Double Recognition and Selective Extraction of Glycoprotein Based

Feb 13, 2017 - Boronic acid functionalized graphene oxide (GO-APBA) was first prepared and a template glycoprotein (ovalbumin, OVA) was then immobiliz...
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Double recognition and selective extraction of glycoprotein based on the molecular imprinted graphene oxide and boronate affinity Jing Luo, Jing Huang, Jiaojiao Cong, Wei Wei, and Xiaoya Liu ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.6b14733 • Publication Date (Web): 13 Feb 2017 Downloaded from http://pubs.acs.org on February 14, 2017

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Double Recognition and Selective Extraction of Glycoprotein Based on the Molecular Imprinted Graphene Oxide and Boronate Affinity Jing Luo∗, Jing Huang, Jiaojiao Cong, Wei Wei, Xiaoya Liu The Key Laboratory of Food Colloids and Biotechnology, Ministry of Education, School of Chemical and Material Engineering, Jiangnan University, Lihu Street 1800, Wuxi 214122, Jiangsu, China

Abstract: Specific recognition and separation of glycoproteins from complex biological solutions is very important in clinical diagnostics considering the close relationship between glycoproteins with the occurrence of diverse diseases, but the lack of materials with high selectivity and superior capture capacity still makes it a challenge. In this work, graphene oxide (GO) based molecularly imprinted polymers (MIPs) possessing double recognition abilities have been synthesized and applied as highly efficient adsorbents for glycoprotein recognition and separation. Boronic acid functionalized graphene oxide (GO-APBA) was first prepared and a template glycoprotein (ovalbumin, OVA) was then immobilized onto the surface of GO-APBA through boronate affinity. An imprinting layer was subsequently deposited onto GO-APBA surface by a sol-gel polymerization of organic silanes in aqueous solution. After the removal of the template glycoprotein, 3D cavities with double recognition abilities toward OVA were obtained in the as-prepared imprinted materials (GO-APBA/MIPs) owing to the combination of boronate affinity and molecularly imprinted spatial matched cavities. The obtained GO-APBA/MIPs exhibited superior specific recognition toward OVA with imprinted factor (α) as high as 9.5, significantly higher than the corresponding value (4.0) of GO/MIPs without the introduction of boronic acid groups. Meanwhile, owing to the synergetic effect of large surface area of graphene and surface imprinting, high binding capacity and fast adsorption/elution rate of GO-APBA/MIPs toward OVA were demonstrated and the saturation binding capacity of GO-APBA/MIPs could reach 278 mg/g within 40 min. The outstanding *

Corresponding author. Tel: Telephone: 86-510-85917763. Fax: 86-510-85917763. E-mail: [email protected] (J.Luo). 1

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recognizing behavior (high adsorption capacity, highly specific recognition, and rapid binding rate) coupled to the facile and environmental-friendly preparation procedure makes GO-APBA/MIPs promising in the recognition, separation, and analysis of glycoproteins in clinics in the future. Keywords: Molecular imprinting, boronate affinity, double recognition, glycoprotein separation, graphene oxide, sol-gel

Introduction Protein glycosylation is one of the most popular post-translational modifications, which functions critical roles in numerous crucial biological events, including cell– cell interaction, signal transduction, protein folding, and molecular recognition.1,2 It has been demonstrated that glycoproteins are closely associated with many diseases, such as diabetes, cancer, and immune disorders, and so on. To date, glycoproteins have been frequently employed as clinical biomarkers and therapeutic targets in clinical diagnostics. Therefore it is highly valued to discover and detect glycoproteins in biological samples such as serum for the discovery of disease. However, serious matrix interferences and low concentrations of the complex biological samples put great obstacles for the recognition and detection of glycoproteins. Mass spectrometry (MS) has been recognized as a powerful tool for the analysis of glycoprotein, but it is very difficult to recognize glycoproteins directly without pretreating samples owing to the low abundance and poor ionization. Several strategies, hydrazine chemistry,3 hydrophilic interaction chromatography,4 lectin based affinity chromatography5 and boronic acid-based affinity chromatography (BAC)

6-10

, have been developed to

achieve the enrichment of glycoproteins. Although some success has been achieved employing these methods, several limitations still need to be resolved. For example, hydrazine chemistry is only applicable for N-linked glycoproteins, whereas hydrophilic interaction chromatography is significantly minimized by its deficient selectivity and recovery. Boronic acid-based affinity chromatography (BAC) has attracted more and more attention in glycoprotein enrichment. Boronic acid affinity 2

