and Qin, 2006; Zhang et al., 2003) or nanoparticles (Pinijsuwan et al., 2008; Liu and Lin, 2007; Yu et al., 2010). The first electrochemical DNA hybridization biosensor was reported using the minor groove binding redox active label Co(Phen)33+, which gave different electrochemical signals depending on whether the DNA was double- or single-stranded (Millan and Mikkelsen, 1993). The subsequent developments in this area include the synthesis of a new cobalt groove binder for the detection of sequences from the HIV DNA (Niu et al., 2006). Barton reported to use intercalators such as methylene blue or Daunomycin for DNA hybridization (Boon et al., 2000). Ozsoz and co-workers used the redox marker methylene blue for the detection of sequences related to the hepatitis B virus (Erdem et al., 2000), which is one of the best developed examples of the preferential binding strategy. Physical changes in DNA from the flexible ss-DNA to the rigid rod-like dsDNA can be used for detection by covalently attaching a ferrocene tag to the probe (Anne and Demaille, 2006). Heeger and coworkers improved these ideas using a DNA stem-loop structure labeled with ferrocene for DNA hybridization detection (Lai et al., 2006). Alternatively, the use of metal/semiconductor nanoparticle labels has become attractive. These nanoparticles can be oxidized

H. Gao et al. / Biosensors and Bioelectronics 48 (2013) 210–215

at a planar electrode or can be first dissolved and then measured by anodic stripping voltammetry (ASV), a technique that imparts high sensitivity due to the pre-concentration step (Won et al., 2013; Pinijsuwan et al., 2008; Merkoci, 2010; Perez-Lopez and Merkoci, 2011; Rosi and Mirkin, 2005; Yu et al., 2010). Dendrimer-encapsulated metal nanoparticles are of scientific and technological interest because of their unique structure and functionality (Balogh and Tomalia, 1998; Crooks et al., 2001). These nanoparticles have been primarily used in catalysis (Scott et al., 2005; Myers et al., 2012; Maity et al., 2013; Ye and Crooks, 2005), optics (Zheng et al., 2004), and other applications (Herrero et al., 2010). Typically, dendrimer-encapsulated copper nanoparticles were firstly studied because Cu2+ complexes with PAMAM dendrimers are very well behaved and have easily interpretable UV– vis spectra (Zhao et al., 1998). Recently, dendrimer-entrapped gold or silver nanoparticles have also been used for cancer-cell targeting and imaging (Shi et al., 2007) or sensitive immunosensors (Stofik et al., 2009). Due to their uniform compositions and unique structures, dendrimers have also caused the increasing attention in sensing applications. For instance, dendrimers were used as oligonucleotide labels (Zhu et al., 2009a) or surface confinement of probe DNA (Zhu et al., 2010) to enhance the hybridization signal of electrochemical DNA biosensor and to achieve the good sensitivities. The dendrimers/carbon nanotubes-modified electrodes were also used for developing electrochemical sensors (Zhu et al., 2009b; Shen et al., 2009). In this study, we demonstrate a new and effective strategy for electrochemical stripping detection of DNA hybridization based on dendrimer-encapsulated copper (Cu-DNCs) as labels. The complexation interaction between dendrimers and Cu2+ is strong. By optimizing the ratios of Cu2+/dendrimer, the stable nanocomposites with maximal copper loading in the interior of a polymeric shell were obtained. Due to a large amount of copper ions released from DNA hybrid and the highly sensitive anodic-stripping voltammetry (ASV) technique for the measurement of Cu2+ in solution, the detection limit down to pM level for measuring the full complementary sequence is obtained. This approach could be generalized to prepare other dendrimer-encapsulated metal (Ag) or semiconductor nanoparticles (CdS, PbS) tags for electrochemical monitoring of DNA hybridization, and this may pave a way for the simultaneous detection of multiple DNA targets in one sample.

