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Field and Pretreatment-Free Detection of Heavy-Metal Ions in Organic Polluted Water through an Alkyne-Coded SERS Test Kit Yi Zeng,† Jiaqiang Ren,† Aiguo Shen,* and Jiming Hu* Key Laboratory of Analytical Chemistry for Biology and Medicine (Ministry of Education), College of Chemistry and Molecular Sciences, Wuhan University, Wuhan 430072, P. R. China S Supporting Information *
ABSTRACT: Field and pretreatment-free detection of heavymetal ions in organic polluted water is important but still challenging in current water pollution emergency response systems. Here we report a polyadenine−DNA-mediated approach for a rationally designed alkyne-coded surfaceenhanced Raman scattering (SERS) test kit, enabling rapid and simultaneous detection of Hg2+ and Ag+ by a portable spectrometer, impervious to organic interferences. Because of the formation of thymine (T)−Hg2+−T and cytosine (C)−Ag+− C, highly recognizable SERS signals are rapidly detected when two different alkyne-labeled gold nanoparticles (AuNPs) are induced to undergo controllable bridging upon the addition of low-volume targets. For multiplex detection through a portable spectrometer, the limits of detection reach 0.77 and 0.86 nM for Hg2+ and Ag+, respectively. Of particular significance, the proposed CC-containing Raman reporters provide an extremely effective solution for multiplex sensing in a spectral silent region, when the hyperspectral and fairly intense optical noises originating from lower wavenumber region (1800 cm−1) with nearly only one characteristic peak (2212 cm−1 for OPE1 and 2158 cm−1 for OPE2) has been achieved (Figure S1), which is essential for multiplex labeling to avoid signal overlap of the tags. From the DFT calculation results, the assignment of the alkynyls’ vibration bands in different reporters has been reliably approved (Figure S2). First of all, a comparison between a conventional Raman reporter and a novel alkyne reporter was performed, highlighting the special analytical capability of alkyne. As shown in Figure 1a, the abundant signal from five typical conventional Raman reporters overlapped severely in the fingerprint region [p-mercaptobenzoic acid (4-MBA), malachite green, rhodamine 6G, rhodamine B, and methyl orange]. 4-MBA was chosen because it possesses the simplest emission peaks among those above. A series of control tests were proposed by the simultaneous use of common 4-MBA and OPE1. Here, four dyes were selected as typical organic pollutants in water samples: malachite green, methylene blue, methyl orange, and rhodamine 6G.17−22 The concentration was set as 5 μM for each dye. According to the literature,17−22 even after purification, the concentrations of the remaining dyes in the sample are still around 20−100 μM for each organic pollutant. As shown in Figure 1a, generally, both characteristic bands of 4MBA at 1076 and 1580 cm−1 in the fingerprint region can be used for quantification. However, once an organic-rich sample was added, a high optical response of the dyes largely masked the signals of 4-MBA. The conventional reporter immediately became incapable of reliable quantitative assay. Actually, the original SERS spectra before baseline correction can confirm this point more convincingly because the broad high fluorescence emission of interferences has already disabled the specific bands of 4-MBA by totally covering them up (Figure S3). The specific band intensity turned out to be meaningless and invalid. Furthermore, it can be easily inferred that, except for these dyes, plenty of organics that possess a spectroscopic signature would also certainly impede spectral collection in various optical methods. Meanwhile, because the conventional quantified ability was suppressed in the fingerprint region, the OPE1-coded SERS test kit can still complete satisfying quantification in the spectral-silent region (Figure 1b). The result proved that, for metal-ion detection in organicrich samples, conventional probes were helpless while an alkyne-coded test kit was impervious to interferences. The DFT calculation results in a silent region, and the designs of alkyne-coded SERS NPs are shown in Figure 2a,b. We present a fairly simple and one-step synthetic route for this alkyne-coded SERS test kit. With the alkyne-containing reporters attaching on the surface of the AuNPs, bivalent DNA−AuNPs are facilely prepared using unmodified diblock
Figure 2. (a) DFT-calculated spectra of OPE1 and OPE2. The insets represent corresponding ball-and-stick models (the gray ball stands for C, white for H, and yellow for S). (b) Schematic illustration for the design of alkyne-coded SERS NPs for metal-ion detection. (c) Typical TEM images of SERS NPs with increasing concentrations of metal ions.
