Multifunctional MIP nanogels
To develop multifunctional MIP nanogels and grant enhanced stability to CsPbBr3 perovskites toward water and oxygen, three HEMA derivatives were synthesized. The HEMA derivatives GA-HEMA, CA-HEMA, and OA-HEMA have been synthesized through Steglich esterification method as shown in Fig. 1a. GA-HEMA, CA-HEMA, and OA-HEMA were obtained in form of brown, yellow, and transparent oils, respectively. Three monomers were characterized by 1H-NMR and FT-IR techniques with the discussion presented in supporting information and Figure S1. Multifunctional MIP nanogels were prepared via surfactant-free emulsion polymerization method using four HEMA derivatives: GA-HEMA, CA-HEMA, OA-HEMA, and PEG-HEMA in the presence of ROX as a template, as shown in Fig. 1b. Afterward, CsPbBr3 perovskite nanoparticles were loaded via in-situ synthesis for selective detection of ROX, as shown in Fig. 1c-d. The FT-IR spectrum of MIP nanogels showed a strong absorption peak at 1730 cm− 1, attributed to the characteristic C = O stretch of the ester group and a strong peak located at 1640 cm− 1, associated with conjugated C = C group of the aromatic groups as shown in Fig. 2a. Moreover, the two absorption peaks at 1250 cm-1 and 1167 cm-1 are attributed to the characteristic C-O stretch of ester and PEG. Finally, the relatively strong peak at 2940 cm-1 is caused by sp3 C-H stretching from oleic acid. To confirm a successful polymerization, the molecular weight of nanogel was measured via gel permeation chromatography (GPC) technique, as illustrated in Figure S2. The average molecular weight (Mw) of MIP nanogels were found to be 14,820 g/mol with a polydispersity (Mw/Mn) equal to 1.25. Whereas, a non-imprinted polymer had an average Mw of 15,850 g/mol, and polydispersity (Mw/Mn) was 1.21. This result confirms that the presence of ROX during the polymerization step does not affect the polymerization rate and the template was successfully removed. To confirm the successful imprinting of ROX in nanogels, UV–vis absorption was measured. As illustrated in Fig. 2b, ROX has an absorption peak located at 211 nm which was greatly decreased after washing MIP with water several times. These results confirm the successful removal of the ROX template from MIP. The morphology and the size of MIP nanogels were studied through FE-SEM images (Fig. 2c-d). MIP nanogels have a well-defined spherical shape with a size distribution of 300 nm. It is worthy to note that the majority of MIP nanogels are aggregated together in a well-dried state, as shown in Figure S3a, due to polymeric nature. However, after the addition of ethanol/water mixture, these MIP nanogels disperse and swell to reach a size ranged from 700 nm to 900 nm (Figure S3b).
Perovskite-loaded MIP nanogels
Cesium lead bromide perovskite nanoparticles were loaded into MIP nanogel through in-situ synthesis by hot-injection approach. First MIP nanogels were dispersed in ethanol and added to the PbBr2 precursor solution and heated at 120°C to evaporate ethanol and to distribute the PbBr2 precursor in nanogel cavities. In the second step, the cesium oleate precursor solution was mixed with MIP/PbBr2 via hot-injection method to form CsPbBr3 perovskite nanoparticles loaded in hydrophobic cavities. To confirm the successful formation of CsPbBr3 perovskite nanoparticles, X-ray diffraction (XRD) analysis was performed, as shown in Fig. 3a. The synthesized perovskites have a crystal structure in which a tetragonal system is present [mp-1014168, Materials Project data repository]. The diffraction peaks at 2θ = 14.94°, 21.05°, 29.95°, 36.90°, and 42.61° are ascribed to the tetragonal CsPbBr3 lattices planes (110), (112), (220), (312), and (224), respectively. However, XRD patterns also indicate the transformation of the small quantity of CsPbBr3 into a CsBr-rich non-perovskite rhombohedral Cs4PbBr6 (JCPDS card no. 01-073- 2478) phase probably due to the accumulation of Cs ions on more accessible locations during the second step of hot-injection method. This transformation has been already observed during the synthesis with an excess of CsBr [8]. Due to fast reaction rate, the CsCO3 precursor does not have enough time to spread evenly, which results in the accumulation of Cs ions in a certain location, leading to the formation of CsBr-rich non-perovskite rhombohedral Cs4PbBr6 nanoparticles. To study the morphology of MIP/CsPbBr3, FE-SEM analysis was performed. As shown in Fig. 3b and Figure S4, MIP nanogels were successfully loaded with perovskite nanoparticles with a size distribution of 200 nm while the size of nanogels has increased to around 900 nm due to swelling properties. In addition, we could confirm the successful loading of CsPbBr3 nanoparticles into the nanogel by the presence of cesium, lead, and bromine atoms in energy dispersive X-ray spectrum, as shown in Figure S5. Initially, nanogels were synthesized only from CA-HEMA, GA-HEMA, and OA-HEMA monomers. However, the formed nanogels showed low water dispersibility which further influenced the fluorescence stability of MIP/CsPbBr3. As shown in Fig. 3c, the loading of perovskite nanoparticles in such nanogels has increased the water stability as expected, but the absence of hydrophilic function resulted in low dispersibility and fast aggregation of MIP/CsPbBr3. To increase the dispersibility and the accuracy of photoluminescence measurements, commercially available PEG-HEMA monomer was introduced. Owing to hydrophilic property and low non-specific interaction of PEG-HEMA, it enabled a better dispersibility and lower aggregation rate of nanogel. Moreover, the water stability of perovskite was further improved, and the intensity of fluorescence was increased during the first hour (Fig. 3d). A similar phenomenon was observed by Qixuan Zhong et al. after coating perovskite nanoparticles with silica shell which allowed better dispersion in water resulting in higher photoluminescence 32. The good stability of MIP/CsPbBr3 can be explained by the insertion of perovskite nanoparticles in hydrophobic cavities of nanogel during the hot-injection method. Moreover, nanogels are composed of three-dimensional polymeric networks that absorb the water thus lowering the contact of water molecules with perovskite nanoparticles. Finally, GA-HEMA and CA-HEMA act as an antioxidant to reduce the oxidation of perovskite by oxygen species.
