One-Step Synthesis of Water-Dispersible and Biocompatible Silicon

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One-step synthesis of water-dispersible and biocompatible silicon nanoparticles for selective heparin sensing and cell imaging Sudai Ma, Yonglei Chen, Jie Feng, Juanjuan Liu, Xianwei Zuo, and Xingguo Chen Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.6b02448 • Publication Date (Web): 30 Sep 2016 Downloaded from http://pubs.acs.org on October 1, 2016

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Analytical Chemistry

One-step synthesis of water-dispersible and biocompatible silicon nanoparticles for selective heparin sensing and cell imaging Su-dai Ma1,2, Yong-lei Chen1,2, Jie Feng1,2, Juan-juan Liu1,2, Xian-wei Zuo1,2, Xing-guo Chen1,2,3

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State Key Laboratory of Applied Organic Chemistry, Lanzhou University,

Lanzhou, 730000, China 2

Department of Chemistry, Lanzhou University, Lanzhou, 730000, China

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Key Laboratory of Nonferrous Metal Chemistry and Resources Utilization of

Gansu Province, Lanzhou University, Lanzhou, 730000, China * Corresponding author E-mail address: [email protected] (X-G Chen) Tel: 86-931-8912763 Fax: 86-931-8912582

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ABSTRACT: A sensitive and selective fluorescence “turn-off” sensor to detect heparin using water-soluble silicon nanoparticles (Si NPs) was developed for the first time. The Si NPs were synthesized by a simple one-step procedure, which did not need high-temperature and complex modification. The as-prepared Si NPs featured strong fluorescence, favorable biocompatibility and robust photo- and pH-stability. Significantly, the Si NPs were induced to assemble or aggregate via hydrogenbonding, which resulted in the fluorescence of Si NPs quenched. Under the optimized conditions, the linear range was obtained from 0.02 to 2.0 μg/mL, with a limit of detection of 18 ng/mL (equal to 0.004 U/mL). It was lower than the proper therapeutic level of heparin during cardiovascular surgery and long-term therapy. This proposed method was relatively free of interference from heparin analogues, which commonly existed in heparin samples and could possibly affect heparin detection. Moreover, it did not need to introduce any control medium. As expected, the method was successfully applied to detect heparin in human serum samples with satisfactory recovery ranged from 98.8-102.5%. The Si NPs were superbly suitable for cell imaging owing to the negligible cytotoxicity and excellent biocompatibility.

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INTRODUCTION Silicon, known as the second most abundant element on earth, is primarily known for its applications in the modern microelectronic and semiconductor industry.1 In the past decade, there is an ever growing interest looking at its nanoscale properties, promoting potentially revolution silicon-related basic research and practical applications. Among them, silicon nanoparticles (Si NPs) have attracted great attention in chemical and biological fields owing to their intrinsic advantages, such as low cost, strong fluorescence, ultrahigh photostability and great stability.2-6 Moreover, the Si NPs exhibit unique dominances such as low toxicity, favorable biocompatibility and biodegradability, which make Si NPs widely accepted as promising alternatives to the toxic heavy-metal-containing II-VI quantum dots and organic dyes.7-9 Significantly, Sailor et al. discovered that Si NPs could be biodegradable in vivo in a comparatively short time (∼8 h).10,11 Prasad et al. reported that Si NPs tend to be degraded in the liver and spleen of a healthy mouse after two months.12 Therefore, Si NPs exhibit a significant potential in biological field. It is also worthwhile to point out that in order to application in biological systems, Si NPs need to be water-soluble and suitable for the conjugation of biologically interesting compounds. Recently, He et al. introduced a one-pot aqueous synthesis method for water-soluble Si NPs with the help of microwave irradiation at 160 ℃.13 He et al. further developed a photochemical strategy capable of large-quantity synthesis of Si NPs under UV irradiation.14 Li et al. reported fabrication of ultrabright water-dispersible Si NPs through a designed chemical surface modification.6 Wang et al. also reported a method for one-step synthesis of water-dispersible Si NPs for timeresolved imaging of living cells.15 Despite, scientists have made elegant work on the preparation of fluorescent and water-dispersed Si NPs,8 the detection of various

