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A simple one-step pretreatment, highly sensitive and selective sensing of 17β-estradiol in environmental water samples using surface-enhanced Raman spectroscopy
Accepted Manuscript Title: A Simple One-Step Pretreatment, Highly Sensitive and Selective Sensing of 17-Estradiol in Environmental Water Samples Using Surface-enhanced Raman Spectroscopy Authors: Siyao Liu, Ruojie Cheng, Yuqing Chen, Huijie Shi, Guohua Zhao PII: DOI: Reference:
S0925-4005(17)31425-9 http://dx.doi.org/doi:10.1016/j.snb.2017.08.003 SNB 22870
To appear in:
Sensors and Actuators B
Received date: Revised date: Accepted date:
2-5-2017 7-7-2017 1-8-2017
Please cite this article as: Siyao Liu, Ruojie Cheng, Yuqing Chen, Huijie Shi, Guohua Zhao, A Simple One-Step Pretreatment, Highly Sensitive and Selective Sensing of 17-Estradiol in Environmental Water Samples Using Surface-enhanced Raman Spectroscopy, Sensors and Actuators B: Chemicalhttp://dx.doi.org/10.1016/j.snb.2017.08.003 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 proof before it is published in its final 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.
A Simple One-Step Pretreatment, Highly Sensitive and Selective Sensing of 17β-Estradiol in Environmental Water Samples Using Surface-enhanced Raman Spectroscopy
Siyao Liu, Ruojie Cheng, Yuqing Chen, Huijie Shi, Guohua Zhao*
School of Chemical Science and Engineering, and Shanghai Key Lab of Chemical Assessment and Sustainability, Tongji University, 1239 Siping Road, Shanghai 200092, People’s Republic of China
*Corresponding Author: Phone: +(86)-21-65981180. Fax:+(86)-21-65982287 E-mail: [email protected]
Highlights SERS improved the sensitivity of detection of E2 by 4-MBA labelled Au@Ag CS NPs. Aptamers gave the selectivity of SERS method. Aptamer-based SERS biosensing system had the great anti-interference ability. Real water samples were only treated by the simple membrane filtration.
Abstract: A highly sensitive and selective aptamer-based surface-enhanced Raman spectroscopic biosensing system was established for detecting trace amount of 17β-estradiol (E2) for the first time, and applied in the environmental samples based on a simple one-step pretreatment. Raman reporter molecule 4-mercaptobenzoic acid labelled gold-silver core-shell nanoparticles (Au@Ag CS NPs) and E2-aptamer endowed SERS with high sensitivity and selectivity. A wide linear range from 0.1pM to 10 nM was obtained for the detection of E2, with a low detection limit of 0.05 pM. Additionally, this system showed excellent selectivity for E2, where the Raman intensity of E2 was greater than 3.97-fold that of 100-fold concentration of other interfering substances. The high binding affinity of the E2-aptamer towards E2 was further investigated by the UV-vis absorption measurements, whereas E2-aptamer showed no binding affinity to other interferents. Finally, E2 in the environmental water samples collected from the sewer and nearby river of local obstetric hospital were successfully determined by this system, which exhibited superior sensitivity.
This work has provided a new detection biosensing system for trace determination of typical contaminants in the environment. Keywords: Surface-enhanced Raman spectroscopy; 17β-estradiol; Selective analysis; Simple pretreatment; Coexisting system 1. Introduction The most active nature estrogen 17β-estradiol (E2), as a typical environmental endocrine disrupting chemical (EDC)[1-3], possesses a very long residual period and low concentration in the environment. However, it brings many deleterious effects to the human and the aquatic organisms[4-6], due to its strong bioaccumulative toxicity[7], which causes disequilibrium in endocrine function and even results in cancers[6, 8]. E2 in the environment is mainly derived from the excretion of animals and human, and the emissions from breeding industrials, then flows into the surface water through sewage and other methods, which impacts on the normal growth of aquatic organisms directly. Toxicological experiments have shown that the male fishes that exposed to E2 at a concentration of 3.68-184 pM synthesized vitellogenin in vivo[9]. The United States National Environmental Protection Agency proposed the maximum residue of E2 in surface water was 1.47 pM in 2012[10], and Japan also increased the new regulations that E2 in drinking water was limited to 0.294 nM in 2015. Therefore, it cannot be ignored to detect E2 in the environment with low concentration and high toxicity. It is of great significance to protect the environment and human health by establishing a simple, rapid, efficient and sensitive method for the detection of E2.
To date, some new analytical methods such as high performance liquid chromatography (HPLC)[11,12], liquid chromatography combined with mass spectrophotometer (LC-MS)[13], immunoassay[14,15], colorimetric[16] and electrochemical[17-19] methods are used for the detection of E2. These methods have low detection limit of nM or pM for E2. However, for the conventional analytical methods like LC-MS and HPLC, complex pretreatments such as purification, concentration and extraction of the samples are required. Besides, it also reveals the shortcomings including exploring the complex detection conditions of the instrument, the difficulty and the longtime of analysis. Moreover, immunological analysis based on antibody-receptor, such as fluorescence immunoassay, and chemiluminescence immunoassay[20], show high sensitivity and sensitivity in E2 determination, but the biocomponents are expensive, and the requirements of environmental water samples are harsh, which needs to be separated and purified in order to improve the analytical sensitivity. Colorimetric method based on the Au NPs and aptamers, possesses the advantages such as the low experimental cost and simple operation, but the high purity is necessary, so that the water samples in the complex environment must be diluted and purified. In addition, electrochemical and photoelectrochemical[2] methods are not selective in their own, recognition elements are always required to achieve high selectivity, but the stability and reproducibility are still a major challenge, and the environmental water samples are detected after extraction or dilution. Therefore, development of the methods for highly sensitive detection of E2 with simple and rapid pre-treatment are very important.
Compared with the above-mentioned detection methods, it is noticed that surface-enhanced Raman spectroscopy (SERS) as no damage detection method is applied to environmental biology analysis nowadays, such as the detection of protein[21], toxins[22], bacteria[23], heavy metals[24] and etc. Recently, organic pollutants such as PCB77[25], bisphenol A[26,27], pesticide[28,29] have also been detected by SERS, because it can provide rich molecular information and has the characteristics of fastness, sensitivity, non-destruction and simplicity[30]. However, given that he intensity of Raman spectra of organic small molecules E2 is low, E2 only can be detected at high concentrations. While the SERS can greatly enhance the detection sensitivity because the Raman enhancement factors are on the order of 1014 to 1015[31], and substrate labelled with Raman reporter molecule can enhance the sensitivity of SERS to detect E2. More importantly, the environmental water samples are complex systems that contain E2 and various other coexisting contaminants. So it is significant to achieve the selective detection of E2 in complex coexisting system. SERS combined with random forest[32] or immunoassays[33] is used to detect E2 with low detection limit. However, narrow linearity range or high cost becomes its weakness. Aptamers with specific binding ability to the target E2 are capable of endowing SERS with selectivity. Nucleic acid aptamers are single-stranded DNA or RNA, which are found in recent years that can selectively combine specific targets. Because of plenty of advantages such as the lack of immunity in the special physiological environment, good stability, simple preparation method, low cost and easily modification[34,35], aptamers have been used with a variety of signal
converters for the detection or identification of a variety of substances, such as acetamiprid[36], PCBs[37], bisphenol A[38] and heavy metal ions[39], obtaining the excellent specific recognition property. Therefore, it is envisioned that the combination of SERS with aptamers biosensing system can establish a specific assay for highly sensitive and selective detection of E2. Based on the above, we propose a SERS biosensing system to be applied in the complex water sample analysis, which combined the sensitivity of SERS technique with the aptamer to realize specific recognition of E2. It showed high sensitivity and high selectivity in E2 detection. The enhancement effect of Au@Ag CS NPs was investigated by the signal intensity of 4-mercaptobenzoic acid (4-MBA). The sensitivity and selectivity of this method to detect E2 were also studied. Moreover, the binding ability of aptamer towards E2, as well as anti-interference ability and selective performance in the presence of other contaminants were further investigated by UV-vis spectrophotometry. Herein, we report a novel SERS-based biosensing system which was applied to detect the concentration of E2 from the typical water samples in different fields, including medical waste water and surrounding river wastewater. It has a good application prospect to test the trace content of E2, and provides a novel method to detect different environmental pollutants in real water samples.
