Glucose assay based on a fluorescent multi-hydroxyl carbon dots reversible assembly with phenylboronic acid brush grafted magnetic nanoparticles

Journal Pre-proof Glucose Assay Based on a Fluorescent Multi-Hydroxyl Carbon Dots Reversible Assembly with Phenylboronic Acid Brush Grafted Magnetic Nanoparticles Ji Li, Xinjie Li, Rongqin Weng, Taotao Qiang, Xuechuan Wang

PII:

S0925-4005(19)31548-5

DOI:

https://doi.org/10.1016/j.snb.2019.127349

Reference:

SNB 127349

To appear in:

Sensors and Actuators: B. Chemical

Received Date:

19 June 2019

Revised Date:

27 September 2019

Accepted Date:

26 October 2019

Please cite this article as: Li J, Li X, Weng R, Qiang T, Wang X, Glucose Assay Based on a Fluorescent Multi-Hydroxyl Carbon Dots Reversible Assembly with Phenylboronic Acid Brush Grafted Magnetic Nanoparticles, Sensors and Actuators: B. Chemical (2019), doi: https://doi.org/10.1016/j.snb.2019.127349

This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. 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. © 2019 Published by Elsevier.

Glucose Assay Based on a Fluorescent Multi-Hydroxyl Carbon Dots Reversible Assembly with Phenylboronic Acid Brush Grafted Magnetic Nanoparticles Ji Li a, b, *, Xinjie Li a, Rongqin Weng a, Taotao Qiang a, b, Xuechuan Wang a, b, * a

College of Bioresources and Materials Engineering, Shaanxi University of Science & Technology, Xi’an 710021, PR China a

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National Demonstration Center for Experimental Light Chemistry Engineering Education, Shaanxi University of Science & Technology, Xi’an, Shaanxi 710021, PR China *

Corresponding author: College of Bioresources and Materials Engineering, Shaanxi University of Science & Technology, Xi’an 710021, PR China E-mail addresses: [email protected] (J. Li), [email protected] (X. Wang)

E-mail address: [email protected]

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The authors declare no competing financial interest.

A novel composite fluorescent probe was synthesized via assembling multi-hydroxyl carbon dots on phenylboronic acid molecular brush modified magnetic nanoparticles The composite fluorescence probe can be used for glucose detection, and the fluorescence intensity shows a enhance phenomenon with the increase of glucose concentration.

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*Corresponding Authors

ABSTRACT

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A novel composite fluorescent probe was synthesized by assembling multi-hydroxyl carbon dots (CDs) on magnetic nanoparticles modified with phenylboronic acid (PBA) molecular brush by a reversible dynamic covalent bond. Subsequently, the composite fluorescent probe was used for glucose determination, demonstrating that the so-prepared nanoparticles mask the fluorescence of the multi-hydroxyl CDs due to the light adsorption of the black magnetic nanoparticles. The fluorescence can be recovered in the presence of glucose in the solution due to the release of the multi-hydroxyl CDs. The fluorescence intensity of the released multi-hydroxyl CDs exhibits a relatively good linear relationship with the concentration of glucose. The results of the glucose assay show that the composite fluorescent probe possesses high sensitivity and a wide linear response range from 0.2 to 20 mM of glucose, confirming to be promising sensor glucose non1

enzymatic detection. 1. Introduction

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Diabetes mellitus is a disease caused by the defecting in insulin secretion, which leads to glucose accumulation. According to statistics, more than 170 million individuals suffered from diabetes mellitus in the year 2000 worldwide; furthermore, it has been estimated that 366 million people will be suffering from diabetes mellitus in 2030.[1] Therefore, it is urgent to design highly sensitive, selective, and reliable methods for glucose determination to be used in many fields, such as clinical diagnostics, biotechnology, and food industry.[2] So far, various methods and devices have been proposed for glucose assay, such as electrochemical method,[3-5] surface-enhanced Raman scattering,[6] chemiluminescence,[7] electrochemical transistor biosensor,[8] potentiometric biosensor,[9, 10] electrochemiluminescence biosensor,[11] and fluorescence biosensor.[12, 13] Among them, the latter has been recognized as a simple, fast, and convenient device to detect glucose.

