The exploration of quantum dot-molecular beacon based MoS2 fluorescence probing for myeloma-related Mirnas detection

Highly sensitive and reliable detection of multiple myeloma remains a major challenge in liquid biopsy. Herein, for the first time, quantum dot-molecular beacon (QD-MB) functionalized MoS2 (QD-MB @MoS2) fluorescent probes were designed for the dual detection of multiple myeloma (MM)-related miRNA-155 and miRNA-150. The results indicate that the two probes can effectively detect miRNA-155 and miRNA-150 simultaneously with satisfactory recovery rates, and the limit of detections (LODs) of miRNA-155 and miRNA-150 in human serum are low to 7.19 fM and 5.84 fM, respectively. These results indicate that our method is the most sensitive detection so far reported and that the designed fluorescent probes with signal amplification strategies can achieve highly sensitive detection of MM-related miRNAs for MM diagnosis.


Introduction
Fluorescent probes are tracers that connect to the target substances, which are wildly used in the field of biosensing due to their sensitivity and convenience for practical applications [1]. The fluorescence probes generally include three parts: the recognition group, the connecting part, and fluorescence emitters. The function of the recognition group is to identify the target substances. The connecting part connects the recognition group and the fluorescent group, and the fluorescent groups are a component that can emit fluorescence under light excitations. So far, a variety of DNA structures have been utilized to develop DNA-based fluorescent probes, such as single-stranded DNA probes [2], double-stranded DNA probes [3], molecular beacon (MB) [4], and other structures. Among them, MB is a kind of fluorescent probe with a hairpin structure. Comparing with the single-stranded DNA fluorescent probe, MB has the advantages of high sensitivity and strong specificity. MB's hairpin structure can retain good structural stability during the detection process and can be easily designed as a probe structure to restore fluorescence by opening the hairpin structure. The regular MBs are hairpin-structured single-stranded nucleic acids that are usually labeled with fluorescence organic dyes. MBs labeled with organic dyes generally have a broad emission band with a poor photostability and easy photobleaching, which are not desirable for the development of stable and multivariate detection probes. Quantum dot (QD) is a new type of fluorescence materials with distinct advantages of strong intensity, high stability, strong anti-bleaching ability, wide excitation spectrum, narrow emission spectrum, and adjustable emission wavelength [5][6][7][8][9]. The effective bandgap of QDs is increased with the decrease of particle radius, resulting in a blue shift of their absorption and emission [10]. Therefore, the position of their excitation and emission can be adjusted only by changing their size. Moreover, the advantage of a wide excitation spectrum can realize the simultaneous excitation of different QDs under the same excitation. Because the fluorescence spectrum of QDs is relatively narrow, the fluorescence peaks of multiple QDs excited at the same time are not easy to overlap, which can prevent the crosstalk and interruption. Overall, these excellent fluorescence characteristics of QDs make it possible to realize simultaneous detection of biomolecules.
MicroRNA (miRNA) is a type of single-stranded RNA of 21-25 bases [11,12], which is an important regulator of cell proliferation, division, differentiation, and apoptosis since it regulates the expression level of various DNA genes after transcription [13]. The abnormal expression of specific miRNAs is related to many diseases (such as cancer, tumors, and diabetes) [14][15][16]. Therefore, miRNAs are considered to the biomarkers for important diseases like cancer [17][18][19], for example, Shen [20] et al.
showed that miRNA-202 in the bone marrow microenvironment can negatively regulate BAFF by inhibiting multiple myeloma (MM) cell survival, growth, and adhesion. Long [21] et al. found that miRNA-765 plays a carcinogenic role in MM progression by directly targeting Sox6. In addition, a miRNA such as miRNA-155 may be involved in the regulation of a variety of diseases, i.e. not only regulates MM, but also relate to the production of colorectal cancer, cardiovascular and other diseases [22][23][24][25]. Previous studies have shown that a single miRNA marker cannot reflect the specificity of a particular disease because multiple miRNA markers are often presented in the same disease. Therefore, the detection of a single miRNA biomarker is not reliable and accurate for MM diagnosis. This means that the simultaneous detection of multiple MM-related miRNAs with high sensitivity and specificity is required. However, the current high-sensitive probes and strategies for multiple miRNAs detection of MM are relatively limited [2][3][4]26].
Therefore, more and better methods need to be explored. In this paper, two types of quantum dot-molecular beacons (QD-MB) were prepared on MoS 2 monolayer nanosheet to obtain two QD-MB functionalized MoS 2 fluorescent probes (QD 525 -MB 155 @MoS 2 and QD 585 -MB 150 @MoS 2 ). The two probes were used for the detection of MiRNA-155 and miRNA-150 simultaneously by duplex-specific nuclease (DSN)-assisted signal amplification strategy. The design principle and preparation process are shown in Fig. 1. The 3 ′ of MB is labeled with biotin and the 5 ′ was modified with 15 cytosines (PolyC15). The biotin can be coupled with the streptavidin on the surface of QDs, and PolyC15 can absorb on MoS 2 nanosheets by van der Waals force [27] (Fig. 1(a), Step 1). At this time, QD is moving closer to MoS 2 , resulting in significant quenching of QD fluorescence. In the presence of target miRNA, the hybridization of miRNA and MB results in the formation of DNA/RNA heteroduplex, leading to the QD moving away from the MoS 2 nanosheet and partially restoring the fluorescence of QD ( Fig. 1(a), step 2). In the following step with the assistantance of the DSN molecule, the MB in the MB/RNA heteroduplex can be cut into fragments by DSN, resulting in the release of QDs and miRNAs from MoS 2 nanosheets (Fig. 1, step 3), which enhances the fluorescence of the QD and allows the target miRNA circulated in the cycling signal amplification ( Fig. 1(b), step 4). The proposed QD-MB@MoS 2 probes and the corresponding DSN-assisted signal amplification strategy can be used for simultaneous detection of miRNA-155 and miRNA-150 in human serum with high sensitivity and selectivity, which can provide a convenient and reliable tool for clinic MM diagnosis.
A pressure cooker (GI-54DWS, Shanghai Danding International Trade Co., Ltd., China) is used to autoclave all pipette tips and reagents. Fluorescence was measured by a fluorescence spectrometer (F-4600, Hitachi, Japan). The morphology of QD was imaged by a transmission electron microscope (TEM, FEI TECNAI G2 F20). and 600 μL Tris-Hcl buffer, followed by shaking for 2 h. The solution was then transferred to a 100 K ultrafiltration tube centrifugally (4000 rpm for 3 min) to remove the free MBs three times. The pure QD-MBs dispersed in 100 μL TrisHcl buffer and 45 μl blocking solution were obtained and stored in 4 • C for future use. The above operations were carried out at room temperature.