2. Materials and methods 2.1 Materials and reagents
The single-stranded DNA aptamer of E2 was synthesized from Shanghai, containing 76 bases of aptamers[40], and purified by HPLC. 4-MBA and HEPES were purchased from Shanghai Aladdin Biochemical Technology Co., Ltd. Chloroacetic acid (50% Au) and E2 were purchased from Sigma Chemical reagents. All glass instruments were immersed in aqua regia (HCl / HNO3 = 3: 1) for 12 h and rinsed with double distilled water for subsequent experiments. All experiments were performed in 50 mM HEPES buffer solution. All solutions were prepared with double distilled water without special mention. The morphologies of Au @ Ag CS NPs were characterized by field emission electron microscopy (FE-SEM, Hitachi S-4800, Japan) and high resolution transmission electron microscopy (HRTEM, JEM-2100, Japan). The UV absorption spectra of Au @ Ag CS NPs was measured in a 200 μL quartz cuvette using an ultraviolet-visible spectrophotometer (UV-Vis, Agilent, 8453). Raman spectra was performed using Renishaw's Invia Reflex microscopy confocal Raman spectrometer. All spectra were calibrated referring to the 520 cm-1 line of silicon. A 20× objective lens was used to focus a laser spot. A semiconductor laser operating at λ =532 nm was used as the excitation source with a laser power of approximately 5 mW. All the Raman spectra were the results of 5s twice accumulations.
2.2 Establishment and detection performance of E2 SERS biosensing system Au @ Ag CS NPs were fabricated by typical chemical reduction methods and seed-induced growth methods in the light of the previous reports[41]. Briefly, 300 mL
of 0.01% (w / w) of HAuCl4 was boiled and then 4.5 mL of 1% (w / w) of sodium citrate was added, stirring at 100 ° C for 20 min until the solution became bright red. Then 2.7 mL of AgNO3 solution (0.5% w / w) and 3 mL of 1% (w / w) of sodium citrate solution were added to the boiling Au NPs solution, and kept boiling for 1 h. After that, the solution was cooled down to room temperature and stored at 4 °C in the refrigerator. 100 mL solution of Au @ Ag CS NPs was added with 0.5 mL of 1 mM 4-MBA and stirred for 4 h to obtain Au@Ag-4MBA solution. Then, 10 μl of 120nM aptamer and 10 μl of different concentrations E2 solution were mixed and shaken for 10 min, then added to the above-mentioned 200 μl of Au @ Ag-4 MBA solution and finally added to the HEPES solution to 400 μl , after shaken 25min to test the Raman intensity. In addition, the interferons including ethinylestradiol, estriol, DBP, atrazine, bisphenol A, 4-nonyl phenol and humic acids, and D-Dextran and bovine serum albumin were measured exactly as E2 illustrated above. Furthermore, the UV absorption spectra of the aptamers with different concentrations of E2 and different other contaminants were determined by Agilent 8453 UV/vis spectrophotometer. The testing methods in digital substation were as follows: On the one hand, adding 0.01 μmol / L of 200 μL aptamer solution into 6 vials, respectively. Then the different concentrations of E2 were added in the vials, and obtained final E2 concentrations were 0 μmol / L, 0.025 μmol / L, 0.05 μmol / L, 0.1 μmol / L, 0.2 μmol / L, 0.4 μmol / L, 0.5 μmol / L, 1 μmol / L, respectively. On the other hand, 1μmol / L of E2 aptamer solution and the same volume concentration
of 1μmol / L of different contaminants were added into 10 sample vials, respectively. After interacting 15 minutes, the mixtures were treated with UV-visible spectrophotometry and the wavelength was measured from 220 to 320 nm.
2.3 E2 SERS biosensing system of actual samples Environmental water samples were collected from the obstetric hospital sewer and obstetric hospital surrounding river. All the samples were filtrated method by 0.22 μm membrane to remove particles and suspended solids, and the obtained sample was left at a temperature of 4 °C. The sample was tested within 1 day. In the test, the environmental water samples prepared and E2 aptamers were incubated and then detected by the established method prepared and determined by SERS as illustrated above to assist in quantitative analysis of E2 in the environmental water samples.
