Magnetic-graphene oxide based molecularly imprinted polymers for selective extraction of microsystin-LR prior to the determination by HPLC

Accepted Manuscript Magnetic-graphene oxide based molecularly imprinted polymers for selective extraction of microsystin-LR prior to the determination by HPLC

Xingguo Tian, Chuang She, Zhenke Qi, Xiaoyan Xu PII: DOI: Reference:

S0026-265X(18)31788-0 https://doi.org/10.1016/j.microc.2019.02.033 MICROC 3679

To appear in:

Microchemical Journal

Received date: Revised date: Accepted date:

8 December 2018 27 January 2019 11 February 2019

Please cite this article as: X. Tian, C. She, Z. Qi, et al., Magnetic-graphene oxide based molecularly imprinted polymers for selective extraction of microsystin-LR prior to the determination by HPLC, Microchemical Journal, https://doi.org/10.1016/ j.microc.2019.02.033

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ACCEPTED MANUSCRIPT Magnetic-graphene oxide based molecularly imprinted polymers for selective extraction of microsystin-LR prior to the determination by HPLC

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Xingguo Tian, Chuang She, Zhenke Qi, Xiaoyan Xu *

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Guangdong Provincial Key Laboratory of Food Quality and Safety, College of Food Science, South

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China Agricultural University, Guangzhou 510642, China

* Corresponding author. [email protected]; Tel.: +86-020-8528-3448

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Address: No.483, Wushan Road, Tianhe District, Guangzhou City, Guangdong Province,

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China

ABATRACT

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A novel magnetic molecularly imprinted polymer on a magnetic graphene oxide surface, GO@Fe3O4-MIP, was successfully synthesized and applied as adsorbent for the

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extraction of microcystin-LR (MC-LR) from water samples prior to analysis using high performance liquid chromatography (HPLC). A variety of characterizations have proven

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successful preparation of this material. The GO@Fe3O4-MIPs showed high adsorption capacity with maximum adsorption capacity of 1.24 mg/g, fast adsorption rate (25 min) and high selectivity toward MC-LR. The adsorption processes followed the Freundlich isotherm and pseudo-second-order kinetic models. Various extraction conditions were optimized. The linearity of the method was studied from 2 to 10000 μg/L, obtaining a correlation coefficient of 0.9985. The limit of detection was calculated as 0.08 μg/L (S/N=3), and the recoveries from spiked samples were 86%-113%, with relative

ACCEPTED MANUSCRIPT standard deviations of 1.0%-6.8%. The developed method was suitable for the rapid and sensitive determination of MC-LR in water samples. Keywords: Molecularly imprinted polymers; Magnetic graphene oxide; Surface

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imprinting; Solid-phase extraction; MC-LR

ACCEPTED MANUSCRIPT 1. Introduction Microcystins (MCs), a group of monocyclic heptapeptides, generated by the cyanobacterial blooms frequently occurring in freshwater throughout the world [1, 2]. Humans may be exposed to these toxins mainly through consumption of contaminated fish and drinking water [3, 4]. Microcystins are considered to be an invisible

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hepatotoxin due to their inhibitory effects on intracellular serine/threonine phosphatases

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1 and 2 A (PP1 and PP2A) [5-6]. Increasing evidences have demonstrated that they might also target other organs such as kidney [7-9], intestine [10, 11], lungs [12],

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reproductive system [13], and brain [14, 15]. Among the microcystins isolated and identified to date, microcystin-LR (MC-LR) ，with leucine (L) and arginine (R) at the

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two variable positions，is extremely toxic and the most frequently addressed member [16].The World Health Organization (WHO) has stipulated the threshold limit as 1 µg/L

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for MC-LR in drinking water.

So far, various methodologies including enzyme-linked immune sorbent assay

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(ELISA) [17, 18], high-performance liquid chromatography (HPLC) [19, 20], capillary electrophoresis (CE) [21], electrochemical biosensors [22] and protein phosphatase

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inhibition assay (PPIA) [23] have been utilized and developed for the detection of MC-LR. Amongst these analytical methods, HPLC with UV detection is the most

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popular approach. However, detection of MC-LR at extremely low concentrations still remains challenging due to matrix interferences in complex environmental samples.

