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A facile, green synthesis of biomass carbon dots coupled with molecularly imprinted polymers for highly selective detection of oxytetracycline
Accepted Manuscript Title: A facile, green synthesis of biomass carbon dots coupled with molecularly imprinted polymers for highly selective detection of oxytetracycline Authors: Haochi Liu, Lan Ding, Ligang Chen, Yanhua Chen, Tianyu Zhou, Huiyu Li, Yuan Xu, Li Zhao, Ning Huang PII: DOI: Reference:
S1226-086X(18)30982-1 https://doi.org/10.1016/j.jiec.2018.10.007 JIEC 4203
To appear in: Received date: Revised date: Accepted date:
14-4-2018 8-10-2018 8-10-2018
Please cite this article as: Haochi Liu, Lan Ding, Ligang Chen, Yanhua Chen, Tianyu Zhou, Huiyu Li, Yuan Xu, Li Zhao, Ning Huang, A facile, green synthesis of biomass carbon dots coupled with molecularly imprinted polymers for highly selective detection of oxytetracycline, Journal of Industrial and Engineering Chemistry https://doi.org/10.1016/j.jiec.2018.10.007 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
A facile, green synthesis of biomass carbon dots coupled with molecularly imprinted polymers for highly selective detection of oxytetracycline Haochi Liua, Lan Dinga,*Ligang Chenb,* *, Yanhua Chena, Tianyu Zhoua, Huiyu Lia, Yuan Xua, Li Zhaoa, Ning Huanga a
College of Chemistry, Jilin University, 2699 Qianjin Street, Changchun 130012, China
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E-mail: [email protected] (L. Ding) E-mail: [email protected] (L. Chen)
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Corresponding author: *Tel.: +86-431- 85168399 **Tel.: +86-451- 82190679
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Department of Chemistry, College of Science, Northeast Forestry University, 26 Hexing Road, Harbin 150040, China
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Graphical Abstract
Schematic of the preparation of MIP-coated CDs
ABSTRACT: Biomass carbon dots (CDs) prepared by sweet potato peels were superior fluorophores with low toxicity and excellent photostability. A novel designed fluorescence probe for specific recognition 1
and sensitive detection of oxytetracycline (OTC) was fabricated with CDs and molecularly imprinted polymer. The quenching of CDs happened when rebinding with OTC due to electrontransfer-induced fluorescence quenching mechanism. The fluorescence probe was successfully applied in honey with the recoveries ranging from 90.2% to 97.3%. The detection limit of OTC was
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15.3 ng mL-1. This work provides promising perspectives that the development of fluorescent MIP
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will be valuable for rapid analysis in complex samples.
Key words: Biomass carbon dots; Molecularly imprinted polymers; Oxytetracycline; Fluorescence
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probe; Honey
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1. Introduction
Oxytetracycline (OTC) belongs to a kind of antibiotics that can be applied in many fields [1]. It
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can be used as a veterinary drug to prevent animal disease, due to its low cost and superior
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antibacterial properties. However, the widespread use of OTC in foods of animal origin has caused concerns about the level of unsafe residue in food such as honey, meat and milk. The residue of OTC poses a threat to public health. In China, the regulation (GB 14963-2011) makes OTC illegal
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to use in honey [2].Thus, finding a succinct and efficient approach to detect trace OTC in honey is of significance. At present, some conventional methods have been established to detect OTC, including highperformance liquid chromatography (HPLC) [3,4], colorimetric analysis [5], enzyme-linked immunosorbent assay [6] and LC with tandem mass spectrometry (LC-MS) [7,8]. As for HPLC/LC2
MS, they offer good separation and selectivity, but the requirement of professional skill and tedious operating procedure limits their application. Colorimetric analysis is less sensitive and selective. Enzyme-linked immunosorbent assay is still unsatisfactory because of the potential cross-reactivity and high cost. Therefore, it is necessary to develop a fast analysis method with low-cost and high selectivity.
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Recently, fluorescence has become a promising method in analytical fields due to the merits of convenience, time saving and good sensitivity [9]. Gao et al. [10] have synthesized TGA-capped
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CdTe as a fluorescent sensor to detect OTC with good precision and wide linear range. Xu et al.
[11] performed Au nanoclusters as a versatile probe for the detection of OTC with a good linearity and a high selectivity. The above sensors generally require complicated preparation approach or
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expensive reagents. Carbon dots (CDs) as novel fluorescent nanomaterials have good stability, low
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toxicity, tunable and unique photoluminescence properties which attached much attention in recent
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years [12-14]. Recently, many carbon materials have been synthesized from biomass for the
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application of environment and energy [15-20]. From the material synthesis and environment point of views, it is necessary to use inexpensive, eco-friendly and readily available biomass resources to
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prepare CDs. Some researchers used walnut shells to fabricate CDs by high-temperature carbonization at 1000 oC [21]. In addition, a facile hydrothermal method was proposed by using
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waste paper to synthesize CDs at 180 oC for 10 h [22]. However, most of the reported biomass CD
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methods have problems such as high reaction temperature, long reaction time, and limited application range. The exploration of new carbon sources for simple operation, wide application, and green synthesis of CDs is highly desirable.
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Molecularly imprinted polymer (MIP) is an ideal choice for improving fluorescence detection
selectivity [23,24]. Several researchers have synthesized different types of MIP-quantum dots (QDs) composites [25-28]. These sensors can selectively recognize the target molecules because of the MIP as recognition units [29]. Hou et al. compared the selectivity of CDs and CDs@MIP for the target analyte and its analogs under the same experimental conditions. The result showed that the 3
selectivity of CDs@MIP for the target analyte detection was improved significantly [30,31]. Up to now, MIPs as sensors combining with CDs have been reported for recognition of 2,4-dinitrotoluene, trinitrotoluene and glucose [32-34]. However, the synthesis of CDs in MIP-coated CDs often requires using a large amount of toxic and expensive chemicals as well as intricate processes. Herein, we fabricated a novel type of MIP-coated CDs by means of a simple and time-saving sol-
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gel polymerization approach to detect complex samples. Sweet potato peels were used to produce biomass CDs by a one-pot hydrothermal approach without complicated preparation methods or
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using toxic solvents. A new system was generated after OTC was again bonded to MIP-coated CDs, causing fluorescence quenching. Moreover, the eco-friendly fluorescence sensors were successfully applied to detect OTC in honey without additional pollution, showing their potential prospects for
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rapid analysis in complex samples.
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2. Experimental
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2.1. Chemicals
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The standards of OTC, chlorotetracycline (CTC), tetracycline (TC) and bisphenol A (BPA) were purchased from Aladdin (Shanghai, China). TEOS, APTES, ammonia, diethyl ether, Na2HPO4,
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Na2EDTA, citric acid monohydrate, ethanol and methanol were obtained from Kermel (Tianjin, China). The sweet potato and honey samples were purchased from markets in Changchun (China).
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High purity water was got by a Milli-Q water system (Millipore, Billerica, MA, USA). The Na2EDTA-McIlvaine buffer (0.1 mol L-1, pH 4.0) solution was weekly produced by dissolving Na2HPO4 (8.86 g), Na2EDTA (16.81 g) and citric acid monohydrate (5.90 g) in 500 mL of water
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[35].