3. Results and discussion 3.1 Highly sensitive performance of E2 SERS biosensing system Scheme 1 illustrates the schematic representation of the detection of E2 with SERS. The Raman-enhanced substrate Au@Ag CS NPs was prepared by citrate reduction and seed-induced growth methods. 4-MBA, served as Raman reporter molecules, was adsorbed on the surface of Au@Ag CS NPs through the mercapto group. Due to the "hot spot" effect of the Au@Ag CS NPs, narrow Raman peaks of 4-MBA was obtained with high intensity[22,27]. When the aptamers serving as identification elements were added, the aptamers interacted with Au@Ag CS NPs by N atom
attaching to the phosphoric acid. Due to the electrostatic interaction originated from aptamers, the Au@Ag CS NPs were separated, leading to the weakened "hot spot" effect and the reduced 4-MBA Raman intensity[41]. When E2 was added to the system, due to the specific binding effect between E2 and aptamers, E2-aptamer complex was formed and got away from the surface of Au@Ag CS NPs. Therefore, Au@Ag CS NPs lost the protection from aptamer, and more "hot spots" were produced again. Thus the surface enhancement effect of Au@Ag CS NPs recovered, and so as the Raman intensity of 4-MBA. By monitoring the changing of Raman intensity before and after E2 addition, we successfully established a SERS bioanalytical assay for the detection of E2. Among SERS substrate, AuNPs and AgNPs were the most extensively used to enhance the Raman intensity[27]. AgNPs were employed as SERS substrate because of the strong surface enhancement effect, but the poor stability limited its application. AuNPs had good stability, but the SERS effect was relatively weak[42]. Therefore, Au@Ag CS NPs were prepared in this work to achieve a win-win effect. Fig. 1A showes the improved surface enhancement effect of Au@Ag CS NPs. In the present of Au @ Ag CS NPs, a narrow and high intensity Raman peak was obtained for 4-MBA, although the intrinsic Raman intensity of 4-MBA was very weak, and the enhancement factor of Au@Ag CS NPs was about 6.868×104 for 4-MBA(see details in the Supporting Information), while the Raman intensity was lower in the presence of AgNPs or AuNPs(Fig. 2S). Besides, the stability of Au@Ag CS NPs was investigated in Fig. 1B, the SERS enhancement factor of Au@Ag CS NPs was almost
constant when stored at 4 °C for 0, 1, 2, 3 weeks. The UV-vis maps of AuNPs and Au @ Ag CS NPs are shown in Fig. 1C. It was found that AuNPs had a peak at 520 nm, while Au @ Ag CS NPs showes two peaks at 510 nm and 390 nm, respectively, which were the specific peaks of Au and Ag. The UV absorption peak was blue shifted after Au NPs coated by Ag shell, which means Au core was wrapped around by Ag shell[43]. It was further confirmed by SEM and HRTEM characterization as displayed in Fig. 1D. It is notable to find that the AuNPs were surrounded by lots of Ag tiny particles, forming shell on Au surface. It might because silver nitrate solution reduced with sodium citrate around AuNPs. Because the Raman signal intensity, i.e. the sensitivity of the proposed method was highly influenced by the concentration of 4-MBA, the E2-aptamer and the reaction time of the aptamers-E2 complex with the substrate[44], the experiment conditions were optimized at firstly(Fig. S1). With the increment of 4-MBA concentration, the Raman intensity also increased, and reached to the maximum at 5.0 nM, while the stability of the Au @ Ag CS NPs might decline due to the high concentration of 4-MBA[41]. So a Raman reporter molecule with a concentration of 5.0 nM was chosen as a probe. Aptamers with different concentrations and 0.1 nM of E2 were mixed in an equal volume for 40 min. Then 20 μL of the above solution was added to 200 μL of Au @ Ag-4 MBA solution, and the Raman intensity was tested after 25 min. It was found that the Raman intensity was the highest when the concentration of the aptamer was 120 nM, thus determining the optimum concentration of aptamer under this condition. In addition, the incubation time of the aptamers-E2 and Au @
Ag-4MBA was optimized. After 120 nM of aptamer and 0.1 nM of E2 were mixed in equal volume, 200 μL of Au @ Ag-4 MBA was added and incubated at different times. As the incubation time prolonged, the Raman intensity increased. When the incubation time was 25 min, the signal was enhanced to the maximum. So the incubation time of 25 min was determined to be optimal. In order to further demonstrate the sensitivity of the biosensing system, the Raman intensity changes after adding different concentrations of E2 was tested, as exhibited in Fig. 2. The Raman intensity at 1587 cm-1 was selected for quantifying E2. As the E2 concentration increased from 0.1 pM to 10 nM, the peak signal intensity also increased, as shown in Fig 2A and 2B. It noticed that the SERS signal was linear against the logarithm of E2 concentration between 0.1 pM and 10 nM. The fitting equation was obtained as y = 19321.06+ 2692.61lg x (x was the concentration of E2), the determination coefficient R2 was 0.999, the detection limit was 0.05 pM. Besides, the error bar demonstrated that the reproducibility of our biosensing system to detect E2 was great. The results indicates the high sensitivity of the SERS analysis, which was sufficient to meet the detection of E2 requirements in the environmental water samples.
3.2 Highly selective performance of E2 SERS biosensing system Environment is a complex system, in which there are a variety of organic pollutants, sugars, proteins and other substances[45]. And these substances may affect SERS analysis of E2. Therefore, the E2 selectivity and anti-interference ability of this assay
were futher investigated. Fig. 3A and 3B exhibites that if there was no aptamer, no E2 and no other substances, due to the "hot spot" effect of the Au@Ag CS NPs, narrow Raman peaks of 4-MBA were obtained with high intensity. If there was no E2 aptamer in the system, the addition of E2 could not alter the Raman peak intensity at 1587 cm-1 because of the no change of the the "hot spot" effect of the Au@Ag CS NPs. So did the interferents of dibutyl phthalate (DBP) and bovine serum albumin (BSA). On the contrary, if the aptamer was present, because of the electrostatic interaction originated from aptamers, the Raman peak intensity decreased. On this basis, the Raman peak intensity increased evidently after addition of E2, but lower than the initial condition of no aptamer and no E2 because there were part of aptamers interacting with Au@Ag CS NPs, leading to the weakened "hot spot" effect and the reduced 4-MBA Raman intensity.Meanwhile, the coexisted DBP or BSA exhibited no interference on E2 response. It indicates that the biosensing system showed a good selectivity for E2. Fig. 4 displays the relative Raman peak intensity of 10 pM E2 and 1 nM other ten types of interferons, including oestrone, estriol, DBP, atrazine, bisphenol A (BPA), 4-nonyl phenol, humic acids, D-glucan and BSA individually and 50mM NaCl solution. It is found that the assay was only responsive to E2, and the ratio R, which was the ratio of Raman peak intensity between interferons and E2, was less than 0.252, indicating that the excellent selectivity for E2. Furthermore, when it was used to detect E2 in the presence of all the above-mentioned substance solutions, it is concluded that the obtained signal intensity was consistent with the signal intensity detected only in the E2 solution, suggesting that other endocrine disruptors,
sugars, protein and other substances had little effect on the detection of E2. It further confirmed the anti-interference ability of the biosensing system. This high selectivity in E2 detection could be attributed to the strong binding affinity between the aptamers and E2, which was far greater than that with other substances[40]. Thus, the interaction between aptamer and E2 were studied by UV-vis absorption measurements in order to further demonstrate the superior selective performance of the biosensing system. The interaction between aptamer and other interferents were also investigated for comparison. The interaction between aptamer and E2 can be represented by the following equation 1: n E2 + aptamer → E2 (apt) (1) The Ka can be expressed as equation 2: Ka =
[E2(apt)] [E2]n ×[aptamer]
(2)
Here [E2(apt)], [E2] and [aptamer] represent the compound of aptamer with E2, E2 and aptamer, respectively. Equation on both sides of the logarithm was obtained as equation 3: lg
E2(apt) =nlg[E2]+lgKa (3) [aptamer]
The absorbance is proportional to the concentration of the absorption material according to Lambert–Beer law. So the equation 4 was obtained as follows: lg
(A0 -Ax ) = nlg[E2] + lgKa (4) (Ax -A1 )
There A0 , A1 represent the absorbance in the absence of E2 and the absorbance after exceeded addition of E2, respectively. Ax stands for the absorbance after adding a certain concentration of E2 to the aptamer solution. So there was a linear relationship
between logarithm of
(A0 -Ax ) (Ax -A1 )
and lg[E2] . If lg
(A0 -Ax )
=y, lg[E2] = x, the equation 5
(Ax -A1 )
can be obtained: y = nx + lgKa (5) Because of the constants of association constant (Ka) and comprehensive coordination number (n), the calibration plot gives Ka by the index of the intercept and n by the slop in Fig. 5A and B. The obtained linear regression equation was y = 5.8000 + 0.9608x, thus, Ka was 6.310×105/M, and n was 0.9608. Such high Ka value demonstrated that E2 aptamers had a strong recognition ability to E2. This was consistent with the reported Kd = 0.1 ~ 3μM[40]. Additionally, the absorbance of the mixture of aptamers and other pollutants was similar to that of the aptamers only. However, when adding the same concentration of E2, the absorbance decreased, as shown in Fig. 5C, which also displayed that the binding affinity of the aptamers for E2 was much higher than that for the other contaminants.
