A simple highly sensitive and selective aptamer-based colorimetric sensor for environmental toxins microcystin-LR in water samples

Journal of Hazardous Materials 304 (2016) 474–480

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A simple highly sensitive and selective aptamer-based colorimetric sensor for environmental toxins microcystin-LR in water samples Xiuyan Li, Ruojie Cheng, Huijie Shi, Bo Tang, Hanshuang Xiao, Guohua Zhao∗ Department of Chemistry, Tongji University, 1239 Siping Road, Shanghai 200092, People’s Republic of China

h i g h l i g h t s • • • •

A colorimetric aptasensor was fabricated for MC-LR detection for the first time. The colorimetric aptasens or exhibit high sensitivity with a LOD of 0.37 nM. The specificity of the aptamer enhanced the selectivityof the sensor remarkably. MC-LR determination in real water samples exhibits high accuracy and stability.

a r t i c l e

i n f o

Article history: Received 29 June 2015 Received in revised form 29 September 2015 Accepted 9 November 2015 Available online 17 November 2015 Keywords: Microcystin-LR (MC-LR) Aptamer Colorimetric sensor High sensitivity High selectivity

a b s t r a c t A simple and highly sensitive aptamer-based colorimetric sensor was developed for selective detection of Microcystin-LR (MC-LR). The aptamer (ABA) was employed as recognition element which could bind MC-LR with high-affinity, while gold nanoparticles (AuNPs) worked as sensing materials whose plasma resonance absorption peaks red shifted upon binding of the targets at a high concentration of sodium chloride. With the addition of MC-LR, the random coil aptamer adsorbed on Au NPs altered into regulated structure to form MC-LR-aptamer complexes and broke away from the surface of Au NPs, leading to the aggregation of AuNPs, and the color converted from red to blue due to the interparticle plasmon coupling. Results showed that our aptamer-based colorimetric sensor exhibited rapid and sensitive detection performance for MC-LR with linear range from 0.5 nM to 7.5 ␮M and the detection limit reached 0.37 nM. Meanwhile, the pollutants usually coexisting with MC-LR in pollutant water samples had not demonstrated disturbance for detecting of MC-LR. The mechanism was also proposed suggesting that high affinity interaction between aptamer and MC-LR significantly enhanced the sensitivity and selectivity for MC-LR detection. Besides, the established method was utilized in analyzing real water samples and splendid sensitivity and selectivity were obtained as well. © 2015 Elsevier B.V. All rights reserved.

1. Introduction Microcystin-LR (MC-LR) is one of the most poisonous pollutants in Microcystins released by cyanbacteria in the water of eutrophication. As a common toxin, it can severely inhibit the activities of protein phosphatases type 1(PP1) and type 2A (PP2A) even in very low concentration [1,2]. However, MC-LR is a stable chemical substance and almost impossible to be degraded naturally resulted from the existence of ring structure and double bonds in the molecule [3]. So it has become a crucial pollutant in water quality control and environmental monitoring. In 1998, the World Health Organization (WHO) proposed a maximum limit

∗ Corresponding author. Fax: + 86 21 65982287. E-mail address: [email protected] (G. Zhao). http://dx.doi.org/10.1016/j.jhazmat.2015.11.016 0304-3894/© 2015 Elsevier B.V. All rights reserved.

of 1 ␮g/L for MC-LR in drinking water [4]. Therefore, the exploration of new method for MC-LR detection at low concentration has become an increasingly important issue in the field of environmental analysis. Several analytical techniques have been adopted for MC-LR determination, such as whole cell bioassays [5], protein phosphatase inhibition assays (PPIA) [6], immunoassays [7], high-performance liquid chromatography (HPLC) [8], and liquid chromatography-mass spectrometry (LC-MS) [9]. However, these methods call for complex sample preparation, expensive equipment, high professional skills and massive time. Compared with the methods mentioned above, colorimetric method is convenient, rapid, without using large-scale equipment and easy to realize online testing [10–15]. There has been some research work attempting to develop colorimetric method for MC-LR detection [16,17], though the sensitivity of which is far from satisfactory and require to be improved significantly. Also, the selectivity of

