AdsorptionâDesorption Behavior of Black Phosphorus Quantum Dots

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Interfaces: Adsorption, Reactions, Films, Forces, Measurement Techniques, Charge Transfer, Electrochemistry, Electrocatalysis, Energy Production and Storage

Adsorption-Desorption Behavior of Black Phosphorus Quantum Dots on Mucin Surface Yinqiang Xia, Siqi Wang, Renliang Huang, Rongxin Su, Wei Qi, and Zhimin He Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.8b01531 • Publication Date (Web): 28 Jun 2018 Downloaded from http://pubs.acs.org on June 28, 2018

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Adsorption-Desorption Behavior of Black Phosphorus Quantum Dots on Mucins surface

Yinqiang Xia, †, ǁ Siqi Wang,†, ǁ Renliang Huang,*, ‡ Rongxin Su,*, †, §, # Wei Qi,†, §, # Zhimin He†

†

State Key Laboratory of Chemical Engineering, School of Chemical Engineering and

Technology, Tianjin University, Tianjin 300072, P. R. China ‡

Tianjin Key Laboratory of Indoor Air Environmental Quality Control, School of

Environmental Science and Engineering, Tianjin University, Tianjin 300072, P. R. China §

Collaborative Innovation Center of Chemical Science and Engineering (Tianjin), Tianjin

300072, China #

Tianjin Key Laboratory of Membrane Science and Desalination Technology, Tianjin

University, Tianjin 300072, P. R. China

ǁ

Y. Xia and S. Wang contributed equally to this work.

* Author to whom any correspondence should be addressed E-mail: [email protected] (R. H.), [email protected] (R. S.) Tel: +86 22 27407799. Fax: +86 22 27407599.

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ABSTRACT: Black phosphorus quantum dots (BPQDs) as a novel nanomaterial have many potential applications in biomedicine. However, the interaction of BPQDs with proteins and its biological effects and potential risks are still unclear. Mucin, which serves biologically as the physical barrier against foreign substances entering tissues, was chosen as a model substrate here for studying the adsorption-desorption behavior of BPQDs using surface plasmon resonance (SPR) sensing and quartz crystal microbalance with dissipation monitor (QCM-D). We found that surface modification of BPQDs with polyethylene glycol-amine (PEG) reduces the quantum dots’ adsorption rate but increases their adsorbed amount on the mucins surface. The pH value, ionic strength, and ionic valence also had significant effects on the adsorption behavior of BPQDs. Upon increasing pH from 2 to 7, the amount of BPQD adsorption decreased from 14.1 to 3.2 ng/cm2. A high ionic strength and ionic valence (e.g., Mg2+, Al3+) also inhibit the surface adsorption of BPQDs. Furthermore, the adsorption-desorption mechanisms of BPQDs on mucins surface were proposed. The adsorption-desorption behavior under different conditions may be attributed to the steric hindrance of PEG, the electrostatic interaction, and/or charge screening. These findings provide useful insights into the interfacial behavior of BPQDs before they enter the tissues. Keywords: black phosphorus; quantum dots; mucin; adsorption; toxicological evaluation

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INTRODUCTION Since 2014, black phosphorus (BP) as a new nanomaterial has received enormous attention.1 Due to their highly accurate optical response and anisotropic charge transport, BP nanomaterials have been applied in electronics and optoelectronics.2 More recently, some studies have focused on using them in biomedical science. For instance, BP quantum dots (BPQDs) were applied as a photosensitizer for photodynamic therapy of cancer, because of the unique electronic structure of BPQDs for generating efficient single oxygen.3 Based on their good photothermal performance and biocompatibility, BP nanoparticles also successfully served as an agent in photoacoustic imaging and photothermal therapy in vivo.4-5 In addition, BP possesses a high surface-to-volume ratio with its puckered lattice configuration, suggesting great potential as a new drug delivery system for cancer therapy.6-7 Considering these applications, there will be a growing probability of human exposure to BPQDs, and thus it is vital to understand the associated health hazards. However, information on the in-vivo toxicological effects of BPQDs, their interaction, and fate is rare and inconsistent. While a few studies reported little cytotoxicity of BP nanomaterials for various cells,8-9 other researchers obtained contrary results. For example, Latiff et al.10 had shown a dose-dependent response in A549 cells to BP nanomaterials with a generally intermediate level of toxicity (between graphene oxides and transition metal dichalcogenides). Zhang et al.11 showed that layered BP can generate intracellular reactive oxygen species and disrupt cell membrane integrity. Therefore, there is a need for further assessment of their potential toxicity. One important tool for evaluating the potential toxicity of nanomaterials12 is studying their 3