2. Experimental 2.1. Reagents and materials 24-base synthetic oligonucleotides were purchased from Shenggong Bioengineering Ltd. Company (Shanghai, China); probe sequence: 5′-NH2-GAG CGG CGC AAC ATT TCA GGT CGA-3′; the fully complementary target sequence: 5′-NH2-TCG ACC TGA AAT GTT GCG CCG CTC-3′; and the non-complementary oligonucleotide sequence: 5′-NH2-GAG CGG CGC AAC ATT TCA GGT CGA-3′ (the same as the probe sequence). Multi-walled carbon nanotubes (MWNTs) with a diameter of about 10–30 nm were obtained from Shenzhen Nanotech Co. Ltd. (Shenzhen, China). Prior to use, the MWNTs were purified by chemical oxidation of the as-received MWNTs in a mixture of H2SO4 and HNO3 (3:1, v/v) under ultrasonication for approximately 4 h. As reported previously, such a purification procedure also produces carboxyl groups at MWNTs (Zhu et al., 2010). 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) was purchased from Sigma. The following buffers were used: 0.1 M PBS (0.1 M NaCl, 0.01 M sodium phosphate buffer, and pH 7.0), 0.3 M PBS (0.3 M NaCl, 0.01 M sodium phosphate buffer, and pH 7.0), 0.75 M PBS (0.75 M NaCl, 0.01 M sodium phosphate buffer, and pH 7.0) and 0.1 M HAc–NaAc buffer (pH 4.7). The G 4.5

211

carboxyl-terminated poly(amidoamine) dendrimer with a trimesyl core (G 4.5 dendrimer) was obtained from Dr. Yang (Shanghai Normal University) (Zhu et al., 2009a). Other chemicals were of at least analytical grade and used as received. Aqueous solutions were prepared with double distilled water. 2.2. Preparation of Cu-DNCs-labeled DNA probe and its hybridization with target DNA For the synthesis of Cu-DNCs, dendrimers (0.1 mM, 1 ml) were mixed with CuSO4 solution (3 mM, 0.5 ml) in molar ratios 1:15 (dendrimer/Cu2+) at least for 30 min under vigorous stirring to form Cu-DNCs composite. Cu-DNCs DNA probe was prepared by mixing 1.53 ml aforementioned Cu-DNCs with 2 OD (about 66 μg) ssDNA containing 5 mM EDC with gentle stirring for 8 h at ambient temperature (Zhu et al., 2009a). Then ethanol and 3 M acetate buffer (pH 5.2) in the ratio of 20:1 (V:V, 1 ml) were added and the solution was placed in a refrigerator (<−15 1C) for 20 min (Forster et al., 1985). After being centrifuged, the precipitate was washed with 70% ethanol (1 ml
3) and re-centrifuged to remove unreacted Cu-DNCs. The obtained precipitate was dispersed in 0.1 M PBS, and the free DNA was separated from Cu-DNCs DNA complex using Amicon Y100 molecular weight cutoff filters. The filters and solution were spun at 10,000g for 8 min with no more than 300 mL of the Cu-DNCs DNA mixture on the Y100 filter. Thus, the majority of the free DNA sequence not conjugated with CuDNCs passes through the filter and Cu-DNCs DNA complex remains on the filter. The obtained Cu-DNCs DNA probe was dissolved in water for further DNA hybridization immediately or stored in refrigerator (−15 1C). Thus Cu-DNCs DNA probe was prepared via an amide linkage between the -NH2 of ssDNA at 5′ end and the -COOH of dendrimer on the periphery. The glassy carbon electrode (area of 7.07 mm2) was polished with alumina powder (0.3 and 0.05 μm) and sonicated in acetone and double distilled water (each for 3–5 min). MWNT suspension was prepared by dispersing 2 mg purified MWNTs into 1 mL organic solvent N,N–dimethylformamide (DMF) with the aid of ultrasonic agitation. Then 2.0 μL of the abovementioned MWNT dispersion was dip-coated onto freshly smoothed glassy carbon electrode (GCE) surface and the organic solvent was then evaporated under an infrared lamp. The immobilization of target DNA on MWNT-modified electrode was prepared by immersing the MWNT-modified electrode into different concentrations of target ssDNA solution containing 5 mM EDC for 6–8 h at room temperature. The resulting electrode (target/MWNT-modified electrode) was finally rinsed with 0.1 M phosphate buffer and distilled water to remove the physically adsorbed DNA. Hybridization reaction was performed by immersing the target ssDNA (the full complementary target and a non-complementary sequence) captured MWNT/glassy carbon (GC) electrode into a stirred hybridization solution (0.3 M PBS buffer and pH 7.0) containing a certain concentration of Cu-DNCs DNA probe for 60 min at 37 1C. After that, the electrode was rinsed three times with 0.75 M phosphate buffer (0.75 M NaCl and pH 7.0) and water respectively, to remove the non-hybridized physically adsorbed probe DNA. 2.3. Instruments and Measurements Cyclic voltammetry (CV) and differential pulse voltammograms (DPV) were performed using CHI660B electrochemical analyzer (CHI Instrument Corp. Shanghai) with a conventional threeelectrode system: glassy carbon electrode as working electrode, a Pt wire as counter electrode and a saturated calomel electrode (SCE, saturated KCl) as reference electrode. Unless stated otherwise, all measurements were performed at ambient temperature.

