- Email: [email protected]
DNA interaction
Biosensors and Bioelectronics 69 (2015) 174–178
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Highly sensitive colorimetric sensor for Hg2 þ detection based on cationic polymer/DNA interaction Yingyue Zhu a,1, Yilin Cai a,b,1, Yibo Zhu a, Lixue Zheng a, Jianying Ding a, Ying Quan a, Limei Wang a, Bin Qi a,n a b
School of Biotechnology and Food Engineering, Changshu Institute of Technology, Changshu, Jiangsu 215500, China School of Chemical Engineering & Technology, China University of Mining and Technology, Xuzhou, Jiangsu 221116, China
art ic l e i nf o
a b s t r a c t
Article history: Received 19 December 2014 Received in revised form 9 February 2015 Accepted 10 February 2015 Available online 19 February 2015
The detection of ultralow concentrations of mercury is a currently significant challenge. Here, a novel strategy is proposed: the colorimetric detection of Hg2 þ based on the aggregation of gold nanoparticles (AuNPs) driven by a cationic polymer. In this three-component system, DNA combines electrostatically with phthalic diglycol diacrylate (PDDA) in a solution of AuNPs. In the presence of Hg2 þ , thymine (T)-Hg2 þ -T induced hairpin turns are formed in the DNA strands, which then do not interact with PDDA, enabling the freed PDDA to subsequently facilitate aggregation of the AuNPs. Thus, according to the change in color from wine-red to blue-purple upon AuNPs aggregation, a colorimetric sensor is established to detect Hg2 þ . Under optimal conditions, the color change is clearly seen with the naked eye. A linear range of 0.25–500 nM was obtained by absorption spectroscopy with a detection limit of approximately 0.15 nM. Additionally, the proposed method shows high selectivity toward Hg2 þ in the presence of other heavy metal ions. Real sample analysis was evaluated with the use of lake water and the results suggest good potential for practical application. & 2015 Elsevier B.V. All rights reserved.
Keywords: AuNPs aggregation Colorimetric sensor Mercury ion T-Hg2 þ -T
1. Introduction Environmental pollution by heavy metal ions is widespread and the resulting adverse effects to biological systems and human health have generated extensive concern (Needleman, 2004). Mercuric ion (Hg2 þ ) is the best-known metal pollutant; it is the most toxic metal contaminant, even at low concentrations, that exists widely in our environment. Mercury and mercury compounds invade the human body through the respiratory and digestive tracts and the skin, and can potentially damage the central nervous, digestive, and endocrine systems (Baughman, 2006). Hence, effective analytical methods that can monitor and help control the harmful influences of even trace amounts of Hg2 þ in the environment are important. Although a number of research strategies have been developed for the accurate and sensitive detection of Hg2 þ , including inductively coupled plasma-mass spectrometry (ICP-MS), ion-selective electrode or polarography, and atomic absorption/emission spectroscopy (Guo et al., 2009; Wang et al., 2007), most current instrumental methods are inconvenient, requiring sophisticated instrumentation or sample n
Corresponding author. E-mail address: [email protected] (B. Qi). 1 These authors contributed to this paper equally. http://dx.doi.org/10.1016/j.bios.2015.02.018 0956-5663/& 2015 Elsevier B.V. All rights reserved.