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analytes using label-free Si NPs was very limited.16 There is still much room to further explore Si NPs-based applications in the potential field, which is crucial in demand. Heparin is a highly negatively charged linear polysaccharide with a variable length that consists predominantly (>70%) of trisulfated disaccharide repeating units. It has been widely used as a major anticoagulant in various clinical diagnostic and therapeutic processes with about half a billion doses applied annually.17-23 Moreover, heparin plays a vital role in regulating of a range of normal physiological and pathological processes such as venous thromboembolism, inflammation, metabolism, immune defense, cell growth and differentiation, and blood coagulation.24-28 On the contrary, the higher dose and prolonged use of heparin often induce adverse effects, such as thrombocytopenia and hemorrhage or other fatal bleeding complication.24,29-32 Therefore, controlling the amount of heparin during surgery especially for postoperative and long-term care patients in anticoagulant therapy is crucially significant.33-35 However, it is still considered to be a challenge to quantitative measurement of heparin due to its natural polydispersity, chemical heterogeneity, lack of fluorescent properties or significant absorbance and the interference of analogues.36,37 To date, a lot of methods have made great progress in heparin sensing, such as phosphorescence,19 spectrophotometry38 and fluorescence methods39 and colorimetric assays.32,40 However, more simple, rapid and accurate for detection heparin with high sensitivity and selectively are still highly desirable. In this work, water-soluble Si NPs were synthesized by a facile, simple one-step method using 3-aminopropyl triethoxysilane (APTES) as silicon source and Lascorbic acid (AA) as the reduction reagent (Scheme 1A). Compared with previously published reports,13,14 this method avoided high-temperature processing, tedious

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procedures and special conditions or equipment. The as-prepared Si NPs exhibited an intense green fluorescence and strongly photo- and pH-stability, excellent salt stability and favorable biocompatibility. It was found that the Si NPs easily assembled or aggregated in the presence of heparin via hydrogen bonding, resulting in the fluorescence could be quenched by heparin (Scheme 1B). Based on this quenching mechanism, a selective and sensitive method to detect heparin was developed for the first time. The proposed method was successfully applied to detect heparin in human serum samples with satisfactory results. In addition, the Si NPs were superbly suitable for cell imaging owing to the negligible cytotoxicity and excellent biocompatibility. EXPERIMENTAL SECTION Preparation of the Si NPs. The Si NPs were synthesized by a facile one step method.15 Firstly, 1.00 mL of APTES was added to 4.00 mL aqueous solution with stirring at 40 ℃ water bath. Then, 1.25 mL of 0.1 M AA was added to the above mixture by stirring for 20 min. The total synthesis was completed in 30 min. Detection of Heparin in Aqueous Solution. To study the quenching effect of heparin on the fluorescence intensity (FL intensity) of the Si NPs, heparin was dissolved in water to form a 0.200 mg/ml solution. A series of solutions with different concentrations was prepared by adding different amounts of heparin to phosphatebuffered saline solution (PBS: pH 7.4, 10 mM). After different concentrations of heparin (150 μL) and Si NPs solution were incubated in PBS for 15 min (the final volume was 3.00 mL), the fluorescence emission spectra were measured with excitation wavelength at 420 nm. The selectivity experiments were carried out under the same conditions and operation. Preparation of Human Serum Samples. The human serum samples were collected from healthy volunteers, which were centrifuged at 13000 rpm for 3 min to

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obtain the serum. All samples were diluted 50-fold with PBS before detection. The concentrations of heparin in the diluted human serum samples were detected by the standard addition method. Cytotoxicity

Assays.