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adsorption-desorption behavior on protective proteins that function as barrier on cell surfaces, since this is the initial step in the particles’ interaction with cell membrane or permeation into cells.13 Mucins are the main component of the slimy mucus gels that cover epithelial cell surfaces.14 They act as physical barriers against pathogens and noxious substances entering the tissues, and also lubricate cell surfaces in various internal organs.15-16 Further, mucins interact with nutrients and enteric drugs before they are absorbed into organs.17 Given their unique protective functions, mucins have been used in pharmaceutical research18 as a model to investigate the biological effects of nanomaterials19 in order to understand their behavior and possible toxicity in vivo.16, 20 In addition, mucins are associated with inflammation caused by their overproduction or hypersecretion.21 Some nanoparticles, such as silver and titanium dioxide, can induce mucin secretion and thus cause disease.22 Therefore, the interactions between nanomaterials and mucins is important for related toxicological assessments. Quartz crystal microbalance with dissipation (QCM-D) sensor is widely used to study various interactions.23-24 It can monitor the real-time surface adsorption of nanomaterials by measuring changes in the resonant frequency (∆f) and in the energy dissipation (∆D),25-26 which correspond to changes in the wet mass and viscoelastic properties.27-28 The changing signal also provides some information about the structure and conformation of molecules on the surface, which helps to clarify their interactions.29 Additionally, surface plasmon resonance (SPR) sensor as an optical instrument has been applied in biological interactions.30 The mass (dry mass) can be measured this way according to the change in refractive index.31 Therefore, SPR is complementary to QCM-D analysis in the quantitative study of surface interactions.32 Many studies employed both 4


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methods to investigate the toxicity and adsorption behavior of nanomaterials, including the polymer-mucin systems.33-34 However, there are no studies on the interaction between BPQDs and mucin. In this work, we investigated the adsorption-desorption of BPQDs on mucin using QCM-D and SPR sensors. First, BPQDs were prepared using a simple liquid exfoliation technology.35 Then the sensor surfaces were modified with mucin as the substrate. The adsorption-desorption behaviors of BPQDs with and without modification by polyethylene glycol-amine (PEG) on mucins surface were compared. The effects of BPQD concentration, pH, and co-existing ions on the adsorption process were investigated, and the interaction mechanism was further discussed to understand the adsorption behavior and their potential toxicity of BPQDs in the internal environment.

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MATERIALS AND METHODS Materials. Bulk BP (99.998%) was purchased from Nanjing XFNANO Materials Tech Co., Ltd. (Nanjing, China). N-Methyl-2-pyrrolidinone (NMP, 99.5%) and sodium hydroxide (NaOH) were obtained from Shanghai Macklin Biochemical Co., Ltd. (Shanghai, China). Polyethylene glycol-amine (PEG-NH2) was purchased from Shanghai Yare Biotech, Co., Ltd. (Shanghai, China). Mucins from porcine stomach were purchased from Sigma-Aldrich (Shanghai, China). Hydrochloric acid (HCl), phosphate buffered saline (PBS), sodium chloride (NaCl), magnesium chloride (MgCl2), and aluminum chloride (AlCl3) were all obtained from Tianjin Kemiou Chemical Reagent Co., Ltd. (Tianjin, China). Ultrapure deionized (DI) water was produced by a Milli-Q water purification system (Millipore Corporation, Billerica, MA, USA). Preparation of BPQDs and BP-PEG. The BPQDs were synthesized via a top-down route using a simple liquid exfoliation technology. In brief, 5 mg of BP powder was dispersed in 10 mL of NMP in a glass bottle and mixed by sonication for 20 min. Then, the mixture was sonicated with a sonic tip at 1000 Hz and 60% power for 6 hours, during which the ultrasound probe was alternatively turned on for 2 s and off for 4 s. After exfoliation, the resultant dispersion was centrifuged for 20 min at 3000 rpm to remove non-exfoliated bulk BP, and the solution was then centrifuged at 7000 rpm for another 20 min. The supernatant containing BPQDs was pipetted off gently and evaporated during decompression. The precipitate was repeatedly rinsed with water and re-suspended in the aqueous solution. To prepare BP-PEG, 0.8 mL of BPQD solution and 0.2 mL of PEG-NH2 solution (2 mg/mL) were mixed and stirred for 3 h.5 After incubation, the excess PEG-NH2 was removed by 6


