An efficient Horseradish peroxidase chemiluminescence biosensor with surface imprinting based on phenylboronic acid functionalized ionic liquid@magnetic graphene oxide

Accepted Manuscript Title: An efficient Horseradish peroxidase chemiluminescence biosensor with surface imprinting based on phenylboronic acid functionalized ionic liquid@magnetic graphene oxide Author: Huimin Duan Xiaojiao Wang Yanhui Wang Jianbo Li Yuanling Sun Chuannan Luo PII: DOI: Reference:

S0925-4005(16)30667-0 http://dx.doi.org/doi:10.1016/j.snb.2016.05.003 SNB 20150

To appear in:

Sensors and Actuators B

Received date: Revised date: Accepted date:

17-12-2015 9-4-2016 2-5-2016

Please cite this article as: Huimin Duan, Xiaojiao Wang, Yanhui Wang, Jianbo Li, Yuanling Sun, Chuannan Luo, An efficient Horseradish peroxidase chemiluminescence biosensor with surface imprinting based on phenylboronic acid functionalized ionic liquid@magnetic graphene oxide, Sensors and Actuators B: Chemical http://dx.doi.org/10.1016/j.snb.2016.05.003 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

An efficient Horseradish peroxidase chemiluminescence biosensor with surface imprinting based on phenylboronic acid functionalized ionic liquid@magnetic graphene oxide

Huimin Duan, Xiaojiao Wang, Yanhui Wang, Jianbo Li, Yuanling Sun, Chuannan Luo*

Key Laboratory of Chemical Sensing & Analysis in Universities of Shandong (University of Jinan), School of Chemistry and Chemical Engineering, University of Jinan, Jinan 250022, China

* Corresponding author. Tel.: +86 0531 89736065. E-mail address: [email protected].

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Abstract Mainly, ionic liquid (IL) was used as functional bridging agent and surface modifier to obtain phenylboronic acid modified-IL functionalized GO@Fe3O4 nanoparticles (GO@Fe3O4@IL/PBA) with reversible covalent binding properties with cis-diol containing compounds particularly. Then GO@Fe3O4 @IL/PBA with the properties of high surface area and easy separation was designed as supporting material to prepare Horseradish Peroxidase (HRP) SMIP which then was characterized by SEM, XRD and FTIR. The adsorption capacity of GO@Fe3O4@IL/PBA-SMIP to HRP was researched to be 8.8 g/g, and the adsorption model and adsorption kinetics of GO@Fe3O4 @IL/PBA-SMIP to HRP followed Langmuir adsorption isotherm and pseudo second-order model while the washing process was superior with acetic acid than NaOH solutions. Possessing the merit of improving selectivity, GO@Fe3O4@IL/PBA-SMIP was introduced into CL biosensor which could linearly respond to HRP in the range of 1.0 × 10-4 - 8.0 × 10-3 mg/mL with a detection limit 2.9 × 10-5 mg/mL. Finally, the proposed GO@Fe3O4@IL/PBA-SMIP-CL biosensor was used to detect the HRP in samples with satisfied results and the recoveries were ranged from 87% to 107%. Keywords: Phenylboronic acid boronate affinity; Ionic liquid; horseradish peroxidase; surface molecular imprinting; chemiluminescence biosensor; magnetic graphene oxide

1 Introduction Because of specific interactions, easy formation, high affinity and easy elution, boronate affinity has appealed to great researchers for its unique reversible covalent binding property with cis-diol containing compounds particularly which permitted the specific isolation and separation of cis-diol containing compounds [1, 2]. When covalently binding cis-diol containing molecules to afford the corresponding cyclic 2

boronate esters in an alkaline pH, the eventual binding was dissociated at lower pH. Thus, multi-functionalized nanomaterials based on boronate affinity have been applied in many fields ranging from enrichment [3], micro-solid-phase extraction [4], sensing [5] and adsorbing [6] to biologic delivery [7]. At present, surface molecularly imprinted polymers (SMIP) were polymers containing specific recognition sites with a predetermined selectivity for analytes of interest [8]. SMIP could separate template molecules from the other matrix materials to improve the selectivity and further accumulate template molecules on the surface of SMIP to enhance the sensitivity [9]. Now, the imprinting of biological macromolecules was important but rather challenging, because of conformational change during polymerization and slow mass transfer in the polymers [10]. Particularly, boronate affinity-functionalized surface molecular imprinting, which was designed on the basis of common features of a subclass of proteins, has been developed as powerful tools for selective extraction and enrichment of glycoproteins [11, 12]. However, conventional boronate affinity materials based on phenylboronic acid in practical application in SMIP were generally associated with low adsorbing capacity, difficult separation, easy lose, low stability, low efficiency in rebinding and difficult recycle [13]. Therefore, modification of phenylboronic acid in surface molecular imprinting was required, and which would facilitate its applications in proteomics analyses. Graphene oxide (GO), with immense surface area, had captured the attention of the scientific community [14]. Up to now, the large surface area and oxygen-containing groups in GO made it in generations of adsorbent, sensors and backing material [15-17]. Even though, it was still limited due to their defects such as aggregation for its large surface energy, difficult separation and carboxyl in the edge 3

