Synthesis of graphene oxide functionalized surface-imprinted polymer for the preconcentration of tetracycline antibiotics

Abstract

In this work, we synthesized graphene oxide functionalized a surface-imprinted polymer based on the self-polymerization of dopamine to generate the imprinted cavity. Using minocycline as the template molecule, effects of experimental conditions in the imprinting procedure such as γ-MAPS@GO percentage, template concentration, and self-polymerization time of dopamine on the selectivity and performance of the prepared molecularly imprinted polymers were investigated. The characteristics of the prepared surface-imprinted polymer were determined by scanning electron microscope, transmission electron microscope, energy dispersive spectroscopy, Fourier-transformed infrared spectra, X-ray photoelectron spectroscopy, thermal gravimetric analysis, atomic force microscope, and water contact angle. The prepared graphene oxide functionalized surface-imprinted polymer showed good recognition capacity and enrichment performance for tetracycline antibiotics and was successfully applied to the detection of the target analytes in milk samples.

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1. Introduction

Graphene oxide (GO), as a two-dimensional carbon nanomaterial which is generated from graphite oxide, has engendered tremendous attention in the area of materials science and nanotechnology owing to its fascinating physical properties, such as excellent mechanical, electrical, thermal, and optical properties.^1–3 GO contains a range of reactive bearing hydroxyl, carboxyl, and epoxide functional groups on their basal planes, which renders it a good candidate for use in the aforementioned applications through chemical functionalizations.^4,5 Additionally, due to its ultrahigh surface area and excellent chemical properties, GO has been explored as a supporting material of adsorbents for the preconcentration of small molecules.^6,7 Even through these merits, it is difficult to use GO directly to fabricate uniform separation matrix and to retain it in the column, especially under high pressure in chromatographic systems. To avoid the abovementioned problems and still take advantage of the specific features of GO, fabrication of GO functionalized monolithic column is a good choice,⁸ among which monolithic columns emerged in the 1990s and have been widely used in many areas owing to their easy preparation, excellent permeability, and fast convection mass transfer. In our previous research, GO modified poly(glycidyl methacrylate-ethylene dimethacrylate) monolith was successfully synthesized based on the amide reaction between amine-modified monolithic column and carboxyl groups of GO. The obtained GO functionalized monolith exhibited good mechanical property and enrichment performance for the isolation of sarcosine from human urine samples.⁹

Molecular imprinting technology, a very promising and attractive technology, not only offers specific molecular recognition sites for molecular targets,¹⁰ but also exhibits distinct advantages such as high mechanical/chemical stability, easy and inexpensive preparation, and potential reusability.^11,12 Although molecularly imprinted polymers have unique sensitivity and specificity which make them suitable for use in chemical separation, drug-controlled release, and molecular sensing, the obstacles of imperfect removal of template molecules and recognition sites destruction still exist after polymerization.¹³ Therefore, surface imprinting has emerged to overcome such drawbacks by which recognition sites are formed on the material surface.¹⁴ The imprinted film prepared on the surface of solid support substrate via surface imprinting can enable rebinding and extraction of the template molecule, speed the response kinetics, and improve the access to the surface binding sites.

Dopamine, containing catechol and amine groups, can form thin surface adherent polydopamine (PDA) film by self-polymerization onto a wide variety of materials in weak alkaline solution.^15,16 PDA film can be noncovalently attached to the surface of many organic and inorganic materials, of which the pronounced biocompatibility, hydrophilicity, and adhesive capacities make it suitable for forming surface-imprinted film.¹⁷ Recently, PDA film surface-imprinted polymers such as surface molecularly imprinted nanowire,¹⁸ magnetic molecularly imprinted polymers,¹⁹ and molecularly imprinted nano-layer²⁰ have obtained growing research.

