Typical XRD spectra of the bulk MoS₂ used for exfoliation and the subsequently obtained nanosheets are shown in Figure S1. The reflection peaks were assigned to the family lattice planes of bulk MoS₂ (JCPDS card no.77-1716). After probe sonication in N-vinyl-2-pyrrolidone for 8 h, the intensity of the (002) peak decreased dramatically, implying the formation of few-layered MoS₂ nanosheets. No new peaks appeared in the XRD spectrum of the exfoliated MoS₂ nanosheets.
UV-vis absorbance spectra of MoS₂ nanosheets and nano-onion/MoS₂ nanosheet composites with various weight ratios (1:1 to 1:8) were measured in the range from 300 to 900 nm. MoS₂ nanosheets showed excitonic absorption peaks at 624 and 689 nm arising from the direct gap transitions at the K point of the Brillouin zone and induced by energy splitting of the valence band and spin orbital coupling. An additional peak at around 460 nm is attributed to the optical transition between the density of states peak in the valence and conduction bands [34]. The first peak in the longer wavelength region corresponds to the lowest optical band gap of ~ 1.8 eV of the MoS₂ nanosheets, which is higher than that of bulk MoS₂ (~ 1.2 eV) [35, 36]. This increase is a clear indication of quantum confinement in the nanosheets, with that of a few layers of MoS₂ being close to that of its single-layer counterpart. Excitonic absorption by the nano-onion/MoS₂ nanosheet composites was preserved since the positions of the peaks at 624 and 689 nm were similar. Although as the weight ratio of MoS₂ in the nano-onion/MoS₂ nanosheet composite was increased, their intensities became stronger. These results indicate that the optical characteristics of MoS2 were well preserved in the nano-onion/MoS₂ nanosheet composites. Moreover, chemical conjugation of the nano-onions to the MoS2 nanosheet surfaces did not deteriorate the optical properties of MoS2.
The chemical structures of nano-onion-COOH, MoS2 nanosheets, and nano-onion/MoS2 nanosheet composites were investigated by using XPS, the results of which are shown in Fig. 2. The C 1s spectra were deconvoluted to provide evidence for the formation of the nano-onion/MoS2 nanosheet composites. Nano-onions derived from nanodiamonds have an unsaturated vinyl C 1 peak at 284.6 eV (C = C), indicating that onion-like amorphous carbon was generated during thermal annealing (Fig. 2(a)). Surface oxidation of the nano-onions treated by using Hummer’s method was confirmed by the strong oxidation peak intensities at 287.2 eV (C-O) and 289 eV (COOH). High oxygen content is indicated by the carbonyl and carboxyl peaks at 531.6 and 532.8 eV (Fig. 2(c)), thereby proving that the oxygen-terminated surface functional groups were generated on the outer nano-onion shells, which could be chemically conjugated with the MoS2 nanosheets. Nano-onion-COOH has a high chemical composition of C = C sp2 bonds (approx. 45.1%), while the MoS2 nanosheets showed weak peak attribution.
The XPS C 1s spectra of the nano-onion/MoS2 nanosheet composites in Fig. 2(e–l) indicate that the C = C peak became smaller as the amount of MoS2 nanosheets was increased, which indicates the formation of a composite comprising nano-onions and MoS2 nanosheets. In addition, the C = O/C-N ratio of the nano-onion/MoS2 nanosheet composites increased as the amount of MoS2 nanosheets was increased, with ratios of 1:2, 1:4, and 1:8 achieving 11.2%, 9.1%, and 14.2%, respectively. And C = O/C-N peak position of the nano-onion/MoS2 nanosheet composites was accompanied by a shift to a higher energy binding energy (from 287.2 to 287.5eV). Semi-electron-rich MoS2 nanosheets on the nano-onions facilitate electron transfer into the nano-onion graphitic shell layer, resulting in increased electron mobility between the nano-onions and MoS2 nanosheets. The C = O/C-N composition of nano-onion-COOH and MoS2 nanosheets were 21.8%, and 3.9%, respectively. The deconvoluted O 1s spectra of the nano-onions and nano-onion/MoS2 nanosheet composites show peaks at 531.6 eV for C = O and 532.8 eV for C-OH. As the amount of MoS2 nanosheets was increased, the peak intensity of C-OH at 532.8 eV lessened, indicating that the carboxylic groups in the nano-onions had chemically conjugated with the MoS2 nanosheets.
