pH-responsive polymer assisted aptamer functionalized magnetic nanoparticles for specific recognition and adsorption of proteins

Analytica Chimica Acta xxx (xxxx) xxx

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pH-responsive polymer assisted aptamer functionalized magnetic nanoparticles for specific recognition and adsorption of proteins Liping Zhao, Linsen Li, Chao Zhu, Murtaza Ghulam, Feng Qu* Key Laboratory of Molecular Medicine and Biotherapy, School of Life Science, Beijing Institute of Technology, 5 South Zhongguancun Street, Beijing, 100081, China

h i g h l i g h t s

g r a p h i c a l a b s t r a c t


A new adsorbent based on pHresponsive polymer assisted aptamer functionalized magnetic nanoparticles was developed for recognition and adsorption of proteins.
The adsorbent exhibited excellent adsorption capacity because of the synergistic effect of and pH-responsive polymer.
The adsorption and desorption process could be regulated through varying environmental pH.

a r t i c l e i n f o

a b s t r a c t

Article history: Received 19 July 2019 Received in revised form 7 October 2019 Accepted 2 November 2019 Available online xxx

A new adsorbent based on pH-responsive polymer assisted aptamer functionalized magnetic nanoparticles was developed for specific recognition and efficient adsorption of proteins. Arising from the synergistic effect of specific affinity of apatamer on protein and tunable hydrophobic/hydrophilic property of pH-responsive polymer, the adsorbent exhibited excellent adsorption capacity for target protein. Notably, because of the pH-responsive property of the polymer, the adsorption and desorption process could be regulated through varying environmental pH. The resultant adsorbent that immobilized with lysozyme binding aptamer was successfully applied in specific recognition and efficient adsorption of lysozyme in egg white samples and good recovery results in the range of 95.2e103.2% were obtained. Moreover, the adsorbent immobilized with cytochrome C binding aptamer also exhibited satisfactory adsorption to cytochrome C. The synergistic effect of pH-responsive polymer and aptamer promoted the recognition selectivity and adsorption capacity to target protein, illustrating a facile way for construction of more specific protein adsorbents. © 2019 Elsevier B.V. All rights reserved.

Keywords: Aptamer functionalized magnetic nanoparticles pH-responsive polymer Protein recognition and adsorption Lysozyme

1. Introduction Proteins are essential building blocks in human life and closely associated with various life activities [1]. Some proteins have

* Corresponding author. E-mail address: [email protected] (F. Qu).

important physiological functions, such as lysozyme plays a key role in mediating protection against microbial invasion, and cytochrome C, an intermediate in apoptosis, is indispensable in electron transport chain [2]. There is an ongoing need for distinguishing particular proteins with special functions from the complex matrices such as cells, tissues and serum in various applications [3]. The development of diverse methods and materials for efficient

https://doi.org/10.1016/j.aca.2019.11.001 0003-2670/© 2019 Elsevier B.V. All rights reserved.