preparation, time-consuming analysis, and technical expertize. Colorimetric (Li et al., 2009b; Wang et al., 2010b), electrochemical (Zhao and Zhou, 2012), and other methods (Li et al. 2011; Stobiecka et al., 2012) have also been established because of their simple operation, excellent sensitivity, and time-saving process features. Among the various colorimetric transducers for Hg2 þ in aqueous solution, gold nanoparticles (AuNPs)-based colorimetric assays are particularly convenient and attractive; AuNPs have proven to be excellent signal reporters as a result of color changes upon aggregation (He et al., 2011; Liu et al., 2008). Generally, AuNPs are of great interest because of their strong distance- and size-dependent optical properties, rapid colorimetric reactions for target detection, and changes in the surface plasmon resonance (SPR) absorption peaks and intensities (Liu et al., 2012; Xie et al., 2011). Hence, in a AuNPs-based colorimetric system, target recognition signals display distinct color variations which can be easily read by the naked eye or distinguished using UV–vis absorption spectrometry (Daniel and Astruc, 2004; Xu et al., 2012). In recent years, a great deal of AuNPs-based research utilizing the specific binding between thymine (T)-containing oligonucleotides and mercury ions has been performed for the detection of Hg2 þ (Gao et al., 2014; He et al., 2011; Wu et al., 2011). Hg2 þ can combine with two T residues in DNA to mediate a T–T mismatch (T-Hg2 þ -T), which has been proven by NMR spectroscopy, and transform its
Y. Zhu et al. / Biosensors and Bioelectronics 69 (2015) 174–178
configuration into a hairpin structure (Miyake et al., 2006; Tanaka et al., 2007; Torigoe et al., 2010). Dramatically, the T–T mismatch has been found to exhibit high specificity and stability, and is only induced by Hg2 þ rather than other ions; the properties of T-Hg2 þ -T have been used to stabilize duplex DNA and convert single-stranded DNAs into duplexes (Lou et al., 2012; Wang et al., 2010a; Zhao and Zhou, 2012). Moreover, this specific binding has also been applied in the folding of single-stranded DNA with abundant T bases (Li et al., 2011b; Stobiecka et al., 2012). Herein, a novel, rapid, and ultrasensitive colorimetric sensor is presented based on well-dispersed AuNPs, specific single-stranded T-rich DNA (ssDNA), and a good water-soluble cationic polymer, phthalic diglycol diacrylate (PDDA). According to previous reports, in comparison to salt, PDDA is highly efficient in inducing the aggregation of AuNPs (Song et al., 2011; Zheng et al., 2011), and, in particular, can interact with ssDNA via electrostatic attractions (Ofir et al., 2008; Peng et al., 2006). In this paper, we demonstrate the first use of PDDA instead of salt to develop a colorimetric sensor for the ultrasensitive and selective detection of Hg2 þ . The general principles used in this method are based on the interaction of PDDA and ssDNA, the strong specific T-Hg2 þ -T binding, and the changes in Ultraviolet–visible (UV–vis) absorption peaks and intensities caused by AuNPs aggregation.
175
and diluted to afford sample solutions at different Hg2 þ concentrations. The detection procedure was conducted through the following steps. First, Tris–HCl buffer solution (470 μL, 0.02 mM, pH 7.41) was added into a 2 mL plastic vial containing ssDNA solution (15 μL, at final concentration 30 nM). The Hg2 þ solution (5 μL, at final concentration 0.05, 0.25, 0.5, 5, 50, 250, 500, 1000, 2000 and 5000 nM) was added and thoroughly mixed, and the mixture was incubated for 10 min at room temperature. Then, PDDA (10 μL, at final concentration 25 nM) was added into the vial and reacted for 10 min after mixing. Afterwards, the prepared AuNPs solution (1000 μL) was added into the vial and mixed well. After 3 min, 1500 μL of the mixed solution was transferred into a quartz microplate for the analysis by UV–vis spectroscopy at wavelengths from 400 to 800 nm. Changes in the absorption peaks and intensities were related to the AuNPs aggregation. To investigate the selectivity of the colorimetric sensor, the concentration of Hg2 þ and other metal ions including Zn2 þ , Mg2 þ , Cu2 þ , Mn2 þ , Cd2 þ , Ni2 þ , and Pb2 þ were added into the vial at 500 nM. Thereafter, all the assays were detected via the foregoing procedure. To determine the practical applicability of this Hg2 þ detecting sensor, the influence of the solvent environment on Hg2 þ detection was evaluated using lake water. After dilution with lake water, Hg2 þ standard solutions at various concentrations (0.5, 5, 50, and 250 nM) were detected by the same method.
2. Experimental section 2.1. Reagents and apparatus
3. Results and discussion
Chloroauric acid (HAuCl4) was acquired from Sigma-Aldrich (Shanghai, China). Sodium citrate and Trisbase were purchased from Shanghai Chemical Reagents Company. Poly (Diallyldimethylammonium chloride) (PDDA) was obtained from Aladdin Ltd. (Shanghai, China). Mercury, lead, nickel, and other metal salts were purchased from the National Standard Substances Center (Beijing, China). All chemicals were of analytical grade purity. The water (418 MΩ) used throughout the study was purified through a Millipore Milli-Q system. Specific ssDNA with the sequence 5′TTC TTT CTT CCC CTT GTT TGT T-3′, was synthesized by Sangon Biotechnology Co. Ltd. (Shahai, China). UV–vis absorption spectra measurements were made on an UV-2450 spectrophotometer (Shimadzu). All concentrations of added reagents in this article are mentioned in terms of final concentration.
