Author’s Accepted Manuscript DNA-engineered chiroplasmonic heteropyramids for ultrasensitive detection of Mercury ion Wenjing Yan, Yongli Wang, Hong Zhuang, Jianhao Zhang www.elsevier.com/locate/bios
PII: DOI: Reference:
S0956-5663(15)00029-9 http://dx.doi.org/10.1016/j.bios.2015.01.028 BIOS7403
To appear in: Biosensors and Bioelectronic Received date: 21 November 2014 Revised date: 3 January 2015 Accepted date: 12 January 2015 Cite this article as: Wenjing Yan, Yongli Wang, Hong Zhuang and Jianhao Zhang, DNA-engineered chiroplasmonic heteropyramids for ultrasensitive detection of Mercury ion, Biosensors and Bioelectronic, http://dx.doi.org/10.1016/j.bios.2015.01.028 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
DNA-engineered chiroplasmonic heteropyramids for ultrasensitive detection of mercury ion Wenjing Yan a, Yongli Wang a, Hong Zhuang b, Jianhao Zhang a, a
b
National Center of Meat Quality & Safety Control, Nanjing Agricultural University, Nanjing, 210095, China
Quality and Safety Assessment Research Unit, Agricultural Research Service, USDA, Athens, GA 30605, USA
ABSTRACT In this study, plasmonic heteropyramids (HPs) made from two different sized gold nanoparticles (Au NPs) and five ssDNA sequences and their application for ultrasensitive detection of mercury ion (Hg2+) were demonstrated. Four ssDNA sequences were used as building blocks to form a pyramidal DNA frame, which contains a T-rich probe DNA at one vertex and three sulfhydryl groups modified with 10 nm Au NPs at the other three vertices. Another T-rich DNA sequence was modified and attached to a 25nm Au NP. In the presence of Hg2+ ions, 25 nm Au NPs hybridized with pyramidal DNA frame to build the plasmonic HPs based on T–Hg2+–T interaction, which exhibits unprecedented circular dichroism (CD) signal in the visible region. Based on this mechanism, a simple, high sensitive and selective chiroplasmonic HPs-based probe was constructed and demonstrated for Hg2+ ions detection. Under optimized conditions, Hg2+ ions could be selectively detected in a concentration range from 1 to 500 pg mL-1 with a limit of detection as 0.2 pg mL-1, which is much lower than the strictest Hg2+ safety requirement of 1 ng mL-1 in water.
Keywords: Gold nanoparticles; Heteropyramids; Circular dichroism; Mercury ion;
Corresponding authors. E-mail addresses: [email protected] (J. Zhang). 1
1. Introduction Hg2+ ions are highly toxic heavy metal ions produced from the industrial wastes and natural activities. The ions can be transformed into methyl mercuric compounds, concentrate through biological cycles and cause a series of diseases, such as pneumonia, enteritis, and bronchitis in human beings (Zhou et al. 2010). Mercury pollution is widely found in tap water and food and has been one of food safety concerns to consumers in the entire world. Obviously, developing an effective method for detecting Hg2+ ions in water and food is urgently needed. Among various Hg2+ ions detection methods, metal nanomaterial-enabled approaches offer an excellent opportunity for low-concentration, highly selective, and rapid detection of Hg2+ ions due to their unique size effect and tunable optical properties (Du et al. 2013; Guo et al. 2013). The predominant approaches for Hg2+ ions determination using metal nanomaterial as the signal conductor are surface enhanced Raman scattering (SERS) (Chen et al. 2014; Ma et al. 2013b; Ren et al. 2012), colorimetric (Chen et al. 2014; Wu et al. 2012; Xu et al. 2014), and fluorescence (Cui et al. 2015; Hao et al. 2012; Zhang et al. 2011). Considering the ultrahigh extinction coefficient (108-109) of the surface plasmon resonance (SPR) absorption of metal nanoparticles (NPs), these assays may achieve high sensitivity in the detection of Hg2+ ions (Guo et al. 2013; Ye and Yin 2008). However, they still have many constraints, such as high cost, easy contamination, complexity and non-repeatability (Kim et al. 2012; Zhang et al. 2013). More importantly, most of these assays rely on an aggregate of NPs or nanostars (NSs) to enhance the responding signal, increasing the sensitivity in the detection of Hg2+ ions (Kanayama et al. 2011). However, NPs are fragile to the complex environment and easy to form an aggregate or random homo-nanoassemblies (homodimers, trimers or chains), which is likely to cause false-positive 2