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(boronate affinity) relates to its special reversible covalent interaction between boronic acid and cis-diol-containing compounds (for example, glycoproteins). The boronic acid can covalently bind with glycoproteins under alkaline conditions and form five or six-membered cyclic ester, which dissociate reversibly when the pH of the environmental medium becomes acidic.11,12 Therefore it is easier for boronic acid to adsorb and desorb glycoproteins reversibly with the pH change. Although the boronic acid based materials could capture glycoproteins from general compounds (non-glycoproteins), they could not recognize a specific glycoprotein from its analogs (other glycoproteins or glycopeptides). It is thus highly desirable to develop a new and general sample pretreating approach with high selectivity and excellent tolerance for interference for isolating glycoproteins from real biological samples. Molecularly imprinting has become an outstanding tool for fabricating tailor-made materials with good recognition capacity and high selectivity, and has been frequently employed as a reliable sample pretreatment in the past decade.13 However, the poor performance of imprinting biomacromolecules, such as proteins and polypeptides, has significantly restricted its application in glycoprotein extraction. The inherent problems of imprinting protein are mainly related to the structural complexity and large size of the protein, difficulty in transfer and eluting, complex construction condition and solubility. In spite of these problems, a review reported the recent progress achieved in protein imprinting14, where surface imprinting, epitope imprinting15 and metal-chelating imprinting16 are highlighted. Surface imprinting with the recognition cavities close to or at the surface of MIPs, has been considered as one of the most promising strategies for protein imprinting, which enables easy elution and re-adsorption of the target molecules.17,18 Up to now, various materials, including nanoparticles19,20, nanotubes/nanowires21 and quantum dots (QDs)22, have been used as supporting materials for surface imprinting protein. Recently, graphene23-26 and its derivative GO27-31 have been considered as ideal candidates as supporters for the preparation of surface molecularly imprinted materials.32 Its two-dimensional structure endows a dominant surface-to-volume ratio compared to spherical structures33, which can accommodate more recognition sites. In fact, several MIPs 3

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using graphene/graphene oxide (GO) as supporters for the recognition of proteins have been successfully fabricated,33–36 which demonstrated not only large binding capacity but also fast adsorption dynamics toward the template protein. However, the selectivity of the obtained MIPs towards the target protein is still far from satisfactory. The possible reason is that the target proteins are normally complexed with the imprinted sites via weak non-covalent interactions such as electrostatic, hydrophobic and hydrogen bonding interactions. It has been reported that some imprinted sites in the MIPs via non-covalent bonding easily underwent irreversible shrink or collapse, which had negative effects on their selectivity. Covalently imprinted polymers have demonstrated a better selectivity combining the shape-matching of the cavities and specific recognition groups contained in the MIPs. However, the formed covalent bonds between the template and MIPs were hard to break and normally required harsh washing condition as well as long washing time, which is quite disadvantageous for the removal of the template. Considering the advantages of surface imprinting and boronate affinity, great efforts have been dedicated to the integration of surface imprinting and boronate affinity into one system for developing a novel kind of recognition element, which could significantly improve the specific recognition performance for glycoproteins through a synergistic effect. Rick et al.

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and Bonini et al.

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prepared phenylboronic

acid functionalized MIPs on various supporting matrixes for protein recognition. Lin et al.

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fabricated boronate-functionalized molecularly imprinted monolithic column

for recognizing and enriching glycoprotein. Recently, a novel kind of core-shell structured HRP-imprinted boronic acid-functionalized silica nanoparticles was also prepared by the same group combining surface imprinting on nano-sized supporters with boronate affinity.40 Liu’s group developed a clever boronate affinity-based controllable oriented surface imprinting strategy for both an intact glycoprotein and its characteristic fragments.41-44 The prepared imprinted materials could recognize the template glycoprotein not only from nonglycoproteins, but also from another glycoprotein, showing preferential adsorption toward the template glycoprotein over the non-template glycoproteins. Zhang et al. developed a fluorescence nanosensor 4

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based on the molecularly imprinted spatial structure and boronate affinity for selectively monitoring glycoproteins.45 However, most of the above-mentioned materials suffer from a limited surface, which greatly restricts their capability to capture glycoprotein. In addition, the exact role of boronate affinity was not sufficiently investigated and explained as the recognition performance was not compared with the corresponding imprinted material without the introduction of boronate affinity. Therefore, it is desirable to gain more knowledge and further explore new boronate affinity-based molecular imprinting materials. In this work, combining the merits of surface imprinting, two-dimensional graphene unit and boronate affinity, a new kind of boronate affinity-based glycoprotein imprinting materials using boronic acid functionalized graphene oxide (GO-APBA) as supporting materials was prepared and applied to the recognition and separation of glycoprotein. As far as we are aware, the two-dimensional boronic acid functionalized–graphene based MIPs for glycoproteins have not been reported. The adsorption kinetics, the adsorption capacity, selectivity and the recovery of the resultant imprinted materials towards the template glycoprotein were investigated. The well-designed GO-APBA/MIPs possess the following attractive features: (1) The covalent cyclic ester formed between boronic acids and glycoprotein combining with the molecular imprinted cavities complementary to the shape of glycoprotein endows a double recognition and thus a special affinity for the template glycoprotein, which is highly suitable for the specific recognition and separation of the template glycoprotein from complex biological systems because a number of glycoproteins and non-glycoproteins normally coexists in real samples; (2) The extremely large specific surface area of graphene endows GO-APBA/MIPs a large adsorption capacity (278 mg/g) towards glycoproteins; (3) The reversible covalent interaction between boronic acid and glycoprotein and the thin MIPs layer make the removal and rebinding of template easier, ensuring the fast adsorption/desorption process of glycoprotein and high recovery efficiency; (4) The whole preparation procedure of GO-APBA/MIP composite is simple, robust, environmental friendly, time-saving and of low cost. 5