3.1. Mechanism of the sensing system
2.2. Synthesis of AuNPs Colloidal AuNPs were prepared by reducing HAuCl4 with sodium citrate according to the method reported by Storhoff (Storhoff et al., 1998). All glassware was immersed in aqua regia before use. An aqueous solution of HAuCl4 (0.01 wt%, 200 mL) was added into a conical flask and heated to boiling with gentle agitation. Subsequently, freshly prepared sodium citrate solution (1 wt%, 4 mL) was added quickly, and heating was continued until a color change from pale yellow to a stable dark red was observed. Thereafter, the solution was heated for a further 10 min and then cooled to room temperature with gentle agitation. The prepared AuNPs solution was stored at 4 °C. Based on the UV–vis absorption spectrum (as presented in Fig. S1, Supplementary data), the diameter and concentration of the AuNPs were calculated to be 17 nm and 2.1 nM, respectively, according to Haiss’ law (Haiss et al., 2007).
The proposed strategy for the colorimetric detection of Hg2 þ is illustrated in Scheme 1. The ssDNA in the system can not only bind specifically with Hg2 þ via the T-Hg2 þ -T mismatch, but also hybridize with PDDA through strong electrostatic attractions. Importantly, the binding force in T-Hg2 þ -T is much stronger than the interchain binding power between ssDNA and PDDA. Hence, under the near-equilibrium conditions in the sensing system, i.e., with either no unbound PDDA or insufficient PDDA to induce the aggregation of the AuNPs, the absorbance of the system remains unchanged. However, in the presence of Hg2 þ , ssDNA specifically recognizes Hg2 þ and its random coil structure is converted to a hairpin structure, preventing its interaction with PDDA. As a consequence, the free PDDA in the solution acts to aggregate the AuNPs. Therefore, owing to the highly selective reaction between ssDNA and Hg2 þ , the absorption intensity is different depending on the concentration of Hg2 þ , and color changes from claret-red to blue-purple are observed. Consequently, a colorimetric sensor can be established for the quantifiable detection of Hg2 þ . Fig. S2 shows the absorption intensities of the sensing system in the absence (curve a) and presence of 500 nM Hg2 þ (curve b).
2.3. Colorimetric sensing of Hg2 þ A Hg2 þ standard solution (100 μg/mL) was prepared in water
Scheme 1. Description of the optical mercury sensing mechanism.
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Y. Zhu et al. / Biosensors and Bioelectronics 69 (2015) 174–178
Fig. 1. A: Ultraviolet absorptions (A680/A520) of AuNPs solution after addition of different concentrations of PDDA (from 0 to 40 nM in 5 nM steps). B: The A680/A520 values obtained for a fixed concentration of PDDA (25 nM) in the presence of ssDNA (from 0 to 40 nM in 5 nM steps).
3.2. Optimization of detection conditions Parameter ascertainment and optimization of the present method are the key factors to its performance in terms of effectiveness and sensitivity, which strongly depend on the PDDA and ssDNA concentrations. Therefore, PDDA at different concentrations (0-40 nM, in 5 nM increments) was mixed in a plastic vial with Tris–HCl buffer solution (500 μL), and the resulting solution was reacted with the AuNPs solution (1000 μL). Then, the UV–vis absorbance values were measured. As seen in Fig. 1A, as the amount of PDDA rises, the degree of AuNPs aggregation increases, as is reflected by the ratio of absorbance values at 680 and 520 nm (A680/A520). Furthermore, the A680/A520 values stay constant as the concentration reaches 25 nM, and the color changes completely to blue. Hence, subsequent assays were performed with 25 nM PDDA. To optimize the DNA content, a similar series of DNA dilutions (0-40 nM, in 5 nM increments) was prepared. The DNA solutions were mixed with PDDA (25 nM) and incubated 10 min at room temperature before treatment with the AuNPs solution (1000 μL). The resulting UV–vis absorbance and A680/A520 values are shown in Fig. 1B. The minimum A680/A520 value was obtained at an ssDNA concentration of 30 nM. Consequently, 30 nM ssDNA was applied as the optimal concentration in subsequent experiments.
Fig. 2. UV–vis absorption spectra of the proposed method in the presence of different amounts of Hg2 þ . The inset shows the corresponding photographic images.