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As novel 0-dimension materials with the diameter ranging from 1-10 nm, carbon dots (CDs) possess excellent photostability, good solubility in aqueous and resistance to photobleaching.[14] Comparing with other fluorescence materials, CDs have the advantages of low cost, easy operability, mild preparation condition, low toxicity, biocompatibility as well as a wide range of sources. [15, 16] Therefore, it has been widely used in optoelectronic devices,[17] photocatalysis,[18, 19] biomedical,[20] optical imaging [21, 22] and especially in analytical chemistry as a fluorescence probe.[23, 24] For example, Chi and co-workers [25] developed nitrogen-doped CDs for Cu(II) sensing by co-pyrolysis citric acid and branched poly(ethylenimine): the so-prepared CDs can detect Cu(II) in the substrate concentration from 10 to 1100 nM. Kansal and co-workers [26] employed a cost-effective and straightforward thermal pyrolysis technique to fabricate high-performance CDs using lemon juice for the 2,4,6-trinitrophenol determination. The result indicated that the obtained CDs showed good performance. However, the selectivity of CDs is not excellent, thus limiting their application. To improve the selectivity of CDs, specific ligands should be grafted on the surface of CDs, such as dopamine for Fe3+ determination, [27] aptamer for ochratoxin and aflatoxin B1.[28, 29] With excellent selectivity and low limit of detection, these functionalized CDs can be used in assays of as real samples.

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Since phenylboronic acid (PBA) can bind reversibly with cis-1,2- or cis-1,3-diols, forming five- or sixmembered cyclic, it presents a high affinity to carbohydrates, vitamins, and coenzymes.[30] Thus, PBA-modified CDs have been fabricated for non-enzymatic glucose detection.[31] Shi and coworkers[32] synthesized PBA-functionalized CDs that were used as a fluorescence probe for glucose detection. Via the microdialysis technique, they successfully monitored glucose in the striatum of rats. Haider and co-workers [33] developed a novel optical diffuser for glucose sensing by incorporating PBA in hydrogels. The hydrogel-assembled micro-lenses can collect light at different focal lengths and incident directions due to the volumetric expansion. Thus, the variation in glucose concentrations was easily tested by a physiological method that was used to measure spread and intensity of the transmitted light through the sensor. In this present work, a novel glucose composite probe with good sensitivity and selectivity was designed using multi-hydroxyl CDs that were assembled on PBA-molecular-brush-grafted magnetic nanoparticles (Fe3O4@APBA) via a reversible dynamic covalent bond to mask the fluorescence of 2

multi-hydroxyl CDs. When the glucose co-exists with the obtained composite probe, the multihydroxyl CDs dissociate from the magnetic nanoparticles, and the fluorescence signal can be recovered. This assay has the advantages of a wide linear range, low limit of detection, good selectivity, and relatively high sensitivity. Furthermore, these features render our probe suitable for further applications in the glucose determination and monitoring in real samples. 2. Experimental Section 2.1. Materials

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Glucose, 3-amino phenylboronic acid (APBA), FeCl3·6H2O, tetraethyl orthosilicate, γ(aminopropyl)triethoxysilane and anhydrous sodium acetate were obtained from Sigma-Aldrich. Ethylene glycol, N-hydroxysuccinimide (NHS), 2-bromoisobutyryl bromide (BiB), CuBr, ethylenediamine, 1-[3-(dime-thylamino)propyl]-3-ethylcarbodiimide hydrochloride, tris(hydroxylmethyl)aminomethane, and N,N,N′,N″,N″-pentamethyldiethylenetriamine, were purchased from Sinopharm Chemical Reagent Co., Ltd. The 2-bromo-2-methyl-N-(3-(triethoxysilyl) propanamide) was synthesized according to a previous report [34]. All chemicals used in the experiment were analytical grade. The water used in the whole experiment was reverse osmosis water.

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2.2. Equipment

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Ultraviolet-visible and fluorescence spectra were recorded by Cary 5000 spectrometer and FS5 fluorescence spectrophotometer, respectively. The morphology of multi-hydroxyl CDs, magnetic nanoparticles, and the composite probe was observed by the FEI Tecnai G2 F20 S-TWIN transmission electron microscope. The compositions and structure of the obtained materials were assessed by Kratos AXIS Ultra DLD X-ray photoelectron spectroscopy (XPS) and measured by VERTE70 Fourier-transform infrared spectrometry (FTIR). 2.3. Preparation of the Composite Probe