2.2. Instrumentation The carbon, hydrogen, nitrogen, sulphur and oxygen content of the sweet potato peels were determined using a Flash 2000 CHNS/O automatic elemental analyser (Thermo Fisher Scientific, USA). The proximate analysis of sweet potato peels was characterized by a themogravimetric 4
analyser (TGA; Pyris 1, Perkin Elmer, Waltham, MA, USA). The morphology and size of MIPcoated CDs were investigated by a transmission electron microscopy (TEM, Hitachi H-7650, Japan). The XRD spectrum of the MIP-coated CDs was characterized by a Shimadzu XRD-6100 spectrometer (Shimadzu, Japan). The XPS spectra were recorded on an ESCALAB250 electron spectrometer (Thermo Electron, USA). Fourier transform infrared (FT-IR) spectrum was observed
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with an FT-IR360 spectrometer (Nicolet, Madison, WI, USA). The UV spectrum was collected by a TU-1901 spectrometer (PERSEE, Beijing, China). The nanosecond fluorescence lifetime was
an F-4600 fluorescence spectrophotometer (Hitachi, Japan). 2.3. Preparation and surface modification of CDs
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investigated by an FLS 920 instrument (Edinburgh, UK). Fluorescence detection was carried out on
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Sweet potato peels were cut into small pieces to prepare biomass CDs by a hydrothermal method.
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The mixture of sweet potato pieces (2 g) and high-purity water (30 mL) was added into a 50 mL
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autoclave and heated to 200 °C for 3 h. The resulting dark brown solution is filtered through a 0.22
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μm membrane. Then the suspension was filtered though a dialysis membrane (MWCO:1000) for 24 h.
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In briefly, 40 mL of CDs (1 mg mL-1) was used for the surface modification, to which 2 mL of
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APTES is dropwise added. The resulting solution is stirred for 24 h at 60 °C. Finally, the modified CDs were extracted by diethyl ether twice.
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The quantum yield (QY) of CDs was calculated with excitation wavelength at 350 nm according to the equation.
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QCD QR
2 I CD AR CD 2 I R ACD R
(1)
where I is the luminescent spectra intensity, A is the absorbance at exited wavelength, η is the
solvent refractive index. Quinine sulfate in 0.1 M H2SO4 solution (QY=54%) was chosen as the reference. The subscript R designate reference in this equation. [36]. 2.4. Preparation of MIP-coated CDs The MIP-coated CDs were synthesized via a sol-gel process based on previously reported with a 5
minor modification [37]. Firstly, 50 mg OTC and the modified CDs (40 mL) were added to the reaction system by using sonication to make OTC completely dissolve. Then, TEOS (0.5 mL), APTES (100 μL) and NH3·H2O (200 μL) were added into the mixture with stirring for 24 h at room temperature. The synthesized product was washed with ethanol and water several times to remove the reagents. The OTC template was extracted with acetic acid and methanol (5:95, v/v), until no
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OTC was detected. In the meantime, the non-imprinted polymer-coated CDs (NIP-coated CDs) were synthesized consistent with the described method without adding OTC (template molecule).
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2.5. Fluorescent Measurement
The fluorescent spectra were obtained under a wavelength of 400-700 nm with excitation at 350 nm. The parameters such as excitation voltage (750 V), scan speed (240 nm min-1) and slit widths
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(10 nm) were set. The spectra were analyzed after three parallel scans. The OTC standard solution
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was mixed well with 0.1 mg mL-1 MIP-coated CDs (1:1, v/v) at room temperature at pH=8 adjusted
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by phosphate buffer (0.1 mol L-1). After full mixing, the fluorescent measurement was recorded by
2.6. The pretreatment of honey sample
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a fluorescence spectrophotometer.
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The honey sample (5 g) was dissolved completely in 20 mL of 0.1 mol L-1 Na2EDTA-McIlvaine
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buffer (pH 4.0). The spiked honey samples were prepared by adding OTC solutions. The diluted honey spiked with OTC was mixed with the 0.1 mg mL-1 MIP-coated CDs (1:1, v/v) at room
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temperature under the condition of pH=8 adjusted by phosphate buffer (0.1 mol L-1). After fully reacting, the solution was centrifuged to remove the supernatant, and MIP-coated CDs were
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redispersed into phosphate buffer (pH=8) for fluorescent measurement.
3. Results and discussion 3.1. Synthesis process of MIP-coated CDs Biomass CDs were successfully synthesized using sweet potato peels which were readily available, green and low cost. The proximate analysis and ultimate analysis of the dried sweet 6
potato peels are presented in Table S1. Sweet potato peels contain carbohydrates like ascorbic acid, protein, and sugar as carbon precursors. As shown in Fig.1, the possible mechanism for CDs formation from sweet potato peels is illustrated. The mechanism is based on the previous literature about carbonization of carbohydrates [38]. Under the presence of ascorbic acid, the hydrolysis, dehydration and decomposition of different macromolecule compounds and carbohydrates occur
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and produce soluble compounds such as ketones, furfural and several organic acids. These organic acids such as acetic, formic acid and levulinic can be used as acid resources when acid catalyzed
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reactions took place. The condensation and polymerization process converted the products to
soluble polymers. Aromatization and carbonization are subsequently carried out by condensation and cycloaddition reactions. After that, these aromatic clusters at a critical concentration at the point
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of supersaturation can synthesize CDs by means of a possible nuclear burst. In addition, the
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ascorbic acid existed in sweet potato peels can promote the reaction to occur in a short period of
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time. The synthesized CDs are dispersed in water and look transparent.
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The major preparation steps of MIP-coated CDs were presented in Fig. 2A. Before polymerization, the surface of CDs was chemically modified by the APTES which was used as the
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precursor of silica layer. The versatile organosilane APTES can form bonds to connect self-
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assembled monolayer on different substrates [39]. Typically, the Si-O-Si bond can be formed between the silanol groups and the surface to effect organosilane self-assembly [40]. In addition, the
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extensive extension of effective chemical functional groups at the silane molecules has flexible adaptation of the surface. Therefore, this coating method can make the cross-linked silica shell become a strong hybrid nanocomposite to protect CDs from the external disturbance and improve
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the reaction between CDs and MIP [36]. Then, the MIP-coated CDs nanoparticles were synthesized by polymerization reaction among APTES (functional monomer), TEOS (crosslinker), and NH3·H2O (catalyst) in the presence of CDs-APTES (fluorescent materials) and OTC (template). The APTES with amino groups can be conjugated with OTC molecules and made them place into the polymer matrix [41]. When the template molecule OTC is absent in the complex, the 7
recombination of electrons and holes can lead to fluorescence (Fig. 2B). The OTC molecules can be introduced as efficient electron acceptor for the excited-state electron from the CDs-APTES composite, and prevent the electron-hole recombination at the CDs-APTES composite interface, which will cause fluorescence quenching. The selective fluorescence probes (MIP-coated CDs) showed a potential application in the field of separation and detection.
