Fabrication of Yb3+-immobilized hydrophilic phytic acid-coated

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Characterization, Synthesis, and Modifications

Fabrication of Yb3+ - immobilized hydrophilic phytic acid - coated magnetic nanocomposites for the selective separation of bovine hemoglobin from bovine serum Jundong Wang, Huiyuan Guan, Qiang Han, Siyuan Tan, Qionglin Liang, and Ming-Yu Ding ACS Biomater. Sci. Eng., Just Accepted Manuscript • DOI: 10.1021/acsbiomaterials.9b00074 • Publication Date (Web): 02 May 2019 Downloaded from http://pubs.acs.org on May 2, 2019

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ACS Biomaterials Science & Engineering

Fabrication of Yb3+ - immobilized hydrophilic phytic acid - coated magnetic nanocomposites for the selective separation of bovine hemoglobin from bovine serum Jundong Wang, Huiyuan Guan, Qiang Han, Siyuan Tan, Qionglin Liang* and Mingyu Ding* MOE Key Laboratory of Bioorganic Phosphorus Chemistry and Chemical Biology, Ministry of Education, Department of Chemistry, Tsinghua University, Beijing 100084, China *Corresponding author: Prof. Qionglin Liang, Department of Chemistry, Tsinghua University, Beijing 100084, China Email:[email protected], Tel: +86-10-62772263 Prof.Mingyu Ding, Department of Chemistry, Tsinghua University, Beijing 100084, China Email:[email protected], Tel: +86-10-62797087

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Abstract: In this work, Yb3+ - immobilized hydrophilic phytic acid - coated magnetic nanocomposites were prepared through a facile route and used it to selective separation of bovine hemoglobin. Hydrophilic phytic acid (PA) was coated onto the magnetic Fe3O4-PEI via electrostatic interactions, followed by finally chelating with Yb3+ ions which could produce specific protein binding sites at room temperature in water, and complex instrumentation was not necessary. The performance of as-prepared hybrids (Fe3O4-PEI-PA-Yb3+) was assessed by selectively isolating bovine hemoglobin (BHb). The obtained maximum binding capacity was 347.3 mg g-1. The retained BHb could be eluted under simple elution via using 0.1 M of Na2CO3, giving a recovery of 83%. Moreover, the generation of nanocomposites was demonstrated. In addition, the PA and PEI could improve the hydrophilicity of nanoparticles and further reduce the nonspecific adsorption. Therefore, such nanocomposites were successfully employed to selectively bind and separate BHb from bovine serum as verified by SDS-PAGE and MALDITOF MS analysis, providing a new perspective for the isolation of heme proteins in proteomics. Keywords: Ytterbium; phytic acid; bovine hemoglobin; separation; metal affinity

1. Introduction Proteins, one of the biomacromolecules, play an important role in physiological and biotechnological process. Annoyingly, the accurate monitoring and analysis of proteins is an intractable problem due to the complexity of proteins itself and intricate matrix environment. Therefore, the development of efficient protocols for the selective purification and isolation of specific proteins from biological systems has sparked considerable attentions with the progress of proteomics 1,2. Hemoglobin (Hb) is one of the histidine-rich proteins. Hb is well known for its role, being a carrier of oxygen and carbon dioxide, as well as maintaining the acid-base balance of blood


2

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3,4.

Therefore, establishment of powerful techniques for selective separation of hemoglobin from

complex matrixes, has been highly desired in the therapeutic and diagnostic field. Currently, various strategies have been developed for purifying hemoglobin from biological samples, including Molecular imprinting technology (MIT) chromatography (IMAC)

8-11,

5-7,

immobilized metal affinity

metal oxide affinity chromatography (MOAC)

12,

liquid-liquid

extraction (LLE) 13, and so on. Among the existing isolation methods, immobilized metal affinity chromatography (IMAC) is the most powerful technique for the separation and enrichment of target proteins or peptides based on the coordination between the immobilized metal ions and specific amino acids of proteins. For example, Du and co-workers fabricated a novel affinity material of Cu2+ chelated cellulose/magnetic hydroxyapatite particles hybrid beads for adsorption of histidine-rich proteins 14. Chen etc synthesized a series of affinity adsorbent of IDA immobilized MNPs, and then chelated with Ni2+ / Cu2+ for selective depletion of histidine-rich proreins 15,16. Xu etc developed nickle-nitrilotriacetic acid (NTA)-based magnetic nanoparticles for the highly specific separation of His-rich bovine hemoglobin

17.

Ding etc prepared a novel material (Cu2+

immobilized ethylenediamineteraacetic acid modified magnetic nanoparticles) for selective removal hemogloin from blood samples 18. However, traditional chelating ligands, nitrilotriacetic acid (NTA), tris(carboxymethyl) ethylenediamine (TED) and iminodiacetic acid (IDA), always result in the undesired loss of the bound metal ions during the washing steps (each metal ion coordinates with only one chelating ligands )

19.

