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Ultra-Sensitive, Specific, Recyclable and Reproducible Detection of Lead Ions in Real Systems through a Poly AdenineAssisted Surface-Enhanced Raman Scattering Silicon Chip Yu Shi, Houyu Wang, Xiangxu Jiang, Bin Sun, Bin Song, Yuanyuan Su, and Yao He Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.5b04551 • Publication Date (Web): 26 Feb 2016 Downloaded from http://pubs.acs.org on March 3, 2016
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Analytical Chemistry
Ultra-Sensitive, Specific, Recyclable and Reproducible Detection of Lead Ions in Real Systems through a Poly AdenineAssisted Surface-Enhanced Raman Scattering Silicon Chip Yu Shi,‡ Houyu Wang,‡ Xiangxu Jiang, Bin Sun, Bin Song, Yuanyuan Su, and Yao He* Institute of Functional Nano & Soft Materials (FUNSOM) and Collaborative Innovation Center of Suzhou Nano Science and Technology, Soochow University, Suzhou, Jiangsu 215123, China. ABSTRACT: It is of great significance to accurately and reliably detect trace lead (II) (Pb2+) ions, preferably at sub-nM level, due to the possible long-term accumulation of Pb2+ in human body, which may cause serious threats to human health. However, a suitable Pb2+ sensor meeting the demands is still scanty. Herein, we develop a Poly Adenine (A)-assisted surface-enhanced Raman scattering (SERS) silicon chip (0.5 cm × 0.5 cm) composed of core (Ag)-satellite (Au) nanoparticles (Ag-Au NPs)-decorated silicon wafers (Ag-Au NPs@Si) for high-performance Pb2+ detection. Typically, strong SERS signals could be measured when DNAzyme conjugated on the SERS silicon chip is specifically activated by Pb2+, cleaving the substrate strand into two free DNA strands. A good linearity exists between the normalized Raman intensities and the logarithmic concentrations of Pb2+ ranging from 10 pM to 1 µM with a good correlation coefficient, R2 of 0.997. Remarkably, Pb2+ ions with a low concentration of 8.9 × 10−12 M can be readily determined via the SERS silicon chip ascribed to its superior SERS enhancement, much lower than those (~nM) reported by other SERS sensors. Additionally, the developed chip features good selectivity and recyclability (e.g., ~11.1% loss of Raman intensity after three cycles). More importantly, the as-prepared chip can be used for accurate and reliable determination of unknown Pb2+ ions in real systems including lake water, tap water and industrial waste water, with the RSD value less than 12%.
Lead (II) (Pb2+) ions, known as one of the most hazardous heavy metal ions, exist in a variety of sources (e.g., food, blood, drinking water, industrial waste water, etc.), which have triggered a series of serious problems concerning environmental contamination.1 For instance, long-term Pb2+ exposure can induce severe damages on nervous system, urinary system and intellectual development through food chains and drinking water, becoming a main threat to human health, especially for children.2-4 The United States Environmental Protection Agency (U. S. EPA) defines that a safe threshold of Pb2+ amount is 483 nM (100 ppb) in blood, and 72.4 nM (15 ppb) in drinking water, respectively.5,6 More seriously, Pb2+ cannot be biodegradable, and thus even a small amount of Pb2+ may also cause terrible harm to human health due to the long-term accumulation in human body. For example, recent study has proven that the intellectual capacity of children could greatly diminish ascribed to long-term and low-level Pb2+ exposure.4 With this regard, there is an increasing interest in the development of analytical methods for Pb2+ assay with ultrahigh sensitivity. To achieve this, numerous analytical strategies such as atomic absorption spectrometry (AAS),7 inductively coupled plasma-mass spectrometry (ICP-MS),8 inductively coupled plasma-atomic emission spectrometry (ICP-AES)9 are conventionally employed for the determination of Pb2+. These methods are well established, they nevertheless require specialized instruments or tedious sample preparation process. During the past decade, newly developed Pb2+ sensors with high sensitivity, short response time, portability, minimal equipment requirements, and low cost have attracted extensive attention.1034 Most of them are primarily relied on a “8-17” DNAzyme,