DNA, which contains 100 adenines (polyA100) at its 5′ end, while the recognition blocks lift up at the surface to capture metal ions.33,34 The introduction of metal ions triggers the formation of T−Hg2+−T and C−Ag+−C complexes35 and induces the chainlike aggregation of NPs in a controlled manner. The lengths of the nanochains grow longer with increasing concentration of metal ions, which results in a corresponding increase of the SERS signals. The TEM images in Figure 2c visually confirm this. Before assay of the heavy-metal ions, the synthesized SERS NPs are fully evaluated on the signal reproducibility (10 times random test from 3 different batches of SERS NPs), the NP stability against time (10 times random test in 5 days), and the pH (3−12). As shown in Figure 3, each image stands for 10 spectra merged together from bottom to top, which are collected from the test kit under different testing conditions. The bright-yellow zone represents the location of the quantifiable peaks, specifically at 2212 cm−1 for the top three images and at 2158 cm−1 for the bottom three images. According to the color lump distribution, these two vital emission peaks are both highly uniform and reproducible. For OPE2, some unexpected intensity fluctuation around 2000 cm−1 appears in stability against time and pH values. C
DOI: 10.1021/acsami.6b09722 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX
Research Article
ACS Applied Materials & Interfaces
Gaussian 09 program, and the geometric structures are optimized via spin-restricted calculations by the B3LYP hybrid functional. For S and C atoms, the 6-311+G* basis set is adopted. The calculated spectra are not exactly the same but approximately agree with the experimental results. Specific peaks turn out to be 2158 and 2206 cm−1 in Figure S2. For the unstable broad band around 2000 cm−1, normally thiol from signal molecules directly contacts with the metal surface; if part of the alkynes did that instead, a single broad peak at 2096 cm−1 would be generated. The corresponding computational SERS spectra are shown in Figure S4. It is clear that this phenomenon arises from different binding motifs for the molecule at the particle surface. Thus, for reporter 2, the peak randomly arises, but for reporter 1, the internal alkynyl has much more difficulty making direct contact with the metal surface because of higher steric hindrance from two adjacent bulky benzene rings, so no peak in the lower-wavenumber region can be observed. For a single component, highly sensitive and selective detection of Hg2+ is achieved by monitoring the intensity changes of the SERS peak of OPE1. The freshly prepared OPE1-labeled AuNPs are functionalized with a pair of T-rich DNA sequences, which consist of partially complementary sequences to specifically recognize Hg2+. Here, five mismatched T−T base pairs are designed for the ligation of Hg2+ ions, and six intervening matched C−G base pairs are used to enhance the affinity, as well as to avoid self-folding of the single-stranded DNA. Specifically, polyA is utilized instead of the thiolated DNA to achieve a relatively controllable aggregation degree and subsequently to get better reproducible SERS signals. Generally, the thiolated DNA is difficult to be precisely modify on the surface of AuNPs in some quantity. The SERS NPs with lots of DNA strands would fiercely aggregate into a dense block
Figure 3. Performance tests of the OPE1-coded (a) and OPE2-coded (b) SERS NPs: substrate signal reproducibility, stability against time, and pH values.
To figure out this phenomenon and ensure the feasibility and accuracy of this test kit, the Raman shifts of alkynyls in different substitution patterns are evaluated and speculated with the aid of complementary DFT calculation. It is performed using the
Figure 4. SERS spectra, linear plots, and selectivity tests of the alkyne-coded single-component SERS test kit: OPE1-coded SERS test kit for Hg2+ (a−c). SERS spectra and the corresponding linear plots for multiplex detection of Hg2+ and Ag+ (d−f). D
DOI: 10.1021/acsami.6b09722 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX
Research Article
ACS Applied Materials & Interfaces in the presence of Hg2+ ions, resulting in a tremendous and unstable increase of the Raman signals. In contrast, DNA with consecutive adenines can strongly bind to AuNPs by the intrinsic high-affinity absorption of polyA, whereas the recognition block adopts an upright conformation that favors DNA hybridization. Besides, the surface density of DNA on AuNPs can be systematically modulated by adjusting the length of polyA.33 Inspired by these, a single 20 nm AuNP accommodating only two or three strands of DNA is designed when the length of polyA is sufficiently long as polyA100. Because most conjugates are bivalent DNA−AuNPs, the addition of Hg2+ or Ag+ ions therefore induces the formation of a uniform nanochain rather than a random block,36 which could be further confirmed by typical TEM images in Figure 2c. From this figure, a good yield of dimers has been observed from the starting point, and then the nanochains gradually grow longer with increasing concentrations of Hg2+ or Ag+ ions. Thus, the polyA−DNA-assisted fabrication method allows only two or three strands of DNA to attach to the surface of a single AuNP, subsequently ensuring better reproducibility of the SERS assay than in the previous work.37 In this assay, the highly tunable hybridization ability forms the basis of a rapid plasmonic DNA probe. Figure 4a presents highly recognizable SERS signals that are rapidly detected when OPE1-labeled AuNPs are induced to undergo controllable bridging upon the addition of a series of gradient concentrations of Hg2+ ions, respectively. With increasing concentrations of target, SERS bands of OPE1 at 2212 cm−1 unambiguously increase in intensity, which indicates that Hg2+ ions make the OPE1-labeled AuNPs aggregate by means of stabilizing the mismatched T−T pairs. The Raman spectra are processed through baseline