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2. Experimental Section 2.1Materials Graphite powder, tetramethoxysilane (TMOS), phenyltriethoxysilane (PTEOS), bovine serum albumin (BSA; Mw 66.4 kDa, pI 4.9), horseradish peroxidase (HRP; Mw 44 kDa, pI 7.2), and bovine hemoglobin (BHb; Mw 64 kDa, pI 6.8) were bought from Sinopharm Chemical Reagent Co., Ltd.. Ovalbumin (OVA; Mw 45 kDa, pI 4.7) was purchased from Sigma-Aldrich. 3-Aminophenylboronic acid hydrochloride (APBA) and bromelain (Mw 33 kDa, pI 9.5) were purchased from Beijing J&K Chemical Technology Co., Ltd. Deionized water was used for all experiments in the work. Other chemicals were of analytical grade and used as received. 2.2 Characterization Fourier transform infrared (FTIR) spectroscopy measurement was performed on a FTIR spectrometer (FTLA 2000-104). UV−vis spectroscopy measurement was carried out on a TU-1901 spectrophotometer (Beijing Purkinje General Instrument Co., Ltd.). Scanning electron microscopy (SEM) measurements were carried out on a Hitachi S-4800 field-emission scanning electron microscope. X-ray photoelectron spectroscopy (XPS) measurement was performed on a VG ESCALAB MkII spectrometer. Nitrogen adsorption−desorption isotherms were determined using a Micromeritics ASAP 2050 system. Gel electrophoresis for protein extraction was conducted by regular sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) according to the manual introduction (Bio-Rad, Hercules, CA, USA). Proteins were stained with Coomasie Brilliant Blue R-250. 2.3 Synthesis of boronic acid functionalized graphene oxide (GO-APBA) Graphene oxide (GO) was prepared from graphite powder according to a modified Hummers method. 46 GO dispersion was first prepared by dispersing GO (200 mg) in deionized water (200 mL) under ultrasonic vibration for 60 min. APBA was subsequently added to the above solution. The obtained mixture was heated to 80oC for 24 h under stirring. The resulting boronic acid functionalized graphene oxide (GO-APBA) was separated via centrifugation, washed by deionized water and ethanol 6

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for several times, and dried. 2.4 Synthesis of GO-APBA/MIPs composite GO-APBA/MIPs composites with OVA as the template glycoprotein were prepared by the sol-gel polymerization of organic silanes (TMOS and PTEOS) on the surface of GO-APBA. Typically, 20 mg of GO-APBA was dispersed in 40 mL of PBS buffer (pH 8.5, 10 mM) under ultrasonication and 20 mg of OVA was added, and then the solution was shaken for the immobilization of OVA on GO surface through the formation of covalent cyclic ester at room temperature for 3 h. After the addition of the organic silane monomers (20 mg of TMOS and 60 mg of PTEOS), the pH of the obtained solution was regulated to ∼9.3 with NH3·H2O (28 wt %) and stirred at room temperature for 12 h. The resulted product was collected by centrifugation and washed with deionized water to remove unreacted monomers and adsorbed oligomers. The OVA imprinted GO-APBA/MIPs were subsequently washed with HAc/NaAc solution (pH = 4.0) for several times to extract the embedded glycoprotein until no OVA was detected in the supernatant. The obtained product was then washed by water for several times and finally dried for further use. As a control sample, the corresponding non-imprinted material (GO-APBA/NIPs) was also prepared under the same conditions in the absence of template glycoprotein. In addition, to investigate the roles of the introduced boronic acid group, GO/MIPs composite was prepared using unmodified GO as the supporting material instead of GO-APBA. The other conditions are kept the same with those of GO-APBA/MIPs. 2.5 Binding experiments Kinetics adsorption experiments: GO-APBA/MIPs (or GO-APBA/NIPs and GO/MIPs, 10 mg) was added to OVA solution (0.2 mmol/mL, 10 mL) and incubated for different interval time (2 to 130 min). After centrifugation, the concentrations of OVA in the supernatant were measured on a UV/vis spectrometer at 208 nm. The amount of OVA (Q, mg/g) adsorbed by GO-APBA/MIPs or the control GO-APBA/NIPs and GO/MIPs composite was calculated using the following formula: Q = (C0 − Ce)V/W,

(1)

where C0 and Ce (mg mL−1) are the solution concentrations before and after 7

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adsorption, respectively. V represents the initial solution volume (mL), and W (g) represents the mass of GO-APBA/MIPs or GO-APBA/NIPs, GO/MIPs. Isothermal adsorption experiments: the initial concentration of OVA solutions changed from 0.1 to 1.0 mg mL-1. The adsorption time was kept constant at 40 min. The Langmuir equation was employed to investigate the binding properties of the GO-APBA/MIPs, GO-APBA/NIPs and GO/MIPs, respectively. 2.6 Selectivity experiments The selectivity of the GO-APBA/MIPs or GO-APBA/NIPs and GO/MIPs was evaluated using horseradish peroxidase (HRP), bromelain, bovine serum albumin (BSA), and bovine hemoglobin (BHb) as competitors. Imprinting factor (IF) was used to estimate the recognition capability according to the following equation: IF = QMIP/QNIP

(2)

where QMIP and QNIP represent the adsorption amounts of the template or the competitive proteins on the GO-APBA/MIPs and GO-APBA/NIPs, respectively. 2.7 Regeneration/reuse of GO-APBA/MIPs 10 mg of GO-APBA/MIPs was mixed with 10 mL of OVA solution (1 mg mL-1), and the mixture was shaken at room temperature for 40 min. The recovered GO-APBA/MIPs separated by centrifugation was washed by HAc/NaAc solution (pH = 4.0, 5 × 1 mL) followed by water, dried in vacuum, and reused in the next cycle of adsorption experiments. The adsorption–desorption cycles were repeated five times using the same GO-APBA/MIPs.