results. Thus, a simple, reliable and highly sensitive plasmonic method is still needed for the detection of Hg2+ ions (Aragay et al. 2011). Chiral nanomaterials represent one of the most rapidly developing research fields. Chiral nanomaterials could affect the rate of absorbance of the left- and right-circularly polarized light (CPR) and produce a significant signal in the visible region of the CD spectrum (Han et al. 2014; Mark et al. 2013; Ye and Yin 2008). DNA-engineered chiral nanostructures have fascinated the chemistry and engineering world due to their myriad geometries and distinctive optical properties (Lan et al. 2013; Li et al. 2012; Wu et al. 2013; Zhao et al. 2014). By designing the sequences of DNA molecules, single NPs can be assembled into nanostructures with asymmetrical geometry, represented by helix (Chen et al. 2008; Kuzyk et al. 2012; Song et al. 2013), pyramids (Mastroianni et al. 2009; Yan et al. 2012), and tetrahedron (Fan and Govorov 2010), in which pyramidal nanostructures represent a unique subset of DNA-engineered chiral nanomaterials. They exhibit large chiroptical responses of pure NPs at the corresponding plasmonic wavelength. The tunable light-matter interaction optical response of the complex is critically dependent on the interparticle spacing, shapes, and composition (Mastroianni et al. 2009), which is completely different from single chiral NPs (Gautier and Burgi 2009). Furthermore, diverse elements and engineered three-dimensional space structure endow NPs pyramids with unique optical properties and wide application potentials. In our previous work, metal NPs and/or quantum dots (QDs) modified with four ssDNA were used to construct a series of geometry controlled chiral NP pyramids in high yields. It has been demonstrated that the geometry parameters of nanostructures play a significant role in the origin of chirality of NPs pyramids (Yan et al. 2012). Specifically, once achiral NPs are assembled into 3
three-dimensional nanostructures in an asymmetric manner, the complex can display adjustable and multiple chiroptical activities in the 300-550 nm range due to the chiral arrangement of NPs in space predetermined by predesigned DNA scaffolds and asymmetric shapes of single NP (Fan et al. 2013; Ma et al. 2013a). This method provides a new approach for the formation of chiral materials and endows NPs pyramids with the ability to be used as a chiral sensor for target detection (Yan et al. 2014). Here, chiroplasmonic HPs were assembled using DNA scaffold modified with two different sized Au NPs as building blocks and the application in Hg2+ ions detection were reported. In the presence of Hg2+ ions, the T-rich region in DNA sequence selectively combines with Hg2+ ions to form a stable covalent T-Hg2+-T structure, resulting in the assembly of heterogeneous nanoassemblies with significant CD signal in the visible range. The assembly of HPs and the CD signal were dependent on the concentration of Hg2+ ions. Based on this principle, the approach was successfully used for the detection of Hg2+ ions in solution and in tap water samples with high sensitivity and prominent selectivity.
2. Materials and methods 2.1 Materials and reagents Metal ion standards used in this study (Hg2+, Zn2+, Mg2+, Fe3+, Cd2+, Pb2+, Mn2+, Fe2+, Ca2+, Cu2+) were purchased from the National Institute of Metrology P.R.C. (Beijing, China).Unless stated otherwise, all chemicals used were purchased from Sigma-Aldrich. Deionized water from a Milli-Q device (18.2 MΩ, Millipore, Molsheim, France) was used throughout this work. All glassware was cleaned with freshly prepared aqua regia and rinsed thoroughly by deionized water prior to use. Thiolated DNA oligonucleotides, which were purified by polyacrylamide gel 4
electrophoresis (PAGE), were purchased from Shanghai Sangon Biological Engineering Technology & Services Co. Ltd. (Shanghai, P.R. China) and suspended in DI water to a final concentration of 100μM. The DNA sequences are as follows: H1:5’-SH-AAA GCC TGG AGA TAC ATG CAC ATT ACG GCC CCC CCT ATT AGA AGG TCT CAG GTG CGC GCC CCG GTA AGT AGA CGG GAC CAG TTC GCC-3’ H2:5’-SH-AAA CGC GCA CCT GAG ACC TTC TAA TAG GGC CCG CGA CAG TCG TTC AAC TAG AAT GCC CCC CGG GCT GTT CCG GGT GTG GCT CGT CGG-3’ H3: 5’-SH-AAA GGC CGA GGA CTC CTG CTC CGC TGC GGC CCG GCG AAC TGG TCC CGT CTA CTT ACC GCC CCC GAC GAG CCA CAC CCG GAA CAG CCC-3’ H4: 5’-ACC TTT TTT TCA GTG AAA GCC GTA ATG TGC ATG TAT CTC CAG GCC CCC CGC AGC GGA GCA GGA GTC CTC GGC CCC CGG GCA TTC TAG TTG AAC GAC TGT CGC-3’ H5: 5’- SH-CAC TGT TTT TTT GGT-3’ 2.2 Synthesis of Au NPs Au NPs with a diameter of 10 nm (Au1) were prepared as follows: 80mL, 0.0125% HAuCl4 was prepared as solution A. Then, sodium citrate (1%, 4 mL), tannic acid (1%, 0.1 mL), K2CO3 (25 mM, 0.1 mL), and 15.8 mL of deionized water were mixed together, named as solution B. Then, A and B were heated to 60 °C for 30 min before being mixed under high-speed stirring. Au NPs with a diameter of 25 nm (Au2) were synthesized by reduction of HAuCl4 with citrate. Briefly, aqueous trisodium citrate solution (1.6 mL, 1%) was quickly added to a boiling aqueous solution of HAuCl4 (100 mL, 0.25 mM) under vigorous stirring. After the color of the solution changed from blue to wine red (about 2 min), the heat source was removed and the solution was cooled to room temperature.