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adsorption and recognition of proteins is increasingly significant for scientific researchers and application developers. Numerous materials including molecularly imprinted polymers, carbon nanotubes, magnetic nanoparticles (MNP), silica-based materials and many others have been developed as adsorbents for proteins adsorption and recognition [4e7]. Among them, MNP is prominent for its easy preparation, high specific surface area and fast separation by magnetic force [8,9]. Many efforts have been carried out on functional modifications of MNP to improve its selectivity and efficiency. Polymer modified MNP has also been proposed and used for different targets adsorption for it can provide many different functional groups to increase the adsorption capacity through electrostatic, hydrogen bond and hydrophilic/ hydrophobic interactions [10e14]. The stimuli-responsive polymer can change its properties with external stimuli, when it is modified on MNP for protein adsorption, remote control of the adsorption capacity can be realized [15e18]. A thermo-responsive polymer MNP was synthesized by Zhang’s group, in which high adsorption capacity was achieved at lower temperature with thermo-sensitive polymer poly (N-isopropylacrylamide) [17]. Guo et al. realized gascontrolled protein adsorption with CO2-responsive polymer brushes poly(N,N-diethylamino-ethylmethacrylate) modified MNP [19]. Poly (2-diisopropylaminoethyl methacrylate) (pDIPAMA), a pH-responsive polymer, can change its hydrophobic/hydrophilic property in different pH [20], which exhibits potential in tunable interaction with protein, and there is seldom related report about pDIPAMA modified MNP for protein adsorption. Aptamers, viewed as “chemical antibodies”, are short singlestranded oligonucleotides, which offer advantages of high affinity, easy synthesis, high purity, low cost as well as facile modification with different groups (-NH2, eSH) [21,22]. Aptamers can specifically interact with their relevant target molecules with high affinity through van der Waals forces, hydrogen bonds, and electron acceptor-donor interactions [23]. What’s more, aptamers have adaptable two or three-dimensional structures when binding with target molecules, which make them much more effective and selective for target recognition [24]. Thus, aptamer based materials can be used as alternative adsorbents for targets recognition. In addition, the applications of aptamer-functionalized MNP as adsorbents for thrombin, vascular endothelial growth factor, human neutrophil elastase and plenty of other proteins have been reported [25e29]. Although, the introduction of aptamer to MNP can improve the specific recognition of target proteins, there are increasing demands for adsorption capacity improving considering practical applications. Recently, Elham Shoghi et al. combined aptamer based MNP and polymer together for high throughput protein recognition in which nanosized aptameric cavities was imprinted on the surface of MNP [30]. In that, due to the extra polymerization procedures after the immobilization of aptamers, the synthesis process was a little complex, also cost lots of protein template, and especially spent about 12 h for protein completely desorption. In fact, simple and easy operated methods with no extra cost of protein template and fast desorption of protein are definitely welcomed. In this work, we aimed to develop new adsorbent based on aptamer functionalized MNPs with stimuli-responsive polymer assisted interaction for protein specific recognition and effective adsorption. Lysozyme was selected as the model target, and its binding aptamer reported in literature was employed to improve the specific recognition ability [31]. A pH-responsive polymer poly (allyl glycidyl ether-2-diisopropylaminoethyl methacrylate) (p(AGE-DIPAMA)) was utilized to increase the tunable interaction. Lysozyme binding aptamer (LBApt) was immobilized on pHresponsive polymer p(AGE-DIPAMA) modified MNP (pMNP) which was synthesized through the easy operated free radical polymerization method to construct the adsorbent LBApt-pMNP.

The resultant adsorbent was comprehensively characterized and its recognition selectivity and tunable adsorption capacity and desorption capability were studied. Meanwhile, it was successfully applied to the recognition and adsorption of lysozyme in egg white samples. Furthermore, the constructed adsorbent immobilized with cytochrome C binding aptamer (CBApt-pMNP) also displayed good results of Cyt C on adsorption capacity and recognition selectivity. Our results demonstrated that the specific recognition was realized by the immobilized aptamer and the adsorption capacity was increased with the assistant of the tunable hydrophobic/ hydrophilic property of pH-responsive polymer. The controllable adsorption and desorption of protein providing a sensitive and efficient apatmer-based and pH-responsive polymer assisted adsorbents for protein recognition in real biological samples. 2. Materials and methods 2.1. Materials Lysozyme (Lys), trypsin (Try), cytochrome C (Cyt C), human serum albumin (HSA), conalbumin (Con), pepsin (Pep) and 2diisopropylaminoethyl methacrylate (DIPAMA) were provided by Sigma-Aldrich (St. Louis, USA). Allyl glycidyl ether (AGE), 3-(Trimethoxysilyl) propyl methacrylate (MPS), iron chloride hexahydrate (FeCl3▪6H2O) and azodiisobutyronitrile (AIBN) were bought from Aladdin (Shanghai, China). Ferrous chloride tetrahydrate (FeCl2▪4H2O), methanol and trifluoroacetic acid (TFA) were obtained from InnoChem (Beijing, China). Ammonium hydroxide (NH3▪H2O), tetraethyl orthosilicate (TEOS), sodium chloride (NaCl), disodium tetraborate decahydrate (Na2B4O7▪10H2O), boric acid (H3BO3), magnesium chloride hexahydrate (MgCl2▪6H2O), hydrochloric acid (HCl), tetrahydrofuran (THF), alcohol and glycerol were obtained from Beijing Chemical Corporation (Beijing, China). Ovalbumin (OVA) was provided by Solarbio (Beijing, China). Tris (hydroxymethyl) aminomethane (Tris) was obtained from Amresco (Lardner,USA) and acetonitrile was bought from Fisher Scientific (Massachusetts, USA). Egg samples were purchased from local market. Milli Q (Millipore Co., Massachusetts U.S.A) water was used for the preparation of solutions. All solutions for HPLC analysis was filtered with a 0.45 mm filtration membrane. The 50 -amino-modified ssDNA oligonucleotides (50 -ATC TAC GAA TTC ATC AGG GCT AAA GAG TGC AGA GTT ACT TAG-30 for Lys aptamer and 50 -AGT GTG AAA TAT CTA AAC TAA ATG TGG AGG GTG GGA CGG GAA GAA GTT TAT TTT CAC ACT-30 for Cyt C [31,32]) with a C6 spacer arm were synthesized and purified by Sangon Biological Engineering Technology and Services (Shanghai, China). 2.2. Instruments and conditions Fourier transform infrared (FT-IR) spectra characterization of the synthesized materials was applied on a Shimadzu IRTracer-100 spectrophotometer (wave numbers range was 4000-400 cm1). The morphology of the synthesized pMNP was obtained by JEM2010 Transmission Electron Microscope. Pyris 1 thermogravimetric analyzer (PerkinElmer, Akron, OH) was used for thermogravimetric analysis (TGA) with the temperature ranging from 25 to 900  C and the ramp rate was 25  C min1. The results of vibrating sample magnetometry (VSM) were obtained from a Lakeshore 7307 vibrating sample magnetometer. Dynamic light scattering (DLS) was performed on a Zetasizer Nano ZS (Malvern Instruments). Immobilization amount of LBApt was carried out on Agilent 7100 capillary electrophoresis system (Agilent Technology, USA) with DAD detector. A 75 mm i.d.  50 cm (41.5 cm effective) capillary (Xinnuo Optical Fiber Co., Ltd., Hebei, China) was utilized for the experiments. 50.0 mM boric acid-borax at pH 8.7 was used as