The Mo and S chemical composition and bonding states in the MoS2 nanosheets and nano-onion/MoS2 nanosheet composites were investigated by using appropriate deconvoluted XPS spectra (Fig. 3). The deconvoluted XPS Mo 3d spectra show two dominant peaks and a smaller peak at a higher binding energy for both the MoS2 nanosheets and nano-onion/MoS2 nanosheet composites. In the nano-onion/MoS2 nanosheet composites, the chemical composition of Mo 3d was relatively strengthened as the amount of MoS2 nanosheets was increased. Moreover, the peaks were slightly shifted to lower binding energies in the nano-onion/MoS2 nanosheet composites, compared to the MoS2 nanosheets. This infers that complexation of the nano-onions and MoS2 nanosheets occurred and resulted in a small rise in the electron density around Mo in the nanocomposites, a phenomenon that is similar to N-doped MoS2 [37]. The deconvoluted XPS S 2p spectra in Fig. 3(b) provide the same results as for Mo 3d with the highest composition ratio in the MoS2 nanosheet and the lowest in the 1:1 nano-onion/MoS2 nanosheet composite. The S 2p doublet peaks were assigned as S 2p1/2 at 163.8 and S 2p3/2 at 162.6 eV, that is attributed to the binding energy of divalent sulfide (S2−) ions in stoichiometric MoS2 [38, 39]. Downshift of S 2p binding energy and decrease of binding energy indicate that sulfur may be reduced than the valance state of divalent sulfide (S2−), providing election-rich S chemical environment [40]. Similar to the Mo 3d spectra of composite, shifting of the peaks to lower binding energies in the nano-onion/MoS2 nanosheet composites indicates that nano-onion complexation with MoS2 nanosheets provided higher electron densities around the Mo and S atoms
FT-IR spectroscopy was used to identify functional groups in the various samples (Fig. 4). The FT-IR spectra of nano-onion-COOH and nano-onion-acyl chloride show a strong absorption band at 3,400 cm− 1 hydroxyl bond (OH) stretching and one at 1,625 cm− 1 attributed to C = C bond bending. Especially, the peak at 593 cm− 1 was due to acyl chloride bond (C-Cl) stretching in nano-onion-acyl chloride. The FT-IR spectrum of nano-onion-COOH showed strong absorption bands for the OH, -COOH, and C = C functional groups but none for C-Cl.
The FT-IR spectra of nano-onion-acyl chloride, aminated MoS₂ nanosheets, and nano-onion/MoS₂ nanosheet composites were compared to confirm conjugation of the amide bonds (NH-C = O) between nano-onion-acyl chloride and the amine-functionalized MoS₂ nanosheets. The latter shows an N − H deformation vibration peak at 1604 cm− 1 and an NH2 stretching peak at 3390 cm− 1. Meanwhile, the nano-onion/MoS₂ nanosheet composites had the characteristics of both the nano-onions and MoS₂ nanosheets with the disappearance of the C-Cl stretching peak at 593 cm− 1. The nano-onion/MoS₂ nanosheet composites provided absorption bands attributed to hydroxyl bond stretching at 3,400 cm− 1. Furthermore, the peak at 1632 cm− 1 corresponding to the NH-C = O group in the nano-onion/MoS₂ nanosheet composites was attributed to overlapping C = C vibrations and C = O stretching, indicating that successful chemical conjugation between the materials formed amide bonds.
Structural visualization of bulk MoS₂, MoS₂ nanosheets, nanodiamonds, nano-onions, and nano-onion/MoS₂ nanosheet composites are revealed in the HRTEM images in Fig. 5. The MoS2 nanosheets were quite transparent, indicating their nanoscale thickness. The lateral size of the MoS2 nanosheet ranged from 400–500 nm and its stripe-like grain lattice can be observed on the edge of the nanosheet at high magnification, thereby indicating the regular orientation of the elements therein. The onion-like curved layer shown in the HRTEM images of nano-onions verified the sp2 structure of the carbon atoms in both hexagonal and pentagonal arrangements, which is unlike planar graphene. The curved layer in the nano-onions resulted in a greater lattice spacing in their molecular layer (0.34 nm) than in nanodiamonds (0.2 nm). In the nano-onion/MoS2 nanosheet composites with ratios from 1:1 to 1:8, agglomerates of nano-onions were well enveloped in the MoS2 nanosheets, thereby indicating their stable binding together.