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these methods demands prominent enhancement as real aquatic environment consisting of MC-LR with other numerous organic contaminants such as atrazine, glyphosate, trichlorfon, clofentezine. Therefore, establishing a simple, sensitive colorimetric method with high selectivity for MC-LR detection is sorely needed and of great importance. It is recognized that aptamer obtained by systematic evolution of ligands employing exponential enrichment (SELEX) technology can bind its targets with high affinity, specificity and selectivity. Moreover, aptamer is inexpensive, easy to be synthesized, small in size and of excellent chemical stability. More importantly, aptamer has displayed remarkable flexibility, in other words, it can specifically bind various target molecules with its structure changing from random coil structure to hairpin structure or G-quadruplex structure automatically [18,19]. Thus it has been widely used for environmental monitoring as “chemical antibodies”. The aptamer of MC-LR has been successfully synthesized and implemented as a biological recognition element in electrochemical sensing technology [20–22]. As far as we know, there is still no report on applying aptamer to assemble colorimetric sensor for fast and accurate MC-LR detection. Therefore we attempt to develop an aptamer-based colorimetric sensor by combing the specificity of the aptamer to MC-LR and the sensitivity of colorimetric methods for detecting low level of MC-LR in water sample. In this work, the aptamer-based colorimetric sensor was established to detect MC-LR in environmental at the first time. The colorimetric sensor employed the aptamer as recognition element and gold nanoparticles as sensing materials. As is known, aptamer can be absorbed on the surface of AuNPs by coordination between Au atoms and the nitrogen atoms of exposed bases, which protects AuNPs from salt-induced aggregation as aptamer carries more negative charges. Once aptamer interacted with MC-LR, the random coil structure of aptamer would transform into regular 3D structure leading to unexposed bases. And the MC-LR-conjuncted aptamer no longer protected AuNPs from salt-induced aggregation and as a result the color of AuNPs turned into blue from red. Based on the mechanism above, a label-free colorimetric aptasensor was fabricated for MC-LR quantitative analysis. The sensitivity and selectivity of the established colorimetric sensor was discussed in detail. Moreover, the mechanism for the high sensitivity and selectivity of the assay was also investigated by calculating the association constant between MC-LR and selected aptamer. The colorimetric aptasensor was further applied in the determination of MC-LR in tab water and pond water samples.

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2.2. Colorimetric detection of MC-LR Au NPs was fabricated by typical chemical reduction methods in the light of the previous reports [23]. Briefly, 100 mL of 1 mM aqueous solution of HAuCl4 was heated to boiling and stirred in a round-bottom flask. Then 10 mL of 38.8 mM trisodium citrate was added fleetly and the solution was boiled for another 30 min with the color of which turned wine red from yellow. Later the solution was stirred to cool down to room temperature, filtrated with 0.22 ␮m filter membrane and stored at 4 ◦ C in the refrigerator. The concentration of as prepared Au NPs was 4.4 nM measured by absorption of 521 nm with an extinction coefficient of 2.7 × 108 M−1 cm−1 [24]. 32 ␮L of 2 ␮M aptamer and 24 ␮L of 500 mM sodium chloride solution were added into as prepared 433 ␮L Au NPs solution successively and afterwards incubated for 15 min. Then, the preceding solution was mixed with 11 ␮L MC-LR solution with different concentration respectively, incubated for another 15 min. The ultimate concentration of MC-LR was 0.5 nM, 1.0 nM, 5.0 nM, 50 nM, 150 nM and 7500 nM respectively. The absorption spectra of the mixed solution were determined by UV–vis spectroscopy. Accordingly, the interfering substances including acetamiprid, glyphosate, trichlorfon, clofentezine and atrazine were measured exactly as MC-LR illustrated above. 2.3. Determination of the association constant and coordination number The association constant (Ka ) and comprehensive coordination number (n) between aptamer and MC-LR were estimated by UV spectra measurements. The concentrations of MC-LR were determined by Agilent 8453 UV/vis spectrophotometer at 260 nm at 20 ◦ C with an extinction coefficient of 38,728.4 M−1 cm−1 , then different volume of MC-LR was added into the aptamer solution and stirred rapidly for at least 10 min until it was well-proportioned. Afterwards, the solutions with different concentration of MC-LR were tested at 1 nm intervals from 230 to 305 nm at 20 ◦ C. Volume effect has not been taken into consideration due to the slight amount of MC-LR addition. After adding different volume MC-LR, corresponding spectra was attained by subtracting the spectrum of aptamer from adsorption spectra of solutions with different concentration of MC-LR. Subsequently, the linear equation between the absorbance and concentration of MC-LR were attained as well, then n and Ka between aptamer and MC-LR could be determined as the slope and vertical intercept of obtained linear equation, respectively.

