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Separation and Recycling of Functional Nanoparticles Using Reversible Boronate Ester and Boroxine Bonds Chen Liu, Haiyue Gong, Weifeng Liu, Bin Lu, and Lei Ye Ind. Eng. Chem. Res., Just Accepted Manuscript • DOI: 10.1021/acs.iecr.9b00253 • Publication Date (Web): 27 Feb 2019 Downloaded from http://pubs.acs.org on March 1, 2019

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Industrial & Engineering Chemistry Research

Separation and Recycling of Functional Nanoparticles Using Reversible Boronate Ester and Boroxine Bonds Chen Liu, 1 Haiyue Gong, 2 Weifeng Liu, 2† Bin Lu, 1* Lei Ye2*

1 Key

Laboratory of Environment and Health, Ministry of Education & Ministry of

Environmental Protection, State Key Laboratory of Environmental Health (Incubating), School of Public Health, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei 430030, China

2 Division

of Pure and Applied Biochemistry, Department of Chemistry, Lund University, Box

124, 221 00 Lund, Sweden

KEYWORDS Nanoparticles; recycling; molecular recognition; boronic acid; poly(vinyl alcohol)

ACS Paragon Plus Environment

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ABSTRACT

The sustainable and green chemistry concept calls for effective separation and recycling of valuable functional nanoparticles. In this work, a viable approach to separate and recover synthetic nanoparticles without involving heavy-duty instrument was established. The nanoparticle separation was enabled by using reversible boronate ester and boroxine bonds formed between boronic acid-functionalized nanoparticles and poly(vinyl alcohol), and between the nanoparticles themselves. The reversible covalent bonds were controlled by simple adjustment of solvent pH. To demonstrate the viability of the approach, two types of nanoparticles, inorganic silica nanoparticles and organic molecularly imprinted nanoparticles functionalized with boronic acid on surface were used as models. Upon addition of poly(vinyl alcohol) and adjustment to basic pH, the nanoparticles formed aggregates and readily settled from solution. After changing to an acidic solvent, the boronate ester bonds formed between boronic acid-functionalized nanoparticles and poly(vinyl alcohol) were hydrolyzed, and poly(vinyl alcohol) was released from the nanoparticle aggregates. The particles remained as aggregates due to


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the formation of new, inter-particle boroxine bonds. Using pH-controlled dynamic particle aggregation, propranolol-imprinted nanoparticles could be easily recovered and used repetitively without centrifugation. This work provides a new approach for recovery of valuable functional nanomaterials for potentially large scale industrial applications.

1. INTRODUCTION

With the fast development of nanoscience and nanotechnology, more and more nanoparticles featuring high surface area and multi-functionalities have been developed for a large variety of applications. From inorganic nanoparticles (such as silver, gold and silica nanoparticles) to organic nanoparticles (such as nanoparticle-supported polymer brushes, covalent organic frameworks and molecularly imprinted polymers (MIPs)), new nanomaterials are widely used in catalysis,1 biomedical detection,2 drug delivery,3,4 engineering5, and imaging.6 Previous studies have shown that nanoparticles can maintain their excellent functionality after being recycled for multiple times.7-10 Besides, the concept of sustainable and green chemistry calls for effective separation and recycling of


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nanoparticles.11 For large scale industrial applications, new cost-effective techniques to recover valuable nanoparticles deserve further exploration. So far, for applications involving a liquid phase, solid nanoparticles are usually separated from solution by filtration or centrifugation. These methods of nanoparticle separation are widely used, but have certain limitations because of time- and energyconsuming operations, blockage of filtration units and necessity of heavy-duty instruments. For large-scale industrial applications, the conventional methods for nanoparticle separation are not economically viable, which hinders the widespread use of functional nanoparticles for commercial applications.12,13 To solve the problem of recovering nanoparticles, magnetic nanoparticles have attracted great interests in the past years. Different composite magnetic particles have been designed to simplify nanoparticle separation: the composite particles are usually composed of a magnetic core and a non-magnetic shell to offer multiple functionalities. The composite particles can be easily separated from liquid phase by applying an external magnetic field.14,15 However, it is often difficult to synthesize composite magnetic nanoparticles with well-controlled particle size and narrow size distribution. Besides, the instability and high cost are also