212

H. Gao et al. / Biosensors and Bioelectronics 48 (2013) 210–215

A Varian Cary 100 Ultraviolet–visible spectrophotometer (Varian Comp. US) and a JB-1 stirring machine (Branson, Shanghai, China) were used. Cu-DNCs linked to the hybrids on the MWNT/GCE surface were immersed in a 10 ml analytical cell containing 200 mL 50% nitric acid solution for 5 min and Cu2+ ions release from the dendrimer interior into solution. Then 3 mL 0.1 M HAc–NaAc (pH 4.7) buffer was added to the cell as a supporting electrolyte, the released Cu2+ ions were quantified by ASV. For ASV experiments, the threeelectrode system consists of a freshly treated GC working electrode, a Pt wire counter electrode and a reference electrode (SCE). The GC working electrode was cathodically electrodeposited at −0.4 V for 300 s in the analytical cell containing the released Cu2+, then followed by a positive differential pulse voltammetry (DPV) scan from −0.2 to 0.2 V (vs. SCE), with amplitude 50 mV, pulse width 50 ms, and pulse period 0.2 s. The anodic stripping peak current located at ∼+0.010 V (vs. SCE) was taken as the analytical response.

Fig. 2 shows absorption spectra that illustrate the pH dependence of 0.1 mM G 4.5 dendrimer+3 mM CuSO4. The bands arising from complexation between G 4.5 dendrimer and Cu2+ at 556 nm and 240 nm decrease with decreasing pH from 9.0 (Fig. 2 —) to 3.0 (Fig. 2 -  -). When the pH of the solution is adjusted to 2.0 (Fig. 2    ), the band at 556 nm essentially disappear and a broad, weak band corresponding to free Cu2+ appears at around 810 nm (Fig. 2 inset,    ), implying that no Cu2+ ions can bind to dendrimer at low pH. In addition, considering that the hydroxylation of Cu2+ increases with pH increasing from 6.0 to 9.0, so the complexation interaction between dendrimers and Cu2+ was carried out at pH 6.0 (the original pH value of the CuSO4 and G 4.5 dendrimer mixture) in this study. The number of Cu2+ ions extracted into each G 4.5 dendrimer by spectrophotometric titration was also assessed quantitatively. Spectra of a 0.1 mM G 4.5 dendrimer solution containing different amounts of Cu2+ ions are given in Fig. 3. The absorbance at 556 nm increases with the ratio of [Cu2+]/[G

