MoS2 quantum dots featured fluorescent biosensor for multiple detection of cancer

Transition metal ions, such as those generated through MoS2 material, possess an intrinsic fluorescence quenching property towards organic dye molecules; thus, they can be used to construct biosensors as quenchers. However, we found that the conventional bulk MoS2 blocks the view of fluorescence imaging, and is incapable of tracing and visualizing mucin 1-overexpression cancer cells. Herein, a FAM fluorophore-labeled ssDNA fluorescent probe (P0-FAM) stacked on the surface of MoS2 quantum dots (QDs) was used to construct a MoS2 QDs–P0-FAM biosensor. MoS2 QDs exhibit a high fluorescence quenching ability towards fluorescent dyes, possess large specific surface area and a large number of active sites to adsorb and quench more fluorescent probes, promoting sensitivity between quenching and the recovery signal. In addition, the lighter color of unstack-MoS2 QDs is beneficial to define the location of cancer cells compared to MoS2 nanosheets. The novel MoS2 QDs-based biosensor demonstrates high sensitivity to MUC1 with a detection limit of 0.5 nM, and may become an important tool toward the detection of cancer cells.


Introduction
Breast cancer is, by far, the most frequent cause of cancer deaths in women. 1 For this reason, early detection and early diagnosis are critical factors to guarantee treatability and curability. 2 As studies have reported, MUC1 is a transmembrane glycoprotein, which is abnormally expressed in all stages of development of human adenocarcinomas. [3][4][5] Overexpression of MUC1 in mammary glandular cells, compared with normal cells, is likely to alter its function and affect the behavior of cancer cells. Therefore, MUC1, as a biomarker indicator, is widely used for early detection of breast cancer. 6,7 General techniques for detecting MUC1 and cancer cells include ow cytometry, DNA chip technology and PCR techniques; however, these methods usually involve complex instruments and lack efficiency. 8 In addition, the uorescence method has characteristics of high sensitivity, strong antiinterference and rapid response, as well as tracking and visualizing analytes via uorescence imaging. [9][10][11] To overcome the drawbacks of time-consumption, high cost and complexity of uorescent biosensors, [12][13][14] we require to construct a simple uorescent biosensor with sensitive signal and strong speci-city recognition abilities to detect MUC1 and MCF-7. Recently, transient metal quantum dots were employed as nanoprobes for biological applications due to their uorescent, paramagnetic properties, radio-opacity, and quenching ability. [15][16][17] As far as we know, transition metal ions possess an intrinsic uorescence quenching property towards organic dye molecules. 18 Typically, MoS 2 , as an ultrathin direct bandgap semiconductor, has found wide spread applications in optoelectronics, nanoelectronics, and energy harvesting, [19][20][21] and can act as a uorescence quencher due to its capacity of quenching dye-labeled singlestranded DNA (ssDNA) via van der Waals force or coordination, 22-24 which opens new analytical opportunities. 25 However, uorescent imaging applications of frequently-used quencher MoS 2 nanosheets remain signicantly challenging. The multilayer stack of MoS 2 nanosheets blocks the view of uorescence imaging making it hard to clearly observe the location of cancer cells. In this study, we chose MoS 2 QDs to solve this problem.
Herein, we constructed a novel, simple and sensitive MoS 2 QDs-based sensing platform for the assay of MUC1. The biosensor is composed of a uorescent probe (FAM uorophore-labeled ssDNA, dened as P 0 -FAM) and a quencher (MoS 2 QDs). MoS 2 QDs can recognize complementary oligonucleotides or aptamers as recognition units. 26,27 In addition, they can spontaneously adsorb P 0 -FAM via van der Waals force between the nucleobases of ssDNA and the surface of MoS 2 QDs. The intrinsic uorescent quenching property of MoS 2 to organic dye molecules causes uorescence quenching of P 0 -FAM when P 0 -FAM is adsorbed on MoS 2 QDs, while the uorescence recovery of P 0 -FAM occurs under the attack of MUC1, which is attributed to the exposure of P 0 -FAM due to the detachment of P 0 -FAM from MoS 2 QDs with a stronger affinity between P 0 -FAM and MUC1. [28][29][30][31] The employment of MoS 2 QDs is benecial in uorescence imaging to detect the location, the size of the tumor and the treatment region due to larger specic surface area, more active sites to adsorb more uorescent probes, and the lighter color of unstack-MoS 2 QDs compared to MoS 2 nanosheets. The MoS 2 QDs-P 0 -FAM-based uorescent biosensor with sensitive signal and strong specicity recognition abilities to MUC1 is expected to provide a new perspective for the detection and diagnosis of breast cancer.

