Affinity-Modulated Molecular Beacons on MoS2 Nanosheets for

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Biological and Medical Applications of Materials and Interfaces

Affinity-Modulated Molecular Beacons on MoS2 Nanosheets for MicroRNA Detection Mingshu Xiao, Arun Richard Chandrasekaran, Wei Ji, Fan Li, Tiantian Man, Changfeng Zhu, Xizhong Shen, Hao Pei, Qian Li, and Li Li ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b14035 • Publication Date (Web): 02 Oct 2018 Downloaded from http://pubs.acs.org on October 3, 2018

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ACS Applied Materials & Interfaces

Affinity-Modulated Molecular Beacons on MoS2 Nanosheets for MicroRNA Detection Mingshu Xiao1, Arun Richard Chandrasekaran2, Wei Ji1, Fan Li1, Tiantian Man1, Changfeng Zhu3, Xizhong Shen3, Hao Pei1, Qian Li4* and Li Li1* 1

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 2

The RNA Institute, University at Albany, State University of New York, Albany, NY 12222,

USA 3

Department of Gastroenterology, Zhongshan Hospital, Fudan University, Shanghai, 200032, P.R

China 4

School of Chemistry and Chemical Engineering, Shanghai Jiao Tong University, Shanghai

200240, China KEYWORDS: Nanoprobe, TMDs, polyC, MoS2, microRNA, biosensors

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ABSTRACT: DNA functionalized layered two-dimensional transition metal dichalcogenides have attracted tremendous interest for constructing biosensors in recent years. In this work, we report diblock molecular beacons with poly-cytosine (polyC) tails anchored on molybdenum disulfide (MoS2) nanosheets as probes for microRNA detection. The polyC block is adsorbed on MoS2 and the molecular beacon block is available for hybridization to the target; duplex-specific nuclease provides signal amplification by target recycling. By changing the length of polyC, we regulate the density of probes on MoS2 and inhibit the adsorption of enzyme-cleaved oligonucleotides, thereby leading to higher quenching efficiency. PolyC-mediated molecular beacons on MoS2 have very low background signal, ultrahigh sensitivity (LOD ∼3.4 fM), specificity to detect a single nucleotide mismatch, and selectivity to detect target microRNA from serum samples. This detection platform holds great potential for quantitative analysis of miRNAs in clinical diagnosis and biomedical research.

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INTRODUCTION Layered two-dimensional materials such as transition metal dichalcogenides have widespread usage across many disciplines due to their unique crystal structures, the physical and chemical properties.1-2 Of these, molybdenum disulfide (MoS2) nanosheets are now frequently used to develop sensors owing to their special optical properties.3-4 With the advent of DNA nanotechnology, researchers have shown DNA to be a powerful tool to functionalize nanomaterials.5 Using DNA to functionalize MoS2 via noncovalent adsorption is a popular approach that has several distinctive advantages over covalent functionalization.6-7 For example, in noncovalent adsorption, the activity of DNA is retained for hybridization, whereas covalent linkages (e.g., thiolated DNA) lead to low hybridization efficiency.8 Noncovalent adsorption may also be a low-cost method for DNA conjugation. However, noncovalent functionalization is compromised by its easiness to induce nonspecific competitive substitution. Moreover, probes confined to the surface of MoS2 often decrease their recognition capability to the target. Recent studies showed that poly-cytosine (polyC) DNA can be stably adsorbed on MoS2,9 and the probe density can be fine-tuned by varying the polyC length.10 These findings indicate that polyC DNA is a good anchor to functionalize MoS2, overcoming the aforementioned deficiencies related to noncovalent functionalization. Here we present diblock molecular beacons with a polyC block functionalized on layered MoS2 for use as nanoprobes in biological sensing. MicroRNA (miRNA), a class of biomarker in clinical diagnosis and therapy,11-13 is selected as model target here. Our system consists of polyC-containing molecular beacons (polyC-MB) with a stem-loop structure, in which a polyC block binds at 5’ end and a fluorophore binds at 3’ end, as well as a loop complementary to the target oligonucleotide (Figure 1). When incubated with layered MoS2,