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the main drawbacks of magnetic functional nanoparticles.16 Consequently, more alternative methods for separation and recycling of nanoparticles need to be developed, especially for large scale industrial applications. In a previous work, we demonstrated that molecularly imprinted nanoparticles modified with boronic acid can react with cis-diol modified nanoparticles to form reversible boronate ester bond. The dynamic aggregation of nanoparticles can respond to pH variation and specific chemical stimuli, making it possible to control the nanoparticle assembly independent of specific molecular binding and release.17 Based on these results, we conceived the possibility of using flexible cis-diol polymers to assist separation and recovery of boronic acid-coated functional nanoparticles.18,19 The idea was to achieve reliable nanoparticle separation without involving heavy-duty instrument. In other words, we intended to separate surface functionalized nanoparticles by controlled aggregation and sedimentation. Poly(vinyl alcohol) (PVA), a synthetic polymer with repeating pendant cis-diol groups is able to react with boronic acid to form boronate ester bond, which can be hydrolyzed at acidic pH. PVA is inexpensive, stable, and highly water-soluble. It has a wide range of industrial, commercial, and medical applications.20 For these reasons, we


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decided in this work to use PVA as a particle agglomeration reagent to realize controlled nanoparticle separation. To demonstrate the feasibility of boronic acid-mediated particle separation, we used inorganic silica nanoparticles and MIP nanoparticles in our proof-ofconcept investigation. These two types of nanoparticles represent hard and soft materials decorated with different levels of boronic acid on their surfaces, therefore are good staring examples to cover a wide range of functional nanoparticles.

2. EXPERIMENTAL SECTION

2.1. Materials

Poly(viny alcohol) (MW = 9000-10000; 13000-23000; 76000; 85000-124000; 146000186000),

ammonia,

tetraethylorthosilicate

(TEOS),

(3-aminopropyl)triethoxysilane

(APTES), 4-formylphenylboronic acid (FPBA), horseradish peroxidase (HRP), Alizarin Red S (ARS), fructose, methanol and acetonitrile were supplied by Sigma-Aldrich (Dorset, UK). (R,S)-Propranolol hydrochloride (99%) was purchased from Fluka (Dorset, UK).


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2.2. Boronic acid-modified silica nanoparticles (Si@BA) Silica nanoparticles were synthesized using one-step Stöber method.21 Briefly, ammonia (25%, 22.4 mL), water (33 mL) and methanol (100 mL) were mixed in a 1 L glass beaker and stirred. Then, a mixture of 130 mL of methanol and 13.8 mL of TEOS was quickly added. After the reaction, the mixture was stirred at ambient temperature for 8 h. The product was separated by centrifugation, washed with water (30 mL, 3 times) and methanol (30 mL, 3 times), and dried in a vacuum chamber. Silica nanoparticles (3.0 g) and 1% APTES solution (0.5 mL APTES added in 49.5 mL anhydrous toluene) were mixed in a 100 mL flask. The mixture was stirred at 110°C for 24 h. The product (Si@NH2) obtained was separated by centrifugation, washed with water (30 mL, 3 times) and methanol (30 mL, 3 times), and dried in a vacuum chamber. The obtained Si@NH2 was added into 160 mL of FPBA solution in methanol (1 mg/mL) at ambient temperature and agitated for 10 h. The nanoparticles were separated by centrifugation (12000 rpm, 13680 g, 10 min), and then immersed in 160 mL methanol containing 1 mg/mL sodium cyanoborohydride. The mixture was stirred at room temperature for 10 h. Finally, the product (Si@BA) was separated by centrifugation


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(12000 rpm, 13680 g, 10 min), washed with water (30 mL, 3 times) and methanol (30 mL, 3 times), and dried in a vacuum chamber.