rather than the whole basal plane which may decrease the efficiency when combined with large size-composites [18]. Thereby, restraining the aggregation of GO nanosheets and modifying the carboxyl in the edge of GO would made its application wider [19-20]. Over the past years, in order to overcome the drawbacks of GO, one type of excellent modifier materials was emerged as Fe3O4 nanoparticles (Fe3O4 NPs) [21]. In general, Fe3O4 NPs were of great importance for their good magnetic properties, which endowed them with potential applications in sensor, magnetic recording media and soft magnetic materials [22]. On the other hand, as a new type of nanomaterial, Fe3O4 NPs itself could be used as backbone material in the preparing of SMIP because of its low toxicity, high tensile strength and water in solubility [23]. Although Fe3O4 NPs decorated GO (GO@Fe3O4) could restrain the aggregation of GO nanosheets effectively, some disadvantages were still there. Hence, it was significant to develop another green and commonly used material to modify the surface of GO. Herein, ionic liquid (IL) was preferentially employed as surface modifier to synthesis GO@Fe3O4 based supporting material (GO@Fe3O4@IL). Ionic liquid (IL), in recent years, have been used as the functional monomer in the preparation of MIP which can be used in aqueous media based on the π–π interactions between the analyte and ionic liquids [24, 25]. It should be noted that not only functional monomer but also solvent and porogen can IL did in the preparation of MIP [26]. As surface modifier of GO@Fe3O4, IL with amino could combined with phenylboronic acid to accelerate the synthesis process and improve the selectivity and adsorption of SMIP, and the use of the IL as the unique functional bridging agent for preparation of selective SMIPs was very meaningful. In this report, IL was firstly used as functional bridging agent and surface modifier to obtain GO@Fe3O4@IL/PBA with reversible covalent binding properties 4

with cis-diol containing compounds particularly on surface. Then GO@Fe3O4@IL/PBA with the properties of high surface area and easy separation was designed as supporting material for preparation of SMIP. Subsequently, GO@Fe3O4@IL/PBA-SMIP was introduced into chemiluminescence (CL) biosensor to improve its selectivity. Finally, the proposed GO@Fe3O4@IL/PBA-SMIP-CL biosensor was used to detect the Horsereddish Peroxidase (HRP) in samples with satisfied results.

2. Experimental 2.1 Chemicals and materials Horsereddish Peroxidase (HRP, RZ: > 2.5, activity: > 200 units/mg), Dimethylaminoethyl acrylate (DMAEMA, 99%), N,N,N',N'-tetramethyl ethylenediamine (TEMED, A.R), N-N methylene double acrylamide (MBA, A.R), Sodium cyanoborohydride were purchased from Aladdin Industrial Co. (China); N-Hydroxysuccinimide (NHS, 99%) , were supplied by Shanghai Civi Chemical Technology Co. Ltd (China), 1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide (EDC, 95%), 3-Bromopropylamine Hydrobromide (98%), 4-Amino-5-imidazolecarboxamide (98%), Ammonium persulphate (APS, AR) were supplied by Sinopharm Chemical Reagent Co. Ltd (China); 4-Formylphenylboronic acid (FBA, 98%), Sodium cyanoborohydride (95%) were purchased from Macklin Chemical Reagent Co. Ltd. Graphite powder, Potassium permanganate, Acetic acid,

Ethanol, Luminol and all

the other chemicals unless specified were of analytical reagent grade and used without further purification. Phosphate buffer solution (PBS, pH = 8.5, 0.01 mol/L) solution was used in all the experiment and stored in refrigerator (4°C).