3-(4,5-dimethylthia-zol-2-yl)-2,5-diphenyltetrazolium

bromide (MTT) was used to measure the cytotoxicity effect of the Si NPs on the human hepatocellular carcinoma (SMMC-7721) cell. Briefly, 1×104 cells were incubated with the Si NPs in triplicate in a 96-well plate for 24 h at 37°C in a final volume of 100 μL. The Si NPs were added to the wells with increasing concentrations at 10, 50, 100, 150 and 200 μg/mL, respectively. Then, 10 μL of MTT solution (5 mg/mL) was added into each well and incubated for an additional 4 h at 37 °C. Finally, the optical densities (O.D) was measured at 520 nm using a RT-6100 microplate reader. The cell viability was by comparing the O.D value with that of untreated control cells in wells of the same plate ( Itreated / I control ). Cell Imaging Experiments. The SMMC-7721 cells (~ 106 cells) were seeded in a 12-well plate and cultured in RPMI-1640 medium (1 ml/well) at 37 °C in a humidified atmosphere of 5% CO2 overnight. The Si NPs (0.2 mg/mL) were added to the cell culture, and the cells were incubated for another 1 h at 37°C. The cells were rinsed with PBS (10 mM, pH= 7.4) for three times to remove the unbound compounds. The cell samples were then ready for imaging measurements. RESULTS AND DISCUSSION Optimization of Parameters in the Synthesis of the Si NPs. The waterdispersible Si NPs were one step synthesized by facile mixing and stirring APTES and AA for 30 min. In this method, APTES served as the silicon source and AA was deemed as a reduction reagent.15 As shown in Figure S1, several other reduction reagents (i.e., AS, BSA, NaBH4, and GSH) were applied instead of AA for synthesis

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of the Si NPs. The results showed that there were no fluorescence emissions at 520 nm using BSA, NaBH4, and GSH as reduction reagent, respectively, indicating that only AS and AA were effective in the formation of the Si NPs. As the Si NPs exhibited relatively strong FL intensity with AA as reduction reagent, AA was chosen in the synthesis of Si NPs. It was found that the temperature had a great influence on the preparation of Si NPs. As shown in Figure S2A, the Si NPs prepared under 30 to 40℃ exhibited the higher FL intensity. This phenomenon maybe due to the Si NPs did not form at low temperature. However, the oxidation of AA was accelerated under high temperature, which leaded to the reduction of AA was suppressed and the Si NPs maybe generated in part of the aggregation. Accordingly, with the increase of temperature, the color of Si NPs solution gradual changed from pink to jacinth (Figure S2B). Characterization of the Si NPs. A transmission electron microscopy (TEM) image of the Si NPs was shown in Figure 1A, in which the Si NPs appeared as spherical particles with good monodispersibility. The size distribution in Figure 1B showed an average diameter of 1.6 ± 0.3 nm. The elemental analysis of the Si NPs indicated that Si NPs contained Si, C, O, and N elements (Figure 1C). Besides, a typical XRD analysis confirmed that Si NPs were in the amorphous phase (Figure S3). To confirm the presence of various functional groups in Si NPs, FT-IR experiment was measured (Figure 1D). Typically, the sharp absorbance peak at∼1022 cm-1 corresponded to the vibrational stretch of Si−O−Si bonding. The absorbance in the range of 2867-2930 cm-1 was attributed to the deformation and stretch vibration of the O–H bond. The strong signals at 1609-1662 and 3422 cm-1 were, respectively, assigned to the N−H bending vibration and the N−H stretching vibration.6,14 The FT-

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IR results demonstrated that the resultant Si NPs have a large amount of amino groups.15 Furthermore, XPS measurement was performed for the surface composition and element analysis of as-prepared Si NPs. The full range XPS analysis (Figure 2A) of Si NPs clearly showed five peaks at 100.2, 152.3, 283.1, 399.0 and 531.2 eV, which were attributed to Si 2p, Si 2s, C 1s, N 1s, and O 1s, respectively. 41,42 The highresolution C1s analysis revealed the following five types of carbon atoms: the fitted signals at approximately 284.1, 284.6, 285.2, 286.1 and 287.4 eV implied the presence of C–Si, C–C, C–N, C–O and C=O (Figure 2B). The N 1s spectrum (Figure 2C) manifested that the peaks at 397.3 and 399.4 eV were for N–(C)3 and N–H.43-45 This indicated that the as-prepared Si NPs were rich in amino groups on the surfaces, which was consistent with the corresponding FT-IR spectrum. The three fitted peaks at 531.0, 532.5 and 533.7 eV in O 1s spectrum (Figure 2D) were assigned to C=O, C– OH/C–O–C and Si–O groups, respectively.42,45 The Si 2p peaks at 100.1, 100.7, and 101.5 eV in Figure 2E could be ascribed to Si-C, Si-N, and Si-O bond, respectively.46 The surface components of the Si NPs determined by the XPS were in good agreement with FT-IR results. The optical UV-Vis and fluorescence spectra of the Si NPs were shown in Figure 2F. There was no absorption detected for the wavelength region > 450 nm. Broad absorption was observed for the wavelength region < 400 nm. Under excitation at 420 nm, the Si NPs solution exhibited maximum symmetrical fluorescence spectra profiles at 520 nm. The aqueous solution of Si NPs exhibited green fluorescence under UV irradiation, which was strong enough to be seen with the naked eye. The absolute photoluminescence quantum yield of the synthesized Si NPs was ~8.2%.

