Copper-Decorated Titanate Nanosheets: Novel Homogeneous

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Copper-Decorated Titanate Nanosheets: Novel Homogeneous Monolayers with a Superior Capacity for Selective Isolation of Hemoglobin Peng-Fei Guo, Dan-Dan Zhang, Zhiyong Guo, Ming-Li Chen, and Jian-Hua Wang ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b08942 • Publication Date (Web): 08 Aug 2017 Downloaded from http://pubs.acs.org on August 9, 2017

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ACS Applied Materials & Interfaces

Copper-Decorated Titanate Nanosheets: Novel Homogeneous Monolayers with a Superior Capacity for Selective Isolation of Hemoglobin Peng-Fei Guo, Dan-Dan Zhang, Zhi-Yong Guo, Ming-Li Chen* and Jian-Hua Wang* Research Center for Analytical Sciences, Department of Chemistry, College of Sciences, Northeastern University, Box 332, Shenyang 110819, China

Keywords: unilamellar nanomaterials; titanate nanosheets; copper-decorated nanosheets; selective isolation; hemoglobin Abstract: Novel unilamellar and homogeneous titanate nanosheets were prepared by anchoring of (3-aminopropyl)triethoxysilane (APTES) and chelating of copper ions, shortly as Cu-APTES-TiNSs. The nanosheets are uniform 2D lamellas/monolayers with a thickness of 1.9 nm, and they were further characterized by AFM, SEM, TEM, FT-IR, XRD, XPS, ICP-MS and N2 adsorption-desorption. The copper-decorated titanate nanosheets possess a copper content of 4.28%±0.14% and exhibit a favorable selectivity to the adsorption of hemoglobin with a considerable capacity of 5314.2 mg g-1. The adsorbed hemoglobin is easily collected with a recovery rate of 91.3% by using 0.5% (m/v) sodium dodecyl sulfate (SDS) as the eluent. Circular dichroism (CD) spectra confirmed that virtually no conformational alteration is observed for hemoglobin. Cu-APTES-TiNSs are further applied for the selective adsorption of hemoglobin from human whole blood.

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INTRODUCTION During the last years, the preparation and application of multifarious two-dimensional (2D) nanosheets have caught extensive attentions due to the excellent properties and the vast application prospects of graphene.1-5 Titanate nanosheets, as novel exfoliated 2D titanate nanostructures, have attracted intense research interest due to their exceptional physiochemical properties 6 and various applications as adsorbents,7 photocatalysts8 and nanobio materials.9 It has been demonstrated that layered titanate nanosheets with an abundant interlayer surface exhibit highly selective adsorption capability toward ciprofloxacin and corresponding fluoroquinolone antimicrobial molecules.10 In addition, the exfoliated monolayer titanate provides tremendous reversible intercalation capability for hemoglobin and exhibits the potential for improving the hemoglobin biocatalytic behavior in organic solvents.11-12 In this respect, it is thus imaginable that titanate nanosheets could be potential novel nanoadsorbents for the interaction with biomacromolecules/proteins and facilitate their selective adsorption/isolation from complex biological systems/fluids. Over the past decade, proteomics have gained tremendous interest, whereas the efficient separation and purification of proteins/proteome from real biological systems still remain the key premise and propose great challenge.13-15 It is known that immobilized metal affinity chromatography (IMAC) is widely employed for the isolation/enrichment of specific proteins/proteomes or peptides in virtue of the affinity interaction between specific amino acids of protein and metal ions immobilized on a