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3.2. Photoluminescence properties of biomass CDs The fluorescent intensity of CDs was detected when the excitation wavelength is in the range of
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300-420 nm (Fig. S1A). The maximum fluorescence signal occurs when excitation wavelength is at 350 nm. As the excitation wavelength increased, the emission peak showed a red shift, indicating fluorescence intensity is related to the excitation wavelength. A possible mechanism of the CDs
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photoluminescent behavior is the existence of surface energy trap distribution and different particle
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sizes. The QY of CDs was measured using quinine sulfate (QY=54%) as reference, and the
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measurement result was 8.9%. The QY of CDs is comparable to those prepared by other biomass
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carbon sources [42,43], which is lower than the CDs derived from small molecules [44]. However, the following analysis results indicated that the fluorescence intensity of the CDs synthesized by the
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biomass carbon source can meet the analysis requirement. Therefore, in order to reduce the
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synthesis cost, we used biomass materials as precursors for the fabrication of CDs. The effects of pH, irradiation time, and ionic strength were also investigated in the
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Supplementary materials
3.3. Characterization of CDs and MIP-coated CDs The TEM images of CDs and MIP-coated CDs were shown in Fig. 3A,B. The image of CDs
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from sweet potato peels by hydrothermal synthesis indicates that they compose of monodispersed nanodots. The particle diameter of CDs was 2.0 ± 0.6 nm. The average particle size of the MIPcoated CDs was around 31 ± 7 nm which was larger than that of CDs. The result indicated that the CDs were well coated with MIP. The XRD pattern of MIP-coated CDs was shown in Fig. 3C. The broad peak of MIP-coated CDs 8
centered at 2θ=24o can correspond to the C (002) plane. The XRD peak showed the amorphous nature of CDs [45]. Fig. 3D shows the FT-IR spectra of CDs and MIP-coated CDs particles. For CDs, a broad absorption band at 3400 cm-1 is related to the O-H stretching vibrations. The absorption bands at 1710, 1610 and 1420 cm-1 correspond to stretching vibrations of C=O, C=C and C-O-C groups,
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respecctively. For MIP-coated CDs, the peak at 1060 cm-1 was attributed to the O-Si-O stretching vibration, and the peak at 780 cm-1 was ascribed to the Si-O stretching vibration. Several common
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characteristic peaks were the broad adsorption of -OH groups between 3300 cm-1 and 3550 cm-1 and the adsorption band of C=O stretching vibration at 1651 cm-1, which are the same as the previous literature [46]. In addition to the characteristic peaks of CDs, the O-Si-O stretching vibration and
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Si-O stretching vibration can be found in the FT-IR spectrum of MIP-coated CDs. It indicated that
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the imprinted polymers incorporating CDs have been successfully synthesized by sol-gel
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condensation.
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The surface composition and elemental analysis of CDs and MIP-coated CDs were obtained by XPS technique. The XPS result of CDs (Fig. S2) shows that the biomass CDs were equipped with
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functional groups such as amino, carboxyl and hydroxyl groups, which can promote the CDs
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solubility without additional chemical modification [47]. In addition, the four peaks at 288.8, 403.4, 536.3, and 106.7 eV were seen in the XPS spectrum of MIP-coated CDs (Fig. S3A) that are
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assigned to C1s, N1s, O1s, and Si2p, respectively. Except the element of CDs, the XPS spectra of MIP-coated CDs confirmed the presence of Si element. The result supported that the MIP-coated CDs have been successfully synthesized.
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3.4 Photoluminescence properties of MIP-coated CDs After adding MIP-coated CDs into the OTC solution, the effect of the reacting time is
investigated (Fig. S4A). The fluorescent intensity of the system reached equilibrium within 5 min, and remained stable for at least 1.5 h. The pH of solution has a great influence on the fluorescence properties. The pH value in the 9
range of 3-12 was investigated in Fig. S4B and Fig. S4C. Different pH values can change the surface charge of MIP-coated CDs and OTC, thus affecting the interaction between them. The quenching efficiency achieves the best effect at pH = 8. The result illustrates that the quenching of the system fluorescence depends not only on the electrostatic force but also on other interactions such as hydrogen bonding.
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To prove the fluorescent stability, the fluorescence emission measurement was performed by testing the fluorescence intensity in each hour. The result indicated that the emission of MIP-coated
3.5 Mechanism of MIP-coated CDs for the detection of OTC
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CDs was stable with a relative standard deviation (RSD) of 1.0% (Fig. S4D).
To study the fluorescence quenching mechanism of MIP-coated CDs, the UV absorption peaks of
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OTC, MIP-coated CDs, CDs and MIP-coated CDs with OTC were displayed in Fig.4A. The UV
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absorption peaks of OTC are located at 275 nm and 352 nm, and the UV absorption spectrum of
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MIP-coated CDs is weak. After mixing with OTC, the system has two obvious peaks. Compared
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with the UV absorption peaks of OTC, the UV absorption peaks of the MIP-coated CDs with OTC changed slightly. The UV absorption peaks of OTC at 352 nm red shifted to 357 nm. It indicated
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that the MIP-coated CDs nanoparticles have an interaction with OTC. The result indicated that the
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binding of MIP-coated CDs with OTC was attributed to static quenching. Furthermore, the fluorescence lifetime experiment was also performed to investigate the
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fluorescence attenuation properties of MIP-coated CDs in the presence and absence of OTC. The fluorescence lifetime of MIP-coated CDs was 4.67 ns (Fig.4B). After binding with OTC, the fluorescence lifetime was 4.38 ns with almost unchanged. It can be considered that fluorescence
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resonance energy transfer (FRET) can be excluded, because FRET would result in a significant change of fluorescence lifetime. In addition, the emission spectrum of MIP-coated CDs and UV absorption spectra of OTC have no obvious overlap (Fig.4A). It further indicated that FRET was not a possible mechanism to explain the fluorescent quenching of MIP-coated CDs. Furthermore, molecular orbital theory was adopted to explain the quenching mechanism, as 10
depicted in Fig. 4C. The electrons of CDs can be excited from the ground state to the excited state after accepting UV photon. Then, the excited electrons of CDs return to the valence band with producing the fluorescence signal. Additionally, APTES has triethylene silane group which is introduced into the polymer matrix. After adding OTC, the Meisenheimer complex could be formed between the OTC and amino groups of APTES. Our experiment is performed at pH = 8, while the
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OTC molecules carry negative charge and the surface charge of MIP-coated CDs keeps positive. There is strong electrostatic attraction between the OTC and MIP-coated CDs. The interaction force
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would lead to electron transfer between CDs and OTC. The UV and visible absorption peaks of OTC are at about 275 and 352 nm, very close to the conduction band of CDs (Fig. 4A), so the excited electron could directly jump into the LUMO level layer of the Meisenheimer complex,
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leading to the quenching of CDs fluorescence [48,49].
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The thermodynamic experiment was also applied to explain the fluorescence quenching
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mechanism by the Stern-Volmer equation at different temperatures (293 K, 303 K and 313 K).
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When the temperature increased, the quenching constant decreased (Table S2). The results showed that the possible fluorescence quenching mechanism of MIP-coated CDs-OTC system is static
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quenching [50].