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capillary electrophoresis running buffer. Capillary electrophoresis conditions were described as follows: wavelength: 260 nm; separation voltage: 20 kV; injection: 50 mbar for 15 s. High performance liquid chromatography (HPLC) was performed on a Shimadzu Prominence LC-20A HPLC system (Tokyo, Japan), and an Agela Technologies Venusil XBP C18 column (Tianjin, China, 5 mm, 100 Å, 4.6  150 mm) was used for sample analysis. Mobile phase A and B were composed of water with 0.2% TFA, (v/v) and acetonitrile with 0.1% TFA (v/v), respectively. HPLC conditions were described as follows: wavelength: 280 nm; flow rate: 0.5 mL min1; mobile phases solvent A: solvent B (64.2:35.8, v/v); injection volume: 15.0 mL. 2.3. Synthesis of pMNP Fe3O4 MNP was synthesized according to literature with some modifications [33]. First, FeCl3▪6H2O (5.4 g) and FeCl2▪4H2O (2.0 g) were dissolved in water (100.0 mL) in a round bottom flask. Then 110.0 mL NH3▪H2O was added dropwise in 30 min to adjust pH to 12.0, then the reaction was achieved with stirring for 3 h in an oil bath at 70  C. Finally, Fe3O4 MNP was obtained by washing of the products through a magnet with water and alcohol to remove the unreacted solvent until the pH value changed to 7.0. The synthesized products were dried under 45  C and then collected for further use. In order to increase the dispersity of Fe3O4 MNP, Fe3O4@SiO2 was synthesized as follows: 0.5 g Fe3O4 MNP, 100.0 mL alcohol, 25.0 mL water, 2.5 mL NH3▪H2O and 5.0 mL TEOS were added into a round bottom flask and stirred for 24 h. And then Fe3O4@SiO2 was obtained after washing with water and alcohol until the pH value changed to 7.0, and dried at 45  C. MPS was used to introduce vinyl groups on Fe3O4@SiO2. 200.0 mg Fe3O4@SiO2, 100.0 mL alcohol and 4.0 mL MPS were added into the flask and reacted 24 h under stirring. Fe3O4@SiO2@MPS was obtained by using water and alcohol to wash the product in turn and dried under 45  C. pMNP was synthesized through free radical polymerization method with the initiator AIBN. The reaction occurred in a 3-neck flask with the protection of nitrogen. 100.0 mg Fe3O4@SiO2@MPS, 940.0 mg AGE, 200.0 mg DIPAMA, 4.0 mg AIBN and 20.0 mL THF were put into the flask with magnetic stirring at 60  C for 24 h. Then the product was rinsed with water and alcohol in turn and dried in the vacuum oven at 50  C. pMNP-1-7 represented different mass ratio between DIPAMA and AGE (Table 1), and the synthesis process was same as described above.