Meanwhile, nonselective adsorption of acidic

proteins and deterioration of adsorption efficiency are also inevitably. In addition, the synthetic routes relying on these chelating ligands are tedious and time-consuming. Other chelating ligands based on polymer need complex synthesis condition such as oxygen-free, organic solvent and complex instrumentation. These disadvantages hamper their practical utilization in proteins


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isolation. To overcome this problem, it is imperative to develop new approaches to ameliorate the selective isolation of heme proteins from complicated biological samples. Thus, discovering a robust chelating ligand with desirable chelating ability is of great significance. Hydrophilic phytic acid (PA) is a natural and eco-friendly compound that is obtained from grains in plants. It is widely used as food antioxidant and additive. The PA molecule, possessing six phosphate groups in its structure, could coordinate with numerous metal ions 20-22. Moreover, the excellent hydrophilicity of PA could further reduce the nonspecific adsorption. At this point, PA molecule would be considered as an ideal chelating ligand to fabricate IMAC adsorbents. Magnetic nanoparticles (MNPs) have attracted an extensive amount of attention in proteomic analysis due to their convenient magnetic separation, uniform morphology and high surface to volume ratio reduction

23-25.

26-28.

They can be synthesized hydrothermal synthesis, coprecipitation and thermal

In recent years, rare-earth materials have attracted intense interest owing to their

high number of coordination sites, remarkable catalytic activity and luminescence 29-31. Rare–earth materials can coordinate with aliphatic nitrogen, phosphor and oxygen containing ligands

32.

Ytterbium (Yb3+), one of the lanthanide element, has been extensively investigated in the fields of photoacoustic imaging 33, luminescent probe 34 and electrochemiluminescent aptasensor 35 due to the low cytotoxicity, tunable optical property and biocompatibility. As far as we know, preparation of IMAC adsorbents using Ytterbium (Yb3+) as binding sites for selective isolation of heme proteins has not been reported. Therefore, exploration of efficient and stable IMAC materials relying on ytterbium (Yb3+) for separation of heme proteins is highly desirable. Herein, Yb3+ - immobilized hydrophilic phytic acid - coated magnetic nanocomposites (denoted as Fe3O4-PEI-PA-Yb3+) were synthesized through a facile method. Firstly, PEI (polyethylenimine)-modified magneitic microspheres were synthesized via one-pot solvothermal


4

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synthesis. Then, PA and Yb3+ were introduced onto the surface on the Fe3O4-PEI nanospheres by electrostatic interactions at room temperature, and the complex instrumentation is not necessary. Remarkably, the resultant adsorbents exhibits favorable selectivity for the adsorption of bovine hemoglobin. The properties of Fe3O4-PEI, Fe3O4-PEI-PA and Fe3O4-PEI-PA-Yb3+ were characterized by transmission electron microscopy (TEM), X-ray photoelectron spectroscopy (XPS), Dynamic light scattering (DLS) measurement, x-ray powder diffraction(XRD), Fourier transform infrared spectroscopy (FT-IR), zeta potential and vibrating sample magnetometer (VSM). Furthermore, diluted blood were applied to evaluate the adsorption performance of the resultant adsorbents as corroborated by SDS-PAGE and MALDI-TOF MS analysis. The results of this work would be expected to open a new avenue for specific separation of biomacromolecules from complex biological samples. 2. Experimental section 2.1 Chemicals Ferric chloride (FeCl3·6H2O) was attained from Xilong Chemical Co., Ltd (Guangdong, China). Ytterbium nitrate pentahydrate (Yb(NO3)3) was bought from Shanghai Macklin Biochemical Co., Ltd (Shanghai, China). Ethylene glycol (EG), sodium acetate (C2H5ONa), Ammonia solution (NH3·H2O), sodium carbonate anhydrous (Na2CO3), sodium hydroxide (NaOH) and hydrochloric acid (HCl) were acquired from Beijing Chemical Works (Beijing, China). polyethylenimine (PEI, 50%, Mw=7000), phytic acid (PA, 70%) were purchased from Aladdin (Shanghai, China). sodium dodecyl sulfonate (SDS), Sodium phosphate tribasic (Na3PO4) were received from Tianjin Fuchen chemical reagents factory (Tianjin, China). Bovine serum albumin (BSA, MW 67kDa, pI 4.7), lysozyme (Lyz, MW14kDa, pI11.2), bovine hemoglobin (BHb, MW64.5kDa, pI6.8) and ovalbumin (OVA, MW 45kDa, pI4.7) were obtained


