Magnetic Nanospheres Encapsulated by Mesoporous Copper Oxide

Subscriber access provided by The University of Liverpool

Article

Magnetic Nanospheres Encapsulated by Mesoporous Copper Oxide Shell for Selective Isolation of Hemoglobin zhiyong guo, Yue Zhang, Dan-Dan Zhang, Yang Shu, Xu-Wei Chen, and Jian-Hua Wang ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.6b11158 • Publication Date (Web): 12 Oct 2016 Downloaded from http://pubs.acs.org on October 20, 2016

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

ACS Applied Materials & Interfaces is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 31

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

ACS Applied Materials & Interfaces

Magnetic Nanospheres Encapsulated by Mesoporous Copper Oxide Shell for Selective Isolation of Hemoglobin

Zhi-Yong Guoa, Yue Zhanga, Dan-Dan Zhanga, Yang Shub, Xu-Wei Chena* and Jian-Hua Wanga* a

Research Center for Analytical Sciences, Department of Chemistry, College of

Sciences, Northeastern University, Box 332, Shenyang 110819, China b

Institute of Biotechnology, College of Life and Health Sciences, Northeastern

University, Box H006, Shenyang 110169

Keywords: mesoporous CuO; core-shell structure; magnetic Fe3O4 nanospheres; hemoglobin; isolation. Abstract: A novel strategy for the preparation of magnetic nanospheres encapsulated by mesoporous copper oxide shell, shortly termed as Fe3O4@mCuO, is reported via the calcination of Cu(NH3)4(NO3)2 into continuous mesoporous CuO shell onto the surface of Fe3O4 nanoparticles. The magnetic nanospheres are characterized to possess stable core-shell structure with a crystalline mesoporous CuO layer, exhibiting a CuO loading content of 25.2±1.1% along with a favorable magnetic susceptibility. Fe3O4@mCuO nanospheres exhibit favorable selectivity on the adsorption of hemoglobin with a high adsorption capacity of up to 1162.5 mg g-1. After adsorption, the high magnetic susceptibility allows convenient separation of the nanospheres by an external magnet. The retained hemoglobin could be readily 1

ACS Paragon Plus Environment


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

recovered by using 0.5% (m/v) SDS as stripping reagent, providing a recovery of 78%. Circular dichroism (CD) spectra illustrate virtually no change on the conformation of hemoglobin after the process of adsorption/desorption. Fe3O4@mCuO nanospheres are further applied for the selective isolation of hemoglobin from human whole blood, achieving high purity hemoglobin as demonstrated by SDS-PAGE assays.

2


Page 2 of 31

Page 3 of 31

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


INTRODUCTION The designed core-shell nanostructures with tailored morphologies and biological properties have attracted extensive attentions.1-5 The integration of various functional modules may endow the core-shell nanostructures with enhanced performance. Magnetic composites with prominent magnetic responsive nature and favorable biocompatibility are important components in the fabrication of novel multifunctional core-shell nano-configurations and have gained wide applications in various fields, e.g., separation,6 catalysis,7 biomedicine,8 optics,9 drug storage and delivery.10 When using magnetic nanostructures as adsorption media, many of the complex manipulations in conventional extraction protocols, e.g., centrifugation and filtration, may be avoided by using an external magnetic field.11 Thus, magnetic nanostructure-based extraction/adsorption protocols have received extensive popularity in the pretreatment of biological samples,12-18 in which simple and fast manipulations are favorable to maintain the original activities of the target biological molecules/analytes. Recently, proteomics and protein-based biotechnologies have gained increasing focus, whereas the efficient separation and enrichment of proteins or peptides from biological systems is a pre-requisite.19-21 Immobilized metal affinity chromatography (IMAC) has been widely adopted in the isolation and enrichment of specific proteins or peptides relying on the affinity interaction between specific amino acids of protein and metal cations immobilized on a substrate.22-25 Iminodiacetic acid (IDA) and nitrilotriacetic acid (NTA) are the commonly used linkers between the metal cations