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3.6 MIP-coated CDs with OTC at different concentrations The feasibility of using MIP-coated CDs for the detection of OTC is evaluated. The fluorescent
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intensities were investigated after equilibrium and plotted against the different concentrations of OTC in the range of 50 - 900 ng mL−1. The fluorescent intensity of MIP-coated CDs decreases notably with an increased OTC concentration (Fig.5). The phenomenon is satisfied by the Stern-
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Volmer equation as follows: F0
F
K SV C q 1
(1)
Where Ksv means the Stern-Volmer constant and Cq means the addition of OTC concentration, F0 and F are the fluorescence intensities in the absence and presence of OTC, respectively. The Stern-Volmer plot of MIP-coated CDs satisfies a linear relationship with different OTC 11
concentrations in Fig.5. The regression equations were shown as follows: F0/F = 0.00196Cq + 1.0797 (MIP-coated CDs, R2=0.999), F0/F = 0.000409Cq + 1.0528 (NIP-coated CDs, R2=0.962). The limit of detection (LOD) was 15.3 ng mL-1 which can be calculated by 3σ/slope, where σ stands for the deviation of blank measurements. Repeated availability is a significant factor for the use of fluorescent MIP. As shown in Fig. S5,
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these sensors can be used at least five times by using methanol-acetic acid (95:5,v/v) as eluent. 3.7 Specificity and selectivity of MIP-coated CDs
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The recognition ability of MIP-coated CDs was investigated by testing other tetracycline analogs, such as CTC and TC. The chemical structures of OTC, CTC and TC are very similar (Fig. S6) so that CTC and TC can enter into the recognition sites and result in fluorescence quenching. The
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imprinting factor (IF) was investigated based on KSV, MIP/KSV, NIP. The IF values of OTC, CTC and
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TC were 4.8, 2.5, 2.3, respectively (Table S3), which indicated that MIP-coated CDs had specific
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recognition cavities towards OTC and its analogues, but had the highest affinity for template OTC.
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We also investigated the effects of OTC, CTC and TC on CDs quenching. The quenching efficiency of CDs has no significant difference for the three compounds. It is obvious that MIP can improve
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selectivity of CDs for the template molecule OTC detection. We also studied BPA as a reference
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compound for investigating the recognition ability of prepared materials (Fig. S6). The IF value for BPA was 1.1 (Table S3), indicating that MIP-coated CDs had no specific imprint affinity for BPA.
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3.8 Practical application of the sensor in honey sample In this work, a series of different OTC concentrations (50, 250, and 500 ng mL−1) were added into the honey to test the applicability of MIP-coated CDs. The main problems for detecting OTC in
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food samples are its low concentration and the coexisting interference. The established MIP-coated CDs method could selectively and sensitively detect OTC in honey samples. The RSDs were ranged from 2.3% to 4.1% and the quantitative recoveries were from 90.2% to 97.3%. The results showed that the established MIP-coated CDs method has good accuracy, wide dynamic range and low detection limit. Therefore, the MIP-coated CDs were practically feasible for rapid OTC analysis in 12
honey. 3.9 Method performance comparison The established MIP-coated CDs method was compared with classical methods that have been established to detect OTC, as seen in Table 1. The RSDs and recoveries of the reported method were comparable with these methods in the literature. However, some disadvantages still exist that
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the LOD is higher than some other methods. It is well known that different methods have their own advantages and disadvantages in terms of analysis time, specific identification and precision. The
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MIP-coated CDs method can avoid complex preseparation procedure and improve the selective
recognition ability. Furthermore, this method can shorten the detection time and easy to operate. In the meantime, more efforts should be made to enhance the sensitivity of MIP-coated CDs
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fluorescent probe with a good selectivity.
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4. Conclusion
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In summary, a novel MIP-coated CDs fluorescent probe was prepared to detect OTC through
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electron-transfer-induced fluorescence quenching. Compared with classical methods, the application of biomass CDs has solved the problem of high cost and toxicity. The combination of
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imprinting technique and fluorescence detection enhanced the sensitivity and selectivity of the sensor. The possible fluorescence quenching mechanism is static quenching. The stability and
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practicability of the MIP-coated CDs have obtained good results. This eco-friendly sensor showed satisfactory accuracy and a low detection limit in complex sample analysis. With the rapid development of multifunctional MIPs and various signal units such as CDs, we can speculate that
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synergistic effects will provide broad perspectives to explore the potential applications of composite materials.
Acknowledgement This work was supported by the Development Program of the Ministry of Science and Technology 13
of Jilin Province, China ((No. 20180201011GX), the Fundamental Research Funds for the Central Universities (No. 2572017EB08), Natural Science Foundation of Heilongjiang Province ((No. JJ2018ZR0081) and Harbin science and technology innovation talent research special funds ((No. 2016RAQXJ151).
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[2] GB 14963-2011. National Standard of the People's Republic of China.
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[33] S. Xu, H. Lu, Biosens. Bioelectron. 85 (2016) 950 [34] H. Wang, J. Yi, D. Velado, Y. Yu, S. Zhou, ACS Appl. Mater. Interfaces 7 (2015) 15735 [35] U. Koesukwiwat, S. Jayanta, N. Leepipatpiboon, J. Chromatogr. A 1140 (2007) 147. [36] M. Grabolle, M. Spieles, V. Lesnyak, N.Gaponik, A. Eychmuller, U. Resch-Genger, Anal. Chem. 81 (2009) 6285 15
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[56] A. Pena, N. Pelantova, C.M. Lino, M.I.N. Silveira, P. Solich, J. Agric. Food Chem. 53 (2005)
Figure captions:
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3784.
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Fig. 1 Possible mechanism for the formation of CDs Fig. 2 Illustration of the preparation of MIP-coated CDs (A) and mode of interaction of MIP-coated CDs with OTC (B).
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Fig.3 TEM images of CDs (A) and MIP-coated CDs (B), XRD pattern of MIP-coated CDs (C) and FT-IR spectra of CDs and MIP-coated CDs (D). Fig.4 UV spectra of OTC (curve 1), MIP-coated CDs nanocomposite with OTC (curve 2), CDs (curve 3), and MIP-coated CDs nanocomposite (curve 4), the fluorescence emission spectra of MIPcoated CDs nanocomposite (curve 5) (A). Fluorescence decay profile of the MIP-coated CDs in the 17
absence and presence of OTC (B), Schematic of the CDs fluorescence quenching mechanism about molecular orbital theory (C). Fig. 5 Fluorescence emission spectra of MIP-coated CDs with OTC (A, inset: Stern-Volmer plots from MIP-coated CDs with OTC) and NIP-coated CDs with OTC (B, inset: Stern-Volmer plots
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from NIP-coated CDs with OTC).
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A
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Fig. 1
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B
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Fig. 2
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Fig.4
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Fig. 5
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Table 1 Comparison with other methods for determination of OTC in honey samples.