5
2.3 Preparation of ssDNA-modified Au NPs Before use, Au NPs (Au1 and Au2) were stabilized with bis (p-sulfonatophenyl) phenylphosphine dihydrate, dipotassium salt (BPS). Aqueous solutions of Au NPs (Au1 and Au2) were concentrated 10-fold. Then 10 mL of Au NPs was stirred with an excess of BPS (20 mg/mL) at room temperature for more than 10 h. The solutions were then centrifuged at different speeds (13,000 r/min for Au1 or 7,000 r/min for Au2) for 10 min and resuspended in 0.5×TBE buffer to obtain a final concentration of 20 nM. Four different Au-DNA conjugates were: Au1-H1, Au1-H2, Au1-H3, and Au2-H5. One μL of 10μM DNA was added to 100 μL of 20nM Au NPs with a nanoparticle/DNA molar ratio of 1/5. The DNA-Au NPs mixture was incubated in 0.5×TBE buffer with 50 mM NaCl for 2h, and then centrifuged at 13,000 (Au1) or 7,000 (Au2) r/min for 10 min. The supernatant was removed and the pellet was resuspended in 1×TBE buffer to obtain a final concentration of 20 nM. 2.4 Preparation of chiral plasmonic HPs Five different solutions including Au1-H1, Au1-H2, Au1-H3, H4, Au2-H5 with the same volume (50 µL) were added to 1×TBE buffer with 100mM NaCl. The hybridization mixture was heated at 90°C for 5 min and then slowly cooled to room temperature. Hg2+ was added into the hybrid mixture and incubated for 50 min at room temperature with constant shaking. 2.5 Fabrication of chiral sensor for Hg2+detection The mixture of Au1-trimers and Au2-DNA conjugate prepared in 2.4 was transferred to seven separated tubes. For each tube, Hg2+ standard with different concentrations (0, 1, 5, 10, 50, 100, 500 pg mL-1) was added. The mixture were incubated for 50 min at room temperature with constant 6
shaking. The samples were characterized by transmission electron microscopy (TEM), circular dichroism (CD) and ultraviolet–visible (UV-vis) spectroscopy. 2.6 Specificity testing The specificity of the HP probe was investigated by mixing ssDNA-engineered nanostructures with ten metal ions, Hg2+, Pb2+, Mg2+，Mn2+, Zn2+，Fe3+, Cd2+, Fe2+, Ca2+, Cu2+,respectively. The final concentration of the metal ions was 1 ng mL-1 and CD spectrum of the samples was collected. 2.7 Analysis of water samples Spiked tap water samples were used for recovery analysis. Three concentrations of Hg2+ ions (5, 10 and 50 pg mL-1) were spiked in tap water. All samples were analyzed using CD spectroscopy and the same sample was repeated at three times for calculation standard deviation (s.d.) value.
2.8 Characterization The CD spectroscopy was performed on Bio-Logic MOS-450/AF circular dichroism. The UV/vis spectra were acquired on a UNICO 2100 PC UV/vis spectrophotometer and processed with the Origin Lab software. TEM images were collected on a JEOL JEM-2100 transmission electron microscope with an acceleration voltage of 200 kV. The sample was prepared by dropping an aqueous pyramid solution onto a copper grid coated with the carbon film, and then was dried in air.
3. Results and discussion 3.1 Sensing strategy Fig. 1 illustrates a chiroptical sensor that was fabricated in this study for the detection of Hg2+ ions. As shown in Fig.1A, five ssDNA sequences (H1, H2, H3, H4 and H5, see 2.1 Materials and 7
reagents) were used to assembly of heterogeneous nanostructures. Four ssDNA (H1, H2, H3 and H4) can complementarily hybridize with each other to form a pyramidal DNA frame. Each ssDNA can be segmented into three parts; each part is complementary to a third of each of the other strands. So, each strand of DNA consists of one face of the pyramid. Three ssDNA (H1, H2 and H3) modified with sulfhydryl group at 5’-end can connect with three Au1 NPs (10 nm) to form a NP trimer, one ssDNA (H4) contains an extended T-rich region at 5’-end to form a recognition site at one apex. The fifth DNA (H5) attached on the surface of Au2 NP (25 nm) contains another T-rich region. In the presence of Hg2+, H5 hybridizes with the pyramidal DNA frame by the T-Hg2+-T interaction. A heterogeneous nanostructure was fabricated using Au2 NPs and three Au1 NPs (Fig. 1A). Dipolar interactions between two different sized Au NPs within each HPs could affect the rate of absorption of left circularly polarized (LCP) and right circularly polarized (RCP) light (Kuzyk et al. 2012), and produce strong CD signal around the plasmon resonance frequency (Fig. 1B). Based on changes in the CD intensity of the solution, Hg2+ ions can be sensitively and specifically detected in the low concentration. Fig. 1 3.2 Self-assembly of chiroplasmonic HPs The morphology of two Au NPs (Au1 and Au2) is shown in Fig. S1. To test the impact of the arrangement of NPs and the geometry of assemblies on the CD signal in the visible range, the assembly process of chiroplasmonic HPs was examined with TEM and CD spectra. As shown in Fig.2A, Au1NPs formed Au1 NPs trimers with high yields of 85% based on the hybridization of ssDNA (Fig. S2, black). With the addition of Au2-DNA conjugates, the percentage of single NPs in solution was increased from 10% to 51% (Fig. S2, red) and the number of HPs was still negligible 8
(Fig. 2B). With the addition of Hg2+ ions, Au2 NPs and Au1 NPs trimers were assembled based on the T-Hg2+-T interaction to form HPs as shown in Fig. 2C, thus the number of HPs in solution was increased from 1% to 83% (Fig. S2, blue). The typical size distribution of NPs assemblies was determined by DLS data also indicating the success assembly of HPs, as shown in Fig.2D. Fig. 2 3.3 Chiroptical properties of HPs CD spectrum was used to determine the chiroptical properties of the structures. As shown in Fig. 2E, Au1-trimers and the mixture of Au1-trimers and Au2 NPs explored CD peak only in the range of 200-300 nm due to nucleotides in DNA molecules, but no chiroptical activity was observed in the plasmon wavelength of Au NPs because of all-identical Au1NPs and symmetric frame in trimers (Fan and Govorov 2010). However, once HPs were formed in the presence of Hg2+ ions, the solution exhibited remarkable CD signal at 522 nm with (-) 42 mdeg. The signal has been attributed to the asymmetric arrangement of NPs determined by three-dimensional DNA scaffold and high inequality in the size and shape of Au NPs (Mastroianni et al. 2009; Yan et al. 2012). This explanation was also further demonstrated in our control study. In the control study, homopyramids consisted of four uniform Au1 NPs with the addition of Hg2+ions were prepared and examined with TEM and CD spectra. As shown in Fig. S3, after binding with Hg2+ions, the number of homopyramids was increased (Fig. S3A-C), but no chirality was observed in the CD spectra in the 450-550 nm range (Fig. S3D). In order to understand the effects of the interaction time on HP formation in the nanoassemblies and Hg2+ mixture and relationship between HP formation and CD values, dynamic assembly process of HPs (Fig. S4) and CD spectra of the mixture (Fig. S5) in the presence of 1 ng 9