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centrifugation at 12000 rpm. After repeated washing with water, the resultant BP-PEG was re-suspended in aqueous solution and stored at 4 °C. Characterization. The morphology and size distribution of BPQDs were characterized using a transmission electron microscope (TEM, JEOL 1200EX, Japan) operated at 80 kV. An atomic force microscope (AFM, Agilent-5500 USA) was used to analyze the topography and thickness of BPQDs. The sample concentrations were determined by inductively coupled plasma-optical emission spectrometry (ICP-OES, VISTA-MPX, Varian, USA). The optical adsorption was determined on a TU-1810 UV/via spectrophotometer (Shanghai Metash Instruments, China) using a quartz cuvette with 2 mm path length. A scanning probe microscopy system (NTEGRA Spectra, Russia) was used to obtain the Raman data of BPQDs and BP-PEG at room temperature. The hydrodynamic diameter and zeta potential of BPQDs before and after PEG-NH2 modification were measured by dynamic light scattering (DLS, Zetasizer Nano ZS, Malvern Instruments, UK) at 25 °C. Modification of Sensor Chips. Prior to the modification, Au chips were cleaned following the recommended protocols.12 Briefly, the chips were treated in a UV-ozone chamber (Novascan PSD, USA) for 15 min, and then soaked in a mixed liquid (ammonium hydroxide, hydrogen peroxide, and DI water, v/v/v, 1:1:5) at 75 °C for 5 min. Afterwards, the chips were rinsed with DI water, dried with nitrogen gas, treated in the UV-ozone chamber for another 30 min, and then stored for use. The mucin solution (0.2 mg/mL) was injected into the SPR or QCM-D instruments for immobilization. The immobilization process was repeated several times to ensure coverage on the sensor chips. After modification, the chips were washed with PBS buffer to 7


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remove the weakly bound mucin molecules. All the processes were performed online with the flow rate of 30 µL/min. Adsorption-Desorption of BPQDs and BP-PEG on Mucins surface. We used SPR and QCM-D to monitor the interaction between BPQDs and mucin in real time. The SPR sensor measures changes in the refractive index at the sensor surface, which are related to changes in mass, in order to trace the interaction with the sensor surface.30 The SPR experiments used a Biacore X100 system (GE Healthcare, USA), and the flow rate was maintained at 30 µL/min. In addition, a QCM-D sensor (E1, Q-Sense, Vastra Frolunda, Sweden) was used to determine the adsorption behavior, and examine the influence of PEG, pH, and electrolytes on the interactions. Before adsorption, PBS buffer was flowed to remove the weakly bonded mucin. Then background electrolytes were injected until the baseline (for SPR) or the frequency and dissipation (for QCM-D) signals became stable (defined as response fluctuation < 5 RU per 2 min in SPR, or drift of normalized frequency < 0.3 Hz within 10 min in QCM-D). After obtaining the stable baseline, the BPQDs or BP-PEG dispersed in background solution was injected for detection. A BPQD solution prepared with PBS buffer (8 µg/mL) was used. In QCM-D, the adsorption process lasted at least 20 min to ensure a balance of ∆f and ∆D, and then the background electrolyte was introduced again to investigate the release of BPQDs from mucin. In SPR, considering the volume limit of single injection, we set the injection time to 300 s (the max injection volume) to observe the adsorption of BPQDs, and a long desorption time was also adopted using background electrolyte. Similar procedures were repeated with BP-PEG. In addition, the adsorption-desorption of BPQDs was performed in different conditions, 8


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including the different concentrations, pH values, ionic strength and valences (the details were shown in supporting information).

