ZnS nanocomposites towards ultrasensitive resonant Raman scattering-based immunoassays

Materials Letters 253 (2019) 354–357

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Fabrication of Fe3O4/ZnS nanocomposites towards ultrasensitive resonant Raman scattering-based immunoassays Yadan Ding a, Yuwei Sun a, Xia Hong a,⇑, Tie Cong a, Xiaokun Wen a, Huiying Zhao b,⇑ a b

Key Laboratory of UV-Emitting Materials and Technology (Northeast Normal University), Ministry of Education, Changchun 130024, China Department of Basic Medicine, Gerontology Department of First Bethune Hospital, Jilin University, Changchun, Jilin 130021, China

a r t i c l e

i n f o

Article history: Received 30 January 2019 Received in revised form 25 May 2019 Accepted 29 June 2019 Available online 1 July 2019 Keywords: Nanocomposite Semiconductor Magnetic material Resonant Raman scattering Immunoassay

a b s t r a c t Magnetic-optical nanocomposites, which consist of iron oxide nanoparticles and quantum dots, have aroused considerable research interest owing to their unique magnetic and luminescent properties. However, the luminescence of quantum dots tends to be quenched by the magnetic component. Resonant Raman scattering (RRS) of semiconductor nanocrystals, which originates from a strong electron-phonon interaction, provides a new candidate property to be exploited as an analytical tool. Herein, Fe3O4/ZnS nanocomposites were prepared with the seed-mediated growth approach. The nanocomposites exhibited superparamagnetism and characteristic multi-order phonon resonant Raman lines. By using biofunctionalized Fe3O4/ZnS nanocomposites as RRS probes, a femtomolar immunoassay of a model protein, human IgG, was realized with the assistance of an external magnetic field. The results provide guidance for the broad applications of magnetic-optical nanocomposites. Ó 2019 Elsevier B.V. All rights reserved.

1. Introduction Magnetic optical nanocomposites that consist of iron oxide nanoparticles and quantum dots have shown great potential in a wide range of applications, such as bioimaging, cell separation, theranostics and biochemical sensing [1,2]. The magnetism of the iron oxide component allows for magnetic separation, magnetic targeting, magnetic resonance imaging (MRI) and magnetic hyperthermia [3,4]. The unique optical properties of the quantum dots render the nanocomposites attractive as fluorescent tags [5]. Despite the great advances, the photoluminescence of quantum dots tends to be quenched by the magnetic component [6], which limits the applications of magnetic-optical nanocomposites. Resonant Raman scattering (RRS) of semiconductor nanocrystals originates from a strong electron-phonon interaction under resonance excitation conditions. Compared with non-resonant conditions, the resonant Raman intensities can be enhanced by up to six orders of magnitude [7]. The evenly-spaced multiphonon resonant Raman lines are several orders of magnitude narrower than their fluorescence bands [8]. More importantly, the RRS property is less susceptible to the surroundings than the fluorescence property. Unfortunately, the magnetic-RRS bifunctionalities ⇑ Corresponding authors. E-mail addresses: [email protected] (X. Hong), [email protected] (H. Zhao). https://doi.org/10.1016/j.matlet.2019.06.092 0167-577X/Ó 2019 Elsevier B.V. All rights reserved.

of iron oxide-quantum dot nanocomposites have been rarely explored. Fe3O4/ZnS nanocomposites have been intensively studied in various applications due to their magnetic and fluorescent properties [9,10]. Herein, Fe3O4/ZnS nanocomposites were prepared and the magnetic-RRS bifunctionalities were explored. Oleate-capped Fe3O4 nanoparticles served as the seeds, and ZnS was grown on the surface by thermolysis of Zn-oleate in the presence of sulfur. A sandwich structured immunoassay protocol was further designed and applied for ultrasensitive detection of a model protein with the biofunctionalized magnetic-RRS nanocomposites. 2. Materials and methods The details of the chemicals and instruments have been included in the Electronic Supplementary Material. 2.1. Preparation of the Fe3O4/ZnS nanocomposites Fe3O4 nanoparticles were prepared by thermal decomposition of Fe(acac)3 [11]. Zn-oleate was prepared through the reaction of Na-oleate and ZnCl2 [12]. To prepare Fe3O4/ZnS nanocomposites, oleic acid (4.5 mL), oleylamine (15 mL) and hexane-dispersed Fe3O4 nanoparticles (10 mg/mL, 0.6 mL) were mixed and heated to 100 °C under N2 flow to first remove hexane, and then Zn-oleate and sulfur with a molar ratio of 1:1.70 were added.