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2.2 Apparatus The IFFM-E flow injection CL analyser (Xi’an Remex Electronic instrument High-Tech Ltd., China) was equipped with an automatic injection system and a detection system. A certain amount of GO@Fe3O4@IL/PBA-SMIP and nonimprinting polymer (GO@Fe3O4@IL/PBA-SNIP) filled in capillary was placed in front of the CL analyser as shown in Fig. 1. When HRP solution ran through the capillary, HRP molecules could be absorbed by GO@Fe3O4 @IL/PBA-SMIP via boron affinity selectively, a CL signal I1 could be obtained, while GO@Fe3O4@IL/PBA-SNIP could not absorb HRP molecules, another CL signal I2 was obtained. The difference ∆I = I2 - I1 was the concentration of HRP in a linear relationship. In this approach, a specific recognition and measurement system was obtained. XRD measurement was made on a D8 focus spectrometer (Brooke AXS, Germany). FT-IR spectrometer (PerkinElmer, USA) was employed to confirm the products. The morphology of nanoparticles was analyzed with a Scanning Electron Microscope (SEM, GUANTA FEG 250, America). 2.3 Preparation of GO@Fe3O4 GO was prepared from nature graphite powders by a modified Hummers method [27] and our group. Briefly, 180 mL of H2SO4 was added into a 500 mL flask containing 5.0 g of graphite powder and 2.5 g of NaNO3. Then, 15 g of KMnO4 was added under stirring, followed by addition of 30% H2O2. Finally, the mixture was filtered, followed by washing till the pH = 7.0. The GO was obtained after drying. GO@Fe3O4 was synthesized according to a modified procedure described in our group [28]. Firstly, 0.5 g GO was suspended in 200 mL of solution containing 1.7 g (NH4)2Fe(SO4)2·6H2O and 2.5 g NH4Fe(SO4)2·12H2O. Then, NH4OH (8 mol/L) aqueous solution was added till the pH = 11.5. The reaction was maintained at 80°C 6

for 30 min. The black precipitation GO@Fe3O4 was separated and washed, and dried. 2.4 Preparation of GO@Fe3O4@IL/PBA Firstly, 10 mL ethanol was added into 100 mL flask containing 0.1261 g 4-amino-5-imidazolecarboxamid and 0.2500 g 3-bromopropylamine hydrobromide under the protection of N2. Subsequently, the solution was reacted at 80°C for 24 h under N2 atmosphere while stirring. After that, the ethanol was removed by rotary evaporation. The viscous liquid was collected and vacuum dried. The above synthesized 0.1 g GO@Fe3O4 and 0.05 g IL were well dispersed in acid solutions (pH=5.5). Then 0.2 g EDC was added into the solution by stirring. The activation was maintained at 90 °C for 15 min. Subsequently, 0.5 g NHS was added under stirring and the pH of above solution was adjusted to 8.0, followed by heat at 90 °C for 12 h. Finally, the black gray solid was separated and washed, and dried. As prepared 0.1 g GO@Fe3O4 @IL was dissolved in 50 mL PBA ethanol solutions (10 mg/mL). The whole solution was sealed and slightly shaken at 30 °C for 6 h. After that, 20 mL sodium cyanoborohydride solution dissolved in anhydrous ethanol (10 mg/mL) was added. Then, the reaction was sealed and slightly shaken for 12 h. The black GO@Fe3O4@IL/PBA was separated, washed and dried. 2.6 Preparation of HRP GO@Fe3O4@IL/PBA-SMIP HRP GO@Fe3O4@IL/PBA-SMIP was synthesized according to a modified procedure described in literature [9]. The detail preparing process was shown in Fig. 2. By suspending 0.5 mg/mL HRP in 50 mL PBS solution, which was supplemented with 150 mg 100 mL well-dispersed GO@Fe3O4@IL/PBA, the system was adjusted to pH=8.5, sealed and shaken. Then, the solution of 30 mg MBA and 5 mL aniline was added, sealed and shaken. After supplemented with 30 mg APS and 32 mg TEMED dissolved in 10 mL PBS, the mixture was sealed and shaken for 1 h. After 7