2. Materials and methods 2.4. Colorimetric sensing of actual samples 2.1. Reagents and apparatus Microcystin-LR (MC-LR), chloroauric acid (HAuCl4 ), sodium chloride (NaCl), acetamiprid, glyphosate, trichlorfon, clofentezine and atrazine were purchased from Sigma–Aldrich (USA). The aptamer of MC-LR was chosen according to reported literatures [20] with sequences of 5 -GGCGCCAAACAGGACCACCATGACAATTA CCCATACCACCTCATTATGCCCCATCTCCGC-3 , and synthesized by Shanghai Sangon Biotechnology Co., Ltd., (Shanghai, China) and purified by the method of HPLC. The concentration of the aptamer was determined by ultraviolet absorption intensity at 260 nm with an extinction coefficient of 38,728.4 M−1 cm−1 . All the solutions were prepared by double distilled water. Field emission scanning electron microscopy (FE-SEM, HitachiS4800, Japan) and high resolution transmission electron microscope (TEM, JEM-2100, Japan) were used to characterize the size and morphology of Au NPs. UV–vis spectroscopy (UV–vis, Agilent, 8453, USA) was applied to measure the absorption of the Au NPs.

The water samples were collected from tap water and pond water. Then the filter paper and 0.22 ␮m membrane were consecutively employed for samples purification. The preprocessed samples were detected by as prepared colorimetric sensor and determined by UV–vis spectroscopy as illustrated above. 3. Results and discussion 3.1. Construction and characterization of the MC-LR colorimetric sensor As shown in Scheme 1A, Au NPs aggregation was induced by concentrated salt solutions due to the electrostatic screening effect [25], leading to the surface plasma resonance absorption peak at 521 nm red shifting to 646 nm, accompanied by color change of the solution from wine red to blue (Scheme 1A). As the aptamer with certain base sequence was added to the Au NPs solution, it

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Scheme 1. Schematic illustration for the colorimetric sensor and the mechanism for the analytic determination of MC-LR.

could be absorbed on the Au NPs surface through the coordination interaction between the N atom of exposed bases and Au NPs [26], which was strong enough to overcome the electrostatic repulsion between electronegative phosphate backbone and electronegative Au NPs. Owing to more negative charges the aptamer carried, the Au NPs would remain stable and dispersed against salt-induced aggregation as their color maintained wine red (Scheme 1B). When MC-LR was added into the aptamer protected Au NPs solution, the aptamer would specifically bind with MC-LR forming aptamer–MCLR complexes. It caused conformation changes from random coil structure to regular 3D structure [27], which significantly reduced the exposure of the bases, and thus resulting in the aptamer no longer protecting Au NPs from salt-induced aggregation and the color of the Au NPs turned to violet blue (Scheme 1C). However, the interfering substances could not bind with the aptamer and conduce to the folding of the aptamer owing the high recogni-

tion selectivity towards the targets, so the Au NPs would maintain the dispersive state even when salt solution was concentrated (Scheme 1D). Fig. 1 utilized the Transmission Electron Microscope (TEM) technology to characterize the morphology change of Au NPs before and after addition of 10−3 M MC-LR. It was clearly seen in Fig. 1A that the Au NPs was well dispersed in the presence of 24 mM NaCl and 0.128 ␮M aptamer. However, the Au NPs aggregation arose with MC-LR added (Fig. 1B). And the color of Au NPs turned from wine red to violet blue. To further investigate the Au NPs aggregation, a series of corresponding UV–vis absorption spectra are represented in Fig. 2. It was noted that the pure Au NPs had a characteristic plasma resonance absorption peak at 521 nm, which would red shift sensitively when the dispersion state changed. The characteristic peak of Au NPs maintained at 521 nm in the presence of 24 mM NaCl and the aptamer, indicating the good dispersion of

Fig. 1. TEM images of Au NPs in the presence of 24 mM NaCl and 0.128 ␮M aptamer before (A) and after (B) the addition of MC-LR.