3. Results and Discussion 3.1 Synthesis and characterization of GO-APBA/MIPs The general scheme for the synthesis of GO-APBA/MIPs is illustrated in Fig. 1. First, boronic acid as an affinity ligand for glycoprotein immobilization was attached to the surface of GO through the synergistic effect of the π-π interaction and an amide reaction between the amino group of APBA and the epoxy group of GO, leading to boronic acid functionalized graphene oxide (GO-APBA). Secondly, OVA was immobilized onto GO-APBA surface via the formation of cyclic boronate complexes 8

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between boronic acid and the cis-diol group of OVA at the rationally designed pH value of 8.5. After the sol–gel polymerization of TMOS and PTEOS, a polymeric network around OVA was produced on the surface of GO-APBA, leading to the formation of glycoprotein-embedded composite. Finally, washing by HAc/NaAc solution, the cyclic boronate complexes between boronic acid and OVA was destroyed, which facilitated the extraction of the template protein from the sol-gel network. A thin sol-gel coating with recognition sites complementary to OVA in size, shape, as well as functional group orientation was then generated on GO-APBA surface. The created OVA-imprinted cavities could function as favorable OVA recognition sites and thus provide highly specific recognition toward OVA. Such designed glycoprotein-imprinted hybrid materials (GO-APBA/MIPs) will combine several key merits into a single one, including highly selective recognition toward template glycoprotein, large adsorption capacity, fast adsorption/desorption process, simple and environmental-friendly preparation, excellent structural stability and chemical inertness.

Fig. 1 Schematic illustration of the synthesis of GO-APBA/MIPs. As described in many previous works, the template recognition capability is strongly dependent on the preparation conditions of MIPs such as the type and 9

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amount of monomer, crosslinker, and template. In addition, it should be noted that during the sol-gel polymerization process, the bulk polymerization of silane monomers in solution competed with their surface polymerization on GO-APBA, which has a great influence on the thickness as well as homogeneity of the MIPs layer on GO-APBA surface. It is thus significant to tune preparation conditions for constructing thin and homogeneous MIPs layer on GO-APBA surface. By optimizing the preparation conditions, the effect of bulk polymerization has been minimized and a sol-gel MIPs layer with reasonable (150–300 nm) thickness could be obtained on the surface of GO-APBA. The molecular recognition capability can also be adjusted through this optimization. 3.2 Optimization of the synthesis conditions To investigate the influence of monomer amount on the molecular recognition capability, 20 mg of GO-APBA, 10 mg of OVA, and varying monomer amounts (PTEOS + TMOS) were mixed to prepare GO-APBA/MIPs. The mass ratio of TMOS:PTEOS was set as 1:3 to obtain a stable and intact sol-gel MIPs layer based on our experience.

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Their binding capacities were measured and exhibited in Fig. 2A.

It was found that the adsorption capacities (Q) of GO-APBA/MIPs increased with the decreasing total amount of monomer from 200 to 80 mg, suggesting that thinner imprinted polymer layer is beneficial to improve the adsorption capacity. However, the Q value dropped when the amounts of monomer was lower than 80 mg. The possible reason may be that lower amount of sol-gel monomer could not accommodate adequate template molecules and thus induce fewer binding sites in GO-APBA/MIPs. As a result, 80 mg monomer was chosen for preparing GO-APBA/MIPs.

10

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Fig. 2 The effect of the monomer amount (PTEOS + TMOS) (A), the ratio of GO/APBA (B), template protein (OVA) amount (C), and reaction time (D) on the adsorption capacity of GO-APBA/MIPs. As it is supposed that boronic acid of APBA directs the immobilization of OVA on GO surface, the amount of APBA should have a great influence on the amount of the template glycoprotein imbedded in the so-gel network, which in turn affect the adsorption capacity of the final GO-APBA/MIPs. To evaluate the effect of APBA, GO-APBA was prepared with different amount ratios of GO/APBA, which was then used to prepare GO-APBA/MIPs. As shown in Fig. 2B, the adsorption amount gradually enhances with the increasing mass ratios of APBA/GO and reaches equilibrium value when the mass ratio of APBA/GO is 3. It is presumed that the increasing amount ratios of APBA/GO increase the amount of boronic acid functional groups on GO surface, which thus could immobilize more OVA molecules on the surface of GO-APBA, resulting in the increasing number of recognition cavities. The effect of template amount and reaction time was also studied. As shown in Fig. 2C and 2D, the adsorption amount increases with the increasing template amount and a maximum binding amount is obtained at the added amount of 20 mg OVA. As for 11