which can be specifically activated by Pb2+, leading to the change of detection signals, including colorimetry,10-13 fluorescence,14-19 electrochemical signals20-22 and so forth (see Table S1). Notwithstanding, the detection limits of these sensors are normally limited in ~nM levels. Thereby, developing new sensors for detecting Pb2+ ions in real samples in a more sensitive manner (e.g., sub-nM level) is still desirable. Surface-enhanced Raman scattering (SERS) sensors, emerged as a highly promising analytical tool,35-37 have been utilized for detection of Pb2+ in rapid, sensitive and low-cost manners, mainly taking advantages of their three merits: (1) ultrahigh sensitivity due to the huge SERS enhancement factor (EF) up to ~106-9, (2) narrow and sharp Raman bands leading to minimal background noise, and (3) robust stability in diverse environments inert to humidity, oxygen and foreign species.23,24 Although these SERS sensors are workable, the sensitivity (e.g., ~1 nM of detection limit) and reproducibility are still unsatisfactory, limiting their applications in the detection of trace Pb2+, especially in real systems.23,24 Very recently, He et al. have proposed a series of novel silicon nanohybrids-based SERS substrates (e.g., gold nanoparticles modified silicon nanowire array (Au NPs@SiNWAr), silver nanoparticles decorated silicon wafer (Ag NPs@Si), etc), featuring large SERS EF (~106-7) attributed to the highefficacy coupled SERS hot spots produced between the metallic nanoparticles and the dielectric silicon substrate.38-44 More significantly, the developed substrates show adaptable reproducibility (relative standard deviation, RSD < 20%) stem from the steady immobilization of metallic nanoparticles on the silicon substrates, effectively preventing the aggregation of
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free nanoparticles.38-44 Consequently, silicon-based SERS substrates have been used for the detection of chemical and biological species at the molecular and cellular levels. For instance, Wang et al. have utilized Ag NPs@Si-based SERS substrates for sensitive and reproducible detection of the deafness-causing mutations in the clinical samples.39 More recently, the same group developed a multifunctional SERS substrate made of Ag NPs@Si, integrating three abilities of capture, analysis and sterilization of bacteria.43 In order to further improve sensitivity and reliability of silicon nanohybrids-based SERS sensors, more recently, He et al. presented a kind of SERS substrates composed of core (Ag)-satellite (Au) nanoassemblies (Ag-Au NPs) decorated silicon wafers (Ag-Au NPs@Si), by using Poly Adenine (A) (consecutive adenines at the 5’ end of DNA sequence) as the anchor and mediator instead of thiolated DNA.44 Of particular significance, the asprepared Ag-Au NPs@Si exhibits stronger EF (~107) and better reproducibility (RSD: ~12.4%) in optimum conditions.44 Inspired by these previous studies, in this article, we for the first time present a Poly A-assisted SERS silicon chip (0.5 cm × 0.5 cm) for Pb2+ ions detection, which is assembled by AgAu NPs@Si functionalized with Pb2+-specific DNAzyme. In principle, the rigid double-stranded DNA structure can be cracked into flexible single-stranded structure in the presence of Pb2+ ions. Thus, the Cy5-tagged DNA strand would drop down to the SERS substrate, generating significant Raman signals. As a result, Pb2+ ions with an extremely low concentration down to 8.9 × 10−12 M can be easily discriminated, which is around three orders of magnitude lower than the value defined by U. S. EPA.6 Moreover, Pb2+ ions can be readily identified from the mixture solution containing other eleven kinds of interfering metal ions, indicating excellent selectivity. In addition, the developed SERS chip can be regenerated by supplementation of DNAzyme substrate strands, retaining stable SERS signals during 3-time cycle. More significantly, the novel silicon SERS chip is demonstrated to be highly suitable for determination of unknown Pb2+ in various real systems, including tap water, lake water and industrial waste water, featuring a high accuracy (e.g., recoveries of 92.4% to 95.9%), a robust reliability (e.g., RSD values of < 12%) and a good correlation with the results detected by conventional ICP-AES method.