2.3. Boronic acid-modified MIP nanoparticles (MIP@BA) Core-shell MIP nanoparticles containing epoxide groups (MIP@epo) and MIP nanoparticles modified with boronic acid (MIP@BA) were prepared following the procedures described in our previous work.17, 22

2.4. Detection of boronic acid on nanoparticles with ARS Si@BA particles (5 mg) were mixed with 2 mL of ARS solution (0.1 mM dissolved in 20 mM, pH 7.4 phosphate buffer) and sonicated for several minutes. The fluorescence spectra of the samples were collected using an excitation wavelength at 469 nm. As a control, Si@NH2 was tested under the same condition.

2.5. Sedimentation of boronic acid-modified nanoparticles


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The sedimentation of boronic acid-modified nanoparticles was investigated under different conditions:

2.5.1. The effect of pH on nanoparticle sedimentation Si@BA particles (1 mg) were added into 1 mL of 10 mM phosphate buffer (pH = 6, 8.5, 9) and 10 mM acetate buffer (pH = 4), respectively. The mixture was sonicated for 30 min. A solution of PVA (1 mL, 0.5 mg/mL) of different molecular weight prepared in 10 mM phosphate buffer (pH = 6, 8.5, 9) were mixed with the Si@BA suspension at the same pH. The sedimentation of nanoparticles was monitored by measuring the absorbance of visible light at 600 nm of the sample every 10 s, using a UV-Vis spectrometer.

2.5.2. The effect of PVA concentration on nanoparticle sedimentation Si@BA (1 mg) was added into 1 mL of 10 mM phosphate buffer (pH 9). The mixture was sonicated for 30 min. Solutions of PVA (MW = 9000-10000; 13000-23000; 76000; 85000-124000; 146000-186000) at different concentrations were prepared in 10 mM phosphate buffer (pH 9). The PVA solution (1 mL, at different concentration) was mixed with 1 mL of the Si@BA suspension. The sedimentation of nanoparticles was monitored by measuring the absorbance of visible light at 600 nm of the sample every 10 s.


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MIP@BA suspension (1 mg/mL) was prepared by adding MIP@BA particles into a mixture of acetonitrile and 10 mM phosphate buffer, pH 9 (1:1, v/v) and sonicated for 30 min. Solutions of PVA (MW = 9000-10000; 13000-23000; 76000; 85000-124000; 146000186000) at different concentrations were prepared in a mixture of acetonitrile and 10 mM phosphate buffer, pH 9 (1:1, v/v). The PVA solution (1 mL, of different concentration) was mixed with 1 mL of the MIP @BA suspension. The sedimentation of nanoparticles was monitored by measuring the absorbance of visible light at 600 nm of the sample every 10 s.

2.6. Evaluation of accessible boronic acid groups on MIP@BA and NIP @BA MIP@BA and NIP@BA (3 mg) were added to 1 mL of HRP solution (100 μg/mL) prepared in phosphate buffer (20 mM phosphate, pH 9, containing 0.5 M NaCl). The mixtures were gently shaken at room temperature overnight and then centrifuged (10000 rpm, 5590 g, 10 min). The free HRP in the supernatant was determined by measuring fluorescence emission at 340 nm using an excitation wavelength at 284 nm.


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2.7. Verification of reversible boronate ester bond Si@BA suspension (1 mg/mL) was prepared in 10 mM phosphate buffer (pH 9) and sonicated for 30 min. PVA solution (1 mL, 0.5 mg/mL, MW = 9000-10000) prepared in PBS buffer (pH 9) was mixed with 1 mL of the Si@BA suspension. Sedimentation of nanoparticles in the mixture was monitored by measuring the absorbance at 600 nm every 10 s. Subsequently, fructose (3 mg) was dissolved into the particle suspension, and the mixture was stirred on a rocking table for 1 h. After this step, the absorbance of visible light at 600 nm was monitored. For comparison, the visible light absorbance of the Si@BA suspension was also measured in phosphate buffer at pH 6 following the same procedure.