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the solution (10.0 mM aptamer in 10.0 mM Tris, 0.1 M NaCl and 5.0 mM MgCl2, pH 7.4) at 95  C for 5 min and leaving it to stand at room temperature. 15.0 mg pMNP and 940.0 mL 10.0 mM aptamer solution were mixed and stirred for 24 h to construct LBApt-pMNP. Then 2.0 mL 1.5 mg mL1 HSA solution was mixed with LBAptpMNP to block the unreacted epoxide groups. Finally the obtained LBApt-pMNP was stored in 10.0 mM pH 7.4 PBS at 4  C for further use. Aptamer immobilization amount was calculated through the aptamer concentration before and after the immobilizing reaction which was measured by capillary electrophoresis. 2.5. Preparation and analysis of egg samples Eggs were purchased from local market. Egg white was collected and diluted 40 folds with the buffer containing 10.0 mM Tris, 0.1 M NaCl and 5.0 mM MgCl2 (pH 6.7) and stored as sample at 4  C for following experimental use. The disposed egg white samples were spiked with 20.0, 50.0 and 80.0 mg mL1 Lys, respectively. LBAptpMNP and each 2.0 mL sample solution were mixed and stirred for 2 h at room temperature for protein adsorption. After adsorption process, LBApt-pMNP was collected through an external magnet, and the supernatants were saved for further analysis. Subsequently, the adsorbent materials were washed by 2.0 mL 1.0 M NaCl solution (pH 7.0) for 10 min to desorb Lys. The Lys in supernatants and washed solution were analyzed by UVevis spectra at 280 nm, which represented the amount of unadsorbed and adsorbed Lys, respectively. 2.6. Adsorption and desorption efficiency calculation The adsorbents capacity was expressed by adsorption efficiency for comparison, which was calculated by the following equation:

Adsorption efficiency ¼

A0 A1  100 % A0

(1)

A0 was the absorbance of Lys before adsorption, and A1 was the absorbance of the supernatants after adsorption process. Desorption efficiency was evaluated by recovery, which was calculated by:

Recovery ¼

A2  100 % A0 A1

(2)

A2 was Lys absorbance of the supernatants regenerated after desorption. All the absorbance was determined by UVeVis spectroscopy under 280 nm.

2.4. Construction of LBApt-pMNP

3. Results and discussion

Amino group modified LBApt was immobilized on pMNP based on the reaction of eNH2 and epoxide group in AGE. Prior to immobilization, the oligonucleotides were renatured by heating

3.1. Construction and characterization of LBApt-pMNP pMNP was synthesized through free radical polymerization

Table 1 Effect of the pMNP composition on adsorption efficiency.a LBApt-pMNP

Fe3O4 (mg)

AGE (mg)

DIPAMA (mg)

Adsorption efficiency (%)

LBApt-pMNP-1 LBApt-pMNP-2 LBApt-pMNP-3 LBApt-pMNP-4 LBApt-pMNP-5 LBApt-pMNP-6 LBApt-pMNP-7

100.0 100.0 100.0 100.0 100.0 100.0 100.0

313.0 313.0 313.0 313.0 156.0 616.0 939.0

0 195.0 282.0 585.0 195.0 195.0 195.0

48.8 ± 0.1 68.7 ± 0.1 52.9 ± 0.2 61.0 ± 0.1 47.3 ± 0.1 59.0 ± 4.2 91.8 ± 1.3

a Experimental conditions: adsorption conditions: Lys concentration was 0.2 mg mL1, and the adsoprtion was occurred at pH 6.7 buffer (10.0 mM Tris-HCl with 0.1 M NaCl and 5.0 mM MgCl2) for 2.0 h; desorption conditions: 1.0 M NaCl (pH 7.0) for 10 min.