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polyC-MB binds to MoS2 via adsorption of polyC on MoS2 via van der Waals forces,14 concomitantly quenching the fluorescence of dye due to long-range energy transfer.15-16 As a result of steric hindrance, the MB block is away from MoS2 surface, exposed for hybridization with the target. PolyC-MB hybridizes to target upon addition of target miRNA forming a DNA/RNA heteroduplex, which then becomes the digested target for duplex-specific nuclease (DSN). Previous studies have demonstrated that DSN can selectively cleave DNA in DNA/RNA heteroduplexes with at least 15 bp or double-stranded DNA with at least 10 bp.17-21 Here, we therefore deliberately designed the stem of MB with 6 bp, which could effectively prevent the stem from cleavage by DSN. Cleavage of PolyC-MB releases the target miRNAs from the heteroduplex, and the released miRNA subsequently hybridizes to another PolyC-MB, enabling the initiation of the next cycle. The DSN-based cleavage of the MB provides signal amplification for the trace detection of target miRNAs. Notably, both the weak affinity between the cleaved short fluorophore-labeled oligonucleotides and MoS2, and the ability of polyC block to inhibit nonspecific adsorption of enzyme product on MoS2 contribute to the retention of the fluorescence.


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Figure 1. Schematic illustration of polyC-mediated molecular beacons on MoS2 nanosheets for microRNA detection. PolyC-MB/MoS2 nanoprobes start with quenched fluorescence (left). Hybridization of target microRNA opens the loop of the molecular beacon to give rise to a DNA/RNA heteroduplex, allowing the fluorophore to drift away from the MoS2 surface and increasing the fluorescence intensity. Addition of duplex-specific nuclease (DSN) causes cleavage of the DNA strand in the DNA/RNA heteroduplex, releasing the miRNA that can then bind to other polyC-MB, causing signal amplification. EXPERIMENTAL SECTION Materials. RNase inhibitor and diethyl pyrocarbonate (DEPC)-treated water were purchased from Takara Biotechnology Co. Ltd. (Dalian, China). Duplex-specific nuclease (DSN) was supplied by Newborn Co. Ltd. (Shenzhen, China). MiRNAs and FAM-labeled probes listed in Table S2 were synthesized by Sangon Biotech Co. Ltd. (Shanghai, China) with standard desalting and purified with HPLC. The monolayer molybdenum disulfide (MoS2) was supplied by Nanjing XFNANO Materials Tech Co., Ltd. All other chemicals were of analytical reagent grade and obtained from Sigma-Aldrich (St. Louis, MO). Ultrapure water (18.2 MΩ) purified with a Milli-Q Integral water purification system (Millipore Corp., Bedford, MA) was used throughout the study. To create and maintain an RNase-free environment, all aqueous solutions used in this work were treated with 0.1% DEPC and autoclaved. The tubes and tips are RNase-free without need for pretreatment to inactivate RNases. Healthy human real serum was donated from Zhongshan Hospital, Fudan University, Shanghai, China. A set of serum samples were prepared by mixing serum with various concentrations of miRNA let-7b (0.01 pM, 0.05 pM, 0.1 pM, 1 pM, and 10 pM). Practical sample detection was conducted in accordance with the guidelines for the care



and use of laboratory animals proved by the Animal Ethics Committee of East China Normal University. Instrumentation. The fluorescence tests were performed with a HITACHI F-7000 fluorophotometer (Hitachi, Japan) at ambient temperature. The excitation wavelength was fixed at 485 nm with a 3 nm bandwidth and 0.3 s integration time. The emission spectra were collected from 505 nm to 650 nm with a wavelength step of 2 nm/s. DNA adsorption. Typically, the preparation of PolyC-MB/MoS2 complex is as follows: All used DNA samples were dissolved in HEPES buffer (5 mM, pH = 7.5) with a stock solution of 100 µM according to the quantity of DNA supplied by the vendor. Subsequent solutions were made by diluting the DNA stock. To adsorb DNA, 100 nM of PolyC-MB with different length (PolyC5-MB, PolyC10-MB, PolyC15-MB, PolyC20-MB or PolyC30-MB) was mixed with MoS2 (500 µg/mL) in 500 µL buffer A (25 mM HEPES, pH = 7.6, 150 mM NaCl, and 1 mM MgCl2) in dark at room temperature for 1 h. The fluorescence of samples was monitored by fluorophotometer for evaluating the quenching efficiency of MoS2 toward PolyC-MB. DSN-assisted signal amplification for miRNA Detection. All fluorescence measurements were performed in 1×DSN buffer (50 mM Tris-HCl pH=8.0, 1 mM dithiothreitol, and 5 mM MgCl2). After preparing probes by incubating poly-MB with MoS2 for 60 min, signal amplification proceeded in 100 µL solution (10 nM probes, 2 U/mL DSN, and different concentrations of target miRNA from 10 fM to 10 nM) at 55 °C for 30 min and then their fluorescence was collected. RESULTS AND DISCUSSION