Sensitivity of ROX Detection
The fluorescence response of the MIP/CsPbBr3 (0.001 ppm) was studied upon the addition of increasing the concentrations of ROX between 1 × 10− 6 and 1 × 10− 10 M and shown in Fig. 4a. The fluorescence intensities of perovskite solution decreased gradually with an increase in ROX concentration, because of the tailor-made recognition sites of the MIP/CsPbBr3 specific to ROX. As illustrated in Fig. 4b, the F0/F value represents a linear relationship with the concentration of ROX ranged from 1 × 10− 6 M to 1 × 10− 10 M with a good linear correlation coefficient (0.995) and low detection limit (2.06 × 10− 11 M). The limit of detection was determined by the following equation. LOD=\({10}^{\left[\frac{\text{log}\left(3{\sigma }+{\text{y}}_{0}\right)-\text{a}}{b}\right]}\), where a is the intercept of fitted line, b is the slope of fitted line, σ is the standard deviation of the blank intensities of the perovskite solution and y0 is the mean of blank intensities of the perovskite solution (n = 3). In order to confirm the formation of specific recognition sites to ROX within MIP nanogels, detection of the different concentrations of ROX using the perovskite-loaded nonimprinted polymer (NIP) nanogels was performed. In the case of NIP, a slight change in the fluorescence intensity of perovskite has been observed, which is explained by minor quenching of more accessible perovskite particles located near the surface by water or by ROX. However, compared to the MIP, the changes in fluorescence intensity were insignificant according to the concentration of the ROX (Fig. 4c). These results demonstrate that the developed MIP/CsPbBr3 particles have great properties to detect ROX with high sensitivity.
Selectivity of ROX Detection
Selectivity tests were performed to evaluate whether the developed MIP/perovskite can selectively detect only ROX among various antibiotics and common tripeptide Glutathione (Fig. 4d). Four different antibiotics (Azithromycin, Chloramphenicol, Ciprofloxacin, and ROX) were selected as analytes and prepared in ethanol/water at a concentration of 1 mM (Fig. 4d). As shown in Fig. 4e, non-macrolide antibiotics, such as chloramphenicol and ciprofloxacin did not affect the fluorescence intensity of the developed sensor, while azithromycin which has structural similarities to ROX induced a slight decrease in fluorescence intensity. Bearing in mind that this sensor has a potential in the analysis of animal-derived food products that contain different proteins, the selectivity to common tripeptide glutathione was investigated. A slight quenching of MIP/CsPbBr3 observed in the presence of glutathione can be explained by the transformation of CsPbBr3 into non-luminescent phase. A similar phenomenon was observed in previously reported work, where CsPbBr3 was transformed into non-luminescent Cs4PbBr6 in the presence of thiol-alkyl and residual oleylamine 33. In contrast to tested molecules, ROX exhibited significant quenching efficiency more than 2-fold, thus confirming the efficient selectivity of the developed sensor towards ROX.