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Fig. 2. UV–vis absorption spectra of Au NPs under different experimental conditions, c(Au NPs) = 4.4 nM, c(aptamer) = 0.128 ␮M, c(NaCl) = 24 mM, c(MC-LR) = 750 nM, T = 298 K.

Fig. 3. Different absorbance ratio  (A646 /A521 ) of AuNPs under different concentrations of NaCl and aptamer, the concentration of NaCl altered in the range of 20 and 28 mM, the concentration of aptamer changed from 0.120 to 0.136 ␮M.

aptamer protected Au NPs. However, upon the addition of MC-LR (10−3 M), the absorbance at 521 nm decreased dramatically and a new absorption peak appeared at 646 nm, manifesting the aggregation of Au NPs. It indicated that the added MC-LR may interact with the aptamer, making the Au NPs released and aggregate in the presence of salt. 3.2. Optimization of experimental conditions It was noted that the concentration of sodium chloride and aptamer had a great impact on the sensitivity of the established colorimetric sensors.  (A646 /A521 ) was adopted as evaluation parameter to optimize the experimental conditions,  (A646 /A521 ) = the absorption ratio A646 /A521 in the presence of MCLR—the absorption ratio A646 /A521 in the absence of MC-LR. As shown in Fig. 3, the concentration of NaCl in the range of 20–28 mM and the concentration of aptamer in the range from 0.120 to 0.136 ␮M were studied respectively. The results showed that  (A646 /A521 ) obtained the maximum value when the concentration of NaCl was 24 mM, thus the optimized concentration of sodium chloride was confirmed as 24 mM. In addition,  (A646 /A521 ) arrived

Fig. 4. Highly sensitive performance of the MC-LR colorimetric sensor. (A) UV–vis absorption spectra of the Au NPs with different concentrations of MC-LR (B) The linear calibration curve of A646 /A521 versus the logarithm of MC-LR concentrations from 0.5 nM to 7.5 ␮M. (C) The corresponding color changes of Au NPs under different concentrations of MC-LR (0.5 nM to 7.5 ␮M).

at the maximum value when the concentration of aptamer was 0.128 ␮M, therefore, the concentration of aptamer was determined as 0.128 ␮M for further experiments in this work. Ionic concentration is also very important to the measurement results. However, our colorimetric method is carried out in a high salt system, red dispersed (repulsion) state of Au NPs form purplecolored aggregates (attraction) in the presence of NaCl with a high concentration [10] (in this work, the concentration was 24 mM). It enabled that when the colorimetric method was applied in real water sample analysis, the low concentration of inorganic salts can hardly influence the MC-LR detection. 3.3. Highly sensitive and selective performance of the MC-LR colorimetric sensor Under optimized conditions, MC-LR with different concentration was added respectively and their UV–vis absorption spectra are given in Fig. 4A. With the addition of MC-LR, the absorbance value at 521 nm decreased constantly whereas the absorbance value at 646 nm increased gradually. The color change of Au NPs with different concentration of MC-LR addition are corresponded to the

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Fig. 5. Highly selective performance of the MC-LR colorimetric sensor. (A) UV–vis absorption spectra of 4.4 nM Au NPs in the presence of 0.128 M aptamer, 1 nM MC-LR and 50 nM other interfering substances after the addition of 24 mM NaCl, (B) Relative signal of MC-LR and other interfering substances. The insert are the structural formulas of acetaminprid, glyphosate, dylox, atrazine, and clofentezine (from a to e), respectively.

