High-capacity boronate affinity-based template-immobilized surface imprinted silica nanoparticles for rapid, selective, and efficient extraction and determination of lincomycin in milk and chicken

Background: Lincomycin, a natural antibiotic, is widely used by animal and fishery husbandries to prevent infections and treat diseases. It endangers people’s health when they eat foods containing lincomycin residue, especially the frequent consumption of milk and chicken products containing lincomycin. Hence, it is extremely important to evaluate the content of lincomycin in food samples. However, a direct analysis of lincomycin in milk and chicken is quite difficult because of its very low concentration level and the presence of undesirable matrix effects. Therefore, selective and efficient extraction of lincomycin from complex food samples prior to its quantification is required.

Results: In this study, lincomycin-imprinted silica nanoparticles were prepared according to boronate affinity-based template-immobilized surface imprinting. Silica nanoparticles and boronic acid ligands 3-fluoro-4-formylphenylboronic acid were used as supporting materials and functional monomers, respectively. The prepared lincomycin-imprinted silica nanoparticles exhibited several significant results, such as good specificity, high binding capacity (19.45 mg/g), fast kinetics (6 min), and low binding pH (pH 5.0) toward lincomycin. The reproducibility of lincomycin-imprinted silica nanoparticles was satisfactory. The lincomycin-imprinted silica nanoparticles could still be reused after seven adsorption–desorption cycles, which indicated high chemical stability. In addition, the recoveries of the proposed method for lincomycin at three spiked levels of analysis in milk and chicken were 93.3–103.3% and 90.0–100.0%, respectively.

Conclusion: The prepared lincomycin-imprinted silica nanoparticles are feasible for the recognition of target lincomycin with low concentrations in real food samples such as milk and chicken. Our approach makes sample pre-preparation simple, fast, selective, and efficient.

1 Introduction

Antimicrobial agents are widely used to prevent and treat diseases and promote growth in animal husbandry (Ding et al., 2021; Khafi et al., 2023). However, food safety issue by veterinary drug residues has become a worldwide public health concern. As a lincosamide antibiotic, lincomycin (Lin) is widely used in human and veterinary medicine due to its excellent antibacterial effect (Dasenaki and Thomaidis, 2015; Koike et al., 2021). Meanwhile, Lin is also used in association with other antimicrobial drugs to treat livestock infections caused by Bacteroides fragilis, as well as diseases of the respiratory tract in different animal species (Maddaleno et al., 2019). However, it also has an adverse impact on human products and life because of the irrational use of Lin. Antibiotic residues may lead to disturbances in ecological functions and promote the development and distribution of resistant genes, which pose a potential risk to the environment (Bengtsson-Palme and Larsson, 2016; Andrade et al., 2020). Among the most important effects of Lin getting released into the environment are the transfer of Lin-resistant genes (LRGs) and the alteration of the microbial community, such as the impact on food safety and human health due to crop plant uptake (Mehrtens et al., 2021). It affects people’s health when they eat foods containing Lin residue, especially the frequent consumption of milk and chicken products containing Lin. The acceptable maximum residual level (MRL) of Lin in milk and chicken was set at 150 and 200 μg kg⁻¹ by the Codex Alimentarius Commission and the European Union, respectively (Du et al., 2019). Therefore, it is important to have in place an efficient way to evaluate the levels of Lin in animal-derived food samples such as milk and chicken.

Thus far, a variety of analytical methods have been used to evaluate Lin in different real samples including GC–MS (Tao et al., 2011), LC–MS (Khadim et al., 2023), LC–MS/MS (Fernandes-Cunha et al., 2015; Maddaleno et al., 2019; Du et al., 2021; Li et al., 2021), and immunoassay (Zhou et al., 2014; Cao et al., 2015). Although these methods have many advantages, especially good sensitivity and accuracy, most of them suffer from several drawbacks, such as costly instruments, complicated sample preparation, long time consumption, and poor storage stability. This compels the need for a simple, fast, and sensitive quantitative method that absorbs Lin in food samples. However, the low content of Lin and high content of complex interfering substances add to the difficulty and challenges in the direct determination of Lin in food samples using UV detection. Therefore, an efficient sample pre-preparation prior to detection becomes very necessary, including separation and enrichment of Lin in food samples.

Molecularly imprinted polymers (MIPs) have been used as efficient solid phase extraction (SPE) adsorbents on account of their high specificity, easy preparation, low cost, and good physicochemical stability (Vlatakis et al., 1993; Li et al., 2015; Hao et al., 2016). To the best of our knowledge, although there are several reports on the preparation of MIPs for Lin to date (Li et al., 2017; Zhang et al., 2019; Dong et al., 2020), the molecular imprinting used in these studies was performed through a one-pot process. In fact, such an imprinting methodology is bulk imprinting in essence because the thickness of the imprinted coating cannot be calibrated to the size of the template, which thereby greatly affects the imprinting effect. To address these issues, the template-immobilized surface imprinting approach (Li et al., 2015; Hao et al., 2016; Zhang et al., 2022) should be used to prepare imprinting coating on the surface of silica because it can be adjusted to the size of the template effectively. This method of surface imprinting offers several advantages, including easy template removal, high binding capacity, and fast mass transfer. Recently, we used boronate affinity-based oriented surface imprinting to prepare chlorogenic acid-imprinted magnetic nanomaterials and catecholamine-imprinted magnetic nanomaterials, which were applied, respectively, for the selective recognition of chlorogenic acid in fruit juices and for trace analysis of catecholamine in human urine (Li et al., 2020; Li P. et al., 2022). However, the binding capacity of the above imprinted magnetic nanomaterials was relatively low. In addition, this kind of template-immobilized surface imprinting has not been used to develop MIPs for Lin for food safety in previous reports. The pesticide residue of Lin is one of the most important food safety concerns. In this study, a novel boronic acid ligand, FFPBA, has been used for the first time as a functional monomer, and its pKa value has also been measured for the first time. Although boronic acid functionalized magnetic nanoparticles (MNP) were developed for highly efficient capture of lincomycin (Zhang et al., 2023), the selective ability of the boronic acid functionalized magnetic nanoparticles for Lin was weak due to the absence of an imprinting site. To improve its selectivity and binding, boronate affinity-based imprinted nanoparticles specific to Lin were prepared.

In the template-immobilized surface imprinting strategy, template immobilization and removal are the key steps. Fortunately, Lin has a cis-diols structure; boronate affinity materials can covalently bind compounds with cis-diol at high pH values, and boronate esters can dissociate at relatively low pH values under mild ambient conditions (Liu et al., 2012; Qu et al., 2012; Li D. et al., 2022). In addition, 3-fluoro-4-formylphenylboronic acid (FFPBA) has been used for the first time as boronic acid ligands to immobilize and imprint Lin. Silica nanoparticles (SiO₂), due to their good biocompatibility, low toxicity, easy post-modification, high yield synthesis, and easy preparation, can be used successfully as solid substrates (He et al., 2010; Lin et al., 2012; Arabi et al., 2016). Therefore, high-density boronic acid ligands can be formed on the surface of SiO₂ by post-modification reactions. Furthermore, the imprinted coating is important for the binding properties of the imprinted materials. The poly (2-anilinoethanol) with a more hydrophilic domain can be used as a good imprinting coating.

In this study, we attempted to prepare novel Lin-imprinted silica nanoparticles (Lin-imprinted SiO₂@APTES@FFPBA) using a boronate affinity-based template-immobilized surface imprinting approach. As depicted in Figure 1, the imprinting process included three steps. First, Lin was immobilized onto FFPBA-functionalized SiO₂ (SiO₂@APTES@FFPBA) through boronate affinity interaction. Then, the 2-anilinoethanol was self-polymerized on the surface of SiO₂@APTES@FFPBA to form an imprinting coating with appropriate thickness. Generally, the thickness of the imprinting coating should be adjusted to 1/3 to 2/3 of the molecular size of the template in one of the three dimensions. Finally, the Lin template was removed by an acidic solution with SDS to form the MIPs with an imprinting cavity containing FFPBA. Because the obtained imprinting coating could cover excessive binding sites, non-specific adsorption could be effectively eliminated. The prepared Lin-imprinted SiO₂@APTES@FFPBA exhibited several significant advantages, such as good specificity, high binding capacity, fast kinetics, and low binding pH toward Lin. In addition, the recoveries of the proposed method for Lin at three spiked levels of analysis in milk and chicken were 95.3–102.7% and 96.8–104.5%, respectively, which indicated the successful detection of Lin in real samples.

FIGURE 1

Figure 1. The formation mechanism of Lin-imprinted silica nanoparticles by boronate affinity-based template-immobilized surface imprinting.

2 Experimental materials and methods

2.1 Materials

Adenosine (A), deoxyadenosine (DA), guanosine (G), 2’-O-methylguanosine (Gm), dopamine (Dop), rutin (Rut), quercertin (Que), Baicalein (Bai), Vitamin b12, genistein (Gen), kaempferol (Kae), Lin, desonide (Des), 2-anilinoethanol, tetraethoxysilane (TEOS), (3-amino-propyl) triethoxysilane (APTES), ammonium persulfate (APS), FFPBA, Sodium cyanoborohydride, and anhydrous methanol were purchased from J&K Scientific Ltd. (Shanghai, China). All other reagents were of analytical grade or above and used without further treatment.

2.2 Instruments

Transmission electron microscopy (TEM) characterization was performed on a JEM-1010 system (JEOL, Tokyo, Japan). UV absorbance and the adsorption isotherm measurements were carried out using a U-3010 UV spectrophotometer equipped with a 1-cm cuvette (Kyoto, Japan). The X-ray photoelectron spectroscopy (XPS) was performed with an ESCALAB 250Xi X-ray photoelectron spectrometer (Thermo, USA) with Al Kα radiation (hv = 1486.6 eV). The instrument was calibrated against the C1s band at 284.8 eV. Powder X-ray diffraction (XRD) analyses were carried out using a Bruker D8 Advance diffractometer with Cu Kα radiation, and the scanning angle ranged from 10° to 80° of 2θ.

2.3 Preparation of FFPBA-functionalized silica nanoparticles (SiO₂@APTES@FFPBA)

FFPBA-functionalized MNPs were prepared by the following three-step reaction (Figure 1): (1) synthesis of silica nanoparticles (SiO₂), (2) functionalization with APTES (SiO₂@APTES), and (3) functionalization of SiO₂@APTES using FFPBA by the Schiff base reaction (SiO₂@APTES@FFPBA). The bare SiO₂ was synthesized according to a modified previously reported method (Lin et al., 2014). Briefly, 6 mL of TEOS was gradually added to a mixture of 100 mL ethanol, 4 mL deionized water, and 3.2 mL aqueous solution of 25–28% ammonium. The mixture solutions were vigorously stirred at 30°C for 24 h. The resulting SiO₂ was rinsed with water and ethanol three times in sequence and then vacuum-dried at 40°C overnight. Then, 1.0 mL of APTES was added dropwise to 20 mL anhydrous methanol containing 120 mg SiO_2, and the SiO₂@APTES was obtained by stirring the mixture for 24 h. The resultant SiO₂@APTES was purified by three cycles of centrifugation, separation, and resuspension in ethanol by ultrasonication and dried at room temperature under vacuum for further use. The third step was to functionalize SiO₂@APTES with FFPBA. 100 mg of SiO₂@APTES was added to 150 mL anhydrous methanol containing 2.0 g FFPBA, and the obtained mixture was stirred for 12 h at 30°C. After that, sodium cyanoborohydride was added into the above solution (400 mg every 6 h) and kept for 24 h at 30°C. The SiO₂@APTES@FFPBA was separated from the mixtures by a magnet and washed with water and ethanol in turn. The dried SiO₂@APTES@FFPBA was obtained using vacuum drying (40°C) and needed cold preservation.

2.4 Selectivity of SiO₂@APTES@FFPBA

The selectivity of SiO₂@APTES@FFPBA was evaluated using A, G, and Lin as cis-diol compounds with DA and Gm as non-cis-diol analogs. The cis-diol or non-cis-diol solution (1 mg/mL) was obtained after they were dissolved in 50 mM PBS (pH 7.0), respectively. SiO₂@APTES@FFPBA of 3 mg were dispersed into 1 mL above solution respectively, and the mixture was shaken at room temperature for 30 min. Then, the obtained targets-treated SiO₂@APTES@FFPBA were collected by magnetic force and rinsed with 500 μL PBS (pH 7.0) three times. After that, the target-treated SiO₂@APTES@FFPBA were eluted with an acetic acid solution for 1 h, and the obtained eluates containing targets were collected.

2.5 Preparation of Lin-imprinted SiO₂@APTES@FFPBA

The Lin-imprinted SiO₂@APTES@FFPBA were prepared according to the boronate affinity-based template-immobilized surface imprinting approach. As depicted in Figure 1, Lin templates were first immobilized onto SiO₂@APTES@FFPBA. Specifically, 100 mg of SiO₂@APTES@FFPBA were dispersed into 20 mL phosphate buffer solution (pH 7.0) containing 20 mg Lin, and the obtained suspension was shaken at 25°C for 1 h. The obtained Lin-immobilized SiO₂@APTES@FFPBA were collected and washed with 50 mM phosphate buffer solution (pH 7.0). Then, 80 mg Lin-immobilized SiO₂@APTES@FFPBA were dispersed into 10 mL 2-anilinoethanol solution (100 mM in pH 7.0 PBS) and shaken at 25°C for 5 min. Subsequently, 10 mL APS solution of 50 mM was added into the above-obtained suspension. The mixture was rapidly sealed and shaken at 25°C for 30 min. The imprinted polymer layer was formed by the self-polymerization of 2-anilinoethanol. Finally, the Lin templates were removed using 100 mM acetic acid and then washed with water and ethanol. The same process was followed for the preparation of non-imprinted SiO₂@APTES@FFPBA, except it did not involve Lin templates.

2.6 Optimization of imprinting conditions

As key imprinting conditions, the concentration of 2-anilinoethanol and polymerization time were investigated by measuring the imprinting effect. The Lin solution of 1 mg/mL in 50 mM phosphate (pH 7.0) was applied as a template solution. An equivalent Lin-imprinted SiO₂@APTES@FFPBA was added to each centrifuge tube. Then, 500 μL of Lin template solution was added to a centrifuge tube with Lin-imprinted SiO₂@APTES@FFPBA and shaken for 1 h at 25°C. After the Lin-immobilized SiO₂@APTES@FFPBA was washed with phosphate buffer of pH 7.0 two to three times, an equivalent 10 mL of 2-anilinoethanol and APS solutions at different concentrations was added in sequence to obtain the concentration of 2-anilinoethanol at 20, 40, 60, 80, and 100 mM. Additionally, the process of polymerization was carried out for a duration of 5–60 min.

The imprinting factor (IF) was calculated by the ratio of Q_MIPs to Q_NIPs, which was used to evaluate the imprinting effect of Lin-immobilized SiO₂@APTES@FFPBA toward Lin. Q_MIPs and Q_NIPs (mg/g) represent the adsorption capacities of Lin-immobilized SiO₂@APTES@FFPBA and non-imprinted SiO₂@APTES@FFPBA for Lin.

2.7 Specificity of Lin-imprinted SiO₂@APTES@FFPBA

The specificity of the Lin-immobilized SiO₂@APTES@FFPBA for Lin was evaluated using nine samples, including Lin, Dop, Rut, Que., Bai, VB12, Gen, Des, and Kae. First, an equivalent amount of Lin-imprinted SiO₂@APTES@FFPBA was added to each centrifugal tube of 1.5 mL. Each sample solution (1 mg/mL) of 500 μL was added to each centrifuge tube and shaken at 25°C for 1 h. After being washed with phosphate buffer three times, the Lin-immobilized SiO₂@APTES@FFPBA were eluted using 100 μL of acetic acid solution (pH 2.7) for 2 h. The eluent was measured with UV absorbance at maximum absorption wavelength. The measurement was repeated three times.

2.8 Determination of the pK_a value of FFPBA

The pK_a value of FFPBA was measured according to the previously reported method. UV absorption changes were measured by the titration of 0.1 mM solution of FFPBA in 100 mM phosphate buffer with 1 M sodium hydroxide. The wavelength of the spectrophotometer was set at 268 nm, and a pH meter was fixed in the solution to continuously record the pH of the solution.

2.9 Binding isotherm and Scatchard analysis

The dissociation constant (K_d) and maximum binding capacity (Q_max) were determined according to a previously reported method (Li et al., 2015). An equivalent quantity of Lin-immobilized SiO₂@APTES@FFPBA (3 mg each) was mixed with 500 μL of Lin solutions at different concentrations (0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.08, 0.10, and 0.12 mg/mL) in centrifuge tubes. Then, the obtained mixture solutions were shaken on a rotator for 1 h at room temperature. The Lin-imprinted SiO₂@APTES@FFPBA were collected, rinsed, and eluted with PBS (pH 7.0) and acetic acid solution, respectively. The obtained eluates were used to measure the Lin in the eluates. The K_d and Q_max were calculated based on the following Scatchard equation (Li et al., 2015):

$\frac{Q_e}{C_s}=\frac{Q_{\text{max}}}{K_d}-\frac{Q_e}{K_d}$

Q_e and C_s are the binding capacity and the free concentration of the Lin-imprinted SiO₂@APTES@FFPBA for Lin at equilibrium, respectively. K_d and Qmax can be calculated from the slope and the intercept of the plots of Qe/Cs versus Qe.

2.10 Analysis of Lin in real milk and chicken samples

70 mL of milk was placed in a 100 mL centrifugal tube and shaken with ultrasound for 10 min to achieve a uniform milk sample (Li and Bie, 2017). To determine Lin in milk, 30 mg of Lin-imprinted SiO₂@APTES@FFPBA was added to 15 mL of the milk sample and shaken for 10 min at room temperature. The Lin-imprinted SiO₂@APTES@FFPBA, which absorbed the Lin, was eluted with acetic acid, and the eluent was collected and measured. To evaluate the recoveries of Lin in the milk sample, different amounts of Lin were mixed into milk to obtain milk solutions containing Lin at different concentrations (0.05, 0.15, and 0.30 μg/g). Then, an equivalent quantity of Lin-imprinted SiO₂@APTES@FFPBA was placed in the above-prepared 15 mL milk solution. After washing, Lin-imprinted SiO₂@APTES@FFPBA were eluted with 2 mL acetic acid solution (pH 2.7) each time for three times, and 6 mL of eluent was collected. The feasibility of pretreatment of Lin in milk by Lin-imprinted SiO₂@APTES@FFPBA was analyzed by HPLC-UV.