Pretreatment method
Detection method
Detection limit (ng mL-1)
Recovery (%)
SPE with phenyl cartridges
HPLC-PDA
9.0
92.1-96.1
SPE with MIP monolithic column
HPLC-DAD
10.2
64.8-78.8
5.4-9.3
52
Fluorescent probe based BODIPY
Fluorescence spectrophotometer
33.8
101.9-107.9
0.8-1.5
53
SPE by C8
HPLC-FD
24.0
90.0-109.0
1.0-9.0
54
HPLC-MS/MS
3.0
68.7-82.5
-
55
HPLC -FD
25.0
86.0-93.0
4.0-8.0
56
Fluorescence spectrophotometer
15.3
90.2- 97.3
2.3-4.1
This workss
Reference
24
51
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1.5-3.0
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SPE with sulfobetaine polymer resin SPE with polymeric and ion exchange cartridges Fluorescence probe based MIP@CDs
RSD (%)
S1226-086X(18)30982-1 https://doi.org/10.1016/j.jiec.2018.10.007 JIEC 4203
To appear in: Received date: Revised date: Accepted date:
14-4-2018 8-10-2018 8-10-2018
Please cite this article as: Haochi Liu, Lan Ding, Ligang Chen, Yanhua Chen, Tianyu Zhou, Huiyu Li, Yuan Xu, Li Zhao, Ning Huang, A facile, green synthesis of biomass carbon dots coupled with molecularly imprinted polymers for highly selective detection of oxytetracycline, Journal of Industrial and Engineering Chemistry https://doi.org/10.1016/j.jiec.2018.10.007 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
A facile, green synthesis of biomass carbon dots coupled with molecularly imprinted polymers for highly selective detection of oxytetracycline Haochi Liua, Lan Dinga,*Ligang Chenb,* *, Yanhua Chena, Tianyu Zhoua, Huiyu Lia, Yuan Xua, Li Zhaoa, Ning Huanga a
College of Chemistry, Jilin University, 2699 Qianjin Street, Changchun 130012, China
b
E-mail: [email protected] (L. Ding) E-mail: [email protected] (L. Chen)
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Corresponding author: *Tel.: +86-431- 85168399 **Tel.: +86-451- 82190679
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Department of Chemistry, College of Science, Northeast Forestry University, 26 Hexing Road, Harbin 150040, China
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Graphical Abstract
Schematic of the preparation of MIP-coated CDs
ABSTRACT: Biomass carbon dots (CDs) prepared by sweet potato peels were superior fluorophores with low toxicity and excellent photostability. A novel designed fluorescence probe for specific recognition 1
and sensitive detection of oxytetracycline (OTC) was fabricated with CDs and molecularly imprinted polymer. The quenching of CDs happened when rebinding with OTC due to electrontransfer-induced fluorescence quenching mechanism. The fluorescence probe was successfully applied in honey with the recoveries ranging from 90.2% to 97.3%. The detection limit of OTC was
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15.3 ng mL-1. This work provides promising perspectives that the development of fluorescent MIP
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will be valuable for rapid analysis in complex samples.
Key words: Biomass carbon dots; Molecularly imprinted polymers; Oxytetracycline; Fluorescence
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probe; Honey
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1. Introduction
Oxytetracycline (OTC) belongs to a kind of antibiotics that can be applied in many fields [1]. It
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can be used as a veterinary drug to prevent animal disease, due to its low cost and superior
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antibacterial properties. However, the widespread use of OTC in foods of animal origin has caused concerns about the level of unsafe residue in food such as honey, meat and milk. The residue of OTC poses a threat to public health. In China, the regulation (GB 14963-2011) makes OTC illegal
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to use in honey [2].Thus, finding a succinct and efficient approach to detect trace OTC in honey is of significance. At present, some conventional methods have been established to detect OTC, including highperformance liquid chromatography (HPLC) [3,4], colorimetric analysis [5], enzyme-linked immunosorbent assay [6] and LC with tandem mass spectrometry (LC-MS) [7,8]. As for HPLC/LC2
MS, they offer good separation and selectivity, but the requirement of professional skill and tedious operating procedure limits their application. Colorimetric analysis is less sensitive and selective. Enzyme-linked immunosorbent assay is still unsatisfactory because of the potential cross-reactivity and high cost. Therefore, it is necessary to develop a fast analysis method with low-cost and high selectivity.
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Recently, fluorescence has become a promising method in analytical fields due to the merits of convenience, time saving and good sensitivity [9]. Gao et al. [10] have synthesized TGA-capped
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CdTe as a fluorescent sensor to detect OTC with good precision and wide linear range. Xu et al.
[11] performed Au nanoclusters as a versatile probe for the detection of OTC with a good linearity and a high selectivity. The above sensors generally require complicated preparation approach or
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expensive reagents. Carbon dots (CDs) as novel fluorescent nanomaterials have good stability, low
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toxicity, tunable and unique photoluminescence properties which attached much attention in recent
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years [12-14]. Recently, many carbon materials have been synthesized from biomass for the
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application of environment and energy [15-20]. From the material synthesis and environment point of views, it is necessary to use inexpensive, eco-friendly and readily available biomass resources to
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prepare CDs. Some researchers used walnut shells to fabricate CDs by high-temperature carbonization at 1000 oC [21]. In addition, a facile hydrothermal method was proposed by using
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waste paper to synthesize CDs at 180 oC for 10 h [22]. However, most of the reported biomass CD
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methods have problems such as high reaction temperature, long reaction time, and limited application range. The exploration of new carbon sources for simple operation, wide application, and green synthesis of CDs is highly desirable.
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Molecularly imprinted polymer (MIP) is an ideal choice for improving fluorescence detection
selectivity [23,24]. Several researchers have synthesized different types of MIP-quantum dots (QDs) composites [25-28]. These sensors can selectively recognize the target molecules because of the MIP as recognition units [29]. Hou et al. compared the selectivity of CDs and CDs@MIP for the target analyte and its analogs under the same experimental conditions. The result showed that the 3
selectivity of CDs@MIP for the target analyte detection was improved significantly [30,31]. Up to now, MIPs as sensors combining with CDs have been reported for recognition of 2,4-dinitrotoluene, trinitrotoluene and glucose [32-34]. However, the synthesis of CDs in MIP-coated CDs often requires using a large amount of toxic and expensive chemicals as well as intricate processes. Herein, we fabricated a novel type of MIP-coated CDs by means of a simple and time-saving sol-
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gel polymerization approach to detect complex samples. Sweet potato peels were used to produce biomass CDs by a one-pot hydrothermal approach without complicated preparation methods or
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using toxic solvents. A new system was generated after OTC was again bonded to MIP-coated CDs, causing fluorescence quenching. Moreover, the eco-friendly fluorescence sensors were successfully applied to detect OTC in honey without additional pollution, showing their potential prospects for
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rapid analysis in complex samples.
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2. Experimental
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2.1. Chemicals
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The standards of OTC, chlorotetracycline (CTC), tetracycline (TC) and bisphenol A (BPA) were purchased from Aladdin (Shanghai, China). TEOS, APTES, ammonia, diethyl ether, Na2HPO4,
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Na2EDTA, citric acid monohydrate, ethanol and methanol were obtained from Kermel (Tianjin, China). The sweet potato and honey samples were purchased from markets in Changchun (China).