2.8. Separation and recycling of MIP nanoparticles The process of nanoparticle separation and recycling is illustrated in Scheme 1. Firstly, MIP@BA (1 mg) was added into 1 mL of 10 μM propranolol dissolved in a mixture of acetonitrile and phosphate buffer, pH 9 (1:1, v/v). The sample was stirred on a rocking table overnight. Thereafter, the particle suspension was mixed with 1 mL solution of PVA


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(MW = 146000-186000, 0.5 g/mL prepared in a mixture of acetonitrile and phosphate buffer, pH 9 (1:1, v/v)). The mixture was let to stand for 15 min before the supernatant was removed. The amount of propranolol in the supernatant was detected using a florescence spectrometer (Ex: 292 nm, Em: 334 nm). The particles were then resuspended in 1 mL of mixture of acetonitrile and 10 mM acetate buffer, pH 4 (1:1, v/v), and the mixture was stirred on a rocking table overnight. After this step, the particle suspension was let to stand for 15 min before the supernatant was removed. The amount of propranolol in the supernatant was detected using the same florescence spectrometer.

Scheme 1. Boronic acid-mediated molecular separation using MIP@BA particles, and recovery of the MIP@BA particles by simple pH control.


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2.9. Characterization Attenuated total reflection (ATR) FT-IR analysis was conducted on a Nicolet IS5 Spectrophotometer (Thermo Fisher-Scientific Inc., Waltham, USA). All the spectra were collected at room temperature with 32 scans within the 4000 - 500 cm-1 range at a resolution of 4 cm-1. Hydrodynamic diameters and zeta potential of the nanoparticles were measured at 20C using a Zetasizer Nano ZS instrument (Malvern Instruments, United Kingdom). The morphologies of particles were observed using a scanning electron microscope (SEM, JSM-6700F, JEOL, Japan). UV-Vis absorption spectra were recorded


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using a Cary 60 UV/Vis spectrophotometer (Agilent Technologies，USA). Fluorescence emission was measured using a Quanta Master C-60/2000 spectrofluorometer (Photon Technology International, Lawrenceville, NJ, USA).

3. RESULTS AND DISCUSSION 3.1. Synthesis and characterization of nanoparticles In this work, to investigate the boronic acid-mediated separation and recovery of nanoparticles, we prepared two types of nanoparticles that were surface-modified with boronic acid. The inorganic silica nanoparticles were synthesized using the one-step Stöber method.21 The Si nanoparticles were first modified with APTES to introduce amino groups, which were used to immobilize FPBA to give the boronic acid-modified Si@BA particles. In Figure 1, the FT-IR spectra illustrated the characteristics of the modified silica. The obvious absorption band at 1051 cm-1 is associated with the asymmetric stretching vibration of Si-O-Si.23 After modification with APTES, the silanol signal at 3400 cm-1 (due to the O-H) became weaker, and a new adsorption band related to the amino group at 1534 cm-1 emerged.24


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Si Si@NH 2

1534

4000

3500

3000

2500

2000

1500

1000

500

-1

W avenumber (cm )

Figure 1. FT-IR spectra of silica and Si@NH2 particles.

To prove successful introduction of boronic acid into the Si@NH2 particles via reaction with FPBA, the product Si@BA particles were treated with a cis-diol containing fluorogenic dye, ARS to reveal the fluorescence emission characteristic of the particles. It is well known that ARS displays a dramatic increase in fluorescence intensity when it binds to a boronic acid. ARS is widely used as a general fluorescent reporter to detect boronic acid.25 Figure 2 shows that Si@BA indeed displayed a strong fluorescence emission (Ex: 469 nm, Em: 580 nm) after reacting with ARS. For comparison, no


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fluorescence was observed from Si@NH2 after it was treated with ARS. Therefore, we could conclude that boronic acid has been successfully introduced into Si@BA nanoparticles.

120000

ARS Si@NH2+ARS

100000

Fluorescence Intensity

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Si@BA+ARS

80000 60000 40000 20000 0 540

560

580

600

620

640

Wavelength(nm)

Figure 2. Fluorescence emission spectra obtained from a mixture of Si@BA and ARS (blue line), Si@NH2 and ARS (red line), and ARS alone (black line).