3.3 E2 SERS analysis of actual samples In order to further study the application of the biosensing system in environmental water samples, wastewater samples were collected from the sewer and nearby river of local obstetric hospital, where E2 was always present. All the samples were used after filtration by 0.22 μm membrane to remove particles and suspended solids. The environmental water samples, and that spiked with the standard E2 solution of 0.5, 1 nM were tested. The results are shown in Table 1. The recovery of the E2 SERS method was ranging from 93.9% to 104.6%, and the relative standard deviation was
between 7.4% and 27.6% (n = 3). Such excellent recovery and relative standard deviation owned to the selectivity of E2 SERS biosensing system Besides, it is found that E2 concentration of the obstetric hospital sewer was 10.8pM, while that in the hospital surrounding river was 1.3 pM. The high level of E2 present in the obstetric hospital sewer might be because that plenty of estrogen drugs were used to treat female patients and the excretion of pregnant women produced. The level of E2 in the obstetric hospital surrounding river was low perhaps because the water samples were related with water treatment before discharge. Besides, the recovery rate of the environmental spiked water samples was close to that of the standard HPLC method. It was worth noting that a complex multi-step pretreatment was required in detecting E2 by HPLC, while only a simple pre-filtration process was carried out for E2 SERS analysis. In addition, the E2 SERS assay was sensitive enough to detect the trace level of E2 in the water sample, whereas it could not be detected by HPLC. This approach was also more convenient for detecting E2 in environmental water samples considering the simple pre-treatment method compared to other literatures (Table S1). It was clear to find that one step of simple pretreatment based on the membrane filtration was needed in the detection of the actual sample, whereas different pretreatments such as purification, concentration, extraction and derivation of the samples were required in other analytical methods. Besides, it was the best detection limit of E2 in real water samples only by membrane filtration pretreatment, while a lower detection limit of 0.023pM was obtained by the fluorescence immunoassay after solid-phase extraction pretreatment[46]. In summary, the E2 SERS biosensing
system could achieve the goal of high sensitivity, high selectivity, simple pretreatment and easy operation.
4. Conclusion In this work, an aptamer-based SERS approach was successfully established for the detection of E2 with high sensitivity and high selectivity in complex environmental water samples for the first time. The biosensing system was based on Au @ Ag CS NPs as carrier, 4-MBA as a Raman reporter molecule, and aptamer of E2 as a recognition element. The detection of E2 exhibited a high sensitivity, with the linear range from 10-4 to 10 nM, and the detection limit of 0.05 pM. Moreover, this system exhibited a high selectivity to E2, achieving the trace detection of E2 in complex environmental water samples system. It is worth noting that this analytical method could be carried out following only by one step of simple pretreatment in the detection of the actual sample, which greatly reduced the complex, pre-processing operations in the instrumental detection of environmental water samples. Our work has provided a new analytical approach for the detection of endocrine disruptors in complex water samples.
Acknowledgements This work is supported by National Natural Science Foundation of China (Nos. 21537003), and the Science & Technology Commission of Shanghai Municipality (14DZ2261100).
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Author Biographies Siyao Liu is a master student in School of Chemistry Science and Engineering, Tongji University, Shanghai, China. Her research interest is biosensor. Ruojie Cheng is a master student in School of Chemistry Science and Engineering, Tongji University, Shanghai, China. Her research interest is biosensor. Yuqing Chen is a master student in School of Chemistry Science and Engineering, Tongji University, Shanghai, China. Her research interest is biosensor. Huijie Shi is an associate professor in School of Chemistry Science and Engineering, Tongji University, Shanghai, China. Her research interests are photoelectrocatalysis and its application in environmental analysis and contaminant control. Guohua Zhao is a professor in School of Chemistry Science and Engineering, Tongji University, Shanghai, China. His research interests are environmental electrochemistry, photoelectrochemistry, environmental/biology photoelectrochemical sensors, environmental energy chemistry.
Figure Captions Fig. 1. The characterization of Au@Ag CS NPs. (A) SERS spectra of 4-MBA absorbed on or not absorbed on the surface of Au@Ag CS NPs. (B) The raman enhancement of Au@Ag CS NPs after the synthesis of 0, 1, 2, 3 weeks. (C) UV-vis absorption spectra of Au@Ag CS NPs and Au NPs. (D) SEM images of Au@Ag CS NPs. The insert displays HRTEM images of Au@Ag CS NPs. Fig. 2. Highly sensitive performance of the E2 SERS detection. (A) SERS spectra with different concentrations of E2 (B) The linear calibration curve of SERS intensity at 1587cm-1 peak versus the concentrations of E2 from 0 pM to 10 nM. The inset exhibits The linear calibration curve of SERS intensity at 1587cm-1 peak against natural logarithm over concentration of E2. Fig. 3. Anti-interference ability and selectivity performance of the E2 SERS detection. (A) SERS spectra with different interferences in the absence of aptamers and the change of SERS spectra intensity in the presence of aptamers. (B)The change of raman intensity at 1587 cm-1 with different interferences in the absence of aptamers and in the presence of aptamers (here A, B, C, D, E, F, G, H represent no aptamer + no E2 + no others, no aptamer + E2 +BSA, no aptamer + E2 +DBP, no aptamer + E2, aptamer + no E2 + no others, aptamer + E2 +BSA, aptamer + E2 +DBP, aptamer + E2, respectively). Fig. 4. Highly selectivity performance of the E2 SERS detection. SERS intensity at 1587cm-1 peak relative signals of E2 in the presence of different interferences in the concentration. The insert shows the structural formulas of E2, ethinylestradiol, estriol,
dibutyl phthalate, atrazine, bisphenol A, 4-nonyl phenol, humic acids and D-glucan(from left to right), respectively. Fig. 5. UV spectra of aptamer after interacting with E2. (A) UV spectra of aptamer interacting with different concentrations of E2. (B) The linear calibration curve of lg(A0-Ax)/(Ax-A1) versus the logarithm of E2 concentration. (C) UV spectra of 1 nM aptamer interacting with different kinds of 1 nM interferences in the same volume. Scheme 1. Schematic illustration for the E2 SERS detection; the inset showed SERS spectra under different experimental conditions
Fig. 1.
Fig. 2.
Fig. 3.
Fig. 4.
Fig. 5.
32
Scheme 1.