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The mixture was further heated to 300 °C and kept for 1 h. Ethanol was added to facilitate the precipitation of the nanocomposites, and the final product was dispersed in chloroform after washing and magnetic separation. 2.2. Sandwich structured immunoassay protocol Fe3O4/ZnS nanocomposites (10 mg/mL, 2 mL) were mixed with poly(acrylic acid sodium salt) (PAA, 10 mg/mL, 20 mL) and stirred for 48 h. The nanocomposites were transferred to phosphate buffered saline (PBS) after magnetic separation and washing. Then, 1 mL of N-(3-dimethylaminopropyl)-N0 -ethylcarbodiimide hydrochloride (EDC)/N-hydroxysulfylsuccinimide sodium salt (NHS) (EDC/NHS, 0.05 M/0.01 M) was added to the PAA-capped Fe3O4/ZnS nanocomposites (8 mg/mL, 1 mL), and reacted for 1.5 h. After magnetic separation and washing, goat-anti-human IgG solution (660 nM, 0.5 mL) was added, and incubated at 37 °C for 1 h. After magnetic separation and PBS scrubbing, bovine serum albumin (BSA) solution (1%, 3 mL) was added and incubated at 37 °C for 1 h. Fe3O4/ZnS RRS probes were obtained after magnetic separation and PBS scrubbing, and dispersed in PBS (2 mL). The immunoassay substrates were prepared by immersing goldcoated silicon wafers in ethanol solution of thioctic acid (2%) for 24 h, EDC/NHS solution (0.1 M/0.02 M) for 1.5 h, and then goatanti-human IgG solution (660 nM) for 2 h at 37 °C with gentle shaking. After rinsing the substrates with water and blowing dry with N2 gas, BSA solution (1%) was incubated with the substrates at 37 °C for 2 h. To construct the sandwich structure, the Fe3O4/ZnS RRS probes (100 mL) were incubated with different concentrations of human IgG (the analyte, from 1 fM to 1 nM) at 37 °C for 2 h, and washed with PBS and magnetically separated. The immunoassay substrates were then immersed in the RRS probe/analyte complex solution, and incubated at 37 °C for 1 h with a permanent magnet placed under the substrates. Finally, the substrates were thoroughly rinsed with PBS and blown dry with N2 gas. RRS spectra of the substrates were then recorded under an excitation of 325 nm. 3. Results and discussion 3.1. Composition and morphology of the Fe3O4/ZnS nanocomposites The synthesis strategy of the Fe3O4/ZnS nanocomposites is depicted schematically in Scheme S1. The strategy was initiated by the adsorption of Zn-oleate onto the surface of oleate-capped Fe3O4 nanoparticles through hydrophobic-hydrophobic interaction, hydrogen bonding and dispersion force. Thermal decomposition of Zn-oleate then produces Fe3O4/ZnO nanocomposites. When sulfur, which has been widely applied in the sulfuration reaction of various organic compounds and inorganic oxides [13], is added along with Zn-oleate, Fe3O4/ZnS nanocomposites can be obtained. If the amount of sulfur is not sufficient, Fe3O4/ZnO/ZnS nanocomposites can be generated. XRD patterns of the nanocomposites prepared with different amounts of sulfur are shown in Fig. 1a. As expected, Fe3O4/ZnO was formed in the absence of sulfur, evidenced by the coexistence of diffraction peaks of spinel structured Fe3O4 (JCPDS card No. 75-1610) and hexagonal wurtzite structured ZnO (JCPDS card no. 65-3411). When a small amount of sulfur was added, additional diffraction peaks from cubic zinc-blende structured ZnS (JCPDS card no. 65-9585) appeared, indicating the formation of Fe3O4/ZnO/ZnS nanocomposites. When the amount of sulfur was increased, the diffraction peaks of ZnO disappeared, and Fe3O4/ZnS was obtained. A typical transmission electron microscopy (TEM) image of the Fe3O4/ZnS nanocomposites is presented in Fig. 1b. The Fe3O4/ZnS nanocomposites exhibited a quasi-spherical shape with an average size of approximately 10.5 ± 2.1 nm. The lattice fringes corresponding to the (1 1 1)

Fig. 1. (a) XRD patterns of the nanocomposites prepared with different molar ratios of Zn-oleate to sulfur: (i) 1:0, (ii) 1:0.85, and (iii) 1:1.70. (b) TEM and (c) HRTEM images of Fe3O4/ZnS nanocomposites.