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Stability of the Si NPs. When the Si NPs were explored for practical sensing applications, the material must be water-soluble and stable toward ambient environments. Due to the amino groups in structure of the Si NPs, the synthesized Si NPs performed inherently water-solubility. To investigate the stability, FL intensity of the Si NPs toward high ionic strengths in solution, extreme pH and strong light exposure were measured. As shown in Figure 3A, even in 100 mM NaCl solution, the FL intensity of the Si NPs remained almost constant with the one without NaCl in a solution. It suggested the outstanding stability of the Si NPs toward high ionic conditions. As shown in Figure 3B, Si NPs had strong and relatively stable fluorescent activities in the range of pH 3 to 10. Furthermore, the FL intensity of the Si NPs maintained stable after exposure of light illumination at 420 nm for 30 min, indicating that they have excellent photostability (Figure 3C). Taking the results above, these findings suggested outstanding stability of the Si NPs toward ambient environment. Sensitivity of the Si NPs for Heparin Detection. On the basis of above significant advantages of the Si NPs, a sensitive and selective fluorescent heparin sensor was developed. The relevant experimental parameters have been investigated. The Figure S4A depicted that the FL intensity of Si NPs had a remarkable quenching after adding heparin in the pH from 7 to 10. The FL intensity of Si NPs became steady in the pH from 7 to 8. Hence, the physiological condition pH 7.4 was chosen as the ideal reaction pH. As illustrated in Figure S4B, the fluorescence quenching efficiency reached a maximum and continued stable when the incubation time reached to 15 min. Therefore, 15 min was chosen as the ideal incubation time. The Figure 4A demonstrated the corresponding heparin concentration-dependent fluorescence spectra of Si NPs. The concentrations of heparin from top to down were

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0, 0.02, 0.1, 0.2, 0.6, 1.0, 1.4, 1.8, 2.0 μg/mL, respectively. The FL intensity at 520 nm gradually decreased with the increasing of concentrations of heparin. As plotted in Figure 4B, a good linear behavior was displayed between the fluorescence quenching efficiency ( F  F0 ) / F0 and the concentration of heparin, where F0 and F were FL intensities at 520 nm in the absence and presence of heparin, respectively. The linear regression equation was ( F0  F ) / F0 =0.023 + 0.16 Cheparin (μg/ml), with a correlation coefficient of 0.996. Moreover, the limit of detection (LOD) of 18 ng/ml (equal to 0.004 U/mL) was calculated based on a 3σ/S (σ was the standard deviation of the blank signal, and S was the slope of the calibration curve). The LOD was much lower than the proper therapeutic level of heparin during cardiovascular surgery (2-8 U/mL) and postoperation and long-term therapy (0.2-1.2 U/mL). Selectivity of the Si NPs for Heparin Detection. Selectivity has been considered to be one of the greatest challenges for heparin detection operating in biological media. In this work, we investigated the influence of three heparin analogues, i.e., hyaluronic acid (HA), chondroitin sulfate (Chs) and dextran (Dex), which commonly existed in heparin samples (chemical structure in Figure S5). As shown in Figure 5, HA, Chs and Dex hardly produced distinct interference for heparin detection even at 10-fold concentration of heparin. Compared with reported works,29,47-50 this method could meet the selective requirements for detecting heparin in complex biological samples. In additional, the effects of relevant ions and compounds, such as Cl- , SO2-4 , HCO3- , HPO2-4 , AA, BSA and glucose were studied. Figure 5 demonstrated that the

interference from these species could be neglected. These results revealed that the Si NPs sensor presented an excellent selectivity and strong tolerance for heparin sensing. It was significant to compare the present approach with other reported methods for heparin determination.19,39,47,50-54 Firstly, this method could directly detect heparin. 10


