Microcystins (MCs) is very dangerous toxins, which is generated by the cyanobacteria in the diversified natural environment. In 1878, it was first reported that cyanobacteria toxins caused poisoning in Australia lake . Among of the microcystins variants, microcystin-LR (MC-LR) is the most common, the most toxic, the most widely distributed, the most serious harm to fresh water in the cyanobacteria toxins currently known. MC-LR has a lot of characteristics, such as soluble in water, methanol or acetone, heat-resistant, non-volatile, anti-pH changes. Its presence is a very serious threat on water supplies all over the world . It is one of the main reasons for massive poisoning and pollution . In China, the incidence of primary liver cancer is the highest in areas with cyanobacteria pollution [4, 5]. Dianchi, Chaohu, Taihu have occurred serious cyanobacteria contamination phenomenon. The survey showed that cyanobacteria contamination have been detected not only in the "three lakes", but also in the lakes and reservoirs in the middle and lower reaches of the Yangtze River and the Yellow River. It posed a grave threat for health of the residents [6, 7]. World Health Organization (WHO) considered that the microcystin are carcinogen to humans . From 1998, WHO set up the guideline level of MC-LR in drinking water (less than 1 mg/L) . MC-LR acts on hepatocytes and Kupffer cells and inhibits the protein phosphatase, activates protein kinase and cyclooxygenase in hepatocytes. Moreover, it induced tumor necrosis factor and interleukin in macrophages, which can cause liver inflammation, liver damage and even liver necrosis. Thus, it is very important to develop a rapid and sensitive detection method of MCs in water.
In recent years, there were a lot of reported analytical methods for the detection of MC-LR, such as liquid chromatography-mass spectrometry (LC–MS) [10, 11], thin layer chromatography (TLC) , enzyme-linked immunosorbent assay (ELISA)  and fluorescent immunoassay . Although those methods have high sensitivity, they are easy to be disturbed by matrix effects, and the instrument is expensive and the sample pretreatment is tedious which do not meet the demand of the field measurements because they require skilled operator. Recently, electrochemical sensor was given special attention due to its high sensitivity, lowcost, fast operation, and miniaturization. For example, Qin et al. successfully constructed a sensitive sandwich-type electrochemical immunosensor, using a graphene platform combined with mesoporous PtRu alloy as a label for signal amplification to catalytic H2O2 . Ping et al. constructed a label-free immunosensor of rapid detection MCs by modifying gold nanoparticles on the electrode .
Metal-organic frameworks (MOFs) are organic-inorganic hybrid solids with infinite, uniform framework structures built from self-assembly of organic linkers and inorganic metal nodes or metal-containing cluster . MOFs have attracted researchers widespread concern and became popular research in past few years because of its diversity, large surface area, adjustable pore size, internal adjustable . Many varieties of MOFs have been discovered and used in different areas such as gas storage, material separation, sensing, optics or catalyst [19-24]. MIL-101 is a porous MOFs material based on trivalent chromium skeleton, and consisted by the Cr3O trimers and 1, 4-benzene dicarboxylic acid. It was first synthesized by the Ferey et al. in 2005 (CCDC number is 605510) . MIL-101 has many characteristics such as large volume, large surface area, stable chemical properties . Therefore, it has perfect prospect in gas adsorption, separation, catalysis, etc. Structure of the MIL-101 was shown in Fig. S1 (Supporting information). Wang et al. have successfully synthesized a series of efficient ruthenium chloride (RuCl3)-anchored MOF (MIL-101-NH2) catalysts as heterogeneous catalyst for hydrogenation of CO2 to formic acid .
There were many reports of MOFs materials used for electrochemical measurement in recent years. Dong et al. synthesized the Cu3(BTC)2 and modified it on a carbon paste electrode for detecting 2, 4-dichlorophenol . Yang et al. prepared Cu-MOF materials and modified it on glassy carbon electrode for detecting hydrogen peroxide . To the best of my knowledge, there is no report using MIL-101 as label to construct an immunosensor for detecting MCs.
