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A MoS nanoprobe for microRNAs quantification based on duplex-specific nuclease signal amplification Mingshu Xiao, Tiantian Man, Changfeng Zhu, Hao Pei, Jiye Shi, Li Li, Xiangmeng Qu, Xizhong Shen, and Jiang Li ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b18984 • Publication Date (Web): 12 Feb 2018 Downloaded from http://pubs.acs.org on February 12, 2018

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ACS Applied Materials & Interfaces

A MoS2 nanoprobe for microRNAs quantification based on duplex-specific nuclease signal amplification Mingshu Xiao1,2, Tiantian Man2, Changfeng Zhu1*, Hao Pei2, Jiye Shi3, Li Li2, Xiangmeng Qu2, Xizhong Shen1,4 and Jiang Li5* 1

Department of Gastroenterology, Zhongshan Hospital, Fudan University, 180 Fenglin Rd.,

Shanghai 200032, China 2

Shanghai Key Laboratory of Green Chemistry and Chemical Processes, School of Chemistry

and Molecular Engineering, East China Normal University, 500 Dongchuan Road, Shanghai, 200241, P. R. China 3

UCB Pharma, 208 Bath Road, Slough, SL1 3WE, UK

4

Shanghai Institute of Liver Diseases, Zhongshan Hospital, Fudan University, 180 Fenglin

Rd., Shanghai 200032, China 5

Division of Physical Biology & Bioimaging Center, Shanghai Synchrotron Radiation

Facility, Shanghai Institute of Applied Physics, Chinese Academy of Sciences, Shanghai 201800, P. R. China

KEYWORDS: Multiplexed detection, microRNA, MoS2, molecular beacons, DSN

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ABSTRACT: MicroRNAs (miRNAs) play significant regulatory roles in physiologic and pathologic processes and are considered as important biomarkers for disease diagnostics and therapeutics. Simple, fast, sensitive, and selective detection of miRNAs, however, is challenged by their short length, low abundance, susceptibility to degradation, and homogenous sequence. Here we report a novel design of nanoprobes for highly sensitive and selective detection of miRNAs based on MoS2-loaded molecular beacons (MBs) and duplex-specific nuclease-mediated signal amplification (DSNMSA). We show that MoS2 nanosheets not only exhibit high affinity toward MBs but also act as an efficient quencher for absorbed MBs. The strong fluorescence quenching ability of MoS2 in combination with cyclic DSNMSA contributes to the superior sensitivity of our method, with a limit of detection (LOD) 4 orders of magnitude lower than that of traditional hybridization methods. Moreover, the nanoprobes also show high selectivity for discriminating homogenous miRNA sequences with one-base differences due to the discrimination ability of MBs and DSN. Furthermore, we demonstrate that the MoS2-loaded MBs nanoprobes can be utilized for multiplexed detection of miRNAs. Given its high sensitivity and specificity, as well as the multiplexed function, this novel method as an effective tool shows great promise for simultaneous quantitative analysis of multiple miRNAs in biomedical research and clinical diagnosis.

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INTRODUCTION MicroRNAs (miRNAs) play critical roles in regulating physiologic and pathologic processes via target mRNAs cleavage or translational repression1-2. Accumulating evidence has revealed that the dysregulated expression of miRNAs is frequently related to the development of various diseases3. Moreover, miRNAs have been regarded as candidate biomarkers that hold promise in medical diagnosis and therapy4. For example, Mitchell et al.5 found that miRNA-141 could be used as biomarkers for detection of prostate cancer. Hence, miRNA detection with high sensitivity and selectivity is extremely important for miRNA profiling, scientific study6, as well as clinical diagnosis7-8. Nevertheless, it is still a significant challenge to detect the miRNAs as a result of their short length, low abundance, and susceptibility to degradation9-12. Besides quantitative analysis, specific detection of miRNAs is also plagued by their high homogeneity in terms of both size and sequence. Currently, various techniques have been developed for miRNA analysis, but some intrinsic drawbacks attached to these techniques cannot be overlooked. For example, the northern blotting is considered as the gold standard of miRNA quantification, but with low sensitivity and a tedious procedure13; the most widely used real-time PCR (RT-PCR) requires complex experiment and high cost in spite of its high sensitivity and specificity14; isothermal amplification with high amplification efficiency is compromised by a nonspecific background amplification15-17. In addition, considerable research efforts have been devoted to multiplexed detection of miRNAs as some pathologies are ascribed to dysregulated expression of several miRNAs18-21. Therefore, a facile, sensitive, and selective method for multiplexed detection of miRNAs is still urgently in demand. Single or few-layered transition metal dichalcogenides (TMD) (e.g., MoS2, TiS2, WS2 etc.), a large family of two-dimensional layered nanomaterials analogous to graphene, have received extensive attention recently because of their carrier mobility, unique optical, 3



electrochemical, and catalytic properties22-26. Notably, TMD possesses several nonnegligible advantages9,