correction and the intensities normalized with respect to the intensity of the blank sample. The corresponding quantitative calibration curve against the Hg2+ concentration ranging from 1 nM to 10 μM has been plotted and further summarized in Figure 4b. Above 10 μM, the intensity still slowly increases but deviates from linearity. The regression linear equation is determined as y = 654 log x + 208 (10 < x < 10000) with the squared correlation coefficient R2 = 0.99, where y is the normalized SERS intensity and x is the concentration of Hg2+ (nM). The result shows a limit of detection (LOD) based on 3σ of 0.36 nM. For the selectivity of this assay (Figure 4c), the separate addition of each kind of ion, including Ag+, Cu2+, Fe3+, Pb2+, Zn2+, Cr3+, Mn2+, Ca2+, Cd2+, Mg2+, and Ni2+, into the aqueous samples (5 times over the Hg2+ concentration) does not result in obvious changes of the SERS band at 2212 cm−1, whereas Hg2+ causes a very intense one. Furthermore, the detection of Hg2+ ions is also investigated in the presence of all of these mixed ions, which, to a certain extent, demonstrates the interference-free ability of this test kit. Correspondingly, C-rich DNA is employed for the detection of Ag+ ions basically in the the same way as Hg2+, with a detected range from 0.1 nM to 10 μM and a LOD of 0.06 nM (Figure S5). Successful selective analysis of a single component enables multiplex analysis. Simultaneously, dual-component detection is first carried out by a confocal micro-Raman spectrometer. Two groups of an alkyne-coded Raman test kit are mixed together in a 1:1 proportion. Mixed metal ions are separately recognized by specific sequences simultaneously. The two specific bands at 2212 and 2158 cm−1, originating from OPE1 and OPE2, respectively, increase, which implies successful application of
the simultaneous detection of both ions (Figure 4d−f). The LODs of Hg2+ and Ag+ are 0.47 and 0.38 nM, respectively. Dual-component detection led by a portable Raman spectrometer further applies this strategy to real and field detection. The simple and rapid detection and SERS spectra are illustrated in Figure 5, and the corresponding logistic curves of
Figure 5. Schematic illustration of portable multiplex detection with an alkyne-coded SERS test kit for metal ions and the corresponding SERS spectra.
the normalized intensities are shown in Figure S6. This multiplex assay for Hg2+ and Ag+ exhibits a 5 orders of magnitude dynamic range and LODs of Hg2+ and Ag+ that turn out to be 0.77 and 0.86 nM, respectively. Notably, real sample detection is carried out by spiking dyes and dual metal ions in water. The as-prepared organic-rich water samples are determined by both a portable Raman spectrometer and an atomic absorption spectrophotometer, in order to compare the developed SERS test kit with a conventional AAS method. Especially, a nitric acid digesting sample pretreatment is necessary to remove organics before delivery into the atomic absorption spectrophotometer, which might bring more interference into the system. As a result, the alkyne-coded SERS test kit provides better correlation with spiked statistics (Table S2).
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CONCLUSION Detection methods along with nanomaterials adapt to allow miniaturization and in-field applications. In parallel with other metal-ion-sensing platforms, this protocol presents tremendous significance and creativity in on-spot detection. Coupling the spectroscopic signature of alkyne with Raman microscopy yields a new probe modality beyond fluorescence and label-free microscopies. An alkyne-coded SERS test kit should be recognized as an ideal SERS probe for metal-ion quantitative analysis for the following reasons: (1) the extremely valuable analytical feature of being unaware of an organic matrix during spectral collection; (2) its ability to manage simultaneously multicomponent detection; (3) its ability to achieve rapid, direct, and field detection by portable Raman instruments. In summary, toward Hg2+ and Ag+ quantification in an organic matrix, we first utilize the specific alkyne vibration in the silent region to get rid of confused information in the fingerprint region from interferences. Given that this alkyne-coded SERS E
DOI: 10.1021/acsami.6b09722 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX
Research Article
ACS Applied Materials & Interfaces test kit is facile, reliable, and field-detection-available, we thus envision that it may serve as a practical analytical tool for precisely probing metal ions and can be further developed for biological probes.
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ASSOCIATED CONTENT
* Supporting Information S
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsami.6b09722. Additional tables and figures including sequences of DNA, SERS spectra for feasibility, DFT calculation for OPE1 and OPE2, comparison of SERS spectra by MBA, a computational SERS spectrum of 4-ethynylbenzenethiol, SERS spectra, linear plots and selectivity tests for Ag+, logistic calibration curves for multiplex quantitative assay by a portable Raman spectrometer, and an organicrich water sample determined by AAS and the SERS test kit (PDF)
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AUTHOR INFORMATION
Corresponding Authors
*E-mail:
[email protected]. *E-mail:
[email protected]. Author Contributions †
These authors contributed equally.
Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS We express our sincere thanks to Prof. Deyin Wu and Xiaguang Zhang for their general help in providing DFT calculations. We gratefully acknowledge financial support from the National Natural Science Foundation of China (Grants 21475100, 81471696, 41273093, and 21175101), Foundation of China Geological Survey (Grant 12120113015200), and Natural Science Foundation of Hubei Province of China (Grant 2014CFA002).
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DOI: 10.1021/acsami.6b09722 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX
