Preparation of UCMPs@MIL-100@MIP
This study integrates the fluorescence characteristics of UCMPs, the high specific surface area of MIL-100 material, and the specific recognition capability of thermo-sensitive MIP to develop a fluorescence strategy for the specific detection of β-LG. Scheme 1A shows the synthesis process of NaYF4: Yb3+, Er3+ UCMPs with green fluorescence by solvothermal method. Through the ligand exchanging process between OA and PAA, the prepared UCMPs were transformed into hydrophilic UCMPs (Scheme 1B). Because of the interaction between Fe3+ and the carboxyl groups of UCMPs and H3BTC, MIL-100 framework was wrapped around the hydrophilic UCMPs. The template protein β-LG can be fixed onto the surface of UCMPs@MIL-100 by non-covalent interaction, and the MIP layer was prepared by polymerizing NIPAM and MBA in aqueous solution. After elution of template protein β-LG, UCMPs@MIL-100@MIP with specific recognition sites was obtained.
Due to the use of thermo-sensitive monomer NIPAM, the adsorption and desorption performance of UCMPs@MIL-100@MIP can be achieved by controlling the external temperature. When the temperature is lower than lower critical solution temperature (LCST), NIPAM exhibits hydrophilicity, and the expanded imprinting cavities have no complementary affinity to the template protein functionally and spatially. When the temperature is higher than the LCST, the NIPAM is in a hydrophobic state, and the imprinting cavity shrinks. At this time, although the imprinting cavity is not complementary to the target protein, the hydrophobic interaction plays a leading role and the protein will also be captured.
Characterization of UCMPs@MIL-100@MIP
SEM and TEM analysis. SEM and TEM were used to observe the surface morphology and size of the synthesized UCMPs, UCMPs@MIL-100 and UCMPs@MIL-100@MIP (Fig. 1). Evidently, the bare UCMPs showed regular hexagonal with a particle size of about 1.5 μm and a thickness of about 188 nm (Fig. 1A,B). When MIL-100 film was formed on the surface, the size of the UCMPs@MIL-100 increased significantly and the coating thickness of MIL-100 film was about 80 nm (Fig. 1C,D). Accompanied by the growth of MIP upon the surface of UCMPs@MIL-100, the size of the coating layer has expanded to 162 - 188 nm (Fig. 1E,F), and the shape was irregular, confirming the successful preparation of UCMPs@MIL-100@MIP.
FT-IR spectra analysis. The UCMPs-PAA, UCMPs@MIL-100 and H3BTC were characterized by FT-IR spectroscopy. At 2850 and 2918 cm-1, there were symmetrical stretching vibration peaks and anti-symmetric stretching vibration peaks, respectively, which suggested the stretching vibration of the -CH2- on PAA (Fig. 2A (a)). The characteristic peaks at 1421 and 1636 cm-1 were attributed to the stretching vibration of -COOH groups. These results have demonstrated that the UCMPs with PAA ligand were successfully synthesized.
The characteristic peaks at 2664, 1720 and 918 cm-1 in Fig. 2A(c) correspond to the stretching vibration of the O-H, C=O, and the bending vibration of the O-H in H3BTC, respectively. In the FT-IR spectra of UCMPs@MIL-100 (Fig. 2A(b)), these three main characteristic peaks disappeared, and two significant absorption peaks appeared at 1622 and 1379 cm-1, which related to the symmetric and asymmetric stretching vibrations of ionized -COO-, respectively. This indicated that the -COOH groups of H3BTC dissociated into -COO- anions and formed coordination bonds with Fe3+. In addition, the fingerprint peaks derived from the vibration of the benzene ring were observed at 760 and 712 cm-1. These FT-IR spectra results were in full agreement with the step-by-step assembly process of MIL-100, indicating the surface of UCMPs has been successfully coated by MIL-100 framework.
XRD and XPS analysis. Fig. 2B has illustrated the crystal phase structure of UCMPs, UCMPs@MIL-100 and UCMPs@MIL-100@MIP determined by powder Xray diffraction. In the powder XRD diagram of UCMPS@MIL-100, UCMPS diffraction peaks were observed to be well preserved, and part of the characteristic peaks were consistent with those of MIL-100 previously reported, suggesting that the composite material was composed of UCMPS and MIL-100. In addition, the peak intensity of UCMPS@MIL-100@MIP was significantly reduced compared with that UCMPS@MIL-100. The peak intensity reflected the crystallization of the material, so it is speculated that the reason for the weakening was the formation of the MIP film on the surface. These results confirmed the successful prepare of UCMPS@MIL-100@MIP, which was consistent with the results of above characterization results.
To further demonstrate the component of UCMPs@MIL-100 and UCMPs@MIL-100@MIP, the composites obtained were characterized using XPS. In Fig. 2C, the primary signals of C1s at 284.81 and 288.6 eV, O1s at 531.71 eV and Fe2p at 712.09 eV can be clearly observed, illustrating the MIL-100 was successfully coated on the surface of the UCMPs. Compared with Fig. 2C, the N1s peak was clearly observed at 397.02 eV in Fig. 2D. The N source mainly comes from the N elements carried by β-LG, NIPAM, and MBA during the formation of the MIP layer, indicating the MIP was successfully coated on the surface of UCMPs@MIL-100.
Fluorescence quenching mechanism of β-LG to UCMPs@MIL-100@MIP.
In the study, the prepared UCMPs and UCMPs@MIL-100 materials (1.0 mg) were dispersed in 2.0 mL of water to investigate the fluorescence properties. As shown in (Additional file 1: Fig. S1A), under the excitation of the external 980 nm laser, UCMPs and UCMPs@MIL-100 appear green fluorescence emission peaks at 529 nm and 544 nm, respectively, which is due to the transition of Er3+ between the 2H11/2→4I15/2 and 4S3/2→4I15/2 energy levels. Therefore, the maximum emission peak at 544 nm was selected as a marker to evaluate the fluorescence characteristics of the synthesized materials. The fluorescence intensity of UCMPs@MIL-100 was significantly lower than that of UCMPs, which is due to fluorescence quenching of UCMPs caused by MIL-100 coating. These results preliminarily prove the successful synthesis of UCMPs@MIL-100 composites.
As shown in (Additional file 1: Fig. S1B), compared with UCMPs@MIL-100@NIP (a), UCMPs@MIL-100@MIP without removing β-LG has lower fluorescence intensity (c). While β-LG was removed, the fluorescence intensity of UCMPs@MIL-100@MIP (b) was significantly enhanced, almost close to that of NIP, which verified the quenching effect of β-LG on the fluorescence of UCMPs. Current studies have proved that the main mechanisms that cause fluorescence quenching are fluorescence resonance energy transfer (FRET) and photoinduced electron transfer (PET). Nevertheless, FRET occurs when the excitation band of the fluorescent acceptor and the emission band of the donor overlap in the analysis system. According to (Additional file 1: Fig. S1C), the absorption peak of β-LG at 280 nm did not overlap with the emission peak of the fluorophore. Therefore, the fluorescence quenching effect of β-LG on UCMPs is probably caused by electron transfer.
Thermo-sensitive property of the UCMPs@MIL-100@MIP.
As is known to all that NIPAM-based polymers exhibit both hydrophilic and hydrophobic state at different temperatures, simultaneously, the volume of polymers would change with the external temperature. As a result, the influence of temperature on the adsorption capacity of the prepared UCMPs@MIL-100@MIP was investigated. (Additional file 1: Fig. S2) showed the fluorescence intensity of the UCMPs@MIL-100@MIP without adding the template protein β-LG at 20 °C and 44 °C. Fig. 3A showed the fluorescence intensity of the UCMPs@MIL-100@MIP in adsorption to β-LG at 20 °C and 44 °C, indicating a significant temperature dependence of their interactions. After five cycles, the fluorescence intensity of the thermo-sensitive UCMPs@MIL-100@MIP was almost unchanged, indicating its good fluorescence anti-attenuation ability. These results demonstrated that β-LG adsorption and desorption of UCMPs@MIL-100@MIP can be realized by controlling the external temperature, which provides the basis for its repeatable use in fluorescence sensing.
Furthermore, the adsorption capacity (Q, mg g-1) of UCMPs@MIL-100@MIP and NIP for β-LG was calculated by the following equation.
In which, C0 and C represents the initial and residual concentration of β-LG, mg mL-1, respectively; V is the volume of β-LG solution, mL; and m represents the mass of MIP or NIP, g.
As shown in Fig. 3B, the adsorption capacity of the prepared UCMPs@MIL-100@MIP for β-LG reached 183.0 mg g-1 at 32 °C, which was significantly higher than that at 20 °C (47.0 mg g-1) and 44 °C (90.9 mg g-1). This is because at the temperature, the shape and size of the imprinted sites or cavies formed in the polymer were complementary to β-LG. At 20 °C, the NIPAM monomer is hydrophilic and forms a large number of hydrogen bonds in water, which enlarged the imprinting cavity of the polymer and resulted in most β-LG molecules entering and leaving unrecognized. At high temperature (44 °C), the hydrogen bonds formed by NIPAM were destroyed and the hydrophobic action dominated, leading to shrinkage of the polymer cavity in the aqueous phase. This hydrophobic effect also leads to a significant increase in non-specific adsorption, making its adsorption capacity higher than 20 °C, which is consistent with the study of Zhou et al[33]. In addition, UCMPs@MIL-100 material had a larger specific surface area than traditional carrier materials, reaching 637.38 m2 g-1, measured by nitrogen adsorption/desorption isotherm. This was also one important reason why UCMPs@MIL-100@MIP had stronger adsorption performance for the target protein.
Optimization of UCMPs@MIL-100@MIP preparation conditions.
In the study, the amount of UCMPs@MIL-100, the molar ratio of functional monomer and cross-linker, and the adsorption environment (pH) were investigated to obtain the optimal adsorption performance of UCMPs@MIL-100@MIP. The variable control method was adopted, and the imprinting factor (IF) was used as the final judgment result. The amount of UCMPS@MIL-100 prepared as the fluorescence source is closely related to the sensitivity of the constructed fluorescence sensor. (Additional file 1: Fig. S3A) showed the fluorescence responses of MIP and NIP prepared using different amounts of UCMPS@MIL-100. It can be observed that the addition amount of UCMPS@MIL-100 significantly affects the fluorescence response of the prepared MIP and NIP. When the addition amount is 50 mg, the maximum value of IF is 2.465. The cross-linker can form a stable rigid structure, which is conducive to the curing of the functional monomer in the polymerization layer, and then forming cavities or binding sites that match the template molecules. When the cross-linker is insufficient, the network structure of imprinting layer cannot be well connected, which affects the adsorption of β-LG molecule by MIP. Nevertheless, superfluous cross-linker will increase the thickness of the imprinted layer, resulting in mass transfer barrier, which will not only affect the mass transfer speed of β-LG in the imprinted layer, but also hinder its interaction with the fluorescence source UCMPS@MIL-100, thus reducing the detection sensitivity of the fluorescence sensor. By comparing the fluorescence response of UCMPs@MIL-100@MIP and NIP prepared under different ratios of functional monomers and cross-linker (Additional file 1: Fig. S3B), the IF reaches the maximum value (2.790) at the molar ratio of 2/3, which was chosen for further experiments. In addition, when the adsorption environment pH is 7.4 (Additional file 1: Fig. S3C), the best IF value of 3.208 is obtained. This is because when the pH of the solution is lower than 7.4, the positive charge on the surface of β-LG is less, while the alkalinity of UCMPs@MIL-100@MIP and the solution environment is relatively weak. When pH = 7.4, the surface positive charge of β-LG increases, and the alkalinity of UCMPs@MIL-100@MIP was stronger than that of solution system, indicating that UCMPs@MIL-100@MIP plays an important role in the recognition and retention of β-LG. With the continuous increase of pH value, the affinity of the solution system to β-LG gradually dominates, leading to gradually lost the recognition ability of UCMPS@MIL-100@MIP.
Fluorescence response of UCMPs@MIL-100@MIP to β-LG.
In this work, the fluorescence response of prepared UCMPs@MIL-100@MIP and NIP to different concentrations of β-LG allergen was evaluated. As shown in Fig. 4C, the fluorescence response value (F0/F) of UCMPs@MIL-100@MIP is significantly correlated with the concentration of β-LG in the range of 0.1 - 0.8 mg mL-1, in line with the following Stern-Volmer equation.
In which, F0 and F respectively represent the fluorescence intensity before and after the adsorption of β-LG, KSV is the quenching constant, and C represents the β-LG concentration (mg mL-1).
The fluorescence quenching equation of UCMPs@MIL-100@MIP is F0/F = 1.4423 C + 0.9538 with R2 of 0.9881, and the LOD was calculated as 0.043 mg mL-1. Compared with the fluorescence spectra of NIP at the same β-LG concentration, the quenching degree of UCMPs@MIL-100@MIP is obviously higher than that of NIP (Fig. 4A and 4B). This is because more binding cavies or recognition sites matching the size and shape of β-LG protein are formed in the imprinting layer. By comparing the slope of the fluorescence quenching equation (Fig. 4C), the IF was calculated as 3.415, indicating that the prepared UCMPs@MIL-100@MIP had good selectivity and specificity for β-LG recognition.
Kinetics evaluation of UCMPs@MIL-100@MIP.
To evaluate the kinetic properties of the prepared UCMPs@MIL-100@MIP and NIP, the equilibrium binding analysis was performed at a β-LG concentration of 0.4 mg mL-1. As can be seen from (Additional file 1: Fig. S4), the adsorption rate of UCMPs@MIL-100@MIP increases within 30 min and almost reached the adsorption equilibrium within 60 min. In the same period of adsorption, the F0/F change of UCMPs@MIL-100@MIP for β-LG was more significant than that of UCMPs@MIL-100@NIP. This is because UCMPs@MIL-100@MIP generates imprinting sites with respect to β-LG during the preparation process and has specific and non-specific binding during the adsorption process. However, UCMPs@MIL-100@NIP only existed non-specific adsorption. In addition, these results also indicated that the introduction of MIL-100 material not only increased the number of β-LG specific recognition sites in imprinting system, but also arranged the specific recognition sites in order, which was beneficial to the rapid binding of β-LG. This verifies the merits of this work in improving the adsorption capacity and efficiency of the molecularly imprinted system.
Selectivity Study.
The selectivity of UCMPs@MIL-100@MIP was evaluated using ALa, Lf, and Cas as competitive proteins at 0.4 mg mL-1 concentration. As illustrated in Fig. 5A, it was clearly observed that the F0/F of UCMPs@MIL-100@MIP for β-LG changes more significantly than ALa, Lf, and Cas. However, there was no significant difference in F0/F of the selected proteins for UCMPs@MIL-100@NIP. The IF was calculated as 2.19, 1.27, 1.21, and 1.15, respectively. This is because the specific cavies or recognition sites are formed that complement the size, shape, and functional groups of β-LG protein during the preparation of UCMPs@MIL-100@MIP. However, due to the lack of template protein β-LG, UCMPs@MIL-100@NIP only forms non-specific adsorption sites, resulting in the fluorescence of UCMPs not being significantly quenched by the target protein.
Equal amounts of the interfering proteins were added into the β-LG solution (0.4 mg mL-1) to further investigate the anti-interference ability of the developed UCMPs@MIL-100@MIP. As shown in Fig. 5B, the fluorescence response of UCMPs@MIL-100@MIP showed no significant changes in the three-protein mixed system compared to β-LG, indicating that its specific recognition ability for β-LG was not affected by the interfering proteins. UCMPs@MIL-100@NIP obtained a higher fluorescence response in a mixed protein system than each single interfering protein. These results indicate that the prepared UCMPs@MIL-100@MIP has significant specificity for β-LG recognition and can be applied under the hindrance of the interferents in complex samples.
Sample analysis and method validation.
To evaluate the application capability of the developed fluorescence sensor for analyzing β-LG in actual samples, raw milk and infant formula were selected and spiked with β-LG at three levels (0.1, 0.2, and 0.4 mg mL-1). After simple sample treatment, the β-LG content of the resulting extracts was measured using the molecularly imprinted fluorescence sensor and validated by standard HPLC method. Table 1 illustrates the measurement results of β-LG content obtained. The data listed is from a 10-fold dilution of raw milk extract and a 5-fold dilution of infant formula milk powder.
Obviously, at all concentrations tested, the proposed fluorescence sensor yielded β-LG content similar to those obtained using HPLC, with a correlation coefficient achieving 0.9949 (Additional file 1: Fig. S5). This means that the fluorescence sensor prepared in this study can be used for reliable and accurate analysis of β-LG. A comparison of the results of the reported strategies for β-LG analysis in various matrices was provided in Table 2, highlighting the merits of the developed UCMPs@MIL-100@MIP fluorescence sensor.