3. Results and discussion 3.1. Preparation of Cu-DNCs-labeled DNA probe Fig. 1 shows absorption spectra of 0.1 mM G 4.5 dendrimer (c), 3 mM CuSO4 solution before (a) and after (b) adding 0.1 mM G 4.5 dendrimer. As can be seen, G 4.5 dendrimer solution shows a strong and a weak absorption band located at ∼225 nm (E2 band) and ∼290 nm (B band), respectively (Fig. 1c), which may be attributed to benzene ring of trimesyl core in G 4.5 dendrimer. In the absence of dendrimers, Cu2+ exists primarily as [Cu(H2O)6]2 + , which gives rise to a broad, weak absorption band centered at 810 nm (Crooks et al., 2001) (Fig. 1 inset, a). This corresponds to the well-known d–d transition for Cu2+ in a tetragonally distorted octahedral or square-planar ligand field; and in the presence of dendrimers, λmax for d–d transition shifts to 556 nm (Fig. 1 inset b). In addition, a stronger absorption band centered at 240 nm emerges (Fig. 1b), which can be assigned to the ligand-to-metalcharge-transfer (LMCT) transition (Zhao et al., 1998). The d–d transition band and the LMCT transition do not decrease significantly even after 36 h of dialysis against pure water, indicating that the complexation interaction between dendrimers and Cu2+ is strong. The binding between Cu2+ and dendrimers is pH dependent.

Fig. 1. Absorption spectra of 3 mM CuSO4 in the absence (a) and in the presence (b) of 0.1 mM G 4.5 dendrimer. The absorption spectrum of 0.1 mM G 4.5 dendrimer solution is also shown (c).

Fig. 2. Absorption spectra of 0.1 mM G 4.5 dendrimer+3 mM CuSO4 at pH 9.0 (—), 6.0 (—), 3.0 (-  -) and 2.0 (    ).

Fig. 3. Absorption spectra as a function of the Cu2+/G 4.5 ratio. The inset is a spectrophotometric titration plot showing absorbance at the peak maximum of 556 nm as a function of number of Cu2+ ions per G 4.5 dendrimer. Error bars show the standard deviations of three measurements.

H. Gao et al. / Biosensors and Bioelectronics 48 (2013) 210–215

4.5] from 0 to 15, but only slowly when the ratio is larger than 15 (from 15 to 25), and meanwhile a gradual red-shifting from 556 nm to 601 nm was observed, which could be ascribed to the presence of free Cu2+ ions in solution containing Cu-DNCs. The titration results are given in the inset of Fig. 3. We estimated the titration endpoint by extrapolating the two linear regions of the curve, and this treatment indicates that each G 4.5 dendrimer can strongly sorb up to 15 Cu2+ ions. A G 4.5 dendrimer used in this study contains 24 outermost amines, thus, it was estimated that each Cu2+ is coordinated to two amine groups, and the remaining positions of the ligand field are likely to be occupied by more weakly binding ligands such as amide groups or water (Crooks et al., 2001). The 24-base 5′-NH2-capped oligonucleotide was attached onto the surface of Cu-DNCs via an amide linkage between the free amino groups of ssDNA at 5′end and the carboxyl of G 4.5 dendrimer on the periphery (Hermanson, 1996). The UV–vis spectrum of Cu-DNCs–DNA probe, obtained with a Varian Cary 100 UV–vis spectrophotometer using Cu-DNCs as a blank was obtained (Supplementary data, Fig. S1). An obvious absorption band appeared at about 282 nm, which corresponded to the characteristic absorption of DNA base. The shift of the adsorption peak compared with pure DNA solution (about 264 nm) may be due to the interaction between dendrimer and DNA, suggesting that the DNA molecules were successfully conjugated onto the Cu-DNCs. 3.2. Electrochemical stripping detection of DNA hybridization involving Cu-DNCs label Single-stranded (ss) DNA target attachment onto MWNT transducers was verified by the changes in cyclic voltammetric (CV) response at the bare, MWNT-modified and ssDNA/MWNT-modified GC electrodes in 0.1 M HAc–NaAc (pH 4.7) supporting electrolyte. As displayed in Fig. 4, the background currents obtained at the MWNT-modified electrodes (—) were obviously larger than those at the bare GC electrodes (    ) in 0.1 M HAc– NaAc solution, which were attributed to the fact that MWNT could significantly increase the active area of electrode. In addition, a pair of redox waves with the cathodic and anodic peak potentials of 0.10 V and 0.14 V (vs SCE) (Fig. 4, inset) was observed, respectively. Similar to the case with the SWNT-modified GC electrode