Mechanism of ROX Detection
Different quenching mechanisms have been considered, including molecular interactions by electrostatic or hydrogen bonding between analyte and perovskite, Förster resonance energy transfer (FRET), inner filter effect (IFE), and perovskite phase transformation or oxidation. In the FRET mechanism, the collision during dynamic quenching between the fluorescent material in an excited state and the quencher molecule results in loss of energy and return to the ground state. Moreover, it requires that the emission spectrum of the energy donor must overlap with the absorption spectrum of the energy acceptor. The synthesized MIP/CsPbBr3 have a typical peak emission of cesium lead bromide at 520 nm, whereas the ROX absorption peak is at 221 nm, as shown in Fig. 5a. These results suggest the absence of spectral overlap between perovskite and ROX; therefore, the probability of energy transfer in the fluorescence quenching mechanism is very minuscule. Thus, possible quenching mechanisms are either phase transformation or the oxidation of perovskite by ROX. In the case of NIP to which ROX is not added as a template molecule in the polymerization process, no cavity complementary to ROX exists in the polymer. Therefore, even if ROX is added during the detection process, it cannot bind to ROX, and the fluorescent emission of perovskite is not affected. However, in the case of MIP to which ROX is added as a template molecule in the polymerization process, a cavity complementary to ROX exists within the nanogel. Thereby, during the detection process, ROX binds to the cavity in the polymer, and structural decomposition of perovskite is induced by the N-oxime functional group having the oxidative property of ROX. Subsequently, the decomposition of CsPbBr3 nanoparticles results in a decrease in fluorescence (Fig. 5b).
Practical Application of the Sensor for Animal-derived Food Products
The practical relevance of perovskite-loaded MIP nanogels for the detection of ROX was investigated in three animal derived-food products: meat, milk, and egg. Pork meat, eggs, and milk products were acquired from local grocery stores in Changwon, Republic of Korea. Prior analysis, samples were extracted by experimental procedure reported in previous works and spiked with ROX standard solution 34. Acceptable recoveries and relative standard deviations (RSDs) of ROX spiked in milk, porcine muscle, and egg samples using MIP/CsPbBr3 have been achieved, as shown in Table 1. The recoveries of ROX ranged from 99.2 ~ 100 %, 101 ~ 102 %, and 98.2 ~ 99.1 %, with RSDs, ranged from 6.32 ~ 11.7 %, 3.66 ~ 6.99 %, and 5.84 ~ 6.46 % for milk, pork, and eggs, respectively. These findings demonstrate the accuracy of the developed sensor for selective and sensitive detection of ROX in animal-derived food samples, thus revealing the good potential for practical application. The analytical performances of the synthesized MIP/CsPbBr3 for detecting ROX were compared to several methods previously reported. As shown in Table 2, most reported methods require sophisticated sample treatment, good technicians, and long analysis time. By contrast, our developed fluorescence sensor does not require expensive instruments and long analysis time, and shows excellent analytical performance with wide dynamic range from 8.4 × 10− 5 to 8.4 × 10− 1 µg/mL and lower detection limit of 1.7 × 10− 5 µg/mL (20.6 pM) compared with those in the previously reported sensing methods.
Table 1
Results of ROX detection in milk, pork, and eggs real samples by developed MIP/perovskites.
Samples | Added (×10− 5 M) | Found (×10− 5 M) | Recovery (%) | RSD (n = 3, %) |
Milk | 1.00 × 10− 3 | 0.99 ± 0.09 × 103 | 99.3 | 9.32 |
1.00 × 10− 1 | 0.99 ± 0.11 × 101 | 99.2 | 11.7 |
1.00 × 10 | 1.00 ± 0.06 × 10 | 100 | 6.32 |
Pork | 1.00 × 10− 3 | 1.02 ± 0.03 × 103 | 102 | 3.66 |
1.00 × 10− 1 | 1.03 ± 0.07 × 101 | 102 | 6.99 |
1.00 × 10 | 1.01 ± 0.04 × 10 | 101 | 4.72 |
Eggs | 1.00 × 10− 3 | 0.99 ± 0.06 × 103 | 99.1 | 6.46 |
1.00 × 10− 1 | 0.99 ± 0.06 × 101 | 98.7 | 6.07 |
1.00 × 10 | 0.98 ± 0.05 × 10 | 98.2 | 5.84 |
Table 2
Comparison of CsPbBr3-loaded MIP nanogels with other general methods for the detection of ROX.
| Analytical technique | Linear Range (µg/mL) | LOD (µg/mL) | References |
1 | High-performance liquid chromatography (HPLC) | 0.05–20.0 | 5.0 × 10− 2 | 37 |
2 | Electrochemistry (EC) | 4.2–84 | 4.0 × 10− 1 | 38 |
3 | Fluorescence using CdTe quantum dots (FL) | 25.0-350.0 | 4.6 | 39 |
4 | Aqueous two-phase system extraction (ATPSE) | 1.0–20.0 | 3.0 × 10− 2 | 40 |
5 | Capillary electrophoresis (CE) | 0.02–201.0 | 7.0 × 10− 3 | 29 |
6 | Fluorescence using MIP/CsPbBr3 (FL) | 8.4 × 10− 5-8.4 × 10− 1 | 1.7 × 10− 5 | This Study |