mL-1 Hg2+ ions were determined at time intervals from 0 to 50 min using typical TEM images and the CD spectroscopy, respectively. It was found that with increased interaction time, the number of HPs increased (Fig. S4) and the CD values at 522 nm were also gradually increased (Fig. S5). These results demonstrate that the HP formation from the nanoassemblies and Hg2+ ions reaction can be affected by the interaction time and there is a positive relationship between the number of HPs and CD values. 3.4 Chiroplasmonic HPs-based probe for Hg2+ detection The above results indicate that a plasmonic HPs-based chiral device could be potentially used to determinate Hg2+ ions. Fig. 3 displays representative CD spectra after mixing the plasmonic nanoassemblies with different concentrations of Hg2+ ions. It is demonstrated that, in addition to strong absorbance in the UV range of CD spectra, the mixture displays significant CD signal in the visible range (450-550 nm); and with the concentration of Hg2+ ions increasing from 0 pg mL-1 to 500 pg mL-1, the CD value at 522 nm was decreased from 0.2 mdeg to (-) 46.3 mdeg (Fig. 3). The high CD value around 522 nm could be attributed to its strong plasmonic interactions between heterogeneous NPs in the well-controlled nanostructure space (Zhao et al. 2014). In contrast, UV-vis spectra did not exhibit any significant changes in the range of 450-550 nm when the concentration of Hg2+ ions was changed, as shown in Fig. S6. This characteristic makes CD measurement a powerful technique for monitoring of Hg2+ ions. As shown in Fig. 4, the linear range of Hg2+ ions detection was between 1 pg mL-1 to 500 pg mL-1, and the limit of detection (LOD) was 0.2 pg mL-1, which is approximately 100 times lower than the chiroplasmonic method based on gold nanorod complex (Zhu et al. 2012) and other NPs-based biosensing approaches (Wu et al. 2012). 10
Fig.3 Fig.4 In addition to the high sensitivity, the specificity of this method was also evaluated with other metal ions (Pb2+, Mg2+, Mn2+, Zn2+, Fe3+, Cd2+, Fe2+, Ca2+, Cu2+) commonly found in water and results are shown in Fig 5. The CD signal of the solution containing 1 ng mL-1 Hg2+ is approximately 9 times higher than those of Mg2+, Cd2+, Cu2+ and 30 times higher than those of Fe3+, Fe2+, Pb2+, Mn2+, Zn2+and Ca2+. This result demonstrates the excellent specificity of the chiroplasmonic method developed in the present study (Fig. S7). Besides sensitivity and specificity, the practical application of the developed approach for tap water samples was also investigated. The recovery was measured by spiking water samples with different concentrations of Hg2+ ions. Each test was repeated at least three times. As shown in Table S1, our HPs probe displays excellent recoveries (95.6%-108.2%). Fig.5
4. Conclusions In the present study, we demonstrated a novel chiral-aptamer sensor for the detection of Hg2+ ions based on DNA-engineered heterogeneous nanoparticle assembly. The approach exhibited high sensitivity and selectivity in the detection of Hg2+ ions, and the limit of detection is 0.2 pg mL-1 in the low concentration linear response. Since the method is dependent on DNA-templated self-assembled of nanostructures instead of disordered NP aggregates, it can be reused by heating the system at 90 ℃ for 5min, and then was cooling down to the room temperature. this characteristic is not available for some Au NPs aggregates-based colorimetric method. It can also avoid false positive results resulting from nonspecific adsorption, it is promising for the sensitive determination of Hg2+ ions in real samples. More importantly, the approach should be highly 11
adaptable to the determination of other analytes following the same principle.
Acknowledgments This study was sponsored by the National Technology Support Program of China (Grant No. 2012BAD28B01), the International Technology Cooperation Program of Jiangsu province, China (Grant NO. BZ2014034).
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Captions: Fig.1. Schematic of NPs heteropyramids assembly-based circular dichroism method for the detection of Hg2+ ions Fig.2. Representative TEM images for (A) Au1-trimers, (B) mixture of Au1-trimers and Au2 NPs without and (C) with the addition of Hg2+ ions. (D) Dynamic light scattering size distributions and (E) CD spectra of NPs heteropyramids. Fig.3.
CD spectra of plasmonic heteropyramids in the presence of Hg2+ ions at different concentrations, the concentration is 0 pg mL-1, 1 pg mL-1, 5 pg mL-1, 10 pg mL-1, 50 pg mL-1, 100 pg mL-1 and 500 pg mL-1.
Fig.4 The calibration curve relating the CD intensity at 522 nm of HPs and the concentration of Hg2+ ions. Fig.5 CD intensity of nanoparticles HPs-based sensor in the presence of Hg2+ ion and other metal ions, the concentration of each analyte was 1 ng mL-1.
Highlights A chiral-aptamer sensor was fabricated for Hg2+ ions detection based on heterogeneous nanostructures. This method exhibited high selectivity for Hg2+ ions and a limit of detection as low as 0.2 pg mL-1. The approach was successfully used for the detection of Hg2+ ions in tap water samples.