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the influence of reaction time, the adsorption capacity initially enhances with the elongation of the polymerization time, reaches maximum at 24 h, and then significantly decreases. It is presumed that elongating reaction time might result in an over-thick MIPs layer, and OVA templates are probably embedded too deep and thus become difficult to be removed. 3.3 Characterization of GO-APBA/MIPs FTIR spectra provide a direct evidence for successful preparation of GO-APBA/MIPs, which are illustrated in Fig.3A. GO exhibits strong OH vibration band from 3600 to 3000 cm-1, C=O stretch vibration at 1725 cm-1, C=C skeletal vibration from graphitic domains at 1640 cm-1, C-OH of carboxyl groups at 1421 cm−1, C-O-C at 1225 cm−1 and C–O of epoxy or alkoxy groups at 1065 cm−1. For GO-APBA, the characteristic peaks of B–O stretching mode (1340 cm−1) and C–N stretching at 1243 cm−1 appear in its spectrum, indicating that APBA has been successfully modified onto GO. After the sol-gel polymerization, the new bands in the range of 1140~950 cm-1 characteristic of Si-O-Si vibrations appeared in the spectrum of GO-APBA/MIPs, providing the evidence of silicon network on GO-APBA surface.25 In addition, the sharp peak at 1430 cm−1 was assigned to the characteristic peak of benzene ring at PTEOS. All these peaks confirmed the successful preparation of GO-APBA/MIPs.

Fig. 3 FTIR (A) and XPS (B) spectra of GO, GO-APBA and GO-APBA/MIPs The boron element loading on GO was further investigated by X-ray photoelectron spectroscopy (XPS) and its composition was also provided. Fig. 3B displays the XPS spectra of GO-APBA and GO-APBA/MIPs. XPS spectrum of GO was also provided 12

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for comparison. For GO-APBA, the appearance of characteristic boron peak at 190.4 eV, which was not observed for GO, confirmed the successful modification of GO by APBA. And the mass concentration percentage of boron element was calculated to be about 3.45 %. Successful formation of sol-gel inorganic MIP layer onto GO-APBA surface was indicated by the appearance of Si 1s signal at 103.5 eV together with the almost disappearance of B peak in the XPS spectrum of GO-APBA/MIPs. The combination of FTIR and XPS spectra results corroborated the successful synthesis of GO-APBA/MIPs. Nitrogen sorption and desorption was used to evaluate the surface area of GO-APBA/MIPs. Fig. 4 shows the nitrogen adsorption desorption isotherms of GO-APBA/MIPs

and

GO-APBA/NIPs.

The

specific

surface

areas

of

GO-APBA/MIPs and GO-APBA/NIPs were calculated to be 79.9 m2 g−1 and 3.6 m2 g−1, respectively. In addition, the total pore volume and average pore diameter of GO-APBA/MIPs were 0.39 cm3 g−1 and 19.8 nm, which were much higher than those of GO-APBA/NIPs. The above results showed that GO-APBA/MIPs featured of greater specific surface area, pore volume, and pore size, further evidencing the presence of numerous imprinting cavities in GO-APBA/MIPs.

Fig.4 Nitrogen adsorption desorption isotherms of GO-APBA/MIPs (-■-) and GO-APBA/NIPs (-●-) In addition, the morphological structures of GO, GO-APBA and GO-APBA/MIPs were determined by SEM as shown in Fig. 5. GO exhibits a curved, layerlike structure 13

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with a fairly smooth surface (Fig. 5A) similar to previous literatures. GO-APBA exhibits a similar structure to GO (Fig. 5B). However, after the sol-gel polymerization of PTEOS and TMOS, a rather rough and dense morphology was demonstrated for GO-APBA/MIPs (Fig. 5C), indicating that a MIPs layer has been successfully deposited on the surface of GO-APBA.

Fig.5 SEM images of GO (A), GO-APBA (B) and GO-APBA/MIPs (C) 3.4. Binding properties of GO-APBA/MIPs The binding performance of GO-APBA/MIPs is first studied by measuring the adsorption amount of OVA at different concentrations ranging from 0.1 to 1.0 mg mL-1. As shown in Fig. 6A, the adsorption capacity of GO-APBA/MIPs increases rapidly with the increasing OVA concentrations from 0.1 to 0.6 mg mL-1, and achieves saturation value (278 mg/g) with the concentration value of OVA at 0.8 mg mL-1. Therefore 0.8 mg/mL was selected as the optimum concentration in the following experiments.