Instrumentation
A draught drying cabinet, numerical control ultrasonic cleaners and a medical centrifuge were used to prepare the MoS 2 QDs. Transmission electron micrograph (TEM) was obtained using an H-7650 TEM instrument (Hitachi, Japan). The X-ray diffraction (XRD) pattern was recorded on a D/max 2005VL/PC X-ray diffractometer (Rigaku, Germany). X-ray photoelectron spectroscopy (XPS) was conducted with PHI Quantera II. Zeta potential analysis was performed on a dynamic light scatter (DLS, NANO-ZS920, Malvern, UK). Fluorescence spectra were recorded on an F-4600 spectrouorometer (HITACHI, Japan) equipped with a xenon lamp, l ex ¼ 490 nm, l em ¼ 520 nm. The PMT voltage was 620 V and the slits for both the excitation and the emission were set at 10 nm. The MTT assay was obtained using a Varioskan ash microplate reader (Thermo Scientic) at 490 nm. The confocal microscopy experiments were conducted using a MRC-1024 (Bio-Rad, Ltd., USA).

Synthesis of water-soluble MoS 2 QDs
MoS 2 QDs were prepared by a modied mixed solvent strategy for liquid exfoliation. 32 Initially, 60 mg MoS 2 powder was mixed with 20 mL of ethanol/water with an ethanol volume fraction of 45% in a 50 mL ask. The sealed ask with the mixture was ultrasonicated for 24 h and a dark green suspension was obtained. The dispersion was centrifuged at 2000 rpm for 10 min three times to remove the aggregates. Following this, the supernatant was centrifuged at 10 000 rpm for 10 min and collected at 60 C in a drying cabinet to remove the ethanol and water absolutely. Next, the product was dissolved in deionized water and centrifuged at 2000 rpm for 10 min to remove the larger MoS 2 nanoparticles. Finally, the supernatant was ltered through a 0.22 mm Millipore membrane lter and collected at 60 C in a drying cabinet.