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High purity water was got by a Milli-Q water system (Millipore, Billerica, MA, USA). The Na2EDTA-McIlvaine buffer (0.1 mol L-1, pH 4.0) solution was weekly produced by dissolving Na2HPO4 (8.86 g), Na2EDTA (16.81 g) and citric acid monohydrate (5.90 g) in 500 mL of water
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[35].
2.2. Instrumentation The carbon, hydrogen, nitrogen, sulphur and oxygen content of the sweet potato peels were determined using a Flash 2000 CHNS/O automatic elemental analyser (Thermo Fisher Scientific, USA). The proximate analysis of sweet potato peels was characterized by a themogravimetric 4
analyser (TGA; Pyris 1, Perkin Elmer, Waltham, MA, USA). The morphology and size of MIPcoated CDs were investigated by a transmission electron microscopy (TEM, Hitachi H-7650, Japan). The XRD spectrum of the MIP-coated CDs was characterized by a Shimadzu XRD-6100 spectrometer (Shimadzu, Japan). The XPS spectra were recorded on an ESCALAB250 electron spectrometer (Thermo Electron, USA). Fourier transform infrared (FT-IR) spectrum was observed
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with an FT-IR360 spectrometer (Nicolet, Madison, WI, USA). The UV spectrum was collected by a TU-1901 spectrometer (PERSEE, Beijing, China). The nanosecond fluorescence lifetime was
an F-4600 fluorescence spectrophotometer (Hitachi, Japan). 2.3. Preparation and surface modification of CDs
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investigated by an FLS 920 instrument (Edinburgh, UK). Fluorescence detection was carried out on
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Sweet potato peels were cut into small pieces to prepare biomass CDs by a hydrothermal method.
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The mixture of sweet potato pieces (2 g) and high-purity water (30 mL) was added into a 50 mL
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autoclave and heated to 200 °C for 3 h. The resulting dark brown solution is filtered through a 0.22
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μm membrane. Then the suspension was filtered though a dialysis membrane (MWCO:1000) for 24 h.
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In briefly, 40 mL of CDs (1 mg mL-1) was used for the surface modification, to which 2 mL of
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APTES is dropwise added. The resulting solution is stirred for 24 h at 60 °C. Finally, the modified CDs were extracted by diethyl ether twice.
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The quantum yield (QY) of CDs was calculated with excitation wavelength at 350 nm according to the equation.
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QCD QR
2 I CD AR CD 2 I R ACD R
(1)
where I is the luminescent spectra intensity, A is the absorbance at exited wavelength, η is the
solvent refractive index. Quinine sulfate in 0.1 M H2SO4 solution (QY=54%) was chosen as the reference. The subscript R designate reference in this equation. [36]. 2.4. Preparation of MIP-coated CDs The MIP-coated CDs were synthesized via a sol-gel process based on previously reported with a 5
minor modification [37]. Firstly, 50 mg OTC and the modified CDs (40 mL) were added to the reaction system by using sonication to make OTC completely dissolve. Then, TEOS (0.5 mL), APTES (100 μL) and NH3·H2O (200 μL) were added into the mixture with stirring for 24 h at room temperature. The synthesized product was washed with ethanol and water several times to remove the reagents. The OTC template was extracted with acetic acid and methanol (5:95, v/v), until no
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OTC was detected. In the meantime, the non-imprinted polymer-coated CDs (NIP-coated CDs) were synthesized consistent with the described method without adding OTC (template molecule).
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2.5. Fluorescent Measurement
The fluorescent spectra were obtained under a wavelength of 400-700 nm with excitation at 350 nm. The parameters such as excitation voltage (750 V), scan speed (240 nm min-1) and slit widths
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(10 nm) were set. The spectra were analyzed after three parallel scans. The OTC standard solution
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was mixed well with 0.1 mg mL-1 MIP-coated CDs (1:1, v/v) at room temperature at pH=8 adjusted
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by phosphate buffer (0.1 mol L-1). After full mixing, the fluorescent measurement was recorded by
2.6. The pretreatment of honey sample
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a fluorescence spectrophotometer.
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The honey sample (5 g) was dissolved completely in 20 mL of 0.1 mol L-1 Na2EDTA-McIlvaine
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buffer (pH 4.0). The spiked honey samples were prepared by adding OTC solutions. The diluted honey spiked with OTC was mixed with the 0.1 mg mL-1 MIP-coated CDs (1:1, v/v) at room
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temperature under the condition of pH=8 adjusted by phosphate buffer (0.1 mol L-1). After fully reacting, the solution was centrifuged to remove the supernatant, and MIP-coated CDs were
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redispersed into phosphate buffer (pH=8) for fluorescent measurement.
3. Results and discussion 3.1. Synthesis process of MIP-coated CDs Biomass CDs were successfully synthesized using sweet potato peels which were readily available, green and low cost. The proximate analysis and ultimate analysis of the dried sweet 6
potato peels are presented in Table S1. Sweet potato peels contain carbohydrates like ascorbic acid, protein, and sugar as carbon precursors. As shown in Fig.1, the possible mechanism for CDs formation from sweet potato peels is illustrated. The mechanism is based on the previous literature about carbonization of carbohydrates [38]. Under the presence of ascorbic acid, the hydrolysis, dehydration and decomposition of different macromolecule compounds and carbohydrates occur
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and produce soluble compounds such as ketones, furfural and several organic acids. These organic acids such as acetic, formic acid and levulinic can be used as acid resources when acid catalyzed
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reactions took place. The condensation and polymerization process converted the products to
soluble polymers. Aromatization and carbonization are subsequently carried out by condensation and cycloaddition reactions. After that, these aromatic clusters at a critical concentration at the point
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of supersaturation can synthesize CDs by means of a possible nuclear burst. In addition, the
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ascorbic acid existed in sweet potato peels can promote the reaction to occur in a short period of
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time. The synthesized CDs are dispersed in water and look transparent.
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The major preparation steps of MIP-coated CDs were presented in Fig. 2A. Before polymerization, the surface of CDs was chemically modified by the APTES which was used as the
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precursor of silica layer. The versatile organosilane APTES can form bonds to connect self-
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assembled monolayer on different substrates [39]. Typically, the Si-O-Si bond can be formed between the silanol groups and the surface to effect organosilane self-assembly [40]. In addition, the
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extensive extension of effective chemical functional groups at the silane molecules has flexible adaptation of the surface. Therefore, this coating method can make the cross-linked silica shell become a strong hybrid nanocomposite to protect CDs from the external disturbance and improve
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the reaction between CDs and MIP [36]. Then, the MIP-coated CDs nanoparticles were synthesized by polymerization reaction among APTES (functional monomer), TEOS (crosslinker), and NH3·H2O (catalyst) in the presence of CDs-APTES (fluorescent materials) and OTC (template). The APTES with amino groups can be conjugated with OTC molecules and made them place into the polymer matrix [41]. When the template molecule OTC is absent in the complex, the 7
recombination of electrons and holes can lead to fluorescence (Fig. 2B). The OTC molecules can be introduced as efficient electron acceptor for the excited-state electron from the CDs-APTES composite, and prevent the electron-hole recombination at the CDs-APTES composite interface, which will cause fluorescence quenching. The selective fluorescence probes (MIP-coated CDs) showed a potential application in the field of separation and detection.