33
Table 1. Performances of E2 SERS biosensing system and HPLC for E2 determination in real water
The SERS aptasensor Sample
HPLC
Add(nM) Mean±SD(pM)
CV(%)
Recovery(%)
Mean±SD(pM)
CV(%)
Recovery(%)
obstetric hospital
0
10.8±0.8
7.4
0
undetected
-
-
sewer
500
523.0±103.5
19.8
102
527.8±20.4
3.9
106
1000
1057.3±153.3
14.5
105
1012.2±15.0
1.5
101
obstetric hospital
0
1.3±0.2
11.5
0
undetected
-
-
surrounding river
500
470.8±34.1
7.2
94
512.4±28.5
5.6
103
1000
1002.9±276.7
27.6
100
999.8±19.5
2.0
100
S0925-4005(17)31425-9 http://dx.doi.org/doi:10.1016/j.snb.2017.08.003 SNB 22870
To appear in:
Sensors and Actuators B
Received date: Revised date: Accepted date:
2-5-2017 7-7-2017 1-8-2017
Please cite this article as: Siyao Liu, Ruojie Cheng, Yuqing Chen, Huijie Shi, Guohua Zhao, A Simple One-Step Pretreatment, Highly Sensitive and Selective Sensing of 17-Estradiol in Environmental Water Samples Using Surface-enhanced Raman Spectroscopy, Sensors and Actuators B: Chemicalhttp://dx.doi.org/10.1016/j.snb.2017.08.003 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 proof before it is published in its final 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.
A Simple One-Step Pretreatment, Highly Sensitive and Selective Sensing of 17β-Estradiol in Environmental Water Samples Using Surface-enhanced Raman Spectroscopy
Siyao Liu, Ruojie Cheng, Yuqing Chen, Huijie Shi, Guohua Zhao*
School of Chemical Science and Engineering, and Shanghai Key Lab of Chemical Assessment and Sustainability, Tongji University, 1239 Siping Road, Shanghai 200092, People’s Republic of China
*Corresponding Author: Phone: +(86)-21-65981180. Fax:+(86)-21-65982287 E-mail: [email protected]
Highlights SERS improved the sensitivity of detection of E2 by 4-MBA labelled Au@Ag CS NPs. Aptamers gave the selectivity of SERS method. Aptamer-based SERS biosensing system had the great anti-interference ability. Real water samples were only treated by the simple membrane filtration.
Abstract: A highly sensitive and selective aptamer-based surface-enhanced Raman spectroscopic biosensing system was established for detecting trace amount of 17β-estradiol (E2) for the first time, and applied in the environmental samples based on a simple one-step pretreatment. Raman reporter molecule 4-mercaptobenzoic acid labelled gold-silver core-shell nanoparticles (Au@Ag CS NPs) and E2-aptamer endowed SERS with high sensitivity and selectivity. A wide linear range from 0.1pM to 10 nM was obtained for the detection of E2, with a low detection limit of 0.05 pM. Additionally, this system showed excellent selectivity for E2, where the Raman intensity of E2 was greater than 3.97-fold that of 100-fold concentration of other interfering substances. The high binding affinity of the E2-aptamer towards E2 was further investigated by the UV-vis absorption measurements, whereas E2-aptamer showed no binding affinity to other interferents. Finally, E2 in the environmental water samples collected from the sewer and nearby river of local obstetric hospital were successfully determined by this system, which exhibited superior sensitivity.
This work has provided a new detection biosensing system for trace determination of typical contaminants in the environment. Keywords: Surface-enhanced Raman spectroscopy; 17β-estradiol; Selective analysis; Simple pretreatment; Coexisting system 1. Introduction The most active nature estrogen 17β-estradiol (E2), as a typical environmental endocrine disrupting chemical (EDC)[1-3], possesses a very long residual period and low concentration in the environment. However, it brings many deleterious effects to the human and the aquatic organisms[4-6], due to its strong bioaccumulative toxicity[7], which causes disequilibrium in endocrine function and even results in cancers[6, 8]. E2 in the environment is mainly derived from the excretion of animals and human, and the emissions from breeding industrials, then flows into the surface water through sewage and other methods, which impacts on the normal growth of aquatic organisms directly. Toxicological experiments have shown that the male fishes that exposed to E2 at a concentration of 3.68-184 pM synthesized vitellogenin in vivo[9]. The United States National Environmental Protection Agency proposed the maximum residue of E2 in surface water was 1.47 pM in 2012[10], and Japan also increased the new regulations that E2 in drinking water was limited to 0.294 nM in 2015. Therefore, it cannot be ignored to detect E2 in the environment with low concentration and high toxicity. It is of great significance to protect the environment and human health by establishing a simple, rapid, efficient and sensitive method for the detection of E2.
To date, some new analytical methods such as high performance liquid chromatography (HPLC)[11,12], liquid chromatography combined with mass spectrophotometer (LC-MS)[13], immunoassay[14,15], colorimetric[16] and electrochemical[17-19] methods are used for the detection of E2. These methods have low detection limit of nM or pM for E2. However, for the conventional analytical methods like LC-MS and HPLC, complex pretreatments such as purification, concentration and extraction of the samples are required. Besides, it also reveals the shortcomings including exploring the complex detection conditions of the instrument, the difficulty and the longtime of analysis. Moreover, immunological analysis based on antibody-receptor, such as fluorescence immunoassay, and chemiluminescence immunoassay[20], show high sensitivity and sensitivity in E2 determination, but the biocomponents are expensive, and the requirements of environmental water samples are harsh, which needs to be separated and purified in order to improve the analytical sensitivity. Colorimetric method based on the Au NPs and aptamers, possesses the advantages such as the low experimental cost and simple operation, but the high purity is necessary, so that the water samples in the complex environment must be diluted and purified. In addition, electrochemical and photoelectrochemical[2] methods are not selective in their own, recognition elements are always required to achieve high selectivity, but the stability and reproducibility are still a major challenge, and the environmental water samples are detected after extraction or dilution. Therefore, development of the methods for highly sensitive detection of E2 with simple and rapid pre-treatment are very important.
Compared with the above-mentioned detection methods, it is noticed that surface-enhanced Raman spectroscopy (SERS) as no damage detection method is applied to environmental biology analysis nowadays, such as the detection of protein[21], toxins[22], bacteria[23], heavy metals[24] and etc. Recently, organic pollutants such as PCB77[25], bisphenol A[26,27], pesticide[28,29] have also been detected by SERS, because it can provide rich molecular information and has the characteristics of fastness, sensitivity, non-destruction and simplicity[30]. However, given that he intensity of Raman spectra of organic small molecules E2 is low, E2 only can be detected at high concentrations. While the SERS can greatly enhance the detection sensitivity because the Raman enhancement factors are on the order of 1014 to 1015[31], and substrate labelled with Raman reporter molecule can enhance the sensitivity of SERS to detect E2. More importantly, the environmental water samples are complex systems that contain E2 and various other coexisting contaminants. So it is significant to achieve the selective detection of E2 in complex coexisting system. SERS combined with random forest[32] or immunoassays[33] is used to detect E2 with low detection limit. However, narrow linearity range or high cost becomes its weakness. Aptamers with specific binding ability to the target E2 are capable of endowing SERS with selectivity. Nucleic acid aptamers are single-stranded DNA or RNA, which are found in recent years that can selectively combine specific targets. Because of plenty of advantages such as the lack of immunity in the special physiological environment, good stability, simple preparation method, low cost and easily modification[34,35], aptamers have been used with a variety of signal
converters for the detection or identification of a variety of substances, such as acetamiprid[36], PCBs[37], bisphenol A[38] and heavy metal ions[39], obtaining the excellent specific recognition property. Therefore, it is envisioned that the combination of SERS with aptamers biosensing system can establish a specific assay for highly sensitive and selective detection of E2. Based on the above, we propose a SERS biosensing system to be applied in the complex water sample analysis, which combined the sensitivity of SERS technique with the aptamer to realize specific recognition of E2. It showed high sensitivity and high selectivity in E2 detection. The enhancement effect of Au@Ag CS NPs was investigated by the signal intensity of 4-mercaptobenzoic acid (4-MBA). The sensitivity and selectivity of this method to detect E2 were also studied. Moreover, the binding ability of aptamer towards E2, as well as anti-interference ability and selective performance in the presence of other contaminants were further investigated by UV-vis spectrophotometry. Herein, we report a novel SERS-based biosensing system which was applied to detect the concentration of E2 from the typical water samples in different fields, including medical waste water and surrounding river wastewater. It has a good application prospect to test the trace content of E2, and provides a novel method to detect different environmental pollutants in real water samples.