Selection of MoS 2 QDs concentration and kinetic assay
The MoS 2 QDs solution was diluted by PBS buffer (pH 7.4) to a nal concentration of 500 mg mL À1 . Different volumes (0-200 mL) of MoS 2 QDs solution (500 mg mL À1 ) were mixed with 100 mL of 100 nM P 0 -FAM in a 2.0 mL centrifugal tube; different volumes of PBS buffer were added to make 500 mL of each solution. The nal P 0 -FAM concentration was 20 nM, and the concentrations of MoS 2 QDs were 0, 5, 10, 15, 20, 30, 50, 100, and 200 mg mL À1 . Then, these mixtures were allowed to react for 30 min at 37 C. Finally, uorescence measurements were performed at room temperature.
Kinetic assay was performed on the uorescence quenching and uorescence recovery. For uorescence quenching, 100 mL of MoS 2 QDs solution (500 mg mL À1 ) was mixed with 100 mL of 100 nM P 0 -FAM in a cuvette. The uorescence measurements were performed at different times (0-6 min) at room temperature. For uorescence recovery, 100 mL of MoS 2 QDs solution (500 mg mL À1 ) was mixed with 100 mL of 100 nM P 0 -FAM in the cuvette; aer 15 min at room temperature, 250 mL MUC1 and 50 mL PBS were added into the reaction mixture. The uorescence measurements were performed at different times (0-10 min) at room temperature.
Assay for MUC1 in aqueous buffer 100 mL of MoS 2 QDs solution (500 mg mL À1 ) was mixed with 100 mL of 100 nM P 0 -FAM in a test tube; then, the mixed solution was allowed to react for 6 min at room temperature. Following this, different volumes of MUC1 (20 mM) in PBS buffer (0-250 mL) were added. Finally, different volumes of PBS buffer (pH 7.4) were introduced to prepare 500 mL of each reaction solution and the nal MUC1 concentration (0, 0.001, 0.005, 0.010, 0.050, 0.100, 0.500, 2, 3, 4, 5, 6, 7, 8, 9, and 10 mM). The nal mixed solution was allowed to react for 10 min at 37 C. The uorescence spectra were measured at room temperature.
Cell culture and MTT experiments MCF-7 cells, PBMSC and L929 cells were cultured in a cell culture ask in Dulbecco's modied Eagle's medium (DMEM)/ high glucose containing 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin solution (100Â) at 37 C under an incubator containing 5% CO 2 .
MCF-7 was incubated with different concentrations of MoS 2 QDs (10-200 mg mL À1 ) at 37 C and in 5% CO 2 for 24 h. Further, the cell viability experiments were conducted using a Varioskan ash microplate reader (Thermo Scientic) at 490 nm. The cell viability was then assessed using the equation below: Cell viability ð%Þ ¼ OD value of treatment group OD value of control group Â 100%

Assay for MCF-7 cells in DMEM
Initially, 1 mL of MoS 2 QDs solution (500 mg mL À1 ) was mixed with 1 mL of 100 nM P 0 -FAM in a test tube; the mixed solution was allowed to react for 6 min at room temperature. Then, 200 mL of the mixed solution was added to different concentrations of MCF-7 (0 to 5 Â 10 5 cells per mL). Aer incubation at 37 C for 30 min, the uorescence spectra were measured at room temperature.

Selectivity assays
The selectivity assays were tested by comparing the uorescence signal changes of samples containing glucose oxidase (GOD), cytochrome c (CyC), myoglobin (Mb), Lys and bovine serum albumin (BSA). Initially, 1 mL of MoS 2 QDs solution (500 mg mL À1 ) was mixed with 1 mL of P 0 -FAM (100 nM) in a test tube; the mixed solution was allowed to react for 10 min at room temperature. Then, 200 mL of the mixed solution was added to the sensing systems containing 10 mM MUC1, 100 mM glucose oxidase (GOD), 100 mM cytochrome c (CyC), 100 mM myoglobin (Mb), 100 mM Lys and 100 mM bovine serum albumin (BSA). Aer incubation at 37 C for 15 min, the uorescence spectra were measured at room temperature.