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from Beijing Keao Biological Pharmaceutical Co., Ltd (Beijing, China). α-Lactalbumin (α-Lac, MW14kDa, pI4.2), β-Lactoglobulin (β-Lac, MW18kDa, pI5.2) and Fetuin (MW48kDa, pI3.8) were acquired from Sigma-Aldrich (St. Louis, MO, USA). Bovine plasma was from Beijing Keao Biological Pharmaceutical Co., Ltd (Beijing, China). 2.2 Synthesis of Fe3O4-PEI-PA-Yb3+ microspsheres 2.2.1 Preparation of PEI-functionalized Fe3O4 microspheres Fe3O4-PEI nanoparticles were prepared via one pot solvothermal method according to literature with a few modifications 36. Briefly, 1.0 g of FeCl3·6H2O, 2.0 g of C2H5ONa and 2.0 g of PEI were first dissolved in 60 mL of ethylene glycol to form a transparent solution under ultrasonic treatment. Then, the resulting solution was stirred vigorously for 1 h at 60 ℃. Next, the dispersion was sealed in a 100 mL Teflon-lined stainless stell autoclave and heated to 200 ℃ for 10 h. Finally, the obtained nanoparticles were washed with deionied water several times and lyophilized to dryness for the next reaction. 2.2.2 Fabrication of phytic acid - coated magnetic nanocomposites Phytic acid was deposited onto the surface of the Fe3O4-PEI microspheres via electrostatic interactions. The dried 100 mg of Fe3O4-PEI microspheres were dispersed in 100 mL of water, and then 4.0 g of PA (70%) added by ultrasonic treatment. Then, the above intermixture was mechanically stirred for 6 h at room temperature, finally the particles were collected using a magnet and washed several times with distilled water for further use. 2.2.3 Synthesis of Yb3+ - immobilized IMAC materials The above Fe3O4-PEI-PA particles were added into 0.1 M of Yb(NO3)3 (100 mL). The mixture was mechanically stirred for 2 h at ambient temperature. The obtained products were


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separated with an external magnet and washed with deionized water several times and lyophilized to dryness. 2.3 Characterization An H-7650B transmission electron microscopy (TEM, Hitachi, Japan) was employed to examine the morphologies of the obtained nanoparticles. X-ray photoelectron spectroscopy (XPS) data were recorded with a PHI-5300 ESCA X-ray photoelectron spectrometer (PHI, USA). Fourier transform infrared (FT-IR) spectra in KBr was recorded using a Bruker Fourier Transform Infrared Spectrometer (Horiba, Germany) from 4000-400cm-1. The magnetization curves of samples were investigated by vibrating sample magnetometer (VSM) (Quantum Design, USA). The Dynamic light scattering (DLS) and Zeta potential data were carried out using the SZ-100 Nanoparticle Analyzer (Horiba, Japan). The crystalline structure of the synthesized materials was analyzed by X’Pert Pro MPD (Peak, Japan). MALDI-TOF MS analysis was performed on a 4800 Plus MALDI TOT/TOF MS (Applied Biosystems) with a repetition rate of 200 Hz, the Nd-YAG laser at 355 nm. Linear mid mass internal was employed for the analysis of proteins. 0.5 μL of protein sample was spotted on a stainless steel target followed by the addition of 0.5μL SA (10 mg/mL) as the matrix. Circular dichroism (CD) spectrometer (Applied photophysis, UK) was utilized to determine the secondary structure of protein. Protein samples were desalted prior to MALDI-TOF MS analysis and circular dichroism spectroscopy. 2.4 Protein adsorption behavior with Fe3O4-PEI-PA-Yb3+ nanospheres In the present work, to estimate the performance of as-prepared hybrids on the adsorption of protein, seven proteins with different structure, i.e. BHb, BSA, Lyz, α-Lac, β-Lac, Fetuin and OVA, were chosen as the model proteins for evaluating the adsorption behavior.


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Typically, 1.0 mg of the Fe3O4-PEI-PA-Yb3+ nanospheres were mixed with 1.0 mL of protein solution in a centrifuge tube. After incubation for a certain time at room temperature, the reaction mixture was isolated by placing an external magnet and the supernatant was collected for the determination of residual protein content. The influence of pH, initial concentration, incubation time, and ionic strength on the adsorption efficiency of protein were investigated. After adsorption, 0.1 M of Na2CO3, NaOH, Na3PO4, Tris-HCl, 0.5% SDS and 1% NH3·H2O were used for eluting the retained protein. The adsorption experiments were conducted using the same Fe3O4-PEI-PAYb3+ nanospheres for investigating the recyclability of the resultant adsorbents. The adsorption capacity (Q, mg/g) was determined by the following formula. (1) Q = (C0-Ce)*V/ m

(1)

The adsorption efficiency (η,%) was calculated according to the following equation. (2) η= [1-(C1/C0)]*100%

(2)

The experimental adsorption data were fitted by Langmuir model. (3) Ce / Qe= 1 / bQm + Ce / Qm

(3)

The adsorption kinetic were fitted by the Preudo-second-order models. (4) t / Qt= 1 / k2Qe2 + t / Q

(4)

Where C0 (mg/mL), Ce (mg/mL), and C1(mg/mL) are the initial, equilibrium and supernatant proteins concentrations, respectively; Q(mg/g), Qm (mg/g) and Qt (mg/g) are adsorption capacity, equilibrium adsorption capacity and adsorption capacity at a real time for proteins, respectively; b is the Langmuir constant; k2 is the Preudo-second-order rate constant.