CV profiles of electrodes comprising MoS2 nanosheets or nano-onion/MoS2 nanosheet composites with weight ratios from 1:1 to 1:8 were obtained at scan rates of 0.05, 0.1, and 0.5 V/s (Fig. 6). The CV profile of the MoS2 nanosheets shows the typical rectangular shape and the small shoulder of redox peak, indicating a double-layer reaction and pseudocapacitive behavior with a fast-reversible redox reaction, respectively, as previous the reported literature [41–43]. The peak shoulder may be contributed from redox reaction of Mo active chemical site on the composite edge. The CV curves of MoS2 nanosheets at 0.05 V/s in the potential range from − 0.8 to 0.2 V suggest high electrochemical stability at the active sites in MoS2 and good reversibility due to fast electrolyte ion diffusion. The current density of the 1:1 nano-onion/MoS2 nanosheet composite electrode was significantly improved by the presence of nano-onions compared to the MoS2 nanosheet electrode. The enhanced high current density of 1:1 nano-onions/MoS2 nanosheeet complex electrode resulted from high conductivity and effectiveness of MoS2 active sites that has occurred by intercalation and deintercalation of K + ion during reduction and oxidation process [44]. The possible two mechanism of redox reaction of surface could be suggested from peaks in the CV profiles of MoS2- based electrodes (Eqs. 1 and 2) [44–46]. The non-faradaic process due to double layer formation may occurred during the adsorption of cation on the MoS2 nanosheet on the electrode, as following equation.
(MoS2)Surface + K+ + e −↔(MoS2 − K+)Surface (1)
Based on the pseudo-capacitive behavior of CV profile, alkali cation (K+) diffuse in the interlayer of MoS2 nanosheet with nanoonion interface as following equation.
MoS2 + xe− + xK+↔(MoS – SK+) (2)
The improved current density of the nano-onion/MoS2 nanosheet composite electrodes could be due to better facilitation of electron transfer. The nano-onion/MoS2 nanosheet composite electrodes with ratios from 1:2 to 1:8 had lower current densities than the 1:1 nano-onion/MoS2 nanosheet composite electrode but higher ones than the MoS2 nanosheet electrode. This could be ascribed to the fact that the adsorption and desorption processes of electrolyte ions (K+) on the surface and MoS2 intra/interlayers could be compromised by the electron transfer capability of the nano-onions.
CV curves was also performed at different scan rate of 0.05, 0.1, 0.5 V/s. NO complex formation provides effective intercalation of K + ion by generating additional conductive paths, showing that enhanced charge storage and additional kinetics of oxidation and reduction pathways. Nano-onion composite with MoS2 nanosheet would facilitate heterogeneous charge transfer and ion diffusion and capacity by providing platform of surface defect and surface area of active sites. Nano-onion may be contribute to the distanced distribution of Mo active site and edge, resulting enhanced distance of ion diffusion and facilitated electron transfer and capability [41, 44, 47]. The nano-onion/MoS2 nanosheet composites facilitated electron transfer at the interface and enhanced the electrochemical functionality of the electrode. As expected, the MoS2 nanosheet electrode with a higher amount of MoS2 provided a smaller area in its CV profiles, indicating that the nano-onions provide the dual roles of electronic transition and a conductive electron donor. The complexation of MoS₂ nanosheets with nano-onions overcomes the low conductivity of the former. The 1:1 nano-onion/MoS2 nanosheet composite was chosen as the DNA linking platform substrate.
DNA hybridization with a DNA probe and target DNA (HPV-16 or HPV-18) was investigated on the 1:1 nano-onion/MoS2 surface platform. MB as a redox indicator for electron transfer reactions intercalates with dsDNA via electrostatic adsorption for target DNA detection. DNA hybridization and MB intercalation was performed at reaction time of 10 min [11, 48]. The redox reaction of MB transfers one hydrogen ion and two electrons to the elongated and rigid hybridized dsDNA with the aid of a planar aromatic ring, thereby making it possible to examine the current charge via highly sensitive DPV with low limit of detection [49]. The current peaks via DPV appeared at ~ 1.1 V on the MoS2 nanosheet and 1:1 nano-onion/MoS2 electrodes.