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As shown in Table S1, it did not need to introduce control medium, such as protamine, polybrene, peptide and GO, which simplified the assay procedure. Secondly, the Si NPs-based sensor did not need complex surface modification. Finally, the LOD was lower than or comparable to most of the previously reported methods for heparin detection (Table S1). It was clearly demonstrated that the Si NPs-based FL sensor toward heparin was simple and highly efficient. Possible Mechanism of the Si NPs for Heparin Detection. The possible mechanism of FL response of the Si NPs to heparin was explored. It can be seen from Figure 6, the Si NPs were well dispersed with slight random agglomerations. However, the introduction of heparin caused the aggregation of the Si NPs and they assembled to form larger clusters.20,55 It was speculated that the fluorescence quenching of Si NPs by heparin main stem from aggregation of the Si NPs. The aggregation of the Si NPs may be attributed to the hydrogen bonding or electrostatic interactions between heparin and Si NPs. Since the Si NPs were abundant in amino groups, heparin consisted of sulfated glycosaminoglycan segments. The hydrogen-bond was easy to form between the molecules. In order to confirm that this fluorescence quenching resulted from the hydrogen-bond interaction, FT-IR spectroscopy was employed to investigate the functional groups. It could be seen from Figure 7A that the band at 1662, 3422 cm-1 were related to the N−H bending vibrations and stretching vibration of Si NPs, while for the Si NPs + heparin, these peaks were shifted to 1657, 3432 cm-1. Meanwhile, the bands assigned to the stretching vibration of S=O, O−H, N−H and C−O for heparin, which were at ~1421, 3438, 1059 and 1237 cm-1, shifted to 1425, 3433, 1029 and 1312 cm-1 for the Si NPs + heparin.21,55 These spectral changes could be explicated by the strong hydrogen bonding interactions between the Si NPs and heparin. In addition,

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the mixing of the Si NPs and heparin led to the changes in UV-Vis absorption spectrum of Si NPs (Figure 7B), which was possible to result from the interaction between Si NPs and heparin. It was in good agreement with FT-IR results. Furthermore, the changes of zeta-potential and steady-state fluorescence lifetime were performed to discuss the mechanism. As shown in Figure 7C, the zeta potentials of the Si NPs and heparin were -2.16 mV and -20.20 mV respectively, indicating that heparin possessed more abundant negative charges than the Si NPs. When heparin was added into the Si NPs solution, the zeta potential became to be -6.57 mV, suggesting that the binding between Si NPs and heparin was not caused by electrostatic interaction.19,26 On the other hand, there was no obvious change in the average lifetimes of the Si NPs without and with heparin, which just decreased from 9.32 to 9.29 ns (Figure 7D). Therefore, it was extremely likely to be statically quenched by aggregation.55-57 These results demonstrated that the Si NPs were induced to assemble or aggregate via hydrogen-bonding, which resulted in the fluorescence of Si NPs quenched. Detection of Heparin in Human Serum Samples. With excellent sensitivity and selectivity of the method, the proposed method was used to determine the amount of heparin in human serum samples. As Table 1 listed, the average recoveries of heparin in human serum samples reached 98.8-102.5% with the relative standard deviation (RSD) less than 5%, which were acceptable for quantitative assays performed in biological samples. It was indicated that the proposed method showed excellent potential applicability. Cytotoxicity Assays and Cell Imaging. For further biological applications, toxicity was a great concern. MTT assays were carried out to evaluate the cytotoxicity of the Si NPs to SMMC-7721 cells. As expected, the Si NPs exhibited favorable