Characterization of MoS 2 QDs
The morphology of MoS 2 was studied using TEM (Fig. 1). The synthetic MoS 2 QDs ( Fig. 1B and C) are dispersed evenly in aqueous solution compared to MoS 2 nanosheets in aqueous solution (Fig. 1A). TEM images revealed that the average size of smaller MoS 2 QDs, which were used for further experiments, were about 3 nm ( Fig. 1C and D). The XRD patterns ( Fig. 2A) of the samples matched well with that of 2H-MoS 2 (JCPDS: 24-513). As can be observed, the primary diffraction peaks at 14.4 , 33.2 and 58.4 were attributed to the (002), (100) and (110) planes of the hexagonal MoS 2 , respectively, indicating the high purity of the obtained smaller MoS 2 QDs. 33 Raman spectrum was used to further conrm that smaller MoS 2 QDs were obtained (Fig. 2B). The Raman spectrum of bulk MoS 2 was well-known with two main modes, the A 1g and E 2g , corresponding to the out-plane vibrations and in-plane vibrations as located at 408 and 382 cm À1 , respectively. 34 This was represented by the black line shown in Fig. 2B. It could be observed that the A 1g mode of MoS 2 QDs slightly blue shied to 405.5 cm À1 , which proved that we had successfully prepared MoS 2 QDs. 35 As shown in Fig (Fig. 2D). The XPS spectra were consistent with those in previously reported literatures, indicating the dominant 2H MoS 2 phase in the MoS 2 QDs. 36,37 The zeta potential of the MoS 2 QDs were determined to be À27.8 mV (Fig. S1 †), suggesting the great colloidal stability of the MoS 2 QDs in aqueous media.   electrons or energy between the closely connected dye molecules and the MoS 2 QDs (Fig. S2 †). Interestingly, in the presence of MUC1, the uorescence recovery of P 0 -FAM could be observed because MoS 2 -P 0 -FAM adopted a rigid and denite tertiary structure owing to the specic binding between ssDNA and MUC1. The affinity of ssDNA with MUC1 was stronger than that of MoS 2 QDs, resulting in the release of the P 0 -FAM from the QDs surface and recovery of the uorescence signal. In contrast, without MoS 2 QDs, P 0 -FAM was primarily in the unfolded and exible state in the presence of MUC1. The FL signal did not drastically change, indicating that MoS 2 QDs as a quencher played a crucial role in turn-on FL biosensor for the sensitive detection of MUC1 in cancer cells.

Optimization of detection conditions
To evaluate the uorescence-quenching ability of MoS 2 QDs toward P 0 -FAM, the uorescence signal changes were recorded upon mixing P 0 -FAM and the prepared MoS 2 QDs. As shown in Fig. 3A, the quenching of FAM uorescence by MoS 2 depended on the concentration of the quenchers. In the presence of 100 mg mL À1 MoS 2 QDs, the emission of the FAM was almost quenched with 90% quenching efficiency (Fig. 3B), revealing a high quenching efficiency of MoS 2 QDs toward the aptamer biosensor. The observed background uorescence, as shown in Fig. 3B, corresponding to the uorescence of 200 mg mL À1 MoS 2 QDs, could be attributed to the existence of the secondary structure of P 0 -FAM at the detection conditions. Fig. 4A and B show the adsorption kinetics of the dye-labeled aptamer biosensor on the MoS 2 QDs. The quenching was rapid and achieved equilibrium in about 4 min. This suggested that the interaction of P 0 -FAM with MoS 2 QDs was quite strong and the MoS 2 QDs possessed a high uorescence-quenching ability. The MoS 2 QDs exhibited robust quenching efficiency possibly because of the better water dispersivity. Furthermore, the uorescence recovery kinetics was performed and the best uorescence recovery efficiency was obtained within 8 min when MUC1 was added into the mixture solution ( Fig. 4C and D). This suggested that the designed MUC1 biosensor system works successfully and can deliver high performance. In order to achieve more effective detection, 15 min was chosen as the optimal reaction time.

Quantitative analysis of MUC1 in aqueous buffer
As shown in Fig. 5, a new simple and sensitive assay for MUC1 was successfully designed. The uorescence intensity depended on the concentration of MUC1 over a range of 0-10 mM when the concentration of MoS 2 QDs was 100 mg mL À1 (Fig. 5A and B). As shown in Fig. 5C, the uorescence intensity increases rapidly as the concentration of MUC1 increases from 0 mM to 0.5 mM (R 2 ¼ 0.9978). However, it exhibited another linear relationship as shown in Fig. 5D (R 2 ¼ 0.997) when the concentration of MUC1 changed from 0.5 mM to 10 mM, where the uorescence intensity increased more slowly with the increase in MUC1 concentration. The reason for this phenomenon was probably that P 0 -FAM in the state of random coil single strand was correspondingly abundant when MUC1 was less than 0.5 mM in the system, and MoS 2 QDs could strongly adsorb these random coil singlestrands FAM-ssDNA onto its surface owing to the weak   adsorption competition among the free FAM-ssDNA. When MUC1 exceeded 0.5 mM in the system, free FAM-ssDNA in the state of random coil single strand became scarce. The adsorption competition among them became more intense, so the uorescence intensity changed more slowly as the MUC1 concentration increased. This method could be applied to detect MUC1 concentrations as low as 0.5 nM (3 times the standard deviation rule) in aqueous buffer. Moreover, the detection range was wide, ranging from 0 mM to 10 mM.
Combined with the data listed in Table S1, † the results demonstrated that the uorescent detection of biosensor for MUC1 was feasible for a relatively broad detection range and low detection limit. From this perspective, the proposed method towards MUC1 detection had its own uniqueness, that is, the present method was much simpler and more effective to detect MUC1.