Figure 7 shows DPV profiles for the MB electrochemical oxidation on the 1:1 nano-onion/MoS2 nanosheet composite and MoS2 nanosheet electrodes. Functional groups on the electrode surface were chemically conjugated with the HPV target probe, for which DPV measurements were performed sequentially. The chemisorption of the HPV-16 and HPV-18 target DNA probes was sensed in HPV-16-positive Siha cells (Fig. 7(a)) and HPV-18-positive Hela cells (Fig. 7(b)), respectively. The average DPV current peaks from both electrodes were measured via continuous and sequential DNA adsorption and HPV target DNA hybridization on each electrode surface with 1 µM MB and 0.1 M KCl (pH 7.0). Comparatively, the 1:1 nano-onion/MoS2 nanosheet composite electrode attained a higher current peak than the MoS2 nanosheet electrode, thereby confirming the synergistic effect of the nano-onions combined with MoS2 nanosheets. The current peaks obtained with both electrodes indicate the occurrence of the MB redox reaction at the electrode-buffer interface and probe. The nano-onions have catalytic properties and provide conductivity to the nano-onion/MoS2 nanosheet composite electrode. The hexagonal and pentagonal graphitic onion-like layers in the nano-onions provide excellent electron transfer [21, 50, 51]. The DNA probes for HPV-16 and HPV-18 were thiolated during their chemisorption on both the MoS2 nanosheet and 1:1 nano-onion/MoS2 nanosheet composite electrodes. The electronically conductive carboxylated nano-onions and amine-functionalized MoS2 nanosheets enable the electrode surface to chemisorb the DNA probe, which reduces the DPV current peak. Notably, the DPV current peak of the DNA probe on the 1:1 nano-onion/MoS2 nanosheet composite electrode was larger than that of the MoS2 nanosheet electrode during all sequential process of DNA detection. The addition of Siha and Hela positive HPV target ssDNA fragments lowered the DPV current peak, indicating that the oligonucleotides had successfully hybridized with the capture probes. MB intercalation into the hybridized DNA did not effectively elevate the current peak because other DNA fragments from the Siha and Hela cells could hybridize with MB, thereby reducing the accessibility of the guanine bases in the dsDNA on the surface [52]. A larger reduction in the DPV current peak for MB was induced by target DNA detection, and thus greater amplification of the differential sensing signal for both HPV cell lines was confirmed with the 1:1 nano-onion/MoS2 nanosheet composite electrode, compared to the MoS2 electrode. The excellent electronic conductivity and chemical functionalization of the 1:1 nano-onion/MoS2 nanosheet composite makes it potentially applicable to a wide variety of biological molecules.
To confirm the specificity of the DNA targeting by the sensor, non-complementary DNA was added to the HPV16 and HPV18 probe DNA surface electrodes, and then the current change by DPV was measured on 1:1 nano-onion/MoS2 nanosheet composite and MoS2 electrodes. Figure 8(a) and 8(b) show the current changes due to the binding of non-complementary DNA to the sensor probes for detecting HPV-16 and HPV-18, respectively. Increased DPV peak current amplitude was observed for both HPV-16 and HPV-18, confirming a reduction in the DPV current signal. The electrical coupling between the groove in ss DNA due to guanine and MB led a large DPV peak. When the non-complementary DNA was added to the sensor chamber, the DPV current peak increased because a double helix was not formed and MB could intercalate into the groove of ssDNA.
DPV responses were recorded after the addition of target DNA at various concentrations ranging from 5 pg to 5 ng with 1 µM MB in KCl (pH 7.0) (Fig. 9). The anodic peak current in presence of MB decreased after the target DNA was increased. The results indicate that the intercalation of MB between dsDNA bases was restricted and thus resulted in reduced oxidation due to steric hindrance of the bonded bases. For the HPV-16 probe DNA in the 1:1 nano-onion (NO)/MoS2 nanosheet composite sensor (Fig. 9(a)), the increased concentration of HPV-16 positive DNA in Siha cells decreased the anodic peak current due to the more restricted intercalation of MB. Similar results for detecting HPV-18 positive DNA in Hela cells (Fig. 9 (b)) also shows that the anodic peak current decreases as the concentration of target DNA increases. It can be confirmed that the DNA sensor sensitivity for both viruses was as low as 5 pg using low limit DPV measurement.