Superparamagnetic MoS2@Fe3O4 nanoflowers for rapid resonance-Raman scattering biodetection

Sensors for rapid and reliable detection of biomolecules are crucial for clinical medical diagnoses. Here, a rapid, ultra-sensitive, magnetic-assisted biosensor based on resonance Raman scattering at MoS2@Fe3O4 composite nanoflowers is presented. Raman shifts and X-ray photoelectron spectra indicated that the composite was formed via Fe–S covalent bonds. Convenient magnetic separations could be performed because of the superparamagnetic Fe3O4 nanoparticles. MoS2 E12g and A1g Raman peaks were used as probe signals for anti-interference immunoassays. The probe unit of the immunoassay also included goat anti-human IgG molecules that were used as the target analyte. Au substrates coupled with the goat anti-human IgG were used as capture units to form sandwich biosensors. Because of the magnetic enrichment, the detection limit was improved by three orders-of-magnitude and the detection time was reduced from 1.5 h to 1 min. Sandwich biosensors using MoS2@Fe3O4 nanoflowers as Raman probes could be very promising sensors for proteins, antigens, and other immunogenic biopolymers, as well as for corpuscular viruses and cells.


Introduction
Rapid and accurate biological detection of pathogenic biological materials has been particularly important since the outbreak of COVID-19. Challenges facing most detection methods are complex, time-consuming procedures, and low target concentrations [1]. Biological Raman detection is attractive because of its fingerprint characteristics, narrow half-widths, noninterference from water, and no photobleaching [2].
These offer the possibility of accurate and simple low-concentration bioassays [3]. With regard to shortening detection times, superparamagnetic nanoparticles (NPs) benefit from sensitive responses to external magnetic fields, no coercivity, and no remanence. Superparamagnetic Fe 3 O 4 NPs are biocompatible and can be used in clinical medicine. Its high saturation magnetization enables rapid magnetic enrichment and separations [4,5]. Fe 3 O 4 NPs can be accurately accumulated at required sites by applying an external magnetic field for rapid enrichment of target molecules. Thus, they improve the detection limit and shorten the detection time [6][7][8].
Magnetic-Raman dual-functional bioassays have demonstrated improved detection limits for miRNA [9], antigens [10], and malachite green isothiocyanate [11]. Moreover, Fe 3 O 4 superparamagnetic nanoparticles have significantly shortened detection times [12], and magnetic separations are more convenient. Previous studies were mainly focused on composites of superparamagnetic nanoparticles and noble metal nanostructures for magnetically assisted surface-enhanced Raman scattering [13,14].
In contrast to surface-enhanced Raman scattering, resonance Raman scattering (RRS) from inorganic nanomaterials does not require the assistance of metallic nanostructures. The target signals also do not require organic Raman reporters or contrast enhancers, which simplifies the procedure [15][16][17]. Previous reports have used RRS signals from semiconductors (ZnO and ZnS) for femto-molar-level immunological detection [18]. Unlike those for zincbased semiconductors, the MoS 2 bandgap is in the visible light region. Thus, MoS 2 RRS can be excited with a 532-nm laser source, instead of the ultraviolet light required for ZnO or ZnS. This makes detection safer, less costly, and no autofluorescence interference has been observed from bulk MoS 2 [19][20][21]. Therefore, RRS of MoS 2 is promising for biosensing. However, the complexation of Fe 3 O 4 and MoS 2 for RRS-based biological detection has not been reported. MoS 2 @Fe 3 O 4 nanocomposites were fabricated, and the E 1 2g and A 1g vibrational modes of MoS 2 were used as RRS probe signals for immunoassays. The magnetic separation and enrichment improved the detection limit and shortened the detection time. This magnetic-assisted rapid RRS detection strategy was simple, low cost, environmentally safe, and could be used for early detection of various pathogens, infectious diseases, and cancers.

Characterization
X-ray diffraction (XRD) patterns were acquired with a Rigaku D/MAX using Cu-Ka radiation (Rigaku Corporation, Japan). The sample morphologies were imaged with scanning electron microscopy (SEM, Quanta FEG-250, FEI Co. Ltd., USA) and transmission electron microscopy (TEM, TecnaiG220S-Twin, USA). X-ray photoelectron spectra (XPS) were acquired with a Thermo Escalate 250Xi XPS spectrometer (Thermo Scientific, USA). RR spectra were acquired with a confocal Raman spectrometer (Horiba Lab Ram HR evolution, Japan). Vibrating sample magnetometry measurements were performed with a 736-VSM Controller (Ohio, USA).