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method with AGE and DIPAMA as monomers and AIBN as the initiator, and the schematic diagram of the synthesis process was exhibited in Fig. S1. The FT-IR spectra results were shown in Fig. 1. FeeO stretching was found at 588.3 cm1 in all these three spectrums, demonstrating that the structure of the substrate Fe3O4 MNP was not affected after the modifications (Fig. 1a). The peak at 1093.6 cm1 established that SiO2 was coated on the substrate nanoparticles. C]C stretch of the Fe3O4@SiO2@MPS found at 1506.4 cm1 was due to the introduction of vinyl groups on to Fe3O4@SiO2 (Fig. 1b). In FT-IR spectrum of pMNP (Fig. 1c), other than the absorption peaks described above, the peaks found in 1469.8 and 2933.7 cm1 were ether group in AGE, and the peaks in 1267.2 cm1 and 1730.1 cm1 were attributed to the asymmetrical stretching vibration of epoxy group in AGE and stretching vibration absorption of eC]O in DIPAMA, respectively, which demonstrated the successfully conjugated of the monomer onto the nanoparticles. The diameter results were obtained from DLS measurement and shown in Fig. S2, which exhibited that the diameter of pMNP (126.0 ± 20.7 nm) was much larger than that of Fe3O4 MNP (83.8 ± 1.1 nm), proving that the polymer was successfully modified on MNP. The morphology of pMNP was displayed in Fig. S3, demonstrating that pMNP exhibited a uniform spherical morphology. After the synthesis of pMNP, LBApt was immobilized on pMNP to construct LBApt-pMNP (Fig. S4). The immobilization amount of LBApt was determined through capillary electrophoresis. Different concentrations of aptamer solutions (0.2, 2.0, 3.0, 5.0, 8.0, and 10.0 mM) were used for obtaining the calibration curve (Fig. S5). The concentration of the aptamer solution before and after immobilization were 10.0 mM and 2.4 mM, respectively, showing that 7.6 mM aptamer was immobilized on pMNP. Normally, the content of Fe3O4 in LBApt-pMNP is positively proportional to the magnetic response of LBApt-pMNP. The VSM results in Fig. 2 displayed that magnetization curves of the synthesized nanoparticles were all in the shape “S”, and were all symmetric through origin, illustrating that they were all superparamagnetic. Saturated magnetizing strength of Fe3O4, Fe3O4@SiO2@MPS, pMNP and LBApt-pMNP were 66.9, 38.6, 36.5 and 34.4 emu g1, respectively, and the decrease was caused by the modification of monomers and immobilization of aptamers onto their surface, but the retained magnetism was enough to realize the fast separation under magnetic field. Thermogravimetric analysis (TGA) was utilized to identify the preparation of Fe3O4, Fe3O4@SiO2@MPS, pMNP and LBApt-pMNP (Fig. S6). Because of the small amount of residual water, there was a slight weight loss of all of them at around 100  C. Then

Fig. 1. FT-IR spectra of Fe3O4 (a), Fe3O4@SiO2@MPS (b), pMNP (c).

Fig. 2. Magnetization curves obtained by VSM: Fe3O4 (a), Fe3O4@SiO2@MPS (b), pMNP (c), LBApt-pMNP (d).

obvious weight loss of pMNP and LBApt-pMNP were observed at 500e600  C, which resulted from the degradation of polymer. At 900  C, the weight loss was more pronounced for LBApt-pMNP and it was reasonable due to the degradation of the immobilized aptamer. The results of TGA further proved the successful construction of LBApt-pMNP. 3.2. Effects on LBApt-pMNP adsorption capacity Some metal ions which have effect on aptamers structure, were often used in aptamer selection process. Here two kinds of metal ions 0.1 M NaCl and 5.0 mM MgCl2 were used to increase aptamer affinity [34] and improve adsorption efficiency. In order to obtain high adsorption efficiency of LBApt-pMNP for Lys, key factors of pMNP composition, amount of pMNP, concentration of Lys, adsorption time and adsorption pH were investigated. Considering the pH-sensitive polymer p(AGE-DIPAMA) in LBApt-pMNP could increase the interaction between Lys and LBApt-pMNP: DIPAMA block could tune the interaction between LBApt-pMNP and Lys and AGE block could influence the aptamer immobilization amount, the influence of different compositions of DIPAMA and AGE in pMNP on adsorption capacity were studied. Seven kinds of pMNP were synthesized based on different mass ratio of DIPAMA and AGE. 15.0 mg of each pMNP was added in 940.0 mL aptamer solution (10.0 mM) with stirring for 24 h to prepare LBApt-pMNPs (LBApt-pMNP 1e7), their adsorption efficiency were calculated and given in Table 1. Comparing the adsorption efficiency of LBApt-pMNP 1e4 in which AGE amount was kept at 313.0 mg and DIPAMA amount was increased from 0 to 585.0 mg gradually, it was found that LBAptpMNP-2 with DIPAMA weight of 195.0 mg gave the highest adsorption efficiency 68.7%. Whereas LBApt-pMNP-1 without the monomer DIPAMA exhibited a less pronounced adsorption efficiency (48.8%), which indicated that DIPAMA indeed had a significant function in adsorption of Lys. The impact of AGE amount was also studied by keeping the amount of DIPAMA at 195.0 mg, and increasing the amount of AGE from 156.0 to 939.0 mg (LBApt-pMNP 5, LBApt-pMNP 2 and LBApt-pMNP 6e7). The results showed that the adsorption efficiency reached peak value 91.8% with AGE amount of 939.0 mg in LBApt-pMNP-7, which was obviously superior to the other three. Thus LBApt-pMNP-7 was utilized, and if there was no special illustration, LBApt-pMNP was LBApt-pMNP-7 in following experiments. In addition, the influence of amount of pMNP on Lys adsorption efficiency was also investigated, which was displayed in Fig. 3A. The amount of pMNP also had a significant influence on adsorption efficiency, for it provided the epoxide groups which influence the amount of the immobilized aptamer. The adsorption efficiency