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biocompatibility. As shown in Figure 8, when the incubation concentration of the Si NPs increased to 200 μg/mL, the slight damage just began and the cell viability was estimated to be greater than 85%. High cell viability confirmed the low toxic, excellent biocompatibility, and the great potential of the as-prepared Si NPs for cell imaging. As shown in Figure 9, by incubating the SMMC-7721 cells with the Si NPs (200 μg/mL) for 1 h at 37°C, a significant green emission from the intracellular region could be observed. In addition, the cells were stained with a nucleus staining dye (Hoechst 33342) for another 15 min. Then, the cell image of Hoechst 33342 and that of Si NPs were overlapped (Figure 10), which indicated that Si NPs were specifically targeted to the nuclei of SMMC-7721 cells. The Si NPs could be expected as a platform to facilitate a myriad of biological applications. CONCLUSION The water-soluble and biocompatible Si NPs were synthesized by a facile and quick one-pot method. With the good properties of the Si NPs, a sensitive and selective method for the determination of heparin was established. The proposed method was simple, convenient, time-saving and easy-to-operate. Owing to the negligible cytotoxicity and excellent biocompatibility, the Si NPs were superbly suitable for cell imaging. It was promised Si NPs as an efficient platform for a wide range of biological applications.

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ACKNOWLEDGEMENTS The authors are grateful for financial support from the National Natural Science Foundation of China (No. 21375053) and the Specialized Research Fund for the Doctoral Program of Higher Education (No. 20130211110039). SUPPORTING INFORMATION The Supporting Information was available free of charge on the ACS Publications website. Details about the Materials and Apparatus. Additional figures (S1 to S5) and Table S1 were provided.

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Figure Captions Scheme 1. (A) The one-step synthetic strategy of the Si NPs. (B) The schematic illustration of fabricating Si NPs for heparin detection. Figure 1. (A) The TEM image of the Si NPs (scale bar 20 nm). (B) The diameter distribution of the Si NPs measured by TEM. (C) The EDS pattern of the Si NPs. (D) The FT-IR spectra of the Si NPs. 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 Si NPs, respectively. (F) UV-Vis absorption (red curve) and fluorescent excitation/emission spectra (black/green curve) of the Si NPs. Inset is the photographs of the Si NPs solutions under visible light and UV light illumination. Figure 3. (A) Normalized FL intensity of the Si NPs in pH 7.4 PBS containing various concentrations of NaCl. (B) FL intensity of the Si NPs at different pH values. (C) FL intensity variation of the Si NPs as a function of time under 420 nm light illumination. Figure 4. (A) Fluorescence emission spectra of the Si NPs solution upon addition of various concentrations of heparin (from top to bottom, 0, 0.02, 0.1, 0.2, 0.6, 1.0, 1.4, 1.8 and 2.0 μg/mL. (B) The linear calibration plot between fluorescence quenching efficiency ( F0  F ) / F0 and the concentration of heparin (0.02-2.0 µg/mL). Error bars are the standard deviation of three independent experiments. All experiments were conducted in 10 mM PBS (pH 7.4). Figure 5. (A) Fluorescence quenching efficiency ( F0  F ) / F0 response of 1.0 µg/mL Si NPs toward heparin and other interferents (10-fold concentration of heparin). (B) Fluorescence quenching efficiency response of Si NPs toward heparin and other interferents in the presence of heparin. Figure 6. The TEM images of the Si NPs (A) and Si NPs + heparin (B). 19



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Figure 7. (A) FT-IR spectra of the Si NPs, heparin and Si NPs + heparin. (B) UV-Vis absorption spectra of the Si NPs and Si NPs + heparin. (C) The zeta potential histogram of Si NPs, heparin, Si NPs + heparin. (D) Lifetimes of steady-state fluorescence of Si NPs and Si NPs + heparin. Figure 8. Cell viability of SMMC-7721 cells in presence of different concentrations of the Si NPs. Figure 9. Fluorescence microscopy image (A) and their corresponding bright-field transmission images (B) of SMMC-7721 cells. Figure 10. Fluorescence microscopy image of SMMC-7721 cells with Si NPs and Hoechst 33342. Table Caption Table 1. Results of heparin determination by this method in human serum samples.

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Scheme 1

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Figure 1

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Figure 2

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Figure 3

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Figure 4

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Figure 5

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Figure 6

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Figure 7

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Figure 8

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Figure 9

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Figure 10

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Table 1 Found

Added

Total found

RSD

Recovery

(μg/mL)

(μg/mL)

(μg/mL)

(%) (n=3)

(%)

1

Not found

0.40

0.41

1.6

102.5

2

Not found

0.60

0.62

1.9

103.3

3

Not found

0.80

0.79

1.3

98.8

Sample

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For TOC only

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One-Step Synthesis of Water-Dispersible and Biocompatible Silicon

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