26-29

, such as, ability to directly disperse in aqueous solution, facile surface

modification as well as large-scale production. These features endow TMD with great potential in the field of optics, catalysis, sensors, energy storage etc30-36. Moreover, previous study has demonstrated that MoS2 nanosheets are capable to discriminate single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA)17. In addition, MoS2 nanosheets also display high adsorption and fluorescence-quenching abilities toward dye-labeled ssDNA37-40. On the basis of these findings, Zhang group developed single-layer MoS2-based nanoprobes for the detection of DNA and small molecules41. On the other hand, molecular beacons (MBs), which are single-stranded oligonucleotides with a hairpin structure, have emerged as a useful tool in biosensing, and RNA and DNA monitoring due to its excellent selectivity capable to differentiate one-base mismatched targets42-44. Hence, we propose a formulation that an efficient sensing platform was constructed for nucleic acid detection, even multiplexed detection by integrating TMD and MBs.

Figure 1. Schematic illustration of MoS2-loaded MBs nanoprobes for detection of microRNA based on DSNMSA.

In this work, we construct a platform based on MoS2-loaded MBs and duplex-specific 4


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nuclease-mediated signal amplification (DSNMSA) for miRNAs detection with high sensitivity and remarkable specificity. Duplex-specific nuclease (DSN) exhibits a strong preference for hydrolyzing dsDNA or DNA strand in DNA/RNA heteroduplex, but remains inactive toward ssDNA or RNA9, 19. In addition, this enzyme can differentiate perfectly and nonperfectly matched short duplexes2. Therefore, DNSMSA with various advantages over other amplification strategies has inspired growing interest in miRNAs detection. As shown in Figure 1, MBs are elaborately designed as DNA hairpin structures with a fluorophore. Fan et al previously reported that MBs could tightly bind on the surface of two-dimensional materials and their fluorescence was quenched with 98% efficiency45. Fluorophore-labeled MBs thus were expected to be absorbed on the surface of MoS2 via the van der Waals force41 when mixed with MoS2 nanosheets, simultaneously quenching the fluorescence of the dye. Upon the addition of target miRNAs, target miRNAs will open the stem-loop structure of MBs, followed by MBs hybridizing with miRNAs and thus forming DNA/RNA heteroduplexes. Because of their densely negative charge, the formed DNA/RNA heteroduplexes will exfoliate from the MoS2 surface, giving rise to retention of the fluorescence of MBs41. The features of DSN enzyme make certain the cleavage of ssDNA rather than miRNAs in the duplexes. As a result, the target miRNA is released from duplexes and then hybridize with another MB, initiating the next round of cleavage, releasing, and hybridization2, 9, 19. As the cleaved dye-linked oligonucleotide fragments are incapable to absorb on MoS2, this cyclic reaction causes fluorescent signal amplified. This DSNMSA strategy attaches our MoS2-loaded MBs nanoprobes to highly sensitive detection of miRNA. Additionally, the remarkable selectivity of MBs (discriminating single-nucleotide mismatched nucleic acids) and DSN (discriminating perfectly and nonperfectly matched nucleic acid duplexes) contributes to the specificity of our MoS2-based nanoprobes to distinguish highly homologous miRNAs with one-base variations. Hence, it is envisioned that 5