As a large class of antibiotics, tetracycline antibiotics (TCs) have been used for the treatment of lactating dairy cattle for several contagious diseases because of their broad spectrum antibacterial activity and cost effectiveness.²¹ However, relatively high levels of antibiotic residues in edible animal tissues can be toxic and dangerous for human health and potentially cause allergic reactions.²² Even more importantly, the presence of trace levels of residues in foodstuffs has led to the drug resistance.²³ Therefore, it is a challenging task to determine TCs in foodstuff samples without sample pretreatment because of their low concentration levels and the very complex sample matrix.^24,25 Polymer monolith microextraction (PMME), based on capillary monolithic column, is an effective method to preconcentrate trace analytes from environmental and biological samples before the analysis step. Monolithic materials have been introduced as useful extraction media due to their several attractive features such as frit-free construction, good biocompatibility, and easy preparation.²⁶ However, the lack of specific recognition sites of the synthetic crosslinked polymer may lead to the nonspecific recognition of specific substances. Surface-imprinted polymer monolithic column, a kind of artificial synthetic material, not only possesses overriding advantages of monolithic column but also exhibits specific recognition performance for template molecules, leading to its wide attention in the field of protein recognition, wastewaters treatment, and food analysis.^27–29

Herein, a surface-imprinted polymer was prepared by self-polymerization of dopamine on the surface of poly(methacrylic acid-3-(trimethoxysilyl) propylmethacrylate@GO-N,N′-methylenebisacrylamide) (poly(MAA-γ-MAPS@GO-MBA)) monolithic column skeletons after reversible immobilization of the template minocycline and was used for the preconcentration and determination of TCs combined with high performance liquid chromatographic (HPLC). The morphology of the surface-imprinted polymer was characterized by several kinds of characterization techniques.

2. Experimental section

2.1 Chemicals and instruments

Tetracycline antibiotics, tetracycline (TC), oxitetracycline (OTC), chlortetracycline (CTC), and minocycline (MC), were obtained from Jilin Institute for Drug Control (Changchun, China). Methacrylic acid (MAA), polyethylene glycol (PEG, M_n = 6000), dimethylsulfoxide (DMSO), ammonium persulphate (APS), γ-methacryloxypropyl trimethoxysilane (γ-MAPS), dopamine, and tris(hydroxymethyl)aminomethane (Tris) were purchased from Aladdin Reagent (Shanghai, China). N,N′-Methylenebisacrylamide (MBA) was obtained from Sigma-Aldrich (USA). Silica capillaries (530 μm i.d. × 690 μm o.d.) were purchased from Hebei Yongnian Optical Conductive Fiber Plant (Handan, China). All other reagents were obtained from various commercial sources and were of analytical or HPLC grade.

Scanning electron microscope (SEM) images and energy dispersive spectroscopy (EDS) were recorded on a JSM-6700F analyzer (JEOL Company, Japan). X-ray photoelectron spectroscopy (XPS) data were obtained with an Escalab 250Xi electron spectrometer (Thermo Fisher Scientific, UK). Fourier-transformed infrared spectra (FT-IR) were performed using Thermo Nicolet 670 FT-IR spectrometer (Thermo Nicolet Corporation, USA). Thermal gravimetric analysis (TGA) was carried out on a Q500 apparatus (TA, USA) from 25 to 700 °C with a heating rate of 10 °C min⁻¹ under air atmosphere. Contact angle goniometer was obtained from OCA20 machine (DataPhysics, Germany). Transmission electron microscope (TEM) measurement was carried out on a Hitachi H-800 facility with an accelerating voltage of 100 kV. Atomic force microscopic (AFM) image was taken by using a SPA300 instrument (SEIKO, Japan).

2.2 Standard solutions and separation conditions

Stock standard solutions of 1 mg mL⁻¹ TCs were prepared by dissolving 10 mg as-prepared product in 10 mL methanol, stored at 4 °C in the dark. Working solutions were freshly prepared by appropriate dilution with ultrapure water.

HPLC analysis was performed on a Waters 2489 series LC system (Waters, USA) equipped with binary pumps (Waters 1525), a column oven, and a dual-wavelength UV/Visible detector (Waters 2489). Breeze software was used for acquiring and processing data. An RP Symmetry C18 column (4.6 mm × 75 mm, 3.5 μm) was used for the separation of the target analytes, which was protected by a phenomenex C18 security guard column (4.0 mm × 3.0 mm, Phenomenex, Torrance, Canada). 0.01 mol L⁻¹ oxalic acid (pH 4.0), methanol, and acetonitrile (70 : 10 : 20, v/v/v) were used as the mobile phase. The injection volumes for all samples and standards were 5 μL. The column was kept at room temperature. The detection wavelength was set at 350 nm.