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matrix.16 For IMAC sorbents, the metal ions, e.g., Ti4+, Ni2+, Zr4+ and Cu2+ have modest affinity constants and higher ligand stability compared to biospecific affinity ligands.17-20 Cu2+ is a sort of special metal ion extensively used for the separation and purification of proteins from complex biological systems, as the strong affinity interaction between copper and histidine residues in proteins provides excellent selective absorption toward histidine-rich proteins, particularly hemoglobin.21-22 Hemoglobin is not only the chief component of red blood cells but also the foremost respiratory protein of vertebrates by virtue of its capability in the transportation of oxygen and carbon dioxide. In the construction of efficient IMAC sorbents, (3-aminopropyl)triethoxysilane (APTES) is a widely used coupling agent with favorable branching capacity, outstanding flexibility and solubility between the metal ions and the substrate.23 It is most suitable for the insertion of titanate owing to the proper molecular size and the formation of Ti-O-Si bonds between APTES and titanate.9 Besides, APTES can also be used for the immobilization of copper via the coordination with Cu2+.24-25 In this study, a novel copper-decorated titanate nanosheets is prepared by anchoring of (3-aminopropyl)triethoxysilane (APTES) and chelation of copper on the surface of titanate nanosheets. Cu2+ ions were chelated with amino groups and silicon oxygen groups of APTES-anchored titanate nanosheets (APTES-TiNSs), which ensures the homogeneity of the exfoliated single-layer titanate nanosheets lateral. The copper-decorated titanate nanosheets are merited with a 2D monolayer structure. As a novel adsorption nanosorbent, Cu-APTES-anchored titanate nanosheets

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(Cu-APTES-TiNSs) selectively adsorb hemoglobin by virtue of the specific metal-affinity. It also exhibits favorable biocompatibility which causes virtually no conformational variation for hemoglobin after adsorption and desorption. The adsorption of hemoglobin with Cu-APTES-TiNSs from human whole blood was further performed, which clearly presents tremendous capacity and potential for biological applications.

RESULTS AND DISCUSSION Characterizations. The copper-decorated monolayer titanate nanosheets Cu-APTES-TiNSs were achieved by chelating copper ions on the surface of the titanate nanosheets, as illustrated in Scheme 1. Prior to the chelation with copper ions, the interlayer cesium ions in the pristine titanate (Csx[Ti2−x/4□x/4]O4 (□:vacancy; x~0.7)) were replaced with proton exchange, followed by sequential intercalation of dodecylamine and APTES into the protonated titanate. APTES-TiNSs were obtained by ultrasonication of the APTES-anchored titanate aqueous suspension. Ultimately, Cu-APTES-TiNSs were achieved by the chelation/complexation of copper with amino groups and silicon oxygen groups.

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Scheme 1. The preparation schematics for copper-decorated monolayer titanate nanosheets Cu-APTES-TiNSs.

As shown in Figure 1, AFM images demonstrated that Cu-APTES-TiNSs are homogeneous 2D unilamellar nanosheets with a lateral dimension of ca. 400 nm and a thickness of 1.9 nm. The bare titanate monolayer was reported to have a thickness of about 0.75 nm 26 and the molecular length of APTES molecule was estimated to be about 0.75 nm. The measured thickness of Cu-APTES-TiNSs was well consistent with the calculated value from the assumption that both faces of the TiNSs were modified with APTES molecule (Scheme 1). The lateral homogenization of Cu-APTES-TiNSs is caused by the chelation/complexation of copper with amino groups and silicon oxygen groups on the surface of the titanate nanosheets.

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Figure 1. AFM images and height profile of the Cu-APTES-TiNSs

SEM images in Figures 2a-2b indicated that the Cu-APTES-TiNSs are aggregates of a large number of irregular monolayer nanosheets, lying or standing with each other and exhibiting layered nanostructure. While TEM images in Figures 2c-2d further demonstrated the irregular monolayer nanosheets structure of Cu-APTES-TiNSs. The surface variation of titanate nanosheets corresponds to the functionalization of copper and APTES. This observation is consistent with that observed by AFM images.

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Figure 2. SEM images (a, b) and TEM images (c, d) of the Cu-APTES-TiNSs

As presented in Figure S1, the suspensions of Cu-APTES-TiNSs and APTES-TiNSs are light green and white respectively. The colour change is attributed to the functionalization of APTES-TiNSs with copper. The XPS spectrum in Figure 3a showed two strong satellite peaks and a main peak corresponding to Cu2+ 2p3/2 at 942.4, 938.2 and 931.8 eV. This clearly demonstrated the characteristic feature of the Cu2+ species.27 The binding energies at 529.1, 457.5, 400.4, 284.6 and 102.0 eV were assigned to O 1s, Ti 2p, N 1s, C 1s and Si 2p, which confirms the successful anchoring of APTES. 7