our constructed MoS2-based platform can be capable of detecting miRNAs with high sensitivity and selectivity. EXPERIMENTAL SECTION Materials. Few-layer MoS2 nanosheet was purchased from Nanjing XFNANO Materials Tech Co. Ltd. RNase inhibitor, HPLC-purified miRNAs, and DEPC-treated water were obtained from Takara Biotechnology Co. Ltd. (Dalian, China). DSN was provided by Newborn Co. Ltd. (Shenzhen, China). The FAM-labeled MB1, TRAMA-labeled MB2, and Cy5-labeled MB3 were synthesized by Sangon Biotech Co. Ltd. (Shanghai, China) with standard desalting and without further purification. The sequences of these MBs and miRNAs are listed in Table S1. All chemicals were obtained from Sigma-Aldrich (St. Louis, MO) and used without further purification. All aqueous solutions were prepared using deionized water purified by a Milli-Q water purification system (Millipore Corp., Bedford, MA) with a resistivity of 18.2 MΩ⋅cm−1 unless otherwise stated. To create and maintain an RNase-free environment, all aqueous used in DSNMSA method were treated with 0.1% DEPC and autoclaved. The tips and tubes used in this work are RNase-free and don’t require pretreatment to inactivate RNases. Instrumentation. The fluorescence emission spectra were recorded by a HITACHI F-7000 fluorophotometer with different excitation wavelength according to fluorophore. The measurement settings were λex= 485 nm for FAM dye, λex = 560 nm for TAMRA dye, λex = 643 nm for Cy5 dye. DSNMSA Method for miRNAs Detection. All experiments were conducted in the 1×DSN buffer containing 50 mM Tris-HCl (pH 8.0), 5mM MgCl2 and 1mM DTT. After MB incubated with MoS2 for 20 min, the amplified detection of miRNA was carried out in 200 µL solution with 10 nM MB, 0.1 U DSN and different concentrations of target miRNA at 65 ffi 6


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for 30 min, followed by detecting fluorescence intensities. The miRNA concentration in samples ranged from 10 fM to10 nM. RESULTS AND DISCUSSION MoS2 nanosheets as layered two-dimensional nanomaterials analogous to graphene possess many unique properties and show great promising in biological analysis. Therefore, few-layers MoS2 nanosheets were exploited for constructing sensing platform, and characterized by atomic force microscopy (AFM). The AFM image in Figure 2a illustrates that the thickness of MoS2 nanosheet is ∼2 nm, verifying that few-layers MoS2 nanosheet was yielded, which is in good agreement with the previous work26, 46. The result also is supported by transmission electron microscopy (TEM) image and energy-dispersive X-ray spectroscope (EDS) (Figure S1). Then, we developed MoS2-loaded MBs nanoprobes and carried out the feasibility assay to confirm our MoS2-loaded MBs nanoprobes capable for miRNA detection, in which the let-7a miRNA was selected as model targets because their expression level is closely associated with human cancer. Additionally, the MB1 was designed with stem and loop structure: 5′-FAM-CGAGCTAACTATACAACCTACTACCTCAAGCTCG-3′ (stem is underlined), which is complementary to let-7a. As shown in Figure 2b, the fluorescence of MB1 was almost entirely quenched when mixed with MoS2 nanosheets (MB1/MoS2). Moreover, it is found that the quenching efficiency is up to 97% for MB1/MoS2 (Figure 2c). The high quenching efficiency is attributed to strong affinity of MB1 to MoS2 nanosheets45 and nanoscale surface energy transfer between MB1 and MoS226. When an equal amount of target miRNA (5′-UGAGGUAGUAGGUUGUAUAGUU-3′) was added to open loop and hybridize with MB1 to form RNA/DNA heteroduplex, the weak interaction between heteroduplex and MoS2 induced heteroduplex to exfoliate from the surface of MoS2, and thus the fluorescence was partially recovered. However, when 7



incubating MB1 with DSN, the signal of MB1/MoS2 is nearly equal to that without DSN (Figure 2b, inset), implying that MB1 absorbed on MoS2 can be prevented from the cleavage by DSN due to the steric effect47, which avoids leakage signals. In the presence of both target RNA and DSN, the MB1 was hybridized to target RNA and the formed heteroduplex would be cleaved by DSN. As a result, the miRNAs in heteroduplex were released for the next cycle of hybridization, cleavage, and releasing. In addition, as the short FAM-labeled oligonucleotide fragments have weak affinity to MoS2, DSNMSA gives rise to remarkable fluorescence enhancement, whose signal-to-background ratio is ∼21. Notably, the DSNMSA was triggered by the target RNA. Given these results, it is concluded that our MoS2-loaded MBs nanoprobes for miRNA detection on the basis of DSNMSA is feasible.

Figure 2. (a) AFM image of single layer MoS2. (b) Fluorescence emission spectra of MB1, MB1/MoS2, MB1/MoS2, MB1/MoS2 with miRNA in the absence of DSN, MB1/MoS2 in the presence of DSN, MB1/MoS2 with miRNA in the presence of DSN. Inset: the amplification of the fluorescence emission spectra of MB1/MoS2 with miRNA in the absence of DSN and MB1/MoS2 in the presence of DSN. (c) Relative fluorescence intensity of (b).