From the SEM images (Figure S1a and S1b), it is clear that the silica and Si@BA particles are very uniform and have well-defined spherical shape. The particle sizes estimated from the SEM images are in agreement with the hydrodynamic diameters of


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the silica particles (268.4 nm, PDI = 0.015) and Si@BA (297.8 nm, PDI = 0.080) measured by dynamic light scattering (DLS). For the organic MIP nanoparticles, boronic acid was introduced into the outer layer of the particles by post-imprinting modification. The propranolol-imprinted core particles were synthesized by precipitation polymerization, and a loosely cross-linked shell was added in a subsequent polymerization step. In the last step the boronic acid was introduced using Cu(I)-catalyzed click reaction.17 Based on the SEM images (Figure S1c and S1d), the size of MIP@BA and NIP@BA particles are estimated to be 170 ± 3 nm and 520 ± 76 nm, respectively. All the core-shell particles have a spherical shape, and a rough surface most likely due to the loosely cross-linked shell.

3.2. Boronic acid-mediated nanoparticle sedimentation For boronic acid-modified nanoparticles, we assumed that a flexible polymer bearing multiple pendant cis-diol groups will be able to induce particle-particle aggregation and lead to an accelerated particle sedimentation. Apparently, the speed of particle


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sedimentation will be affected by solution pH, the molecular weight and concentration of PVA and the type of the nanoparticles. To study the colloidal stability of Si@BA suspension at different pH, the particles were dispersed in aqueous buffer adjusted to different pH values before the change of visible absorbance of the suspension was monitored. As shown in Figure 3, in phosphate buffer adjusted to three different pH values (6, 8.5, 9), Si@BA particles exhibited a very slow sedimentation. The relatively high colloidal stability can be explained by the apparent surface charges of the particles, as reflected from the Zeta potential measured at the different pH values (Table 1). Interestingly, when dispersed in acetate buffer at pH 4, Si@BA particles aggregated and settled quickly to the bottom. This fast particle sedimentation can be explained as a result of inter-particle cross-linking through formation of boroxine bond, as has been discussed in the previous literature.17, 26, 27


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pH=4 pH=8.5

0.5

pH=6 pH=9

0.4

Absorbance

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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0.3

0.2

0.1

0.0 0

200

400

600

800

1000

1200

Time (s)

Figure 3. Sedimentation of Si@BA particles (CSi@BA = 1 mg/mL) in buffer at different pH monitored by measurement of absorbance of visible light at 600 nm.

Table 1. Zeta potential of Si@BA nanoparticles in phosphate buffer at different pH measured at 20 °C.

pH

Zeta potential (mV)

6

14.7 ± 0.3

8.5

-13.7 ± 0.9

9

-12 ± 0.8


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It is well known that boronic acids on the surface of nanoparticles are able to react with cis-diols in alkaline solution.28 To accelerate sedimentation of Si@BA nanoparticles, PVA was therefore added to crosslink the nanoparticles by reacting with the boronic acid located on the nanoparticle surface. As can be seen from Figure 4a, at pH 9 the nanoparticles settled most quickly after addition of PVA. Decreasing the pH to 8.5 caused the particle sedimentation to become slower, however the speed of sedimentation caused by PVA is still much faster than in the absence of PVA. In contrast to the alkaline buffer, in the slightly acidic buffer (pH 6), the presence of PVA made the nanoparticle suspension to become more stable, most likely due to the increased viscosity caused by the PVA polymer. Figure 4b shows the snap shots of the particle suspension in PVA solution before and after the absorbance measurements. In pH 9 buffer, the nanoparticles settled almost completely and the supernatant became transparent after 1500 s. In pH 6 buffer, however, most of the nanoparticles remained in the top liquid phase after 1500 s. From the SEM images (Figure S1e and S1f), it is clear that the Si@BA nanoparticles are welldistributed in the PVA film isolated from the pH 9 buffer. Therefore, we could conclude that the optimal pH for the boronic acid-mediated particle sedimentation is pH 9.


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a

pH=6

0.5

pH=8.5

b

pH=9

0.4 Absorbance

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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0.3

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0.0

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1400

Time (s)

Figure 4. (a) Particle sedimentation monitored by UV-Vis measurement at 600 nm. (b) Photo imagines of Si@BA nanoparticles (CSi@BA = 1 mg/mL) crosslinked with PVA (CPVA = 0.05 mg/mL) in phosphate buffer at different pH values.