3



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

and matrix.26, 27 However, the preparation of IDA-/NTA-based supports is usually complex and tedious. Meanwhile, the loss of metal cations during analysis steps in IMAC strategy usually results in more adsorption of acidic proteins and the deterioration of separation/enrichment efficiency.19 At this point, quadrivalent metal (IV) phosphate chemistry and metal oxide nanocomposites, employing Ti4+, Zr4+, TiO2 and ZrO2 28-31 to provide the binding sites, have been demonstrated to be more efficient and specific in trapping the target proteins with respect to the conventional IMAC. The core-shell nanostructures combining a magnetic core and a metal oxide shell might simultaneously achieve rapid isolation by magnetic nanospheres and high recovery and selectivity. However, the preparation of core-shell nanospheres is difficult as it is not easy to form a pure metal oxide interface, and meanwhile the silica or carbon layer outside of the core-shell microspheres are prone to cause non-specific adsorption.19, 30 Therefore, the effective fabrication of magnetic core-shell nanospheres with purity covers is highly demanded. In the present work, we propose a new approach for the preparation of high-loading mesoporous CuO magnetic core-shell nanospheres (Fe3O4@mCuO) via the transition process firstly to form a tight coordination compound layer of Cu(NH3)4(NO3)2 outside Fe3O4 nanoparticles, followed by calcination to produce a continuous and crystalline mesoporous CuO shell. CuO is a kind of special metal oxide widely adopted in the IMAC strategy for the enrichment and isolation of proteins and peptides from complex system, as the hydrophobic interaction and metal-affinity interactions

4


Page 4 of 31

Page 5 of 31

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


between CuO and histidine residues of protein provide CuO excellent adsorption specificity towards histidine-included protein, especially hemoglobin. 32, 33 The well defined core-shell structure and highly crystalline mesoporous CuO layer offer the Fe3O4@mCuO nanospheres fast separation from the adsorption medium and high adsorption capacity. The favorable performance of Fe3O4@mCuO nanospheres in the selective isolation of hemoglobin from human whole blood demonstrated its promising potentials in the pretreatment of biological samples.

EXPERIMENTAL SECTION Chemicals and Reagents. Iron(III) chloride hexahydrate (FeCl3·6H2O), copper nitrate trihydrate (Cu(NO3)2·3H2O), sodium acetate (C2H5ONa), trisodium citrate, ethylene glycol (EG), anhydrous ethanol (≥99.7%), acetonitrile (ACN, ≥99.8%), sodium dodecyl sulfate (SDS), histidine (His), ammonium hydroxide (NH3·H2O, 25-28%), glacial acetic acid, phosphoric acid, nitric acid, hydrogen peroxide, boric acid and sodium chloride (NaCl) are purchased from Sinopharm Chemical Reagent Co. Ltd (Shanghai, China). Hemoglobin (Hb, >95%), bovine serum albumin (BSA, >98%), cytochrome c (cyt-c, >95%) are obtained from Sigma-Aldrich (St. Louis, USA). The protein marker is the product from Takara Biotechnology Co. Ltd. (Dalian, China). All these reagents are at least AR level and used without further purification. Human whole blood sample is obtained from the Hospital of Northeastern University and anti-coagulated with sodium citrate. Preparation of magnetic Fe3O4 nanoparticles (Fe3O4 NPs) and Fe3O4@Cu(NH3)4(NO3)2 core-shell microspheres. Magnetic Fe3O4 NPs are 5