2.3 Synthesis of γ-MAPS@GO

GO was synthesized from graphite by a modified Hummers method.⁸ γ-MAPS@GO was prepared according to precious work.³⁰ Briefly, 50 mg GO was dispersed into 50 mL water and sonicated at room temperature for 3 h. Subsequently, 0.1 mL γ-MAPS was added into GO solution with stirring and the silanization process was carried out at room temperature for 24 h. Finally, the γ-MAPS@GO nanosheets were purified and collected. The silanization procedure of GO into γ-MAPS@GO was illustrated in Fig. 1a.

Fig. 1 Illustration of the preparation of (a) γ-MAPS@GO and (b) minocycline-imprinted poly(MAA-γ-MAPS@GO-MBA) monolith.

2.4 Preparation poly(MAA-γ-MAPS@GO-MBA) monolith

Prior to polymerization, the inner wall of capillary was vinylized with γ-MAPS to enable covalent attachment of the monolith.³¹ Subsequently, polymerization solution was made by the mixture of MAA (15 μL), γ-MAPS@GO (0.005 g), MBA (0.03 g), a binary porogenic solvent (0.11 g PEG + 375 μL DMSO), and AIBN (5 mg). The mixture was degassed by ultrasonication for about 30 min. Then the homogeneous solution was filled into the pretreated capillary and sealed at both ends with rubber stoppers. The polymerization was initiated at 55 °C for 24 h. After the polymerization was completed, the prepared monolithic column was washed with methanol to remove the unreacted components.

2.5 Preparation of minocycline-imprinted poly(MAA-γ-MAPS@GO-MBA) monolith

Minocycline (7 μg mL⁻¹) was pumped through the prepared poly(MAA-γ-MAPS@GO-MBA) monolith at the flow rate of 0.005 mL min⁻¹ for 2 h. Subsequently, the monolithic column was equilibrated with Tris–HCl (10 mmol L⁻¹, pH 8.0) for 30 min to remove the overloading minocycline. The obtained monolith was denoted as minocycline-immobilized monolith.

The post-modification of PDA film on the minocycline-immobilized monolith was made as follows: 1 mL Tris–HCl (10 mmol L⁻¹, pH 8.0) containing dopamine (20 mmol L⁻¹) and APS (10 mmol L⁻¹) was pumped through the minocycline-immobilized monolith at 0.005 mL min⁻¹ for 15 min. After placed at 4 °C for 4 h, the resultant monolith was washed with the mixture of 0.01 mol L⁻¹ oxalic acid (pH 4.0), methanol, and acetonitrile (70 : 10 : 20, v/v/v) to remove the template molecule. The minocycline-imprinted poly(MAA-γ-MAPS@GO-MBA) monolith (MIP monolith) was thus obtained. The non-imprinted monolithic column (NIP monolith) was also prepared using the same polymerization procedure as described above except minocycline. The preparation process of MIP monolith was illustrated in Fig. 1b.

2.6 MIP monolith extraction of TCs from milk samples

Milk samples were randomly obtained from local supermarkets and stored at 4 °C in dark until use. 0.5 mL milk samples spiked with known quantitative amounts of TCs standard solution were vortexed with 0.5 mL ACN for 5.0 min. After being equilibrated at room temperature for 15 min, the mixtures were centrifuged for 5 min at 10 000 rpm. The supernatants were totally pipetted and diluted with water. Blank samples were prepared in the same way as above but without the TCs spiking step.