Chicken samples (20 g) were added to 80 mL acetonitrile and extracted by ultrasonics for 40 min (Li et al., 2017). Then, the extracted solutions were transferred into a 100 mL flask for evaporation and drying on a rotary evaporator. The final residue was dissolved in 20 mL PBS (100 mM in pH 7.0) to form chicken sample solutions containing Lin. Then, 20 mg of Lin-imprinted SiO₂@APTES@FFPBA was added to 5 mL of the chicken sample solutions and shaken for 10 min at room temperature. The Lin-imprinted SiO₂@APTES@FFPBA, which absorbed the Lin, was eluted with acetic acid, and the eluent was collected and measured by absorbance. To evaluate the recoveries of Lin in chicken samples, equivalent amounts of Lin-imprinted SiO₂@APTES@FFPBA were placed in the chicken sample solutions containing Lin at different concentrations (0.10, 0.20, and 0.40 μg/g). Lin-imprinted SiO₂@APTES@FFPBA after adsorption were collected and washed with pH 7.0 PBS. Next, Lin-imprinted SiO₂@APTES@FFPBA were eluted with 2 mL acetic acid solution (pH 2.7) each time for three times, and 6 mL of eluent was collected and analyzed by HPLC-UV.

3 Results and discussion

3.1 Characterization of Lin-imprinted SiO₂@APTES@FFPBA

Figure 2A represents the TEM images of Lin-imprinted SiO₂@APTES@FFPBA. It can be observed clearly that Lin-imprinted SiO₂@APTES@FFPBA exhibits approximately spherical morphologies and relatively narrow size distributions with a diameter of about 100 nm. This result indicated that the Lin-imprinted SiO₂@APTES@FFPBA had satisfactory dispersibility, which is highly advantageous for the selective recognition of Lin.

FIGURE 2

Figure 2. TEM images of Lin-imprinted SiO₂@APTES@FFPBA (A), XPS spectra (B), and XRD spectra (C) of bare SiO₂ (A) and Lin-imprinted SiO₂@APTES@FFPBA (B).

To verify the successful preparation of Lin-imprinted SiO₂@APTES@FFPBA, X-ray photoelectron survey spectrometry (XPS) of bare SiO₂ and Lin-imprinted SiO₂@APTES@FFPBA was investigated. As shown in Figure 2B, the XPS spectrum exhibited an O 1 s peak at 531 eV and a Si 2p peak at 105 eV in bare SiO₂. While the XPS spectrum exhibited a C 1 s peak at 286 eV, O 1 s peak at 531 eV, Si 2p peak at 105 eV, N 1 s peak at 399 eV, and B 1 s peak at 191 eV in Lin-imprinted SiO₂@APTES@FFPBA. The peak at a binding energy of 191 eV could be assigned to B atoms in the form of Lin-imprinted SiO₂@APTES@FFPBA, which proved successful preparation of Lin-imprinted SiO₂@APTES@FFPBA. In addition, the crystalline nature of bare SiO₂ and Lin-imprinted SiO₂@APTES@FFPBA could be confirmed by XRD analysis. As depicted in Figure 2C, the relatively discernible strong diffraction peaks corresponding to SiO₂ (2θ = 24.2°) were observed in the curves of two samples, which highly corresponded to the crystalline planes of cubic spinel nanostructure of the silica nanoparticles. This result indicated that the structure of the carrier SiO₂ was not changed during the coating process of the imprinted layer.

3.2 Selectivity of SiO₂@FFPBA

The post-modification of SiO₂ with boronic acid is a key for boronate affinity-based template-immobilized surface imprinting. The post-modification of boronic acids can be confirmed by investigating the selectivity of the FFPBA-functionalized SiO₂ for cis-diols. A and G were tested as cis-diol-containing compounds, while DA and Gm were tested as non-cis-diol analogs. As seen in Figure 3, the boronic acid-modified SiO₂ (SiO₂@FFPBA) exhibited a higher binding amount for A or G than DA or Gm under neutral pH conditions (pH 7.0). Obviously, this result indicated that the SiO₂@FFPBA provided excellent selectivity. Because Lin contains one cis-diol, the SiO₂@FFPBA exhibited a relatively high binding capacity for Lin. Clearly, the boronic acid-functionalized SiO₂ exhibited selective binding to cis-diol-containing compounds. These results also confirmed the successful immobilization of boronic acid FFPBA onto the SiO₂.