Next, we investigated the performance of MoS2-loaded MBs-based platform for quantitative analysis of miRNA based on the aforementioned findings. As shown in Figure 3a, the fluorescence intensity is dramatically increased along with the increasing concentration of let-7a from 10 fM to 10 nM. Figure 3b indicates the correlation between the fluorescence 8


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ratio (F/F0-1) and let-7a concentration in logarithmic scale. It is found that over 21-fold fluorescence enhancement is achieved in the presence of 10 nM target. Moreover, the (F/F0-1) value demonstrates a good linear relationship with the logarithm of let-7a concentration ranging from 1 pM to 10 nM. The calibration equation was (F/F0-1)=52.6+4.0lg C, with a correlation coefficient R2 = 0.9939. The limit of detection (LOD) was calculated to be 10 fM, which is better than or comparable to that of many reported miRNA detection strategies (Table 1)2, 9, 18-19, 48-50. It should be pointed out that the low LOD was achieved within 30 min. To sum up, the excellent sensitivity is assigned to the strong fluorescence quenching ability of MoS2 and DSNMSA. To assess our MoS2-loaded MBs nanoprobes for distinguishing different miRNA family members, a series of contrast experiments were carried out. It is well established that the miRNA family members often are highly homogeneous sequence different as few as only one-base, so we chose let-7f, let-7e, let7-i, and miRNA-429 to evaluate the selectivity of our strategy. Figure 3c shows the comparison of fluorescence readouts corresponding to diverse miRNAs (sequences were listed in Figure 3d). Compared with target let-7a, there was much weaker fluorescence ratio in the presence of single-base-mismatched let-7f or let-7d. Moreover, there were no noticeable fluorescence signals along with the addition of let-7i and miRNA-429 compared with the background. Taken together, our constructed sensing platform based on DSNMSA displays high specificity capable to discriminate homologous miRNAs with single-nucleotide difference. Here, the single-base-mismatch discriminability of MB1 and DSN contribute to the high specificity of our method to detect miRNAs.

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Figure 3. (a) Fluorescence spectra in the presence of different concentrations of let-7a. (b) Fluorescence ratio (F/F0−1) versus miRNA concentrations in logarithmic scale, where F0 and F are the FAM fluorescence intensity of MB1/MoS2 and MB1/MoS2with miRNA in the presence DSN. Inset: linear relationship between fluorescence ratio (F/F0−1) and the logarithm of miRNA concentration. Error bars are standard deviation of three repetitive experiments. (c) Fluorescence ratio (F/F0−1) with various miRNAs. Inset: the fluorescence spectra of the various miRNAs. (d) Sequences of let-7a, let-7f, let-7e, let-7i, and miRNA-429. The bases marked in red are variant with those in let-7a.

To confirm the reliability of the proposed MoS2-loaded MBs-based sensing platform for miRNAs detection, we performed recovery experiments, in which different concentrations of let-7a were added into the 100-fold diluted healthy human real serum sample (provided by Zhongshan Hospital, Fudan University, Shanghai, China), and five repetitive experiments for each sample was carried out. Exhibited from Table S2, it is found that good recoveries were yielded from 98.54% to 106.3%. In addition, the RSD values are ranging from 1.2 to 4.3%. 10


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The results suggest that this platform holds great promise in clinical application.

Table 1. Comparison of different methods for miRNA detection Detection target

Detection method

Linear range

LOD

Selectivity

Ref.