The extraction procedure included preconditioning, sampling, evacuation, and desorption. Prior to extraction, 0.2 mL methanol was passed through the polymer monolithic column for 10 min at 0.02 mL min⁻¹, and then the sample solution was injected to realize the adsorption at 0.02 mL min⁻¹ for 25 min. Subsequently, an empty and clean syringe was employed for driving out the residual solution in the polymer monolithic column. For the desorption step, TCs adsorbed on the monolithic column were eluted with 0.05 mL 0.01 mol L⁻¹ oxalic acid (pH 4.0), methanol, and acetonitrile (70 : 10 : 20, v/v/v) at 0.03 mL min⁻¹, and the eluent was collected into a vial for HPLC analysis.

3. Results and discussion

3.1 Synthesis of γ-MAPS@GO

The morphology and property of the γ-MAPS@GO were investigated by AFM, FT-IR, XPS, and water contact angle. AFM images were used to characterize the 2D surface morphology and the thickness of the pristine GO and γ-MAPS@GO. As shown in Fig. 2, the representative AFM image indicated GO nanosheets with an average thickness of about 1.14 nm (Fig. 2a). Compared with pristine GO, the γ-MAPS@GO presented a slightly increased thickness of about 1.42 nm because the silanization reaction was successfully conducted onto GO (Fig. 2b). The results indicated that the single layer of GO and γ-MAPS@GO nanosheets were obtained. The characteristic functional groups present in pristine GO was confirmed by FT-IR and shown in Fig. S1(a),† such as carboxyl C=O (1715 cm⁻¹), C–O (1402 cm⁻¹), epoxy C–O (1123 cm⁻¹), C=C (1633 cm⁻¹), and –OH (3425 cm⁻¹).³⁰ When the silanization reaction was conducted on GO, the peaks at 1048 cm⁻¹ and 816 cm⁻¹ corresponded to methoxy groups and Si–O of γ-MAPS, respectively. Experimental results also suggested that γ-MAPS was successfully introduced onto the surface of GO nanosheets. Elemental analysis provided a direct proof for the synthetic process of γ-MAPS@GO. The XPS spectrum for γ-MAPS@GO contained Si 2p peaks at 102 eV with percentage about 1.58% (Fig. S1(b)†), which further confirmed that γ-MAPS was covalently bonded onto the surface of GO. In Fig. 3, the surface hydrophilicities of GO and γ-MAPS@GO nanosheets were characterized on the basis of static water contact angle. The water contact angle for the GO nanosheets was about 100.91° while decreased to about 60.74° after modified with γ-MAPS, demonstrating that the hydrophilic γ-MAPS@GO nanosheets were prepared.

Fig. 2 AFM images of (a) GO and (b) γ-MAPS@GO.

Fig. 3 Water contact angles of (a) GO and (b) γ-MAPS@GO.

3.2 Preparation and characterization of MIP monolith

In the preparation of MIP monolith, good permeability and high imprinted factor are basic experimental factors. γ-MAPS@GO percentage, template concentration, and the self-polymerization time of dopamine are key parameters which can affect imprinting efficiency. The poly(MAA-γ-MAPS@GO-MBA) monolith was fabricated through copolymerization of γ-MAPS@GO with the monomer and cross-linker. The percentage of γ-MAPS@GO was investigated in the range of 0.1–5 wt%. The polymerization solution led to ropy and eventually generated precipitate continually when the percentage of γ-MAPS@GO exceed 1 wt%. Thus, 1 wt% γ-MAPS@GO was selected to prepare the poly(MAA-γ-MAPS@GO-MBA) monolith.

Similarly, the effect of template concentration was also investigated in the range from 1 to 10 μg mL⁻¹. Results were given in Fig. S2(a).† The peak areas increased with increasing template molecule contents because of increasing in the number of recognition cavities. A maximum peak area achieved at the minocycline concentration of 7 μg mL⁻¹. When the minocycline concentration was greater than 7 μg mL⁻¹, the peak areas of minocycline were apparently weakened, which was because template molecules with higher concentration in the process of MIP monolith fabrication may cause a decrease of valid recognition cavities.¹⁶ Thus, the optimized template concentration was selected to be 7 μg mL⁻¹. To create more imprinted sites and get rapid response, the PDA film thickness on monolith surface was adjusted via controlling the self-polymerization time of dopamine in the range of 2–8 h. As shown in Fig. S2(b),† the peak areas of minocycline slightly increased at first and then decreased when the polymerization time exceeded 4 h. This may be because long time would increase the thickness of the PDA film on the polymer surface. Thick film may lead to deep burial of the template molecules, making it difficult to form effective recognition sites.²⁰ In order to examine the MIP monolith permeability of different dopamine self-polymerization time, the permeability was investigated when the flow rate was set at 0.5 mL min⁻¹. The values of permeability (K_F) were estimated by the following equation.