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PII: DOI: Reference:
S0956-5663(15)00029-9 http://dx.doi.org/10.1016/j.bios.2015.01.028 BIOS7403
To appear in: Biosensors and Bioelectronic Received date: 21 November 2014 Revised date: 3 January 2015 Accepted date: 12 January 2015 Cite this article as: Wenjing Yan, Yongli Wang, Hong Zhuang and Jianhao Zhang, DNA-engineered chiroplasmonic heteropyramids for ultrasensitive detection of Mercury ion, Biosensors and Bioelectronic, http://dx.doi.org/10.1016/j.bios.2015.01.028 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
DNA-engineered chiroplasmonic heteropyramids for ultrasensitive detection of mercury ion Wenjing Yan a, Yongli Wang a, Hong Zhuang b, Jianhao Zhang a, a
b
National Center of Meat Quality & Safety Control, Nanjing Agricultural University, Nanjing, 210095, China
Quality and Safety Assessment Research Unit, Agricultural Research Service, USDA, Athens, GA 30605, USA
ABSTRACT In this study, plasmonic heteropyramids (HPs) made from two different sized gold nanoparticles (Au NPs) and five ssDNA sequences and their application for ultrasensitive detection of mercury ion (Hg2+) were demonstrated. Four ssDNA sequences were used as building blocks to form a pyramidal DNA frame, which contains a T-rich probe DNA at one vertex and three sulfhydryl groups modified with 10 nm Au NPs at the other three vertices. Another T-rich DNA sequence was modified and attached to a 25nm Au NP. In the presence of Hg2+ ions, 25 nm Au NPs hybridized with pyramidal DNA frame to build the plasmonic HPs based on T–Hg2+–T interaction, which exhibits unprecedented circular dichroism (CD) signal in the visible region. Based on this mechanism, a simple, high sensitive and selective chiroplasmonic HPs-based probe was constructed and demonstrated for Hg2+ ions detection. Under optimized conditions, Hg2+ ions could be selectively detected in a concentration range from 1 to 500 pg mL-1 with a limit of detection as 0.2 pg mL-1, which is much lower than the strictest Hg2+ safety requirement of 1 ng mL-1 in water.
Keywords: Gold nanoparticles; Heteropyramids; Circular dichroism; Mercury ion;
Corresponding authors. E-mail addresses: [email protected] (J. Zhang). 1
1. Introduction Hg2+ ions are highly toxic heavy metal ions produced from the industrial wastes and natural activities. The ions can be transformed into methyl mercuric compounds, concentrate through biological cycles and cause a series of diseases, such as pneumonia, enteritis, and bronchitis in human beings (Zhou et al. 2010). Mercury pollution is widely found in tap water and food and has been one of food safety concerns to consumers in the entire world. Obviously, developing an effective method for detecting Hg2+ ions in water and food is urgently needed. Among various Hg2+ ions detection methods, metal nanomaterial-enabled approaches offer an excellent opportunity for low-concentration, highly selective, and rapid detection of Hg2+ ions due to their unique size effect and tunable optical properties (Du et al. 2013; Guo et al. 2013). The predominant approaches for Hg2+ ions determination using metal nanomaterial as the signal conductor are surface enhanced Raman scattering (SERS) (Chen et al. 2014; Ma et al. 2013b; Ren et al. 2012), colorimetric (Chen et al. 2014; Wu et al. 2012; Xu et al. 2014), and fluorescence (Cui et al. 2015; Hao et al. 2012; Zhang et al. 2011). Considering the ultrahigh extinction coefficient (108-109) of the surface plasmon resonance (SPR) absorption of metal nanoparticles (NPs), these assays may achieve high sensitivity in the detection of Hg2+ ions (Guo et al. 2013; Ye and Yin 2008). However, they still have many constraints, such as high cost, easy contamination, complexity and non-repeatability (Kim et al. 2012; Zhang et al. 2013). More importantly, most of these assays rely on an aggregate of NPs or nanostars (NSs) to enhance the responding signal, increasing the sensitivity in the detection of Hg2+ ions (Kanayama et al. 2011). However, NPs are fragile to the complex environment and easy to form an aggregate or random homo-nanoassemblies (homodimers, trimers or chains), which is likely to cause false-positive 2
results. Thus, a simple, reliable and highly sensitive plasmonic method is still needed for the detection of Hg2+ ions (Aragay et al. 2011). Chiral nanomaterials represent one of the most rapidly developing research fields. Chiral nanomaterials could affect the rate of absorbance of the left- and right-circularly polarized light (CPR) and produce a significant signal in the visible region of the CD spectrum (Han et al. 2014; Mark et al. 2013; Ye and Yin 2008). DNA-engineered chiral nanostructures have fascinated the chemistry and engineering world due to their myriad geometries and distinctive optical properties (Lan et al. 2013; Li et al. 2012; Wu et al. 2013; Zhao et al. 2014). By designing the sequences of DNA molecules, single NPs can be assembled into nanostructures with asymmetrical geometry, represented by helix (Chen et al. 2008; Kuzyk et al. 2012; Song et al. 2013), pyramids (Mastroianni et al. 2009; Yan et al. 2012), and tetrahedron (Fan and Govorov 2010), in which pyramidal nanostructures represent a unique subset of DNA-engineered chiral nanomaterials. They exhibit large chiroptical responses of pure NPs at the corresponding plasmonic wavelength. The tunable light-matter interaction optical response of the complex is critically dependent on the interparticle spacing, shapes, and composition (Mastroianni et al. 2009), which is completely different from single chiral NPs (Gautier and Burgi 2009). Furthermore, diverse elements and engineered three-dimensional space structure endow NPs pyramids with unique optical properties and wide application potentials. In our previous work, metal NPs and/or quantum dots (QDs) modified with four ssDNA were used to construct a series of geometry controlled chiral NP pyramids in high yields. It has been demonstrated that the geometry parameters of nanostructures play a significant role in the origin of chirality of NPs pyramids (Yan et al. 2012). Specifically, once achiral NPs are assembled into 3
three-dimensional nanostructures in an asymmetric manner, the complex can display adjustable and multiple chiroptical activities in the 300-550 nm range due to the chiral arrangement of NPs in space predetermined by predesigned DNA scaffolds and asymmetric shapes of single NP (Fan et al. 2013; Ma et al. 2013a). This method provides a new approach for the formation of chiral materials and endows NPs pyramids with the ability to be used as a chiral sensor for target detection (Yan et al. 2014). Here, chiroplasmonic HPs were assembled using DNA scaffold modified with two different sized Au NPs as building blocks and the application in Hg2+ ions detection were reported. In the presence of Hg2+ ions, the T-rich region in DNA sequence selectively combines with Hg2+ ions to form a stable covalent T-Hg2+-T structure, resulting in the assembly of heterogeneous nanoassemblies with significant CD signal in the visible range. The assembly of HPs and the CD signal were dependent on the concentration of Hg2+ ions. Based on this principle, the approach was successfully used for the detection of Hg2+ ions in solution and in tap water samples with high sensitivity and prominent selectivity.
