A Novel Magnetoelastic Nanobiosensor for Highly Sensitive Detection of Atrazine
- 287 Downloads
Here, we firstly report a wireless magnetoelastic (ME) nanobiosensor, based on ME materials and gold nanoparticles (AuNPs), for highly sensitive detection of atrazine employing the competitive immunoassay. In response to a time-varying magnetic field, the ME material longitudinally vibrates at its resonance frequency which can be affected by its mass loading. The layer of AuNPs coating on the ME material contributes to its biocompatibility, stability, and sensitivity. The atrazine antibody was oriented immobilized on the AuNPs-coated ME material surface through protein A, improving the nanobiosensor’s performance. Atomic force microscope (AFM) analysis proved that the immobilization of atrazine antibody was successful. Furthermore, to enhance the sensitivity, atrazine–albumin conjugate (Atr–BSA) was induced to compete with atrazine for binding with atrazine antibody, amplifying the signal response. The resonance frequency shift is inversely and linearly proportional to the logarithm of atrazine concentrations ranging from 1 ng/mL to 100 μg/mL, with the sensitivity of 3.43 Hz/μg mL−1 and the detection limit of 1 ng/mL, which is significantly lower than the standard established by US Environmental Protection Agency (EPA). The experimental results indicated that the ME nanobiosensor displayed strong specificity and stability toward atrazine. This study provides a new convenient method for rapid, selective, and highly sensitive detection of atrazine, which has implications for its applications in water quality monitoring and other environmental detection fields.
KeywordsME nanobiosensor ME materials AuNPs Atrazine detection
Atom force microscope
Atrazine–albumin conjugate antigen
Bovine serum albumin
1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride
Scanning electron microscope
- US EPA
Environmental Protection Agency in the United States
With the rapid development of industry and agriculture, more and more environmental contaminants were released into the ecological environment , which caused widespread concern about the relevant researches [2, 3]. In recent years, herbicides have been used in increasing amounts to improve quality and yield in agriculture fields, but many herbicides can remain active in water and soils for years causing serious environmental pollution . Herbicide pollution has attracted considerable attention due to its ecological contamination in water or in agriculture products . Among herbicides, atrazine (2-chloro-4-ethylamino-6-isopropylamino-1, 3, 5-triazine) is the most extensively used for broad-leaf plants and grassy weeds control around the world .
Although atrazine has certain inhibitory effect on some perennial weeds, as the environmental contaminant, it is highly toxic  and may cause health risks for humans and other animal species . Long-term high concentrations of atrazine intake can impair animal or human health, such as cancer, birth defects, and damage to the heart and liver [9, 10]. The USA, the European Union, and Japan have all included atrazine in the list of endocrine-disrupting chemicals . In the USA, the Environmental Protection Agency (EPA) allows the permissible limit 3 μg/L (Lifetime Health Advisory Level) of atrazine in drinking water . Thus, it is necessary to accurately quantify atrazine at low concentrations.
Many conventional analytical techniques have been developed for atrazine detection, including LC coupled to mass spectrometry (LC–MS) , high-performance liquid chromatography (HPLC) , and gas chromatography coupled also with mass spectrometry(GC–MS) , but these methods also have some limitations, such as high-cost, need of large instruments, poor selectivity, and time-consuming .
Based on above unique properties of the ME material, the resonance frequency of the ME material decreases with an increase of the extra mass load. Thus, through their functionalization with a sensing film, the ME materials have been developed for physical, chemical, and biological analysis, such as the detection of stress/pressure , temperature/humidity , carbon dioxide , endotoxin , Salmonella typhimurium/Bacillus anthracis spores , and Escherichia coli O157:H7 . To our knowledge, however, no application of the ME material has been applied on the atrazine detection.
In this research, utilizing its excellent properties and advantages, we firstly proposed a wireless ME nanobiosensor employing the ME material as the substrate and gold nanoparticles (AuNPs) as the coating layer, for atrazine detection at ppb level on the basis of the direct competitive immunoassay procedures. Compared with the covalent-random antibody immobilization, the covalent-oriented strategy is more beneficial to improve the sensitivity of the nanobiosensor. Because the protein A is an interesting alternative to specifically bind with the Fc immunoglobulin region of the antibody, it was employed for oriented immobilization of the atrazine antibody , giving the highest immobilization density, to exhibit better antigen binding efficiency and improve nanobiosensor’s performance . The direct competitive immunoassay for atrazine was constructed by oriented immobilization of atrazine antibody to protein A covalently modified on the AuNPs-coated ME material surface, followed by the competitive reaction of atrazine–albumin conjugate (Atr–BSA) and atrazine with the atrazine antibody. Atr–BSA was induced to amplify the signal responses, in turn significantly increasing the sensitivity of the nanobiosensor. The efficiency of the ME nanobiosensor was evaluated, demonstrating that a novel ME nanobiosensor for the detection of trace concentrations of atrazine was successfully developed.
Materials and Methods
Atrazine antibody, atrazine–albumin conjugate antigen (Atr–BSA), atrazine, and protein A were purchased from EastCoast Bio (Maine, USA). Simazine, prometryn, and dichlorodiphenyltrichloroethane (DDT) were obtained from Chengdu Huaxia Chemical Reagent Co., Ltd. Cysteamine, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC), N-hydroxysulfosuccinimide (NHS), bovine serum albumin (BSA, 99%), and phosphate-buffered saline(PBS buffer, pH = 7.4) were purchased from Sigma-Aldrich Corporation (Saint Louis, MO, USA).