213

(Luo et al., 2001), both the cathodic and anodic peak potentials relating to the redox of carboxylic groups negatively shifted with increasing pH, further indicating that carboxylic groups were present on the surface of the MWNT. The immersion of the MWNT-modified electrodes into the solution of target ssDNA results in disappearance of redox couple of carboxylic group (—), suggesting that the target ssDNA was successfully linked onto the MWNT-modified electrodes to form ssDNA/MWNT-modified electrodes. After the ssDNA/MWNT-modified electrodes were incubated into 0.30 M PBS buffer (pH 7.0) containing Cu-DNCs ssDNA probe at 37 1C for 60 min, the obtained electrode was cathodically preconcentrated at −0.4 V for 300 s in 0.10 M HAc–NaAc (pH 4.7), then followed by a CV scan from −0.4 to 0.4 V. As displayed in Fig. 4 (-  -), an obviously anodic stripping peak current at about+0.01 V was observed, which corresponds to the oxidation peak of Cu0–Cu2 + , implying the formation of the Cu-DNCs hybrids on the electrode surface through the hybridization reaction. 3.3. Optimization of voltammetric parameters To detect the Cu2+ by ASDPV in the final step of the assay, the Cu2+ must be released from the Cu-DNCs linked onto hybrids on the electrode, which could be achieved with the use of a 50% HNO3 solution. We have demonstrated that, at or below pH 2.0, the bands arising from complexation between G 4.5 dendrimer and Cu2+ at 556 nm disappeared and a broad, weak band corresponding to free Cu2+ appears at around 810 nm (Fig. 2), which could be explained that the interior tertiary amines are protonated below pH 2, and the dendrimer release Cu2+ at a low pH. In this study, Cu-DNCs linked to the hybrids on the MWNT/GCE surface were immersed in 200 mL 50% nitric acid solution for 5 min to ensure the complete release of Cu2+ from the interior of dendrimer. ASDPV has proved to be a very sensitive method for trace determination of metal ions. In ASDPV, the metal is cathodically electro-deposited onto the surface of an electrode during a preconcentration period, and then it is stripped from the electrode by anodic oxidation. The parameters that effect ASDPV determination of Cu2+ have been optimized. The electro-deposition potential was investigated by negatively varying the Ed from −0.3 V to −0.7 V. The anodic peak current reached a maximum at −0.4 V. Therefore, a deposition potential of −0.4 V was chosen for use in the further studies. The electro-deposition time has strong effect on the ASDPV signal of Cu2+. The peak current increased significantly in the interval from 50 to 300 s, then more slowly in the region up to 350 s. A 300 s electro-deposition time was used in further experiments. 3.4. Analytical properties

Fig. 4. CVs obtained at bare (    ), MWNT-modified (—, inset), ssDNA/MWNTmodified (—) and dsDNA/MWNT-modified (-  -) GC electrodes in 0.10 M HAc–NaAc (pH 4.7) buffer solution. Scan rate, 100 mV/s.