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Multi-hydroxyl CDs and Fe3O4@APBA were synthesized according to the previous reports [35-37] with modifications presented in supporting information. The Fe3O4@APBA/multi-hydroxyl CDs composite probe (referred as ‘composite probe’) was prepared as follows. The multi-hydroxyl CDs with a concentration of 2.5 μg·mL-1 and 5 mg·mL-1 of Fe3O4@APBA in 20 mM phosphate buffered saline (PBS, pH 7.8) were mixed and incubated in a shaker for 2 h at room temperature. Then, the composite probe was collected using a magnet, and rinsed at least three times with 20 mM of PBS (pH, 7.8). Finally, the composite probe was redispersed in 20 mM of PBS (pH, 7.8) and stored at 4 °C. 2.4. Procedure for Glucose Determination One mL of composite probe suspension (5 mg·mL-1) was mixed with a solution of glucose with a concentration of 0–100 mM (5 mL). Their fluorescence spectra were obtained using an FS5 fluorescence spectrophotometer. The calibration curves were fitted based on the relationship between the values of fluorescence intensity varying under maximum emission wavelength of CDs and the glucose concentration. To investigate the selectivity of the composite probe, other saccharides (such as galactose, fructose, xylose, ribose, lactose, maltose, sucrose, and mannose), ascorbic acid, acetaminophen, dopamine, and uric acid were considered as possible interference 3

substances. The fluorescence spectra were acquired at the excitation wavelength of 332 nm. Three parallel tests were conducted for all experiment data acquisition 2.5. Real Sample Assay

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We followed the procedure for treating blood samples described in the literature.[11] Firstly, the plasma in the blood samples was removed by centrifugation at 10,000 rpm for 10 min. Then, the upper supernatant was collected and mixed with 1.5-folds of acetonitrile. After vigorously shaking, the mixture was treated by centrifugation at 10,000 rpm for 10 min to remove the proteins in the serum. The PBS solution (pH 8.0) was added to adjust the pH value of the supernatant. Different glucose concentrations were spiked to the diluted serum samples. Finally, 100 μL of treated human serum was diluted for 20-folds by PBS (pH 8.0), followed by the addition of 1 mL composite probe suspension (5 mg·mL-1). The fluorescence spectra of each solution were recorded after 5 min shaking and external magnet separation. The glucose level of serum samples was calculated with the above-mentioned calibration curves. 3. Results and Discussion 3.1. Assay Strategy

Scheme 1.

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PBA-molecular brushes possess a high affinity to saccharides due to dynamic reversible covalent interactions. Thus, the materials conjugated with PBA polymer brushes have been used for glycoproteins sensor chips fabrication,[38] glucose detection,[39] and regulation of cell adhesion.[40] Here, we have prepared Fe3O4@APBA nanoparticles. The multi-hydroxyl CDs can be easily assembled on the Fe3O4@APBA by dynamic reversible covalent interactions, which lead to the formation of the CDs/Fe3O4@APBA composite probe and masking the fluorescence of multihydroxyl CDs. Subsequently, with the addition of glucose, which binds to the PBA-molecular brushes, multi-hydroxyl CDs are dissociated, re-activating the fluorescence signal that can be collected after being separated by an external magnetic field. The basic principle of the glucose assay is shown in Scheme 1.

3.2. Synthesis and Characterization of the Composite Probe

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Synthesis and Characterization of multi-hydroxyl CDs: The multi-hydroxyl CDs were prepared via the pyrolysis of citric acid and tris(hydroxymethyl)aminomethane at the same time (the route of the process is shown 1 in Scheme.2). The so-prepared multi-hydroxyl CDs were characterized via TEM, XPS, and FTIR to confirm their structure and properties. Fig.1A displays the TEM images of multi-hydroxyl CDs. It can be observed that the size of the multi-hydroxyl CDs is homogeneously distributed in the range from 1 to 3 nm, with the average size being 2.3 nm (see Fig.1B). This result suggests that such uniform particle size might significantly enhance the photoluminescence properties. Moreover, the characteristic fingers of the lattice can also be observed via a highresolution transmission electron microscope (HRTEM) (see inset of Fig.1A): the lattice spacing is of 0.21 nm corresponds to the lattice spacing of graphite (002 facet).[41] The chemical composition 4

of multi-hydroxyl CDs was identified via XPS and FTIR. As shown in Fig. 1C, the XPS spectrum shows that the multi-hydroxyl CDs are mainly composed of three elements: carbon (53.70%), oxygen (45.24%) and nitrogen (1.06%). The high-resolution XPS spectrum of O 1s demonstrates that the multi-hydroxyl CDs mainly contain C–O (529.08 eV), C=O (530.29 eV) and –OH (531.43 eV) functional groups (as shown in the inset of Fig. 1C). The atomic percentage of various oxidation states of oxygen are 2.61%, 71.56%, and 25.82%, respectively. The FTIR spectrum in Fig. 1D further confirms the presence of C=O (1660 cm-1), O–H (3395 cm-1), and N–H (1571 cm-1) functional groups. These results indicate that –OH functional groups abound on the surface of the so-prepared CDs.