let-7a

Fluorescence method

1 pMto 10 nM

10fM

one-base difference

This work

let-7a

Fluorescence method

0.5 pM to 500 pM

0.4 pM

one-base difference

2

let-7b

Electrochemical method

2.0 fM to 2.0 pM

1.0 fM

one-base difference

51

miRNA-21

Fluorescence method

1 pM to 10 nM

300 fM

one-base difference

9

miRNA-21

Fluorescence method



2.0 pM

other RNA family members

48

miRNA-21

Electrochemiluminescence method

1.0 fM to 1.0 nM

0.5 fM

one-base difference

49

miRNA-21

Surface plasmon resonance

10 fM to 100 pM

3 fM

two-base difference

50

5.0 fM to 50 pM

4.2 fM four-base difference

18

miRNA-141

5.0 fM to 50 pM

3.0 fM

miRNA-141

100 pM to 100 nM

∼100 fM






19





miRNA-21 Electrochemical method

miRNA-21

Fluorescence method

let-7d miRNA-141

photoelectrochemical method

0.25 fM to 25 pM

83.3 aM


52

miRNA-141

Electrochemiluminescence method

10 aM to 100 pM

2.1 aM


53

miRNA-15a

Surface plasma resonance imaging

5 fM to 0.5 nM

0.5 fM



54

miRNA 155

SERS

0.1 fM to 100 pM

0.083 fM

one-base difference

55

The demand for simultaneous quantitative analysis of multiple miRNAs is becoming more and more urgent in clinical diagnostics as a result of some pathologies often associated with abnormal expressions of several miRNAs. Hence, to demonstrate our sensing platform with multiplexed capability to detect miRNAs, miRNA-21 and miRNA-141 together with let-7a were selected as targets. As shown in Figure 4a, we designed three nanoprobes, MB1 (labeled with FAM) /MoS2, MB2 (TAMRA) /MoS2, and MB3 (Cy5) /MoS2, which were complementary to let-7a, miRNA-21, and miRNA-141, respectively (Table S1). Then, seven sample (Table S3) containing different combinations of the three miRNAs targets were added into the mixture containing 0.1 U DSN enzyme , 1× DSN buffer, and the three nanoprobes, followed by analyzing with fluorophotometer. Figure 4b displayed that the fluorescence 11



readout could be obtained in the corresponding detection channel as only one kind of miRNA was added. However, when all targets were added, the solution emitted all fluorescence spectra. That is to say: the fluorescence signal of each nanoprobe was orthogonally triggered by the specific miRNA target, thus the ingredients of each sample can be obtained directly from the multi-channel fluorescence readouts. Moreover, we observed no crosstalk between the channels that interfered the signal response of each miRNA.

Figure 4. (a) The schematic illustration of MoS2-base sensing platform for multiplexed detection of miRNAs. (b) Multiplexing miRNAs detection. Let-7a (FAM channel, emission at 518 nm, shown with green columns); miR-21 (TAMRA channel, emission at 580 nm, blue); miR-141 (Cy5 channel, emission at 663 nm, red).

CONCLUSION In summary, we have developed a novel target signal-amplifying method on the basis of MoS2-loaded MBs and duplex-specific nuclease, and demonstrated its application for rapid, ultrasensitive, and selective detection of miRNAs. In contrast with previous methods for miRNA detection, this MoS2-loaded MBs-based sensor possesses several distinctive advantages. First, it is very simple and fast to detect miRNAs, just simply mixing of MBs, 12


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MoS2, DSN enzyme, and target miRNAs in the solution and then incubating within 30 min, without any separation and tedious procedure. Second, MoS2’s fluorescence quenching ability combined with cyclic DSNMSA contributes to the ultrasensitivity of our method, which is 4 orders of magnitude lower than that of the conventional hybridization-based strategies. Third, owing to the remarkable discrimination abilities of MB and DSN, our method shows remarkable selectivity for distinguishing homologous miRNA sequences with single nucleotide variations. Moreover, our method allows multiplexed miRNAs detection by designing different dye-labeled nanoprobes. We envision that in our future study, the combination of the MoS2-based platform with well-defined DNA nanostructures at the similar scale (e.g. DNA origami) may provide better programmability and addressability, thus enables high-throughput and/or smart analytical applications56-61. In addition, the MoS2 nanosheets can be readily synthesized on a large scale and the homogeneous fluorometric assay is easy to be automated. These features make our method a promising platform for in

vitro detection of miRNAs in clinical samples. ASSOCIATED CONTENT Supporting Information. This material is available free of charge on the ACS Publications website. This material includes the TEM image and EDS spectrum of MoS2 (Figure S1), the sequences of oligonucleotides (Table S1), the recovery results of the proposed method in human serum (Table S2),and the samples used in the multiplexed miRNAs detection (Table S3). AUTHOR INFORMATION Corresponding Authors *E-mail: [email protected]; [email protected]. 13



Notes The authors declare no competing financial interest. ACKNOWLEDGMENT This work was supported by the National Science Foundation of China (grant numbers 21722502, 21505045, 21705048, 21605026, 81672720, U1532119), the Shanghai Pujiang Talent Project (16PJ1402700), China Postdoctoral Science Foundation (2015M581565, 2017T100283).

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