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Therefore, proper sample pretreatments processes to isolate and enrich target analytes are indispensable prior to HPLC analysis. The most commonly used sample preparation

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technology is solid-phase extraction (SPE) due to its simplicity and rapidness. However, regardless of the mode of SPE, the choice of sorbent is a key factor influencing its performance. Molecular imprinting polymers (MIPs) have been widely exploited as one of the most attractive sorbents for SPE in the past decade because of their high adsorption capacity, high selectivity, low cost, and ease of preparation [24-26]. MIPs are tailor-made polymeric materials with three-dimensional cavities complementary in shape, size and functional groups to the template molecules, so they can offer refabricated structures, specific recognition, and universal application [27-29]. Nevertheless, MIPs also have some inevitable shortcomings in practical applications

ACCEPTED MANUSCRIPT like slow mass transfer, heterogeneous binding sites and complicated after-treatment workup. It is gratifying that surface molecular imprinting can overcome the abovementioned problems because it can form binding sites at or approximate to the supports surface. Surface imprinting over nanosized support materials with large specific surface area is suitable for achievement of high binding capacity [30]. Graphene oxide (GO) would

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therefore propose to be an excellent supporting material for synthesis of imprinted

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polymers owing to its unique properties such as large surface area, extraordinary

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mechanical properties, and abundant oxygen-containing functional groups (e.g. epoxide, hydroxyl, and carboxylic groups). In practical situations, functionalization of GO is

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important because it can help to improve aqueous dispersibility, reduce aggregation, decrease toxicity, and increase selectivity of GO [32]. Dopamine, a unique

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mussel-adhesive-protein inspired biomolecule that contains catechol and amine functional groups, can self-polymerization to form polydopamine (PDA) in weak

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alkaline solution, which can modify almost all material surfaces [33]. Taking advantage of its structural characteristics and fascinating properties (self-polymerization,

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reduction, and adhesion), dopamine is expected to show excellent performance in the preparation of functionalized GO [34]. One deficiency of the MIPs on a GO surface that

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can not be ignored is the nanoparticle sizes make it hard to be separated by traditional centrifugation and filtration methods. The marriage of the GO and magnetite

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nanoparticles (Fe3O4) can endow the resulting polymer material (Fe3O4@GO-MIPs) with magnetic susceptibility and therefore easily separated by external magnetic fields

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after adsorption and recognition. The aim of the study was to synthesize and apply a novel and selective adsorbent for SPE coupled with HPLC to extract and detect MC-LR in natural and public drinking water. For the purpose, A MIP was prepared based on a one-pot method, in which Fe3O4@GO was used as a supporting material and dopamine was used to functionalize GO. Meanwhile, dopamine acted as a functional monomer as well as cross-linker in molecular imprinting. The obtained MIP was thoroughly characterized, and its adsorption performance for MC-LR was evaluated. In addition, taking the advantages of Fe3O4@GO-MIPs as magnetic adsorbent, a facile and sensitive method for MC-LR

ACCEPTED MANUSCRIPT analysis based on HPLC with magnetic solid-phase extraction (MSPE) pretreatment was proposed and further applied in practical samples.

2. Experimental 2.1. Materials and reagents The microcystin standard (MC- LR, RR, YR, respectively, ≥95%) were purchased

Reagent

Factory

(Tianjin,

China).

Dopamine

hydrochloride,

tris

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Chemical

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from Algal Science lnc. (Taiwan, China). Graphite powder was obtained from Damao

(hydroxymethyl)aminomethane (Tris) and 2, 2-azobisisobutyronitrile (AIBN) were

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purchased from Macklin Biochemical Co. Ltd. (Shanghai, China). All other chemicals were of analytical grade and were used directly without further treatment. Ultrapure

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water (18.2 MΩ) was prepared using a Milli-Q water purification system (Millipore, Bedford, USA).