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2.5 Evaluation of separation selectivity and real sample analysis To further investigate the selectivity of the designed method for the isolation of BHb, a standard protein mixture of BHb and BSA with a mass ratio of 1:1, 1:10, 1:20 and 1:30 was adopted. Typically, 1.0 mg of the Fe3O4-PEI-PA-Yb3+ nanospheres were mixed with 1.0 mL of protein mixture solution for 2 h at pH 6.0 at room temperature. After separation using a magnetic field, the particles-protein conjugates were washed with Tris-HCl (pH=6) buffer solution three times. The captured proteins were eluted with 0.1 M Na2CO3. Then, the original protein solution, the supernatants and eluents were analyzed by SDS-PAGE and MALDI-TOF MS analysis. The practical usefulness of the Fe3O4-PEI-PA-Yb3+ nanospheres was demonstrated by the selective isolation of BHb from 50-fold dilution of bovine blood. Nanospheres (1.0 mg) were incubated with 1.0 mL of the diluted blood samples for 2 h, then separated from the solution by external magnet and washed with Tris-HCl (pH=6) buffer solution three times. Subsequently, 0.5 mL of eluent (0.1 M Na2CO3) was added and the mixture was then oscillated for 1 h. Finally, the evaluation of separation selectivity from real sample was determined by SDS-PAGE and MALDITOF MS analysis. 3. Results and discussion 3.1 Characterizations of the Fe3O4-PEI-PA-Yb3+ nanospheres The preparation procedure for the novel hybrid MNPs (Fe3O4-PEI-PA-Yb3+) was displayed in Figure 1a. Firstly, Fe3O4-PEI MNPs were directly prepared through one-pot solvothermal reaction. Then, the PEI was coated on the surface of Fe3O4-PEI MNPs with widely existing amino groups on branched PEI via electrostatic interactions. Finally, the chelation between Yb3+ and PA occured in water at room temperature, and complex instrumentation is not necessary. Meanwhile,


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the novel hybrid nanospheres themselves are readily available and inexpensive. The typical process for separation of BHb with Fe3O4-PEI-PA-Yb3+ MNPs was shown in Figure 1b. Transmission electron microscope (TEM) images of the Fe3O4-PEI MNPs and Fe3O4-PEI-PAYb3+ MNPs were shown in Figure S1. It is obvious that the synthesized Fe3O4-PEI MNPs were nearly spherical in shape (Figure S1a). Meanwhile, the uniformly spherical morphologies of the as-prepared Fe3O4-PEI-PA-Yb3+ MNPs were also obtained after functionalized with PEI and Yb3+ via electrostatic interactions (Figure S1b).

Figure 1 Schematic illustration of the route for preparation of the Fe3O4-PEI-PA-Yb3+ MNPs (a) and protein adsorption (b) The size distribution and alteration of the nanospheres were monitored using a dynamic light scattering (DLS) technique. The results of size distribution of the Fe3O4-PEI MNPs and Fe3O4PEI-PA-Yb3+ MNPs were exhibited in Figure S1c and Figure S1d, respectively. All the particles


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were dispersed in ultrapure water by sonication for 30 min. It could be seen that the most Fe3O4PEI MNPs have a particle size of 85 nm. Analogously, the main size value of the Fe3O4-PEI-PAYb3+ MNPs were 105 nm. From DLS experiments, it could be seen that the coating components formed on the surface of Fe3O4-PEI MNPs successfully. X-ray photoelectron spectroscopy (XPS) was performed to determine the surface composition of the resultant MNPs. Wide range scans of Fe3O4-PEI MNPs, Fe3O4-PEI-PA MNPs and Fe3O4PEI-PA-Yb3+ MNPs were shown in Figure S2a, and the all elements were clearly detected. The high-resolution scans of Fe 2p, C 1s, O 1s, N 1s, P 2p and Yb 4d were illustrated in Figure S2b-g. The high resolution spectrum of Fe 2p showed strong Fe 2p 2/3 and Fe 2p 1/2 bands at 711.3 and 724.8 eV. The N 1s at 400 eV corresponded to -NH2 group of PEI 37. The spectrum of C 1s where the peaks were identified at 284.8 eV, 285.6 eV, 286.6 eV and 288.8 eV, which were assigned to C-C, C-N, C-O and C=O

38.

The peaks at 134.5 eV was attributed to P 2p in PA. The high

resolution spectrum of O 1s exhibited four peaks at 530.8 eV, 532.5eV, 533 eV and 533.8 eV, which were ascribed to Fe-O, H-O, P-O and C-O

39,22.

The 4d spectrum of Yb3+ was strongly

affected by Coulomb and exchange interaction, so the LS coupling divided the final state of 4d94f13 into the 3(HGFDP) and 1(HGFDP) states 40. The peak at 185 eV was ascribed to 3H6 and 1G4. The peaks at 187.3 eV and 191.8 eV were assigned to 1D2, 3H5 and 3G6, 1F3. The peaks at 201 eV and 207 eV were attributed to 3G4, 3D3 and 3P2, 1H5. The crystal structures of the Fe3O4-PEI MNPs and Fe3O4-PEI-PA-Yb3+ MNPs were determined by powder X-ray diffraction (XRD). As shown in Figure S3a, the six characteristic peaks of Fe3O4 (2θ=18.27°, 30.05°, 35.39°, 43.01°, 56.88° and 62.46°) were (111), (220), (311) ,(400), (511) and (440),respectively, which agreed well with that of the standard magnetite (JCPDS 89-0688). The same XRD peaks also manifests that Fe3O4-PEI-PA-Yb3+ was highly