MC-LR could be accomplished within 30 min with the advantage of convenient operation. Reasons for such a high sensitivity of the estabilshed colorimetric method were also discussed. The color of the AuNPs altered sensitively to the interparticle distance of the AuNPs, when AuNPs transformed from dispersive to aggregated state, an obviously color change from red to blue could be recorded by UV–vis spectroscopy. Besides, the aptamer which combined MC-LR with high specificity and affinity was adopted as recognition elements, thus the developed colorimetric sensor could bind MC-LR selectively and reduce the interference signal remarkable. In addition, the concentration of aptamer and NaCl which could affect the sensitivity of the fabricated colorimetric sensor had been optimized before MC-LR detection. The specificity of the established colorimetric sensor was also investigated by employing atrazine, clofentezine, acetamiprid, dylox and glyphosate, as interfering substances which may coexist with MC-LR in the polluted water. The corresponding UV–vis absorption spectra are illustrated in Fig. 5A and an obvious change of UV–vis spectrum in the presence of MC-LR was obtained while a slight or little change of UV–vis spectrum in the presence of other interfering substances were attained. The concentration of interfering samples is 50 times as the concentration of target. The relative signal values of the corresponding Au NPs was selected to evaluate the selectivity of our colorimetric sensor (Fig. 5B), i.e., relative signal = [(A646 /A521 , interfering substances) − (A646 /A521 , Au NPs)]/[(A646 /A521 , MC-LR) − (A646 /A521 , Au NPs)]. The results demonstrated that when those five pollutants were detected with the established colorimetric method respectively, the relative signal from UV–vis spectra of Au NPs after adding MC-LR was evidently higher than that of other interfering substances. The reason can be explained that aptamer bound to MC-LR and no longer protected Au NPs, which elicited the aggregation of Au NPs and turned the color of Au NPs from wine red to dark purple. Thus, a significant change of the relative signal can be obtained. However, the change of the relative signal caused by the addition of other interfering substances was much lower with the reason that the aptamer would not combine with other interfering substances. All results revealed that the selectivity of the developed colorimetric method was improved through the specific recognition between aptamer and MC-LR. Hence, the established colorimetric sensor exhibited splendid selectivity towards MC-LR among six detected pollutants (Table 1).

3.4. High-affinity interaction between aptamer and MC-LR different color in the Fig. 4(C). As shown in Fig. 4C, with the addition of MC-LR, the value of A646 /A521 becomes higher. More and more Au NPs get aggregated, so the corresponding color changes from wine red to purple or blue just like the colorimetric card. A linear relationship between A646 /A521 and the logarithm value of MC-LR concentrations ranging from 0.5 nM to 7.5 ␮M was obtained with a correction coefficient of 0.9970 (Fig. 4B), and the regression equation is A646 /A521 = 0.2533 + 0.1662 lgc (MC-LR, nM). The detection limit of the established colorimetric sensor was determined as 0.37 nM (S/N = 3) which was lower than the maximum concentration recommended by WHO in drinking water for humans. The developed colorimetric sensor had a better sensitivity towards MC-LR compared to other reported colorimetric methods [16,17]. Moreover, the binding constant of aptamer becomes higher; the corresponding binding capacity becomes stronger. We can use aptamer to bind to the target molecules from the complex environment, so as to achieve a good selectivity [28–31]. In addition, it was simpler, cheaper and much more stable compared with other reported analytic methods for MC-LR detection, such as HPLC, ELISA and MC-LR immunosensor [32,33]. Furthermore, visual analysis of

As known, the binding affinity between the aptamer and MC-LC may have significant influence on the sensitivity of the colorimetric aptasensor. Therefore, UV–vis absorption measurement was adopted to study the interaction between aptamer and MC-LR and calculate the association constant (Ka ) and comprehensive coordination number (n) [34]. The interaction between aptamer and MC-LR can be represented by the following Eq. (1) nMC-LR + Aptamer → MC-LR × Aptamer

(1)

The Ka can be calculated by Eq. (2): Ka =

[MC-LR × Aptamer] [MC-LR] n × [Aptamer]

(2)

Taking logarithm of Eqs. (2) and (3) is obtained as follows: lg

[MC-LR × Aptamer] = nlg [MC-LR] + 1g Ka [Aptamer]

(3)

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Table 1 Comparison of the results obtained by the present method and other reported methods for the determination of MC-LR. Measurement

The limit of detection

Reference

Colorimetric aptasensor Colorimetric PPIA with ELIPA Chromatic immunosensor LC-MS Electrochemical immunosensor Electrochemical immunosensor Amperometric immunosensor

0.37 nM 3.9 nM 1 nM 0.02–5.0 ␮g/g 0.05–20 ␮M 1.0 × 10−7 –8.0×10−6 nM 0.5 nM

This work Environ. Sci. Technol. [16] Sensor Actuat. B-Chem. [17] J. Chromatogr. B [28] Anal. Chem. [29] Biosens. Bioelectron. [30] Environ. Sci. Technol. [31]

Table 2 Performances of colorimetric sensor for MC-LR determination in real water samples. NO.