Fig. 6. The influence of OVA concentrations (A) and incubation time (B) on the capacities of GO-APBA/MIPs, GO/MIPs and GO-APBA/NIPs. The experimental data from adsorption isotherms were further treated by Langmuir model, which can be expressed according to the following equation: 14

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Ce/Qe=Ce/Qmax+ 1/KLQmax

(3)

where Ce (mg mL-1) is the concentration of OVA at equilibrium in the supernatant after adsorption, and Qe and Qmax (mg g-1) are the adsorption capacity at equilibrium and the maximum adsorption capacity of GO-APBA/MIPs composite, respectively. KL (mL mg-1) is the Langmuir constant associated with the energy of adsorption median binding affinity. The linear fitting of the experimental data to the Langmuir equation yielded a good fit for GO-APBA/MIPs with the regression coefficient (r) higher than 0.98. It is well-known that Langmuir isotherm model is suitable for monolayer adsorption on a surface. It is thus concluded that the adsorption of OVA onto GO-APBA/MIPs may be monolayer adsorption. The binding rate of OVA onto GO-APBA/MIPs was also investigated. The binding kinetics experiments were performed using 0.8 mg/mL OVA solution at different time intervals from 1 to 130 min and the results were presented in Fig. 6B. It was observed that GO-APBA/MIPs showed a fast adsorption profile and the adsorption equilibrium was reached after 40 min. At the first stage of the adsorption process, GO-APBA/MIPs had a large number of empty recognition sites on its surface, enabling the easy and fast adsorption of OVA with less resistance. With the prolonged adsorption time, the adsorption rate slowed down because most of the binding sites had been occupied by OVA molecules, and the adsorption reached equilibrium finally. Previous literatures show that about 10–120 min is needed to achieve rebinding equilibrium for surface imprinted thin films, whereas bulk imprinted materials usually require 12-24 h to achieve adsorption equilibrium. Here, OVA molecules easily approached and diffused into the imprinting cavities which are at the surface or close to the surface of the composites and thus took less time to reach adsorption saturation. In addition to fast rebinding rate, GO-APBA/MIPs also possessed short extraction time. The removal of proteins with high molecular weight is normally difficult for analyst. In our work, the elution process was accomplished using an acidic solution (five times, 5 min each time), which is a great advance compared to the conventional MIPs. This short washing time should be attributed to the easy breaking of boronic ester between OVA and GO-APBA in acidic solution and the advantages of surface 15

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imprinting, which reduces the elution difficulty effectively. The binding experiment of GO-APBA/NIPs was also performed and only a small amount of OVA was adsorbed by GO-APBA/NIPs, which was ascribed to nonspecific adsorption. In the whole investigated adsorption time and OVA concentration, GO-APBA/MIPs adsorbed much more OVA molecules than GO-APBA/NIPs, demonstrating the successful generation of recognition cavities on graphene surface in the sol-gel polymerization reaction, which could specifically recognize OVA. In contrast, there were no recognition sites in GO-APBA/NIPs and nonspecific adsorption thus played a dominant effect. Thus, the binding capacity of GO-APBA/NIPs

was

much

lower.

The

maximum

binding

capacity

of

GO-APBA/MIPs was 278 mg g-1, nearly eight times higher than 32.3 mg g-1 of GO-APBA/NIPs. Such a large adsorption capacity is ascribed to the big surface area of graphene which can accommodate a great number of template glycoproteins and thus create more recognition sites. On the other hand, the thin coatings created over graphene surface and the pH induced switch property of boronic acid in reversibly adsorbing and desorbing glycoproteins ensure a sufficient removal of the template protein molecules. 3.5 Comparison of two imprinted nanoparticles (GO-APBA/MIPs and GO/MIPs) In addition, to fully clarify the role of the introduced boronic acid in the binding performance of GO-APBA/MIPs, GO/MIPs using GO instead of GO-APBA as the supporting matrix were prepared as control samples. The binding performance of GO/MIPs versus incubation time as well as OVA concentration was investigated. As shown in Fig. 6, the amount of OVA adsorbed onto GO/MIPs is significantly lower than that of GO-APBA/MIPs. The binding capacity of GO/MIPs at adsorption equilibrium is only 105 mg g-1, almost one third that of GO-APBA/MIPs (278 mg/g), reflecting that GO-APBA/MIPs possess remarkably higher affinity toward the template glycoprotein than GO/MIPs. The much larger binding capacity of GO-APBA/MIPs fully demonstrated that the boronic acid played an important role in the protein recognition process. The explanation is that the boronic acid on GO-APBA 16

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surface could orientate the template glycoproteins in an orderly fashion and create more homogenous binding sites in the sol-gel inorganic polymer matrix. The role of boronate affinity can be further reflected by the different recognition capability of GO-APBA/MIPs at different pH values. It is well-known that the pH value is crucial for the interaction of boronic acid with glycoprotein. Boronic acid group forms covalent bond with cis-diol of glycoprotein in alkaline solution media, whereas reversible covalent bonds dissociate when the pH value of the medium changes to acidic solution. Fig. 7 shows the binding capacities of GO-APBA/MIPs and GO/MIPs composite at different pH values from 5.0 to 9.0. It can be found that the binding capacity of GO-APBA/MIPs was greatly associated with the pH value, whereas GO-APBA/NIPs and GO/MIPs exhibit a limited change in the binding capacity which could be almost neglected. The enhanced binding capacity of GO-APBA/MIPs with the increasing pH values is in agreement with the stabilization of the covalent bonding between boronic acid and cis-diol moieties of OVA at higher pH values. In contrast, the binding capacities of GO-APBA/NIPs and GO/MIPs did not show any obvious change with different pH values. This is mainly attributed to the absence of boronic acid groups in GO/MIPs and much fewer boronic acid groups in GO-APBA/NIPs exposed on its surface, which resulted in their smaller binding capacities and lack of pH sensitivity.