3.3. Linearity and sensitivity of the colorimetric sensor After introducing the mechanistic rationale and ascertaining the optimum conditions, the essential parameters of Hg2 þ detection were determined. Different concentrations of Hg2 þ were added via the same procedure and UV–vis spectra were recorded (Fig. 2). As the concentration of Hg2 þ increases from 0 to 5 μM, the initial absorption peak (A520) decreases while a new absorption peak (A680) grows, with a gradual concomitant color change from claret-red to blue–purple, which is visible to the naked eye. As presented in Fig. 3, the A680/A520 absorption ratio is proportional to the logarithm of the concentration of Hg2 þ , with a wide linear range of 0.25–500 nM and a linear correlation coefficient of 0.9948. Furthermore, the naked eye can discern a concentration as low as 5 nM, and the detection limit can be as low as 0.15 nM (three times the signal-to-noise ratio). However, in the absence of Hg2 þ , the AuNPs are dispersed due to the interactions of ssDNA with PDDA. Correspondingly, the color of the sensing system presents an obvious change with increasing Hg2 þ concentration, implying that Hg2 þ binding to ssDNA by T-Hg2 þ -T leads to more free PDDA in the solution which then induces the
Fig. 3. Relationship between A680/A520 values and Hg2 þ concentration. The inset shows the linear calibration plot for Hg2 þ detection. The error bars illustrate the standard deviations of three independent measurements.
Y. Zhu et al. / Biosensors and Bioelectronics 69 (2015) 174–178
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spectrometry (ICP-OES). As shown in Table 1, the results obtained by the present sensing system are comparable to the absorbance values from the known amounts of Hg2 þ , and recoveries were in the range of 97.9–109.8%. Table S2 lists the concentration determination of Hg2 þ using ICP-OES. Based on the comparison, the results obtained by the developed method are in good agreement with those from ICP-OES. In conclusion, these data indicate that this sensor will be potentially applicable in aquatic environments without the need for sample pretreatment.
4. Conclusion
Fig. 4. Specificity of the colorimetric sensor towards Hg2 þ : the concentrations of Hg2 þ and the other interferents were 500 nM; error bars show the standard deviations of three independent experiments. The inset shows the corresponding photographic images.
Table 1 Sensing of Hg2 þ in lake water samples by the developed colorimetric method. Samples
Spiked (nM)
The present methoda mean 7 SD (nM)
Recovery (%)
1 2 3 4
0.5 5 50 250
0.497 0.041 5.217 0.45 54.9 7 3.85 244.55 7 11.7
98.0 104.0 109.8 97.9
a
A strategy was successfully applied for the ultrasensitive detection of Hg2 þ by taking advantage of the aggregation of AuNPs induced by PDDA, the specific binding of Hg2 þ with ssDNA, and the electrostatic interaction of PDDA and ssDNA. The proposed method could discriminatively detect Hg2 þ with a low detection limit of 0.15 nM by UV–vis spectroscopy. The Hg2 þ detection limit by the naked eye was as low as 5 nM, which could be used to rapidly monitor the presence of the heavy metal ion. Moreover, the whole sensing process can be implemented at room temperature without sophisticated operation, with an analysis time as short as 20 min. Based on these advantages, we can conclude that this new analytical method merits serious consideration because of its high sensitivity, excellent selectivity, and promising practical applicability over other current Hg2 þ detection techniques.
Acknowledgments
Mean of three determinations; SD: standard deviation
aggregation of the AuNPs. All these observations indicate that a highly sensitive sensing method was established with good linearity and a low limit of detection. As indicated in Table S1, we compared the characteristics of the developed colorimetric sensor with other reported AuNPs-based sensors (Ding et al., 2012; Gao et al., 2014; Gao et al., 2015; Li et al. 2009a; Wang et al., 2015; Xu et al., 2009). The detection sensitivity in this work is higher than in many previous reports, and furthermore, the time required for detection is shorter than or comparable to some previous reports. Therefore, the developed method is simple, fast, and highly sensitive. 3.4. Analytical specificity of the sensing system Specific recognition is an important criterion by which to evaluate the performance of the colorimetric sensor for Hg2 þ detection. To this end, a variety of competitive substances such as Zn2 þ , Mg2 þ , Cu2 þ , Mn2 þ , Cd2 þ , Ni2 þ , and Pb2 þ were investigated. As demonstrated in Fig. 4, the corresponding histograms (standard deviation from the mean, n ¼3) display significant differences in the absorbance intensities of solutions containing Hg2 þ at the same concentration as the other metal ions, meaning that the sensor exhibits promising selectivity toward Hg2 þ against these interferents. These results are consistent with the fact that ssDNA specifically responds to Hg2 þ to form a stabilized hairpin structure. 3.5. Reliability in practical applications To further evaluate the applicability of this system in practical sample analysis, we evaluated its efficacy using lake water spiked with Hg2 þ and made a comparison between the current sensing system and inductively coupled plasma-optical emission
This work is financially supported by NSF of Jiangsu Province (BK20130379, 13KJB550001, BK20140416), and the Suzhou Science and Technology Committee Program (SS201335, SYN201210)
Appendix A. Supplementary Information Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.bios.2015.02.018.
