Core-shell HA-AuNPs@SiNPs nanoprobe for sensitive fluorescence

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Core-shell HA-AuNPs@SiNPs nanoprobe for sensitive fluorescence hyaluronidase detection and cell imaging Jia Ge, Ren Cai, Lu Yang, Liangliang Zhang, Ying Jiang, Yu Yang, Cheng Cui, Shuo Wan, Xia Chu, and Weihong Tan ACS Sustainable Chem. Eng., Just Accepted Manuscript • DOI: 10.1021/ acssuschemeng.8b03684 • Publication Date (Web): 31 Oct 2018 Downloaded from http://pubs.acs.org on October 31, 2018

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ACS Sustainable Chemistry & Engineering

Core-shell HA-AuNPs@SiNPs nanoprobe for sensitive fluorescence hyaluronidase detection and cell imaging Jia Ge, †,‡,§ Ren Cai, ‡,§Lu Yang, ‡ Liangliang Zhang, ‡,⊥ Ying Jiang, ‡ Yu Yang, ‡ Cheng Cui, ‡ Shuo Wan, ‡ Xia Chu,*,‖ Weihong Tan*,‡,‖ †

College of Chemistry and Molecular Engineering, Zhengzhou University, 101 Kexue

Road, Zhengzhou, 450001, China. ‡

Center for Research at Bio/Nano Interface, Department of Chemistry and Department of

Physiology and Functional Genomics, Shands Cancer Center, UF Genetics Institute and McKnight Brain Institute, University of Florida, Gainesville, Florida 32611-7200, United States. ‖

State Key Laboratory for Chemo/Bio-Sensing and Chemometrics, and College of

Chemistry and Chemical Engineering, Hunan University, 2 Lushan Road, Changsha, 410082, China. ⊥

State Key Laboratory for the Chemistry and Molecular Engineering of Medicinal

Resources, School of Chemistry and Pharmaceutical Sciences, Guangxi Normal University, 15 Yucai Road, Guilin, 541004, China.

* Corresponding authors. E-mail address: [email protected], [email protected].

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ABSTRACT A highly sensitive and selective fluorescence strategy for hyaluronidase assay was developed by coupling silicon nanoparticles (SiNPs) as fluorescence indicator with hyaluronic acid-functionalized gold nanoparticles (HA-AuNPs) as quencher. With the formation of a core-shell HA-AuNPs@SiNPs nanoprobe by self assembly, the fluorescence of SiNPs could be quenched by HA-AuNPs through fluorescence resonance energy transfer (FRET). Addition of hyaluronidase leads to cleavage of HA into small fragments and SiNPs are released. As a result, an immediately fluorescent recovery appears when HAase is introduced. The proposed fluorescence method showed a linear response ranged for HAase from 0.01 to 10 U/mL, and a detection limit of 0.004 U/mL (S/N=3) was obtained. Furthermore, it can be employed as an enzyme activatable nanoprobe for cell imaging applied in diagnostic and related biological studies.

Keywods: Silicon nanoparticles; Fluorescence sensing; Hyaluronidase detection; Gold nanoparticles; Live-cell imaging


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INTRODUCTION Hyaluronic acid (HA) is a macromolecule of polysaccharide with a repeating Dglucuronic acid and N-acetyl-D-glucosamine.1,2 HA directly regulate several important biological processes, such as cell adhesion, migration, and proliferation. Hyaluronidase (HAase) is an enzyme that degrades HA into the small fragments.3 HAase is related with a wide range of physiological and pathological processes that include fertilization, embryogenesis, inflammation, and tumor growth.3,4 Recently, increasing evidence has shown that HAase is overexpressed in many cancers, including colon, bladder, prostate, and so on.