polymerization, the solid was separated and washed with 5% HAC containing 0.05% Tween 20, and water. The prepared HRP GO@Fe3O4@IL/PBA-SMIP was dried. For the preparation of SNIP, PBS was added instead of HRP solution while the other steps were the same. 2.7 Adsorbing performance of GO@Fe3O4@IL/PBA-SMIP and GO@Fe3O4@IL/PBA-SNIP Adsorption experiments were conducted to investigate the effect of the initial HRP concentration and the time of equilibrium when GO@Fe3O4@IL/PBA-SMIP binding HRP. Binding Isotherm: 100 mg GO@Fe3O4@IL/PBA-SMIP and GO@Fe3O4@IL/PBA-SNIP NPs were placed into 5 mL centrifuge tubes, respectively. The HRP solutions were prepared at different initial concentration: 0.05 mg/mL, 0.1 mg/mL, 0.25 mg/mL, 0.4 mg/mL, 0.5 mg/mL and 0.75 mg/mL. Then, 4.0 mL of each solution was added into the tube and thoroughly mixed. The sealed liquid was incubated at 25 ◦C for 1 h. After magnetic separation, the concentration of the supernatant in the tube was determined by CL instrument to estimate the HRP amount captured by the GO@Fe3O4@IL/PBA-SMIP. Binding Equilibrium: 100 mg GO@Fe3O4@IL/PBA-SMIP and GO@Fe3O4@IL/PBA-SNIP NPs were soaked in 4.0 mL 1.0 mg/mL HRP solution which then was sealed and incubated at 25 ◦C for 1 min, 5 min, 8 min, 10 min, 15 min and 20 min respectively. After magnetic separation, the HRP concentration of the supernatant in the tube was determined by CL instrument. The amount of protein adsorbed by GO@Fe3O4@IL/PBA-SMIP was calculated from the following formula.

𝑄 = (𝑐0 − 𝑐e )𝑉/𝑚 8

Where Q (g/g) was the mass of HRP adsorbed by unit GO@Fe3O4@IL/PBA-SMIP, c0 (mg/mL) and ce (mg/mL) were the concentrations of the initial and final solution, respectively, V (mL) was the total volume of the adsorption mixture, and m (g) was the mass of GO@Fe3O4@IL/PBA-SMIP (GO@Fe3O4@IL/PBA-SNIP) used. 2.7 Compete binding experiment of the GO@Fe3O4@IL/PBA-SMIP The competing binding experiment of the GO@Fe3O4@IL/PBA-SMIP was carried out with other three kinds of proteins: Immunoglobulin G (IgG), Bovine serum albumin (BSA) and Tryptophan (Try). 100 mg HRP GO@Fe3O4@IL/PBA-SMIP (GO@Fe3O4@IL/PBA-SNIP) was well dispersed in 4 mL 1.0 mg/mL HRP, IgG, BSA and Try in 5 mL centrifuge tubes, respectively. Subsequently, the sealed system was incubated for 1 h at 25 ◦C. After magnetic separation, the concentration of the supernatant in the tube was determined by CL instrument to estimate the HRP, IgG, BSA and Try amount captured by the GO@Fe3O4@IL/PBA-SMIP. 2.8 Effects of adsorption pH and washing conditions To investigate the dependence of target binding capability of the GO@Fe3O4@IL/PBA-SMIP on pH, adsorption process at different pH was carried out. Firstly, 10.0 mL 2.0 mg/mL HRP dissolved in 20 mL ultrapure water were employed in the experiment. Then the pH of the solutions was adjusted to 6.0, 6.5, 7.0, 7.5, 8.0 and 8.5 respectively using 0.1 mol/L HCl and NaOH solutions. Finally, the sealed system was incubated for 1 h at 25 ◦C. After magnetic separation, the concentration of the supernatant in the tube was determined by CL instrument to estimate the HRP amount absorbed by the GO@Fe3O4@IL/PBA-SMIP. Extraction condition: To reuse the GO@Fe3O4 @IL/PBA-SMIP, a washing step was required to remove the HRP to evaluate the advantages of SMIP. PBS containing 9

5% HAC, 0.05% Tween 20 and 0.05 mol/L NaCl were used as washing solvent respectively. The HRP in used GO@Fe3O4@IL/PBA-SMIP was extracted with washing solvent 3 times and water washed 3 times respectively. The sorption experiment was carried out as mentioned above for 5 times for better evaluation. 2.9 Interference Experiment of the GO@Fe3O4@IL/PBA-SMIP-CLbiosensor The interference experiments of the GO@Fe3O4@IL/PBA-SMIP-CL biosensor was carried out with other three kinds of proteins: IgG, BSA and Try. A certain concentration of common substances and proteins were added into HRP standard solution (5.0 × 10−4 mg/mL) to research the effects on the CL intensity. Firstly, 5.0 × 10−4 mg/mL HRP standard solutions were prepared. Then, different concentration times of 5.0 × 10−4 mg/mL IgG solutions was added into HRP standard solution. While the CL signal of mixed solutions was different from the simple HRP solutions, the interference was existed.