Fig. 5 displays the ASDPV plots obtained with the complementary target ssDNA/MWNT-modified electrodes in 0.10 M HAc–NaAc (pH 4.7) after the electrodes were immersed into the hybridization buffer containing various concentrations of Cu-DNCs DNA probe. The Cu stripping signal increases with increasing the concentration of the target DNA on the MWNT-modified electrodes (Fig. 5 inset). The average anodic currents of Cu was linear with the concentration of target DNA within a concentration range from 1 to 75 pM. The linear regression equation was I¼ 0.04462C +0.2050 (C was the concentration of target DNA, pM; I is the anodic currents of Cu, μA) with a correlation coefficient γ¼0.9998. The detection limit of the method was estimated to be 0.5 pM using 3s (where s is the standard deviation of the blank solution and n¼ 10). Previous labeled-based methods for electrochemical detection of DNA hybridization are summarized (Supplementary data, Table 1). It can be seen that the limit of detection achieved here is lower than the other methods of using Cu@Au

214

H. Gao et al. / Biosensors and Bioelectronics 48 (2013) 210–215

Fig. 5. ASDPV curves obtained at a bare GCE after 0.3 M PBS hybridization buffer containing Cu-DNCs DNA probe was incubated with MWNT-modified electrodes containing different concentration of complementary target for 60 min at 37 1C with a gentle stirring and released in 50% HNO3. Ed ¼−0.4 V (vs. SCE); tacc ¼ 300 s; DPV parameters: amplitude 50 mV, pulse period 0.2 s, pulse width 50 ms. Inset: The corresponding calibration curve. Error bars show the standard deviations of three measurements.

nanoparticles (Cai et al., 2003), Au nanoparticles (Wang et al., 2001), PAMAM (Zhu et al., 2009b) and AuNP-latex label (Kawde and Wang, 2004) for DNA detection. It is also lower than electrochemical methods using hematoxylin label (Nasirizadeh et al., 2011). The methods based on latex/gold nanoparticle-assisted signal amplification (Pinijsuwan et al., 2008) or based on signal dual-amplification with Au NPs and cadmium-loaded apoferritin NPs (Yu et al., 2010) reveal better sensitivities. However, in the former method, complete dissolution of latex/gold tag requires more severe conditions (1 M HBr/0.1 M Br2), and the electrode might be damaged in this medium. The need of HgCl2 and an accumulation at −1.4 V for electrochemical detection as well as the complexities inherent in their fabrication and use, in the later method, have tended to limit their applicability. We also investigated the responses of the Cu-DNCs electrochemical hybridization assay by incubating the hybridization buffer containing Cu-DNCs DNA probe with MWNT-modified electrodes containing the target DNA (complementary sequence and non-complementary sequence, 100 pM) and target-free MWNT/GC. As shown in Fig. 6, no visible Cu stripping signal was observed after the target-free MWNT-modified or noncomplementary target ssDNA/MWNT-modified electrodes were incubated in the hybridization buffer containing Cu-DNCs DNA probe, revealing that the stripping signal caused by the nonspecific adsorption of non-complementary DNA or target-free MWNTmodified electrodes was negligible. On contrast, a well-defined Cu stripping signal (∼+0.010 V vs. SCE) was observed after the complementary target ssDNA/MWNT-modified electrodes were incubated in the hybridization buffer containing Cu-DNCs DNA probe. These results demonstrate that the anodic stripping signals were induced by the specific hybridization reaction between the Cu-DNCs probe DNA in solution and the complementary target DNA confined onto electrode surface. The reproducibility of the prepared Cu-DNCs electrochemical hybridization assay was estimated by making repetitive hybridizations with the same concentration of the complementary DNA according to the procedures described above. The relative standard deviation (RSD) based on 7 measurements for Cu-DNCs DNA probe

Fig. 6. The stripping currents of released copper ions at a bare GCE after Cu-DNCs DNA probe incubating with MWNT-modified electrodes containing 75 pM of non-complementary DNA, complementary DNA or target-free (meaning: the MWNT/GCE not containing DNA was incubated in Cu-DNCs DNA probe solution for 60 min and then immersed in 50% HNO3) for 60 min at 37 1C with a gentle stirring. Error bars show the standard deviations of three measurements. Conditions are as same as Fig. 5.

hybridization to the complementary ssDNA with the same electrode was about 9.3%. Moreover, the Cu stripping signal almost remained 95% of the original value after the electrodes containing hybrids linked to Cu-DNCs were stored in the refrigerator for 5 days, demonstrating that the present method for DNA determination has a relatively good reproducibility and stability.