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

obtained according to a modified thermal protocol.34 1.35 g of FeCl3·6H2O is dissolved in 70 mL of ethylene glycol, followed by addition of 3.85 g of C2H5ONa and 0.4 g of trisodium citrate, and the mixture is afterwards stirred vigorously under heating at 80oC until a homogeneous claybank solution is achieved. The solution is transferred into a stainless-steel autoclave for heating at 200oC for 16 h. After cooling down to room temperature, the black product is collected under a magnet and washed for five times with ethanol. The final product is dried at 60oC in a vacuum for further use. Fe3O4@Cu(NH3)4(NO3)2 microspheres are prepared by indirectly in-situ deposition of a Cu(NH3)4(NO3)2 layer on the surface of Fe3O4 NPs. Typically, 50 mg of Fe3O4 NPs are dispersed into a mixture solution containing 90 mL of ethanol and 30 mL of acetonitrile. The mixture undergoes ultrasonic dispersing to achieve a uniformed suspension. Afterwards, different amount of Cu(NO3)2 (with Fe3O4/Cu(NO3)2 mass ratios of 1:0.2, 1:1, 1:2 and 1:6) and 1mL of NH3·H2O are added into the above suspension under stirring. The reaction mixture is heated in an oil bath at 90oC for 2 h. The final product is then collected by a magnet, washed with ethanol and dried at 60 oC at vacuum. Preparation of Fe3O4@mCuO core-shell nanospheres. Fe3O4@mCuO core-shell nanospheres are prepared with calcination from Fe3O4@Cu(NH3)4(NO3)2. 100 mg of dried Fe3O4@Cu(NH3)4(NO3)2 microspheres are put into a combustion boat, which is placed in a tube furnace and calcined at 280oC under air for 2 h. Afterwards, the product is cooled down to room temperature, followed by washing with ethanol

6


Page 6 of 31

Page 7 of 31

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


and water alternatively for five times and then dried at vacuum. Characterizations. Transmission electron microscopy (TEM) images are recorded on a Tecnai G220 microscope (Philips, Holland). X-ray diffraction (XRD) patterns are carried out on a X’Pert Pro MPD X-ray diffractometer (PANalytical BV, Holland) diffraction meter with Cu Kα λ=1.5406 Å. Energy dispersive X-ray (EDX) spectrums are implemented on a SSX-550 scanning electron microscope (Shimadzu, Japan). FT-IR spectra from 4100 to 500 cm-1 are performed on a Nicolet 6700 spectrophotometer (Thermo Electron, USA) with resolution 2.0 cm-1. Thermo gravimetric analysis (TGA) is carried out with a TGA 290C analyzer (Netzsch, Germany) with a heating rate of 10oC min-1 under protection with nitrogen. Nitrogen sorption/desorption experiments are carried out at a Micromeritcs Tristar 3000 analyzer (USA). Magnetic characterizations are measured with a Vibrating sample magnetometer (VSM) on a Model 6000 physical property measurement system (Quantum, USA) at 300 K. The contents of Cu are determined by an Agilent 7500 ICP-MS spectrometer (Agilent Technologies, USA). The circular dichroism (CD) spectras record with a MOS-450 spectrometer/polarimeter (Bio-logic Science Instrument, France) by using a 0.5 cm quartz cell under argon protection. pH values are adjusted by a BSA 224S pH Meter (Beijing Sartorius Instruments Co., Ltd., China). Protein adsorption behavior with Fe3O4@mCuO core-shell nanospheres. Typically, the Fe3O4@mCuO nanospheres are pre-washed with deionized water for three times and then a suspension is achieved with a concentration of 10 mg mL-1. 100

7



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

µL of Fe3O4@mCuO suspension is mixed with 1.0 mL of protein solution in a centrifuge tube. After incubating for 10 min at room temperature, the mixture is undergoing separation by a magnet and the supernatant is collected for the determination of residual protein content. The Fe3O4@mCuO nanospheres are then washed with 1.0 mL of deionized water to remove the non-specific adsorbed proteins. After adsorption of protein, the Fe3O4@mCuO nanospheres are mixed with 1.0 mL of 0.5% SDS solution and shaking on an oscillator for 30 min at room temperature to recover the retained protein. The adsorption and recovery manipulations are repeated for three times.