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2.2. Apparatus

Fourier transform infrared (FT-IR) spectra were recorded using an Fourier transform

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infrared (FT-IR) spectra (4000-500 cm-1) were recorded using a Vertex 70 spectrometer (Bruker, Ettlingen, Germany) with KBr pellets and a resolution of 0.4 cm -1. X-ray

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diffraction (XRD) patterns were acquired on a D8-ADVANCE X-ray diffractometer (Bruker, Karlsruhe, Germany). Transmission electron microscope (TEM) images were

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captured using a JEM-2100F instrument (JEOL, Peabody, MA, USA). Thermal gravimetric analysis (TGA) was performed on a STA449 F3 instrument (Netzsch,

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Hanau, Germany) under argon atmosphere at a heating rate of 10 ℃/min. The magnetic properties were examined using an EZ7 vibrating sample magnetometer (VSM)

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(MicroSense, Lowell, MA, USA). Physisorption analysis was performed on a Brunauer-Emett-Teller (BET) V-Sorb 2800 Series Analyzer (ASAP2020HD88, MIKE, America) at 77k using N 2 as adsorbate molecules. 2.3. Preparation of Fe3O4@GO Graphene oxide (GO) was first prepared from a natural graphite powder by a modified Hummers method [35] and then Fe3O4@GO composites were prepared. Briefly, 50 mg of GO was completely dispersed in 100mL ultrapure water by sonication for 1 h. Then, 4 mmol of FeCl3•6H2O and 2 mmol of FeSO4•7H2O were added to GO solution in sequence. After dissolved thoroughly, 2 mL NH 4OH (25%) was quickly

ACCEPTED MANUSCRIPT added into the solution under violent stirring. The solution was stirred in water bath at 85 °C for approximately 30 min followed by adding 50 mg of dopamine hydrochloride. After being continuously stirred for another1 h, the solution was cooled down to room temperature. The resulting black Fe3O4@GO composites were separated by external magnetic field and washed repeatedly with ultrapure water to pH=7，then washed with

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anhydrous ethanol for three times and dried in vacuum at 70 °C for 12 h.

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2.4. Synthesis of Fe3O4@GO-MIPs

The Fe3O4@GO-MIPs were prepared via surface molecular imprinting technique.

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Firstly, 3 mL of 10 μg/mL MC-LR and 5 mg of dopamine hydrochloride were mixed for 1 h in 200 mL of Tris-HCl buffer solution (pH=8.5) for preassembly. Then 50 mg of

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Fe3O4@GO and 100 mg of AIBN were added to the mixture to initiate the polymerization reaction. The reaction was allowed to proceed at 70 ℃ for 12 h under

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nitrogen stream. The obtained product was washed with ethanol for several times and collected by magnetic separation. Afterward, the template molecules were eluted with

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acetic acid /methanol mixture (2:8, v/v) until no MC-LR was detected in eluent by HPLC. the particles were dried in a vacuum oven at 70 ℃ for further use. For

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comparison, non-imprinted polymers (Fe3O4@GO-NIPs) were prepared in the same way except for the omission of the template.

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2.5. Procedures of MSPE

The tap water sample was collected from the laboratory. The lake water sample was

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collected from Ningyin Lake of South China Agricultural University in China. The samples were filtered through a 0.45 μm microporous membrane before extraction.

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10 mg of Fe3O4@GO-MIPs were added into a sample solution to absorb the analyte MC-LR. After performing the extraction by an ultrasonic water bath for 25 min at room temperature, the Fe3O4@GO-MIPs rebound with analyte were separated with the help of a magnet easily and the supernatant was discarded. Washed with acetic acid /methanol mixture (2:8, v/v), the rebound analyte was eluted from the Fe3O4@GO-MIPs. Finally, the collected eluate was dried with a gentle stream of nitrogen and redissolved in 1mL of methanol for HPLC analysis. To evaluate the reusability of the Fe3O4@GO-MIPs, the used adsorbent was washed with acetic acid /methanol mixture (2:8, v/v), and then was used for the next extraction.