Please cite this article as: L. Zhao et al., pH-responsive polymer assisted aptamer functionalized magnetic nanoparticles for specific recognition and adsorption of proteins, Analytica Chimica Acta, https://doi.org/10.1016/j.aca.2019.11.001

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Fig. 3. Influence on adsorption efficiency: pMNP amount in LBApt-pMNP (A), Lys concentration (B), adsorption time (C) and solution pH (D). Other experimental conditions were same as Table 1.

increased with the improvement of the amount of pMNP from 0 to 15.0 mg, reaching the highest at 15.0 mg. When pMNP amount exceeded 15.0 mg, the adsorption efficiency reduced. Therefore, 15.0 mg pMNP was used in the subsequent studies. Fig. 3B displays the adsorption isotherm of LBApt-pMNP. It was evaluated that the adsorption efficiency increased when Lys concentration increased from 0.0 to 0.2 mg mL1, while a slight change was obtained when the concentration exceeded 0.2 mg mL1. Considering a higher adsorption efficiency, 0.2 mg mL1 was utilized finally. The adsorption kinetics of LBApt-pMNP was shown in Fig. 3C. More than half of Lys was captured by LBApt-pMNP within 20 min, which was attributed to the high affinity between aptamer and Lys. The maximum adsorption efficiency for Lys was obtained within 120 min, and did not increase anymore with the increase of adsorption time, which could be attributed to the assistant of the polymer. For some reported polymer based adsorbents that based on the hydrophobic or hydrophilic weak interaction, the adsorption time was as long as 60e3600 min [35e38]. The interaction between aptamer and Lys was strengthened under the assistant of hydrophilic property of the polymer, thus the adsorption efficiency increased with time gradually. Finally, the adsorption time 120 min was selected for the following experiments. In our view, due to the pH-responsive polymer existing in LBApt-pMNP, tunable adsorption capacity could be realized with changing of different pH values. Thus, the influence of solution pH on adsorption efficiency was further studied. Fig. 3D illustrates that when pH changed from 5.9 to 6.7, a significant adsorption efficiency increase was observed, which changed from 57.8% to 91.8% sharply. When pH was at 7.1, the adsorption efficiency decreased obviously till pH 8.1. This reflected the strong function of pH-responsive property of the monomer DIPAMA in LBApt-pMNP. Since DIPAMA had a narrow protonated hydrophilic pH range of 6.5e6.8, when pH was lower than 6.8, tertiary amino groups in DIPAMA block could be protonated, and the polymer became hydrophilic. When pH increased, the tertiary amino groups were deprotonated, and

turned the polymer to be hydrophobic, hence when pH was higher than 6.8, the shrinking polymer might prevent the interaction between the immobilized aptamer and Lys (Scheme 1). However, when pH was lower than 6.8, the polymer appeared flexible, and the synergistic effect of the hydrophilic property of polymer and high affinity of aptamer on Lys increased the adsorption efficiency. This illustrated that by tuning of solution pH, the adsorption efficiency of Lys could be tuned. Moreover, LBApt-pMNP-1 without DIPAMA monomer showed much lower adsorption efficiency (Table 1) proving that pH-responsive polymer indeed contributed to the improvement of adsorption efficiency. So when the proposed adsorbent is applied in real sample analysis, pH which could tune the adsorption efficiency is a key factor that needs to be taken into consideration.

3.3. Effects on LBApt-pMNP desorption capability Desorption of target protein from the adsorbent is a significant step in the entire protein separation process. Thus, the influence of desorption conditions of desorption solvent, desorption time and solution pH on the recovery of Lys was investigated. Five kinds of

Scheme 1. Illustration of LBApt-pMNP for Lys adsorption under different pH.