Preparation of QD-MB@MoS2 fluorescent probes
The QD-MBs were mixed with monolayer MoS 2 nanosheets to prepare QD-MB@MoS 2 fluorescent probes. Briefly, 22.5 μL QD-MBs, 67.5 μL blocking solution, and 200 μL 0.64 μM MoS 2 nanosheets were added into 250 μL buffer (5 mM Tris-HCl, 1 M NaCl, pH = 7.4), followed by diluting the mixture to 500 μL with ultrapure water and shaking it for 1 h. The obtained mixture was centrifugally purified (4000 rpm for 3 min) to obtain pure QD-MB@MoS 2 by re-dispersing the sediments in 125 μL TrisHcl buffer and 75 μL blocking solution. The QD-MB@MoS 2 solution was stored at 4 • C. The above operations are all carried out at room temperature. The fluorescent probe QD 525 -MB 155 @MoS 2 of miRNA-155 and the fluorescent probe QD 585 -MB 150 @MoS 2 of miRNA-150 are produced according to the above steps, separately.

Dual detection of miRNA-155 and miRNA-150 in buffer
The two fluorescence probes obtained in the previous step were mixed in a volume ratio of 1:1 to prepare QD-MB@MoS 2 probes for dual detection of miRNA-155 and miRNA-150 ( Fig. 1(b), step 3). Using HEPES buffer as the diluent, miRNA-155 and miRNA-150 were diluted into different concentrations (10 fM, 100 fM, 1 pM, 10 pM, 100 pM, and 1 nM, respectively). Then, 20 μL miRNA-155 and 20 μL miRNA-150 solutions with the same concentration were mixed to prepare the miR-NAs mixture for the test. Subsequently, the miRNAs mixture was added into 100 μL QD-MB@MoS 2 probes. After incubating for 30 min, 0.3 U of DSN was added into the mixture and incubated at 55 • C for 40 min. Finally, the fluorescence of the mixture was recorded.