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Biosensors and Bioelectronics journal homepage: www.elsevier.com/locate/bios
Highly sensitive colorimetric sensor for Hg2 þ detection based on cationic polymer/DNA interaction Yingyue Zhu a,1, Yilin Cai a,b,1, Yibo Zhu a, Lixue Zheng a, Jianying Ding a, Ying Quan a, Limei Wang a, Bin Qi a,n a b
School of Biotechnology and Food Engineering, Changshu Institute of Technology, Changshu, Jiangsu 215500, China School of Chemical Engineering & Technology, China University of Mining and Technology, Xuzhou, Jiangsu 221116, China
art ic l e i nf o
a b s t r a c t
Article history: Received 19 December 2014 Received in revised form 9 February 2015 Accepted 10 February 2015 Available online 19 February 2015
The detection of ultralow concentrations of mercury is a currently significant challenge. Here, a novel strategy is proposed: the colorimetric detection of Hg2 þ based on the aggregation of gold nanoparticles (AuNPs) driven by a cationic polymer. In this three-component system, DNA combines electrostatically with phthalic diglycol diacrylate (PDDA) in a solution of AuNPs. In the presence of Hg2 þ , thymine (T)-Hg2 þ -T induced hairpin turns are formed in the DNA strands, which then do not interact with PDDA, enabling the freed PDDA to subsequently facilitate aggregation of the AuNPs. Thus, according to the change in color from wine-red to blue-purple upon AuNPs aggregation, a colorimetric sensor is established to detect Hg2 þ . Under optimal conditions, the color change is clearly seen with the naked eye. A linear range of 0.25–500 nM was obtained by absorption spectroscopy with a detection limit of approximately 0.15 nM. Additionally, the proposed method shows high selectivity toward Hg2 þ in the presence of other heavy metal ions. Real sample analysis was evaluated with the use of lake water and the results suggest good potential for practical application. & 2015 Elsevier B.V. All rights reserved.
Keywords: AuNPs aggregation Colorimetric sensor Mercury ion T-Hg2 þ -T
1. Introduction Environmental pollution by heavy metal ions is widespread and the resulting adverse effects to biological systems and human health have generated extensive concern (Needleman, 2004). Mercuric ion (Hg2 þ ) is the best-known metal pollutant; it is the most toxic metal contaminant, even at low concentrations, that exists widely in our environment. Mercury and mercury compounds invade the human body through the respiratory and digestive tracts and the skin, and can potentially damage the central nervous, digestive, and endocrine systems (Baughman, 2006). Hence, effective analytical methods that can monitor and help control the harmful influences of even trace amounts of Hg2 þ in the environment are important. Although a number of research strategies have been developed for the accurate and sensitive detection of Hg2 þ , including inductively coupled plasma-mass spectrometry (ICP-MS), ion-selective electrode or polarography, and atomic absorption/emission spectroscopy (Guo et al., 2009; Wang et al., 2007), most current instrumental methods are inconvenient, requiring sophisticated instrumentation or sample n
Corresponding author. E-mail address: [email protected] (B. Qi). 1 These authors contributed to this paper equally. http://dx.doi.org/10.1016/j.bios.2015.02.018 0956-5663/& 2015 Elsevier B.V. All rights reserved.