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FT-IR spectra of cesium titanate, protonated titanate, dodecylamine-intercalated titanate, APTES-TiNSs and Cu-APTES-TiNSs were presented in Figure 3b. The absorptions at 515 cm-1 observed in all the spectra were assigned to Ti–O vibration in titanate. In the spectrum of protonated titanate, the bands at 3400 and 1630 cm-1 were accounted for the stretching and bending vibrations of H2O (H3O+).28 For the dodecylamine-intercalated titanate, the absorptions at 1370, 1470 and 1630 cm-1 were ascribed to C-N stretching, C-H bending and N-H bending, while those at 2920 and 2850 cm-1 were considered as C-H stretching vibrations.29 Those bands at 1631 and 1108 cm-1 observed on APTES-TiNSs were ascribed to N-H bending and SiO-C stretching vibrations respectively, which further confirms the successful anchoring of APTES.9 The absorptions at 1628 and 1104 cm-1 stem from N-H bending vibration and SiO-C stretching vibration were also observed in the spectrum of Cu-APTES-TiNSs. However, slight shifts in the peak positions for these two absorptions were identified in Cu-APTES-TiNSs with respect to those observed in APTES-TiNSs. This observation could be explained by the coordination of copper with amine groups and silicon oxygen groups.25 XRD patterns of cesium titanate, protonated titanate, dodecylamine-intercalated titanate, APTES-TiNSs and Cu-APTES-TiNSs were illustrated in Figure 3c. The 2θ diffraction peaks of cesium titanate at 10.5°, 20.8°, 28.2°, 31.4°, 35.3°, 40.4°, 44.0° and 47.7° correspond to (020), (040), (130), (060), (150), (121), (170) and (200) planes of Csx[Ti2−x/4□x/4]O4 (□:vacancy).30 The diffraction peaks of protonated titanate at 9.8°, 19.5°, 30.7°, 33.8°,39.3°, 46.4° and 48.2° might be assigned to the

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(020), (040), (060), (031), (051), (071) and (200) planes of Hx[Ti2−x/4□x/4]O4·H2O (□:vacancy; x~0.7).31 These diffraction peaks of dodecylamine-intercalated titanate at 4.2°, 6.2°, 12.5° and 48.5° are consistent with the (010), (020), (030) and (200) planes of the octylamine-intercalated titanate. These observations well indicated successful intercalation of dodecylamine.9 The diffraction peaks of APTES-TiNSs at 5.3°, 10.4° and 48.1° are identified for (020), (030) and (200) planes of the APTES-intercalated titanate, associating with the successful anchoring of APTES.9 These peaks are slightly shifted to 6.2°,12.5° and 48.5° in Cu-APTES-TiNSs with respect to those observed in APTES-TiNSs, which might be due to the complexation/chelation of copper with amino groups and silicon oxygen groups. This observation is well agreed with the results derived from the FT-IR spectra. The BET surface area of Cu-APTES-TiNSs was derived to be 163.1 m2 g-1. The nitrogen adsorption/desorption isotherms in Figure 3d are identified as type IV isotherm with a type H3 hysteresis loop,32 which is a characteristic feature in mesoporous structures comprising of aggregates of nanosheets providing slit-shaped pores with inhomogeneous sizes.33 This observation is well agreed with those derived from the SEM images.

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Figure 3. XPS spectrum (a) of Cu-APTES-TiNSs. FT-IR spectra (b) and XRD patterns (c) of cesium titanate, protonated titanate, dodecylamine-intercalated titanate, APTES-TiNSs and Cu-APTES-TiNSs. N2 adsorption/desorption isotherms (d) of Cu-APTES-TiNSs.