5–7

Accordingly, it is extremely important to develop facile and sensitive

methods to detect HAase, which would be crucial importance for clinical diagnosis and early therapy. Various methods for HAase detection have been developed, such as turbidimetric,8 viscometric,9

zymographic,10

colorimetric,11,12

spectrophotometric,13

and

immunoassays,14 and plate assays.15-18 Among the strategies for HAase sensing, these methods are often time-consuming, require expensive equipment, and lack the selectivity required for practical applications. Thus, it is necessary to exploit more simple and highly sensitive assay to detect HAase. Taking the advantage of good sensitivity, real-time response, and operational simplicity, fluorescence methods are becoming useful analytical techniques in the field of bioanalysis and bioimaging.19,20 Some fluorescent method have recently been reported for the detection of HAase by fluorophore-labeled HA,21,22 organic dyes,23,24 or upconversion nanoparticles.25 Although these fluorescence methods are more sensitive than other previous traditional methods, their shortcomings, such as gold nanoparticles are not stable in high salt biological environments, and


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fluorophore or biotin-labeled HA might affect the activity of HAase. Hence, the development of novel fluorescent nanoprobes with high sensitivity, facile operation, and good biocompatibility has become increasingly important and urgent. Recently, nanomaterial-based probes has attracted lots of attention for bioimaging, biological, and biomedical applications based on their inherent advantages, such as simplicity, sensitivity, and good photostability.

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In the past few years, quantum dots

have become optical sensing nanomaterials commonly applied in bioimaging, biological labeling, and biomedical research owing to their high quantum yields, good photostability, and size-tunable emission. 31 However, serious toxicity of quantum dots and the need for organic solvent to observe their superior photophysical features have restricted their further analytical application for biological fields.

32,33

Thus, researchers are developing

methods to make quantum dots water-soluble and biocompatible. Si nanostructures are one of the most favored and important materials in biomedicine and nanotechnology.34-38 In particular, fluorescent silicon nanoparticles (SiNPs) are attracting interest for a variety of optical applications because of the abundant supply of silicon, controllable surface modification, high fluorescence, robust photostability, and good biocompatibility.39-42 Despite the significant progress in the design new kinds of fluorescent SiNPs sensors featuring outstanding optical properties and low toxicity, the detection of various analytes using SiNPs is still very limited. To the best of our knowledge, the exploration of SiNPs for monitoring targeted species in living cells or biological applications still remains at a very early stage. Herein, we propose a novel fluorescent method for HAase detection based on SiNPs as fluorescent probes and HA-functionalized gold nanoparticles (HA-AuNPs) as


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quenchers. As shown in Scheme 1, SiNPs were prepared by a facile method using N-[3(trimethoxysilyl) propyl] ethylenediamine (DAMO) as the silicon source and sodium ascorbate as the reduction reagent, respectively. HA could be used as a stabilizer for the synthesis of AuNPs. With the formation of the core-shell HA-AuNPs@SiNPs nanoprobe by self-assembly of positive SiNPs and negative HA-AuNPs via electrostatic adsorption, the fluorescence of SiNPs is quenched by HA-AuNPs through FRET. With the introduce of HAase, HA is degraded into small fragments, which leads to the release of SiNPs and the aggregation of AuNPs without protection from HA, resulting in the recovery of quenched SiNPs fluorescence. This novel assay has three major advantages. First, the preparation procedure of SiNPs is simple, and the photostability and biocompatibility are good. Second, HAase can be detected high selectively and sensitivity. Third, a cell imaging assay can be used for the detection of intracellular HAase activity.

EXPERIMENTAL Preparation of the SiNPs. SiNPs were prepared via a microwave irradiation method. In brief, 0.11 g of sodium ascorbate was added into Ar-saturated glycerol solution (3.2 mL). The mixture was stirred for 20 min until a homogeneous solution was formed. Then, DAMO (0.8 mL) was slowly added into the above mixture, and the resulting suspension kept stirring for 10 min. Thereafter, the resultant precursor was transferred into a vitreous vessel with a volume of 10 mL. After microwave irradiation at 160 °C for 30 min, the resultant SiNPs were removed when the temperature cooled to 25 °C naturally, and a light brown solution was obtained, indicating that the SiNPs had been synthesized. Finally, the as-prepared SiNPs were purified by a combination of centrifugation at 10000