ME Nanobiosensor Fabrication
Preparation of the ME Nanosensor Platform
Atrazine Antibody Immobilization
The AuNPs-coated nanosensor platforms were ultrasonically cleaned with acetone, isopropanol, deionized water, and ethanol each for 5 min, and dried under a stream of nitrogen. Then, the nanosensor platforms were immersed into cysteamine solution (10 mM) for 12 h at room temperature to obtain a self-assembled monolayer (SAM) (Fig. 1c). The protein A (1 mg/mL) was activated with 4 mg/mL EDC-4 mg/mL NHS for 30 min at room temperature. After that, the activated protein A was incubated on the SAM-modified nanosensors for 30 min at 37 °C and rinsed with PBS buffer (Fig. 1d). The nanosensor platforms were then incubated with atrazine antibody for 50 min and washed with PBS buffer (Fig. 1e). To prevent non-specific adsorption, the atrazine antibody-coated nanosensors were further treated with 0.5% BSA for 30 min, and then rinsed with PBS buffer to remove any unbound BSA and dried under a nitrogen stream. Finally, the ME nanobiosensors were fabricated for atrazine detection (Fig. 1f).
Results and Discussion
Characterization of the Nanobiosensor Surface Morphology
Optimization for Concentration of Atrazine Antibody
Optimization for Concentration of Atr–BSA
Since atrazine is the small molecule, direct competitive immunoassay approach was employed to improve the sensitivity of the ME nanobiosensor. In the direct competitive immunoassay, the antibody is modified on the sensor surface and the signal response results from the binding of Atr–BSA molecule. Conversely, in the indirect competitive immunoassay, Atr–BSA is immobilized on the sensor surface and the response results from the binding of antibody molecule. According to the literature researches  and our results, the direct competitive immunoassay is feasible for small molecules monitoring. The indirect competitive immunoassay is highly sensitive to the analyte sample with trace concentration . Although the indirect competitive immunoassay has a higher sensitivity [40, 41], it may be complicated to operate and difficult to implement for repeated reliable use . However, the direct competitive immunoassay is very fast, simple to use, and self-contained—no additional reagents needed . Thus, for the future development, the direct competitive immunoassay may be the most promising method.
ME Nanobiosensor Specificity
ME Nanobiosensor Stability
A wireless ME nanobiosensor based on ME materials and AuNPs was successfully developed for real-time and highly sensitive detection of atrazine employing the competitive immunoassay. The oriented immobilization of atrazine antibody through protein A improved the nanobiosensor’s performance. Atr–BSA with heavy molecule mass and atrazine competitively combined with atrazine antibody on the nanobiosensor surface, amplifying the signal responses, which in turn improved the sensitivity. The resonance frequency shift mainly induced by the bound Atr–BSA is inversely proportional to the target atrazine concentration. Besides, the working concentrations of atrazine antibody and Atr–BSA were optimized to be 50 μg/mL and 40 μg/mL, respectively. Under the optimum conditions, the ME nanobiosensor displays widely linear determination ranges for atrazine from 1 ng/mL to 100 μg/mL, with the satisfactory sensitivity of 3.43 Hz/μg mL−1 and the detection limit of 1 ng/mL which is sufficient for the legislative requirements and is lower than other reported methods. AFM images verified that the atrazine antibody was successfully immobilized on the nanobiosensor surface in an oriented manner. The experimental results demonstrate that the ME nanobiosensor has high specificity and stability toward atrazine. Benefiting from its effects on detection limits, simplicity, disposable property, and wireless nature, the study not only proposed a new method for highly sensitive detection of atrazine but also indicated its potential practicability for other environmental contaminants detection and water quality monitoring.
The authors are grateful for the financial support from the National Natural Science Foundation of China (No. 51622507, 61471255, 61474079, 61501316, 51505324), Excellent Talents Technology Innovation Program of Shanxi Province of China (201605D211023).
Availability of Data and Materials
All data generated or analyzed during this study are included in this published article.
SS and YZ designed the experiments; XG, RL, and JW performed the experiments; XG and JG analyzed the data; XG wrote the paper; and ZY and WZ discussed the results and commented on the manuscript. All authors read and approved the final manuscript.
The authors declare that they have no competing interests.
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
- 1.Huang X, Hou X, Zhang X, Rosso K, Zhang L (2018) Facet-dependent contaminant removal properties of hematite nanocrystals and their environmental implications. Environ Sci NanoGoogle Scholar
- 2.Chen X, Pu H, Fu Z, Sui X, Chang J, Chen J, Mao S (2018) Real-time and selective detection of nitrates in water using graphene-based field-effect transistor sensors. Environ Sci NanoGoogle Scholar
- 3.Li Z, Jiang Y, Liu C, Wang Z, Cao Z, Yuan Y, Li M, Wang Y, Fang D, Guo Z (2018) Emerging investigator series: dispersed transition metal on nitrogen doped carbon nanoframework for environmental hydrogen peroxide detection. Environ Sci NanoGoogle Scholar
- 13.Koivunen ME, Dettmer K, Vermeulen R, Bakke B, Gee SJ, Hammock BD (2006) Improved methods for urinary atrazine mercapturate analysis—assessment of an enzyme-linked immunosorbent assay (ELISA) and a novel liquid chromatography–mass spectrometry (LC–MS) method utilizing online solid phase extraction (SPE). Anal Chim Acta 572(2):180–189CrossRefGoogle Scholar
- 14.Li YN, Wu HL, Qing XD, Li Q, Li SF, Fu HY (2010) Quantitative analysis of triazine herbicides in environmental samples by using high performance liquid chromatography and diode array detection combined with second-order calibration based on an alternating penalty trilinear decomposition algorithm. Anal Chim Acta 678(1):26–33CrossRefGoogle Scholar
Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made.