ACCEPTED MANUSCRIPT 2.6. HPLC analysis The amounts of analytes were detected by a LC-20A HPLC system (Shimadzu, Kyoto, Japan) with a SPD-20A UV detector. The HPLC conditions were as follows: analytical column (250 mm × 4.6 mm, 5 μm, Waters, USA); column temperature, 40 °C; UV detection, at 238 nm; mobile phase, methanol/ phosphate buffer (pH 2.0) (57:43, v/v); flow rate, 1.0 mL/min; injection volume, 10 μL.

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2.7. Adsorption Experiments

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Static adsorption, kinetic adsorption, and selective adsorption were explored to

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characterize the specific molecular binding properties of Fe3O4@GO-MIPs. Static adsorption experiment was carried out as follows. A series of 10 mg of the

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Fe3O4@GO-MIPs and Fe3O4@GO-NIPs were suspended respectively into 20 mL of MC-LR standard solutions varying from 0.5 to 3.5 μg/mL. After incubation with

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shaking (200 rpm) for 60 min at room temperature, the mixtures were separated by an external magnet and the supernatants were passed through a 0.45 μm microfiltration

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membrane. The residual concentration of MC-LR was detected by HPLC system. The adsorption capacity of Fe3O4@GO-MIPs or Fe3O4@GO-NIPs toward MC-LR was ( C o  C e )V M

(1)

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Qe 

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calculated according to the following equation:

where Qe (mg/g) was the amount of MC-LR adsorbed by the Fe3O4@GO-MIPs or

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Fe3O4@GO-NIPs, Co and Ce were the initial and equilibrium concentration (mol/L) of MC-LR in the eluent solution, respectively; V (mL) were the solution volume; M (mg)

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was the weight of Fe3O4@GO-MIPs or Fe3O4@GO-NIPs. Similarly, in kinetic experiments, 10 mg of Fe3O4@GO-MIPs and Fe3O4@GO-NIPs were spiked with 20 mL of 1.5 μg/mL MC-LR solution. The mixtures were incubated at room temperature under shaking, and the supernatant concentrations were detected at defined time intervals from 2 to 50 min. Selective adsorption experiment was evaluated using MC-RR and MC-YR as structural analogs. 10 mg of the Fe3O4@GO-MIPs and Fe3O4@GO-NIPs were added to 20 mL of of 1.5 μg/mL solution of the above compounds. Then, the mixtures were shaken for 60 min. The Fe3O4@GO-MIPs and Fe3O4@GO-NIPs were collected by an

ACCEPTED MANUSCRIPT external magnet and the supernatant was analyzed by HPLC after filtration through a 0.45 μm membrane filter.

3. Results and discussion 3.1. Synthesis and characterization of Fe3O4@GO-MIPs Scheme 1 illustrated the preparation process of Fe3O4@GO-MIPs. Firstly,

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Fe3O4@GO was synthesized through a chemical deposition method, where iron ions

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was firmly absorbed on GO via the electrostatic and hydrogen interactions. Next, Fe3O4@GO was functionalized with dopamine, which can not only reduce GO into

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graphene but also endow abundant chemical groups for adsorption. Subsequently, a MIP thin film was coated onto the surface of Fe3O4@GO through the unique adhesive effect

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of dopamine. The template MC-LR was imprinted in the polymeric network through the non-covalent interactions of –NH2 and –OH groups on PDA as well as the –COOH and

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–NH groups on MC-LR. Finally, the templates were removed, resulting homologous cavities for the recognition of target MC-LR.

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The successful preparation of Fe3O4@GO-MIPs was evaluated by XRD, FT-IR, VSM and TEM spectroscopy. XRD analyses were used to characterize the phases and

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structures of GO and Fe3O4@GO-MIPs. As shown in Fig. S1(in Supplementary Information, SI), the XRD pattern of the GO showed a sharp peak at 2θ=10.9°, which

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corresponded to a d-spacing at 12.57 nm. Compared to GO, the diffraction peak of Fe3O4@GO-MIPs at 2θ=10.9° almost disappeared and the six characteristic peaks

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observed at 2θ values of 29.9°, 35.4°, 43.2°, 53.6°, 57.2°, 62.7° can be assigned to Fe3O4. These illustrated that GO was reduced by PDA and the coexistence of GO and