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desorption solvents including 1.0 M NaCl solution, acetonitrile, methanol, alcohol and glycerol were utilized. In Fig. S7, the results showed that except for NaCl solution had the highest recovery reaching 102.8%, other four were all lower than 25.0%, revealing that the strong interaction between immobilized aptamer and Lys was sharply reduced under the high concentration of salts which could be attributed to the reduction of charge interactions between aptamer and Lys [39]. Moreover, compared to those organic solvents, NaCl solution had the least influence on protein structure and function. Also, the influence of concentration of NaCl and different kinds of ions on desorption was investigated in detail. Fig. S8 showed that when NaCl concentration was 0.1 M, the recovery was 22.3%, which was far lower than 1.0 M NaCl (102.8%) and 2.0 M NaCl (97.4%). It illustrated that an appropriate ionic strength was beneficial for desorption of Lys. In addition, 1.0 M KCl (68.5%) and 1.0 M MgCl2 (97.1%) were also investigated, while lower recoveries were obtained. Thus, 1.0 M NaCl was selected for Lys desorption. The effect of desorption time in the range of 0e60 min was displayed in Fig. S9, and the recovery had an obvious improvement at 10 min, and did not increase anymore when desorption time increased further, illustrating a high concentration of NaCl could strongly interrupt the interaction between immobilized aptamer and Lys in a short time. Thus, using 1.0 M NaCl as the solvents, most of Lys desorption could be completed in 10 min, which was applied in the following experiments. The influence of solution pH on Lys desorption was studied in detail as well (Fig. S10). When pH of NaCl solution changed from 6.5 to 7.0, the recovery increased obviously from 72.9% to 102.8%. And then it decreased to 84.0%, when pH increased continually to 9.5. This might be attributed to the decrease in hydrophilicity and increase of hydrophobicity of the shrinking polymer at pH 7.0, which interrupted the hydrophilic interaction between the adsorbent and Lys and then resulted in the weakening of interaction between LBApt-pMNP and Lys, so a high recovery was obtained. Therefore, a fast desorption process could be accomplished through

interrupting the interaction of aptamer on protein and tuning the hydrophilicity of the polymer.

3.4. Selectivity, reusability and stability Four proteins including HSA, Pep, Con and OVA were utilized as the competitors of Lys to evaluate the selectivity of the constructed adsorbent LBApt-pMNP. The adsorption efficiency of LBApt-pMNP for HSA, Pep, Con and OVA were all lower than 16%, while Lys reached 91.8% (Fig. 4A), which illustrated the high selectivity of LBApt-pMNP for Lys. It also proved that the immobilized aptamer had a high affinity on its relevant protein. The results displayed that LBApt-pMNP as an adsorbent was capable of selectivity recognition of lysozyme over other proteins. The selectivity of the proposed adsorbent to distinguish Lys from other proteins in complicated real matrix was also investigated by spiking the determined amount of Lys in diluted fetal bovine serum (FBS) (Fig. S11). Gel electrophoresis analysis

Fig. 5. HPLC chromatograms of egg white sample (diluted 40 fold) (a), egg white sample spiked with 80.0 mg mL1 Lys (b), egg white sample after being treated with LBApt-pMNP (c), and egg white Lys deported in release solution (d).

Fig. 4. Selectivity (A), reusability (B) and stability of LBApt-pMNP for Lys adsorption (C) and specific recognition of CBApt-pMNP to Cyt C (D). Experimental conditions were same as Table 1.

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Table 2 Recoveries of Lys in egg white samples. Sample

Detected (mg mL1)

Added (mg mL1)

Found (mg mL1)

Recovery (%)

Egg-1

61.2 ± 10.3

Egg-2

76.8 ± 13.5

Egg-3

79.6 ± 7.2

20.0 50.0 80.0 20.0 50.0 80.0 20.0 50.0 80.0

80.6 ± 0.9 109.1 ± 3.9 143.8 ± 2.5 95.8 ± 2.1 127.0 ± 0.3 155.1 ± 1.9 99.4 ± 7.0 129.5 ± 2.0 155.9 ± 1.4

97.2 ± 1.2 95.7 ± 3.6 103.2 ± 1.8 95.2 ± 2.2 100.3 ± 0.2 97.8 ± 1.2 99.0 ± 7.6 99.8 ± 1.5 95.4 ± 0.9