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rpm for 5 min and an ultrafiltration centrifuge tube (MWCO: 10 kDa) to remove the excess residual reagents. The purified SiNPs was diluted 10 times for further use. Synthesis of HA-AuNPs. The HA-AuNPs were prepared by a modified method described previously.22 First, HAuCl4 (1.2 mL, 40 mg/mL) and HA sodium salt solution (25 mL, 0.1 mg/mL) were mixed and stirred for 3 min. Then, NaBH4 (1 mL, 4 mg/mL) was added into the mixture, and the stock solution gradually changed from yellow to redpurple, indicating HA-AuNPs had been synthesized. At last, the as-prepared HA-AuNPs were purified with a 10 kDa ultrafiltration centrifuge tube. HAase Measurement. In a typical experiment for HAase detection, 20 μL of HAase with different concentrations, 10 μL of SiNPs (0.2 mg/mL), and 20 μL of HA-AuNPs were successively added into 150 μL of phosphate buffer (PB, 10 mM, pH 7.0). The mixture was incubated at 37 °C for 30 min, and the fluorescence emission spectrum of the mixture was recorded for quantitative analysis. To evaluate selectivity, different competing species, including thrombin, BSA, KCl, NaCl, lysozyme, Cys, ALP, glucose, trypsin, and GSH, were employed. Details and the further experimental measurements can be found in the Supporting Information, including HAase activity detection in biological fluid samples, cellular toxicity test, and fluorescence microscopy imaging.

RESULTS AND DISCUSSION Characterization of the SiNPs. The TEM image showed that the as-prepared SiNPs was uniform with well monodispersity (Figure 1A). And, the size distribution of asprepared SiNPs showed an average diameter of 2.8±1.4 nm (Figure 1B). Moreover, the X-ray diffraction analysis confirmed that SiNPs were in the amorphous phase (Figure 1C).


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The FT-IR spectra showed the surface functional groups of the as-prepared SiNPs (Figure 1D). The presence of Si-O-Si was confirmed by the peaks located at 1043 cm-1 (Figure 1D). The peak at 2927 cm-1 was attributed to O-H bond stretching vibration, the peak at 1655 cm-1 showed the N-H bond bending vibration, and the peak at 3415 cm-1 was attributed to the vibrational absorption band of N-H (Figure 1D). Results indicated that the surface of SiNPs are coated with hydroxyl groups and amino groups, implying the excellent aqueous solubility. Furthermore, XPS measurements were performed to confirm the composition analysis of as-prepared SiNPs (Figure 2). The XPS spectra revealed that the as-prepared SiNPs were mainly composed of carbon, nitrogen, oxygen, and silicon. As shown in Figure 2, the XPS survey reveals that SiNPs were mainly composed of Si, C, N, and O elements, corresponding to peaks at 99.3 (Si 2p), 150.08 (Si 1s), 282.05 (C 1s), 395.95 (N 1s), and 528.95 eV (O 1s). The high resolution C 1s analysis revealed four types of carbon bond, which were attributed to C-Si (281.40 eV), C-C (282.24 eV), C-N/C-O (283.01 eV), and C=O (284.85 eV), respectively. The N 1s signal could be fit to three peaks at 395.79, 396.54 and 397.18 eV, which were assigned to N-Si, C-N-C and N-H, respectively. Therefore, the XPS spectra demonstrated the surface of as-prepared SiNPs to be rich in amino groups, which may contribute to the unique properties of SiNPs. The O 1s spectrum could be decomposed into three peaks at 527.81, 528.93, and 529.6 eV, indicating the presence of C=O, C-O-C, and Si-O, respectively. The three peaks at 98.90, 99.26, and 99.71 eV in the Si 2p spectrum were attributed to Si-C, Si-N ,and Si-O groups, respectively. The results of XPS spectra were consistent with the FT-IR spectra.