K_F = FηL/ΔP (1)

where K_F is the permeability (m²), F is the flow rate of the pump (m s⁻¹), η is the solvent viscosity, L is the column length (m), S is the inner cross sectional area of the column, ΔP is the backpressure (Pa). Methanol was used as mobile phase and the corresponding value of viscosity was 0.544 × 10⁻³ Pa s.³² The K_F values obtained at different self-polymerization time of dopamine (2–8 h) were determined in the range of 4.97–2.02 (×10⁻¹²). Although the permeability decreased with the self-polymerization time, the MIP monoliths possessed good permeability when flushed with methanol. Overall consideration, 4 h was chosen as the self-polymerization time of dopamine.

FT-IR spectra of the poly(MAA-γ-MAPS@GO-MBA) monolith and the MIP monolith were compared in Fig. S3(a).† The peak at 1113 cm⁻¹ of the MIP monolith assigned to C–O–C was weaker than that of the poly(MAA-γ-MAPS@GO-MBA) monolith because the surface of the latter was coated by the PDA film. The bonds at 2943 cm⁻¹ and 1652 cm⁻¹ which were due to CH₃ stretch and C=O stretch also became weaker than that of the poly(MAA-γ-MAPS@GO-MBA) monolith. The spectrum of the MIP monolith exhibited a peak at 3420 cm⁻¹ which corresponded to N–H bond and O–H bond.³³ From the FT-IR spectra, it was certain that dopamine was self-polymerized on the surface of the monolith. EDS spectra between the poly(MAA-γ-MAPS@GO-MBA) monolith and MIP monolith were shown in Fig. S3(b).† Accordingly, the atomic compositions were listed in Table S1,† indicating evident differences between the two kinds of monoliths. The C/N molar ratio for MIP monolith altered from 4.09 to 8.24, which approached to the theoretical value of pure dopamine (8).³⁴ The result also suggested that PDA film was successfully coated onto the poly(MAA-γ-MAPS@GO-MBA) monolith. TGA was performed to further estimate the relative composition of the poly(MAA-γ-MAPS@GO-MBA) monolith and MIP monolith in Fig. S3(c).† The weight loss of poly(MAA-γ-MAPS@GO-MBA) monolith from 30 °C to 100 °C was about 40 wt%, which is due to the evaporation of the adsorbed water and the decomposition of labile oxygen. The weight loss of MIP monolith held a slightly greater drop over that of the poly(MAA-γ-MAPS@GO-MBA) monolith at 350 °C (about 0.5 wt%), which is likely because the polymer monolith was adhered by the PDA film.

The size and morphology of the poly(MAA-γ-MAPS@GO-MBA) monolith and MIP monolith were determined by SEM and TEM. As shown in Fig. 4a, it is obvious that the poly(MAA-γ-MAPS@GO-MBA) monolith was composed of a heterogeneous surface of spherical units agglomerated into larger clusters interdispersed by large pore channels characteristic to porous monolithic structures. It could be observed from Fig. 4b that MIP monolith exhibited relatively denser monolithic structures with a lower interstitial porosity and rougher microglobular surface caused by the self-polymerization of dopamine onto the surface of monolith. Compared with TEM images of poly(MAA-γ-MAPS@GO-MBA) monolith and MIP monolith in Fig. 4c and d, MIP monolith with well-defined shape of adherent PDA film and configuration was readily observed on the monolithic network skeleton. The monolithic network skeleton and uniform surface morphology of the MIP monolith were beneficial for the fast diffusion of template molecules to the surface of the MIP monolith.

Fig. 4 SEM images of (a) poly(MAA-γ-MAPS@GO-MBA) monolith and (b) MIP monolith; TEM images of (c) poly(MAA-γ-MAPS@GO-MBA) monolith and (d) MIP monolith.