preparation, time-consuming analysis, and technical expertize. Colorimetric (Li et al., 2009b; Wang et al., 2010b), electrochemical (Zhao and Zhou, 2012), and other methods (Li et al. 2011; Stobiecka et al., 2012) have also been established because of their simple operation, excellent sensitivity, and time-saving process features. Among the various colorimetric transducers for Hg2 þ in aqueous solution, gold nanoparticles (AuNPs)-based colorimetric assays are particularly convenient and attractive; AuNPs have proven to be excellent signal reporters as a result of color changes upon aggregation (He et al., 2011; Liu et al., 2008). Generally, AuNPs are of great interest because of their strong distance- and size-dependent optical properties, rapid colorimetric reactions for target detection, and changes in the surface plasmon resonance (SPR) absorption peaks and intensities (Liu et al., 2012; Xie et al., 2011). Hence, in a AuNPs-based colorimetric system, target recognition signals display distinct color variations which can be easily read by the naked eye or distinguished using UV–vis absorption spectrometry (Daniel and Astruc, 2004; Xu et al., 2012). In recent years, a great deal of AuNPs-based research utilizing the specific binding between thymine (T)-containing oligonucleotides and mercury ions has been performed for the detection of Hg2 þ (Gao et al., 2014; He et al., 2011; Wu et al., 2011). Hg2 þ can combine with two T residues in DNA to mediate a T–T mismatch (T-Hg2 þ -T), which has been proven by NMR spectroscopy, and transform its
Y. Zhu et al. / Biosensors and Bioelectronics 69 (2015) 174–178
configuration into a hairpin structure (Miyake et al., 2006; Tanaka et al., 2007; Torigoe et al., 2010). Dramatically, the T–T mismatch has been found to exhibit high specificity and stability, and is only induced by Hg2 þ rather than other ions; the properties of T-Hg2 þ -T have been used to stabilize duplex DNA and convert single-stranded DNAs into duplexes (Lou et al., 2012; Wang et al., 2010a; Zhao and Zhou, 2012). Moreover, this specific binding has also been applied in the folding of single-stranded DNA with abundant T bases (Li et al., 2011b; Stobiecka et al., 2012). Herein, a novel, rapid, and ultrasensitive colorimetric sensor is presented based on well-dispersed AuNPs, specific single-stranded T-rich DNA (ssDNA), and a good water-soluble cationic polymer, phthalic diglycol diacrylate (PDDA). According to previous reports, in comparison to salt, PDDA is highly efficient in inducing the aggregation of AuNPs (Song et al., 2011; Zheng et al., 2011), and, in particular, can interact with ssDNA via electrostatic attractions (Ofir et al., 2008; Peng et al., 2006). In this paper, we demonstrate the first use of PDDA instead of salt to develop a colorimetric sensor for the ultrasensitive and selective detection of Hg2 þ . The general principles used in this method are based on the interaction of PDDA and ssDNA, the strong specific T-Hg2 þ -T binding, and the changes in Ultraviolet–visible (UV–vis) absorption peaks and intensities caused by AuNPs aggregation.
175
and diluted to afford sample solutions at different Hg2 þ concentrations. The detection procedure was conducted through the following steps. First, Tris–HCl buffer solution (470 μL, 0.02 mM, pH 7.41) was added into a 2 mL plastic vial containing ssDNA solution (15 μL, at final concentration 30 nM). The Hg2 þ solution (5 μL, at final concentration 0.05, 0.25, 0.5, 5, 50, 250, 500, 1000, 2000 and 5000 nM) was added and thoroughly mixed, and the mixture was incubated for 10 min at room temperature. Then, PDDA (10 μL, at final concentration 25 nM) was added into the vial and reacted for 10 min after mixing. Afterwards, the prepared AuNPs solution (1000 μL) was added into the vial and mixed well. After 3 min, 1500 μL of the mixed solution was transferred into a quartz microplate for the analysis by UV–vis spectroscopy at wavelengths from 400 to 800 nm. Changes in the absorption peaks and intensities were related to the AuNPs aggregation. To investigate the selectivity of the colorimetric sensor, the concentration of Hg2 þ and other metal ions including Zn2 þ , Mg2 þ , Cu2 þ , Mn2 þ , Cd2 þ , Ni2 þ , and Pb2 þ were added into the vial at 500 nM. Thereafter, all the assays were detected via the foregoing procedure. To determine the practical applicability of this Hg2 þ detecting sensor, the influence of the solvent environment on Hg2 þ detection was evaluated using lake water. After dilution with lake water, Hg2 þ standard solutions at various concentrations (0.5, 5, 50, and 250 nM) were detected by the same method.
2. Experimental section 2.1. Reagents and apparatus
3. Results and discussion
Chloroauric acid (HAuCl4) was acquired from Sigma-Aldrich (Shanghai, China). Sodium citrate and Trisbase were purchased from Shanghai Chemical Reagents Company. Poly (Diallyldimethylammonium chloride) (PDDA) was obtained from Aladdin Ltd. (Shanghai, China). Mercury, lead, nickel, and other metal salts were purchased from the National Standard Substances Center (Beijing, China). All chemicals were of analytical grade purity. The water (418 MΩ) used throughout the study was purified through a Millipore Milli-Q system. Specific ssDNA with the sequence 5′TTC TTT CTT CCC CTT GTT TGT T-3′, was synthesized by Sangon Biotechnology Co. Ltd. (Shahai, China). UV–vis absorption spectra measurements were made on an UV-2450 spectrophotometer (Shimadzu). All concentrations of added reagents in this article are mentioned in terms of final concentration.