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Optical properties of the SiNPs. The UV−vis absorption peak of as-prepared SiNPs at 368 nm that could be attributed to the n → π* transition of C=O and C=N(Figure 3A, curve a). A high fluorescence intensity of SiNPs was obtained with excitation and emission wavelengths at 370 nm (Figure 3A, curve c) and 440 nm (Figure 3A, curve d), respectively. Using quinine sulfate as a reference, the QY of SiNPs in water at an excitation wavelength of 370 nm was found to be 6.8%. Under UV radiation, a blue luminescence was visible with the naked eye upon excitation at 365 nm, and a strong fluorescence signal was observed at 440 nm (inset in Figure 3A). Meanwhile, when the excitation wavelength gradually shifted from 320 to 385 nm as the emission wavelength changed from 450 to 455 nm, and the SiNPs exhibited a little excitation wavelengthdependent emission property (Figure 3B). It was differ from the apparent excitation wavelength-dependent emission property of carbon quantum dots.37 The optical property indicated that the obtained SiNPs might only contain a single-photon emitter, which could be favorable for the application of SiNPs in quantitative assays and cell imaging. Furthermore, the fluorescence intensity of the SiNPs was relatively stable over a wide range of pH values ranging from 4.0 to 9.0 (Figure S1). SiNPs were also very stable in salt solutions (Figure S2), which is an advantage for using SiNPs in high-ionic strength conditions. Additionally, SiNPs showed excellent photostability, even after continuous excitation with a UV lamp for 60 min (Figure S3). All the properties make SiNPs can be suitable for in vivo imaging or other biomedical applications. Formation and Characterization of the core-shell HA-AuNPs@SiNPs nanoprobe. TEM analysis showed that the size of the as-prepared HA-AuNPs showed an average of 22 nm (Figures 4A). The as-prepared HA-AuNPs exhibited a specific absorption peak at


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521 nm (Figure 3A, curve b). HA-AuNPs have a ζ-potential of -24.2 mV, facilitating their assembly with SiNPs, which have a positive potential of 1.32 mV (Figure S4). The zeta potential of core-shell HA-AuNPs@SiNPs was measured as -6.6 mV, indicating that the positively charged SiNPs could be electrostatically absorbed on negatively charged AuNPs (Figure S4). The formation of the core-shell structured HA-AuNPs@SiNPs was proved using TEM. As shown in Figure 4B, it could be seen that SiNPs were closely held around HA-AuNPs by electrostatic attraction, indicating formation by self-assembly. The EDX analysis shows that the HA-AuNPs@SiNPs are composed of Si, Au, C, and O (Figure S5), which could be consistent with the XPS results. Monitoring of the HAase. To evaluate the performance of our strategy for HAase detection, fluorescence emission spectra under different conditions were investigated. As shown in Figure 5, SiNPs exhibit strong fluorescence emission at 440 nm under 370 nm excitation (Figure 5, curve a). In contrast, the formation of core-shell HA-AuNPs@SiNPs nanoprobe leads to fluorescence quenching because electrostatic interaction could shorten the distance between the donors (SiNPs) and the acceptors (HA-AuNPs) (Figure 5, curve c). Meanwhile, the absorption band of HA-AuNPs significantly overlays the emission band of SiNPs, indicating the fluorescence quenching may be due to FRET (Figure 3A). With introduce of HAase, HA is enzymatically degraded into small fragments, and the aggregation of AuNPs was confirmed by the red-shifted UV-vis absorption (Figure S6). Significant recovery of fluorescence observed in the presence of HAase can be attributed to the specific cleavage of HA by HAase and the release of SiNPs (Figure 5, curve b). These experimental data indicate that the proposed assay can be used to determine the concentration of HAase.


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Optimization of sensing conditions. In order to obtain the best sensing performance of the proposed method, pH value, reaction time, and the concentration of HA-AuNPs were optimized. The effect of the pH on the proposed method was investigated in the range from 4.0 to 9.0. Fluorescence recovery increased gradually with the pH value increased from 4.0 to 7.0, and largest fluorescence enhancement of the core-shell HAAuNPs@SiNPs nanoprobe was observed around pH 7.0, which may have arisen from the highest activity of HAase at the physiological pH value of about 7.0 (Figure S7). A further increase of pH values to 9.0 caused fluorescence signal significant decrease in assay performance (Figure S7). The above results show that the core-shell HAAuNPs@SiNPs nanoprobe is quite stable against pH change under physiological conditions. Reaction time is also an important factor with significant influence over the fluorescence intensity of the proposed method. The recovered fluorescence signal increases gradually with the increase of the reaction time, followed by the absence of obvious changes after 45 min, indicating the attainment of equilibrium within 45 min (Figure S8). Therefore, HAase detection was carried out in PB buffer (10 mM, pH 7.0) and with an incubation time of 45 min in the following experiments. The formation of core-shell HA-AuNPs@SiNPs nanoprobe leads to the quenching of fluorescence, indicating the FRET between donors (SiNPs) and acceptors (HA-AuNPs). The fluorescence intensity decreased gradually with the increase of the concentration of HAAuNPs, suggesting the formation of core-shell HA-AuNPs@SiNPs nanoprobe (Figure S9). The quenching efficacy is about 80% upon addition of 2 nM HA-AuNPs (Figure S9). Thus, 2 nM HA-AuNPs was chosen in the following experiments.