3.3 Effect of sample pH

To study the dependence of pH on the extraction efficiency, different pH values of the sample solution were tested from 2.0 to 12.0. Results in Fig. S4† showed that the peak area reached maximum at nearby pH 4.0 and decreased at lower or higher pH values. TCs exist as hydrochloride in this study which have several ionizable groups such as tricarbonyl group at pK_a1 of 3.30, dimethylamine group at pK_a2 of 7.68, and β-diketone at pK_a3 of 9.69.^35–37 Therefore, these antibiotics may exist in solution as positively and/or negatively charged species with the change of pH values.²⁴ Some researchers have reported that these TCs are unstable and apt to form reversible epimers when pH was lower than 3.0 and higher than 7.0.³⁸ In the solution of pH 4.0, the analytes were predominantly neutral with internal zwitter ion of dimethylamino group protonated and tricarbonyl group ionized. Moreover, MAA residues is present in a negatively charged form at pH > 6.0 due to the pK_a value of carboxylic acid in the MAA molecule is 5.65 and hydrogen bonding between analytes and γ-MAPS@GO is easily conducted in acidic solutions. Therefore, pH 4.0 was favourable for the formation of a strong interaction between TCs and MAA.

3.4 Adsorption behavior of MIP and NIP monoliths

Adsorption equilibrium rate is an important parameter to evaluate MIP and NIP monoliths, which reflects the time required to reach the maximum adsorption of template molecules. The adsorption equilibrium experiments were carried out by using 2 μg mL⁻¹ minocycline on the MIP and NIP monoliths at different time. Fig. S5† demonstrated the relationship between the peak area of minocycline and adsorption time. The adsorption kinetics studies illustrated that the peak area obtained from the MIP monolith increased significantly within the first 20 min and reached about 84% of the maximum adsorption capacity at 100 min. Such efficient adsorption should be contributed to the easy accessibility of the recognition sites on the surface of the MIP monolith and quick diffusion into the MIP monolith cavities. However, only half of the highest peak area was obtained of the NIP monolith at 20 min, which may be due to the nonspecific adsorption in the pore canal of NIP monolith.

To evaluate the selectivity of MIP monolith, both MIP and NIP monoliths were subjected to four kinds of TCs. Generally, the binding capacity (Q) and imprinting factor (IF) were evaluated by the following equations, respectively.²⁹

Q = (C₀ − C_e)V/M (2)

IF = Q_MIP/Q_NIP (3)

where Q (mg g⁻¹) is the equilibrium adsorption capacities, C₀ (mg mL⁻¹) is the initial concentration, C_e (mg mL⁻¹) is the equilibrium concentration, V (mL) is the volume of standard solution, and m (g) is the weight of monolithic column. Q_MIP and Q_NIP are the equilibrium adsorption capacities of minocycline onto MIP and NIP monoliths, respectively. The experimental results were shown in Table S2.† The much larger Q values of MIP monolith (21.28–40.43 μg g⁻¹) than those of the NIP monolith (15.54–21.45 μg g⁻¹) suggested that the former has higher recognition performance for the template structurally related compounds. The IF values were in the range of 1.37–1.89, which were higher than 1, further confirming that a certain amount of minocycline templates have been successfully incorporated into monolith networks by imprinting. The imprinted cavities and specific binding sites in a predetermined orientation have formed after removal of the template.

3.5 Analytical performance

The analytical performance after the enrichment using poly(MAA-γ-MAPS@GO-MBA) MIP monolith (MIP monolith) was compared to that with poly(MAA-MBA) MIP monolith and direct HPLC analysis. Results were shown in Fig. S6(a).† Compared with direct HPLC analysis, the high adsorption selectivity and specificity of the poly(MAA-γ-MAPS@GO-MBA) MIP monolith and poly(MAA-MBA) MIP monolith toward TCs could be attributed to multiple weak interactions (such as hydrogen-bond interaction from amino, carboxyl, and hydroxyl groups, and hydrophilic interaction). In addition, the self-polymerized PDA film can offer strong adsorption affinity to TCs due to their π–π stacking interaction of abundant phenyl groups and imprinted cavities.¹⁶ However, the higher adsorption capacity of γ-MAPS@GO modified MIP monolith than that of poly(MAA-MBA) MIP monolith was primarily because hydrophilic interactions, hydrogen-bond interaction, and ultrahigh surface area of γ-MAPS@GO. As described above, the peak areas of TCs obtained after the enrichment of poly(MAA-γ-MAPS@GO-MBA) MIP monolith were highest.