After optimizing the pH, we studied the effect of molecular weight and concentration of PVA on the nanoparticle sedimentation. Figure 5 shows that in the presence of PVA of both low molecular weight (9000-10000) and high molecular weight (146000-186000), the nanoparticles precipitated most quickly at pH 9. The boronic acids on the surface of the silica formed covalent bonds with the repeating cis-diol groups in PVA, thereby caused the nanoparticles to form crosslinked aggregates to phase-separate from the liquid


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solution. Among the different concentrations and molecular weights of PVA investigated, the concentration of PVA (MW = 9000-10000) of 0.005 mg/mL gave the fastest particle sedimentation. Under this condition it took only 900 s for all the nanoparticles to settle to the bottom. When the concentration and molecular weight of PVA were increased further, the particle sedimentation became slower, most likely due to the increased viscosity. Therefore, the optimal condition to precipitate the Si@BA nanoparticles was determined to be 0.005 mg/mL PVA with a molecular weight of 9000-10000. Most importantly, the inorganic Si@BA nanoparticles could be easily separated from liquid solution by adding a small amount of PVA.

0.55

b

0.50

0.55 0.50

0.45

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0.35

0.30

0.30

Absorbance

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Absorbance

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


0.25 0.20 Si@BA (1mg/mL) Si@BA (1mg/mL)+PVA(0.005mg/mL) Si@BA (1mg/mL)+PVA(0.05mg/mL) Si@BA (1mg/mL)+PVA(0.5mg/mL) Si@BA (1mg/mL)+PVA(1mg/mL) Si@BA (1mg/mL)+PVA(5mg/mL)

0.15 0.10 0.05 0.00 0

200

400

0.25 0.20 0.15

Si@BA (1mg/mL) Si@BA (1mg/mL)+PVA(0.005mg/mL) Si@BA (1mg/mL)+PVA(0.05mg/mL) Si@BA (1mg/mL)+PVA(0.5mg/mL) Si@BA (1mg/mL)+PVA(1mg/mL) Si@BA (1mg/mL)+PVA(5mg/mL)

0.10 0.05 0.00 600

800

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Time (s)

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Time (s)


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d

0.50

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Absorbance

Absorbance

c

0.30 0.25 0.20 Si@BA (1mg/mL) Si@BA (1mg/mL)+PVA(0.005mg/mL) Si@BA (1mg/mL)+PVA(0.05mg/mL) Si@BA (1mg/mL)+PVA(0.5mg/mL) Si@BA (1mg/mL)+PVA(1mg/mL) Si@BA (1mg/mL)+PVA(5mg/mL)

0.15 0.10 0.05

0.30 0.25 0.20 Si@BA (1mg/mL) Si@BA (1mg/mL)+PVA(0.005mg/mL) Si@BA (1mg/mL)+PVA(0.05mg/mL) Si@BA (1mg/mL)+PVA(0.5mg/mL) Si@BA (1mg/mL)+PVA(1mg/mL) Si@BA (1mg/mL)+PVA(5mg/mL)

0.15 0.10 0.05

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e

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Time (s)

0.50 0.45 0.40 0.35

Absorbance

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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0.30 0.25 0.20 0.15

Si@BA (1mg/mL) Si@BA (1mg/mL)+PVA(0.005mg/mL) Si@BA (1mg/mL)+PVA(0.05mg/mL) Si@BA (1mg/mL)+PVA(0.5mg/mL) Si@BA (1mg/mL)+PVA(1mg/mL) Si@BA (1mg/mL)+PVA(5mg/mL)

0.10 0.05 0.00 0

200

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600

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Time (s)

Figure 5. Sedimentation of Si@BA nanoparticles (CSi@BA = 1 mg/mL) in phosphate buffer, pH 9 by adding different amount of PVA of different molecular weight: (a) 9000-10000; (b) 13000-23000; (c) 76000; (d) 85000-124000; (e) 146000-186000. The particle sedimentation was monitored by measurement of absorbance of visible light at 600 nm.