4. Conclusions By taking advantages of dendrimer-encapsulated copper as olignucleotides label and electrochemical stripping analysis for Cu determination, we have developed a new method for electrochemical detection of DNA hybridization. The Cu-DNCs-based DNA probe is sensitive, selective for target DNA with a low detection limit down to 0.5 pM. The good reproducibility and detection sensitivity may expand the potential of Cu-DNCs as entirely new and flexible electrochemical labels for biomolecules. Moreover, the use of Cu-DNCs as an electrochemical label for DNA hybridization detection may be generalized to prepare dendrimer-encapsulated Ag or semiconductor nanoparticles (CdS, PbS) as tags for the simultaneous detection of several DNA targets in one sample.

Acknowledgments This work was supported by the program of the Food Safety and Nutrition Innovation Team of Shanghai Normal University (DXL123).

Appendix A. Supporting information Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.bios.2013.04.021.

References Anne, A., Demaille, C., 2006. Journal of the American Chemical Society 128, 542–557.

H. Gao et al. / Biosensors and Bioelectronics 48 (2013) 210–215

Bakker, E., Qin, Y., 2006. Analytical Chemistry 78, 3965–3984. Balogh, L., Tomalia, D.A., 1998. Journal of the American Chemical Society 120, 7355–7356. Bonanni, A., Pumera, M., 2011. ACS Nano 5, 2356–2361. Boon, E.M., Ceres, D.M., Drummond, T.G., Hill, M.G., Barton, J.K., 2000. Nature Biotechnology 18, 1096–1100. Cai, H., Zhu, N., Jiang, Y., He, P., Fang, Y., 2003. Biosensors and Bioelectronics 18, 1311–1319. Crooks, R.M., Zhao, M., Sun, L., Chechik, V., Yeung, L.K., 2001. Accounts of Chemical Research 34, 181–190. Erdem, A., Kerman, K., Meric, B., Akarca, U.S., Ozsoz, M., 2000. Analytica Chimica Acta 422, 139–149. Forster, A., McInnes, J., Skingle, C., Symons, H., 1985. Nucleic Acids Research 13, 745–750. Golub, T.R., Slonim, D.K., Tamayo, P., Huard, C., Gaasenbeek, M., Mesirov, J.P., Coller, H., Loh, M.L., Downing, J.R., Caligiuri, M.A., Bloomfield, C.D., Lander, E.S., 1999. Science 286, 531–537. Hermanson, G., 1996. Bioconjugate Techniques. Academic Press, New York. Herrero, M.A., Guerra, J., Myers, V.S., Gomez, M.V., Crooks, R.M., Prato, M., 2010. ACS Nano 4, 905–912. Kawde, A.N., Wang, J., 2004. Electroanalysis 16, 101–107. Lai, R.Y., Lagally, E.T., Lee, S.H., Soh, H.T., Plaxco, K.W., Heeger, A.J., 2006. Proceedings of the National Academy of Sciences of the USA 103, 4017–4021. Liu, G.D., Lin, Y.H., 2007. Journal of the American Chemical Society 129 (34), 10394–10401. Liu, Y.H., Li, H.N., Chen, W., Liu, A.L., Lin, X.H., Chen, Y.Z., 2013. Analytical Chemistry 85, 273–277. Luo, H.X., Shi, Z.J., Li, N.Q., Gu, Z.N., Zhuang, Q.K., 2001. Analytical Chemistry 73, 915–920. Maity, P., Yamazoe, S., Tsukuda, T., 2013. ACS Catalysis 3, 182–185. Merkoci, A., 2010. Biosensors and Bioelectronics 26 (4), 1164–1177. Millan, K.M., Mikkelsen, S.R., 1993. Analytical Chemistry 65, 2317–2323. Myers, V.S., Frenkel, A.I., Crooks, R.M., 2012. Langmuir 28, 1596–1603. Nasirizadeh, N., Zare, H.R., Pournaghi-Azar, M.H., Hejazi, M.S., 2011. Biosensors and Bioelectronics 26, 2638–2644. Niu, S.Y., Zhang, S.S., Wang, L., Li, X.M., 2006. Journal of Electroanalytical Chemistry 597, 111–118. Odenthal, K.J., Gooding, J.J., 2007. Analyst 132, 603–610.