displayed that the intensity of Lys band was significantly weaker (line supernatants) after treated by LBApt-pMNP. Therefore, Lys was almost completely captured by LBApt-pMNP and removed from FBS (line crudes), which illustrated LBApt-pMNP had a good adsorption selectivity in the presence of FBS, and also proved that it has the potential in the application of complex matrix. Reusability is also important for the assessment of the Lys adsorption capacity of LBApt-pMNP. Fig. 4B illustrates that repeated adsorption of Lys with the constructed LBApt-pMNP could be accomplished in a pH-responsive manner. After LBApt-pMNP was reused repeatedly for 5 times, the adsorption efficiency was still kept above 90%, illustrating a good reusability. Furthermore, when it was stored in PBS buffer for 4 weeks, the adsorption efficiency remained more than 70% (Fig. 4C), proving a good stability of the proposed LBApt-pMNP. 3.5. Verification of the adsorption of CBApt-pMNP on CytC Due to the immobilization of aptamer on pMNP, the specific recognition of its related protein could be realized. In order to investigate the recognition of immobilized aptamer on its corresponding protein, a new adsorbent based on immobilizing Cyt C binding aptamer (CBApt) on pMNP (CBApt-pMNP) was constructed. The recognition selectivity was studied with four proteins HSA, Pep, Con and OVA as the competitors of Cyt C. Fig. 4D shows that except OVA had an adsorption efficiency of 37.9%, other three were lower than 20.0%, which was far lower than Cyt C, illustrating that CBAptpMNP had a good selectivity on Cyt C and could realize the specific adsorption efficiently. Both adsorbents of LBApt-pMNP and CBAptpMNP showed the specific recognition and high adsorption capacity for the targets Lys and CytC, which are contributed by the synergistic effect of high affinity between given aptamer on its relevant protein and the tunable hydrophobic/hydrophilic property of polymer. 3.6. Application of LBApt-pMNP for adsorption of Lys in egg samples In order to evaluate the feasibility of LBApt-pMNP adsorbents for Lys adsorption in real samples, it was applied in Lys recognition and adsorption of three Lys spiked egg white samples. The amounts of Lys in egg white samples were detected through HPLC method, in which the linear relationship for Lys was 0.01e1.0 mg mL1 (Fig. S12) and limit of detection was 5.0 mg mL1 (S/N ¼ 3), showing an acceptable sensitivity for analysis of Lys in real sample. HPLC chromatograms of the egg white samples were shown in Fig. 5. Fig. 5a exhibits the Lys existed in diluted 40 folds egg white sample, whose peak appeared at 12.0 min. When 80.0 mg mL1 Lys was spiked, the peak height increased obviously (Fig. 5b). After the adsorption by LBApt-pMNP, the peak of Lys disappeared (Fig. 5c), proving that Lys in egg white sample was almost totally absorbed. After desorption by 1.0 M NaCl, Lys in supernatants was then

detected (Fig. 5d), illustrating the effective desorption of Lys by NaCl solution. With LBApt-pMNP efficient adsorption, Lys amounts in three egg white samples were analyzed by HPLC, which were found as 80.8 mg/35 g egg-1, 101.4 mg/40 g egg-2 and 105.7 mg/40 g egg-3, respectively, and it was closed to the reported value of 2.5 mg/g [40]. For three Lys spiked samples, the satisfactory recoveries were obtained in the range of 95.2%e103.2% (Table 2), and the RSD were 7.6% which was in a reasonable range. This demonstrated the feasibility of LBApt-pMNP adsorbent for Lys adsorption in real egg samples and confirmed the accuracy of the proposed method.

4. Conclusion The new adsorbent LBApt-pMNP was synthesized through free radical polymerization method, then the aptamer was immobilized for protein recognition, which is much easy operated and cost no template protein and lower desorption time. Moreover, in LBAptpMNP, the recognition selectivity and high adsorption capacity of Lys could be realized by the high affinity of immobilized aptamer on protein and tunable hydrophobic/hydrophilic property of pHresponsive polymer. A rapid desorption could be achieved by utilizing a high concentration of NaCl and tuning the environmental pH. The proposed adsorbents with high adsorption capacity and recognition selectivity have the potential to be applied in the selective recognition and segregation of different proteins. Employing their selective aptamers, target proteins could be adsorbed with high capacity from the complex matrix by changing pH switch. The usage of pH-responsive polymers assisted aptamer functionalized magnetic nanoparticles paves a promising and facile way for the capture and release of different target proteins in real samples.

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

Acknowledgements This study was financially supported by National Natural Science Foundation of China (Grants NO 21675012, 21874010 and 21827810). We also thank Ms Mingming Zhang for all her kindly help and powerful encouragement.

Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi.org/10.1016/j.aca.2019.11.001.

Please cite this article as: L. Zhao et al., pH-responsive polymer assisted aptamer functionalized magnetic nanoparticles for specific recognition and adsorption of proteins, Analytica Chimica Acta, https://doi.org/10.1016/j.aca.2019.11.001

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Please cite this article as: L. Zhao et al., pH-responsive polymer assisted aptamer functionalized magnetic nanoparticles for specific recognition and adsorption of proteins, Analytica Chimica Acta, https://doi.org/10.1016/j.aca.2019.11.001