Performance of MCF-7 detection and selectivity assays of FL biosensor
The MTT assays of cell viability studies suggested that MoS 2 QDs did not impose a considerable toxicity towards MCF-7 cells as compared to the control (Fig. 6A). The above results indicated that the as-prepared MoS 2 QDs could be promising biosensors in cell detection and imaging. As shown in Fig. 6B, the uorescence intensity was dependent on the concentration of MCF-7 cells over a range of 0 to 5 Â 10 5 cells per mL, when the concentration of MoS 2 QDs was 100 mg mL À1 . As illustrated in Fig. 6C, a linear relationship between peak intensity at 520 nm and MCF-7 cells concentrations was obtained in the concentration range from 10 3 to 5 Â 10 5 cells per mL (R 2 ¼ 0.9949) with a detection limit of 50 cells per mL (according to the rule of three times the standard deviation corresponding to the blank sample detection). Due to the specic binding between P 0 -FAM and MUC1, the MoS 2 QDsbased biosensor was insensitive to the interfering proteins such as GOD, CyC, Mb, Lys and BSA as shown in Fig. S3. † The good selectivity, biocompatibility and the intrinsic optical properties of the biosensor can be used to construct an excellent bioimaging system and recognition system.

Intracellular imaging analysis
Fluorescence microscope images of MCF-7 cells loaded with the MoS 2 -P 0 -FAM (MoS 2 QDs) biosensor for 1 h at 37 C showed green uorescence on cytomembrane (Fig. 7). However, the control experiment on cells without the MoS 2 -P 0 -FAM biosensor gave no green uorescence in the same exposure condition. These results demonstrated the specic recognition of MoS 2 -P 0 -FAM biosensor to MUC1 in MCF-7 cells. In contrast, the uorescence for PBMSC and L929 cells exhibited no green uorescence due to non-overexpression of MUC1 in PBMSC and L929 cells, proving that the MoS 2 QDs-based biosensor can be applied to bioimaging and recognition of MCF-7. By comparison, MCF-7 cells incubated with the MoS 2 sheets-P 0 -FAM biosensor showed extremely weak green uorescence (Fig. S4 †), indicating that the multi-layer stacking of MoS 2 nanosheets would block the view of uorescence imaging and affect the detection of cancer cells. Hence, the MoS 2 QDs-based biosensor was superior to that of MoS 2 sheets.

Conclusion
In summary, this study presented a sensitive MoS 2 QDs based uorescent sensing platform for MUC1. In particular, we applied MoS 2 QDs to MCF-7 detection and cellular imaging, which is extremely rare in the application of MoS 2 . MoS 2 QDs exhibited a high uorescence quenching ability towards uorescent dyes; therefore, they were exploited as carrier and quencher for a uorescent dye-labeled DNA aptamer (P 0 -FAM)  to construct a biosensor. The obtained results showed that the detection range of MUC1 in the solution was in the range of 1 nM to 10 mM and the detection limit was 0.5 nM. The detection range of MCF-7 was in the range of 10 3 to 5 Â 10 5 cells per mL and the detection limit was 50 cells per mL. The biosensor also had the advantages of high sensitivity and specicity. Furthermore, we expect that this strategy based on MoS 2 QDs as a uorescent quencher may offer a new approach in the sensitive and selective detection of a wide spectrum of analytes and cancer cells.

Conflicts of interest
There are no conicts to declare.