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RESULTS AND DISCUSSION Synthesis of BPQDs and Structural Characterization. The BPQDs were prepared from

bulk BP using a simple liquid exfoliation technique. The TEM image in Figure 1A shows a uniform morphology of BPQDs. The high-resolution TEM image in Figure 1B reveals lattice fringes of 0.226 and 0.34 nm, which were ascribed to the (014) and (021) planes of the BP crystal, respectively. In Figure 1C, the average lateral size of BPQDs is about 3.05 ± 0.46 nm based on the statistical analysis. The AFM image (Figure 1D) shows the topographic morphology of BPQDs. The thickness was measured using cross-sectional analysis to be 0.52, 1.32 and 2.32 nm (Figure 1E), which correspond to BPQDs with about ~1, 3 and 4 layers, respectively. And the average thickness is 0.92 ± 0.72 nm (Supporting Information, Figure S1). As shown in Figure S2, the optical photographs of BPQDs and their UV-vis spectra was obtained at different concentrations. A gradual change in the broad absorption band was observed with the change in concentration. Considering that BPQDs are easily oxidized in an aqueous solution in the presence of oxygen under visible light irradiation, as well as the instability of BPQDs in PBS, we modified BPQDs with PEG-NH2 to overcome this drawback. After modification, the hydrodynamic diameter of the nanoparticles increased from 21 to 37 nm (Figure S3), and the zeta potential dropped from -21 to -10 eV, suggesting that the PEG-NH2 was successfully coated on the BPQDs. This was confirmed by Raman scattering experiment . As shown in Figure 1F, three typical Raman peaks can be observed in BPQDs, and assigned to an out-of-plane phonon mode (A1g) at 362.7 cm-1 and two in-plane modes (B2g and A2g) at 438.8 and 462.7 cm-1, respectively. Compared to BPQDs, these three Raman peaks of BP-PEG were red-shifted by 10


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approximately 2.7, 3.7, and 4.7 cm-1, respectively, because the oscillation of P atoms was hindered by PEG.

Figure 1 Characterization of BPQDs. A) TEM image and B) high-resolution TEM image of BPQDs with different lattice fringes. C) Statistical analysis of the sizes of BPQDs measured from TEM images. D) AFM image of BPQDs. E) Height profiles along the lines in (D). F) Raman scatter spectra of BPQDs and BP-PEG. Modification of Mucin on Au Surfaces. The SPR and QCM-D measurements were used to monitor the modification of mucin proteins on Au surfaces. As shown in Figure 2A, after the third injection, the signal response stopped increasing, suggesting that the mucin adsorption became saturated. Figure 2B shows that the resonant frequency dropped sharply with the injection and eventually tended to a stable value, also indicating that the surface was fully covered by mucin. The same trend was observed in the energy dissipation data.

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Figure 2 Real-time signal curves of mucin adsorption on the chips monitored by (A) SPR and (B) QCM-D. Comparison of Adsorption-Desorption of BPQDs and BP-PEG on Mucins surfaces. As shown in Figure 3A, the signal increased quickly upon injection of BPQDs solution, indicative of rapid adsorption of BPQDs on mucins surface. Then, the signal decreased with the flow of washing buffer, corresponding to a rapid desorption of BPQDs. After desorbing for 10 min, the adsorption amount was 3.2 ng/cm2 and the BPQDs was almost washed out in the end of desorption (Figure S4). In comparison, BP-PEG has a higher adsorption amount on mucins surface (12 ng/cm2, Figure 3B). In Figure 3C, after injection of BPQDs, the QCM-D resonant frequency decreased with ∆f = -2.5 Hz, and the energy dissipation increased with ∆D = 1 unit. When PBS was introduced, the resonant frequency and energy dissipation returned to their original levels, suggesting that little BPQDs remained attached on the mucins surface and they had no influence on the viscoelastic properties of the mucin layer. Generally, the deposition/adsorption behavior can improve the ability of the crystal to dissipate energy. Therefore, the result of QCM-D shows that it is difficult 12


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for BPQDs to attach tightly on the surface, which is consistent with the SPR result. As shown in Figure 3D, when BP-PEG was injected, the resonant frequency kept decreasing with a concurrent increase in energy dissipation. Following rinsing with PBS, the final ∆f and ∆D values were about -1.8 Hz and 0.8 units, respectively, indicating that BP-PEG was adsorbed tightly on mucins surface. Additionally, BP-PEG had lower adsorption-desorption rates compared to BPQDs (Figure 3C-D).