EXPERIMENAL SECTION Chemicals and Reagents. DNA oligonucleotides were synthesized and purified by Sangon Biotechnology (Shanghai, China), whose sequences were listed in Table S2. Hydrofluoric acid (HF, ≥ 40%), silver nitrate (AgNO3, ≥ 99.8%), hydrogen peroxide (H2O2, ≥ 30%), sulphuric acid (H2SO4, 98%), trisodium citrate (C6H5Na3O7•2H2O, ≥ 99.0%), gold chloride (HAuCl4, ≥ 47.8%) and lead nitrate (PbNO3, ≥ 99.0%) were purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). Silicon (100) wafers (phosphate-doped, ptype, 0.01-0.02 Ω sensitivity) were bought from Hefei Kejing Materials Technology Co., Ltd. (Hefei, China). All chemicals were used without additional purification. All solutions were prepared in Milli-Q water (18.2 MΩ cm–1) at room temperature. The real samples of tap water, lake water and industrial waste water collected from Suzhou, Jiangsu Province, China. Instruments. The characterization of the as-prepared SERS chip was conducted by a scanning electron microscopy (SEM) (FEI Quanta 200F), a transmission electron microscopy
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(TEM) (Philips CM 200) and an atomic force microscopy (AFM) equipped with Multi Mode V AFM (Vecco Corporation, USA). The UV-vis spectra were recorded by an UV-visnear-infrared spectrophotometer (Perkin-Elmer Lambda 750). Dynamic light-scattering (DLS) measurements were performed by Zetasizer Nano ZS90 (Malvern Instruments). Inductively coupled plasma-atomic emission spectrometer (ICPAES) (Thermo 6300) was utilized for the determination of Pb2+ from real water samples. The Raman spectra were collected by a Raman microscope (HR800, Horiba Jobin Yvon, France) equipped with a He-Ne laser (633 nm, 20 mW, polarized 500:1) and a 100 × objective (NA: 0.9). The collected Raman spectra were further analyzed by the LabSpec5 software. Preparation of SERS Silicon Chip. The standard citrate reduction method was first utilized for the preparation of ~13 nm satellite Au NPs, in which 0.01% (w/v) HAuCl4 were reduced by 1% (w/v) trisodium citrate under high temperature (e.g., 100 oC) for 20 min.45 The core Ag NPs were in situ decorated on the surface of silicon wafer (Ag NPs@Si) through the well-established HF-assisted etching method, in which the clean silicon wafer with a size of (0.5 cm × 0.5 cm) was treated with 5% HF (v/v) followed by immersing the treated silicon wafer into 0.02% (m/v) AgNO3 solution for 90 sec.38,39 Then, the prepared Ag NPs@Si was incubated with phosphate buffer (pH 7.0) containing 50 nM Poly A30-P1 (see Table S2) for 16 h to obtain Poly A30-P1-Ag NPs@Si. Meanwhile, the asprepared 1 nM Au NPs were incubated with phosphate buffer (pH 7.0) containing 50 nM Poly A30-P2 for 16 h to form Poly A30-P2-Au NPs. Afterwards, the asymmetric core-satellite nanoassemblies of Ag-Au NPs@Si were achieved through the hybridizing reaction between P1 and P2 when the two conjugates of Poly A30-P1-Ag NPs@Si and Poly A30-P2-Au NPs were mixed together in hybridization buffer (0.3 M PBS, pH 7.0) for 24 h. Of note, excess DNA or nanoparticles in each assembly step should be removed by rinsing with PBS buffer (pH 7.0) for four times. Functionalization of SERS Silicon Chip. The assembly buffer (0.5 µM enzyme strand, 10 mM phosphate, pH 7.0) of 50 µL was dropped onto the surface of silicon SERS chip for 12 h. In this case, the single enzyme strand was dually labeled with Cy5 and thiol groups (Cy5-17E-SH, see Table S2). Then, the resultant chip was incubated with phosphate buffer (pH 7.0) containing 0.1 M NaCl for 12 h to facilitate the conjugation between DNA and SERS chip. Afterwards, the conjugated enzyme strand were hybridized with 0.5 µM single substrate stand of 17DS for 16 h at 37 °C to form a ds-DNA structure. The final product was washed with PBS buffer (pH 7.0) for four times to remove excess DNA and then dried with a gentle flow of nitrogen. To overcome the negative effects induced by variation of DNA packing density, we strictly control experimental conditions in the fabrication of SERS chip (e.g., incubation time and temperature, substrate size and morphology, etc). Detection of Pb2+ in Distilled Water via SERS Silicon Chip. The developed SERS silicon chip was treated with 100 µL distilled water spiking with Pb2+ ions with different concentrations for 70 min at room temperature. The Raman spectra were measured using a Raman microscope equipped with a 633 nm laser and 100 × objective, employing 1 sec acquisition time, 1000 µm confocal hole, 400 µm slit aperture and 0.2 mW laser power on the sample. The Raman mapping test for each
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Analytical Chemistry
sample was accomplished within ~3.5 min. Other interfering samples containing Zn2+, Ni2+, Na+, Mn2+, Mg2+, Hg2+, Fe2+, Cu2+, Co2+, Ca2+, Ba2+ and the mixture containing all metal ions were also detected using the identical manipulations. Detection of Pb2+ in Real Systems via SERS Silicon Chip. The validity of determination of unknown Pb2+ in real systems by the developed SERS silicon chip was further examined. The test samples included local tap water, industrial waste water, and river water. First, the real water samples were filtered through Millipore filters (0.22 µm) to remove large size particles. Next, the SERS chip was incubated with the treated real water samples of 100 µL for 70 min followed by in situ Raman measurements of Pb2+ in aqueous solutions. In addition, in order to evaluate the relativity between the developed SERS method and the conventional ICP-AES method, the same real water samples were also tested by an ICP-AES.