In this study, an electrochemical immunosensor based on MIL-101 was developed for ultrasensitive detection of MC-LR in water. We focus on developing a controllable and competitive strategy MC-LR immunosensor [30, 31] using Au NPs@MIL-101 as a label and graphene oxide (GO) as matrix to immobilize MCLR antibody. Au nanoparticles were loaded on MIL-101 to prepared Au NPs@MIL-101 composite materials as a marker to label antigen and increase the conductivity and catalytic activity of MIL-101. As a natural enzyme mimics, Au NPs@MIL-101 has strong catalytic properties to the oxidation of ascorbic acid. The principle of reaction was as follows: C6H8O6 - 2e- = C6H6O6 + 2H+. Au NPs@MIL-101 labeled BSA-MC-LR and MC-LR competitively bound with anti-MC-LR immobilized on the surface of electrode. Au NPs@MIL-101 composite materials may catalyze ascorbic acid oxidation and generate response current signal that is inversely proportional with the MC-LR concentration, which can be used to achieve quantitative detection of MC-LR. The high conductivity of GO and high catalytic property of Au NPs@MIL-101 lead to the high sensitivity of the immunosensor. This biosensor with high sensitivity, stability and anti-interference capability exhibits promise applications in environmental monitoring.
MIL-101 was synthesized according to a previously published protocol . 0.1641 g of anhydrous sodium acetate aqueous solution (0.05 mol/L) was put into a beaker. Then, 2 g of chromic nitrate nonahydrate and 0.82 g of p-phthalic acid were added into the beaker and mechanically stirred for 30 min. The above solution was placed to the hydrothermal reactor, the reaction process should be maintained for 12 h at 130 ℃. When it was cooled down to the appropriate temperature, the product was washed with double distilled water for at least three times. At last, green crystals were obtained. The synthesized MIL-101 was soaked in ethanol (95% EtOH with 5% water) at 80 ℃ for 24 h, the above solution was suction filtered and then was washed with water for at least three times. Finally, it was placed in the vacuum drying oven to dry at 80 ℃ for 12 h. The activated MIL-101 was obtained.
A certain amount of PVP was added to 1% HAuCl4 solution (the mole ratio of gold atom and PVP monomer is 1:80). The obtained solution was placed in ice-water bath and mechanically stirred for 1 h. Then 0.1 mol/L new-made NaBH4 solution was dropped to the above solution rapidly. The color of solution turned from yellow to brownish black, which indicated the formation of Au:PVP sol. Then, activated MIL-101 was added and mechanically stirred at 0 ℃ for 4 h. The obtained precipitate was centrifuged and washed with water and vacuum dried at 100 ℃ for 2 h. Finally, Au NPs@MIL-101 was obtained. Graphene oxide (GO) was synthesized using Hummers method .
To prepare Au NPs@MIL-101 labeled-MC-LR-BSA, 0.5 mL of Au NPs@MIL-101 aqueous solution was mixed with 10 mL of 1 mg/mL MC-LR-BSA and shaken overnight at room temperature. The obtained solution was centrifuged and supernatant was removed. Then, the solution was incubated with BSA (5%) for 1 h to block nonspecific binding site. The resultant solution was centrifuged again and washed with PBS for three times and resuspended in 1 mL of PBS to preserve at 4 ℃.
