Breast cancer biomarker detection through the photoluminescence of epitaxial monolayer MoS2 flakes

In this work we report on the characterization and biological functionalization of 2D MoS2 flakes, epitaxially grown on sapphire, to develop an optical biosensor for the breast cancer biomarker miRNA21. The MoS2 flakes were modified with a thiolated DNA probe complementary to the target biomarker. Based on the photoluminescence of MoS2, the hybridization events were analyzed for the target (miRNA21c) and the control non-complementary sequence (miRNA21nc). A specific redshift was observed for the hybridization with miRNA21c, but not for the control, demonstrating the biomarker recognition via PL. The homogeneity of these MoS2 platforms was verified with microscopic maps. The detailed spectroscopic analysis of the spectra reveals changes in the trion to excitation ratio, being the redshift after the hybridization ascribed to both peaks. The results demonstrate the benefits of optical biosensors based on MoS2 monolayer for future commercial devices.


Scientific RepoRtS
| (2020) 10:16039 | https://doi.org/10.1038/s41598-020-73029-9 www.nature.com/scientificreports/ ambient conditions 17,18 , what makes it a perfect candidate for photoluminescence (PL) biosensors. Moreover, after the interaction with the target sequence, the binding events occurring near the surface of MoS 2 produce perturbations in the local dielectric permittivity, enabling detection via PL changes. However, the difficulty to produce high quality MoS 2 flakes has limited the progress in this research line.
With the aim of taking advantage of MoS 2 PL for the development of new biosensors for sensitive breast cancer biomarkers, in this work we use epitaxially grown MoS 2 flakes on sapphire as transducers in a new biosensor for miRNA21 recognition. In order to functionalize these platforms with the bioreceptor (capture probe), we used a specific thiol modified DNA sequence. The thiol functional group is used to fix the biomolecule to the flakes [17][18][19][20] in a disposition that allows hybridization with the target. For the assays, both complementary and non-complementary miRNA21 targets were employed, in order to demonstrate the selectivity of the biosensor. The characterization of the sensing response was carried out by individually monitoring the PL of MoS 2 flakes. In addition, thanks to the microscopic PL system used, both the wavelength and the intensity were mapped across the surface of the flakes, analyzing the homogeneity of the response.

experimental section
Materials. Concerning the transducer, the MoS 2 powder of high purity (99%) used as the precursor for the growth of 2D flakes was obtained from Alfa Aesar. Sodium phosphate and sodium chloride were obtained from Scharlab Co. Synthetic 22-mer oligonucleotides were supplied by Sigma-Aldrich Co. A single-stranded DNA sequence modified at 5′-end with an hexalquilthiol was used as capture probe, and denoted as ss-DNAp-SH. As target analyte a fully complementary sequence (denoted as miRNA21c) and the non-complementary sequence (denoted as miRNA21nc) were used. All these sequences are listed in Table 1. All solutions were prepared just prior to use. Water was purified with a Millipore Milli-Q-system (18.2 MΩ·cm) and was sterilized with a Nüve OT 012 small steam autoclave.
Procedures. Epitaxial growth of MoS 2 monolayers. The MoS 2 monolayers were synthesized by vapor phase growth on double side polished (0001) sapphire substrates, previously cleaned with acetone solution and isopropanol 21 . The growth was carried in a quartz tube fitted in a 3 zone furnace. MoS 2 powder precursor was placed in a quartz boat at the center of the quartz tube. The sapphire substrates were placed downstream at a distance of ~ 10 cm from the precursor. The growth was performed at a pressure of 10 mbar under 20 sccm Ar flow, with the furnace temperature ramped to 970 °C and held there for a duration of 20 min. Afterwards, the furnace was cooled down naturally.
Immobilization of the thiolated capture probe onto MoS 2 flakes. Prior to the capture probe immobilization, the prepared MoS 2 flakes on sapphire were characterized by Raman and PL spectroscopy in order to confirm the monolayer growth and the PL intensity homogeneity. Then, 10 µL of a 10.0 nM thiolated capture probe (ss-DNAp-SH) solution was deposited onto the as-deposited MoS 2 nanoflakes and was kept at 4 °C for 24 h. Afterwards, the functionalized MoS 2 flakes (ss-DNAp-SH-MoS 2 ) were washed with sterile water to remove unspecific adsorbed probe and was dried with N 2 before PL experiments.