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First, we confirmed that the anchorage of MB on layered MoS2 is dependent on poly-C block, as compared to direct interaction of the MB with MoS2. Consistent with previous work,22 the fluorescence of MB is quenched when incubated with MoS2 (Figure 2a). However, the fluorescence of PolyC10-MB/MoS2 is lower than that of MB/MoS2, and their corresponding quenching efficiencies are ∼95% and 89%, respectively (Figure 2b). The difference in quenching efficiencies of polyC10-MB/MoS2 and MB/MoS2 implies higher adsorption affinity of polyC10-MB on MoS2 than that of MB on MoS2. To test our detection platform, we chose let-7 family of miRNA as targets due to their highly similar sequences and expression levels closely related to cell development and human cancer.23 Figure 2c shows that the fluorescence of nanoprobes was significantly quenched when polyC10-MB was incubated with MoS2, and its quenching efficiency was up to 95% (Figure 2d). In the absence of DSN, only a slight fluorescence was recovered because of the 1:1 binding event when target miRNA was added. However, the heteroduplex of polyC10-MB was cleaved upon addition of DSN, producing short fluorophore-labeled DNA fragments and releasing the target miRNA, which subsequently initiated a DSN-mediated cyclic cleavage reaction and significantly increased the fluorescence intensity.



Figure 2. Testing the polyC-MB/MoS2 detection platform. (a) Fluorescence emission spectra of PolyC10-MB (left) and MB (right) before and after anchorage on MoS2. (b) Fluorescence quenching efficiency PolyC10-MB and MB. (c) Fluorescence emission spectra for miRNA detection in the absence or presence of DSN. (d) Relative fluorescence intensity of (c). We then investigated the mechanism of DSN-assisted signal amplification (Figure 3). PolyC10-MB/MoS2-based nanoprobes display low fluorescence as the fluorophore is close to the MoS2 surface (Figure 3a-i). Long-range energy transfer from fluorophore to MoS2 leads to fluorescence quenching of PolyC10-MB, endowing extremely low background with our probes (Figure 3b-c). Target oligonucleotide with single nucleotide polymorphism (SNP), unable to open loop of MB (Figure 3a-ii), resulted in only negligible fluorescence recovery (Figure 3b-c), showing the efficiency of our nanoprobes to discriminate target oligonucleotides with even a single nucleotide mismatch. We then tested fluorescence recovery using a complementary DNA strand to open loop of the polyC10-MB. Addition of complementary DNA (cDNA) induced the


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formation of duplex DNA (Figure 3a-iii), resulting in a slight fluorescence recovery (Figure 3b-c). With a complementary target, DSN is capable of cleaving the formed DNA duplex (Figure 3a-iv), but since both the probe DNA and target DNA are fragmented, there is no cyclic reaction to amplify the signal (Figure 3b-c). On the addition of a miRNA target, however, DSN selectively cleaves the DNA strand in DNA/RNA heteroduplexes (Figure 3a-v), and the miRNAs released from heteroduplexes trigger a DSN-mediated cyclic cleavage reaction resulting in a highly amplified fluorescent signal (Figure 3b-c).

Figure 3. DSN-assisted signal amplification. (a) Schematic illustration of different targets or stages of PolyC10-MB/MoS2 biosensor: (i) PolyC10-MB/MoS2, (ii) PolyC10-MB/MoS2+SNP, (iii) PolyC10-MB/MoS2+cDNA,

(iv)

PolyC10-MB/MoS2+cDNA+DSN

and

(v)

PolyC10-MB/MoS2+miRNA+DSN. (b) Fluorescence emission spectra and (c) relative fluorescence intensity for the different stages shown in (a). We

then

compared

the

performance

of

PolyC10-MB/MoS2

probes

with

the

PolyC10-DNA/MoS2 probes. PolyC10-MB/MoS2 probes incubated with miRNA showed an increase