FIGURE 3

Figure 3. The binding amount of different analytes captured by boronic-acid modified SiO₂ (SiO₂@FFPBA). Binding buffer: 50 mM sodium phosphate buffer (pH 7.0); elution solution: 100 mM HAc (pH 2.7); samples: A, DA, Gm, G, and Lin dissolved in binding buffer (1 mg/mL).

3.3 Investigation of imprinting conditions

For the boronate affinity-based template-immobilized surface imprinting, the thickness of the imprinted coating on the surface of the SiO₂@FFPBA substrate is key for the binding properties of the imprinted materials. Generally, the thickness of the imprinted coating must be calibrated to a smaller size than the template. As we know, the thickness of the imprinted coating is closely related to the concentration of 2-anilinoethanol and polymerization time, and the influence of these two factors on the imprinting effect was systematically evaluated by IF. The thickness of the imprinting coating on SiO₂ is in direct proportion to the concentration of 2-anilinoethanol. The most appropriate 2-anilinoethanol concentration could be evaluated by the binding capacity of MIP and NIP prepared at different concentrations. As observed from Figure 4A, the most appropriate 2-anilinoethanol concentration was 60 mM, with an IF value reaching 7.11. In addition, the influence of polymerization time on the imprinting effect was also investigated. It can be observed from Figure 4B that the binding capacity of MIP for Lin gradually increased with time from 10 to 30 min, while the NIP gradually decreased. However, when the polymerization time was more than 30 min, the binding capacity of MIP for Lin decreased, and NIP remained nearly constant over time. Clearly, the changing trends of the IF were in good accordance with that of the binding amount of MIP. Thus, the optimal imprinting conditions for Lin was the self-polymerization of 60 mM 2-anilinoethanol for 30 min, which provided the best IF of 7.11. The high IF can be attributed to the fact that there were no boronic acid moieties outside of the imprinted cavities in the current imprinting method. For other antibiotic templates, the good choice is to employ a simple and effective method with the preferred optimized conditions reported here and only tune the polymerization time. If they do not work well, a de novo optimization should be carried out.

FIGURE 4

Figure 4. Effects of imprinting conditions of the target amount captured by the Lin-imprinted SiO₂@APTES@FFPBA and the imprinting factor (IF). (A) concentration of 2-anilinoethanol; (B) polymerization time.

3.4 Specificity of Lin-imprinted SiO₂@APTES@FFPBA

To evaluate the specificity of Lin-imprinted SiO₂@APTES@FFPBA for Lin, several compounds, including Dop, Rut, Que., Bai, VB12, Gen, Des, and Kae, were selected as competitive compounds. As depicted in Figure 5, compared to the competitive compounds, Lin-imprinted SiO₂@APTES@FFPBA exhibited a higher binding capacity for Lin. In other words, Lin-imprinted SiO₂@APTES@FFPBA provided a higher affinity for Lin. Although Dop, Rut, Bai, and Que. contain cis-diols, Lin-imprinted SiO₂@APTES@FFPBA exhibited a relatively low binding capacity for these compounds. Therefore, the obtained Lin-imprinted SiO₂@APTES@FFPBA possessed excellent specificity toward Lin. This result implied that the boronate affinity-template immobilized surface imprinting strategy gained great success due to the thickness-controllable imprinting coating generated by the in-water self-polymerization of 2-anilinoethanol. Comparatively, the non-imprinted SiO₂@APTES@FFPBA (NIP) only exhibited a very slight affinity toward Lin and other competitive compounds, indicating that the imprinting coating contained only limited non-specific binding sites.

FIGURE 5

Figure 5. Comparison of the amount of different compounds captured by the Lin-imprinted SiO₂@APTES@FFPBA (MIP) and non-imprinted SiO₂@APTES@FFPBA (NIP). Binding buffer: 50 mM phosphate buffer (pH 7.0); eluate: 100 mM HAc (pH 2.7); samples: 1 mg/mL Lin, Dop, Rut, Que., Bai, VB12, Gen, Des, and Kae dissolved in binding buffer.