RESULTS AND DISCUSSION Preparation and characterization of Fe3O4@mCuO nanospheres. As illustrated in Scheme 1 that the preparation of magnetic Fe3O4@mCuO core-shell nanospheres involves first the preparation and stabilization by citrate via a modified solvothermal reaction.34 Due to the abundant carboxyl groups on the surface, the citrate-stabilized Fe3O4 NPs are heavily negatively charged. After the addition of Cu(NO3)2, the electrostatic attraction between the cation Cu2+ and negatively charged citrate-stabilized Fe3O4 NPs, and the affinity interaction between the free Cu2+ from the added Cu(NO3)2 and the carboxyl groups of citrate-stabilized Fe3O4 NPs, drive the dispersion and precipitation of Cu2+ on the surface of Fe3O4 NPs. Subsequently, a tight Cu(NH3)4(NO3)2 layer is accumulated on the surface of Fe3O4 NPs by nucleation and formation upon increasing the pH value and temperature. The cross-linked matrix with abundant NH3 and NO3 groups provide scaffolds for anchoring more 8


Page 8 of 31

Page 9 of 31

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


Cu(NH3)4(NO3)2 entities as well as the followed coordination process. This interim Cu(NH3)4(NO3)2 layer could be easily decomposed during the calcination process. As illustrated in Scheme 1, the calcination at high temperature leads to the release of NH3 and NO2 from Cu(NH3)4(NO3)2,35 meanwhile, the anchored Cu-O entities react with their neighboring counterparts to constitute a continuous mesoporous CuO shell. The above preparation process ensures the homogeneity of the CuO coating shell which provides more binding sites and reduces the non-specific adsorption of protein by the inside core.

Cu(NO3)2

Calcinations

NH3H2O Fe3O4

Fe3O4@Cu(NH3)4(NO3)2

Fe3O4@CuO

Scheme 1. Schematic illustration for the preparation of Fe3O4@mCuO core-shell nanospheres.

Figure 1a-c illustrate TEM images for the Fe3O4 NPs, Fe3O4@Cu(NH3)4(NO3)2 and Fe3O4@mCuO nanospheres originated from Fe3O4/Cu(NO3)2 mass ratio of 1:2. It is obvious that the uniform Fe3O4 NPs exhibit an average diameter of ca. 220 nm. After coating with the coordination compound layer, the resultant Fe3O4@Cu(NH3)4(NO3)2 microspheres possess an obvious sharpen crystal shell structure and the size of the core-shell microspheres is estimated to be ca. 340 nm.

9



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

The ensuing crystallization of anchored Cu(NH3)4(NO3)2 into a continuous mesoporous CuO shell by calcination results in virtually no change on the size of Fe3O4@mCuO nanospheres, which is estimated to be ca. 350 nm. Dynamic light scattering is adopted to further ascertain the size distribution of the obtained nanospheres. The hydrodynamic diameter of Fe3O4 NPs, Fe3O4@Cu(NH3)4(NO3)2 and Fe3O4@mCuO nanospheres is about 235 nm, 352 nm and 364 nm (Figure S1), respectively, agreeing well with the results from TEM.

Figure 1. (a-c) TEM images of Fe3O4 NPs, Fe3O4@Cu(NH3)4(NO3)2 and Fe3O4@mCuO nanospheres, respectively. (d) Nitrogen adsorption desorption isotherms and BJH pore-size distribution curves (inset) for the Fe3O4@mCuO nanospheres.

BET surface area and total pore volume of the Fe3O4@Cu(NH3)4(NO3)2 and Fe3O4@mCuO nanospheres are derived to be ~11.94 m2 g-1 and 0.039 cm3 g-1, ∼11.99

10


Page 10 of 31

Page 11 of 31

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


m2 g-1 and 0.072 cm3 g-1, respectively. Nitrogen adsorption-desorption isotherms of the Fe3O4@mCuO nanospheres (Figure 1d) show representative type-IIb curves,16 which might be attributed to the slightly distorted mesopore channels and the heterogeneity in the adsorbent-adsorbate interactions. The pore size distribution curve (Figure 1d, inset) derived from the adsorption branch reveals a mesopore size of ca. 2-10 nm.

a

b

c

d

Figure 2. FT-IR spectra (a), XRD patterns (b), TGA curves (c) and VSM curves (d) of Fe3O4 NPs (i), Fe3O4@Cu(NH3)4(NO3)2 nanospheres (ii) and Fe3O4@mCuO nanospheres originated from Fe3O4/Cu(NO3)2 mass ratio of 1:2 (iii).