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crystalline materials, which indicating that the crystalline materials of the magnetite was essentially maintained during the synthetic process. In addition, FT-IR was further employed for qualitative characterization, the results were presented in Figure S3b. From the FT-IR spectra of Fe3O4-PEI MNPs, the peaks around 1375 cm-1 and 1650 cm-1 were ascribed to -NH2- group in PEI, while 2822 cm-1 and 2958 cm-1 were attributed to the asymmetric and symmetric stretching vibrations of -CH2- in PEI, respectively 36. Compared to Fe3O4-PEI MNPs, two new adsorption peaks (1058 and 886 cm-1) could be seen in Fe3O4-PEIPA MNPs attributed to the existence of phosphate group (PO43-). The absorption peak at 564 cm-1 in all FT-IR spectra was the typical characteristic of metal-O bond (including Fe-O, Yb-O) 41. The peaks around 3438 cm-1 was ascribed to the stretching vibration of N-H in PEI. Since the magnetic properties are crucial for fast separation, the magnetization characterization of the Fe3O4-PEI MNPs and Fe3O4-PEI-PA-Yb3+ MNPs was performed via vibrating sample magnetometer (VSM), the consequence was shown in Figure S3c. Compared with that of the Fe3O4-PEI MNPs, the saturation magnetization (Ms) value of the Fe3O4-PEI-PA MNPs and Fe3O4-PEI-PA-Yb3+ MNPs decreased from 74.6 emu g-1 to 53.1 emu g-1 and 45.4 emu g-1. The decrease was attributed to the coating components on the surface of the magnetic nanospheres. As the inset in Figure S3c shows, the well dispersed Fe3O4-PEI-PA-Yb3+ MNPs in Tris-HCl buffer solution could respond quickly to external magnets and were desirable for application in separation of protein. Moreover, zeta potential analysis in Tris-HCl buffer solutions over a pH range from 3.0-9.0 was performed to monitor the changes in surface potential of the Fe3O4-PEI-PA-Yb3+ MNPs. As illustrated in Figure S3d, the Fe3O4-PEI-PA-Yb3+ MNPs took negative charge with the increase of pH value ranging from 3.0-9.0.


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3.2 Performance of BHb separation by using Fe3O4-PEI-PA-Yb3+ MNPs 3.2.1 Influence of pH on adsorption properties Since the specific amino acids binding to an IMAC adsobent at the surface of proteins was greatly affected by the pH value of the reaction system, so the pH value ranging from 3.0 to 9.0 on the adsorption performance of BHb, BSA and Lyz were investigated in detail. As shown in Figure 2a, we could see that the maximun adsorption efficiency for BHb was achieved at pH 6.0, and the adsorption efficiency for BHb decreased rapidly when pH was decreased or increased. This phenomenon was closely related to the following possible mechanisms. Firstly, BHb was a His-rich protein containing 20 surface-exposed His residues 42, and the decline of protonation of the His residues exposed to the surface of the BHb when pH ranging from 3.0 to 6.0, which was beneficial for the metal-affinity interaction between the His residues of BHb and Yb3+. When further increased the pH value, excessive hydroxyl groups would be a potential competitive counterpart to interact with Yb3+, which reduced the possibilities for coordination between the Yb3+ and His residues of BHb. Secondly, the Fe3O4-PEI-PA-Yb3+ MNPs were anionic and BHb was cationic at pH 6.0, further indicating the electrostatic interaction make contribution to the protein adsorption. Thirdly, the protein species became neutral at pH 6.0 which was close to the isoelectric point (pI) of the BHb, so the hydrophobic interaction between the Fe3O4-PEI-PA-Yb3+ MNPs and BHb facilitated the adsorption efficiency 43. Moreover, it should be noticed that the best adsorption efficiency of BSA was obtained at pH 4.0. There were 2 exposed His residues in BSA 44,

so the coordination lead to the adsorption. Also, the electrostatic interaction promoted the

binding because the nanospheres were anionic and BSA was cationic at pH 4.0. In addition, the hydrophobic interaction also generated the adsorption. As far as Lyz was concerned, the best adsorption efficiency was achieved at pH 9.0, which was ascribed to the electrostatic interaction


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and hydrophobic interaction, because the nanospheres were anionic and Lyz was cationic at pH 9.0. Most importantly, favorable adsorption efficiency for BHb was achieved and the lower adsoption capacity for BSA or Lyz at pH 6.0. Based on the obtained results, pH 6.0 was selected for subsequent experiment.