2. Materials and methods 2.1 Materials and reagents Metal ion standards used in this study (Hg2+, Zn2+, Mg2+, Fe3+, Cd2+, Pb2+, Mn2+, Fe2+, Ca2+, Cu2+) were purchased from the National Institute of Metrology P.R.C. (Beijing, China).Unless stated otherwise, all chemicals used were purchased from Sigma-Aldrich. Deionized water from a Milli-Q device (18.2 MΩ, Millipore, Molsheim, France) was used throughout this work. All glassware was cleaned with freshly prepared aqua regia and rinsed thoroughly by deionized water prior to use. Thiolated DNA oligonucleotides, which were purified by polyacrylamide gel 4
electrophoresis (PAGE), were purchased from Shanghai Sangon Biological Engineering Technology & Services Co. Ltd. (Shanghai, P.R. China) and suspended in DI water to a final concentration of 100μM. The DNA sequences are as follows: H1:5’-SH-AAA GCC TGG AGA TAC ATG CAC ATT ACG GCC CCC CCT ATT AGA AGG TCT CAG GTG CGC GCC CCG GTA AGT AGA CGG GAC CAG TTC GCC-3’ H2:5’-SH-AAA CGC GCA CCT GAG ACC TTC TAA TAG GGC CCG CGA CAG TCG TTC AAC TAG AAT GCC CCC CGG GCT GTT CCG GGT GTG GCT CGT CGG-3’ H3: 5’-SH-AAA GGC CGA GGA CTC CTG CTC CGC TGC GGC CCG GCG AAC TGG TCC CGT CTA CTT ACC GCC CCC GAC GAG CCA CAC CCG GAA CAG CCC-3’ H4: 5’-ACC TTT TTT TCA GTG AAA GCC GTA ATG TGC ATG TAT CTC CAG GCC CCC CGC AGC GGA GCA GGA GTC CTC GGC CCC CGG GCA TTC TAG TTG AAC GAC TGT CGC-3’ H5: 5’- SH-CAC TGT TTT TTT GGT-3’ 2.2 Synthesis of Au NPs Au NPs with a diameter of 10 nm (Au1) were prepared as follows: 80mL, 0.0125% HAuCl4 was prepared as solution A. Then, sodium citrate (1%, 4 mL), tannic acid (1%, 0.1 mL), K2CO3 (25 mM, 0.1 mL), and 15.8 mL of deionized water were mixed together, named as solution B. Then, A and B were heated to 60 °C for 30 min before being mixed under high-speed stirring. Au NPs with a diameter of 25 nm (Au2) were synthesized by reduction of HAuCl4 with citrate. Briefly, aqueous trisodium citrate solution (1.6 mL, 1%) was quickly added to a boiling aqueous solution of HAuCl4 (100 mL, 0.25 mM) under vigorous stirring. After the color of the solution changed from blue to wine red (about 2 min), the heat source was removed and the solution was cooled to room temperature.