FT-IR spectra of the Fe3O4 NPs, Fe3O4@Cu(NH3)4(NO3)2 and Fe3O4@mCuO

11



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

nanospheres are illustrated in Figure 2a. The absorptions at 592 cm-1 observed in all the spectra are attributed to the stretching vibration of Fe=O in the Fe3O4 NPs. In the spectrum of citrate-stabilized Fe3O4 NPs, the absorptions at 1421, 1601 and 3433 cm-1 are assigned to the stretching vibrations of symmetric COO-, asymmetric COO- and O-H, respectively. For the Fe3O4@Cu(NH3)4(NO3)2 nanospheres, the new absorption bands at 1565 and 1354 cm-1 are accounted as the symmetric and asymmetric stretching vibration of NO32-, and the sharp band at 3326 cm-1 is considered as the stretching vibration of NH3.36 These absorptions well confirm the effective deposition of Cu(NH3)4(NO3)2 onto the surface of Fe3O4 NPs. The calcination process leads to the decomposition of the Cu(NH3)4(NO3)2 nanospheres, therefore the characteristic adsorptions for NO32- and NH3 are not observed in the spectrum of the Fe3O4@mCuO nanospheres. Figure 2b shows the XRD patterns of Fe3O4 NPs, Fe3O4@Cu(NH3)4(NO3)2 and Fe3O4@mCuO nanospheres. The 2θ diffraction peaks at 30.1°, 35.5°, 43.0°, 53.4°, 56.8° and 62.5° correspond to (220), (311), (400), (422), (511), and (440) planes of Fe3O4 (JCPDS 89-0688). For the Fe3O4@Cu(NH3)4(NO3)2 core-shell nanospheres, a few new diffraction peaks appear at 14.9°, 15.2°, 17.0°, 21.4°, 22.2°, 25.8°, 27.8°, 28.9°, 31.1°, 31.7°, 34.3°, 36.4°, 40.1° and 45.2°. These peaks should correspond to the (040), (101), (140), (141), (240), (320), (301), (321), (142), (341), (242), (421), (103), (282) and (442) planes of the Cu(NH3)4(NO3)2 pattern (JCPDS 70-0195), indicating the successful deposition of Cu(NH3)4(NO3)2 on the surface of Fe3O4 NPs. For the Fe3O4@mCuO core-shell nanospheres, the peaks referring to Cu(NH3)4(NO3)2

12


Page 12 of 31

Page 13 of 31

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


disappear, while new diffraction peaks at 35.4°, 38.6°, 48.9°, 58.2°, 65.7° and 68.0°are observed corresponding to the (002), (-111), (111), (200), (-202), (202), (022) and (220) planes of the CuO pattern (JCPDS 80-0076), this well demonstrated the formation of crystalline mesoporous CuO on the surface of Fe3O4 NPs. Fe3O4@mCuO core-shell nanospheres originated from various Fe3O4/Cu(NO3)2 mass ratios share similar XRD patterns with different intensity (Figure S2), indicating the different amount of crystalline mesoporous CuO formed in the final product under different preparation conditions. The results of energy dispersive X-ray (EDX) are summarized in Table S1. Compared with Fe3O4 NPs, new elements including N and Cu appear in Fe3O4@Cu(NH3)4(NO3)2 nanospheres, demonstrating the successful deposition of Cu(NH3)4(NO3)2 on the surface of Fe3O4 NPs. The calcination leads to the decomposition of the Cu(NH3)4(NO3)2 and the release of NH3 and NO2 from the final product, therefore, no N is determined in the Fe3O4@mCuO nanospheres. Thermo gravimetric analysis (TGA) is carried out to quantitatively confirm the composition of these core-shell nanospheres (Figure 2c). A weight loss of ca. 11.3 wt % for Fe3O4 NPs is attributed to the decomposition of citrate, indicating a Fe3O4 content of 88.7 wt% in the citrate-stabilized Fe3O4 NPs. After the deposition of Cu(NH3)4(NO3)2 layer, the decomposition of citrate and the release of NH3 and NO2 from Cu(NH3)4(NO3)2 offers an obvious weight loss of Fe3O4@Cu(NH3)4(NO3)2 within the temperature range of 180-320oC. A further weight loss at 800-1000oC is contributed to the decomposition of CuO.37 The weight loss at 800-1000oC are also observed for Fe3O4@mCuO nanospheres originated from different Fe3O4/Cu(NO3)2