Protein adsorption behaviors by Cu-APTES-TiNSs. Figure 4 illustrated Hb, BSA and cyt-c adsorption behaviors using APTES-TiNSs and Cu-APTES-TiNSs prepared with various copper contents as adsorbents. It is obvious that Cu-APTES-TiNSs adsorb a certain extent of Hb (>90% for 100 mg L-1 Hb), while cyt-c and BSA are virtually not adsorbed. However, it is interesting to see that at the

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same experimental conditions Cu-APTES-TiNSs exhibit a significant adsorption toward hemoglobin with respect to APTES-TiNSs, and the adsorption efficiency increases significantly by enhacing the copper content in the range studied. On the contrast, however, other proteins, i.e., cyt-c and BSA, are virtually not adsorbed by Cu-APTES-TiNSs similarly to that observed with APTES-TiNSs, and the adsorption remains unchanged by varying the copper content. These observations indicated that there is a dominative copper-protein specific affinity interaction. Considering that the adsorption of hemoglobin is saturated at a copper content of 4.28%±0.14%, further studies are conducted at this level.

Figure 4. The effect of copper content on Hb, BSA and cyt-c adsorption behavior by (A) APTES-TiNSs, (B-F) Cu-APTES-TiNSs with 0.34%±0.01%, 1.35%±0.05%, 4.28%±0.14%, 7.35±0.44% and 7.98±0.22% wt% Cu. Protein solution: 1.0 mL, 100 mg L-1, pH 7. Adsorption time: 30 min. Cu-APTES-TiNSs: 0.1 mg.

Figure 5a illustrated the adsorption of the proteins by Cu-APTES-TiNSs within pH 4-11. It indicated that a favorable adsorption is obtained at the isoelectric point of 11



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each protein. At the isoelectric points, the proteins become neutrally charged and a certain degree of aggregation may occur via metal-affinity interaction between the anchored copper ions and the histidine residues of proteins. It is known that the numbers of the exposed histidine residues are 24, 2 and 1 respectively in the frameworks of Hb, BSA and cyt-c.34 Thus, a remarkably higher adsorption efficiency is obtained for Hb with respect to those achieved for BSA and cyt-c. As a comparison, Figure 5b also illustrated the sorption capabilities of the proteins of interest by APTES-TiNSs, where similar sorption behaviors are encountered as those observed with Cu-APTES-TiNSs as the adsorbent, i.e., the maximum adsorptions are obtained at the isoelectric points. This is due to the fact that as pH value closes to the isoelectric point, the protein species is neutrally charged and could preferentially form hydrogen bonds between APTES-TiNSs and the protein species of interest. The observations herein evidently suggested that metal-affinity interaction makes a great contribution to the adsorption of proteins by anchoring copper ions on the surface of APTES-TiNSs, offering a novel strategy for the surface functionalization of the monolayer titanate nanosheets as adsorbent, providing favorable selectivity for protein adsorption from biological sample systems. These observations demonstrated that the selective adsorption of Hb with coexisting proteins may be easily obtained through manipulation of the pH value in the reaction medium. It is noticed that at pH 7 BSA and cyt-c are almost not adsorbed, meanwhile, however, more than 90% Hb is adsorbed at the same experimental conditions.

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Figure 5. pH-dependent adsorption of Hb, BSA and cyt-c by using Cu-APTES-TiNSs (a) and APTES-TiNSs (b) as adsorbents. Protein solution: 1.0 mL, 100 mg L-1. Adsorption time: 30 min. Mass of the adsorbents, 0.1 mg.

The dependence of Hb adsorption efficiency by Cu-APTES-TiNSs upon ionic strength was investigated by varying NaCl concentration within 0.1-2.0 mol L-1. Figure S2 obviously indicated that changing the ionic strength within a broad range poses virtually no variation on Hb adsorption, and a maximum sorption could be readily achieved even in the presence of 2 mol L-1 NaCl. This observation distinctly illustrated the negligible contribution from electrostatic interaction to the protein adsorption. This further demonstrated vast potentials for Cu-APTES-TiNSs to conduct selective adsorption of hemoglobin from biological systems where relatively high ionic strengths are generally encountered. The adsorption capacity of Hb by Cu-APTES-TiNSs was obtained by carrying out the adsorption isotherm at 25 °C by using a protein concentration of 100-1000 mg L-1. As demonstrated in Figure S3, the sorption behavior of Hb fits Langmuir model

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as showed in the following, with C* as Hb concentration, Q* as the adsorbed amount of Hb by Cu-APTES-TiNSs, Qm as the maximum adsorption capacity, and Kd as the dissociation constant. A theoretical sorption capacity of 5555.6 mg g-1 is obtained, which is well in accordance with the experimental results, i.e., an adsorption capacity of 5314.2 mg g-1.