RESULTS AND DISCUSSION Figure 1a exhibits the schematic illustration for fabrication of the SERS silicon chip. Schematically, the Ag NPs (diameter: ~120 nm) modified on the surface of 0.5 cm × 0.5 cm silicon wafer (Ag NPs@Si) is first facilely prepared assisted by a well-established HF-etching method (see Figure S1).38,39,42,43 Afterwards, two types of single-stranded DNA containing 30 consecutive adenines at the 5’ end of DNA sequence (Poly A30): Poly A30-P1 and Poly A30-P2 are respectively conjugated on the surfaces of core Ag NPs and satellite Au NPs (~13 nm, see Figure S2) based on the strong affinity between Poly A and the surface of Ag or Au NPs.46-48 Then, the asymmetric core-satellite nanostructure of Ag-Au NPs@Si can be achieved owing to the hybridization between P1 and P2 when mixing Poly A30-P1-conjugated Ag NPs@Si with Poly A30P2-conjugated Au NPs. The morphology of the as-prepared SERS silicon chip is characterized by using scanning electronic microscopy (SEM). Particularly, as shown in Figure 1b, the larger core Ag NPs (~120 nm) are uniformly in situ grown on the surface of silicon wafer. Moreover, according to the zoom-in SEM image in Figure 1b, numerous smaller satellite Au NPs (~13 nm) are located on the surface of core Ag NPs. The average number of satellite Au NPs around per core Ag NP is ~20, which is calculated from 10 random core-satellite NPs in each SEM image. According to the Gaussian fittings of inter-particle gap of Au NPs displayed in Figure S3, the gap is 13.2 ± 4.4 nm calculated by measuring 200 random inter-particle gaps in the corresponding SEM images. In principle, the number of Au NPs around each Ag NP can be controlled through changing the length of Poly A block (e.g., Poly A10/30/50) assembled on the core Ag NP. In previous report, we have found that the average number of Au NPs around Ag NP mediated by Poly 50 is found to be ~10, which is much less than ~20 of Poly A30.44 And the average number of Au NPs around each Ag NP is ~60 when using Poly A10 as the mediator. However, in this case, the assembled efficiency of Ag-Au NPs@Si is much lower (e.g., ~49%), nearly half of that of Poly A30 (~90%).44 Consequently, Poly A30 is finally selected as the optimized mediator for the assembly of Ag-Au NPs@Si. In addition, in order to avoid the effect of Raman signals from Poly A30, the feed molar ratio of Poly A30-P1/ Poly A30-P2/ Au NPs is investigated. Typically, Raman signals of Poly A30 are not observed when the amounts of Poly A30-P1, Poly A30-P2, Au NPs are employed in a molar ratio of 50:50:1 (See Figure S4).
Figure 1. (a) Schematic illustration of preparation of SERS silicon chip (Ag-Au NPs@Si) assisted by Poly A (Figure was not to scale). Inset shows the photo of as-prepared chip (0.5 cm × 0.5 cm). (b) The corresponding SEM image of the resultant chip. Inset represents zoom-in SEM image of one core-satellite nanostructure. (c) UV-vis spectra of Au NPs (black), Ag NPs@Si (red), and Ag-Au NPs@Si (blue). (d) Raman spectra of R6G (10−4 M) distributed on pure silicon wafer (black), Ag NPs@Si (red) and Ag-Au NPs@Si (blue). (e) SERS mapping spectra of 10−4 M R6G collected from 50 random spots on the surface of chip. Excitation wavelength: 633 nm; laser power: 0.2 mW.