Figure 2. Effect of pH (a), initial concentration (b), incubation time (c) and ionic strength (d) on adsorption properties of the Fe3O4-PEI-PA-Yb3+ MNPs. Adsorption conditions: V=1.0 mL, m=1.0 mg, c=0.5 mg/mL. 3.2.2 Influence of initial concentration on adsorption properties To evaluate the adsorption efficiency of the Fe3O4-PEI-PA-Yb3+ MNPs for heme protein, BHb, BSA and Lyz were chosen as model proteins. The concentration of protein solution (in the range of 0-1.0 mg g-1) in the process was optimized. As presented in Figure 2b, the adsorbed amount of BHb increased with the increase of the initial concentration, and the adsorption amount reached a plateau when the concentration beyond 0.5 mg mL-1. The saturation adsorption capacities was 347.3 mg g-1 for BHb. However, the adsoption amount changed slightly for BSA


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or Lyz with the increase of initial concentration, and the maximum adsorption capacities for BSA and Lyz were 32.3 mg g-1 and 21.2 mg g-1, respectively. We could see that the nanospheres displayed relatively low affinity to non-BHb protein. In addition, the theoretical adsorption capacity of Fe3O4-PEI-PA-Yb3+ MNPs can be described using Langmuir mode (Table 1 and Figure S4). The all adsorption were well consistent with the Langmuir mode, manifesting the protein could be adsorbed into Fe3O4-PEI-PA-Yb3+ MNPs surface as a monolayer adsorption. Table 1. Summary of the fitted parameters for protein adsorption equilibrium on Fe3O4-PEI-PAYb3+ MNPs Langmuir model protein

Mw(kDa)

pI

Qm ( mg g-1 )

b ( mL g-1 )

R2

BHb

64.5

6.8

344.83

96.66

0.9988

BSA

66.7

4.7

40.98

24.41

0.9835

Lyz

14.3

11.4

29.94

53.86

0.9938

3.2.3 Influence of incubation time on adsorption properties Generally, incubation time was a pivotal parameter which could change the adsorption efficiency drastically. Thus, the adsorption kinetics study were carried out to assess the analyte adsorption rate, as illustrated in Figure 2c. The adsorption capacities of Fe3O4-PEI-PA-Yb3+ MNPs all increased greatly in the first 2 h for BHb, BSA and Lyz, then tended to be stable over time. 2 h was chosen as the most suitable adsorption time. In addition, the results showed that the binding capacity for BHb was much more than that the adsorption performance for BSA or Lyz in the whole period. Furthermore, the Preudo-second-order model was employed to evaluate the dynamic adsorption efficiency of Fe3O4-PEI-PA-Yb3+ MNPs (Table 2 and Figure S5). It could be seen that


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the all adsorption kinetic were fitted by the Preudo-second-order models, indicating that the chemisorption might be the rate-limiting step controlling the adsorption process. Table 2. Summary of the fitted parameters for protein adsorption kinetics on Fe3O4-PEI-PA-Yb3+ MNPs Pseudo-second-order kinetic model protein

Qe ( mg g-1 )

k2 ( g mg-1 h-1 )

R2

BHb

370.37

0.006627

0.9919

BSA

40.16

0.3875

0.9974

Lyz

30.67

0.4832

0.9993

3.2.4 Influence of ionic strength on adsorption properties The influence of ionic strength on the adsorption of BHb was further investigated by the addition of certain amount of NaCl, as illustrated in Figure 2d. The results indicated that the adsorption of three proteins onto the Fe3O4-PEI-PA-Yb3+ MNPs was significantly affected by the increase of ionic strength. This observation demonstrated that electrostatic interaction posed momentous effect on the binding of protein

[45].

The electrostatic interaction could be strongly

shielded with the addition of NaCl, so the negative contribution to the protein adsorption was generated with the increase of NaCl. Therefore, no electrolyte was added in the subsequent experiments. 3.2.5 Comparison of Fe3O4-PEI, Fe3O4-PEI-PA and Fe3O4-PEI-PA-Yb3+ MNPs on adsorption BHb


16

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Figure 3. BHb adsorption on Fe3O4-PEI, Fe3O4-PEI-PA and Fe3O4-PEI-PA-Yb3+ MNPs (a); Recognition of Fe3O4-PEI-PA-Yb3+ MNPs toward different proteins (b). Adsorption conditions: V=1.0 mL, m=1.0 mg, pH=6.0, c=0.5 mg/mL, t=2.0 h. To certify that the affinity binding was the main driving force for the adsorption of BHb onto IMAC materials, Fe3O4-PEI, Fe3O4-PEI-PA and Fe3O4-PEI-PA-Yb3+ MNPs were applied to adsorption experiments. As shown in Figure 3a, a small amount of adsorbed BHb was found for Fe3O4-PEI and Fe3O4-PEI-PA MNPs, which was the result of coordination of unoccupied orbital of iron with His residues in BHb, additionally, the hydrophobic interactions between protein and electrostatic interaction also made the contribution for the adsorption. Although the electrostatic interaction between Fe3O4-PEI-PA MNPs and BHb is stronger (Figure S6), but the steric hindrance reduces the coordination between the unoccupied orbital of iron in Fe3O4-PEI-PA MNPs and BHb. Most importantly, favorable adsorption of BHb for Fe3O4-PEI-PA-Yb3+ MNPs was achieved. Therefore, the high BHb adsorption capacity was attributed to the specific affinity between BHb molecules and adsorbents. 3.2.6 Evaluation of selectivity To investigate the binding specificity of the Fe3O4-PEI-PA-Yb3+ MNPs for BHb, the BSA, Lyz, α-Lac, β-Lac, Fetuin and OVA were chosen as the competition proteins in selectivity studies. The results are shown in Figure 3b. It was obvious that the adsorbents exhibited much larger