2. Materials and methods 2.1 Materials and reagents
The single-stranded DNA aptamer of E2 was synthesized from Shanghai, containing 76 bases of aptamers[40], and purified by HPLC. 4-MBA and HEPES were purchased from Shanghai Aladdin Biochemical Technology Co., Ltd. Chloroacetic acid (50% Au) and E2 were purchased from Sigma Chemical reagents. All glass instruments were immersed in aqua regia (HCl / HNO3 = 3: 1) for 12 h and rinsed with double distilled water for subsequent experiments. All experiments were performed in 50 mM HEPES buffer solution. All solutions were prepared with double distilled water without special mention. The morphologies of Au @ Ag CS NPs were characterized by field emission electron microscopy (FE-SEM, Hitachi S-4800, Japan) and high resolution transmission electron microscopy (HRTEM, JEM-2100, Japan). The UV absorption spectra of Au @ Ag CS NPs was measured in a 200 μL quartz cuvette using an ultraviolet-visible spectrophotometer (UV-Vis, Agilent, 8453). Raman spectra was performed using Renishaw's Invia Reflex microscopy confocal Raman spectrometer. All spectra were calibrated referring to the 520 cm-1 line of silicon. A 20× objective lens was used to focus a laser spot. A semiconductor laser operating at λ =532 nm was used as the excitation source with a laser power of approximately 5 mW. All the Raman spectra were the results of 5s twice accumulations.
2.2 Establishment and detection performance of E2 SERS biosensing system Au @ Ag CS NPs were fabricated by typical chemical reduction methods and seed-induced growth methods in the light of the previous reports[41]. Briefly, 300 mL
of 0.01% (w / w) of HAuCl4 was boiled and then 4.5 mL of 1% (w / w) of sodium citrate was added, stirring at 100 ° C for 20 min until the solution became bright red. Then 2.7 mL of AgNO3 solution (0.5% w / w) and 3 mL of 1% (w / w) of sodium citrate solution were added to the boiling Au NPs solution, and kept boiling for 1 h. After that, the solution was cooled down to room temperature and stored at 4 °C in the refrigerator. 100 mL solution of Au @ Ag CS NPs was added with 0.5 mL of 1 mM 4-MBA and stirred for 4 h to obtain Au@Ag-4MBA solution. Then, 10 μl of 120nM aptamer and 10 μl of different concentrations E2 solution were mixed and shaken for 10 min, then added to the above-mentioned 200 μl of Au @ Ag-4 MBA solution and finally added to the HEPES solution to 400 μl , after shaken 25min to test the Raman intensity. In addition, the interferons including ethinylestradiol, estriol, DBP, atrazine, bisphenol A, 4-nonyl phenol and humic acids, and D-Dextran and bovine serum albumin were measured exactly as E2 illustrated above. Furthermore, the UV absorption spectra of the aptamers with different concentrations of E2 and different other contaminants were determined by Agilent 8453 UV/vis spectrophotometer. The testing methods in digital substation were as follows: On the one hand, adding 0.01 μmol / L of 200 μL aptamer solution into 6 vials, respectively. Then the different concentrations of E2 were added in the vials, and obtained final E2 concentrations were 0 μmol / L, 0.025 μmol / L, 0.05 μmol / L, 0.1 μmol / L, 0.2 μmol / L, 0.4 μmol / L, 0.5 μmol / L, 1 μmol / L, respectively. On the other hand, 1μmol / L of E2 aptamer solution and the same volume concentration
of 1μmol / L of different contaminants were added into 10 sample vials, respectively. After interacting 15 minutes, the mixtures were treated with UV-visible spectrophotometry and the wavelength was measured from 220 to 320 nm.
2.3 E2 SERS biosensing system of actual samples Environmental water samples were collected from the obstetric hospital sewer and obstetric hospital surrounding river. All the samples were filtrated method by 0.22 μm membrane to remove particles and suspended solids, and the obtained sample was left at a temperature of 4 °C. The sample was tested within 1 day. In the test, the environmental water samples prepared and E2 aptamers were incubated and then detected by the established method prepared and determined by SERS as illustrated above to assist in quantitative analysis of E2 in the environmental water samples.