Dual detection of miRNA-155 and miRNA-150 in 10% human serum
To test for practical applications, miRNA-155 and miRNA-150 were spiked in 10% human serum with different concentrations (10 fM, 100 fM, 1 pM, 10 pM, 100 pM, and 1 nM) and tested by the QD-MB@MoS 2 probes following the above-mentioned sensing strategy. Besides, the mixture including an equal concentration of miRNA-155 and miRNA-150 (80 fM, 200 fM, 50 pM, and 540 pM, respectively) were also tested to characterize the recovery rate.

Characterization of quantum dots and MoS 2
The TEM pictures of CdSe/ZnS quantum dots QD 525 and QD 585 are shown in Fig. 2 (a) and (b). It can be seen that the quantum dots QD 525 and QD 585 maintain a relatively good spherical shape and dispersion. According to the information provided by the merchant, the diameters of QD 525 and QD 585 are 47.5 and 48.5 nm, respectively, and the quantum yields are 91% and 89%, respectively. The fluorescence spectra of CdSe/ZnS quantum dots QD 525 and QD 585 are shown in Fig. 2(c). Under the excitation of 365 nm, the fluorescence e mission peaks of QD 525 and  The TEM image is shown in Fig. 3(a) illustrates the monolayer morphology of MoS2 and the average size is about 237 ± 42 nm. The AFM characterizations shown in Fig. 3(b) indicate the monolayer structure of MoS2 nanosheets with height about 1.0 nm. The direct bandgap width of monolayer MoS 2 is about 1.75 eV [28][29][30], and the corresponding cut-off adsorption wavelength can be calculated to be 708.5 nm. This means that MoS 2 can absorb light with a wavelength less than 708.5 nm. The monolayer MoS 2 has five absorption peaks in the Ultraviolet-Visible wave band, which are located near 270 nm, 405 nm, 448 nm, 620 nm, and 670 nm respectively [30,31]. The overlapping spectrum of the four absorption peaks in the visible wave band almost covers the whole visible wave band, resulting in its absorption of the entirety of the visible light. Therefore, the appearance of the monolayer MoS 2 dispersant is black. The positions of the fluorescence emission peaks of QDs selected in this paper are 525 nm and 585 nm, which overlap well with the absorption spectrum of monolayer MoS 2 and meet the conditions of energy transfer quenching [32].   peak is at 542 nm. The peak of QD 525 -MB 155 fluorescence spectrum showed some red shift, which may be caused by the change of surface charge state and charge transfer caused by MB modification [33][34][35][36].