3 Results and discussion 3.1 Characterization of GO@Fe3O4@IL/PBA-SMIP Scanning Electron Microscope (SEM) was used to characterize the surface morphology of the GO nanosheets, GO@Fe3O4 nanoparticles and GO@Fe3O4@IL/PBA which were shown in Fig. 3. Thin, aggregated, crumpled sheets closely together with each other and the single layered GO with wrinkle in the edge exactly showed the large surface area of GO (Fig. 3 A), which was very favorable to provide as basis and supporting material. From Fig. 3 B, it could be clearly seen that Fe3O4 NPs were in the matrix of GO sheets and the Fe3O4 NPs were disorderly distributed on the surface of GO. Apparently, a large amount of Fe3O4 NPs were attached to the surface of GO films. As shown in Fig. 3 C, the obvious difference on the surface of GO@Fe3O4@IL/PBA compared with GO@Fe3O4 proved the 10

successful fabrication of IL and PBA, which did not significantly altered the microstructure of GO@Fe3O4 but the incorporation of IL and PBA on GO@Fe3O4 enabled the imprinting and washing process easy. X-ray diffraction (XRD) measurements were employed to characterize the phase and structure of GO and GO@Fe3O4. As shown in Fig. 4 A, the XRD pattern of the as-prepared GO@Fe3O4 showed a sharp peak at 2 = 10.2◦ corresponding to the reflection of GO, and the other six diffraction peaks at 2  = 30.0◦, 30.0◦, 35.5◦, 43.0◦, 52.9◦, 62.8◦ from the different reflective crystal of Fe3O4, indexing to the reflection of cubic Fe3O4. The XRD pattern of GO@Fe3O4 suggested that the Fe3O4 NPs were effectively deposited on the surface of GO. To confirm the successful grafting of PBA on GO@Fe3O4, Fourier Transform Infrared Spectroscopy (FTIR) shown in Fig. 4 B was employed to characterize the functional groups of conjugation. The characteristic absorption peak of Fe3O4 situated at 595 cm−1 was clearly observed in the spectrum of GO@Fe3O4 and GO@Fe3O4@IL/PBA, demonstrating successful grafting of Fe3O4 on the surface of GO. For GO@Fe3O4@IL/PBA spectrum, the peaks at around 1695cm−1 could be assigned to C=O stretching, and the several bands from 1604 cm−1 to 1581cm−1 were attributed to the stretching of C=C on benzene ring. Several new peaks were visible on the spectrum of GO@Fe3O4 @IL/PBA compared with that of GO@Fe3O4. The complicate bands from 2800 cm−1 to 3500 cm−1 owned to the C-H stretching vibration in IL. The band at 1365 cm−1 was attributed to the B-O peak [29]. In addition, the band at 810 cm−1 was ascribed to absorption to 1,3-disubstituted benzene.

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3.2 Adsorption performance of GO@Fe3O4@IL/PBA-SMIP and GO@Fe3O4@IL/PBA -SNIP The binding capacities of GO@Fe3O4 @IL/PBA-SMIP and GO@Fe3O4@IL/PBA-SNIP composite to HRP was researched shown in Fig. 5 A. The adsorption capacity increased with the increase of initial HRP concentrations, because more HRP molecule was available at higher concentrations, and it provided higher driving force to overcome the mass transfer resistance accordingly. But in the end as shown, it could be verified that the adsorption capacity of GO@Fe3O4@IL/PBA-SMIP composite to HRP reached maximum 8.8 g/g and was higher than that of the SNIP (4.5 g/g) prepared at the same conditions. The binding equilibrium of HRP adsorption on the GO@Fe3O4 @IL/PBA-SMIP and GO@Fe3O4@IL/PBA -SNIP composite were shown in Fig. 5 B. Attributed to the abundant unoccupied imprinting cavities on the surface of GO@Fe3O4@IL/PBA-SMIP, the fast adsorption were observed in the beginning. But dynamically adsorption balance was reached quickly and adsorption process could be accomplished within 10 min. In particular, it was noteworthy that the quickly adsorption process showed an extraordinarily fast adsorption rate. The adsorption isotherms were modeled by Langmuir and Freundlich equations. The Langmuir and Freundlich equations were expressed in Eq (1) and (2) respectively. Eq 1:

𝑐e 𝑞e

𝑐

= 𝑞e + 𝑞 m

1

m 𝑘L

Eq 2: ln𝑞e = ln𝑘F +

ln𝑐e 𝑛

Where ce (mg/mL) is the equilibrium concentration of HRP, qe (g/g) is the adsorption capacity, qm(g/g) is the theoretical saturation adsorption capacity, kL is the Langmuir constant, kF is the binding energy constant and n is the Freundlich constant. As we could see in the linear fitting curves of Langmuir and Freundlich models 12

shown in Fig.5 (C, D) respectively, the relative standard deviation values indicated that the Langmuir isotherm were more appropriate than the Freundlich model, which revealed that the surface energy was the same and correspondingly adsorption process was mainly monolayer uniform adsorption. Therefore, it certificated that the imprinting cavies were on the surface of GO@Fe3 O4@IL/PBA-SMIP. To GO@Fe3O4@IL/PBA-SMIP, the adsorption kinetics which was an important factor for their feasibility of practical applications was explored by the pseudo firstorder model (Fig. 5 E) and second-order model (Fig. 5 F). Obviously, pseudo second-order model was more suitable because the value of R2, which could prove the goodness of the fit of experimental data on the kinetic models, was closer to 1. Therefore, the adsorption process was controlled by the chemical process. 3.3 Compete binding properties of GO@Fe3O4@IL/PBA-SMIP The adsorption capacities of GO@Fe3O4@IL/PBA-SMIP and GO@Fe3O4@IL/PBA-SNIP NPs to HRP, IgG, BSA and Try were shown in Fig. 6 A. Obviously, GO@Fe3O4 @IL/PBA-SMIP exhibited higher adsorption capacity to all biomacromolecule compared to GO@Fe3O4@IL/PBA-SNIP for the imprinting caves on the surface of SMIP which would absorb biomacromolecule more or less while there was no imprinting caves existed in SNIP. Clearly from the figure that GO@Fe3O4@IL/PBA-SMIP adsorbed much more HRP compared to IgG, BSA and Try, which was significant in terms of their selectivity. As it showed, boronate affinity based GO@Fe3O4@IL/PBA-SMIP was aphylactic to HRP particularly. And it was tolerant of interference from all kinds of matrix interference containing cis-diolcontaining compound, protein and amino acid. This result indicated that the as prepared GO@Fe3O4@IL/PBA-SMIP could be applied to recognize HRP in complex biological samples depending on its high selectivity. 13

3.4 Effects of pH on adsorption and washing conditions Effect of adsorption pH on the binding process of GO@Fe3O4 @IL/PBA-SMIP to HRP was researched and shown in Fig. 6 B. It can be seen that the boronate affinity based SMIP toward HRP was pH dependent. Particularly, the surrounding pH of phenylboronic acid was at 8.5 in which the adsorption capacity reached the best. It could be explained that in such pH the cyclic boronate esters could be formed and in such pH binding process was most suitable. Generally, biomolecules adsorption was pH depended, and adjusting the pH of real samples was inconvenient. But while the GO@Fe3O4@IL/PBA-SMIP exhibited significant target binding capability under the pH=8.5, but still binding capability of SMIP to HRP in the range of 6.0-8.5 were observed. Hence, it was confirmed that GO@Fe3O4@IL/PBA-SMIP based on boronate affinity could provide a widely applicable binding pH which made GO@Fe3O4@IL/PBA-SMIP very promising for real sample applications. 3.5 Washing conditions and reusability of the GO@Fe3O4@IL/PBA-SMIP Template washing was important in protein surface imprinting because the well remove of HRP without destroying the imprinted cavities on surface of GO@Fe3O4@IL/PBA-SMIP were difficult. If the template molecules could not be effectively removed, the number of imprinted cavities would not be employed adequately. But if the imprinted cavities were destroyed, the number of imprinted cavities will be decreased greatly. Therefore, template washing process should be proceeded appropriately. PBS containing 5% HAC, 0.05% Tween 20 and 0.05 mol/L NaCl were used to extract HRP in used GO@Fe3O4@IL/PBA-SMIP. As Fig. 6 C showed, PBS containing 5% HAC, 0.05% Tween 20 was much better than 0.05 mol/L NaCl because its adsorption capacity was higher when in its third use. For that the boronate affinity interaction between HRP and PBA in GO@Fe3O4 @IL/PBA-SMIP 14