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Fe3O4 in the composites did not change the XRD phase of Fe3O4. Fig. 1 depicted the FT-IR spectra of the Fe3O4 (curve a), Fe3O4@GO (curve b) Fe3O4@GO-MIPs (curve c), Fe3O4@GO-NIPs (curve d), all of which showed the characteristic Fe-O stretching vibration peak at 589 cm-1. When compared with Fe3O4, the wide peak at approximate 3319 cm-1 (In curve b, c and d) was attributed to the N-H stretching vibrations from PDA, and the two peaks (1545 cm-1 and 1153 cm-1) were assigned to the C=C stretching vibration in the aromatic rings and the C-O-C stretching vibrations from GO, respectively. These results provide the evidence of the successful synthesis of Fe3O4@GO-MIPs.

ACCEPTED MANUSCRIPT The

morphology

and

microstructure

of

Fe3O4,

GO,

Fe3O4@GO

and

Fe3O4@GO-MIPs were fully detected by TEM and SEM observation. The SEM image of GO (Fig. 2a) clearly illustrated its typically crumpled and rippled structure. Fig. 2b showed that Fe3O4 consisted of large quantities of sphere-shaped particles, while the combination of Fe3O4 and GO resulted to a uniformly dispersed Fe 3O4 spheres on the GO layer (Fig. 2c). It can be observed in Fig. 2d that the prepared Fe3O4@GO-MIPs

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had much more porous than Fe3O4@GO, suggesting the formation of recognition sites

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accessible for the template molecules. The TEM image of GO (Fig. 3a) also confirmed

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that GO sheets were exfoliated successfully. Fig. 3b and 3c showed that Fe3O4 nanoparticles formed and were homogeneously anchored onto the GO sheets, and

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Fe3O4@GO possessed a narrow particle size distribution ranging from 16-22 nm. After coating with an imprinting layer, the surface of Fe3O4@GO-MIPs (Fig. 3d) turned

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rough and diameters of the microspheres increased to about 40 nm. The roughness of the surface should be considered as a factor providing an increase in the surface area

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[36], which was beneficial to the high binding capacity and rapid mass transfer. The saturation magnetization of the Fe 3O4, Fe3O4@GO and Fe3O4@GO-MIPs were

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obtained from the magnetization curves. Fig. S2 showed that the saturation magnetization values of Fe 3O4, Fe3O4@GO and Fe3O4@GO-MIPs gradually decreased

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to 88.1, 58.42, 52.38 emu/g, respectively, which was attributed to the layer by layer of modification on the surface of particles. Thus, the obtained Fe3O4@GO-MIPs exhibited

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excellent magnetism ability and could be used for the convenient magnetic separation. BET N2 adsorption/desorption measurement was carried out to certify the porous

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nature of the prepared Fe 3O4@GO-MIPs. As shown in Fig. S3, the isotherm represented the type-IV with an H-1 hysteresis loop at a high relative pressure of 0.6-0.9. The BET surface area and average pore size of Fe3O4@GO-MIPs were 81.92m2/g and 0.31 cm3/g, respectively. 3.2. Adsorption study and evaluation of selectivity 3.2.1. Isothermal adsorption The static binding isotherms of MC-LR on Fe3O4@GO-MIPs and Fe3O4@GO-NIPs were displayed in Fig. 4a. The adsorption capacity of Fe3O4@GO-MIPs increased with the increasing concentration and then gradually reached a plateau when the initial

ACCEPTED MANUSCRIPT concentration of MC-LR was 3 μg/mL. Fe3O4@GO-NIPs showed the same trend but with lower adsorption amounts at the same MC-LR concentration. In addition, the adsorptive maximum capacity of Fe3O4@GO-MIPs and Fe3O4@GO-MIPs were 1.24 mg/g and 0.67 mg/g, respectively, which can be attributed to the specific recognition sites on the surface of the imprinted materials in the polymerization reaction. Two classical isotherm models, namely, Langmuir and Freundlich models were

Ce 1  Qm Qm K L

1 ln Ce n

(2) (3)