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Fig. 7. The effect of buffer solution pH on the adsorption capacities of GO-APBA/MIPs, GO/MIPs and GO-APBA/NIPs 3.5 Recognition specificity of GO-APBA/MIPs

Fig. 8. The recognition ability of GO-APBA/MIPs, GO-APBA, GO/MIPs and GO-APBA/NIPs toward different proteins (OVA, HRP, Bromelain, BSA and BHb). Recognition specificity was one of the most important considerations for the practical applications of imprinted materials. A selectivity experiment was conducted to investigate the selective recognition ability of GO-APBA/MIPs toward OVA. Four types of proteins, including two glycoproteins (HRP and bromelian) and two non-glycoproteins (BSA and BHb), were chosen as the contrast proteins. Fig. 8 displays the binding amounts of GO-APBA/MIPs and GO-APBA/NIPs toward these proteins. The imprinting factor α is estimated from the adsorption capacity ratio of GO-APBA/MIPs to GO-APBA/NIPs. Obviously, GO-APBA/MIPs (the black column) showed a much higher binding capacity toward OVA than other proteins. However, the adsorption capacity of GO-APBA/NIPs (the pink column) toward OVA was quite close to those of other four proteins. The imprinting factor of GO-APBA/MIPs toward OVA was as high as 9.6, where the α values of imprinting factor for HRP, bromelian, BSA and BHb were 2.5, 2.3, 1.5 and 1.4, respectively. In addition, to investigate the influence of the boronate affinity mediated interaction on the imprinting effect, the 18

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binding capacity of GO/NIPs toward OVA was also measured which is quite similar to that of GO-APBA/NIPs. So the imprinting factor of GO/MIPs toward OVA was calculated to be about 4, much lower than that of GO-APBA/MIPs. The above results clearly demonstrated the high selectivity of GO-APBA/MIPs toward the template OVA in comparison to other proteins employed. As another control experiment, the binding performance of GO-APBA was also studied and the results are provided in Fig. 8. Unlike GO-APBA/MIPs which has specific adsorption behavior toward OVA, GO-APBA (the red column) exhibits large adsorption capacity toward all glycoproteins (OVA, HRP and bromelian). It means that GO-APBA cannot recognize a specific glycoprotein from other plycoproteins, confirming the role of imprinting effect in the specific recognition property of GO-APBA/MIPs. So the outstanding recognition specificity of GO-APBA/MIPs toward OVA can be attributed to the combination of boronate affinity imparted by the introduction of APBA and shape memory effect imparted by imprinting effect. The binding capacity and imprinting factor of BSA and BHb on GO-APBA/MIPs is the lowest, which is quite reasonable since they neither possess the cis-diol groups to interaction with boronic acid groups nor did they match the shape of the recognition sites in GO-APBA/MIPs. For HRP and bromelian, although they could form esters with the boronic acid groups, they could not diffuse into the recognition cavities easily since the shape of the recognition sites in the imprinted matrix just fitted the unique molecular structure of OVA, which led to the lower binding capacity and imprinting factor. These results confirmed the excellent imprinting effect of the present strategy whether from the point of imprinting factor or specific adsorption capacity. To be applicable in real applications, imprinted materials should exhibit remarkable affinity for the template molecules, not just in isolation, but when the template protein co-exists with other proteins. To further illustrate the selectivity of GO-APBA/MIPs, competitive binding tests were performed using HRP and BHb as the competing proteins. The binding tests were carried out by keeping the OVA concentration constant and adding an equal amount of HRP and BHb to the OVA solution. As shown in Fig. S1, GO-APBA/MIPs still exhibited high adsorption capacity toward OVA 19

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despite the presence of the competitive proteins. However, it should be noted that the amount of OVA bound to GO-APBA/MIPs in the protein mixture was slightly smaller than that in the single OVA system, mainly because the rebinding of OVA would be somewhat

suppressed

in

the

competitive

environment.

Overall,

when

GO-APBA/MIPs were exposed to the mixture of OVA and the competitor protein, they preferentially adsorb OVA instead of the competitive proteins. Regeneration and real application Desorption and regeneration are of great importance for the practical application of MIPs. To determine the reusability of GO-APBA/MIPs, its adsorption−desorption cycles were repeated six times. After the adsorption process, the GO-APBA/MIPs was regenerated by eluting with HAc/NaAc solution (pH = 4.0) to remove the adsorbed OVA molecules. The GO-APBA/MIPs was quite stable and a marginal decrease of 8.6% in the adsorption capacity is observed after six adsorption– desorption cycles, suggesting the satisfactory reusability of GO-APBA/MIPs. The minor loss should be attributed to the jam or damage of some recognition sites in GO-APBA/MIPs by the repeated adsorption and washing. Encouraged by the outstanding recognizing behavior of GO-APBA/MIPs, the real application of the GO-APBA/MIPs was evaluated by selectively separating OVA from a fresh chicken-egg white sample. Chicken-egg white is a complex biological sample, containing a great deal of OVA. The egg white sample was diluted 250-fold with phosphate buffer solution (20 mM), and then incubated with the GO-APBA/MIPs. The efficiency for the selective extraction of glycoproteins was evaluated by SDS-PAGE analysis. As shown in Fig. 9, the bands of glycoproteins ovotransferrin (76.7 kDa), and ovalbumin (46 kDa) appeared in the egg white sample (lane 1). After being treated by GO-APBA/MIPs, it is found that the band of OVA (46 kDa) became lighter in lane 2, but the ovotransferrin (76.7 kDa) remained almost the same, suggesting that only OVA was well isolated. After eluted by an acidic solution, only one clear band corresponding to OVA (46 kDa) appeared in elution lane (lane 4), further confirming that only OVA has been captured by GO-APBA/MIPs. The results illustrated the specific adsorption of GO-APBA/MIPs toward the template 20

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glycoprotein, which could be employed for the isolation of OVA from complex samples.