3.5 Binding pH

The binding pH is an important binding property that is positively related to the binding affinity of boronic acids toward cis-diols. The binding affinity and binding pH are determined by the structures of the boronic acid ligands and supporting materials (Lin-imprinted SiO₂@APTES@FFPBA). As shown in Figure 6A, the pK_a value of FFPBA was measured to be approximately 5.8 due to the presence of electron-withdrawing groups in FFPBA. Clearly, these specific boronic acid ligands could work under low pH conditions of 6.0 (Figure 6B). In addition, the imprinted cavities in the Lin-imprinted SiO₂@APTES@FFPBA could lead to higher binding affinity, thereby providing lower binding pH. To confirm this, the effect of pH on the binding capacity of Lin-imprinted SiO₂@APTES@FFPBA and non-imprinted SiO₂@APTES@FFPBA was investigated. As depicted in Figure 6B, Lin-imprinted SiO₂@APTES@FFPBA exhibited a lower binding pH value (pH 5.0) as compared with SiO₂@APTES@FFPBA (pH 6.0), while non-imprinted MNPs provided very limited binding capacity for Lin. The binding pH shift was due to the imprinted cavities in the structure of Lin-imprinted SiO₂@APTES@FFPBA. Such a pH shift is beneficial because a lower binding pH value generally results from increased affinity toward cis-diol compounds. These results indicated that Lin-imprinted SiO₂@APTES@FFPBA can enlarge the range of pH when directly applied to real samples without requiring pH adjustment.

FIGURE 6

Figure 6. (A) The pH-dependence of the absorbance at 268 nm of FFPBA and (B) Target binding capability Q of Lin-imprinted SiO₂@APTES@FFPBA (MIP), non-imprinted SiO₂@APTES@FFPBA (NIP), and SiO₂@FFPBA at different pH values. Sample: 1 mg/mL Lin dissolved in 50 mM phosphate buffer (pH 4.0, 5.0, 6.0, and 7.0).

3.6 Binding equilibrium

In order to evaluate the binding equilibrium time of Lin-imprinted SiO₂@APTES@FFPBA toward Lin, the effect of the response time on binding capacity (Q) was investigated (Figure 7). As seen in Figure 7, Lin-imprinted SiO₂@APTES@FFPBA had a faster adsorption rate than non-imprinted MNPs within the first 6 min. When the response time exceeded 6 min, the adsorption rate quickly slowed down, and the binding reaction reached equilibrium at 6 min. This result implied that most of the binding sites had been occupied by Lin in such a situation. Clearly, such an equilibrium time of Lin-imprinted SiO₂@APTES@FFPBA for Lin was lower than that of other imprinted polymers (8–90 min) (Gu et al., 2010; Li et al., 2012, 2017, 2018a; He et al., 2014; Hao et al., 2016). This result indicated that the Lin-imprinted SiO₂@APTES@FFPBA for Lin showed good binding kinetics.

FIGURE 7

Figure 7. Binding equilibrium of Lin-imprinted SiO₂@APTES@FFPBA and non-imprinted SiO₂@APTES@FFPBA. Sample: 1.0 mg/mL of Lin containing 50 mM phosphate, pH 7.0.

3.7 Determination of K_d and Q_max

As we know, the binding affinity of Lin-imprinted SiO₂@APTES@FFPBA can determine how low concentrations of Lin can be extracted by Lin-imprinted SiO₂@APTES@FFPBA. Therefore, the binding isotherm of the Lin-imprinted SiO₂@APTES@FFPBA toward Lin was investigated to evaluate its binding affinity. As shown in Figure 8A, the Lin-imprinted SiO₂@APTES@FFPBA exhibited much higher binding capacity toward Lin as compared with the non-imprinted SiO₂@APTES@FFPBA. According to the binding isotherm, a Scatchard plot was drawn (Figure 8B), which could provide Q_max and K_d values of Lin-imprinted SiO₂@APTES@FFPBA. These were (19.45 ± 1.44) mg/g and (3.65 ± 0.38) × 10⁻⁵ M, respectively. Clearly, Lin-imprinted SiO₂ exhibited much higher binding capacity than other imprinted nanomaterials (Gu et al., 2010; Li et al., 2018a,b,c,d). Such a high-binding capacity could result from the combination of SiO₂ with easy post-modification and FFPBA with low pK_a. Conversely, it also benefited from the high imprinting efficiency. The strong binding strength of the prepared Lin-imprinted SiO₂@APTES@FFPBA for Lin favors the extraction of Lin of trace concentrations.

FIGURE 8

Figure 8. (A) Binding isotherms for binding of the Lin-imprinted SiO₂@APTES@FFPBA and non-imprinted SiO₂@APTES@FFPBA to Lin, and (B) Scatchard plots for the binding of the Lin-imprinted SiO₂@APTES@FFPBA to Lin.