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lnQe  ln K F 

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Qe

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Ce

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studied to comprehend the adsorption isotherms.

where Ce (mg/L) was the equilibrium concentration of template molecules; Qe (mg/g) and Qm (mg/g) were the equilibrium and maximum adsorption capacity, respectively;

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KL (L/mg) and KF (mg/g) were Langmuir and Freundlich binding coefficient, respectively. The corresponding parameters of the both isotherm models were

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calculated and the results were summarized in Table 1. Compared with the Langmuir model, the Freundlich model was more suitable because of the higher value of the

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correlation coefficients (R2). The results suggested that the adsorption of MC-LR onto

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the Fe3O4@GO-MIPs was a multilayer adsorption, and the adsorption occurred on a heterogeneous surface with different binding sites.

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3.2.2. Kinetic adsorption

The kinetic adsorption capacities of Fe3O4@GO-MIPs and Fe3O4@GO-NIPs for MC-LR were investigated as a function of incubation time. As shown in Fig. 4b, the

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adsorption amounts of MC-LR on both Fe3O4@GO-MIPs and Fe3O4@GO-NIPs increased and reached equilibrium within 25 min. However, it is obvious that the Fe3O4@GO-MIPs adsorbed more MC-LR than Fe3O4@GO-NIPs under the same conditions, further validating the specific affinity of Fe3O4@GO-MIPs toward MC-LR. Experimental data were simulated by pseudo-first-order and pseudo-second-order kinetic models as follows:

ln(Qe  Qt )  ln Qe  k1t

(4)

ACCEPTED MANUSCRIPT t 1 t   2 Qt k 2 Qe Qe

(5)

where Qt (mg/g) and Qe (mg/g) were the the adsorbed total amounts of MC-LR at time t and at equilibrium, respectively, and k1 (/min) and k2 (g/mg/min) were the first order and second order rate constants, respectively. The corresponding results were also tabulated in Table 1. As can be seen in Table 1, the kinetics data better fit to the

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pseudo-second-order model. Thus, it can be deduced that the adsorption of MC-LR onto

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Fe3O4@GO-MIPs followed the pseudo-second-order kinetic model and the chemical

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process was a rate-determining step. 3.2.3. Selectivity evaluation

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The binding capacity of template molecule and the structural analogs (Fig. S4) was contrasted to demonstrate selectivity property of desired products. The adsorption

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capacities of Fe3O4@GO-MIPs and Fe3O4@GO-NIPs for MC-LR and its analogs were presented in Fig.5. In addition, to further explore the selectivity and specific recognition

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of Fe3O4@GO-MIPs, the imprinting factor (α) and selectivity factor (β) were calculated as follows:

(6)

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α = QMIP/QNIP β= α template/ α analogue

(7)

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where QMIP and QNIP were the amount of analytes adsorbed by Fe3O4@GO-MIPs and Fe3O4@GO-NIPs, respectively, and αtemplate and αanalogue were imprinting factors of

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MC-LR and other analogs, respectively. The imprinting factor (α) and selectivity factor (β) were listed in Table S1. The images and data in Fig.5 and Table S1 conveyed us the

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expected information that Fe3O4@GO-MIPs displayed a much higher imprinting factor and specific adsorption capacity toward MC-LR than MC-RR and MC-YR. In contrast, the adsorption capacities of Fe3O4@GO-MIPs for MC-RR and MC-YR had no distinct change

compared

with

their

values

of

Fe3O4@GO-NIPs.