The analytical performance results of MIP monolith were investigated. The calibration curves were built by plotting the peak area of TCs extracted from standard solutions. The correlation coefficients (R²) ranging from 0.993 to 0.999 were obtained in the linear concentration range of 0.05–20 μg mL⁻¹. LODs and LOQs were calculated on the basis of S/N ratios of 3 and 10, respectively. LODs for four TCs were found to be 0.030–0.053 μg mL⁻¹ and LOQs to be 0.100–0.176 μg mL⁻¹. As a comparison, some reported extraction methods in recent years for the determination of TCs were summarized in Table S3.† Although the developed method could not provide the best LOD level, it was suitable for TCs analysis when considering the developed surface-imprinted PMME method is very simple to operate and does not need any expensive instruments and large volume of organic solvents.

For MIPs, the synthesis reproducibility is an important issue. In our experiments, the enrichment experiments of 5 batches of MIP monoliths were respectively investigated, and the RSDs of batch-to-batch tests ranged from 2.9 to 9.1%. In Fig. S6(b),† it could be seen that the peak areas of different batches of MIP monoliths for TCs did not have significant changes. A possible explanation may be that the monolith polymerization and template immobilization performed in different steps were conducted under the respectively optimized conditions, which was beneficial for keeping the stability of the MIP monolith. Furthermore, the immobilization of minocycline on polymer could greatly improve the conformation stability of template molecule, ensuring the reproducibility of the MIP monolith.¹⁴

3.6 Application of MIP monolith

In order to evaluate applicability of MIP monolith as PMME materials to determine TCs in milk samples, recovery assays of blank milk samples and spiked with 1 μg mL⁻¹ TCs by treatment above-mentioned were investigated. The typical chromatograms of using HPLC method were illustrated in Fig. 5. It could be observed that a small quantity of TCs residues at detectable levels was found in milk sample and no matrix peaks were found co-migrating with the analytes. The method provides good accuracy in terms of recoveries (from 83.7% to 109.3%) and precision (from 2.9% to 9.1%). The results demonstrated that this new MIP monolith possessed good recognition performance for TCs in milk samples.

Fig. 5 Typical chromatograms of milk samples by the preconcentration procedures: (a) blank sample and (b) sample spiked with 1 μg mL⁻¹ TCs.

4. Conclusions

In this work, γ-MAPS@GO functionalized surface-imprinted polymer based on the self-polymerization of dopamine to generate the imprinted cavity was developed for the first time. Using minocycline as a model target, effects of imprinting conditions such as γ-MAPS@GO percentage, template concentration, and self-polymerization time of dopamine as well as the selectivity and performance of the prepared molecularly imprinted polymers were investigated. Experimental results demonstrated that the γ-MAPS@GO modified MIP monolith had high adsorption efficiency for TCs compared with poly(MAA-MBA) MIP monolith because of multiple interactions, including hydrophilic interactions, hydrogen-bond interaction, and the ultrahigh surface area from γ-MAPS@GO. In addition, the recognition performance of templates has been improved by imprinted cavities and specific binding sites. According to the results obtained in the precision and recovery studies, the surface-imprinted PMME proved itself to be sensitive, accurate, and has promising applications for TCs determinations in milk samples. The developed method could be satisfactory applied as a routine procedure to identify and quantify TCs for food quality and safety control and also for the monitoring of these residues in the future.

Acknowledgements

The project was supported by National Natural Science Foundation of China (No. 21575049) and Jilin Provincial Science & Technology Department (No. 20140101112JC).

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Footnote

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c5ra22462d

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