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3.2. Photoluminescence properties of biomass CDs The fluorescent intensity of CDs was detected when the excitation wavelength is in the range of
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300-420 nm (Fig. S1A). The maximum fluorescence signal occurs when excitation wavelength is at 350 nm. As the excitation wavelength increased, the emission peak showed a red shift, indicating fluorescence intensity is related to the excitation wavelength. A possible mechanism of the CDs
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photoluminescent behavior is the existence of surface energy trap distribution and different particle
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sizes. The QY of CDs was measured using quinine sulfate (QY=54%) as reference, and the
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measurement result was 8.9%. The QY of CDs is comparable to those prepared by other biomass
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carbon sources [42,43], which is lower than the CDs derived from small molecules [44]. However, the following analysis results indicated that the fluorescence intensity of the CDs synthesized by the
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biomass carbon source can meet the analysis requirement. Therefore, in order to reduce the
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synthesis cost, we used biomass materials as precursors for the fabrication of CDs. The effects of pH, irradiation time, and ionic strength were also investigated in the
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Supplementary materials
3.3. Characterization of CDs and MIP-coated CDs The TEM images of CDs and MIP-coated CDs were shown in Fig. 3A,B. The image of CDs
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from sweet potato peels by hydrothermal synthesis indicates that they compose of monodispersed nanodots. The particle diameter of CDs was 2.0 ± 0.6 nm. The average particle size of the MIPcoated CDs was around 31 ± 7 nm which was larger than that of CDs. The result indicated that the CDs were well coated with MIP. The XRD pattern of MIP-coated CDs was shown in Fig. 3C. The broad peak of MIP-coated CDs 8
centered at 2θ=24o can correspond to the C (002) plane. The XRD peak showed the amorphous nature of CDs [45]. Fig. 3D shows the FT-IR spectra of CDs and MIP-coated CDs particles. For CDs, a broad absorption band at 3400 cm-1 is related to the O-H stretching vibrations. The absorption bands at 1710, 1610 and 1420 cm-1 correspond to stretching vibrations of C=O, C=C and C-O-C groups,
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respecctively. For MIP-coated CDs, the peak at 1060 cm-1 was attributed to the O-Si-O stretching vibration, and the peak at 780 cm-1 was ascribed to the Si-O stretching vibration. Several common
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characteristic peaks were the broad adsorption of -OH groups between 3300 cm-1 and 3550 cm-1 and the adsorption band of C=O stretching vibration at 1651 cm-1, which are the same as the previous literature [46]. In addition to the characteristic peaks of CDs, the O-Si-O stretching vibration and
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Si-O stretching vibration can be found in the FT-IR spectrum of MIP-coated CDs. It indicated that
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the imprinted polymers incorporating CDs have been successfully synthesized by sol-gel
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condensation.
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The surface composition and elemental analysis of CDs and MIP-coated CDs were obtained by XPS technique. The XPS result of CDs (Fig. S2) shows that the biomass CDs were equipped with
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functional groups such as amino, carboxyl and hydroxyl groups, which can promote the CDs
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solubility without additional chemical modification [47]. In addition, the four peaks at 288.8, 403.4, 536.3, and 106.7 eV were seen in the XPS spectrum of MIP-coated CDs (Fig. S3A) that are
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assigned to C1s, N1s, O1s, and Si2p, respectively. Except the element of CDs, the XPS spectra of MIP-coated CDs confirmed the presence of Si element. The result supported that the MIP-coated CDs have been successfully synthesized.
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3.4 Photoluminescence properties of MIP-coated CDs After adding MIP-coated CDs into the OTC solution, the effect of the reacting time is
investigated (Fig. S4A). The fluorescent intensity of the system reached equilibrium within 5 min, and remained stable for at least 1.5 h. The pH of solution has a great influence on the fluorescence properties. The pH value in the 9
range of 3-12 was investigated in Fig. S4B and Fig. S4C. Different pH values can change the surface charge of MIP-coated CDs and OTC, thus affecting the interaction between them. The quenching efficiency achieves the best effect at pH = 8. The result illustrates that the quenching of the system fluorescence depends not only on the electrostatic force but also on other interactions such as hydrogen bonding.
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To prove the fluorescent stability, the fluorescence emission measurement was performed by testing the fluorescence intensity in each hour. The result indicated that the emission of MIP-coated
3.5 Mechanism of MIP-coated CDs for the detection of OTC
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CDs was stable with a relative standard deviation (RSD) of 1.0% (Fig. S4D).
To study the fluorescence quenching mechanism of MIP-coated CDs, the UV absorption peaks of
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OTC, MIP-coated CDs, CDs and MIP-coated CDs with OTC were displayed in Fig.4A. The UV
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absorption peaks of OTC are located at 275 nm and 352 nm, and the UV absorption spectrum of
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MIP-coated CDs is weak. After mixing with OTC, the system has two obvious peaks. Compared
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with the UV absorption peaks of OTC, the UV absorption peaks of the MIP-coated CDs with OTC changed slightly. The UV absorption peaks of OTC at 352 nm red shifted to 357 nm. It indicated
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that the MIP-coated CDs nanoparticles have an interaction with OTC. The result indicated that the
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binding of MIP-coated CDs with OTC was attributed to static quenching. Furthermore, the fluorescence lifetime experiment was also performed to investigate the
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fluorescence attenuation properties of MIP-coated CDs in the presence and absence of OTC. The fluorescence lifetime of MIP-coated CDs was 4.67 ns (Fig.4B). After binding with OTC, the fluorescence lifetime was 4.38 ns with almost unchanged. It can be considered that fluorescence
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resonance energy transfer (FRET) can be excluded, because FRET would result in a significant change of fluorescence lifetime. In addition, the emission spectrum of MIP-coated CDs and UV absorption spectra of OTC have no obvious overlap (Fig.4A). It further indicated that FRET was not a possible mechanism to explain the fluorescent quenching of MIP-coated CDs. Furthermore, molecular orbital theory was adopted to explain the quenching mechanism, as 10
depicted in Fig. 4C. The electrons of CDs can be excited from the ground state to the excited state after accepting UV photon. Then, the excited electrons of CDs return to the valence band with producing the fluorescence signal. Additionally, APTES has triethylene silane group which is introduced into the polymer matrix. After adding OTC, the Meisenheimer complex could be formed between the OTC and amino groups of APTES. Our experiment is performed at pH = 8, while the
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OTC molecules carry negative charge and the surface charge of MIP-coated CDs keeps positive. There is strong electrostatic attraction between the OTC and MIP-coated CDs. The interaction force
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would lead to electron transfer between CDs and OTC. The UV and visible absorption peaks of OTC are at about 275 and 352 nm, very close to the conduction band of CDs (Fig. 4A), so the excited electron could directly jump into the LUMO level layer of the Meisenheimer complex,
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leading to the quenching of CDs fluorescence [48,49].
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The thermodynamic experiment was also applied to explain the fluorescence quenching
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mechanism by the Stern-Volmer equation at different temperatures (293 K, 303 K and 313 K).
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When the temperature increased, the quenching constant decreased (Table S2). The results showed that the possible fluorescence quenching mechanism of MIP-coated CDs-OTC system is static
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quenching [50].