Figure 3 (A) SPR sensorgrams for the adsorption-desorption of BPQDs and BP-PEG on mucins surfaces and (B) the corresponding adsorption amount. QCM-D sensorgrams showing the adsorption-desorption of (C) BPQDs and (D) BP-PEG on mucins surfaces.

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Adsorption-Desorption under Different Conditions. Figure S5 shows the real-time curves of BPQD adsorption-desorption on the mucins surface. When the BPQD concentration increased from 1 to 16 µg/mL, its adsorption amount increased from 3.2 to 20.3 ng/mL, indicating that more BPQDs were attached on the surface at a higher concentration. Considering the acidic environment in the stomach, additional solutions with pH 2 and 4 were selected to test the influence of pH on the adsorption-desorption behavior. As shown in Figure 4A, when the pH decreased from 7 to 2, the adsorption amount of BPQDs increased from 3.2 to 14.1 ng/cm2. Figure 4B–D show the QCM-D sensorgrams for the desorption-desorption of BPQDs at pH = 2, 4, and 7, respectively. At pH 2, the resonant frequency significantly decreased, and the energy dissipation increased after the injection of BPQDs. After rinsing with the buffer, the final ∆f and ∆D values are -2.9 Hz and 1.4 units, respectively, indicative of an increase in mass and viscoelasticity and suggesting the adsorption of BPQDs on the mucins surface. According to the ∆D value (1.4 units), we inferred that the terminal regions of mucin may be linked together at pH 2, forming a loose structure that helps to adsorb solvent and BPQDs.17, 36 At pH 4, the final ∆f value (-2.4 Hz) is slightly lower than that at pH 2, but the final ∆D value is only 0.2 units, indicating that despite the adsorption of BPQDs there was little change in the viscoelastic properties of the mucin layer. When the pH value increased to 7, only very little changes were observed in the resonant frequency and energy dissipation, indicating that little BPQDs was attached on the mucins surface.

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Figure 4 (A) Adsorption amount of BPQDs on mucins surface at different pH values. QCM-D sensorgrams showing the desorption-desorption of BPQDs at (B) pH 2, (C) pH 4, and (D) pH 7. The adsorption-desorption behavior of BP-PEG at pH 2 and pH 4 was also inverstigated. As shown in the Figure S6, the adsorption amount of BP-PEG at pH 4 was ~14 ng/cm2, a little more than that of BP (~9 ng/cm2). At pH 2, the adsorption amount of BP-PEG was similar as that of BP. The results indicated that the modification of PEG improve the adsorption of BP on the mucins surface at pH 4 (Figure S6) and pH 7 (Figure 3). Furthermore, the adsorption-desorption behavior of BPQDs at different ionic strengths and ionic valence was investigated. As shown in Figure 5A, with increasing ionic strength (concentration of NaCl from 10 to 100 mM), the adsorption amounts of BPQDs decreased regardless of their concentration in the injected solution. A high ionic strength may induce the 15


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aggregation of BPQDs due to the charge screening,37-38 which reduces the surface energy of BPQDs and thus limits their adsorption on mucins surface. Similar phenomena were previously observed for other nanomaterials: graphene oxide,39 titanate nanowires40, and so on.

Figure 5 Influence of (A) ionic strength and (B) ionic valence on the adsorption amount of BPQDs at different injected concentrations. Figure 5B shows the adsorption amounts in the presence of ions with different valences. From Na+ to Mg2+ and to Al3+, the adsorption amount of BPQDs on mucins surface decreased regardless of its concentration in the solution. Similar to the case of ionic strength, the decrease in adsorption is probably attributed to the enhanced charge screening (the order of charge screening ability is Al3+>Mg2+>Na+).37, 41 In addition, ions with a higher valence may more easily form cation-π interaction with BPQDs,42-43 leading to aggregation and thus decreased surface adsorption of the latter. Mechanism of Adsorption-Desorption of BPQDs and BP-PEG on Mucins surfaces. As mentioned before, BP-PEG exhibited lower adsorption-desorption rates compared to BPQDs, which is probably ascribed to the steric hindrance of the macromolecules (PEG), while the 16