Sandwich Raman immunoassay protocol
The synthesis and working principle of the sandwich biosensor are illustrated in Scheme 1. The biosensor was composed of three parts: a Raman probe signal unit (part A), a Au substrate capture unit (part B), and the immunoassay (part C).
For the preparation of the Raman probe signal unit in part A, a 200-ll solution of MoS 2 @Fe 3 O 4 composites (0.007 mg/ml) was mixed with the same volume of goat anti-human IgG (660 nM), followed by shaking at 37°C for 1.5 h. Then, the resulting MoS 2 @Fe 3-O 4 -antibody complex was magnetically enriched and washed twice to remove excess antibodies. Then, 200 ll (0.2 mg/ml) of BSA in a PBS solution was added and the mixture shaken at 37°C for 1.5 h to block unreacted active sites. Finally, the MoS 2 @Fe 3-O 4 -antibody Raman probe was washed with DI water and then magnetically separated and captured.
The Au substrate capture unit was prepared as described previously [18]. As depicted in part B of Scheme 1, human IgG was used as the target analyte in the immunoassay. In part C of Scheme 1, the goat anti-human-IgG-capture Au substrate was immersed in a human IgG analyte solution with various concentrations. After shaking at 37°C for 1.5 h, the substrate with the captured analyte was rinsed. Here, two strategies were used to study the effects of the external magnetic field on the immunoassay results. In strategy I, the substrate with the captured analyte was immersed in the solution containing the MoS 2 @Fe 3 O 4 Raman probe and gently shaken for various periods of time (6 min, 1 h, or 1.5 h) at 37°C. In strategy II, the substrate was also immersed in the Raman probe solution, but was then placed under an external magnetic field (1.2 T) for several minutes (6 min, 3 min, 1 min, or 0.5 min) instead of being shaken.

Characteristics of the MoS 2 @Fe 3 O 4 composites
XRD was performed to determine the crystal structure of the composites (see Fig. 1 [27], which indicated that the composites were formed with covalent bonds. The peaks at 718.5 eV and 732.5 eV were assigned to Fe (III) and Fe (II) satellites, respectively [28]. The peaks at 530.3 eV and 531.7 eV in the O1s spectra (Fig. 3c) were attributed to Fe-O and O-H in Fe 3 O 4 , respectively. The peaks at 530.9 eV in the O1s spectrum (Fig. 3d) corresponded to Mo-O, which may be attributed to oxidization during the synthesis [29,30]. The characteristic peaks at 228.6 eV and 231.8 eV in the spectra of Mo 3d (Fig. 3e) were attributed to Mo 3d 5/2 and Mo 3d 3/2 , respectively. The component at 225.7 eV corresponded to S 2s in the Mo-S bond [31], and the peak at 235.6 eV corresponded to Mo (VI) [32,33]. In Fig. 3f, the S 2p 1/2 and S 2p 3/2 peaks were located at 162.7 eV and 161.5 eV, respectively, in the composite spectra, which were similar to other reported data or MoS 2 [34].
Characteristic vibrational fingerprints were reflected in the RRS. As noted above, the 1.29-1.9-eV bandgap of MoS 2 enabled resonant 532-nm laser excitation. In Fig. 4a, the Raman peaks at 379.0 cm -1 and 405.1 cm -1 were attributed to the in-plane E1 2 g  and out-of-plane A 1g vibrational modes of MoS 2 , which were consistent with previous reports [35]. Relative to those for MoS 2 , both the E1 2 g and A 1g modes of MoS 2 @Fe 3 O 4 NFs shifted to lower energies (377.1 cm -1 and 403.3 cm -1 , respectively). The shift was attributed to the interaction of MoS 2 with Fe 3 O 4 NPs that formed Fe-S bonds observed in the XPS [36]. This changed the stress in the MoS 2 lattice, which was affected by its multilayer structure, and eventually led to the change in the lattice vibrational frequency [37].  Fig. 4b. Because the NFs were superparamagnetic, the nanoflowers could be redistributed by slight shaking after the external magnetic field was removed, which enabled rapid biological detection.