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3.6 MIP-coated CDs with OTC at different concentrations The feasibility of using MIP-coated CDs for the detection of OTC is evaluated. The fluorescent
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intensities were investigated after equilibrium and plotted against the different concentrations of OTC in the range of 50 - 900 ng mL−1. The fluorescent intensity of MIP-coated CDs decreases notably with an increased OTC concentration (Fig.5). The phenomenon is satisfied by the Stern-
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Volmer equation as follows: F0
F
K SV C q 1
(1)
Where Ksv means the Stern-Volmer constant and Cq means the addition of OTC concentration, F0 and F are the fluorescence intensities in the absence and presence of OTC, respectively. The Stern-Volmer plot of MIP-coated CDs satisfies a linear relationship with different OTC 11
concentrations in Fig.5. The regression equations were shown as follows: F0/F = 0.00196Cq + 1.0797 (MIP-coated CDs, R2=0.999), F0/F = 0.000409Cq + 1.0528 (NIP-coated CDs, R2=0.962). The limit of detection (LOD) was 15.3 ng mL-1 which can be calculated by 3σ/slope, where σ stands for the deviation of blank measurements. Repeated availability is a significant factor for the use of fluorescent MIP. As shown in Fig. S5,
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these sensors can be used at least five times by using methanol-acetic acid (95:5,v/v) as eluent. 3.7 Specificity and selectivity of MIP-coated CDs
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The recognition ability of MIP-coated CDs was investigated by testing other tetracycline analogs, such as CTC and TC. The chemical structures of OTC, CTC and TC are very similar (Fig. S6) so that CTC and TC can enter into the recognition sites and result in fluorescence quenching. The
U
imprinting factor (IF) was investigated based on KSV, MIP/KSV, NIP. The IF values of OTC, CTC and
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TC were 4.8, 2.5, 2.3, respectively (Table S3), which indicated that MIP-coated CDs had specific
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recognition cavities towards OTC and its analogues, but had the highest affinity for template OTC.
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We also investigated the effects of OTC, CTC and TC on CDs quenching. The quenching efficiency of CDs has no significant difference for the three compounds. It is obvious that MIP can improve
ED
selectivity of CDs for the template molecule OTC detection. We also studied BPA as a reference
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compound for investigating the recognition ability of prepared materials (Fig. S6). The IF value for BPA was 1.1 (Table S3), indicating that MIP-coated CDs had no specific imprint affinity for BPA.
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3.8 Practical application of the sensor in honey sample In this work, a series of different OTC concentrations (50, 250, and 500 ng mL−1) were added into the honey to test the applicability of MIP-coated CDs. The main problems for detecting OTC in
A
food samples are its low concentration and the coexisting interference. The established MIP-coated CDs method could selectively and sensitively detect OTC in honey samples. The RSDs were ranged from 2.3% to 4.1% and the quantitative recoveries were from 90.2% to 97.3%. The results showed that the established MIP-coated CDs method has good accuracy, wide dynamic range and low detection limit. Therefore, the MIP-coated CDs were practically feasible for rapid OTC analysis in 12
honey. 3.9 Method performance comparison The established MIP-coated CDs method was compared with classical methods that have been established to detect OTC, as seen in Table 1. The RSDs and recoveries of the reported method were comparable with these methods in the literature. However, some disadvantages still exist that
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the LOD is higher than some other methods. It is well known that different methods have their own advantages and disadvantages in terms of analysis time, specific identification and precision. The
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MIP-coated CDs method can avoid complex preseparation procedure and improve the selective
recognition ability. Furthermore, this method can shorten the detection time and easy to operate. In the meantime, more efforts should be made to enhance the sensitivity of MIP-coated CDs
N
U
fluorescent probe with a good selectivity.
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4. Conclusion
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In summary, a novel MIP-coated CDs fluorescent probe was prepared to detect OTC through
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electron-transfer-induced fluorescence quenching. Compared with classical methods, the application of biomass CDs has solved the problem of high cost and toxicity. The combination of
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imprinting technique and fluorescence detection enhanced the sensitivity and selectivity of the sensor. The possible fluorescence quenching mechanism is static quenching. The stability and
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practicability of the MIP-coated CDs have obtained good results. This eco-friendly sensor showed satisfactory accuracy and a low detection limit in complex sample analysis. With the rapid development of multifunctional MIPs and various signal units such as CDs, we can speculate that
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synergistic effects will provide broad perspectives to explore the potential applications of composite materials.
Acknowledgement This work was supported by the Development Program of the Ministry of Science and Technology 13
of Jilin Province, China ((No. 20180201011GX), the Fundamental Research Funds for the Central Universities (No. 2572017EB08), Natural Science Foundation of Heilongjiang Province ((No. JJ2018ZR0081) and Harbin science and technology innovation talent research special funds ((No. 2016RAQXJ151).
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Figure captions:
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3784.
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Fig. 1 Possible mechanism for the formation of CDs Fig. 2 Illustration of the preparation of MIP-coated CDs (A) and mode of interaction of MIP-coated CDs with OTC (B).
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Fig.3 TEM images of CDs (A) and MIP-coated CDs (B), XRD pattern of MIP-coated CDs (C) and FT-IR spectra of CDs and MIP-coated CDs (D). Fig.4 UV spectra of OTC (curve 1), MIP-coated CDs nanocomposite with OTC (curve 2), CDs (curve 3), and MIP-coated CDs nanocomposite (curve 4), the fluorescence emission spectra of MIPcoated CDs nanocomposite (curve 5) (A). Fluorescence decay profile of the MIP-coated CDs in the 17
absence and presence of OTC (B), Schematic of the CDs fluorescence quenching mechanism about molecular orbital theory (C). Fig. 5 Fluorescence emission spectra of MIP-coated CDs with OTC (A, inset: Stern-Volmer plots from MIP-coated CDs with OTC) and NIP-coated CDs with OTC (B, inset: Stern-Volmer plots
A
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A
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from NIP-coated CDs with OTC).
18
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A
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Fig. 1
19
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U
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B
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Fig. 2
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21
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Fig.4
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Fig. 5
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Table 1 Comparison with other methods for determination of OTC in honey samples.
Pretreatment method
Detection method
Detection limit (ng mL-1)
Recovery (%)
SPE with phenyl cartridges
HPLC-PDA
9.0
92.1-96.1
SPE with MIP monolithic column
HPLC-DAD
10.2
64.8-78.8
5.4-9.3
52
Fluorescent probe based BODIPY
Fluorescence spectrophotometer
33.8
101.9-107.9
0.8-1.5
53
SPE by C8
HPLC-FD
24.0
90.0-109.0
1.0-9.0
54
HPLC-MS/MS
3.0
68.7-82.5
-
55
HPLC -FD
25.0
86.0-93.0
4.0-8.0
56
Fluorescence spectrophotometer
15.3
90.2- 97.3
2.3-4.1
This workss
Reference
24
51
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1.5-3.0
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A
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PT
ED
M
A
N
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SPE with sulfobetaine polymer resin SPE with polymeric and ion exchange cartridges Fluorescence probe based MIP@CDs
RSD (%)