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HAase detection. Figure 6 shows the fluorescence responses of core-shell HAAuNPs@SiNPs nanoprobe upon addition of various concentrations of HAase under the optimized conditions. The quenched fluorescence of SiNPs induced by HA-AuNPs at around 440 nm was gradually recovered with the increasing concentration of HAase from 0 to 10 U/mL owing to the release of SiNPs (Figure 6A). Figure 6B indicates that a linear calibration graph is achieved by plotting Δ F (Δ F =F – F0, where F0 and F are the fluorescence intensity without and with the presence of HAase, respectively) versus the concentration of HAase ranging from 0.01 to 10 U/mL (regression coefficient R2 = 0.9932). The limit of detection (LOD) is estimated to be 0.004 U/mL, which is calculated according to a signal-to-noise ratio of S/N=3. The relative standard deviations of peak fluorescence readings are 2.6%, 3.2%, and 2.8% in three repetitive assays of 0.25 U/mL, 1 U/mL, and 5 U/mL HAase, respectively, which show that this proposed method has a good reproducibility. Compared with other reported strategies for HAase detection (Table 1), this detection sensitivity is comparable, or even superior to, sensitivity as determined by using those previous methods,

12, 22, 23, 43-45

which shows great promise in bioanalysis

and intracellular bioimaging assays. Selectivity of the Nanoprobe for HAase Detection. We also investigated the selectivity of the proposed assay towards HAase. As can be seen from the results in Figure 7, a series of potential interferences, including NaCl, KCl, lysozyme, BSA, glucose, trypsin, Cys, GSH, thrombin, and ALP, were respectively added into the HAAuNPs@SiNPs assay system to incubate for 45 min. It was noted that only HAase was able to effectively induce the fluorescence recovery of HA-AuNPs@SiNPs nanoprobe by its specific ability to degrade HA. The results indicate that the unspecifically reacting


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enzymes did not degrade HA, which could not weak the FRET between SiNPs and HAAuNPs. It was also observed that other proteins had no significant effective influence on our detection of HAase, indicating that the proposed strategy has a remarkable specificity towards HAase. Analysis of real samples. Hyaluronidase is present at high specific activity in human urine. The proposed assay was further applied for the determination of HAase in human urine samples. Different concentrations of HAase were added to the diluted human urine samples to prepare the spiked samples. It was showed that the recovery values of HAase in the real samples were ranged from 97.9 to 100.8% (Table 2). Moreover, we also used a commercial ELISA kit to detect hyaluronidase in the above human urine samples, and the obtained results (Table 2) were in agreement with those obtained by our method. The result indicated the potential of this method could be applied for the detection of HAase in biological samples. Applification of the core-shell HA-AuNPs@SiNPs nanoprobe in cell imaging. In addition to the demonstrated applicability of core-shell HA-AuNPs@SiNPs in our novel assay system, their use in the intracellular imaging of HAase was explored. Before the experiments, the cytotoxicity of the core-shell HA-AuNPs@SiNPs nanoprobe was investigated with conventional MTT assay using the HeLa cell line. Since the absorbance of MTT at 570 nm is dependent upon the degree of cellular activation, the cells incubated with culture medium only was taken as control experiment, and the ratio of the cells incubated with the core-shell HA-AuNPs@SiNPs nanoprobe to the control group was used to evaluate cell viability. SiNPs and core-shell HA-AuNPs@SiNPs nanoprobes show a little cytotoxicity. The cell viability was still greater than 84% upon addition of