13



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

mass ratios(Figure S3), and the content of CuO for the final Fe3O4@mCuO nanospheres is calculated to be ca. 26.5%. The magnetic properties of the core-shell nanospheres are investigated by using a vibrating sample magnetometer (VSM) (Figure 2d). It is seen that no obvious magnetic hysteresis loops (Hc < 30 Oe) are observed in the three nanospheres, indicating that they are superparamagnetic at room temperature. The saturation magnetization (Ms) value of Fe3O4 NPs is 69.6 emu·g-1. The deposition of Cu(NH3)4(NO3)2 layer results in a reduction of the saturation magnetization value to 11.7 emu·g-1 for the Fe3O4@Cu(NH3)4(NO3)2. Afterwards, the calcination process gives rise to a saturation magnetization value of 18.1 emu·g-1 for the Fe3O4@mCuO nanospheres. According to the magnetic behavior, the content of CuO is derived to be ca. 25.8% in the Fe3O4@mCuO nanospheres, which agrees well with that achieved from the results of TGA. Protein adsorption behavior onto Fe3O4@mCuO core-shell nanospheres. Figure 3 illustrates the adsorption behaviors of BSA, cyt-c and Hb onto the Fe3O4@mCuO core-shell nanospheres prepared at various mass ratios of Fe3O4 and Cu(NO3)2. It is interesting to see that all the nanospheres exhibit very low adsorptions on either BSA or cyt-c regardless the content of CuO layer. On the other hand, however, the adsorption efficiency for hemoglobin is improved significantly with the increase of the content of CuO layer in the nanospheres. Considering the fact that over-deposition of CuO layer tend to deteriorate the magnetic properties of the resultant nanospheres, a Fe3O4/Cu (NO3)2 mass ratio of 1:2 is adopted for the preparation of the Fe3O4@mCuO nanospheres, resulting in a mesoporous CuO layer

14


Page 14 of 31

Page 15 of 31

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


content of 25.2%±1.1% in the final product.

Figure 3. Adsorption efficiency of BSA, cyt-c and Hb onto the Fe3O4@mCuO nanospheres prepared at various mass ratios of Fe3O4 and Cu(NO3)2. Protein solution: 1.0 mL, 70 mg L-1, pH 7; adsorption time: 10 min; the mass of Fe3O4@mCuO nanospheres: 1.0 mg. All results are from triplicate determinations.

Figure 4a-b show the adsorption behaviors of BSA, cyt-c and Hb onto the Fe3O4@Cu(NH3)4(NO3)2 and Fe3O4@mCuO core-shell nanospheres at various pH values. It is seen that in an acidic medium, obvious adsorptions for BSA, cyt-c and Hb are observed by using Fe3O4@Cu(NH3)4(NO3)2 nanospheres, attributed to the hydrophobic interaction between the nanospheres and BSA/cyt-c, and probably the coordinating interaction between the iron atom of heme group and the moiety of NH3 or NO32- in the Fe3O4@Cu(NH3)4(NO3)2.38, 39 After the calcination of Cu(NH3)4(NO3)2 into crystalline mesoporous CuO, completely different adsorption behaviors of proteins onto the Fe3O4@mCuO nanospheres are obtained. The 15



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

maximum adsorptions for BSA, cyt-c and Hb are all achieved at pHs close to their pI values, and the deviation of pH from pI leads to the deterioration of adsorption efficiencies for all the three protein species.