Q* =

Qm × C * Kd + C *

Table 1 compares the maximum sorption capacity for hemoglobin with diverse adsorption media. It is obvious that Cu-APTES-TiNSs offer a remarkably superior adsorption capacity for Hb compared with other adsorption matrixes. This might be due to the presence of abundant copper ions anchored on the surface of the unilamellar/monolayer titanate nanosheets, which provide more available binding sites for specific protein adsorption. On the other hand, titanate nanosheets have regularly powdery morphology and layered structure by their perfect layer-to-layer assembly, which could provide an unusual specific intercalation capacity.11 Thus, the considerable adsorption capacity might be facilitated not only by the decorated copper, but also by the nanosheets themselves. In order to demonstrate virtually the adsorption of hemoglobin, TEM image of Cu-APTES-TiNSs composite after a maximum adsorption of Hb was illustrated in Figure S4, where a large amount of hemoglobin was found to retain on the surface of Cu-APTES-TiNSs.

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Table 1. The maximum sorption capacities of Hb achieved by using various adsorption media. Adsorption medium

Adsorption capacity (mg g-1)

Reference

Cu2+-mediated magnetic MIP

232.6

[21]

Cu2+-IDA-SiO2-Fe3O4

418.6

[22]

Fe3O4@mCuO microspheres

1162.5

[34]

Cu2+-MMIPs

116.3

[35]

LaMOF-GO composite

193.0

[36]

P8W48-APTS

355.0

[37]

CuxOy/OMC composite

1666.7

[38]

Cu-APTES-TiNSs

5314.2

This work

Recovery of the retained hemoglobin from Cu-APTES-TiNSs. For further biological applications and proteomic studies/analysis, the recovery of retained Hb from Cu-APTES-TiNSs is highly desired. For this purpose, a series of potential stripping reagents, i.e., 4.0 mmol L-1 BR buffers, Tris, SDS and CTAB, were employed for the collection of the retained hemoglobin. Figure S5 illustrated that both 0.5% SDS and 0.1% CTAB solutions provide favorable recovery rates for the retained Hb, that is, 91.3% and 93.9% of hemoglobin could be recovered by SDS and CTAB solutions respectively. The stripping of Hb from Cu-APTES-TiNSs could probably be due to the association of the hydrophobic amino acid residues in the Hb framework and the hydrophobic dodecyl of SDS or CTAB. Further studies on the conformational variation of protein were conducted as illustrated by the CD spectra in Figure 6a-6b. It is clearly indicated that a 0.5% SDS solution poses no effect on the conformation of the recovered Hb, while 0.1% CTAB solution causes significant conformational 15



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change of the recovered Hb. For further investigations a 0.5% SDS solution is suitable for the collection of the adsorbed hemoglobin from Cu-APTES-TiNSs.

Figure 6. CD spectra of Hb standard solution and that after processing by the Cu-APTES-TiNSs and recovered in a 0.5% SDS medium (a) and 0.1 wt % CTAB solution (b).

In practical applications, the reusability of Cu-APTES-TiNSs in the adsorption and desorption of Hb is an important factor for assaying their performances. As shown in Figure S6, there is almost no variation on the adsorption efficiency of Hb when performing five cycles of continuous adsorption and stripping of hemoglobin. This result clearly illustrated the suitability of Cu-APTES-TiNSs as adsorbent for repetitive processing of hemoglobin adsorption. Hemoglobin adsorption from human whole blood. The practicability of Cu-APTES-TiNSs is further testified by performing isolation of hemoglobin from human whole blood. The blood samples were 400-fold diluted with BR buffer solution (4.0 mmol L-1, pH 7) and then the supernatant was collected by centrifugation at 3000 rpm for 5 min to perform adsorption by Cu-APTES-TiNSs by 16