Hybridization event detection. The ss-DNAp-SH-MoS 2 sensing platform was subsequently hybridized (1 h, 40 °C) with the analyte solution by addition of 10 µL of a 10.0 nM complementary (miRNA21c) or non-complementary (miRNA21nc) sequence in 10 mM phosphate buffer + 0.4 M NaCl pH 7.0 solution. The sensing platform was then immersed in sterile water to remove unspecific adsorbed material and dried with N 2 . Finally, the effect of the hybridization process in the MoS 2 PL was studied.
Optical measurements. Raman and PL measurements were acquired in an Olympus (100 × objective) system with 488 nm laser at a power of 1 mW, a corresponding notch filter and a Jobin-Yvon iHR-320 monochromator (600 L/mm grating) coupled to a Peltier cooled Synapse CCD. With this system, PL maps were acquired with 1 µm steps in 10 × 10 µm 2 areas. Each spectrum was taken at 1 s of integration time and 2 accumulations.
Results and discussion characterization of as-grown MoS 2 /sapphire platforms. In an optical biosensor, the quality of the platform is crucial for the subsequent understanding of the transduction procedure. In this work, the MoS 2 material is epitaxially grown on sapphire by vapor phase transport. This method is commonly used for the deposition of large areas with good crystalline quality 22 . Figure 1a shows the optical image of one of these samples, displaying the well-known triangular morphology of MoS 2 flakes, with dimensions of tens of microns. These flakes are composed of three layers of S-Mo-S in a sandwich-like structure named as monolayer MoS 2 . Different www.nature.com/scientificreports/ triangular isolated flakes (A-D) are identified in the figure. These flakes were analyzed by means of Raman and PL spectroscopy. Figure 1b shows the Raman spectra of the different chosen flakes, which are almost identical and overlapped. The two main peaks are located at 384 cm −1 and 404 cm −1 , which belong to the main phonon vibrations E 1 2g and A 1g , respectively 23 . The wavenumber difference between these two peaks (20 cm −1 ) is used as a fingerprint of the monolayer character of the material 24 , although some differences can arise from using different excitation lasers 23 or substrates 25,26 . The ratio of the intensities A 1g /E 1 2g is 1.927. The PL intensity is another indicator of the monolayer character 27 , and the results obtained in the flakes highlighted are shown in Fig. 1c. The spectra were acquired at four different points in the central regions of the flakes, and all of them display a broad band centered at ~ 673 nm, indicating the 2D character of the layers. The sharp peak around 693 nm in Fig. 1c belongs to impurities of Cr 3+ from the sapphire substrate 28 . Here, the important fact is that the PL intensity barely changes between points (see the inset), confirming the similar quality of the flakes. However, in order to further analyze this homogeneity, we performed a detailed map of PL intensity in flake E (Fig. 1d). The intensity in the central part of the flake is essentially constant, and it increases considerably in the borders due to structural defects, typically Mo vacancies 21,29 . DNA functionalization and miRNA21 detection. The development of the MoS 2 based biosensor is schematically shown in Fig. 2. Figure 2a shows the crystalline structure of the triangular MoS 2 flakes deposited on sapphire. As can be seen in Fig. 2b, the first step is the functionalization of the triangular flakes deposited on sapphire with the DNA capture probe, a ss-DNA sequence totally complementary to the analyte (miRNA21c). Hence, the DNA probe modified at 5′-end with an hexalquilthiol (ss-DNAp-SH) is immobilized on MoS 2 flakes through the thiol group. The organic molecules with -SH group tend to repair or eliminate S vacancies (V S ) of the MoS 2 lattice, resulting in the molecular functionalization with the substrate 19,20,30,31 . After this step, the functionalized platform was tested by hybridization with the complementary (miRNA21c) and the non-complementary (miRNA21nc) sequences, the latter used as control. Figure 2c,d shows these two hybridization assays, which were performed according to the procedure described in the experimental section. PL measurements have been taken in different MoS 2 flakes before and after all these steps. Figure 3 shows the PL spectra obtained for the two routes described before, carried out in two different flakes. As a standard procedure, we tagged different flakes for the analysis prior to the functionalization. Then, we acquired several spectra in different spots of the same flakes to analyze the reproducibility and increase the statistical significance. www.nature.com/scientificreports/ Figure 3a shows the PL measurements for the as-grown MoS 2 flake (grey lines), for the same flake after the immobilization of ss-DNAp-SH (red lines), and for the same flake after the hybridization with miRNA21c (blue line). Figure 3b shows the results for another flake where we used the miRNA21nc control.