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the SiNPs or core-shell HA-AuNPs@SiNPs nanoprobe over a wide concentration range from 10 to 200 μg/mL (Figure S10). The MTT results showed that SiNPs and core-shell HA-AuNPs@SiNPs nanoprobe exhibit the low toxic and good biocompatibility, indicating the great potential of the proposed strategy nanoprobe for bioimaging applications under physiological conditions. Cluster determinant 44 (CD44) is a membrane glycoprotein overexpressed on a variety of tumor cell plasma membrane, including lung, colon, and breast cancer. 46 HA is well known for its high-affinity binding to tumor cells overexpressing CD44 receptors. 47 Therefore, a kind of CD44-positive cell line, HeLa cells, was employed as a model cell line to examine the cancer targeting ability of the core-shell HA-AuNPs@SiNPs nanoprobe. Confocal imaging of HeLa cells treated with/without HA was performed after incubation with the core-shell HA-AuNPs@SiNPs nanoprobe. As shown in Figure 8A, core-shell HA-AuNPs@SiNPs nanoprobes were found to target cancer cells based on the specific affinity of HA to CD44 receptors. Upon incubation of core-shell HAAuNPs@SiNPs nanoprobe, HeLa cells clearly showed remarkable blue fluorescence under fluorescence microscopy, owing to fluorescence emitted from SiNPs. This phenomenon demonstrated that core-shell HA-AuNPs@SiNPs nanoprobes had internalized into HeLa cells, because the HAase overexpressed mainly in the cytoplasm degraded HA and SiNPs were released, resulting in the recovery of SiNPs fluorescence. In comparison, HeLa cells were incubated with free HA (10 mg/mL) to block CD44 before the addition of core-shell HA-AuNPs@SiNPs nanoprobe, which affected the protein's ability to bind core-shell HA-AuNPs@SiNPs nanoprobe, So, only very weak blue fluorescence could be seen in Figure 8B, suggesting a lack of cellular uptake of HA-


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AuNPs@SiNPs nanoprobe. These results suggest that the HA-AuNPs@SiNPs nanoprobe could selectively bind to CD44 and internalize into the cancer cells via receptor active targeting mediated endocytosis. All of the above results demonstrated that the core-shell HA-AuNPs@SiNPs nanoprobe can act as a nanoprobe for targeted tumor cell imaging. CONCLUSION We have developed a novel fluorescence assay for HAase detection based on the core-shell HA-AuNPs@SiNPs nanoprobe, while SiNPs played a role as the fluorescent indicator, and HA-AuNPs acted as quencher. Due to specific HA-CD44 binding and degradation of HA by HAase in tumor cells, the core-shell HA-AuNPs@SiNPs nanoprobe could be employed as a nanoprobe for tumor-targeted cell imaging. All the results suggested that the proposed HAase sensing strategy with great potential for practical applications and with the promise of finding wide applications in early tumor diagnosis and biomedical fields.

ASSOCIATED CONTENT Supporting Information. Additional experimental details and figures. This material is available free of charge via the Internet at http://pubs.acs.org. chemicals and apparatus, stability study of SiNPs with pH, UV irradiation time and NaCl; Zeta potential of SiNPs, AuNPs, and HA-AuNPs@SiNPs nanoprobe; EDX spectrum of HA-AuNPs@SiNPs nanoprobe; UV–visible study; stability study of HA-AuNPs@SiNPs nanoprobe with pH; study of reaction time; study of the amount of HA-AuNPs; study of cytotoxicity.

AUTHOR INFORMATION


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Corresponding Author *E-mail: [email protected], [email protected]. Author Contributions §

These authors contributed equally to this paper.

Notes The authors declare no competing financial interest. ACKNOWLEDGMENT The authors are appreciate Dr. Kathryn Williams for her comments during the preparation of this manuscript. This work is supported by grants awarded by the National Natural Science Foundation of China (21505122, 21525522), the National Institutes of Health (GM079359 and CA133086), the NSFC grants (NSFC 21221003 and NSFC 21327009), the China Scholarship Council, the Education Department of Henan Province, and Zhengzhou University.