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obviously different amounts of adsorption between BPQDs and BP-PEG (Figure 3B) may be attributed to electrostatic double layer forces and hydration force that follows the extended Derjaguin-Landau-Verwey-Overbeek (DLVO) theory. Specifically, the isoelectric point (IEP) of BPQDs is pH 2-3 (Figure S7), and that of mucin is also between pH 2 and 3. Both BPQDs and mucin are negatively charged in PBS buffer (pH 7), and the charge repulsion significantly retards the attachment of BPQDs on mucins surface. Additionally, the mucin molecules strongly interact with water molecules to form a hydrated layer, presenting good antifouling performance against BPQDs. After the BPQDs were modified with PEG-NH2, the zeta potential decreased from -21 to -10 eV, decreasing the electrostatic repulsion and contributing to tighter adsorption of BP-PEG on the surface (Figure 6). On the other hand, the adsorption environment (pH value, ionic strength, and ionic valence) also affects the adsorption-desorption behavior of BPQDs on mucins surfaces. Considering the negative charges localized at the terminal regions of mucin (IEP : pH 2-3) at pH 7, one end of mucin may anchor on the surface (Figure 6).36 When the pH value shifts from 7 to 4 or 2, the dissociation of carboxylic acid moieties (-COOH) is weakened and the potential increased,44 leading to reduced electrostatic repulsion between mucin and BPQDs. In this case, more BPQDs become adsorbed on the mucins surface (Figure 4A). In addition, it is inferred that the attached mucin molecules predominantly have the “tail”-like conformation on the Au surface at pH 4.36 The adsorption of BPQDs on the mucins surface did not change the viscoelastic properties of mucin layer. Due to the weak interaction between mucin and BPQDs, some of the latter may permeate into the mucin layer. At pH 2, which is close to the isoelectric point of mucin, the 17


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terminal regions of mucin may be linked together due to their neutral charges, and a loose structure was formed as evidenced by the ∆D value (Figure 4B),45 making it easier for the BPQDs to become attached (Figure 6). The surface charge of BPQDs decreased with decreasing pH (Figure 6), weakening the repulsion between BPQDs and mucin molecules. Therefore, the adsorption of BPQDs increased when the pH value shifted from 7 to 2. However, with increasing ionic strength and ionic valence, the aggregation of BPQDs may occurred due to the charge screening, which reduced the surface energy of BPQDs and led to lower adsorption on mucins surfaces. (Figure 5).

Figure 6 Schematic illustration of the adsorption mechanism of BPQDs and BP-PEG on mucins surfaces under different conditions.

CONCLUSIONS In summary, the adsorption-desorption of BPQDs on mucins surface was investigated using

SPR and QCM-D measurements, in order to understand the interfacial behavior of BPQDs 18


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before entering tissues. Surface modification of BPQDs with PEG and the environmental conditions (the pH, ionic strength, and ionic valence) had a significant effect on the surface adsorption. The PEG modification generally improves the stability of BPQDs, however it also leads to higher adsorption on the mucins surface. Additionally, less BPQDs is deposited on the mucins surface at high pH (e.g., 3.2 ng/cm2 for pH 7). The adsorption amount also decreases with increases in ionic strength and valence. Hence, the surface adsorption is influenced by the steric hindrance of PEG, the change in weaker electrostatic interaction, the enhanced charge screening at high pH, and high ionic strength and valence. We believe this study provide important information about the interfacial behavior of BPQDs on mucins surface.

ASSOCIATED CONTENT Supporting Information. The experimental details of the adsorption-desorption of BPQDs adsorption in different conditions, height distribution of BPQDs from AFM analysis, UV-vis spectra of BPQDs at different concentrations, size distribution of BPQDs and BP-PEG obtained from DLS analysis, SPR sensorgram showing the adsorption-desorption of BPQDs on mucins surface, the adsorption behavior of BP-PEG at pH 4 and pH 2, the zeta potential of BPQDs at different pH values. This material is available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION Corresponding Author *E-mail: E-mail: [email protected] (R. H.), [email protected] (R. S.) 19


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Tel: +86 22 27407799. Fax: +86 22 27407599. Notes The authors declare no competing financial interest.

ACKNOWLEDGMENTS This work was supported by the Natural Science Foundation of China (Nos. 21777112 and 51473115).

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AdsorptionâDesorption Behavior of Black Phosphorus Quantum Dots

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AdsorptionâDesorption Behavior of Black Phosphorus Quantum Dots