A common feature is the increase of the PL after the immobilization of ss-DNAp-SH. Indeed, the intensity is enhanced by a factor ranging between 2 and 6. This fact indicates the effective anchorage of the probe to the MoS 2 surface as discussed in detailed later on. However, the hybridization process is different for the miRNA21c (Fig. 3a) and for the miRNA21nc (Fig. 3b) targets. Although both of them evidence a decrease in the PL signal, the miRNA21c produces a singular redshift, which is not present in the miRNA21nc. It is important to mention that organic molecules can react with MoS 2 by covalent chemical functionalization 32 or by weak bonds such as Van der Waals 33 , resulting in non-specific reactions, but the redshift in the wavelength clearly points out that the bonding process is significantly different for miRNA21c than for miRNA21nc.
In order to quantify this effect, Fig. 4 shows the wavelength of the PL peak for the different tests performed: miRNA21c (Fig. 4a) and the miRNA21nc (Fig. 4b). A redshift ~ 16 nm (43 meV) occurred for the miRNA21c sequence, whereas the miRNA21nc shows almost no changes. Note that we have performed these tests in 4 different flakes and 4 different spots for each flake, to warrant a good reproducibility of the data within the statistical errors. Therefore, the results demonstrate the specificity of the biosensor and the viability of the recognition of miRNA21 biomarker through PL.
In order to further analyze the homogeneity of the biosensing platforms, we recorded PL wavelength maps for the different steps followed during the assays. Figure 5 shows the maps of the flakes used, where the color scale represents the wavelength of the main peak. Both as-grown MoS 2 flakes (Fig. 5a) and ss-DNAp-SH functionalized flakes (Fig. 5b) show essentially the same PL emission, with maximum variations of 2 nm. When hybridization with the miRNA21c occurs (Fig. 5c), the PL map changes from cyan (670-675 nm) to yellow color (687-689 nm), indicating a homogeneous redshift on the surface of the flake. However, the hybridization with miRNA21nc (Fig. 5d) does not change the wavelength significantly.
It is important to note that the homogeneity is high in the central region, where most of the sulfur vacancies (V s ) are found. At the borders of the MoS 2 flakes the vacancies are predominantly Mo-based, as it has been demonstrated in different works 19,21,29 . Indeed, the borders exhibit p-type doping, typically ascribed to molybdenum vacancies (V Mo ), which results in a blueshift of the PL main peak, an effect confirmed in our maps.
Spectroscopic analysis of the PL peaks. As mentioned before, the functionalization of the MoS 2 with the ss-DNAp-SH depends on the efficiency of the bonding to the as-grown MoS 2 flakes. The presence of intrinsic defects of MoS 2 monolayer has been intensively studied in the last few years, reporting a rich variety of point defects and dislocation cores 34,35 . These defects are known to have a relevant impact on the binding energies www.nature.com/scientificreports/ of the excitons 16 . Some of these defects have been identified in non-resonant PL studies at low temperatures 36 . Indeed, MoS 2 monolayer seems to exhibit at least six optical transitions at 4 K, three of them ascribed to defects. However, at room temperature the remaining PL peaks are typically associated with three main optical states: the A − trion (~ 1.85 eV), the A exciton (~ 1.90 eV), the B exciton (~ 2.03 eV) [36][37][38][39] . The negative trion is normally ascribed to the binding of the photoexcited electron-hole pairs with the excess electrons from the surface, i.e., to the presence of defects (normally producing the n-type behavior of MoS 2 ) 40 . The exact energy values for the states, as well as the existence of multiexcitons are still under debate and may vary also for different substrates and experimental conditions. Multiexciton states, however, might be stable even at room temperature due to the strong Coulomb interaction and reduced dielectric screening of the monolayer flakes 37 . Taking into account this scenario, we have carried out Lorentzian fits of representative PL spectra, shown in Fig. 6 and summarized in Table 2. Figure 6a shows the deconvolution of the PL peak for the as-grown MoS 2 flake. Three main contributions have been identified at ~ 1.80, ~ 1.85, and ~ 2.00 eV, with the one at 1.85 eV the most intense. Thus, the most plausible assignment for the A exciton in our case is the peak at 1.85 eV and, correspondingly, the A − trion at 1.80 eV and the B exciton at 2.00 eV. The values for the A exciton and A − trion agrees well