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2.3 Preparation of ssDNA-modified Au NPs Before use, Au NPs (Au1 and Au2) were stabilized with bis (p-sulfonatophenyl) phenylphosphine dihydrate, dipotassium salt (BPS). Aqueous solutions of Au NPs (Au1 and Au2) were concentrated 10-fold. Then 10 mL of Au NPs was stirred with an excess of BPS (20 mg/mL) at room temperature for more than 10 h. The solutions were then centrifuged at different speeds (13,000 r/min for Au1 or 7,000 r/min for Au2) for 10 min and resuspended in 0.5×TBE buffer to obtain a final concentration of 20 nM. Four different Au-DNA conjugates were: Au1-H1, Au1-H2, Au1-H3, and Au2-H5. One μL of 10μM DNA was added to 100 μL of 20nM Au NPs with a nanoparticle/DNA molar ratio of 1/5. The DNA-Au NPs mixture was incubated in 0.5×TBE buffer with 50 mM NaCl for 2h, and then centrifuged at 13,000 (Au1) or 7,000 (Au2) r/min for 10 min. The supernatant was removed and the pellet was resuspended in 1×TBE buffer to obtain a final concentration of 20 nM. 2.4 Preparation of chiral plasmonic HPs Five different solutions including Au1-H1, Au1-H2, Au1-H3, H4, Au2-H5 with the same volume (50 µL) were added to 1×TBE buffer with 100mM NaCl. The hybridization mixture was heated at 90°C for 5 min and then slowly cooled to room temperature. Hg2+ was added into the hybrid mixture and incubated for 50 min at room temperature with constant shaking. 2.5 Fabrication of chiral sensor for Hg2+detection The mixture of Au1-trimers and Au2-DNA conjugate prepared in 2.4 was transferred to seven separated tubes. For each tube, Hg2+ standard with different concentrations (0, 1, 5, 10, 50, 100, 500 pg mL-1) was added. The mixture were incubated for 50 min at room temperature with constant 6
shaking. The samples were characterized by transmission electron microscopy (TEM), circular dichroism (CD) and ultraviolet–visible (UV-vis) spectroscopy. 2.6 Specificity testing The specificity of the HP probe was investigated by mixing ssDNA-engineered nanostructures with ten metal ions, Hg2+, Pb2+, Mg2+，Mn2+, Zn2+，Fe3+, Cd2+, Fe2+, Ca2+, Cu2+,respectively. The final concentration of the metal ions was 1 ng mL-1 and CD spectrum of the samples was collected. 2.7 Analysis of water samples Spiked tap water samples were used for recovery analysis. Three concentrations of Hg2+ ions (5, 10 and 50 pg mL-1) were spiked in tap water. All samples were analyzed using CD spectroscopy and the same sample was repeated at three times for calculation standard deviation (s.d.) value.
2.8 Characterization The CD spectroscopy was performed on Bio-Logic MOS-450/AF circular dichroism. The UV/vis spectra were acquired on a UNICO 2100 PC UV/vis spectrophotometer and processed with the Origin Lab software. TEM images were collected on a JEOL JEM-2100 transmission electron microscope with an acceleration voltage of 200 kV. The sample was prepared by dropping an aqueous pyramid solution onto a copper grid coated with the carbon film, and then was dried in air.
3. Results and discussion 3.1 Sensing strategy Fig. 1 illustrates a chiroptical sensor that was fabricated in this study for the detection of Hg2+ ions. As shown in Fig.1A, five ssDNA sequences (H1, H2, H3, H4 and H5, see 2.1 Materials and 7
reagents) were used to assembly of heterogeneous nanostructures. Four ssDNA (H1, H2, H3 and H4) can complementarily hybridize with each other to form a pyramidal DNA frame. Each ssDNA can be segmented into three parts; each part is complementary to a third of each of the other strands. So, each strand of DNA consists of one face of the pyramid. Three ssDNA (H1, H2 and H3) modified with sulfhydryl group at 5’-end can connect with three Au1 NPs (10 nm) to form a NP trimer, one ssDNA (H4) contains an extended T-rich region at 5’-end to form a recognition site at one apex. The fifth DNA (H5) attached on the surface of Au2 NP (25 nm) contains another T-rich region. In the presence of Hg2+, H5 hybridizes with the pyramidal DNA frame by the T-Hg2+-T interaction. A heterogeneous nanostructure was fabricated using Au2 NPs and three Au1 NPs (Fig. 1A). Dipolar interactions between two different sized Au NPs within each HPs could affect the rate of absorption of left circularly polarized (LCP) and right circularly polarized (RCP) light (Kuzyk et al. 2012), and produce strong CD signal around the plasmon resonance frequency (Fig. 1B). Based on changes in the CD intensity of the solution, Hg2+ ions can be sensitively and specifically detected in the low concentration. Fig. 1 3.2 Self-assembly of chiroplasmonic HPs The morphology of two Au NPs (Au1 and Au2) is shown in Fig. S1. To test the impact of the arrangement of NPs and the geometry of assemblies on the CD signal in the visible range, the assembly process of chiroplasmonic HPs was examined with TEM and CD spectra. As shown in Fig.2A, Au1NPs formed Au1 NPs trimers with high yields of 85% based on the hybridization of ssDNA (Fig. S2, black). With the addition of Au2-DNA conjugates, the percentage of single NPs in solution was increased from 10% to 51% (Fig. S2, red) and the number of HPs was still negligible 8
(Fig. 2B). With the addition of Hg2+ ions, Au2 NPs and Au1 NPs trimers were assembled based on the T-Hg2+-T interaction to form HPs as shown in Fig. 2C, thus the number of HPs in solution was increased from 1% to 83% (Fig. S2, blue). The typical size distribution of NPs assemblies was determined by DLS data also indicating the success assembly of HPs, as shown in Fig.2D. Fig. 2 3.3 Chiroptical properties of HPs CD spectrum was used to determine the chiroptical properties of the structures. As shown in Fig. 2E, Au1-trimers and the mixture of Au1-trimers and Au2 NPs explored CD peak only in the range of 200-300 nm due to nucleotides in DNA molecules, but no chiroptical activity was observed in the plasmon wavelength of Au NPs because of all-identical Au1NPs and symmetric frame in trimers (Fan and Govorov 2010). However, once HPs were formed in the presence of Hg2+ ions, the solution exhibited remarkable CD signal at 522 nm with (-) 42 mdeg. The signal has been attributed to the asymmetric arrangement of NPs determined by three-dimensional DNA scaffold and high inequality in the size and shape of Au NPs (Mastroianni et al. 2009; Yan et al. 2012). This explanation was also further demonstrated in our control study. In the control study, homopyramids consisted of four uniform Au1 NPs with the addition of Hg2+ions were prepared and examined with TEM and CD spectra. As shown in Fig. S3, after binding with Hg2+ions, the number of homopyramids was increased (Fig. S3A-C), but no chirality was observed in the CD spectra in the 450-550 nm range (Fig. S3D). In order to understand the effects of the interaction time on HP formation in the nanoassemblies and Hg2+ mixture and relationship between HP formation and CD values, dynamic assembly process of HPs (Fig. S4) and CD spectra of the mixture (Fig. S5) in the presence of 1 ng 9