17


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adsorption for BHb than for other competitive proteins, demonstrating good adsorption selectivity. The outstanding specific adsorption efficiency could be explained by the following reason. The experiments were conducted at pH 6.0 which was close to the isoelectric point of BHb, facilitating the exposure of hydrophobic residues in the BHb. Thus, the coordinating interaction and hydrophobic interaction promoted the adsorption for BHb, additionally, the electrostatic interaction also lead to the adsorption. Obviously, the nanospheres displayed the adsorption for all proteins due to the hydrophobic interaction. In the case of Lyz was cationic and microspheres were anionic at pH 6.0, so the Lyz was adsorbed onto the adsorbents due to the electrostatic interaction. A small amount of adsorption for BSA might be ascribed to the affinity interaction due to BSA owned 2 surface-exposed His residues. The adsorption for α-Lac and β-Lac were mainly ascribed to hydrophobic interaction. The higher saccharide content of OVA and Fetuin produced the steric hindrance, showing a lower adsorption efficiency. To elucidate clearly the selectivity of Fe3O4-PEI-PA-Yb3+ MNPs for BHb, a series of binary protein mixtures were made up with different mass ratios (mBHb : mBSA=1:1, 1:10, 1:20) were adopted. Three adsorption experiment were performed via mixing the adsorbents with protein mixtures for adsorption equilibrium, then the samples were analyzed by SDS-PAGE and the results were presented in Figure S7. It was observed that, even at very high ration of BSA (mBHb: mBSA=1:20), the band of BHb was clearly observed in the eluate. The results verified that the Fe3O4-PEI-PA-Yb3+ MNPs possessed excellent selectivity for BHb.


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Figure 4. MALDI-TOF MS data for 1:10 (a), 1:20 (c) and 1:30 (e) of the different mass ratios; (b), (d) and (f) of the eluate. Adsorption conditions: V=1.0 mL, m=1.0 mg, pH=6.0, c= (BHb: BSA, BHb=0.2 mg mL-1) of 1:10, 1:20, 1:30, t=2.0 h. Desorption conditions: VNa2CO3=0.5 mL, t=1.0 h. To further investigate the specificity of Fe3O4-PEI-PA-Yb3+ MNPs for the separation of BHb, the protein mixtures with different mass ratios (mBHb: mBSA=1:10, 1:20 and 1:30) were employed. The protein mixture and eluate were analyzed by MALDI-TOF MS analysis, which could provide sufficient results on the selectivity, as depicted in Figure 4. When the mixture was analyzed directly without separation, the signal of BSA was obvious (Figure 4a, c and e). After selective isolation, BHb could be easily detected and no peak of BSA appeared even the concentration ratio beyond 30:1 (Figure 4b, d and f). There are only BSA remaining in the


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supernatant (Figure S8). These phenomenon indicated that the resultant nanospheres exhibited highly selective separation for BHb. 3.2.7 Eluting of the retained BHb from Fe3O4-PEI-PA-Yb3+ MNPs The recovery of the captured BHb from the Fe3O4-PEI-PA-Yb3+ MNPs for further biological investigation is highly necessary. In this respect, various potential eluting reagents, that is, Na2CO3, NaOH, Na3PO4, SDS and Tris-HCl and 1% NH3·H2O, have been investigated. As shown in Figure 5a, 83% of the BHb was recovered when using 0.1 M of Na2CO3 as the eluting reagent. Furthermore, Circular dichroism (CD) spectra showed that Fe3O4-PEI-PA-Yb3+ MNPs pose no effect on the secondary structure of BHb during the adsorption and ensuing elution (Figure S9).

Figure 5. The elution capacities of BHb by using various stripping reagents (a) and Recyclability of the Fe3O4-PEI-PA-Yb3+ MNPs (b). Adsorption conditions: V=1.0 mL, m=1.0 mg, pH=6.0, c=0.5 mg/mL, t=2.0 h. Desorption conditions: VNa2CO3=1.0 mL, t=1.0 h. 3.2.8 Reusability of Fe3O4-PEI-PA-Yb3+ MNPs The recyclability of the adsorbent is one of the most significant properties for practical applications. Therefore, to obtain further insight into the reusability of the affinity adsorbent, the absorption-desorption recycling of Fe3O4-PEI-PA-Yb3+ MNPs was repeated for five times by using the same adsorbent. The results, as shown in Figure 5b, indicated that the Fe3O4-PEI-PA-Yb3+ MNPs were relatively stable whose adsorption capacity nearly no obvious change.