with the ones reported in similar MoS 2 flakes by Zuo et al. 41 . Figure 6b shows the analysis of a characteristic PL spectrum after the ss-DNAp-SH functionalization. As mentioned in the previous section, the intensity of the PL emission is higher. An increase of the MoS 2 PL emission intensity is often attributed to the chemical adsorption of molecules, although this effect can depend on the particular molecule. The experiments performed with single-stranded and double-stranded DNA proved the good affinity of such biomolecules to MoS 2 via van der Waals forces 11 . More importantly for our case, the thiol groups may repair the S vacancies or transfer electrons to MoS 2 , causing the passivation of defect mediated non-radiative recombination or provide excess electrons in the conduction band of MoS 2 30,42 . Therefore, the PL www.nature.com/scientificreports/  www.nature.com/scientificreports/ enhancement we see can be understood as a result of the effective functionalization of the bioreceptor through both specific (thiol) and weak bonding. From Fig. 6b, we see that the optical mechanism behind the PL change has two clear features: the overall improvement of the intensity and the change in the A − /A ratio. These effects are compatible with screening mechanisms due to the change of the dielectric function produced by the functionalization 39 . At the same time, charge transfer mechanisms between MoS 2 monolayer and other molecules (or layers) have been reported recently 43,44 and validated by means of density functional theory computations 45 . Interestingly, this charge transfer mechanism was found to be substrate-dependent too 26 . Therefore, a combination of these two phenomena (change in the dielectric function and charge transfer) can explain the results found. Figure 6c shows the PL of the hybridized platform with miRNA21c, evidencing an overall decrease of the intensity and a redshift. The decrease in the intensity can be interpreted as the presence of non-radiative channels. The redshift involves both the displacement of A − and A peaks but, also, a new change in the A − /A ratio, which is higher than unity. In addition, the B exciton also experiences a redshift. This behavior does not occur with the miRNA21nc control (Fig. 6d), where the trion peak intensity decreases significantly in the spectrum. Due to the broad features of the spectra, the existence of an additional peak cannot be completely ruled out (a good fit is also possible with 4 peaks in Fig. 6c,d). Hence, the reasons for the observed behavior are not clear and, despite the recent progress 36,37,46 , the current reports does not allow determining a unique origin for that. More research is needed to establish the exact energies of the excitons and multiexcitons, and the specific effect of defects on the optical properties. In this sense, the use of combined and more local techniques could help to shed light on these issues.  www.nature.com/scientificreports/ conclusions In this work we have fabricated an optical biosensor for miRNA21 biomarker of breast cancer taking advantage of the PL of MoS 2 monolayer flakes. This sensor has been produced in three steps: (1) growth of MoS 2 epitaxial layers on sapphire, (2) functionalization with a thiolated DNA probe (ss-DNAp-SH), and (3) hybridization with a complementary and non-complementary miRNA21 sequences (the latter used as a control). The modification with the ss-DNAp-SH increases the native PL from MoS 2 , which diminishes after the recognition assays. A redshift of 16 nm was observed exclusively for the hybridization with miRNA21c, but not for the control miRN-A21nc sequence, demonstrating the specificity of the biosensor and the viability of the recognition via PL. The homogeneity of the biosensing platforms was further verified with microscopic maps. The detailed spectroscopic analysis of the spectra reveals changes in the A − /A trion/exciton ratio, with the redshift after the hybridization ascribed to both peaks. Overall, our results indicate the benefits in terms of sensitivity and selectivity of optical bionsensors based on MoS 2 monolayer. The transduction method through the PL wavelength change, instead of the PL intensity, is a significant achievement for the development of commercial biosensors in the future. Due to the use of individual flakes for the tests only a small area of the sample is needed. However, more research is needed to recover the sensor to the original stage after its usage, since the functionalization of the probe and the miRNA21 modify the original PL intensity of the flakes, preventing the recycling of the same flakes.