a

b

Figure 4. pH dependent adsorption behaviors of BSA, cyt-c and Hb onto the surface of Fe3O4@Cu(NH3)4(NO3)2 (a) and Fe3O4@mCuO nanospheres (b). Protein solution: 1.0 mL, 70 mg L-1, pH 7; adsorption time: 10 min; the mass of nanospheres: 1.0 mg. All results are from triplicate determinations. The above observations might be well explained by the metal-affinity interaction between crystalline mesoporous CuO and histidine residues of protein. At pH close to the pI the electrostatic repulsion between the proteins is minimized, and some degree of aggregation may occur by the metal-affinity interaction. There are 24 exposed histidine residues in Hb, and 2 in BSA and 1 in cyt-c,32 thus the adsorption efficiency of Hb is higher than those of BSA and cyt-c under optimal conditions. Most importantly, favorable adsorption of Hb is achieved at pH 7, while nearly no adsorption of BSA and cyt-c take place at the same condition. This discriminative behavior provides promising potential for achieving selective isolation of Hb. The effect of ionic strength on Hb adsorption is investigated in the presence of 16


Page 16 of 31

Page 17 of 31

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


certain concentration of NaCl (Figure S4). It is obvious that the variation of ionic strength within a wide range poses no effect on the adsorption of Hb, which keeps at a high level NaCl concentration of up to 2 mol L-1. The observation further indicated that electrostatic interaction makes no contribution to the protein adsorption. This observation further illustrates the practical usefulness of the present nanospheres for the treatment of real biological samples wherein relatively high ionic strengths are generally encountered. The adsorption capacity of hemoglobin onto the Fe3O4@mCuO nanospheres is investigated at room temperature within a protein concentration of 50-5000 mg mL-1. Figure S5 illustrated that the adsorption behavior of hemoglobin, and the maximum experimentally adsorption capacity is determined to be 1162.5 mg g-1. The adsorption of hemoglobin onto the Fe3O4@mCuO nanosphere fits well with Langmuir model as expressed in the following equation, where C* is the concentration of Hb, Q* represents the amount of Hb adsorbed by the Fe3O4@mCuO nanospheres, Qm is the maximum adsorption capacity and Kd is the dissociation constant. By fitting the experimental data to this equation, the maximum theoretical adsorption capacity for Hb is derived to be 1470.6 mg g-1.

Q* =

Qm × C * Kd + C *

Table 1 compares the maximum adsorption capacities for Hb by various novel adsorbents.40-46 It is obvious that the Fe3O4@mCuO nanospheres provide a significantly higher adsorption capacity for Hb. This might lie in the fact that the high-loaded crystalline mesoporous CuO on the surface of Fe3O4@mCuO 17



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

Page 18 of 31

nanospheres is able to provide more available binding sites for protein adsorption than other reported novel adsorbents. This superior capacity for Hb should be important for the selective separation of Hb from complex biological sample matrixes. Table 1. The comparison of maximum adsorption capacities for hemoglobin with various magnetic adsorbents. Adsorption capacity

Reference

Adsorbents (mg g-1) Cu2+-IDA-SiO2-Fe3O4

418.6

[40]

Zn2+-IDA-poly(glycidylmethacrylate)-Fe3O4

260.0

[41]

Hb Magnetic MIP

10.5

[42]

Cu2+-mediated Magnetic MIP

232.6

[43]

Cu2+-IDA-poly(glycidylmethacrylate)-Fe3O4

135.7

[44]

LaMOF-GO composite

193.0

[45]

Polymeric ionic liquid Poly(C12vim)Br

205.4

[46]

Fe3O4@mCuO microspheres

1162.5

this work

Recovery of the retained hemoglobin from Fe3O4@mCuO core-shell nanospheres. The stripping of the retained proteins on the solid substrate into an aqueous medium is necessary for the ensuing biological investigations. For this purpose, a series of stripping reagents, i.e., BR buffers, histidine, citrate, tris-HCl and SDS solutions, have been tested (Figure 5a). The results indicated that both 0.5% SDS and 0.5% histidine solutions offer favorable elution efficiency, i.e., 78% for SDS and

18


Page 19 of 31

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


82% for histidine, while the BR buffers at high pH values give rise to low recovery (

Magnetic Nanospheres Encapsulated by Mesoporous Copper Oxide

Recommend Documents