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following the procedure as detailed in Experimental. The adsorbed Hb was then collected with 0.5wt% SDS aqueous solution. Afterwards, SDS-PAGE assays were performed according to the previously reported method.39 More details concerning SDS-PAGE were provided in the Supporting Information. Figure 7 illustrated several protein bands (Lane 2), contributed by serum albumin, carbonic anhydrase and hemoglobin. After treatment with 0.1 mg Cu-APTES-TiNSs for adsorption, these protein bands remained distinctly observable in the original fluid (Lane 3), while the band for hemoglobin was obviously decreased. The result in Lane 4 illustrated a clear single band of Hb at ca. 6.5 kDa by stripping with 0.5% SDS solution. The band for a hemoglobin standard solution (200 mg mL-1) was provided for comparison (Lane 5). These results illustrated that Hb could be selectively isolated from real biological systems with the coexisting proteins and complex matrices.

Figure 7. The results obtained for SDS-PAGE assay. Lane 1: molecular weight standards (kDa); Lane 2: human whole blood after diluted for 400-fold; Lane 3: the

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diluted blood by treatment with Cu-APTES-TiNSs for adsorption; Lane 4: Hb recovered from Cu-APTES-TiNSs by 0.5 wt % SDS; Lane 5: Hb standard solution (200 mg L-1).

CONCLUSIONS In this work, novel 2D unilamellar titanate nanosheets were obtained by surface anchoring of (3-aminopropyl)triethoxysilane and chelating copper ions on the surface of titanate nanosheets. The uniform and homogenous monolayer titanate nanosheets exhibit favorable selectivity toward hemoglobin with a superior adsorption capacity attributed to the metal (copper)-protein affinity interaction. The practical usefulness of copper-decorated titanate nanosheets is well proved by providing selectivity in the isolation of hemoglobin from real biological samples, i.e., blood. The present study indicated that unilamellar titanate nanosheets have great potential for biological applications, and it provides a promising protocol for establishing novel and efficient sorption media for the selective retention of specific protein species.

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ASSOCIATED CONTENT *Supporting Information

AUTHOR INFORMATION *Corresponding author. E-mail address: [email protected]. (M.L.Chen), [email protected] (J.H. Wang). Tel: +86 24 83688944

Notes

The authors declare no competing financial interest.

ACKNOWLEDGEMENTS The authors appreciate for financial supports from the Natural Science Foundation of China (21675019, 21235001, 21375013, 21475017), Fundamental Research Funds for the Central Universities (N150502001, N140505003), and the Open Funds of the State Key Laboratory of Electroanalytical Chemistry (SKLEAC201702).

Supporting Information Available: Experimental section, including Chemicals and reagents; Fabrication of Cu-APTES-TiNSs; Characterizations; Protein adsorption behavior with Cu-APTES-TiNSs. Figure S1: Photographs (a) of Cu-APTES-TiNSs and APTES-TiNSs.

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Figure S2: The variations of Hb adsorption efficiency dependent on the ionic strength of the reaction medium. Protein solution: 1.0 mL, 100 mg L-1, pH 7. Adsorption time: 30 min. Cu-APTES-TiNSs: 0.1 mg. Figure S3: The adsorption isotherm of Hb by Cu-APTES-TiNSs in a 4.0 mmol L-1 BR buffer at pH 7 (Left) and plot of 1/Q*eq against 1/C*eq (Right). Figure S4: TEM image of the Cu-APTES-TiNSs composite after a maximum adsorption of Hb, showing a large amount of hemoglobin retained on the surface of Cu-APTES-TiNSs. Figure S5: The recovery of hemoglobin from Cu-APTES-TiNSs by various stripping reagents. A: 4.0 mmol L-1 BR at pH 4; B: 4.0 mmol L-1 BR at pH 11; C: 0.1 mol L-1 Tris; D: 0.5 wt % SDS; E: 0.1 wt % CTAB. Figure S6: The reusability efficiency of Cu-APTES-TiNSs in the protein adsorption/desorption process. The details concerning SDS-PAGE assay.

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Graphical Abstract

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Copper-Decorated Titanate Nanosheets: Novel Homogeneous

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