3. Results and discussion 3.1 Highly sensitive performance of E2 SERS biosensing system Scheme 1 illustrates the schematic representation of the detection of E2 with SERS. The Raman-enhanced substrate Au@Ag CS NPs was prepared by citrate reduction and seed-induced growth methods. 4-MBA, served as Raman reporter molecules, was adsorbed on the surface of Au@Ag CS NPs through the mercapto group. Due to the "hot spot" effect of the Au@Ag CS NPs, narrow Raman peaks of 4-MBA was obtained with high intensity[22,27]. When the aptamers serving as identification elements were added, the aptamers interacted with Au@Ag CS NPs by N atom
attaching to the phosphoric acid. Due to the electrostatic interaction originated from aptamers, the Au@Ag CS NPs were separated, leading to the weakened "hot spot" effect and the reduced 4-MBA Raman intensity[41]. When E2 was added to the system, due to the specific binding effect between E2 and aptamers, E2-aptamer complex was formed and got away from the surface of Au@Ag CS NPs. Therefore, Au@Ag CS NPs lost the protection from aptamer, and more "hot spots" were produced again. Thus the surface enhancement effect of Au@Ag CS NPs recovered, and so as the Raman intensity of 4-MBA. By monitoring the changing of Raman intensity before and after E2 addition, we successfully established a SERS bioanalytical assay for the detection of E2. Among SERS substrate, AuNPs and AgNPs were the most extensively used to enhance the Raman intensity[27]. AgNPs were employed as SERS substrate because of the strong surface enhancement effect, but the poor stability limited its application. AuNPs had good stability, but the SERS effect was relatively weak[42]. Therefore, Au@Ag CS NPs were prepared in this work to achieve a win-win effect. Fig. 1A showes the improved surface enhancement effect of Au@Ag CS NPs. In the present of Au @ Ag CS NPs, a narrow and high intensity Raman peak was obtained for 4-MBA, although the intrinsic Raman intensity of 4-MBA was very weak, and the enhancement factor of Au@Ag CS NPs was about 6.868×104 for 4-MBA(see details in the Supporting Information), while the Raman intensity was lower in the presence of AgNPs or AuNPs(Fig. 2S). Besides, the stability of Au@Ag CS NPs was investigated in Fig. 1B, the SERS enhancement factor of Au@Ag CS NPs was almost
constant when stored at 4 °C for 0, 1, 2, 3 weeks. The UV-vis maps of AuNPs and Au @ Ag CS NPs are shown in Fig. 1C. It was found that AuNPs had a peak at 520 nm, while Au @ Ag CS NPs showes two peaks at 510 nm and 390 nm, respectively, which were the specific peaks of Au and Ag. The UV absorption peak was blue shifted after Au NPs coated by Ag shell, which means Au core was wrapped around by Ag shell[43]. It was further confirmed by SEM and HRTEM characterization as displayed in Fig. 1D. It is notable to find that the AuNPs were surrounded by lots of Ag tiny particles, forming shell on Au surface. It might because silver nitrate solution reduced with sodium citrate around AuNPs. Because the Raman signal intensity, i.e. the sensitivity of the proposed method was highly influenced by the concentration of 4-MBA, the E2-aptamer and the reaction time of the aptamers-E2 complex with the substrate[44], the experiment conditions were optimized at firstly(Fig. S1). With the increment of 4-MBA concentration, the Raman intensity also increased, and reached to the maximum at 5.0 nM, while the stability of the Au @ Ag CS NPs might decline due to the high concentration of 4-MBA[41]. So a Raman reporter molecule with a concentration of 5.0 nM was chosen as a probe. Aptamers with different concentrations and 0.1 nM of E2 were mixed in an equal volume for 40 min. Then 20 μL of the above solution was added to 200 μL of Au @ Ag-4 MBA solution, and the Raman intensity was tested after 25 min. It was found that the Raman intensity was the highest when the concentration of the aptamer was 120 nM, thus determining the optimum concentration of aptamer under this condition. In addition, the incubation time of the aptamers-E2 and Au @
Ag-4MBA was optimized. After 120 nM of aptamer and 0.1 nM of E2 were mixed in equal volume, 200 μL of Au @ Ag-4 MBA was added and incubated at different times. As the incubation time prolonged, the Raman intensity increased. When the incubation time was 25 min, the signal was enhanced to the maximum. So the incubation time of 25 min was determined to be optimal. In order to further demonstrate the sensitivity of the biosensing system, the Raman intensity changes after adding different concentrations of E2 was tested, as exhibited in Fig. 2. The Raman intensity at 1587 cm-1 was selected for quantifying E2. As the E2 concentration increased from 0.1 pM to 10 nM, the peak signal intensity also increased, as shown in Fig 2A and 2B. It noticed that the SERS signal was linear against the logarithm of E2 concentration between 0.1 pM and 10 nM. The fitting equation was obtained as y = 19321.06+ 2692.61lg x (x was the concentration of E2), the determination coefficient R2 was 0.999, the detection limit was 0.05 pM. Besides, the error bar demonstrated that the reproducibility of our biosensing system to detect E2 was great. The results indicates the high sensitivity of the SERS analysis, which was sufficient to meet the detection of E2 requirements in the environmental water samples.
3.2 Highly selective performance of E2 SERS biosensing system Environment is a complex system, in which there are a variety of organic pollutants, sugars, proteins and other substances[45]. And these substances may affect SERS analysis of E2. Therefore, the E2 selectivity and anti-interference ability of this assay
were futher investigated. Fig. 3A and 3B exhibites that if there was no aptamer, no E2 and no other substances, due to the "hot spot" effect of the Au@Ag CS NPs, narrow Raman peaks of 4-MBA were obtained with high intensity. If there was no E2 aptamer in the system, the addition of E2 could not alter the Raman peak intensity at 1587 cm-1 because of the no change of the the "hot spot" effect of the Au@Ag CS NPs. So did the interferents of dibutyl phthalate (DBP) and bovine serum albumin (BSA). On the contrary, if the aptamer was present, because of the electrostatic interaction originated from aptamers, the Raman peak intensity decreased. On this basis, the Raman peak intensity increased evidently after addition of E2, but lower than the initial condition of no aptamer and no E2 because there were part of aptamers interacting with Au@Ag CS NPs, leading to the weakened "hot spot" effect and the reduced 4-MBA Raman intensity.Meanwhile, the coexisted DBP or BSA exhibited no interference on E2 response. It indicates that the biosensing system showed a good selectivity for E2. Fig. 4 displays the relative Raman peak intensity of 10 pM E2 and 1 nM other ten types of interferons, including oestrone, estriol, DBP, atrazine, bisphenol A (BPA), 4-nonyl phenol, humic acids, D-glucan and BSA individually and 50mM NaCl solution. It is found that the assay was only responsive to E2, and the ratio R, which was the ratio of Raman peak intensity between interferons and E2, was less than 0.252, indicating that the excellent selectivity for E2. Furthermore, when it was used to detect E2 in the presence of all the above-mentioned substance solutions, it is concluded that the obtained signal intensity was consistent with the signal intensity detected only in the E2 solution, suggesting that other endocrine disruptors,
sugars, protein and other substances had little effect on the detection of E2. It further confirmed the anti-interference ability of the biosensing system. This high selectivity in E2 detection could be attributed to the strong binding affinity between the aptamers and E2, which was far greater than that with other substances[40]. Thus, the interaction between aptamer and E2 were studied by UV-vis absorption measurements in order to further demonstrate the superior selective performance of the biosensing system. The interaction between aptamer and other interferents were also investigated for comparison. The interaction between aptamer and E2 can be represented by the following equation 1: n E2 + aptamer → E2 (apt) (1) The Ka can be expressed as equation 2: Ka =
[E2(apt)] [E2]n ×[aptamer]
(2)
Here [E2(apt)], [E2] and [aptamer] represent the compound of aptamer with E2, E2 and aptamer, respectively. Equation on both sides of the logarithm was obtained as equation 3: lg
E2(apt) =nlg[E2]+lgKa (3) [aptamer]
The absorbance is proportional to the concentration of the absorption material according to Lambert–Beer law. So the equation 4 was obtained as follows: lg
(A0 -Ax ) = nlg[E2] + lgKa (4) (Ax -A1 )
There A0 , A1 represent the absorbance in the absence of E2 and the absorbance after exceeded addition of E2, respectively. Ax stands for the absorbance after adding a certain concentration of E2 to the aptamer solution. So there was a linear relationship
between logarithm of
(A0 -Ax ) (Ax -A1 )
and lg[E2] . If lg
(A0 -Ax )
=y, lg[E2] = x, the equation 5
(Ax -A1 )
can be obtained: y = nx + lgKa (5) Because of the constants of association constant (Ka) and comprehensive coordination number (n), the calibration plot gives Ka by the index of the intercept and n by the slop in Fig. 5A and B. The obtained linear regression equation was y = 5.8000 + 0.9608x, thus, Ka was 6.310×105/M, and n was 0.9608. Such high Ka value demonstrated that E2 aptamers had a strong recognition ability to E2. This was consistent with the reported Kd = 0.1 ~ 3μM[40]. Additionally, the absorbance of the mixture of aptamers and other pollutants was similar to that of the aptamers only. However, when adding the same concentration of E2, the absorbance decreased, as shown in Fig. 5C, which also displayed that the binding affinity of the aptamers for E2 was much higher than that for the other contaminants.