Moreover,

the

Fe3O4@GO-NIPs showed similar and little adsorption capacity for the three compounds. The results further revealed that Fe3O4@GO-MIPs surface possessed binding sites with shape and functional groups complementary to the template molecule via the imprinting process. 3.3. Extraction conditions

ACCEPTED MANUSCRIPT 3.3.1. Effect of pH on adsorption Sample pH played an important role in the adsorption process because it may influence the surface change of the adsorbent and the existing form of the analytes. The effect of sample pH on extraction efficiency was investigated in the pH ranged from 3.0 to 9.0. The experimental data (Fig. S5a) showed that the maximum extraction efficiency could

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be observed for the pH values between 6 and 8. This situation may be related to the

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hydrogen bond and π-π interaction between Fe3O4@GO-MIPs and MC-LR [37]. When

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in acidic medium, the increase in pH value could weaken the interference from hydrogen ions and the hydrogen bond affinity was accordingly strengthened. However,

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when pH exceeded 8.0, the electrostatic repulsion between Fe 3O4@GO-MIPs and MC-LR became predominant interaction. The binding to Fe3O4@GO-MIPs surface

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became difficult, thus, the extraction efficiency decreased. So the pH of 7.0 was chosen as the optimum value for subsequent analysis.

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3.3.2. Effect of adsorbent amount

Different amounts of Fe3O4@GO-MIPs (varying in the range of 5-50 mg) were

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applied to extract 20 mL of spiked water samples. Fig. S5b showed that the highest recovery of MC-LR could be achieved when the amount of Fe3O4@GO-MIPs was 10

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mg. Further increasing the amount of Fe3O4@GO-MIPs, no appreciable improvement was obtained for recovery of MC-LR. Herein, 10 mg of Fe3O4@GO-MIPs was selected

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for subsequent experiments.

3.3.3. Effect of desorption solvent

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In order to obtain the content recovery of MC-LR, a series of 20 mL mixture solutions with different volume ratios of methanol/acetic acid (7:3, 8:2, 9:1 and 10:0, v/v) were individually investigated as desorption solvents. From Fig. S5c, the volume ratio of 8:2 showed the highest recovery. Thus, 20 mL of methanol/acetic acid (8:2, v/v) mixture solution was selected as the desorption solution for removing MC-LR from Fe3O4@GO-MIPs. 3.3.4. Effect of extraction time and desorption time The total time for extraction and desorption was an index of great concern to assess the analysis efficiency. Accordingly, different extraction time between 5 and 50 min

ACCEPTED MANUSCRIPT were examined (Fig.S5d), and 25 min was finally selected as the optimal extraction time. The influence of desorption time on the recovery was also studied between 5 and 30 min (Fig.S5e), and 10 min was found as suitable duration to completely recover MC-LR from the adsorbent. 3.4. Reusability of the adsorbent To evaluate the reusability of the obtained Fe3O4@GO-MIPs, they were used as the

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adsorbent to rebind the blank sample spiked with MC-LR in six consecutive

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adsorption-desorption cycles. As shown in Fig. S6, the MC-LR recovery had no

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significant decrease and remained at 82.3% after the sixth cycle. This manifested that Fe3O4@GO-MIPs showed a stable regeneration adsorption efficiency and can be used 3.5. Analytical performance of the Method

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repeatedly

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To demonstrate the practical applicability of Fe3O4@GO-MIPs, water samples including lake water and tap water were analyzed and determined under the optimized conditions. As shown in Table 2, the recoveries of MC-LR spiked at three concentration

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levels varied in the range of 86% –113%, and the relative standard deviation (RSD) was

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less than 6.8%. The calibration curve was obtained in the range of 2-10000 μg/L with a correlation coefficient of 0.9985. Based on the signal-to-noise (S/N) of 3, the limit of

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detection (LOD) of MC-LR was calculated to be 0.08 μg/L. The typical MSPE-HPLC chromatograms of a water sample spiked with MC-LR (100 μg/L) before (curve a) and

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after (curve b) extraction with Fe3O4@GO-MIPs were shown in Fig.6. The performance of the developed procedure was further evaluated by comparing the

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analytical parameters of the method with those of recently reported researches for the extraction and determination of MC-LR. The results in Table 3 supported that the proposed method had wider linear range and acceptable LOD and RSD with more simple and rapid pretreatment. Therefore, the synthesized Fe3O4@GO-MIPs is a selective and reliable adsorbent suitable for preconcentration and determination of MC-LR at trace levels in real samples.