3.8 Reproducibility and reusability

The reproducibility of the obtained Lin-imprinted SiO₂@APTES@FFPBA was evaluated by using six batches of Lin-imprinted SiO₂@APTES@FFPBA prepared on different days, and the measurements replicated three times in parallel. It can be observed from Figure 9A that each individually prepared Lin-imprinted SiO₂@APTES@FFPBA exhibited similar binding capacities for Lin (21.30, 18.89, 17.43, 18.16, 17.85, and 17.46) mg/g, respectively. These results indicated that the reproducibility of Lin-imprinted SiO₂@APTES@FFPBA was satisfactory because the boronate affinity-based template immobilized surface imprinting was beneficial.

FIGURE 9

Figure 9. Batch-to-batch reproducibility (A) and reusability (B) of Lin-imprinted SiO₂@APTES@FFPBA. Sample: 1 mg/mL of Lin containing 50 mM phosphate, pH 7.0.

One of the main advantages of MIPs over native antibodies is their ability to be reused. Thus, the reusability of Lin-imprinted SiO₂@APTES@FFPBA was investigated, and the adsorption–desorption cycle was repeated ten times using the same batch of Lin-imprinted SiO₂@APTES@FFPBA (Figure 9B). Even after six adsorption–desorption cycles, the adsorption capacity of Lin-imprinted SiO₂@APTES@FFPBA = changed very little. Clearly, Lin-imprinted SiO₂@APTES@FFPBA could still be reused after seven adsorption–desorption cycles. Thus, Lin-imprinted SiO₂@APTES@FFPBA possesses high chemical stability.

3.9 Determination of Lin in food samples

In order to investigate the performance of the prepared Lin-imprinted SiO₂@APTES@FFPBA in food samples, the selective separation and determination of Lin from milk and chicken samples by Lin-imprinted SiO₂@APTES@FFPBA was carried out. The obtained results are given in Table 1. Clearly, the content of Lin in chicken was evaluated to be 0.025 μg/g, and no Lin was found in the milk solution. In addition, to investigate the accuracy of the method by selective separation and determination of Lin in real food samples, the evaluation of Lin in milk and chicken was performed. The recoveries were investigated with three standard amounts of Lin added in milk and chicken solutions, and the spiked concentration was fixed at 0.05, 0.15, 0.30 μg/mL, and 0.10, 0.20, 0.40 μg/mL, respectively. By extraction and determination of Lin, the recoveries for milk and chicken solutions were shown in Table 1. The recoveries of Lin for milk and chicken solution were evaluated to be 93.3–103.3% and 90.0–100.0%, respectively. In addition, the RSD for milk and chicken solution ranged from 2.5–5.3% and 3.0–4.5%. The results indicated that the proposed method is accurate, sensitive, and selective for the determination of Lin in animal-derived food samples.

TABLE 1

Table 1. Results of sample assay and recoveries for the determination of Lin (n = 3).

4 Conclusion

In this study, we used boronate affinity-based template-immobilized surface imprinting to prepare the boronate affinity-based Lin-imprinted SiO₂@APTES@FFPBA for the first time. The pKa value of FFPBA was first investigated. The use of boronic acid FFPBA in the MIPs provided several highly attractive features, including high specificity, high binding affinity, and low binding pH. Clearly, the prepared Lin-imprinted SiO₂@APTES@FFPBA are feasible for the recognition of target Lin with low concentrations in real food samples. We foresee rapid development and promising applications of this approach in the future.

Data availability statement

The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

Author contributions

YZ: Data curation, Formal analysis, Investigation, Methodology, Writing – original draft. YD: Data curation, Methodology, Writing – original draft. YM: Conceptualization, Formal analysis, Project administration, Writing – review & editing. ZZ: Formal analysis, Validation, Writing – original draft. YW: Formal analysis, Validation, Writing – original draft. DL: Formal analysis, Project administration, Resources, Supervision, Validation, Writing – review & editing. SW: Formal analysis, Project administration, Resources, Supervision, Validation, Writing – review & editing.

Funding

The author(s) declare financial support was received for the research, authorship, and/or publication of this article. We are grateful to the Key Research Projects of Henan Higher Education Institutions (No. 22A150017) and the Applied Science and Technology Research Fund of Luoyang Normal University (No. RP2100001241) for the financial support of this work. This work was funded by the National Natural Science Foundation of China (Grant No. 21904003), the University Scientific Research Project of Anhui Province (2022AH050296), and the Open Project of Anhui Engineering Technology Research Center of Biochemical Pharmaceutical (Bengbu Medical College) (Grant No. 2022SYKFZ02).

Conflict of interest

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Publisher’s note

All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.

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