mL-1 Hg2+ ions were determined at time intervals from 0 to 50 min using typical TEM images and the CD spectroscopy, respectively. It was found that with increased interaction time, the number of HPs increased (Fig. S4) and the CD values at 522 nm were also gradually increased (Fig. S5). These results demonstrate that the HP formation from the nanoassemblies and Hg2+ ions reaction can be affected by the interaction time and there is a positive relationship between the number of HPs and CD values. 3.4 Chiroplasmonic HPs-based probe for Hg2+ detection The above results indicate that a plasmonic HPs-based chiral device could be potentially used to determinate Hg2+ ions. Fig. 3 displays representative CD spectra after mixing the plasmonic nanoassemblies with different concentrations of Hg2+ ions. It is demonstrated that, in addition to strong absorbance in the UV range of CD spectra, the mixture displays significant CD signal in the visible range (450-550 nm); and with the concentration of Hg2+ ions increasing from 0 pg mL-1 to 500 pg mL-1, the CD value at 522 nm was decreased from 0.2 mdeg to (-) 46.3 mdeg (Fig. 3). The high CD value around 522 nm could be attributed to its strong plasmonic interactions between heterogeneous NPs in the well-controlled nanostructure space (Zhao et al. 2014). In contrast, UV-vis spectra did not exhibit any significant changes in the range of 450-550 nm when the concentration of Hg2+ ions was changed, as shown in Fig. S6. This characteristic makes CD measurement a powerful technique for monitoring of Hg2+ ions. As shown in Fig. 4, the linear range of Hg2+ ions detection was between 1 pg mL-1 to 500 pg mL-1, and the limit of detection (LOD) was 0.2 pg mL-1, which is approximately 100 times lower than the chiroplasmonic method based on gold nanorod complex (Zhu et al. 2012) and other NPs-based biosensing approaches (Wu et al. 2012). 10
Fig.3 Fig.4 In addition to the high sensitivity, the specificity of this method was also evaluated with other metal ions (Pb2+, Mg2+, Mn2+, Zn2+, Fe3+, Cd2+, Fe2+, Ca2+, Cu2+) commonly found in water and results are shown in Fig 5. The CD signal of the solution containing 1 ng mL-1 Hg2+ is approximately 9 times higher than those of Mg2+, Cd2+, Cu2+ and 30 times higher than those of Fe3+, Fe2+, Pb2+, Mn2+, Zn2+and Ca2+. This result demonstrates the excellent specificity of the chiroplasmonic method developed in the present study (Fig. S7). Besides sensitivity and specificity, the practical application of the developed approach for tap water samples was also investigated. The recovery was measured by spiking water samples with different concentrations of Hg2+ ions. Each test was repeated at least three times. As shown in Table S1, our HPs probe displays excellent recoveries (95.6%-108.2%). Fig.5
4. Conclusions In the present study, we demonstrated a novel chiral-aptamer sensor for the detection of Hg2+ ions based on DNA-engineered heterogeneous nanoparticle assembly. The approach exhibited high sensitivity and selectivity in the detection of Hg2+ ions, and the limit of detection is 0.2 pg mL-1 in the low concentration linear response. Since the method is dependent on DNA-templated self-assembled of nanostructures instead of disordered NP aggregates, it can be reused by heating the system at 90 ℃ for 5min, and then was cooling down to the room temperature. this characteristic is not available for some Au NPs aggregates-based colorimetric method. It can also avoid false positive results resulting from nonspecific adsorption, it is promising for the sensitive determination of Hg2+ ions in real samples. More importantly, the approach should be highly 11
adaptable to the determination of other analytes following the same principle.
Acknowledgments This study was sponsored by the National Technology Support Program of China (Grant No. 2012BAD28B01), the International Technology Cooperation Program of Jiangsu province, China (Grant NO. BZ2014034).
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Captions: Fig.1. Schematic of NPs heteropyramids assembly-based circular dichroism method for the detection of Hg2+ ions Fig.2. Representative TEM images for (A) Au1-trimers, (B) mixture of Au1-trimers and Au2 NPs without and (C) with the addition of Hg2+ ions. (D) Dynamic light scattering size distributions and (E) CD spectra of NPs heteropyramids. Fig.3.
CD spectra of plasmonic heteropyramids in the presence of Hg2+ ions at different concentrations, the concentration is 0 pg mL-1, 1 pg mL-1, 5 pg mL-1, 10 pg mL-1, 50 pg mL-1, 100 pg mL-1 and 500 pg mL-1.
Fig.4 The calibration curve relating the CD intensity at 522 nm of HPs and the concentration of Hg2+ ions. Fig.5 CD intensity of nanoparticles HPs-based sensor in the presence of Hg2+ ion and other metal ions, the concentration of each analyte was 1 ng mL-1.
Highlights A chiral-aptamer sensor was fabricated for Hg2+ ions detection based on heterogeneous nanostructures. This method exhibited high selectivity for Hg2+ ions and a limit of detection as low as 0.2 pg mL-1. The approach was successfully used for the detection of Hg2+ ions in tap water samples.
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