Fluorescence characterization of QD
Curve No. 3 (blue) is the fluorescence spectrum obtained after adding MoS 2 to QD 525 -MB 155 , i.e. the fluorescence spectrum of QD 525 -MB 155 @MoS 2 probes. The position of the fluorescence peak sticks at 542 nm, but the peak intensity is very weak, which indicates that the MoS 2 has been coupled to the 5 ′ -end of the MB, and the fluorescence of QD 525 is quenched due to the influence of adjacent MoS 2 . At this time, the maximum distance between QD and MoS 2 is about 8.8 nm (Calculated based on the base sequence number of MB). According to the fluorescence quenching theory, it is judged that the mechanism of MoS 2 quenching QD fluorescence is Förster resonance energy transfer (FRET) which generally occurs between donors (fluorescent group) and acceptors (fluorescent quencher) with a spacing of 1-10 nm. When the vibration frequencies of the two dipoles are the same, the donor oscillates due to the absorption of radiation energy, and the receptor oscillates with the oscillation of the donor, that is, resonance occurs. As a result, the donor transfers the absorbed energy to the receptor and loses the ability to radiate energy, that is, the fluorescence quenching effect occurs. According to the calculation method provided by Cristobal [35], the energy transfer efficiency is calculated to be 0.9675 (The fluorescence intensity of curve 2 is taken as the original fluorescence intensity without acceptors).
Curve No. 4 (green) is the fluorescence spectrum of QD 525 -MB 155 @MoS 2 after incubation with 1 nM miRNA-155 (QD 525 -MB 155 @MoS 2 +miRNA-155). Compared with the fluorescence spectrum of the QD 525 -MB 155 @MoS 2 (No. 3), the fluorescence peak intensity at 542 nm is significantly enhanced. This is due to the hybridization of miRNA-155 and MB on the QD 525 -MB 155 @MoS 2 probe, resulting in the opening of the hairpin structure of MB 155 , which caused QD 525 to move away from the MoS 2 nanosheet and restore part of its fluorescence. According to the base number of MB, it is calculated that the maximum distance between QD and MoS 2 after the MB structure is opened is about 18.7 nm, which is greater than the effective distance of FRET 10 nm. However, the fluorescence of QD has not fully recovered to the original intensity, and analysis suggests that the fluorescence quenching mechanism of energy radiation transfer has occurred [32]. As an energy donor, QD releases the excitation energy in the form of radiation and returns itself to the ground state. MoS 2 , as a receptor, absorbs the photons radiated by QD and is excited, resulting in QD fluorescence quenching. It is calculated that the energy transfer efficiency is 0.62375.
When DSN was added to cut the DNA (i.e. MB base sequence) within the DNA/RNA heteroduplex, the significantly enhanced fluorescence spectrum is shown as curve No. 5 (purple) in Fig. 4(a). The result shows that the DSN selectively and efficiently cut the MB in the double strands, which makes the QDs release from the MoS 2 nanosheet. At the same time, miRNA-155 was also released and reused to hybridize with MBs on QD 525 -MB 155 @MoS 2 probes, so that a DSN-assisted miRNA-155 recycling signal amplification was achieved. Theoretically, the released QD becomes a free state and can restore all fluorescence. However, MoS 2 nanosheets still exist in the solution, and the distance between MoS 2 and QD is far greater than 10 nm. It is believed that there is still some fluorescence quenching process of radiation energy transfer. The efficiency of energy radiation transfer is inversely proportional to the square of the distance R [32], so the recovered fluorescence intensity is much stronger than that of Curve No. 4, but it has not yet reached the recovery of all fluorescence. After being clipped by DSN, the energy transfer efficiency is reduced to 0.125. Based on the above analysis, it is believed that the mechanism of MoS 2 quenching the fluorescence of QDs in this experiment is mainly FRET, followed by radiation energy transfer (when the distance between MoS 2 and QD is greater than 10 nm). And this results also illustrate that the length of the designed MB base sequence has a great influence on the energy transfer efficiency, that is, it has a great influence on the fluorescence quenching effect and the detection sensitivity of fluorescent probes.

Optimization of preparation conditions for fluorescent probes
To optimize the preparation of QD-MB@MoS 2 , different concentra-  Fig. 5(c) and (d). As shown in Fig. 5(d), when the DSN concentration increases from 0 to 0.3 U, the fluorescence intensity gradually increases until it reaches a saturation value, which indicates that the optimal DSN concentration is 0.3 U. Fig. 5(e) plots the fluorescence spectra of the assay of 1 nM miRNA with 0.3 U DSN for different incubation times (0, 10, 20, 30, 40, and 50 min), and Fig. 5(f) plots the fluorescence peak intensity measured at different incubation times. It can be seen clearly that the fluorescence peak intensity increases to a saturation value when the incubation time increases from 0 min to 40 min, indicating that the optimal incubation time of DSN is 40 min.  proposed fluorescence detection strategy.

Specificity
To study the specifics of fluorescent probes for miRNA-155 and   indicates that the fluorescence signal of specific detections can be distinguished from the non-specific detections and blank. The error probabilities during RNA replication and translation are 10 − 10 and 10 − 5 , respectively [37][38][39]. Therefore, the concentration of mature miRNA in the human body is much higher than that of wrong code miRNA. Even if there is a small amount of miscoded miRNA in human serum, it can still provide partial fluorescence. Therefore, we believe that the fluorescence loss caused by miscoded miRNA is less than the error value and does not affect the overall measurement level of miRNA. Therefore, the prepared fluorescent probes and sensing strategy have good specificity.