For the fabrication of MC-LR immunosensor, a glassy carbon electrode (GCE) (φ = 3 mm) was firstly polished with metallographic sandpaper, followed by alumina powder of three particle sizes (1.0, 0.3 and 0.05 mm) to obtain a mirror-like surface. It was then ultrasonically cleaned with nitric acid aqueous solution (1:1), absolute ethanol and double distilled water for 5 min respectively. Graphene oxide was electrochemically deposited using cyclic voltammetry within the scanning range of -1.4 V to 1.2 V at the scanning rate of 10 mV/s. 10 mL of anti-MC-LR (100 mg/mL) was dropped on the surface of GO/GCE and dried overnight at 4 ℃. After washed with PBS (pH 7.4), 10 mL of 1% BSA was added on the GO/ GCE/anti-MC-LR and incubated at 37 ℃ for an hour to block the remaining active groups and dispel the nonspecific binding effect. After Au NPs@MIL-101 labeled-MC-LR-BSA and MC-LR sample (1:1) was mixed evenly, 10 mL of mixed solution was coated on the modified electrode and incubated at 37 ℃ for 1 h. The preparation procedure of immunosensor is shown in Fig. 1.
The immunosensor was placed to the 10 mL of PBS as the working electrode. Then, 0.5 mol/L ascorbic acid was added for chronoamperometry determination. The Au NPs@MIL-101 can catalyze the oxidization of ascorbic acid to deoxidized ascorbic acid. The current response was in inverse proportional to the concentration of MC-LR in the sample to realize the quantitative detection of MC-LR. After the measurement, the modified electrode was cleaned using PBS solution (pH 7.4) followed by stirring it for 30 min in 4 mol/L urea aqueous solution for the regeneration of the electrode. The resulting immunosensor was stored at 4 ℃ in sterile PBS when not in use.
The XRD spectra of synthesized MIL-101was compared with standard. Fig. S2A (Supporting information) confirmed that the synthesized material is the MIL-101. Fig. S2B (Supporting information) shows the SEM image of MIL-101. It is observed that the crystal morphology of samples is regular and the crystallinity is high. Particle dispersion is even with the diameter of 100 nm. Figs. S2C and D (Supporting information) exhibit the TEM images of MIL-101 with different magnification. It can be seen clearly that MIL-101 is octahedral geometry. Fig. 2 shows TEM image of Au NPs@MIL-101. It can be seen that Au NPs with the size of 3 - 5 nm have been loaded in MIL-101 unformly. The above results demonstrate that MIL-101 and Au NPs@MIL-101 were successfully synthesized.
Electrochemical impedance spectroscopy (EIS) is a common method to characterize the modified process of electrode . Fig. 3 shows EIS diagram of different modified electrodes in the solution of 0.1 mol/L KCl containing 5 mmol/L of K3Fe(CN)6/K4Fe (CN)6. It can be seen that bare GCE exhibited an almost straight line (curve a), which was characteristic of a diffusion limiting step of the electrochemical process. A slight change of the electrochemical impedance occurred after the electrodeposition of GO on GCE (Ret = 250 Ω), demonstrating GO was reduced on the surface of electrode (curve b). After anti-MC-LR was modified on the electrode, the interfacial resistance value increased (Ret = 980 Ω) because antibody hindered the transfer of the electron to the electrode surface (curve c). When MC-LR and Au NPs@MIL-101 labeled-MC-LR-BSA mixture were coated on the surface of electrode, the impedance value increased significantly (Ret = 1750 Ω), showing competitive binding behavior took place between MC-LR and Au NPs@MIL-101 labeled-MC-LR-BSA (curve d).
|Fig. 3. Nyquist plots of impedance spectra by using different modified electrodes in 5 mmol/L K3Fe(CN)6/K4Fe(CN)6. (a) bare GCE, (b) GO/GCE, (c) anti-MC-LR/GO/GCE, (d) Au NPs@MIL-101 labeled MC-LR-BSA/MC-LR/anti-MC-LR/GO/GCE.|
The catalytic activity of the electrodes modified with different materials (none, MIL-101, Au NPs, Au NPs@MIL-101) to ascorbic acid (AA) were then investigated. As shown in the Fig. S3 (Supporting information), the bare glassy carbon electrode (curve a) had catalysis effect to the oxidation of AA. The catalysis current of the electrode modified with MIL-101 (curve b), Au NPs (curve c) and Au NPs@MIL-101 (curve d) to AA increased significantly. But the catalysis of the modified electrode Au NPs@MIL-101 was the best, which illustrates that Au NPs and MIL-101 have synergistic catalytic effect to the oxidation of AA.