of

fluorescence,

similar

to

the

fluorescence


signal

obtained

when


PolyC10-DNA/MoS2-based probes were mixed with cDNA (Figure S1). Despite of similar background signal, the fluorescence ratio (F/F0−1) of PolyC10-MB/MoS2 (2.758) was higher than that of PolyC10-DNA/MoS2 (2.406) (Figure S2) after addition of target, which suggests that PolyC10-MB has a stronger signal enhancement. In view of its stronger signal enhancement that is beneficial for sensitive detection, PolyC10-MB/MoS2 was therefore selected to construct nanoprobes for further miRNA detection. In view of the high adsorption affinity of polyC to MoS2, we were inspired to mediate the binding capacity of PolyC-MB on MoS2 by adjusting the polyC length (Figure 4a). The fluorescence intensity of nanoprobes decreases (i.e. higher quenching efficiency) with increasing length of polyC up to 15-nt after which it starts increasing (Figure 4b-c). These results also coincide with previous studies.9-10 Similar to our previous studies,24 the polyC block as an effective anchoring block preferentially binds onto MoS2 with high affinity, and the molecular beacon block as a recognition block adopts an extend and upright conformation that facilitate the target hybridization. The lateral spacing and surface density of polyC-MB (that is adsorption capacity) can therefore be systematically regulated by varying the length of the polyC block. The surface density of probes was found to decrease along with the length of the polyC block. In addition to mediating the density of probes, the polyC decreases competitive substitution while effectively inhibiting the adsorption of enzyme-cleaved oligonucleotides on MoS2, which is beneficial to the detection of target molecules. Optimal length for PolyC-MB also plays a role in higher quenching efficiency of probes, yielding lower background signal. Note that the cost of DNA synthesis also increases with longer polyC. Nanoprobes with the optimal length of polyC15 were selected for further experiments.


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Figure 4. Tuning the length of polyC block. (a) Illustration of PolyCn-MB with different lengths of polyC anchored on MoS2. (b) Fluorescence emission spectra and (c) quenching efficiency of PolyCn-MB, with 5, 10, 15, 20 or 30 cytosines in the polyC block. We then analyzed the performance of PolyC15-MB/MoS2-based nanoprobes for quantitative analysis of miRNA. Figure 5a shows the fluorescence intensity of PolyC15-MB in response to various concentrations of let-7b (from 10 fM to 10 nM). Figure 5b displays the fluorescence ratio (F/F0−1) in response to the concentration of let-7b in logarithmic scale; a 32-fold fluorescence enhancement was obtained at the concentration of 10 nM, with a linear response ranging from 10 fM to 10 pM. This method has a limit of detection (LOD) of ~3.4 fM, which is better or comparable to previous methods for miRNA detection (Table 1).25-37 Note that the high sensitivity of our method was achieved within less than 40 min. The excellent sensitivity and fast response are ascribed to extremely low background signal mediated by polyC and the highly effective cleavage activity of DSN.



Figure 5. MicroRNA detection using polyC-MB/MoS2 nanoprobes. (a) Fluorescence spectra of nanoprobes in response to different concentrations of let-7b. (b) Scatter plot of fluorescence ratio (F/F0−1) as a function of the concentration of miRNA in logarithmic scale, where F0 and F reprent the FAM fluorescence intensity of PolyC15-MB/MoS2 and PolyC15-MB/MoS2+miRNA in the presence of DSN. Inset: linear response range of miRNA concentration. (c) Sequences of let-7b, let-7c, let-7a, and let-7e. Bases that differ from those in let-7b are marked in red. (d) Fluorescence ratio (F/F0−1) in response to different miRNAs targets. There is only negligible fluorescence for even a single nucleotide mismatch in the miRNA sequence. Error bars are standard deviation of three repetitive experiments. The capability to differentiate miRNA family members varying by one or two nucleotides is of great importance in miRNA expression profiling, yet remains challenging. To further assess the specificity of our method, we used the let-7b nanoprobe to detect let-7c, let-7a, and let-7e miRNAs (Figure 5c). There is only a weak signal (F/F0−1) in the presence of 1-nt mismatched


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miRNA (let-7c) in contrast to the perfectly complementary target let-7b (Figure 5d). Negligible fluorescence responses were observed for 2-nt mismatched (let-7a) or 3-nt mismatched (let-7e) miRNAs. In addition, we also tested detection of let-7b in the presence of an equivalent mixture of other miRNAs; the fluorescence ratio was almost the same as that in the presence of only let-7b. These results validate that our detection platform offers high selectivity for miRNA analysis. The high specificity of our method is attributed to both MB with powerful ability to differentiate 1-nt mismatched target and DSN with good capability to discriminate perfectly from imperfectly matched DNA/RNA heteroduplexes. Then, we also conducted recovery experiments. To prepare practical samples, different concentrations of miRNA were added and mixed with 100-fold diluted healthy human serum sample (Table S1). Recovery in serum samples ranged from 98.6% to 110%, showing the feasibility of using this method for clinical applications. Table 1. Comparison of different methods for miRNA detection. Detection target

Detection method

Signal amplification strategy

Linear range

LOD

Ref.