Scheme 2.

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Fig.1.

Fig.2.

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The optical properties of the multi-hydroxyl CDs were characterized by their optical absorption and fluorescence spectra. The solution of multi-hydroxyl CDs is light-yellow under white light and displays a bright blue fluorescence under the emission of 365 nm (inset of Fig.2). Moreover, the multi-hydroxyl CDs show strong absorption in the UV region (such as at 236 nm), which can be attributed to the π-π* transitions of the aromatic structure of multi-hydroxyl CDs. The absorption peak at 332 nm is associated with the trapping of excited state energy by the surface states, resulting in strong fluorescence emission.[42] Indeed, the fluorescence emission spectrum with the maximum wavelength being 407 nm shows that the multi-hydroxyl CDs can emit strong fluoresce under 332 nm excitation. To obtain multi-hydroxyl CDs with excellent optical properties, the following reaction conditions have been optimized: molar ratio of citric acid and tris(hydroxymethyl)aminomethane, reaction temperature, and reaction time (see Fig.1S). The results show that the optimal molar ratio of reactants is 1:1. Furthermore, the reaction temperature and time are also crucial for synthesis of multi-hydroxyl CDs: under the optimized reaction condition, the fluorescence quantum yield of the obtained multi-hydroxyl CDs increases to 58.7 % using quinine sulfate as the reference.

Based on the above-proposed detection mechanism of the composite probe for glucose, the soprepared multi-hydroxyl CDs, as the chromophore, should have good optical stability. It means that multi-hydroxyl CDs are relatively inert toward glucose (i.e., its fluorescence cannot be quenched when the presence of glucose). Therefore, we carried out the quenching experiment of the multihydroxyl CDs by choosing 10, 100 and 500 mM of glucose, respectively. The results are shown in Fig.S2. It can be seen that almost no decrease in the fluorescence intensity of the multi-hydroxyl CDs when the concentration of glucose is below 100mM. Under the relatively high glucose concentration (such as 500 mM), the fluorescence intensity of the multi-hydroxyl CDs is reduced, 5

and its (F-F0)/F0 value is 8.5%. We can conclude that multi-hydroxyl CDs possess high optical stability for glucose detection. Synthesis and Characterization of PBA grafted magnetic nanoparticles: The PBA-molecularbrushes grafted with magnetic nanoparticles were synthesized via the following a versatile procedure. Firstly, the SiO2-covered Fe3O4 nanoparticles were prepared according to a previously reported work.[37] Then, the initiator of atom transfer radical polymerization (ATRP) was modified on the surface of Fe3O4@SiO2. Subsequently, sodium methacrylate (NaMA) was polymerized on the surface of Fe3O4@SiO2 to form poly(methacrylic acid) (PMAA) brushes via surface-initiated ATRP.[43] Finally, the PBA groups were anchored through an amide reaction between APBA and PMAA brushes (the route of the process is shown 2 in Scheme.2).

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Fig.3.

The morphology of magnetic nanoparticles was observed via TEM. The average diameter of

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obtained magnetic Fe3O4 nanoparticles is about ⁓160 nm (Fig.3A). The analysis of the selectedarea electron diffraction shows that the magnetic nanoparticles display a polycrystalline structure (inset of Fig.3A). In Fig. 3B, the Fe3O4@SiO2 nanoparticles present a core-shell structure without free SiO2, indicating that the silica layer has been successfully and evenly coated on the surface of

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Fe3O4. The average thickness of the coating is ⁓20 nm. It can be seen from Fig.3C that there is no evident change in the morphology of Fe3O4@SiO2 modified via ATRP. To confirm the chemical structure of the Fe3O4@BiB, XPS and EDS mapping analyses were performed on the surface (Fig.S3 and S3). EDS mapping analysis shows that Fe, O, C, N, and Br elements are evenly distributed across the Fe3O4@SiO2 surface with the content being 9.45 % (Si), 24.15 % (O), 65.03 % (C), 1.29 % (N), and 0.08 % (Br), respectively. The high-resolution XPS spectrum of Br 3d (Fig.S4) shows that the Br and C atoms exist on the Fe3O4@SiO2 surface in a single bond, which is consistent with previously published results.[36]