The cyclic voltammetry performance of the immunosensor was detected in 10 mL of PBS containing 0.5 mol/L AA with different scanning rate. As shown in the Fig. S4 (Supporting information), the peak current increased with scanning rate increasing. The oxidation peak current had a proportion to the square root of scanning rate, which indicated the current was controlled by diffusion.
It is well known that experimental conditions influence the analytical performance of immunosensors. In order to improve performance of the immunosensor, various experiment conditions have been optimized. From Figs. S5A and B (Supporting information), the optimal conditions could be obtained and summarized as follows: Antigen incubation time is 60 min, immobilized anti-MCLR concentration is 100mg/mL.
Under optimum conditions, the calibration curve for the detection of MC-LR by chronoamperometry was illustrated in Fig. 4. As the concentration of MC-LR increases, the amount of Au NPs@MIL-101 labeled-MC-LR-BSA binding with anti-MC-LR on the surface of electrode decreases, which leads the decrease of oxidation current of AA in buffer solution. Therefore, the response current decreases with the concentration of MC-LR increasing. The linear range is 0.05 - 75, 000 ng/mL, with the linear correlation coefficient of 0.9951. The linear equation is △Ⅰ = -21.16 - 7.976lgC (△Ⅰ=Ⅰ0-ⅠX) with a detection limit of 0.02 ng/mL. Table S1 (Supporting information) displays an overview on recently reported methods and this work for determination of MC-LR [16, 35-38]. From Table S1, it can be seen that the detection limit of this work is lower than that of reported methods.
To investigate selectivity of immunosensor, under the optimized conditions, 20 ng/mL MC-LR were mixed with 20mg/mL of MC-YR and 20mg/mL of MC-RR, respectively. Following this, above mixtures were mixed with the same amount of Au NPs@MIL-101 labeled-MC-LR-BSA and dropped on the surface of the electrode followed by incubating for 60 min at 37 ℃. Then, the modified electrode was put in the beaker with 10 mL of PBS. After adding 0.5 mol/L AA, selectivity of immunosensor was tested using chronoamperometry. The results were shown in Fig. S6 (Supporting information), which indicated MC-YR and MC-RR did not cause interference to the immunosensor. The sensor has a high selectivity for MC-LR.
The stability of the sensors was tested. 95.74% of the initial response of the immunosensor remained after one week. Correspondingly, 85.12% of the initial response of the immunosensor remained after 30 days. Thus, the sensor has a good stability
To detect the response performance of the immunosensor in the real water samples taken from the Swan Lake of the Yunnan Normal University, three different concentrations of MC-LR were added in water samples. After the incubation at 37 ℃, the average recovery of detection by three times was 102.4% (Table S2 in Supporting information). The results are satisfactory, indicating the sensor has a good practicability and can be applied to detect MC-LR in real water samples.
In summary, a novel and sensitive competitive electrochemical immunosensor based on Au NPs@MIL-101 was developed for the detection of MC-LR. Anti-MC-LR was immobilized on the electrode surface modified with GO. MC-LR-BSA was labeled with Au NPs@MIL-101 using competitive method to detect MC-LR. Au NPs@MIL-101 labeled on the antigen has catalysis effect on the oxidation of AA to accomplish the detection of MC-LR. Through optimizing the conditions, the sensitivity and selectivity of the sensor of the MC-LR are enhanced significantly with a wide linear range and a low detection limit. Thus, this strategy provides a new way to detect the MC-LR in real samples.Acknowledgments
This work was supported by the National Natural Science Foundation of China (Nos. 21165023, 21465026, 21765026, 21605130), the National Key Scientific Program of China (Nos. 2011CB911000, 01100205020503104).Appendix A. Supplementary data
Supplementarymaterial related to this article can befound, in the online version, at doi:https://doi.org/10.1016/j.cclet.2018.10.021.
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