let-7b

Fluorescence

DSN-assisted target recycling

10 fM to 10 pM

∼3.4 fM

This work

miR-21

Fluorescence

Rolling circle amplification

10 fM–100 pM

1 fM

[25]

let-7a

Fluorescence

DNAzyme-assisted signal amplification

1.5 fM to 9 fM

1.5 fM

[26]

miR-21

Fluorescence


1 pM to 10 nM

300 fM

[27]

let-7a

Fluorescence

Nicking enzyme-assisted amplification

100 fM to 1 nM

58 fM

[28]

let-7a

Fluorescence

DNase I-assisted target recycling

10 pM to 1 nM

2.3 pM

[29]

let-7a

Fluorescence

Primed and branched rolling-circle amplification

25 fM–1 pM

10 fM

[30]

miR-21

Fluorescence

Polymerase-aided strand-displacement polymerization and exonuclease-assisted template recycling



1 aM

[31]

miR-141

Electro-chemiluminescence

Target-cycling process

10 aM to 100 pM

2.1 aM

[32]

miR-21

Electro-chemiluminescence


1.0 fM to 1.0 nM

0.5 fM

[33]

miR-141

Surface-enhanced spectroscopy

Isothermal cycling signal-amplification

1 fM to 100 nM

0.17 fM

[34]

let-7b

Electrochemical method


2.0 fM to 2.0 pM

1.0 fM

[35]

miR-21

Surface plasmon resonance


10 fM to 100 pM

3 fM

[36]

miR-141

Photoelectrochemical method

DSN-assisted signal amplification

0.25 fM to 25 pM

83.3 aM

[37]

Raman

CONCLUSION



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To summarize, we introduced a novel polyC-MB/MoS2-based nanoprobe with excellent sensitivity and selectivity and demonstrated its potential in miRNA detection. Our strategy involving polyC blocks offers control over surface density of nanoprobes. While earlier studies have used polyA blocks to control DNA probe density on gold nanoparticles,38-39 polyC blocks are useful to program specific sensing platforms.40 Our strategy has several distinctive advantages including simple preparation, control over nanoprobe density on the MoS2 surface, negligible background signal, sensitivity with an LOD of 3.4 fM, and specificity of single nucleotide. Given these advantages, polyC-MB/MoS2-based nanoprobe is expected to provide an excellent biosensing tool with promising applications in biomedicine and clinical diagnosis. ASSOCIATED CONTENT Supporting Information. This material is available free of charge on the ACS Publications website. Fluorescence emission spectra of PolyC10-MB/MoS2 in the absence or presence of miRNA, fluorescence intensities

and

Fluorescence ratio

(F/F0−1) of PolyC10-MB/MoS2

and

PolyC10-DNA/MoS2, recovery results of miRNA let-7b in human serum, and sequences of oligonucleotides used in this work (PDF). AUTHOR INFORMATION Corresponding Author *E-mail: [email protected]; [email protected]. Notes The authors declare no competing financial interest.


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ACKNOWLEDGMENT This work was supported by the National Science Foundation of China (grant numbers 21505045, 21722502), the Shanghai Pujiang Talent Project (16PJ1402700). REFERENCES (1) Wang, Q. H.; Kalantar-Zadeh, K.; Kis, A.; Coleman, J. N.; Strano, M. S. Electronics and Optoelectronics of Two-Dimensional Transition Metal Dichalcogenides. Nat. Nanotechnol. 2012, 7, 699-712. (2) Chen, X.; McDonald, A. R. Functionalization of Two-Dimensional Transition-Metal Dichalcogenides. Adv. Mater. 2016, 28, 5738-5746. (3) Barua, S.; Dutta, H. S.; Gogoi, S.; Devi, R.; Khan, R. Nanostructured MoS2-Based Advanced Biosensors: A Review. ACS Appl. Nano Mater. 2018, 1, 2-25. (4) Kalantar-zadeh, K.; Ou, J. Z. Biosensors Based on Two-Dimensional MoS2. ACS Sensors 2016, 1, 5-16. (5) Xavier, P. L.; Chandrasekaran, A. R. DNA-Based Construction at the Nanoscale: Emerging Trends and Applications. Nanotechnology 2018, 29, 062001. (6) Tan, C. L.; Qi, X. Y.; Huang, X.; Yang, J.; Zheng, B.; An, Z. F.; Chen, R. F.; Wei, J.; Tang, B. Z.; Huang, W.; Zhang, H. Single-Layer Transition Metal Dichalcogenide Nanosheet-Assisted Assembly of Aggregation-Induced Emission Molecules to Form Organic Nanosheets with Enhanced Fluorescence. Adv. Mater. 2014, 26, 1735-1739. (7) Xiang, X.; Shi, J. B.; Huang, F. H.; Zheng, M. M.; Deng, Q. C.; Xu, J. MoS2 Nanosheet-Based Fluorescent Biosensor for Protein Detection via Terminal Protection of



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TOC figure


Affinity-Modulated Molecular Beacons on MoS2 Nanosheets for

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