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To introduce PBA-molecular-brushes on the surface of magnetic nanoparticles, we first used NaMA as a monomer to produce PMAA molecular brushes by surface-initiated ATRP (the carboxyl group of MAA can coordinate with cuprous ions, which hinders the ATRP process). Then, APBA was immobilized to form magnetic nanoparticles containing PBA-molecular-brushes by amide reaction. The chemical composition of the so-prepared Fe3O4@PMAA is shown in Fig.S3. The peak at 577 cm-1 in the FTIR spectrum of Fe3O4 is attributed to Fe-O characteristic absorption peak.[44] After coating the SiO2, the characteristic peaks of Si-O appears at 1100 cm-1. Although no additional absorption peaks appear after the ATRP has been initiator anchored, the results of Fe3O4@BiB of XPS and EDS mapping analyses can be confirmed that the BiB has been successfully modified on the surface of Fe3O4@SiO2 (Fig.S3C-H). The FTIR spectra of Fe3O4@PMAA show that the characteristic absorption peak of the carboxyl group appears at 1740 cm-1, indicating that PMAA molecular brushes are formed on the surface of Fe3O4 by ATRP process. The length and grafting density of the PBA-molecular-brushes can significantly influence the adsorption capacities of the multi-hydroxyl substances and saccharides. To obtain Fe3O4@APBA with excellent binding property towards multi-hydroxyl CDs, the polymerization conditions of 6

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NaMA were optimized. Fig.S5 reports the thermal analysis curve of the magnetic nanoparticles prepared with different concentrations of NaMA. As it can be seen in Fig.S5, the weight loss found from thermogravimetric curve of Fe3O4@PMAA with the temperature varying from room temperature to 105 °C might be caused by the water bonded to SiO2, whereas the weight loss in the 105-300 °C curve can be attributed to the weightlessness of the water bonded to PMAA. A weight loss over 300 °C is caused by the PMAA decomposition. In the preparation process of Fe3O4@PMAA, we changed the concentration of NaMA from 10, 20, 50, 100 to 150 mM during the ATRP. With the concentration increase of NaMA, the weight loss of Fe3O4@PMAA in 360-490 °C gradually increases, revealing that the grafting density of PMAA on the surface of Fe3O4 has increased. Then, the Fe3O4@PMAA reacts with an excess of APBA, namely Fe3O4@APBA-10, Fe3O4@APBA-20, Fe3O4@APBA-50, Fe3O4@APBA-100, and Fe3O4@APBA-150. The adsorption experiments were conducted to evaluate the assembly properties of the above PBA molecular brush grafted magnetic nanoparticles with multi-hydroxyl CDs. The fluorescence spectra of the multi-hydroxyl CDs after adsorption are reported in Fig.S6, where it can be seen that the fluorescence intensity of the multi-hydroxyl CDs has first decreased and then increased with increasing grafting density of the PBA molecular brush on the surface of magnetic nanoparticles. The Fe3O4@APBA-20 spectrum displays the best adsorption capacity for the multi-hydroxyl CDs. These results may be due to the low grafting density that provides a reduced number of binding sites for multi-hydroxyl CDs. However, much higher grafting density values produce a large steric resistance effect, which hinders the multi-hydroxyl CDs entering the inner of the molecular brush. Thus, it can be concluded that Fe3O4@APBA-20 can be used for subsequent studies.

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The morphology of Fe3O4@PMAA (with NaMA used in the preparation process at a concentration of 20 mM) was observed via TEM (Fig.3D). TEM analysis shows that the average thickness of PMAA

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molecular brushes is ⁓10 nm and, thus, can provide carboxyl groups to facilitate the immobilization of APBA. It can be seen from Fig.3E that the functional layer thickness of the Fe3O4@APBA-20 has not increased after the APBA modification. As it is shown in the XPS analysis (inset of the Fig.S4), the characteristic peaks of B 1s appear on the surface of Fe3O4@APBA-20 at 196.3eV, indicating that the molecular brushes containing PBA have been formed on the surface of the magnetic nanoparticles. 3.3. Preparation of the Composite Probe