could be broken down easily in acidic solution, Tween 20 was added to further facilitate template removal in acid pickling. At the same time, the result also showed that there was 8% adsorption capacity loss within 5 times of GO@Fe3O4@IL/PBA-SMIP which powerfully demonstrated that it was reusable. 3.6 Optimization of analytical conditions the CL biosensor To achieve satisfactory trace levels determination of HRP in samples, several parameters of GO@Fe3O4 @IL/PBA-SMIP-CL biosensor were investigated including the peristaltic pumps speed and the concentrations of NaOH, luminol and H 2O2. In consequence, 30 rpm for pump 1 speed and 35 rpm for pump 2 speed were optimized for further work. As anticipated, the concentrations of NaOH, luminol and H2O2 have a great difference on the CL intensity. And the optimization results were shown in Fig. 7 A B C. Apparently, the maximum CL intensity could be obtained at the concentration of NaOH: 0.4 mol/L, luminol: 0.4 × 10-6 mol/L and H2O2: 0. 16 mol/L simultaneously, and they were selected as analytical conditions for further work. 3.7 Analytical performance of GO@Fe3O4@IL/PBA-SMIP-CL biosensor Since HRP was added into the luminol-H2O2-NaOH system, the CL signals were enhanced greatly. And on the other hand, GO@Fe3O4@IL/PBA-SMIP could contribute to higher accuracy and lower detection limit for the template-monomer complex. Under optimal experimental conditions described above (c(NaOH) = 0.4 mol/L, c(luminol) = 0.4 × 10-6 mol/L and c(H2O2) = 0. 16 mol/L), good linearity of HRP was obtained with the concentration of HRP in a range of 1.0 × 10-4 - 8.0 × 10-3 mg/mL with a correlation coefficient was 0.9987 shown in Fig. 7 D. The limit of determination for HRP was 2.9 × 10-5 mg/mL. As shown in Tab. 1, the result showed that our work was superior to simple CL methods.

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To investigate the stability of the proposed biosensor, it was stored at 4 °C and was measured at intervals of 1 day (4 times) and 2 day (4 times). The RSD was 4.5 and 4.7 respectively, which indicated that the GO@Fe3O4@IL/PBA-SMIP-CL biosensor had a good stability. 3.8 Interference Experiment of the GO@Fe3O4@IL/PBA-SMIP-CL biosensor Before direct sample analysis, interference experiments were necessary to investigate the antijamming capability of GO@Fe3O4@IL/PBA-SMIP-CL biosensor. Since glycoproteins could bind PBA, interference matrix was composed of glycoproteins IgG, protein BSA and amino acid Try. Evidently from Fig. 7 D, 2 times IgG concentration (compared with HRP) interfered with the detection of HRP in pure CL system, while in GO@Fe3O4 @IL/PBA-SMIP-CL system, 40 times IgG concentration would interfere the determination for the interesting characteristics of high selectivity and stability of GO@Fe3O4@IL/PBA-SMIP which could recognize and absorb HRP specifically. It was worthy to note that the excellent selectivity was due to not only the excellent binding properties of the boronate affinity ligand but also the matching space in SMIP. Certainly, other substances have little interference on the GO@Fe3O4@IL/PBA-SMIP-CL biosensor. 3.9 Application of the GO@Fe3O4@IL/PBA-SMIP-CL biosensor The powerfulness of this GO@Fe3O4 @IL/PBA-SMIP-CL biosensor, as shown in Fig. 1, was further evaluated with the analysis of HRP in samples. When HRP solution ran through GO@Fe3O4@IL/PBA-SMIP capillary, HRP molecules was absorbed by GO@Fe3O4@IL/PBA-SMIP via boron affinity selectively, a CL signal I1 was obtained, while GO@Fe3O4 @IL/PBA-SNIP could not absorb HRP molecules, another CL signal I2 was obtained. The difference ∆I = I2 - I1 was the concentration of HRP in a linear relationship. In this approach, a specific recognition and measurement 16

system was obtained. Then, solution of different waste water in our Lab was obtained and diluted. Then, the pH of sample solution was adjusted to 8.5 and was proceeded to detect the HRP. And a known amount of HRP standard solution was added to the sample to obtain the desired concentrations, and the results was shown in Tab. 2. The results indicated that the GO@Fe3O4@IL/PBA-SMIP-CL biosensor could recognize and determinate HRP in water sample without sample purification, even though the target was in a complex matrix. Thus the GO@Fe3O4@IL/PBA-SMIP-CL biosensor for the determination of HRP in samples was very practical and of great interest.