Sample

Measured

Recovery (%)

SD (%)

Color

1 2 3 4 5 6

Tab water Tab watera Pond water Pond watera Pond waterb Pond waterc

– 0.95 nM – 0.97 nM 4.93 nM 51.10 nM

– 95 – 97 98.5 102.2

– 7.4 – 9.2 10.7 8.4

Red Slight discoloration Red Slight discoloration Discoloration Large discoloration

–MC-LR was not detected. a 1 nM MC-LR was added into the water sample. b 5 nM MC-LR was added into the water sample. c 50 nM MC-LR was added into the water sample.

of MC-LR concentration. A linear relationship between logarithm of (A0 – Ax )/(Ax – A1 ) and the logarithm concentration of MC-LR was attained in Fig. 6B. The obtained linear regression equation was: [A0 − AX ] = 0.98121 g [MC − LR] + 7.0792 [AX − A1 ]

(5)

Thus, Ka was 1.200 × 107 and n was 0.9812 from the Eq. (5). The Ka value calculated by our method is not so high as that in the literature [22], but both of the values are on the same number of magnitude. The difference may be due to the measurement error or the different experimental methods bringing out the different limits of detection. The comprehensive coordination 0.9812 suggested that the combination ratio was approximately 1:1. High value of Ka indicated the strong affinity between aptamer and MCLR, which would inevitably improve the selectivity and sensitivity of colorimetric method, thus leading to the low detection limit of the established colorimetric aptasensor as 3.7 × 10−10 M. 3.5. Real water sample analysis application of the MC-LR colorimetric sensor Fig. 6. UV spectra of aptamer after interacting with MC-LR (A) UV spectra of aptamer interacting with different concentrations of MC-LR (B) The linear calibration curve of lg(A0 − Ax )/(Ax − A1 ) versus the logarithm of MC-LR concentration.

According to Lambert–Beer law, the absorbance is proportional to the concentration of the absorption material. Eq. (4) can be acquired: lg

[A0 − AX ] = nlg [MC-LR] + 1g Ka [AX −A1 ]

(4)

A0 is the absorbance in the absence of MC-LR, Ax is the absorbance in the presence of MC-LR, and A1 is the absorbance after exceeded addition of MC-LR. Therefore, the association constant (Ka /nmol −1 L) and comprehensive coordination number (n) can be calculated from the intercept and slope of the linear equation. Fig. 6A is the ultraviolet absorption spectrum of aptamer interacting with different concentrations of MC-LR. The insert picture of Fig. 6A was a secondary structure of the selected DNA aptamer used in this work optimized by employing the tool of mfold [35]. It could be obviously noticed that the intensity of characteristic absorption peak at 260 nm of aptamer decreased gradually with the increment

To evaluate the practical application ability of the constructed colorimetric sensor, it was applied to detect MC-LR in real tab water and pond water by the standard addition method. The tab water was added with MC-LR standard sample solution at one concentration and the pond water were added with MC-LR standard sample solution at three concentrations. The measurement results of the two samples were shown in Table 2. The recovery of established colorimetric sensor was ranging from 98.5% to 102.2% and the standard deviation was between 7.4% and 10.7%. It indicated that the developed method had a higher sensitivity and accuracy in the determination of MC-LR in real water samples. The results can be explained by the fact that real water samples had been purified respectively by the filter paper and 0.22 ␮m membrane to remove the particulate matter and suspended matter, thus the interfering signal of the disruptors above was reduced significantly, and the sensitivity and accuracy were improved. Moreover, in the experiment, 11 ␮L of sample water without any pretreatment except filtration was added in the established sensing system and 500 ␮L of solution was obtained eventually. In this process, the water sample were diluted, so the concentrations of coexistence of pollutants, for example, herbicide, insecticide and endocrine