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Effect on pH: Fe3O4@APBA/multi-hydroxyl CDs composite probe was prepared by creating a dynamic reversible covalent bond between PBA-molecular-brushes on the surface of Fe3O4@APBA-20 and cis-diol of multi-hydroxyl CDs (the route of the process is shown 3 in Scheme.2). First, the pH value of the binding media was investigated. It can be seen in Fig.4 that the maximum emission peak of multi-hydroxyl CDs shifts gradually to short-wavelength (blue shift) when the pH increases from 3 to 6. When the pH is above 6, the maximum emission peaks of multihydroxyl CDs remains mostly unchanged: this may be due to the protonation of -COOH (the pKa of citric acid is ⁓3-6), which increases the energy gap between the valence and conduction bands of multi-hydroxyl CDs. We also investigated the correlation between the fluorescence intensity corresponding to the maximum emission wavelength and the pH; the fluorescence intensity increase with the increase of pH. Indeed, when the pH is between 6 and 8, the variation of the fluorescence intensity is negligible (Fig.4 inset), indicating that multi-hydroxyl CDs exhibits a high sensitivity at a high pH level. 7

Fig.4.

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The principle of detection of the composite probe was based on the absorption of the glucose and the simultaneous release of multi-hydroxyl CDs. To increase the sensitivity, it is necessary to load the multi-hydroxyl CDs on Fe3O4@APBA-20 as much as possible: Therefore, the methods employed to improve the loading capacity of Fe3O4@APBA-20 for multi-hydroxyl CDs is critical. The surface of Fe3O4@APBA-20 contains a large number of PBA groups. It is well known that the affinity between PBA groups and cis-diol has a pH dependence. In acidic conditions, the PBA groups and cis-diol mostly generate hydrogen bonds. However, the weak hydrogen bonds can easily be broken by the proton solvents, such as water. Under alkaline conditions, PBA groups turn into tetrahedron structure with a negative charge, leading to the formation of stable reversible dynamic covalent bonds with cis-diol,[30] indicating that alkaline conditions improve the loading capacity of Fe3O4@APBA-20 toward the multi-hydroxyl CDs. Based on the properties of PBA and multi-hydroxyl CDs, we examined the loading capacity of Fe3O4@APBA-20 under pH values ranging from 6 to 8 (Fig.4B). The fluorescence of the multi-hydroxyl CDs has been quenched to varying degrees with the addition of the Fe3O4@APBA-20 in an alkaline medium. Generally, a high fluorescence quenching corresponds to a high loading capacity of Fe3O4@APBA-20 toward multi-hydroxyl CDs. The changing trend of the fluorescence intensity is shown in the inset of Fig.4B, where it can be seen that the fluorescence intensity of the multi-hydroxyl CDs increases with the increase of pH, indicating that the loading capacity of Fe3O4@APBA-20 toward multi-hydroxyl CDs increases gradually. However, Fe3O4@APBA-20 might undergo hydrolysis, which would lead to the abscission of PBA molecular brush when the pH is higher than 9.0. Therefore, pH 8.0 has been selected for the preparation of Fe3O4@APBA/multi-hydroxyl CDs composite probe.

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Characterization of the Composite Probe: Under the optimal conditions, the morphology of Fe3O4@APBA/multi-hydroxyl CDs composite probe was characterized by HRTEM (see Fig.5). A layer of amorphous SiO2 and PBA-molecular-brushes was grafted on the surface of Fe3O4 nanoparticles

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with a thickness of ⁓30 nm via the sol-gel process and surface-initiated polymerization, resulting in the magnetic core-shell Fe3O4@APBA-20 (Fig.5A). In the magnified image in Fig.5B, the shell surface appears more uniform, and no obvious lattice fringes can be found. After loading the multihydroxyl CDs onto Fe3O4@APBA-20, the core-shell structure has not changed significantly (Fig.5C). The TEM images of Fe3O4@APBA/multi-hydroxyl CDs composite probe confirmed that small nanoparticles had been incorporated into the shell of the Fe3O4@APBA-20. Their size and lattice fringe spacing are similar to those of the prepared multi-hydroxyl CDs (see Fig.5D and inset), indicating that multi-hydroxyl CDs have successfully loaded into the shell of Fe3O4@APBA-20. When the so-prepared composite probe contacted with glucose solution, the small nanoparticles in the shell was disappeared (see Fig.5E and F), demonstrating that multi-hydroxyl CDs were displaced by glucose from the surface of Fe3O4@APBA-20 due to the dynamic reversible covalent 8

bonds between PBA and multi-hydroxyl groups. 3.4. Assay Performance 3.4.1 Interference Tests

