Research Paper
A molybdenum disulfide/gold nanorod composite-based electrochemical immunosensor for sensitive and quantitative detection of microcystin-LR in environmental samples

https://doi.org/10.1016/j.snb.2017.01.030Get rights and content

Highlights

  • An electrochemical immunosensor was developed for the detection of MC-LR.

  • MoS2 and AuNRs nanocomposite was employed to provide a large surface area and excellent biocompatibiltiy.

  • HRP-labeled MC-LR antibody was used as an electrocatalytic signal label.

  • The proposed strategy provides a biocompatible immobilization and sensitized recognition platform for MC-LR.

Abstract

A sensitive electrochemical immunosensor based on a molybdenum disulfide (MoS2) and gold nanorods (AuNRs) composite was developed for the detection of microcystin-LR (MC-LR). The immunosensor was constructed by immobilizing MC-LR antibody on the MoS2/AuNRs nanocomposite modified gold electrode. Using a competitive immunoassay format, the coated MC-LR antibody competed for MC-LR antigen with added target MC-LR, forming an antibody-antigen immunocomplex. Subsequently, horseradish peroxidase-labeled anti-MC-LR antibody (HRP-Ab2) was captured and detected. Under optimal conditions, the immunosensor exhibited a linear response to MC-LR ranging from 0.01 to 20 μg L−1 with a detection limit of 5 ng L−1 at a signal to noise of 3. This electrochemical immunosensor showed good performance with high sensitivity, good stability, accepted reusability and promising applications in routine water quality monitoring for various toxins.

Introduction

Cyanobacterial blooms pose a serious threat to public health and the environment due to the release of cyanotoxins into water sources [1]. One of the most common cyanotoxins is cyclic heptapeptide toxin of microcystins (MCs), which may cause mainly functional and structural disturbances of the liver due to a marked inhibition of protein phosphatases [2], [3], [4]. Microcystin-leucinearginine (MC-LR), containing 5 nonproteinogens and 2 substitutions of leucine (L) and arginine (R) at positions 2 and 4, is the most common and widely studied toxic species among the other 90 MCs congeners [1], [5]. MC-LR and other microcystins are considered primary causes of water pollution and outbreaks of toxic biological contaminants [6]. Moreover, MC-LR is particularly stable over wide pH and temperature ranges because of its cyclic structure [7]. The World Health Organization (WHO) has set up a provisional guideline limit of 1.0 μg L−1 for MC-LR in drinking water and daily intake value of 0.04 μg kg−1 MC-LR/body weight because of the toxicity of MC-LR [8]. It is a considerable challenge to quantify MC-LR in environmental samples due to low concentration levels and more than 90 known analogues. Therefore, a sensitive, practicable and convenient analytical method for the identification and quantification of MC-LR is of great importance for public health and environmental monitoring.

Currently, the analytical techniques for MC-LR detection mainly involve high-performance liquid chromatography (HPLC) [9], mass spectrometry (MS) [10], [11], nuclear magnetic resonance (NMR) [12], capillary electrophoresis (CE) [13], and protein phosphatase inhibition assays [14]. Though these methods are well proven and widely accepted, they are complex, costly, and time-consuming. Thus, immunoassays are of great interest for qualitative and quantitative detection of MC-LR due to their highly specific molecular recognition without the need for prior sample concentration and pretreatment. Attempts have been made to develop immunosensors based on fluorescence [15], [16], electrochemiluminescence [17], photoelectrochemical [18], [19], colorimetric [20], [21], and electrochemical [1], [22], [23] techniques. The best candidates for on-site immunoassay of MC-LR are electrochemical immunosensors due to the high sensitivity, simplicity, ease of miniaturization, low cost, and portability. However, in immunoassays, the antibodies are commonly immobilized on the surface of the electrode by physical absorption, which might lead to non-reversible protein denaturation and, thus inhibit the interaction between the antibodies and the analytical device. Alternately, a biocompatible nanomaterial composite for the effective immobilization and detection of small antigen molecules in a highly sensitive and selective immunosensor platform is of considerable interest for MC-LR analysis.