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For the organic MIP nanoparticles, the optimal concentration and molecular weight of PVA to accelerate particle sedimentation was also investigated. Because of the difference in particle structure and plasticity, we considered that the MIP nanoparticles and the inorganic silica nanoparticles may have different optimal parameters for the PVA solution to accelerate particle sedimentation. Besides the different rigidities, the boronic acid on the Si@BA particles are fixed on surface through a short linker, whereas the boronic acid in the MIP@BA particles are located in a loosely crosslinked shell through flexible polymer chains. Our previous work has shown that the boronic acid in the MIP particles are accessible to react with surface-immobilized cis-diols in basic buffer (pH 9), where the MIP maintained its specific target binding.17 Therefore, we decided to optimize the concentration and molecular weight of PVA in a mixture of acetonitrile and phosphate buffer (pH 9). As shown in Figure 6, both the concentration and molecular weight of PVA affected the speed of the nanoparticle sedimentation. For the MIP nanoparticles, the optimal concentration and molecular weight of PVA was found to be 0.5 μg/mL and MW = 146000-186000, respectively.


25


a

0.55

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0.40

0.40

0.35

0.35

0.30 0.25 0.20

MIP@BA(1mg/mL) MIP@BA(1mg/mL)+PVA(0.0005mg/mL) MIP@BA(1mg/mL)+PVA(0.005mg/mL) MIP@BA(1mg/mL)+PVA(0.05mg/mL)

0.50 0.45

Absorbance

Absorbance

b


0.50 0.45

0.30 0.25 0.20

0.15

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Figure 6. Sedimentation of MIP@BA nanoparticles (CMIP@BA = 1 mg/mL) with different amount of PVA of different molecular weight: (a) 9000-10000; (b) 13000-23000; (c) 76000; (d) 85000-124000; (e) 146000-186000 in mixture of acetonitrile and phosphate buffer, pH 9 (1:1, v/v). Sedimentation of NIP@BA nanoparticles (CNIP@BA = 1 mg/mL) with and without PVA (MW = 146000-186000) (f). The particle sedimentation was monitored by measurement of absorbance of visible light at 600 nm.

Under the optimized condition for settling the MIP@BA particles, the NIP@BA particles behaved somewhat differently when their sedimentation in the same solvent was investigated. The addition of PVA showed almost no effect on the sedimentation of NIP@BA, which by itself exhibited a high colloidal stability (Figure 6f). To find out why PVA was not able to accelerate the NIP particle sedimentation, we used a glycoprotein, HRP as a macromolecular probe to compare the amount of accessible boronic acid on both the MIP@BA and NIP@BA particles.29As shown in Figure 7, the MIP@BA particles displayed much higher HRP binding than the NIP@BA particles. Therefore, the MIP@BA particles have more accessible boronic acid groups on surface than the NIP@BA


27


particles. As a result, the more abundant boronic acid groups make MIP@BA particles to react more easily with PVA to cause faster particle sedimentation.

60

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30

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MIP@BA

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Figure 7. Uptake of HRP (100 μg/mL) by 3 mg of MIP@BA and NIP@BA in phosphate buffer at pH 9.

3.3. Reversibility of boronate ester bond To verify that the boronate ester bond formed between the boronic acid-modified nanoparticles and the cis-diol groups in PVA is reversible, we added fructose to Si@BA nanoparticles that have been settled by PVA at pH 9. After addition of fructose, the previously precipitated particles disassembled and was turned into a stable particle


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suspension (Figure 8). This result indicates clearly that the boronate ester bond formed at pH 9 is reversible, as the fructose added was able to displace PVA from the boronic acid-modified nanoparticles. At pH 6, fructose did not show any effect on the colloidal stability of the Si@BA nanoparticles, because no boronate ester bond can form at this pH.

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Figure 8. (a) Sedimentation of Si@BA nanoparticles in PVA solution before and after addition of fructose. (b) Photo imagines of Si@BA nanoparticle suspension (CSi@BA = 1 mg/mL) in PVA solution (CPVA = 0.5 mg/mL) before and after addition of fructose.