4 Conclusion In summary, to obtain high sensitivity and selective CL biosensor, SMIP which was based on the reversible covalent binding properties between phenylboronic acid modified-IL functionalized GO@Fe3O4 nanoparticles (GO@Fe3O4@IL/PBA) with cis-diol containing compounds particularly was introduced. IL was used as functional bridging agent and surface modifier. Then GO@Fe 3O4@IL/PBA with the properties of high surface area and easy separation was designed as supporting material for preparation of SMIP (GO@Fe3O4@IL/PBA-SMIP). Subsequently, the adsorption model and adsorption kinetics of GO@Fe3O4 @IL/PBA-SMIP to HRP was researched and followed Langmuir adsorption isotherm and pseudo second-order model. With the advantage of improving selectivity, GO@Fe3O4@IL/PBA-SMIP was introduced into CL biosensor, and the proposed GO@Fe3O4 @IL/PBA-SMIP-CL biosensor was used to detect HRP in samples with satisfied results. Based on this work, our future work would focus on the preparation of synthetic receptor materials as SMIP elements: (1) on account of reversible covalent binding properties between phenylboronic acid and cis-diol containing compounds, different supporting material as matrix of phenylboronic acid should be tried to obtain higher surface area and more functional groups, and accordingly higher adsorption capacity, (2) seeking for simple, cheap, nonpoisonous and effective supporting material which could recognize biomacromolecule selectively for the fabrication of biomimetic CL biosensors.

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Biography Huimin Duan is a current postgraduate student who is working on synthesis of molecularly imprinted materials and application on chemiluminescence studies in School of Chemistry and Chemical Engineering, University of Jinan. Xiaojiao Wang is a current postgraduate student who is working on constructing electrochemical sensor studies in School of Chemistry and Chemical Engineering, University of Jinan. Yanhui Wang is a current postgraduate student who is working on synthesis and application of new type functional material studies in School of Chemistry and Chemical Engineering, University of Jinan. Jianbo Li is a current postgraduate student, who is working on constructing electrochemical sensor, studies in School of Chemistry and Chemical Engineering, University of Jinan. Yuanling Sun is a current postgraduate student who is working on synthesis of molecularly imprinted materials and application on chemiluminescence studies in School of Chemistry and Chemical Engineering, University of Jinan. Prof. Dr. Chuannan Luo got her Ph.D. from Lanzhou Institute of Chemical Physics, Chinese Academy of Sciences in 2004. Being engaged in analytic chemistry and published many articles in Analytica Chimica Acta, Colloids and Surfaces B: Biointerfaces, Journal of Hazardous Materials,Journal of Luminescence, Sensors and Actuators B: Chemical, etc, she is working on synthesis and application of new type functional material, and exploitation and utilize of biosensor.

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Fig. 1 Schematic diagram of HRP GO@Fe3O4@IL/PBA-SMIP-CL biosensor

21

Fig. 2 The synthesis process of HRP GO@Fe3O4@IL/PBA-SMIP

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Fig. 3 TEM characterization of GO (A), GO@Fe3O4 (B) and GO@Fe3O4@IL/PBA (C)

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Fig. 4 The XRD patterns (A) of GO and GO@Fe3O4, FTIR characterization (B) of GO, GO@Fe3O4, GO@Fe3O4@IL and GO@Fe3O4@IL/PBA

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Fig. 5 Effects of HRP concentration (A) and contact time (B) on adsorption of GO@Fe3O4@IL/PBA to HRP; Langmuir (C) and Freundlich (D) adsorption isotherm models; Pseudo-first-order kinetics (E) and pseudo-second-order kinetics (F)

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Fig. 6 Compete binding properties of GO@Fe3O4@IL/PBA-SMIP (A); Effects of pH on adsorption of GO@Fe3O4@IL/PBA to HRP (B); Washing conditions and reusability (C) and interference study (D) of the GO@Fe3O4@IL/PBA-SMIP

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Fig. 7 Optimization results: Effect of NaOH (A), H2O2 (B) and luminol (C) concentration on CL intensity; The regression equation of the GO@Fe 3O4@IL/PBA-SMIP-CL biosensor (D)

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Tab.1. Comparing results with conventional methods Methods

Liner Range (mg/mL)

Detection Limit(mg/mL)

Our work

1.0 × 10-4 - 8.0 × 10-3

2.9 × 10-5

CL [30]

10 - 500

5

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Tab. 2 Assay of HRP in samples detected by CL biosensor based on GO@Fe3O4@IL/PBA-SMIP

Sample

cHRP in -4 sample(10 mg/mL)

cadded(10-4 mg/mL)

cFound(10-4 mg/mL)(n=6)

RSD/%

Recovery (%)

1#

5.2

10.0

15.0

4.8

98

50.0

58.6

3.9

107

10.0

19.4

4.2

87

50.0

63.3

4.4

105

10.0

24.7

5.1

94

50.0

63.8

4.5

97

2#

3#

10.7

15.3

29