Next, we investigate the local surface plasmon resonance (LSPR) of SERS substrates mediated by Poly A block. As shown in Figure 1c, the UV-vis spectrum of Ag-Au NPs@Si (blue curve) exhibits two distinct absorption peaks at 433 and 555 nm, which are assigned to core Ag NPs and satellite Au NPs, respectively. Compared with the absorption peaks of Ag NPs@Si (red curve) located at 420 nm and Au NPs (black curve) located at 520 nm, the absorption peaks of Ag-Au NPs@Si slightly red-shift, indicating effective surface plasmonic couplings produced between Ag NPs and Au NPs as well as between metallic nanoparticles and dielectric silicon substrate. As a result, the developed SERS chip features much stronger Raman signals than those of pure silicon wafer and Ag NPs@Si (Figure 1d), greatly improving detection sensitivity. Typically, the EF value of Ag-Au NPs@Si is calculated to be ~5.6 × 106, ~two-fold higher than that of Ag NPs@Si (~2.1 × 106) under the same conditions (for a detailed calculation of EF, see the Supporting Information). More significantly, the prepared SERS silicon chip possesses excellent reproducibility owing to tightly anchoring metallic nanoparticles on the silicon support, effectively preventing random movement and
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aggregation of free nanoparticles. As shown in Figure 1e, uniform Raman spectra of R6G with a small RSD value of 15.6% (Raman intensity of R6G at 1364 cm-1) are observed, which are randomly collected from 50 spots distributed on the chip.
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of Pb2+ concentration attributed to the narrowing distance between Cy5 and SERS substrates. In this study, normalized Raman intensity (Iwith Pb2+/IBG) is employed to eliminate systematic errors. Remarkably, a good linear relationship between Iwith Pb2+/IBG and the logarithmic concentration of Pb2+ ranging from 10 pM to 1 µM is observed in Figure 2c. The corresponding regression equation is Y = 58.33 + 5.15 log10X (correlation coefficient, R2= 0.997), where, Y and X represent the normalized Raman intensity and the concentration of Pb2+, respectively. More importantly, this novel kind of SERS chip possesses ultrahigh sensitivity due to its comparatively good SERS enhancement, enabling the determination of trace Pb2+ with limit of detection (LOD) down to 8.9 × 10-12 M by setting the signal-to-noise ratio of 3:1 (for a detailed calculation of LOD, see the Supporting Information), which is around three orders of magnitude lower than the value prescribed by the U. S. EPA,6 and much lower than the best values reported by ever developed SERS sensors.23,24
Scheme 1. Schematic Illustration of Functionalized SERS Silicon Chip for Pb2+ Detection. Then, the as-prepared Ag-Au NPs@Si is employed for constructing SERS sensors for Pb2+ sensing through conjugation with double-stranded DNA (dsDNA), which is composed of Pb2+ ions-specific DNAzyme strand (Cy5-17E-SH) and corresponding substrate strand (17DS). As illustrated in Scheme 1, in the absence of Pb2+ ions, the Raman reporter of Cy5 labeled at one end of enzyme strand (blue font) stays >~6.8 nm away from the SERS substrate, generating feeble Raman signals due to the rigid structure of ds-DNA (Signal off). On the contrary, in the presence of Pb2+, Cy5 is close to the SERS substrate (< 1nm) because of flexible structure of enzyme strand when DNAzyme is activated via the complexation with Pb2+, and then cleaves the substrate strand (black font) into two fragments, resulting in significant Raman signals (Signal on).20, 21 Since the change of Raman intensity exhibits distinct concentration-dependent manner, the unknown Pb2+ can be quantitatively identified according to the corresponding calibration curves. As a proof-of-concept study, we first validate the feasibility of the functionalized Ag-Au NPs@Si for the identification of Pb2+ ions from the distilled water samples. As shown in Figure 2a, obvious Raman signals of Cy5 are observed in both Ag-Au NPs@Si and Ag NP@Si-based SERS-active substrates once addition of Pb2+. On the contrary, weak Raman signals of Cy5 are measured in the corresponding control groups (without Pb2+). Noted that, according to the quantitative intensity of the characteristic Raman peak of Cy5 at 1366 cm-1 (see inset in Figure 2a), the SERS intensity of Poly A-assisted Ag-Au NPs@Si-based SERS substrates is calculated to be ~2.0 times stronger than that of the Ag-Au NPs@Si-based substrates under the same conditions owing to superior SERS enhancement of the Ag-Au NPs@Si, which is in good agreement with the prior discussion. To confirm the SERS reproducibility when sensing Pb2+, we compared SERS signals of Cy5 in the presence of Pb2+ with different concentrations (Figure S5). The SERS mapping spectra together with corresponding RSD values (