Fig. 9 SDS-PAGE analysis of egg white sample before and after treatment with GO-APBA/MIPs. Lane (1) egg white solution; (2) the supernatant after treatment with GO-APBA/MIPs; (3) pure OVA; (4) the eluate. It has been reported that the polymerizing monomers or extracting conditions may have a negative effect on the structural stability of protein, which is destructive for the reusability of the glycoprotein after separation. CD spectrum is an effective tool for studying protein secondary structure, such as a-helix and b-sheet, in the far UV region. Fig.10 shows the CD spectra of original OVA and OVA separated by GO-APBA/MIPs. It can be seen that there was almost no difference between the CD spectra of the original OVA and OVA after separation, demonstrating that secondary structure of the template glycoprotein was well preserved in the preparation of protein imprinted polymers and extraction process.

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Fig.10 Circular dichroism spectra of original OVA (-█-) and OVA separated by GO-APBA/MIPs (-●-) To

demonstrate

the

general

applicability

of

this

approach,

other

glycoproteins—horseradish peroxidase (HRP, 44 kDa; pI 3.0–9.0) and bromelain (33 kDa; pI 9.5) that are distinct in structure and property form OVA, were employed as templates in our ongoing study. HRP as well as bromelain imprinted materials were fabricated employing the same procedure only HRP or bromelain was used instead of OVA as the imprinting glycoprotein and their recognition performances were also investigated. The results are provided in the supporting information (Fig. S2 and S3). It is clearly observed that HRP or bromelain imprinted materials exhibited good specific

recognition

towards

the

corresponding

glycoproteins

templates,

demonstrating that the proposed procedure also works well for HRP and bromelain. Conclusion In this work, a novel kind of molecular imprinting graphene material (GO-APBA/MIPs) with double recognition abilities toward glycoprotein was developed combining surface imprinting, two-dimensional graphene and boronate affinity. GO-APBA/MIPs were prepared using boronic acid functionalized graphene oxide as the supporting matrix, organic silanes as the imprinted monomers in aqueous solution. Three unique features of resulting GO-APBA/MIPs endow the product with excellent recognition capability toward glycoprotein (i.e., good selectivity, large 22

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adsorption capacity, fast rebinding and elution rate). Outstanding specific affinity for the template glycoprotein with the imprinted factor (α) as high as 9.5 was demonstrated, owing to the double recognition ability through the boronate affinity and molecularly imprinted spatial matched cavities. The interference of other proteins (glycoproteins or nonglycoproteins) could be effectively avoided. The synergetic effect of large surface area of graphene and surface imprinting endows GO-APBA/MIPs with high binding capacity and fast adsorption of OVA (the saturation adsorption capacity of imprinted products could reach 278 mg/g within 40 min). All these results proved the feasibility provided by this method to improve the molecular imprinting of glycoprotein and had promising application for the specific recognition and analysis of glycoproteins in clinics in the future.

ACKNOWLEDGMENTS We acknowledge financial support from the National Natural Science Foundation of China (under Grant 51573072), and MOE & SAFEA for the 111 Project (B13025).

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Glycoproteins via Boronate Affinity-Based Controllable Oriented Surface Imprinting. Chem. Sci. 2014, 5, 1135-1140. 43 Bie, Z. J.; Chen, Y.; Ye, J.; Wang, S.; Liu, Z. Boronate-Affinity Glycan-Oriented Surface Imprinting: A New Strategy to Mimic Lectins for the Recognition of an Intact Glycoprotein and Its Characteristic Fragments. Angew. Chem. Int. Ed. 2015, 54, 10211-10215. 44 Li, D.; Chen, Y.; Liu, Z. Boronate Affinity Materials for Separation and Molecular Recognition: Structure, Properties and Applications. Chem. Soc. Rev. 2015, 44, 8097-8123. 45 Zhang, W.; Liu, W.; Li, P.; Xiao, H. B.; Wang, H.; Tang, B. A Fluorescence Nanosensor for Glycoproteins with Activity Based on the Molecularly Imprinted Spatial Structure of the Target and Boronate Affinity, Angew. Chem. Int. Ed. 2014, 53, 12489-12493. 46. Luo, J.; Jiang, S. S.; Zhang, H. Y.; Jiang, J. Q.; Liu, X. Y. A Novel Non-Enzymatic Glucose Sensor Based on Cu Nanoparticle Modified Graphene Sheets Electrode. Analytica. Chimica. Acta. 2012, 709, 47-53.

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