215

Perez-Lopez, B., Merkoci, A., 2011. Analytical and Bioanalytical Chemistry 399 (4), 1577–1590. Pinijsuwan, S., Rijiravanich, P., Somasundrum, M., Surareungchai, W., 2008. Analytical Chemistry 80, 6779–6784. Radhakrishnan, S., Sumathi, C., Dharuman, V., Wilson, J., 2013. Analytical Methods 5, 1010–1015. Rosi, N.L., Mirkin, C.A., 2005. Chemical Reviews 105 (4), 1547–1562. Saiki, R.K., Scharf, S., Faloona, F., Mullis, K., Horn, G.T., Erlich, H.A., Arnheim, N., 1985. Science 230, 1350–1354. Scott, R.W.J., Wilson, O.M., Crooks, R.M., 2005. Journal of Physical Chemistry B 109, 692–704. Shen, Y., Xu, Q., Gao, H., Zhu, N., 2009. Electrochemistry Communications 11, 1329–1332. Shi, X., Wang, S., Meshinchi, S., Van Antwerp, M.E., Bi, X., Lee, I., Baker Jr., J.R., 2007. Small 3, 1245–1252. Shiddiky, M.J.A., Torriero, A.A.J., Zeng, Z., Spiccia, L., Bond, A.M., 2010. Journal of the American Chemical Society 132, 10053–10063. Shuber, A.P., Michalowsky, L.A., Nass, G.S., Skoletsky, J., Hire, L.M., Kotsopoulos, S.K., Phipps, M.F., Barberio, D.M., Klinger, K.W., 1997. Human Molecular Genetics 6, 337–347. Stofik, M., Stryhal, Z., Maly, J., 2009. Biosensors and Bioelectronics 24, 1918–1923. Wang, J., Xu, D., Kawde, A.N., Polsky, R., 2001. Analytical Chemistry 73, 5576–5581. Won, B.Y., Shin, S., Cho, D., Park, H.G., 2013. Biosensors and Bioelectronics 42, 603–607. Xia, F., White, R.J., Zuo, X., Patterson, A., Xiao, Y., Kang, D., Gong, X., Plaxco, K.W., Heeger, A.J., 2010. Journal of the American Chemical Society 132, 14346–14348. Ye, H., Crooks, R.M., 2005. Journal of the American Chemical Society 127, 4930–4934. Yu, F., Li, G., Qu, B., Cao, W., 2010. Biosensors and Bioelectronics 26, 1114–1117. Zhang, Y.C., Kim, H.H., Heller, A., 2003. Analytical Chemistry 75, 3267–3269. Zhao, M., Sun, L., Crooks, R.M., 1998. Journal of the American Chemical Society 120, 4877–4878. Zheng, J., Zhang, C., Dickson, R.M., 2004. Physical Review Letters 93, 077402/ 1–077402/4. Zhu, N., Gao, H., Gu, Y., Xu, Q., He, P., Fang, Y., 2009a. Analyst 134, 860–866. Zhu, N., Gao, H., Xu, Q., Lin, Y., Su, L., Mao, L., 2010. Biosensors and Bioelectronics 25, 1498–1503. Zhu, N., Xu, Q., Li, S., Gao, H., 2009b. Electrochemistry Communications 11, 2308–2311.