3.4. Separation of molecular target and recycling of MIP nanoparticles


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Given that both template binding and aggregation of MIP particles can be controlled by pH adjustment, it was intriguing for us to explore the possibility of achieving selective molecular separation as well as recovery of MIP particles by simply adjusting the solvent pH. Using propranolol as a molecular target, we designed a flow chart shown in Scheme 1 to illustrate the process. The MIP@BA particles were first exposed to propranolol in an aqueous buffer adjusted to pH 9 to allow specific target binding. In the second step the MIP particles were precipitated by adding PVA solution. After removing the supernatant, an acidic buffer was added to the particle sediment to release the bound propranolol. The MIP particles remained as a sediment due to the formation of new, inter-particle boroxine bonds in the acidic buffer. After removing the supernatant, the MIP@BA particles became ready for the next round of molecular separation. Figure 9 shows the propranolol binding measured for the MIP@BA particles after several rounds of recycling. It is clear that the recycled MIP particles exhibited similar propranolol binding at pH 9, and complete release of the bound propranolol when the pH of solvent was adjusted to 4 (Figure 9). The negative value of propranolol binding measured at pH 4 is somewhat odd. The extra propranolol released can be explained as coming from the residual template that has not


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been completely washed away after the MIP preparation. Most importantly, these experimental results confirmed the feasibility to realize selective molecular separation with recycled MIP nanoparticles, and both the molecular separation and nanoparticle recycling can be achieved without involving special filtration or centrifugation instruments. As PVA is a commonly used, low cost industrial material, the new method for selective molecular and nanoparticle separation is deemed to have a potential in future applications.

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Acetonitrile+buffer, pH=9 Acetonitrile+buffer, pH=4

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6 4 2 0 1

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Figure 9. Propranolol binding to recycled MIP@BA particles measured in a mixture of acetonitrile and phosphate buffer, pH 9 and 4 (1:1, v/v).


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4. CONCLUSIONS In this work, we have developed a new approach to facilitate separation and recycling of functional nanoparticles based on pH-controlled reversible covalent bonds between boronic acid-modified nanoparticles and flexible PVA polymer chains. Using inorganic silica nanoparticles and MIP nanoparticles as two examples, we first studied the sedimentation behavior of these nanoparticles upon addition of PVA under different experimental conditions. After establishing the optimal conditions for nanoparticle separation, we demonstrated that the propranolol-imprinted nanoparticles can be used to separate propranolol from aqueous solution by PVA-mediated particle sedimentation. Furthermore, the MIP nanoparticles can be regenerated by removing propranolol and PVA, and reused in a second round of molecular separation. Using pH-induced formation of boronate ester versus boroxine bonds, both hard inorganic nanoparticles and soft MIP particles can be easily recovered through simple particle sedimentation without involving specialized heavy instrument. The new method can be useful when nanoparticle


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separation and recovery needs to be achieved in certain underdeveloped or remote areas, or to handle a large sample volume.

ASSOCIATED CONTENT

Supporting Information. SEM images of nanoparticles.

AUTHOR INFORMATION

Corresponding Author *Bin Lu, Email: [email protected] Tel: +86 27 83691809; *Lei Ye, Email: [email protected] Tel: +46 462229560.

Present Addresses † Key

Laboratory of Interface Science and Engineering in Advanced Materials (Taiyuan

University of Technology), Ministry of Education, Taiyuan 030024, China

ORCID Chen Liu: 0000-0001-7829-6583


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Haiyue Gong: 0000-0002-9357-7364

Weifeng Liu: 0000-0002-1501-7716

Bin Lu: 0000-0003-4691-3326

Lei Ye: 0000-0002-3646-4072

ACKNOWLEDGMENT This work was supported by the Swedish Research Council FORMAS (grant number 2016-00591), the Swedish Foundation for International Cooperation in Research and Higher Education (grant number CH2015-6254) and the National Science Foundation of China (grant number 21611130030). Chen Liu thanks the China Scholarship Council for a visiting scholarship (grant number (201706160072). Chen Liu is an exchange student in Lund University and a PhD candidate in Huazhong University of Science and Technology, China.


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