Recently, layered transition-metal dichalcogenides (such as MoS2, WS2, SnS2 and VS2) have attracted more and more attention in the field of electrochemistry due to their large surface specific area and high electronic conductivity [24]. Molybdenum disulfide (MoS2) is a common transition-metal dichalcogenide. It is composed of a metal Mo layer sandwiched between 2 sulfur layers and stacked together by weak van der Waals interactions [25], [26]. The layered structure of MoS2 is expected to act as an excellent functional material because the 2-dimensional electron–electron correlations among Mo atoms aid in enhancing planar electric transportation properties. Indeed, as a graphene analogue, MoS2 nanosheets have extensive applications as catalysts [27], lubricants [28], lithium batteries [29], super capacitors [30], [31], electrochemical sensors [32], [33], [34], [35], [36], [37], and so on. However, few studies have evaluated MoS2 as an electrode material in sensors because the electronic conductivity of MoS2 is still lower than that of graphene. The combination of MoS2 and other conducting nanomaterials may overcome this deficiency as combined nanocomposites exhibit synergistic effects, such as perfect conductivity, larger surface area and excellent electrochemical performance [38], [39], [40], [41]. The synergistic effects enable these nanocomposites the ability to function as redox probes and enzymes, which provides new perspective in the construction of novel biosensors [42], [43]. Furthermore, like graphene, MoS2 nanosheets tend to form agglomerates through van der Waals interactions, which greatly restrict further applications in electrochemical sensing. Therefore, the functionalization of MoS2 nanosheets to prevent aggregation is very important for expanding its functionality in electrochemical sensors. Surface functionalization with conductive nanomaterials is a potential method of preventing aggregation, because the solubility and dispersibility of MoS2 nanosheets would be greatly improved while the unique properties of MoS2 nanosheets are maintained.

In this work, a novel electrochemical immunosensor, based on a MoS2/AuNRs nanocomposite, was designed for the detection of MC-LR. The application of AuNRs in electrochemical biosensing was attractive because of their biocompatibility, chemical stability, high surface area and fast electron transfer ability [44], [45]. The AuNRs not only improve the electronic conductivity of MoS2 but can also prevent MoS2 aggregation during further treatments. The MoS2/AuNRs nanocomposite was used as an immobilization material to improve the electrochemical performance of the electrode. The immunosensor was constructed by immobilizing MC-LR antibody on a MoS2/AuNRs nanocomposite modified gold electrode. Bovine serum albumin (BSA) was used to block non-specific adsorption sites of the electrode to obtain an immunosensor for MC-LR detection. In a competitive immunoassay format, the coated MC-LR antibody competes for MC-LR antigen with added target MC-LR for form an antibody-antigen immunocomplex. Then, the immunocomplex reacted with HRP-labeled secondary antibody and was subsequently detected by differential pulse voltammetry. The proposed strategy provides biocompatible immobilization and a sensitized recognition platform for MC-LR, and this platform shows great promise in food and environmental monitoring applications.

Section snippets

Materials and reagents

Microcystin-LR (MC-LR) and monoclonal antibody of MC-LR were purchased from Express Technology Co., Ltd. (Beijing, China). A stock solution of MC-LR (1 mg mL−1) was prepared using 0.01 M PBS (pH 7.5) and stored at 4 °C. The blocking buffer was 0.02 M PBS (pH 7.5) containing 2% BSA. Horseradish peroxidase (HRP), bovine serum albumin (BSA), sodium molybdate dehydrate (Na2MoO4·2H2O) and l-cysteine were purchased from Aladdin Industrial Corporation (Shanghai, China). Chloroauric acid trihydrate,