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Scheme1. Schematic illustration of HAase detection based on the core-shell HAAuNPs@SiNPs nanoprobe.


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Figure 1. (A) Typical TEM image and photograph of prepared SiNPs suspension in water. (B) The particle size distribution histograms of SiNPs. (C) XRD patterns of SiNPs. (D) Fourier transform infrared (FT-IR) spectrum of SiNPs.


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Figure 2. High resolution XPS spectra of (A) full range, (B) C 1s, (C) N 1s, (D) O 1s, and (E) Si 2p peak of the SiNPs.


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Figure 3. (A) UV−vis absorption spectra of SiNPs (black line), UV−vis absorption spectra of HA-AuNPs (red line), excitation spectrum of SiNPs at the 440 nm emission wavelength (green line) and emission spectrum of SiNPs at 370 nm excitation wavelength (blue line). (B) Fluorescent spectra of SiNPs under different excitation wavelengths.


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Figure 4. Typical TEM image of (A) as-prepared AuNPs and (B) core-shell HAAuNPs@SiNPs nanoprobe.


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Figure 5. The fluorescence emission spectra of different sensing systems: (a) SiNPs; (b) SiNPs + HA-AuNPs + HAase; (c) SiNPs + HA-AuNPs. Measurements were performed in PB (10 mM, pH 7.0). SiNPs, 0.01 mg/mL; AuNPs, 2 nM; HAase, 10 U/mL.


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Figure 6. (A) Fluorescence spectra of the assay system in the presence of increasing amounts of HAase. The arrow indicates the signal changes with HAase increasing (from bottom to top, 0, 0.01, 0.05, 0.25, 1, 2.5, 5, 7.5, and 10 U/mL). (B) The linear calibration plot between Δ F and the concentration of HAase (0.01-10 U/mL). ΔF is the difference of the fluorescence intensity of the nanoprobe in the presence and absence of HAase. SiNPs, 0.01 mg/mL; AuNPs, 2 nM; Measurements were performed in PB (10 mM, pH 7.0).


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Figure 7. Effect of different ions and coexisting compounds on the fluorescence intensity for sensing HAase (NaCl, 100 mM; KCl, 100 mM; BSA, 10 mg/mL; Glucose, 500 μM; GSH, 500 μM; Cys, 500 μM; ALP, thrombin, lysozyme, trypsin, 100 U/mL). ΔF is the difference of the fluorescence intensity of the nanoprobe in the presence and absence of a species. SiNPs, 0.01 mg/mL; AuNPs, 2 nM; Measurements were performed in PB (10 mM, pH 7.0).


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Figure 8. Fluorescence images of HeLa cells (A) and HeLa cells pretreated with HA (B) after incubation with core-shell HA-AuNPs@SiNPs nanoprobe for 6 h at 37 °C.


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Sensing system

Linear range (HAase, U/mL)

Page 32 of 34

LOD (U/mL)

Reference

colorimetric

0-240

24

12

fluorescence

1.25-50

0.625

22

fluorescence

0.01-10

0.01

23

fluorescence

0.1-8

0.05

43

fluorescence

1-50

0.7

44

zymography

0.625-5

0.625

45

fluorescence

0.01-10

0.004

Our work

Table 1. Comparison of different methods for HAase detection.


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Sample

Initial HAase (U/mL)

Added HAase (U/mL)

Measured HAase mean (U/mL)a

ELISA method (U/mL) a

Recovery(%)

1

6.76

0.5

7.32±0.04

7.25±0.07

100.8%

2

8.41

1

9.28±0.06

9.32±0.08

98.7%

3

12.52

2

14.21±0.12

14.34±0.15

97.9%

Table 2. Determination of HAase in human urine samples using the proposed assay and ELISA method . (a Mean of three separate determinations ±standard deviation).


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For TOC A biocompatible HA-AuNPs@SiNPs nanoprobe prepared for highly sensitive and selective fluorescence turn-on detection of hyaluronidase and cellular imaging.


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Core-shell HA-AuNPs@SiNPs nanoprobe for sensitive fluorescence

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