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The availability of the composite probe towards glucose was investigated using other saccharides (such as galactose, fructose, xylose, ribose, lactose, maltose, sucrose, and mannose), and coexisting compounds (ascorbic acid, acetaminophen, dopamine, and uric acid) with glucose in human blood serum under the optimal conditions. Typically, PBA has a high affinity to cis-diol species including all saccharides and, thus glucose. Therefore, the selective test was performed with addition 0.4 mM of glucose and the same concentration of interfering species. Fig. 6A shows the change of fluorescence intensity ((F-F0)/F0, where F and F0 represent the fluorescence intensity of multi-hydroxyl CDs before and after adding the tested substance, respectively) of composite probe for glucose and interfering species, respectively. As seen in Fig. 6A, the (F-F0)/F0 of glucose is significantly higher than that of the interfering species, suggesting that the composite probe exhibits good sensitivity toward glucose. Considering that the glucose level in human blood is at least 30 times higher than that of these interfering species,[2] the composite probe was used to detect glucose (concentration of 0.4 mM) in the presence of interfering species (each at a

3.4.2 Detection of Glucose

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concentration of 0.04 mM). The (F-F0)/F0 of glucose is only 1.9010-2, while the glucose/interfering species binary mixture range from 1.9110-2 to 1.9410-2 (Fig.6B). The overall (F-F0)/F0 has increased by approximately 1.0-2.0% compared to that of pure glucose. Thus, this novel composite probe is a promising candidate for glucose detection in human blood samples.

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The composite probe was used to detect glucose under the optimal conditions with the varying concentration of the glucose solution for 2 min. The result shows that the fluorescence intensity of the composite probe increases significantly with the increase in glucose concentration. The relationship between glucose concentration and (F-F0)/F0 of composition probe, and the results of the linear fitting are shown in the inset of Fig.7. (F-F0)/F0 has increased significantly in a relatively low range of glucose concentrations and then increased slowly until the concentration is higher than 20.0 mM. Furthermore, the (F-F0)/F0 exhibits a good linear relationship with the glucose concentration in the range from 0.2 to 20 mM. This result can also be derived from the adsorption response curve of the composite probe for different concentrations of glucose (results are shown in Fig.S7). The corresponding linear regression equation is (F-F0)/F0=0.27926Cglucose+0.09395 (Eq. 1) with a high correlation coefficient of 0.9998 (n=5). The limit of detection reduced by the 3δ/S (with δ being the standard deviation of the lowest signal and S the slope of linear regression equation) is 0.15 μM. Compared with the previously reported glucose probes (see Table 1), our composite probe exhibits higher sensitivity and selectivity, which render it a promising tool for glucose detection.

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Table 1 Fig.7.

3.5 Glucose Detection in Real Samples

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We also assessed the suitability of this novel composite probe to assay glucose in human blood samples. Recovery testing was performed to verify the validity of the established method. All blood samples used in the experiment were kindly provided by the Second Hospital Affiliated of Xi’an Medical University (Xi’an, China). The procedure of blood sample treatment was carried out according to literature.[11] The (F-F0)/F0 values of treated serum and spiked serum samples were measured via the composite probe (fluorescence spectra see Fig.8). Then, the glucose levels in the blood samples were estimated via Eq.1. We compared the test results with the results of the hospital analyses (obtained with an Olympus AU2700 automated biochemistry analyzer). As shown in Table 2, the values of glucose determined by the composite probe tested are consistent with the hospital’s results. The average recoveries of glucose are between 96% and 99%, while the relative standard deviation is lower than 2%. These results indicate that the composite probe used for glucose assay possesses good accuracy and, can be used as a fluorescence probe for glucose determination in blood samples or any other complex sample.

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Table 2

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4. Conclusion

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In conclusion, a new sensitive and selective composite fluorescence probe for glucose detection was designed. Multi-hydroxyl CDs were prepared via a one-step pyrolysis of citric acid and tris(hydroxylmethyl)aminomethane. The multi-hydroxyl CDs show good optical properties and have a fluorescence quantum yield up to 58.7%. Then, the multi-hydroxyl CDs were assembled to the PBA-molecular-brushes-grafted magnetic nanoparticles via dynamic reversible covalent bonding and formed the Fe3O4@APBA/multi-hydroxyl CDs composite probe, resulting in multihydroxyl CDs fluorescence masking. The reversible dynamic covalent bond between glucose and the PBA-molecular-brushes caused the fluorescence signal recovery due to the release of the multi-hydroxyl CDs. We found that the intensity of the recovered fluorescence is proportional to the concentration of the glucose in the samples. The proposed composite possesses a wide linear range (0.2-20.0 mM) and low limit of detection (0.15 μM). The composite probe displayed an outstanding accuracy in real human blood samples. Thus, it can be successfully applied in different fields including biosensors, biomedical diagnosis, and environmental monitoring.