Characterization of the MoS2 nanosheets and AuNRs

The morphology of the as-prepared MoS2 sample was characterized by SEM, TEM and HRTEM. Fig. 1A displays the SEM image of the MoS2 nanosheets, illustrating few-layer flexible wrinkled nanosheets and a flower-like shape of MoS2. Many irregular MoS2 nanosheets with thickness of several nanometers aggregated together and assembled into the nanoflowers are clearly shown, indicating that the prepared MoS2 nanosheets have large specific surface areas. Fig. 1B shows a TEM image of the prepared MoS2

Conclusion

In summary, a sensitive and quantitative electrochemical immunosensor was developed for the detection of MC-LR based on a MoS2/AuNRs nanocomposite and HRP-antibody. The MoS2/AuNRs nanocomposite provides a large surface area and excellent biocompatibility. The HRP-antibody complex was used as an electrocatalytic signal transducer. Dual signal amplification was achieved by synergistic effect of the MoS2/AuNRs nanocomposite and HRP-antibody electrocatalyst to H2O2. Under the optimized experimental

Acknowledgements

The authors gratefully acknowledge financial support from the National Natural Science Foundation of China (21565031, 21665027, and 21463028), YMU-DEAKIN International Associated Laboratory on Functional Materials, and the open funding project of the State Key Laboratory of Chemo/Biosensing and Chemometrics (2013014), Hunan University, PR China.

Yanli Zhang received her BSc (2002) degree in chemistry from Yan'an University, MSc and PhD degrees in analytical chemistry from Yunnan University and Hunan University in 2005 and 2008, respectively. Presently, she is an associate professor at Yunnan Minzu University. Her research interests include electrochemical analysis, electrochemical and biological sensors.

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      According to the definition of the International Union of Pure and Applied Chemistry (IUPAC), a biosensor is an integrated receptor-transducer device which is capable of providing specific quantitative or semi-quantitative analytical information using a biological recognition element (biochemical receptor) and a transducer. Up to now, various biological and synthetic recognition elements have been used as bio-receptors for cyanotoxins detection, such as protein phosphatase [16–18], antibodies [19–21], molecularly imprinted polymers [22–24] and aptamers [25–29]. Because of high specificity, antibodies are preferably employed to construct immunosensors.

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    Yanli Zhang received her BSc (2002) degree in chemistry from Yan'an University, MSc and PhD degrees in analytical chemistry from Yunnan University and Hunan University in 2005 and 2008, respectively. Presently, she is an associate professor at Yunnan Minzu University. Her research interests include electrochemical analysis, electrochemical and biological sensors.

    Meng Chen received her BSc (2014) degree in chemistry from Yunnan Minzu University. She is a graduate student at Yunnan Minzu University. Her current researches include electrochemical sensors and biosensors.

    Haiyan Li received her BSc (2014) degree in chemistry from Yunnan Minzu University. She is a graduate student at Yunnan Minzu University. Her current research focuses on electrochemical biosensors.

    Fuqing Yan received her BSc (2014) degree in costume design and engineering from Dezhou University. She is a graduate student at Yunnan Minzu University. Her current research focuses on activated carbons and their application in electrochemical biosensors.

    Pengfei Pang received his BSc (2002) degree in chemistry from Yan'an University, MSc and PhD degrees in analytical chemistry from Hunan University in 2005 and 2008, respectively. He is currently working as an associate professor at Yunnan Minzu University. His research interests include electrochemical chemical and biological sensors.

    Hongbin Wang received his BSc (1989) degree in chemistry from Sichuan University and MSc degree in 2003 from Kunming University of Science and Technology. Presently, he is a professor at Yunnan Minzu University. His research interests include functional materials and application, environmental chemistry and separation and analysis technology.

    Zhan Wu received her PhD degree in chemistry in 2010 from Hunan University. She is currently an assistant professor at Hunan University. Her research is focused on electrochemical biosensors.

    Wenrong Yang received his Ph.D. degree in chemistry in 2002 from the University of New South Wales. He is currently a senior lecture at Deakin University, working on biological and biomedical applications of CNTs and graphene. He also is exploring single-molecule conductivity by scanning probe microscopy.

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