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3.2.9 Comparison with other adsorbents Table 3 was the adsorption comparison of different novel adsorbent materials for BHb isolation. It was remarkable that the adsorption capacity for BHb was greater than many previously reported, and showed the excellent specificity according to the selectivity coefficients, which indicated the adsorbent material was resistant to matrix interference. Furthermore, the adsorbents could be easily prepared and the microspheres exhibited the favorable magnetic property. In addition, we conducted the MALDI-TOF MS analysis which offered the detailed information about selectivity, and the phenomenon indicated that the resultant nanospheres exhibited highly selective separation for BHb. Finally, CD spectrum suggested that the secondary structures of BHb had no change after adsorption and elution. The results revealed this method offered an efficient strategy for selective separation of BHb. Table 3. Comparison on the adsorption capacity and selectivity coefficients of BHb and BSA by various adsorbents

adsorbents

adsorption capacity (mg g-1)

SCa

Reference

BHb

BSA

BHb surface imprinted MPs

37.58

8.24

4.56

46

Cu2+-mediated magnetic imprinted polymer (MIP)

232.6

59.1

3.93

47

MnFe2O4@SiO2@Er2O3

238.2

38.5

6.18

29

Fe3O4@Au NPs BHb-MIP

89.16

20.93

4.26

48

MIPs@SiO2@NH2

90.3

27.19

3.32

49

Fe3O4@SiO2 BHb-MIP

124.86

23.7

5.27

50

Fe3O4-PEI-PA-Yb3+ MNPs

347.3

32.3

10.75

this work

SCa=QBHb / QBSA


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3.2.10 Real sample analysis

Figure 6. SDS-PAGE assay results (b). Lane 1: protein molecular weight marker; Lane 2: bovine plasma diluted 50-fold; Lane 3: remaining bovine plasma after adsorption by Fe3O4-PEIPA-Yb3+ MNPs; Lane 4: BHb recovered from Fe3O4-PEI-PA-Yb3+ MNPs; Lane 5: BHb standard solution of 0.2 mg mL-1. And MALDI-TOF MS data (b) for 50-fold dilution of bovine plasma (1), the eluate (2). Adsorption conditions: V=1.0 mL, m=1.0 mg, pH=6.0, t=2.0 h. Desorption conditions: VNa2CO3=0.5 mL, t=1.0 h. To evaluate the performance of Fe3O4-PEI-PA-Yb3+ MNPs in practical environments, BHb isolation from 50-fold dilution of bovine plasma with Tris-HCl (pH=6.0) was carried out. As presented in Figure 6a, the whole bovine plsma without treatment (Lane 2) revealed many bands corresponding to BSA, BHb, transferrin, and so on. After treatment with Fe3O4-PEI-PA-Yb3+ MNPs, the supernatant (Lane 3) still showed these bands due to their excessive amount. After the washing steps, it is clear that the final protein solution eluted from the adsorbent was BHb (Lane 4) and it was identified at the same position as that of 0.2 mg mL-1 BHb electrophoretogram (Lane


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5). These results indicate that the affinity adsorbent of Fe3O4-PEI-PA-Yb3+ MNPs had the excellent ability to separate BHb from complex proteins mixture. Additionally, isolation process was affirmed with real biosamples (50-fold dilution of bovine plasma) via MALDI-TOF MS analysis. The results are shown in Figure 6b. As we could see, the signal of intensity of BHb was extremely poor and peak for BSA was very obvious in diluted bovine plasma (Figure 6b 1). After selective isolation, BHb could be easily detected with a strong intensity (Figure 6b 2). These results indicated that Fe3O4-PEI-PA-Yb3+ MNPs hold great potential for the selective separation of BHb from complex biological samples. 4. Conclusion In summary, the Yb3+ - immobilized hydrophilic phytic acid - coated magnetic nanocomposites were successfully synthesized by an efficient approach. Thanks to large amount of Yb3+ providing specific binding sites, favorable chemical stability, excellent hydrophilicity of PEI and PA, as well as good magnetic response, the proposed nanocomposites demonstrated significant affinity and selectivity for BHb separation. The obtained maximum binding capacity was 347.3 mg g-1 and the nanospheres showed the excellent specificity according to the selectivity coefficients. The regenerated Fe3O4-PEI-PA-Yb3+ MNPs were efficiently reutilized for BHb separation. Except the current investigated typical BHb, others histidine-rich proteins could be hopefully expanded for the isolation. The remarkable results obtained in this work demonstrate the potential of Fe3O4-PEIPA-Yb3+ MNPs for selective histidine-rich proteins from complex complex systems. Acknowledgments This work was supported by the Natural Science Foundation of China (No. 21575076 and 21621003), the National Key Research and Development Program of China (2016YFA0203101), and the Beijing Municipality Science and Technology Program (D161100002116001).


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Fabrication of Yb3+ - immobilized hydrophilic phytic acid - coated magnetic nanocomposites for the selective separation of bovine hemoglobin from bovine serum Jundong Wang, Huiyuan Guan, Qiang Han, Siyuan Tan, Qionglin Liang* and Mingyu Ding*

Yb3+ - immobilized phytic acid - coated magnetic nanocomposites prepared by facile method were used as novel adsorbents for the selective separation of BHb.


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Fabrication of Yb3+-immobilized hydrophilic phytic acid-coated

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