Declaration of interests 10

☒ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

☐The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:

Acknowledgments

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This work was financially supported by the National Natural Science Foundation of China (Grants 21808134, 51672022, 51302010, and 51671003) and the start-up funding from Shaanxi University of Science & Technology.

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Ji Li received his B.S. degree (2008) and M.S. (2011) in Applied Chemistry at Xi’an University of Architecture and Technology. He obtained his Ph.D. (2016) in Polymer Chemistry and Physics at Northwestern Polytechnical University. After the Ph.D. study, he began his independent career as a faculty member at Shaanxi University of Science & Technology (2016–present). His research interests involve design and synthesis of molecularly

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imprinted polymers, bio-based carbon dots, 2D materials and sensors for biomolecules in aqueous.

Figure captions Scheme 1. Glucose assay strategy

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Scheme 2. The synthesis route of multi-hydroxyl CDs (1), Fe3O4@APBA (2), and the composite probe (3).

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Fig.1. TEM images of multi-hydroxyl CDs (A), inset: corresponding HRTEM images; the sizes distributions curves of multi-hydroxyl CDs (B); and XPS of multi-hydroxyl CDs (C), inset: corresponding high resolution of O 1s. FTIR spectrum of multi-hydroxyl CDs (D).

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Fig.2. Fluorescence (dotted lines) and UV/Vis absorption spectra (solid lines) of the multi-hydroxyl CDs. Inset: Digital photograph of the multi-hydroxyl CDs under irradiation of white light (left) and 365 nm of light (right).

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Fig.3. TEM images of Fe3O4 (A), inset: corresponding selected-area electron diffraction pattern on a separated magnetite particle; TEM images of Fe3O4@SiO2 (B), Fe3O4@BIB (C), Fe3O4@PMAA (D) and Fe3O4@APBA (E).

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Fig.4. Fluorescence spectra of multi-hydroxyl CDs at different pH (A); Fluorescence spectra of the composite at different pH (B).

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Fig.5. TEM images of Fe3O4@APBA-20 (A), (B); TEM images of the composite probe (C), (D), (inset is the TEM image of multi-hydroxyl CDs in PMAA-20); TEM images of composite probe after contact glucose (E), (F). Fig.6. Availability of the Fe3O4@APBA/multi-hydroxyl CDs composite probe (5 mg·mL-1) for glucose and interference species separately (A), and binary mixture of glucose/interference species (B) in pH 8 PBS solution. Fig.7. Fluorescence response of composite probe (5 mg·mL-1) upon addition of various concentrations of glucose (from bottom to top, 0, 0.2, 0.4, 0.6, 0.8, 1.0, 2.0, 4.0, 6.0, 8.0, 10.0, 20.0, 40.0, 60.0, 80.0, and 100 mM) in a pH 8 PBS solution. Inset: semilogarithmic plot of (F-F0)/F0 of composite probe vs the concentration of glucose. 15

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Fig.8. Fluorescence spectra of the composite probe (5 mg·mL-1) and human serum (inset).

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Table captions Table 1 Comparison of the performance of the composite probe with that of reported nonenzymatic glucose sensors.

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Table 2 Results of glucose determination in human blood samples.

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Table 1 Linear range (mM)

Detection limit (μM)

Diamond nanoparticles [5]

0.05-0.7

15.0

NiCo2O4@PANI [45]

0.02-4.7

0.4

CdTe QDs/Au NPs [11]

0.01-10.0

5.3

CdSe QDs/Boronic acids [46]

2.0-20.0

100.0

This work

0.2-20.0

0.15

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Probe

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Table 2 Hospital method

Proposed method

Added

Total founded

Recovery

RSD

founded (mM)

founded (mM)

(mM)

(mM)

(%)

(%, n=3)

1

4.03

4.13

2.00

6.06

96.2

1.36

2

5.10

5.05

2.00

6.92

98.3

1.72

3

4.26

4.19

2.00

6.22

99.2

1.59

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Sample

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