lattice plane of ZnS and (2 2 0) lattice plane of Fe3O4 were distinguished from the high-resolution TEM (HRTEM) image (Fig. 1c). It further demonstrated the successful preparation of Fe3O4/ZnS nanocomposites. 3.2. Magnetic and RRS properties of the Fe3O4/ZnS nanocomposites The magnetic property of the Fe3O4/ZnS nanocomposites was characterized by the hysteresis loop at 300 K. As shown in Fig. 2a, no hysteresis was observed, indicating the superparamagnetic property of the Fe3O4/ZnS nanocomposites. The nanocomposites were enriched rapidly under an external magnetic field within 1 min, and redispersed easily after removing the magnetic field (inset of Fig. 2a). The RRS property of the Fe3O4/ZnS nanocomposites was characterized under excitation at 325 nm. The resonant enhancement effect resulted from the small difference between the photon energy (3.82 eV) of the excitation light and the optical bandgap of zinc-blende structured ZnS (3.72 eV) [14]. Three-order phonon RRS lines of ZnS were observed at around 349.1, 695.9 and 1044.3 cm1 (Fig. 2b). These fingerprint-like evenly-spaced Raman lines were ascribed to the first, second and third-order longitudinal optical (LO) phonon band of zinc-blende structured ZnS, respectively [15]. 3.3. RRS-based immunoassay A sandwich structured immunoassay protocol was designed and shown schematically in Scheme S2. RRS probes were pre-

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Fig. 2. (a) Hysteresis loop of Fe3O4/ZnS nanocomposites. The inset shows that the Fe3O4/ZnS nanocomposites were concentrated or redispersed easily with or without an external magnetic field. (b) RRS spectrum of the Fe3O4/ZnS nanocomposites.

pared by conjugating goat-anti-human IgG antibodies covalently onto PAA-capped Fe3O4/ZnS nanocomposites and blocking the remaining reactive sites with BSA. The biofunctionalization process had little effect on the RRS signals of the Fe3O4/ZnS nanocomposites (Fig. 3a). The analyte (human IgG) was then captured by the RRS probes, and assembled with the goat-antihuman IgG antibodies-functionalized immunoassay substrate to form a sandwich structure with the assistance of an external magnetic field. After removing the unbound Raman probes, the RRS spectra of the sandwich structures with various amounts of the analyte were recorded and are presented in Fig. 3b. The Raman intensity decreased with decreasing concentration of the analyte. When the concentration of the analyte was as low as 1 fM, the 1LO phonon Raman line was still well distinguished, demonstrating the great potential of the magnetic-Raman Fe3O4/ZnS nanocomposites for ultrasensitive biochemical assays. No RRS signals were detected in the absence of the analyte (blank experiment), indicating high specificity of the immunoassay protocol. High selectivity was also demonstrated by the Raman spectrum obtained in the unspecific group, where goat-anti-rabbit IgG

antibodies were used to prepare the Raman probes instead of goat-anti-human IgG antibodies. 4. Conclusions In summary, Fe3O4/ZnS nanocomposites were prepared with Fe3O4 nanoparticles acting as the seeds and Zn-oleate and sulfur acting as the precursors of ZnS. The ratio of Zn-oleate to sulfur played an important role in producing Fe3O4/ZnS nanocomposites. The Fe3O4/ZnS nanocomposites exhibited superparamagnetism with a saturation magnetization of 19.9 emu/g and evenly-spaced multi-phonon resonant Raman lines. Magnetic field assisted ultrasensitive immunoassay of human IgG was realized with biofunctionalized Fe3O4/ZnS as RRS probes. This work opens up a new avenue for the construction and optimization of multifunctional nanoplatforms. Declaration of Competing Interest None.

Fig. 3. (a) RRS spectra of (i) Fe3O4/ZnS nanocomposites and (ii) Raman probes. (b) RRS spectra of the sandwich structures assembled under different concentrations of the analyte.

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Acknowledgements This work was supported by the National Natural Science Foundation of China (Grants No. 11604043 and 51772122), Scientific and Technological Developing Scheme of Jilin Province (No. 20190201243JC), Thirteenth Five-Year Science and Technology Research Project of Education Department of Jilin Province (No. JJKH20170910KJ), and Project funded by China Postdoctoral Science Foundation (No. 2017 M611294). Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi.org/10.1016/j.matlet.2019.06.092. References [1] Q. Ma, Y. Nakane, Y. Mori, M. Hasegawa, Y. Yoshioka, T.M. Watanabe, K. Gonda, N. Ohuchi, T. Jin, Biomaterials 33 (2012) 8486–8494.

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