Rapid and ultra-sensitive detection via MoS 2 @Fe 3 O 4 NF Raman probes
The biological detection of the dual-functional MoS 2 @Fe 3 O 4 NFs was evaluated utilizing the MoS 2 Raman peaks as probe signals and human IgG as the antigen. The detection results using strategy I are shown in Fig. 5a. The RRS intensity decreased with decreasing concentration of the analyte (1 nM, 100 pM, 1 pM, and 100 fM). The weak RRS signal from MoS 2 was observed when the analyte concentration was 100 fM, but was almost invisible when the concentration was 10 fM. Therefore, the experimental detection limit using strategy I was 100 fM. The opportunity of coupling between the Raman probe and the Au substrate was enhanced by shaking. However, that was insufficient for detecting very low concentrations. In strategy II, the immunological substrate immersed in the probe solution was placed above a commercial magnet (* 1.2 T). The ''antibody-analyte-antibody'' sandwich structure was a bridge between the RRS of MoS 2 and the Au substrate. The detection results are shown in Fig. 5b. The RRS signal of the 0.01 fM analyte was negligible, but that for the 0.1 fM analyte was strong. Hence, the detection limit for strategy II was enhanced by three orders-of-magnitude relative to that for strategy I, and was also better than previous reports [38,39]. Standard deviations of the Raman peak intensities are shown in Fig. 5a, b. To verify the detection specificity, RRS measurements were performed in the absence of the analyte (''blank'' in Fig. 5). No MoS 2 fingerprint Raman signal was obtained, indicating that nonspecific adsorption did not occur between the Raman probe and the Au substrate. Another benefit of the magnetic RRS immunoassay was that the detection time was significantly shortened. Because of the magnetic enrichment of the probe, the binding between the capture antibody (goat anti-human IgG on the substrate) and the analyte (human IgG coupled to the probe) was accelerated in a magnetic field during incubation. In strategy I, at least 1.5 h incubation was needed to obtain strong Raman signals (Fig. 5c). However, when a magnet was used to perform the assays (the concentration of the analytes was 100 pM), strong RRS signals were detected after 30 s of incubation, and the signal intensity was stable after 1 min (Fig. 5d). The results thus demonstrated that the Fe 3 O 4 enhanced the detection limit and shortened the detection time.

Conclusions
In summary, magnetic MoS 2 @Fe 3 O 4 NF nanocomposites were fabricated. XPS characterization verified that the composites were covalently bonded. A magnetic-assisted rapid RRS detection method based on MoS 2 @Fe 3 O 4 NFs shortened detection times from 1.5 h to 1 min, and the detection limit was enhanced by three orders-of-magnitude (from 100 fM to 0.1 fM). The sandwich biosensor exhibited high specificity for human IgG. In addition, the MoS 2 @Fe 3 O 4 NF-based immunoassay was robust in various pH solutions and chemically stable. The rapid and ultra-sensitive antibody assay should also be applicable to viruses and early diagnoses of diseases such as cancer. Author contributions TZ contributed toward writing original draft, investigation, data curation, and formal analysis. XC contributed toward conceptualization, manuscript writing, reviewing, and editing, methodology, visualization, project administration, and funding acquisition.
FJ contributed toward investigation, manuscript writing, reviewing, and editing, funding acquisition. MX contributed toward manuscript writing, reviewing, and editing, and funding acquisition. YZ collected resources, and contributed toward manuscript writing, reviewing, and editing, Fig. 5 Raman spectra of the immunoassays: a, b various concentrations of analyte; c, d various incubation times. a and c correspond to strategy I (without magnetic field), b and d correspond to strategy II (1.2-T magnetic field, 100 pM). The insets are error analysis plots of the E1 2 g mode intensity and funding acquisition. JL contributed toward conceptualization, supervision, manuscript writing, reviewing, and editing, and funding acquisition.

Data Availability
The datasets are available from the corresponding author upon reasonable request.

Declarations
Conflict of interest Authors declare no conflicts of interest.
Ethical approval The authors declare that all data in this paper are original and have not been published in any other journal. This paper did not involve unethical treatment of animals.
Consent for participate All authors have provided consent to participate.
Consent for publication All authors have read the manuscript provided for publication.