3.3 E2 SERS analysis of actual samples In order to further study the application of the biosensing system in environmental water samples, wastewater samples were collected from the sewer and nearby river of local obstetric hospital, where E2 was always present. All the samples were used after filtration by 0.22 μm membrane to remove particles and suspended solids. The environmental water samples, and that spiked with the standard E2 solution of 0.5, 1 nM were tested. The results are shown in Table 1. The recovery of the E2 SERS method was ranging from 93.9% to 104.6%, and the relative standard deviation was
between 7.4% and 27.6% (n = 3). Such excellent recovery and relative standard deviation owned to the selectivity of E2 SERS biosensing system Besides, it is found that E2 concentration of the obstetric hospital sewer was 10.8pM, while that in the hospital surrounding river was 1.3 pM. The high level of E2 present in the obstetric hospital sewer might be because that plenty of estrogen drugs were used to treat female patients and the excretion of pregnant women produced. The level of E2 in the obstetric hospital surrounding river was low perhaps because the water samples were related with water treatment before discharge. Besides, the recovery rate of the environmental spiked water samples was close to that of the standard HPLC method. It was worth noting that a complex multi-step pretreatment was required in detecting E2 by HPLC, while only a simple pre-filtration process was carried out for E2 SERS analysis. In addition, the E2 SERS assay was sensitive enough to detect the trace level of E2 in the water sample, whereas it could not be detected by HPLC. This approach was also more convenient for detecting E2 in environmental water samples considering the simple pre-treatment method compared to other literatures (Table S1). It was clear to find that one step of simple pretreatment based on the membrane filtration was needed in the detection of the actual sample, whereas different pretreatments such as purification, concentration, extraction and derivation of the samples were required in other analytical methods. Besides, it was the best detection limit of E2 in real water samples only by membrane filtration pretreatment, while a lower detection limit of 0.023pM was obtained by the fluorescence immunoassay after solid-phase extraction pretreatment[46]. In summary, the E2 SERS biosensing
system could achieve the goal of high sensitivity, high selectivity, simple pretreatment and easy operation.
4. Conclusion In this work, an aptamer-based SERS approach was successfully established for the detection of E2 with high sensitivity and high selectivity in complex environmental water samples for the first time. The biosensing system was based on Au @ Ag CS NPs as carrier, 4-MBA as a Raman reporter molecule, and aptamer of E2 as a recognition element. The detection of E2 exhibited a high sensitivity, with the linear range from 10-4 to 10 nM, and the detection limit of 0.05 pM. Moreover, this system exhibited a high selectivity to E2, achieving the trace detection of E2 in complex environmental water samples system. It is worth noting that this analytical method could be carried out following only by one step of simple pretreatment in the detection of the actual sample, which greatly reduced the complex, pre-processing operations in the instrumental detection of environmental water samples. Our work has provided a new analytical approach for the detection of endocrine disruptors in complex water samples.
Acknowledgements This work is supported by National Natural Science Foundation of China (Nos. 21537003), and the Science & Technology Commission of Shanghai Municipality (14DZ2261100).
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Author Biographies Siyao Liu is a master student in School of Chemistry Science and Engineering, Tongji University, Shanghai, China. Her research interest is biosensor. Ruojie Cheng is a master student in School of Chemistry Science and Engineering, Tongji University, Shanghai, China. Her research interest is biosensor. Yuqing Chen is a master student in School of Chemistry Science and Engineering, Tongji University, Shanghai, China. Her research interest is biosensor. Huijie Shi is an associate professor in School of Chemistry Science and Engineering, Tongji University, Shanghai, China. Her research interests are photoelectrocatalysis and its application in environmental analysis and contaminant control. Guohua Zhao is a professor in School of Chemistry Science and Engineering, Tongji University, Shanghai, China. His research interests are environmental electrochemistry, photoelectrochemistry, environmental/biology photoelectrochemical sensors, environmental energy chemistry.
Figure Captions Fig. 1. The characterization of Au@Ag CS NPs. (A) SERS spectra of 4-MBA absorbed on or not absorbed on the surface of Au@Ag CS NPs. (B) The raman enhancement of Au@Ag CS NPs after the synthesis of 0, 1, 2, 3 weeks. (C) UV-vis absorption spectra of Au@Ag CS NPs and Au NPs. (D) SEM images of Au@Ag CS NPs. The insert displays HRTEM images of Au@Ag CS NPs. Fig. 2. Highly sensitive performance of the E2 SERS detection. (A) SERS spectra with different concentrations of E2 (B) The linear calibration curve of SERS intensity at 1587cm-1 peak versus the concentrations of E2 from 0 pM to 10 nM. The inset exhibits The linear calibration curve of SERS intensity at 1587cm-1 peak against natural logarithm over concentration of E2. Fig. 3. Anti-interference ability and selectivity performance of the E2 SERS detection. (A) SERS spectra with different interferences in the absence of aptamers and the change of SERS spectra intensity in the presence of aptamers. (B)The change of raman intensity at 1587 cm-1 with different interferences in the absence of aptamers and in the presence of aptamers (here A, B, C, D, E, F, G, H represent no aptamer + no E2 + no others, no aptamer + E2 +BSA, no aptamer + E2 +DBP, no aptamer + E2, aptamer + no E2 + no others, aptamer + E2 +BSA, aptamer + E2 +DBP, aptamer + E2, respectively). Fig. 4. Highly selectivity performance of the E2 SERS detection. SERS intensity at 1587cm-1 peak relative signals of E2 in the presence of different interferences in the concentration. The insert shows the structural formulas of E2, ethinylestradiol, estriol,
dibutyl phthalate, atrazine, bisphenol A, 4-nonyl phenol, humic acids and D-glucan(from left to right), respectively. Fig. 5. UV spectra of aptamer after interacting with E2. (A) UV spectra of aptamer interacting with different concentrations of E2. (B) The linear calibration curve of lg(A0-Ax)/(Ax-A1) versus the logarithm of E2 concentration. (C) UV spectra of 1 nM aptamer interacting with different kinds of 1 nM interferences in the same volume. Scheme 1. Schematic illustration for the E2 SERS detection; the inset showed SERS spectra under different experimental conditions
Fig. 1.
Fig. 2.
Fig. 3.
Fig. 4.
Fig. 5.
32
Scheme 1.
33
Table 1. Performances of E2 SERS biosensing system and HPLC for E2 determination in real water
The SERS aptasensor Sample
HPLC
Add(nM) Mean±SD(pM)
CV(%)
Recovery(%)
Mean±SD(pM)
CV(%)
Recovery(%)
obstetric hospital
0
10.8±0.8
7.4
0
undetected
-
-
sewer
500
523.0±103.5
19.8
102
527.8±20.4
3.9
106
1000
1057.3±153.3
14.5
105
1012.2±15.0
1.5
101
obstetric hospital
0
1.3±0.2
11.5
0
undetected
-
-
surrounding river
500
470.8±34.1
7.2
94
512.4±28.5
5.6
103
1000
1002.9±276.7
27.6
100
999.8±19.5
2.0
100