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disruptors were lowered, therefore the interference of complex systems were decreased. In addition, the estabilshed colorimetric sensor employed aptamer as recognition element, which could combine MC-LR with high selecivity, affinity, and specificity, to further improve the sensitivity and accuracy of the detection methods. Finally, our colorimetric method is carried out in a high salt system, but the amount of inorganic salt in the real water sample is very small. So testing the real water sample in the low salt system will not influence its sensitivity. Beyond that, constructed colorimetric sensor has the advantages of simple fabrication, rapid response and easy on-line detection. 4. Conclusion This work constructed a simple aptamer-based colorimetric sensor with high sensitivity and selectivity for quick detecting MCLR. The dection limit of developed colorimetric sensor could reach 0.37 nM with a linear relationship from 0.5 nM to 7.5 ␮M. By adopting aptamer as recognition element, the colorimetric sensor had an excellent selectivity, and the existence of other interfering substances in the water did not affect the determination of MC-LR. The mechanism research indicated that high affinity between aptamer and MC-LR guaranteed the high sensitivity and selectivity of the colorimetric sensor. The developed method can be used to determinate MC-LR in real water samples and the satisfactory recovery had been obtained. The fabricated aptamer-based colorimetric sensor also has the advantages of good stability, low cost and outstanding reproducibility. Therefore, the developed aptamer-based colorimetric sensor has potential application in MC-LR determination in the environment. Acknowledgment This work is supported by National Natural Science Foundation of China (NSFC, No. 21277099, No. 21477085, No. 21307091, No.51208367). References [1] F. Long, M. He, A. Zhu, H. Shi, Portable optical immunosensor for highly sensitive detection of microcystin-LR in water samples, Biosens. Bioelectron. 24 (2009) 2346–2351. [2] S. Singh, A. Srivastava, H.M. Oh, C.Y. Ahn, G.G. Choi, R.K. Asthana, Recent trends in development of biosensors for detection of microcystin, Toxicon 60 (2012) 878–894. [3] K. Chen, M.C. Liu, G.H. Zhao, Fabrication of a novel and simple microcystin-LR photoelectrochemical sensor with high sensitivity and selectivity, Environ. Sci. Technol. 46 (2012) 11955–11961. [4] WHO, Guidelines for drinking-water quality, Health Criteria and Other Supporting Information, 2nd ed., World Health Organization, Geneva, 1998 (addendum to vol. 2). [5] L.N. Sangolkar, S.S. Maske, T. Chakrabarti, Methods for determining microcystins (peptide hepatotoxins) and microcystin-producing cyanobacteria, Water Res. 40 (2006) 3485–3496. [6] R. Dawson, The toxicology of microcystins, Toxicon 36 (1998) 953–962. [7] M. Liu, H. Zhao, S. Chen, H. Yu, X. Quan, Colloidal graphene as a transducer in homogeneous fluorescence-based immunosensor for rapid and sensitive analysis of microcystin-LR, Environ. Sci. Technol. 46 (2012) 12567–12574. [8] E.C. Aguete, A. Gago-Martınez, J.M. Leao, J.A. Rodrıguez-Vázquez, C. Menàrd, J.F. Lawrence, HPLC and HPCE analysis of microcystins RR, LR and YR present in cyanobacteria and water by using immunoaffinity extraction, Talanta 59 (2003) 697–705. [9] V. Ríos, I. Moreno, A.I. Prieto, M. Puerto, D. Gutiérrez-Praena, M.E. Soria-Díaz, A.M. Cameán, Analysis of MC-LR and MC-RR in tissue from freshwater fish (Tinca tinca) and crayfish (Procambarus clarkii) in tench ponds (Cíceres, Spain) by liquid chromatography—mass spectrometry (LC–MS), Food. Chem. Toxicol. 57 (2013) 170–178. [10] C.B. Gopinath, T. Lakshmipriya, K. Awazu, Colorimetric detection of controlled assembly and disassembly of aptamers on unmodified gold nanoparticles, Biosens. Bioelectron. 51 (2014) 115–123.

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