3.1. Mechanism of the sensing system
2.2. Synthesis of AuNPs Colloidal AuNPs were prepared by reducing HAuCl4 with sodium citrate according to the method reported by Storhoff (Storhoff et al., 1998). All glassware was immersed in aqua regia before use. An aqueous solution of HAuCl4 (0.01 wt%, 200 mL) was added into a conical flask and heated to boiling with gentle agitation. Subsequently, freshly prepared sodium citrate solution (1 wt%, 4 mL) was added quickly, and heating was continued until a color change from pale yellow to a stable dark red was observed. Thereafter, the solution was heated for a further 10 min and then cooled to room temperature with gentle agitation. The prepared AuNPs solution was stored at 4 °C. Based on the UV–vis absorption spectrum (as presented in Fig. S1, Supplementary data), the diameter and concentration of the AuNPs were calculated to be 17 nm and 2.1 nM, respectively, according to Haiss’ law (Haiss et al., 2007).
The proposed strategy for the colorimetric detection of Hg2 þ is illustrated in Scheme 1. The ssDNA in the system can not only bind specifically with Hg2 þ via the T-Hg2 þ -T mismatch, but also hybridize with PDDA through strong electrostatic attractions. Importantly, the binding force in T-Hg2 þ -T is much stronger than the interchain binding power between ssDNA and PDDA. Hence, under the near-equilibrium conditions in the sensing system, i.e., with either no unbound PDDA or insufficient PDDA to induce the aggregation of the AuNPs, the absorbance of the system remains unchanged. However, in the presence of Hg2 þ , ssDNA specifically recognizes Hg2 þ and its random coil structure is converted to a hairpin structure, preventing its interaction with PDDA. As a consequence, the free PDDA in the solution acts to aggregate the AuNPs. Therefore, owing to the highly selective reaction between ssDNA and Hg2 þ , the absorption intensity is different depending on the concentration of Hg2 þ , and color changes from claret-red to blue-purple are observed. Consequently, a colorimetric sensor can be established for the quantifiable detection of Hg2 þ . Fig. S2 shows the absorption intensities of the sensing system in the absence (curve a) and presence of 500 nM Hg2 þ (curve b).
2.3. Colorimetric sensing of Hg2 þ A Hg2 þ standard solution (100 μg/mL) was prepared in water
Scheme 1. Description of the optical mercury sensing mechanism.
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Fig. 1. A: Ultraviolet absorptions (A680/A520) of AuNPs solution after addition of different concentrations of PDDA (from 0 to 40 nM in 5 nM steps). B: The A680/A520 values obtained for a fixed concentration of PDDA (25 nM) in the presence of ssDNA (from 0 to 40 nM in 5 nM steps).
3.2. Optimization of detection conditions Parameter ascertainment and optimization of the present method are the key factors to its performance in terms of effectiveness and sensitivity, which strongly depend on the PDDA and ssDNA concentrations. Therefore, PDDA at different concentrations (0-40 nM, in 5 nM increments) was mixed in a plastic vial with Tris–HCl buffer solution (500 μL), and the resulting solution was reacted with the AuNPs solution (1000 μL). Then, the UV–vis absorbance values were measured. As seen in Fig. 1A, as the amount of PDDA rises, the degree of AuNPs aggregation increases, as is reflected by the ratio of absorbance values at 680 and 520 nm (A680/A520). Furthermore, the A680/A520 values stay constant as the concentration reaches 25 nM, and the color changes completely to blue. Hence, subsequent assays were performed with 25 nM PDDA. To optimize the DNA content, a similar series of DNA dilutions (0-40 nM, in 5 nM increments) was prepared. The DNA solutions were mixed with PDDA (25 nM) and incubated 10 min at room temperature before treatment with the AuNPs solution (1000 μL). The resulting UV–vis absorbance and A680/A520 values are shown in Fig. 1B. The minimum A680/A520 value was obtained at an ssDNA concentration of 30 nM. Consequently, 30 nM ssDNA was applied as the optimal concentration in subsequent experiments.
Fig. 2. UV–vis absorption spectra of the proposed method in the presence of different amounts of Hg2 þ . The inset shows the corresponding photographic images.