Dual detections of miRNAs in 10% human serum
To investigate the practical application of the proposed probes and strategy, the 10% human serums including an equal concentration of miRNA-155 and miRNA-150 at different concentrations (10 fM, 100 fM, 1 pM, 10 pM, 100 pM, and 1 nM) are prepared and fluorescence detected respectively. The fluorescence spectra of assays of miRNA-155 and miRNA-150 in human serum with different concentrations (1 nM-10 fM) is shown in Fig. 8(a). Fig. 8(b) plots the miRNA concentrationdependent fluorescence intensity relationships of miRNA-155 and miRNA-150, respectively. The relationship between F 155 /F 0155 and logC miRNA follows a linear relationship, and the calibration curve is F 155 / F 0155 = 53.123 + 3.166 × logC 155 (R 2 = 0.9954). Similarly, for miRNA-150 the linear calibration curve is F 150 /F 0150 = 29.877 + 1.74 × logC 150 (R 2 = 0.9954). The LODs of miRNA-155 and miRNA-150 in human serum are 7.19 fM and 5.84 fM respectively, which is very close to the LODs of miRNAs in the buffer. The results show the good antiinterference ability of probes for detecting miRNAs in complex samples. Liu Ying et al. detected the fluorescence intensity of miRNA-155 in serum of healthy people and MM patients with MB-functionalized monolayer MoS 2 nanosheet fluorescent probe [40]. According to the experimental results, we can infer that the concentration of miRNA-155 in health people serum is about 53.7 pM, and that of miRNA-155 in MM patients serum is about 6.12 nM, which proves that the sensitivity and detection range achieved by our pattern method can meet the clinical detection requirements of miRNA.
The recovery rate was further studied by testing the mixtures including equivalent miRNA-155 and miRNA-150 spiked in 10% human serum, i.e. 80 fM, 200 fM, 50 pM, and 540 pM. According to the found value of the assay shown in Table 2 and Table 3, the recovery rate of miRNA-155 is in the range of 92.14-105.70% with the relative standard deviation (RSD) less than 7.92%, and the recovery rate of miRNA-150 is in the range of 94.03-102.6 2% with the RSD less than 8.22%, which confirms that the proposed probes and strategy can be used for detecting miRNAs in complex serum and has the clinical application prospects.

Conclusions
In this paper, novel quantum dot-molecular beacon functionalized MoS 2 (QD-MB @MoS 2 ) fluorescent probes were designed and fabricated for the dual detection of MM-related miRNA-155 and miRNA-150 via DSN-assisted signal amplification. The QD-MB@MoS 2 fluorescent probes were constructed by linking polyC15-modified MBs to CdSe/ZnS quantum dots and further absorbing on monolayer MoS 2 nanosheets.   The fluorescence quenching and fluorescence recovery mechanism of the QD-MB@MoS 2 probes used in the experiment were analyzed. The results illustrate that the mechanism of MoS 2 quenching the fluorescence of QDs is mainly FRET, followed by radiation energy transfer (when the probes are opened or cut off), and the designed MB length has a great influence on the fluorescence quenching efficiency and the sensitivity of the fluorescent probe. Based on the fluorescent probes, a new scheme was designed and used to achieve high-sensitivity simultaneous detection of dual miRNA (miRNA-155 and miRNA-150). The results indicate that the two probes can sensitively detect miRNA-155 and miRNA-150 simultaneously with a good recovery rate, and the LODs of miRNA-155 and miRNA-150 in human serum are low to 7.19 fM and 5.84 fM respectively. These results indicate that the proposed new MB@MoS 2 fluorescent probe and the DSN-assisted signal amplification strategy for miRNA detection not only has the characteristics of low biological toxicity, selectable wavelength and easy to realize multiplex detection, but also has the advantages of high sensitivity and low detection limit, which provide a valuable tool for clinical diagnosis of cancers and other diseases.