4. Conclusions In summary, a novel water-compatible Fe3O4@GO-MIPs were successfully synthesized by using Fe3O4@GO nanocomposite as the carrier material and then were

ACCEPTED MANUSCRIPT used as adsorbent for the selective separation and preconcentration of trace MC-LR prior to HPLC detection. The results of binding adsorption tests indicated that the Fe3O4@GO-MIPs exhibited a good performance in term of adsorption capacity, equilibration time, selectivity and reusability. The developed method had the outstanding advantages of simple separation, low consumption of organic solvents and high sensitivity. Therefore, the strategy lends itself as an attractive alternative for the

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analysis of MC-LR and other cyanotoxins in environmental and food samples.

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Acknowledgments

31501554),

Guangdong

Provincial

Natural

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This work was supported by the National Natural Science Foundation of China (No. Science

Foundation

of

China

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(2016A030310446), Guangdong Provincial Science and Technology Planning Project of China (2017A020208059).

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ACCEPTED MANUSCRIPT Table 1 Isotherms and kinetic parameters of MC-LR adsorption onto Fe3O4@GO-MIPs at 298 K. Parameters

Value

Langmuir

Qm (mg/g)

3.3003

KL (L/mg)

0.1943

R

KF (mg/g)

0.5195

n (L/mg)

1.2549

R Kinetics models

Pseudo-first-order

0.8312

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Freundlich

2

2

0.9799

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Isotherms models

Model

K1 (/min)

0.1093

R

K2 (g/mg/min) Qe (mg/g)

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Pseudo-second-order

2

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Qe (mg/g)

0.8791 0.05704 1.5760

2

0.9841

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R

1.2057

Table 2

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Accuracy of the proposed method for extraction water samples spiked at different levels. (n=3).

N.D.: Not detected

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a

0.45

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Lake water

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N.D.a

Tap water

Added （μg/L） 0.5 1 2

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Content （μg/L）

Samples

0.5 1 2

Found （μg/L）

Recovery （%）

0.52

104

RSD （%） 1.00

0.86

86

3.06

2.08

104

5.29

1.07

113

4.73

1.37

92

3.79

2.44

100

6.81

ACCEPTED MANUSCRIPT Table 3 Comparison of the proposed method with reported methods for extraction and detection of MC-LR.

[38]

1-5000

0.002

<10.7

[39]

MSPD-HPLC-MS/MS

5-100

13.0

<8.6

[19]

Electrochemical biosensor

0.004-0.512 2-10000

0.0014 0.08

<1.6

[40]

Linear range (μg/L)

LOD (μg/L)

SPE-LC/MS

0.0025-0.08

SPE-LC-MS/MS

<6.8

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MSPE-HPLC/UV

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0.0007

RSD （%） <6.8

Analytical technique

Ref.

This work

ACCEPTED MANUSCRIPT Figure captions Scheme 1. Schematic illustration of preparation of Fe3O4@GO-MIPs by surface imprinting. Fig. 1. FT-IR spectra of Fe3O4 (a), Fe3O4@GO(b), Fe3O4@GO-MIPs (c) and

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Fe3O4@GO-NIPs (d).

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Fig. 2. SEM images of GO (a), Fe3O4 (b), Fe3O4@GO (c) and Fe3O4@GO-MIPs (d). Fig. 3. TEM images of GO (a), Fe3O4 (b), Fe3O4@GO (c) and Fe3O4@GO-MIPs (d).

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Fig. 4. The adsorption isotherms (a) and kinetics (b) of MC-LR on Fe3O4@GO-MIPs and Fe3O4@GO-NIPs.

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Fig. 5. Competitive binding of MC-LR, MC-RR and MC-YR on Fe3O4@GO-MIPs and

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Fe3O4@GO-NIPs.

Fig.6. The typical MSPE-HPLC chromatograms of a water sample spiked with MC-LR

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(100 μg/L) before ((curve a) and after (curve b) extraction with Fe3O4@GO-MIPs.

ACCEPTED MANUSCRIPT Highlights A facile and green imprinting strategy for MC-LR extraction and detection was proposed.



Dopamine was used to functionalize GO, acted as functional monomer and cross-linker.



Fe3O4@GO-MIP had high imprinting efficiency, binding capacity and simple separation.

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

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6