3.3. Linearity and sensitivity of the colorimetric sensor After introducing the mechanistic rationale and ascertaining the optimum conditions, the essential parameters of Hg2 þ detection were determined. Different concentrations of Hg2 þ were added via the same procedure and UV–vis spectra were recorded (Fig. 2). As the concentration of Hg2 þ increases from 0 to 5 μM, the initial absorption peak (A520) decreases while a new absorption peak (A680) grows, with a gradual concomitant color change from claret-red to blue–purple, which is visible to the naked eye. As presented in Fig. 3, the A680/A520 absorption ratio is proportional to the logarithm of the concentration of Hg2 þ , with a wide linear range of 0.25–500 nM and a linear correlation coefficient of 0.9948. Furthermore, the naked eye can discern a concentration as low as 5 nM, and the detection limit can be as low as 0.15 nM (three times the signal-to-noise ratio). However, in the absence of Hg2 þ , the AuNPs are dispersed due to the interactions of ssDNA with PDDA. Correspondingly, the color of the sensing system presents an obvious change with increasing Hg2 þ concentration, implying that Hg2 þ binding to ssDNA by T-Hg2 þ -T leads to more free PDDA in the solution which then induces the
Fig. 3. Relationship between A680/A520 values and Hg2 þ concentration. The inset shows the linear calibration plot for Hg2 þ detection. The error bars illustrate the standard deviations of three independent measurements.
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spectrometry (ICP-OES). As shown in Table 1, the results obtained by the present sensing system are comparable to the absorbance values from the known amounts of Hg2 þ , and recoveries were in the range of 97.9–109.8%. Table S2 lists the concentration determination of Hg2 þ using ICP-OES. Based on the comparison, the results obtained by the developed method are in good agreement with those from ICP-OES. In conclusion, these data indicate that this sensor will be potentially applicable in aquatic environments without the need for sample pretreatment.
4. Conclusion
Fig. 4. Specificity of the colorimetric sensor towards Hg2 þ : the concentrations of Hg2 þ and the other interferents were 500 nM; error bars show the standard deviations of three independent experiments. The inset shows the corresponding photographic images.
Table 1 Sensing of Hg2 þ in lake water samples by the developed colorimetric method. Samples
Spiked (nM)
The present methoda mean 7 SD (nM)
Recovery (%)
1 2 3 4
0.5 5 50 250
0.497 0.041 5.217 0.45 54.9 7 3.85 244.55 7 11.7
98.0 104.0 109.8 97.9
a
A strategy was successfully applied for the ultrasensitive detection of Hg2 þ by taking advantage of the aggregation of AuNPs induced by PDDA, the specific binding of Hg2 þ with ssDNA, and the electrostatic interaction of PDDA and ssDNA. The proposed method could discriminatively detect Hg2 þ with a low detection limit of 0.15 nM by UV–vis spectroscopy. The Hg2 þ detection limit by the naked eye was as low as 5 nM, which could be used to rapidly monitor the presence of the heavy metal ion. Moreover, the whole sensing process can be implemented at room temperature without sophisticated operation, with an analysis time as short as 20 min. Based on these advantages, we can conclude that this new analytical method merits serious consideration because of its high sensitivity, excellent selectivity, and promising practical applicability over other current Hg2 þ detection techniques.
Acknowledgments
Mean of three determinations; SD: standard deviation
aggregation of the AuNPs. All these observations indicate that a highly sensitive sensing method was established with good linearity and a low limit of detection. As indicated in Table S1, we compared the characteristics of the developed colorimetric sensor with other reported AuNPs-based sensors (Ding et al., 2012; Gao et al., 2014; Gao et al., 2015; Li et al. 2009a; Wang et al., 2015; Xu et al., 2009). The detection sensitivity in this work is higher than in many previous reports, and furthermore, the time required for detection is shorter than or comparable to some previous reports. Therefore, the developed method is simple, fast, and highly sensitive. 3.4. Analytical specificity of the sensing system Specific recognition is an important criterion by which to evaluate the performance of the colorimetric sensor for Hg2 þ detection. To this end, a variety of competitive substances such as Zn2 þ , Mg2 þ , Cu2 þ , Mn2 þ , Cd2 þ , Ni2 þ , and Pb2 þ were investigated. As demonstrated in Fig. 4, the corresponding histograms (standard deviation from the mean, n ¼3) display significant differences in the absorbance intensities of solutions containing Hg2 þ at the same concentration as the other metal ions, meaning that the sensor exhibits promising selectivity toward Hg2 þ against these interferents. These results are consistent with the fact that ssDNA specifically responds to Hg2 þ to form a stabilized hairpin structure. 3.5. Reliability in practical applications To further evaluate the applicability of this system in practical sample analysis, we evaluated its efficacy using lake water spiked with Hg2 þ and made a comparison between the current sensing system and inductively coupled plasma-optical emission
This work is financially supported by NSF of Jiangsu Province (BK20130379, 13KJB550001, BK20140416), and the Suzhou Science and Technology Committee Program (SS201335, SYN201210)
Appendix A. Supplementary Information Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.bios.2015.02.018.
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