Establishing biodegradable single-layer MnO₂ nanosheets as a platform for live cell microRNA sensing

Abstract

We have established a simple and effective biosensing platform based on the non-covalent assembly of biodegradable single-layer MnO₂ nanosheets and target-specific oligonucleotide sequences, and demonstrated its application for sensitive and selective sensing of intracellular microRNA in living cells.

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MicroRNAs (miRNAs) are a class of evolutionally conserved, small-sized (approximately 19–23 nucleotides), noncoding, single-stranded RNA molecules that play an important role in controlling the expression of target proteins either via the repression of messenger RNA (mRNA) or the inhibition of mRNA translation in a sequence-specific manner, thereby providing an additional level of gene regulation.^1–3 The changes of miRNA expression levels are associated with various pathological processes, including neuro disease, cardiovascular disease, and cancers.^2,4–6 Therefore, the actual expression level of miRNAs may serve as a reliable diagnostic and prognostic marker in clinical processes. The three currently available common strategies to measure aberrant miRNA expression levels are northern bloting,⁷ microarrays,^8,9 and quantitative real-time PCR.^10–13 Northern blot is considered as the standard method for miRNA detection, since it can locate specific RNA sequences in a solution, and quantitative real-time PCR can easily provide absolute miRNA quantification. However, these sensing techniques do not allow in situ or online monitoring of miRNA in living cells.

With the achievements of nanotechnology and nanoscience, nanoparticle-based probes have been attracting increasing interest for detection of nucleic acids, offering high flexibility, good stability, multiple objective analytics, high sensitivity, and efficient cell internalization.^14–16 Functional nanomaterials have been utilized as nanocarriers and/or fluorescence quenchers of dye-labelled DNA for in situ detection of miRNA in living cells.^17–20 For example, Bian et al. have reported the detection of miRNAs in living human mesenchymal stem cells by using polydopamine-coated gold nanoparticles (Au@PDA).²¹ The PDA shell facilitates the immobilization of fluorescently labelled hairpin DNA that can recognize specific miRNA targets. The gold core and PDA shell quench the fluorescence of the immobilized hairpin DNA, and subsequent binding of the hairpin DNA to the target miRNAs leads to their dissociation from Au@PDA and the recovery of fluorescence signals. In addition, a multiplexed miRNA sensing platform composed of dye-labelled peptide nucleic acid (PNA) and layered graphene oxide (GO) has been developed for real-time monitoring of miRNAs in living cells.²² The miRNA sensor is based on the recovery of the quenched fluorescence of dye-labelled PNA that was tightly bound to the surface of GO, upon addition of target miRNA. Although several nanoparticle-based sensing platforms for miRNA detection have been developed, quantitative monitoring of intracellular miRNA in living cells still remains an important challenge. These reported sensing platforms are considered cytotoxic and nondegradable, and therefore, they are not ideal for intracellular sensors.

Layered transition-metal nanomaterials with single or few atomic layers, recognized as planar covalent-network solids, have attracted growing attention in the past few years owing to their structure analogous to graphene.^23–25 As a low-cost transition-metal oxide nanomaterial with easy preparation, efficient fluorescence quenching ability, good biocompatibility and degradability, manganese dioxide (MnO₂) has shown an increasing potential for application in biosensing and bioimaging.^26,27 Here, we report an intracellular miRNA sensing platform composed of target-specific molecular beacon (MB) and degradable single-layer manganese oxide nanosheets (SLMONs) for sensitive and in situ monitoring of miRNA in living cells (Scheme 1). The miRNA sensor is based on the fluorescence recovery of the MB that was tightly bound to the surface of SLMONs, upon addition of target miRNA. The miRNA sensing strategy utilizing MB as fluorescence probe and SLMONs as nanocarrier and assisted quencher allows high selectivity and specificity toward target miRNA with very low background signal and low cytotoxicity due to (1) advantages of SLMONs involving good redox-responsive degradability in the presence of biothiols, (2) stable binding of MB and SLMONs though the van der Waals force between nucleobases and the basal plane of SLMONs, which decreases enzymatic DNA degradation, and (3) efficient intracellular delivery of the SLMONs/MB complex without assistance of additional transfection reagent.

Scheme 1 Scheme of strategy for miRNA sensing in living cells based on the combination of degradable SLMONs and MB probes.

To realize the above design, SLMONs were synthesized by oxidation of MnCl₂ by H₂O₂ in the presence of tetramethylammonium hydroxide pentahydrate.²⁸ The size and morphology of SLMONs were characterized by transmission electron microscopy (TEM) and atomic force microscopy (AFM) measurements. As shown in Fig. S1 and S2,† the obtained SLMONs presented an obvious single-layer sheet structure, with an average thickness of about 1.3 nm and a width of 20–200 nm. Furthermore, we analyzed SLMONs by zeta potential analysis and UV-vis spectroscopy. The zeta potential value of SLMONs was measured as about −26.7 mV, indicating a negatively charged surface. This charge resulted from the formation of Mn-vacancies on the SLMONs and produced good water dispersibility. The absorption spectrum exhibited a wide band in the range of 250–800 nm with a peak located at 380 nm, characteristic absorption of SLMONs (Fig. S3†). The broad absorption may make the energy acceptor adaptable to diverse fluorophores emitting at different regions. All the analysis data indicate the successful preparation of SLMONs.

Next, we evaluated the adsorption property and fluorescence quenching ability of the prepared SLMONs towards fluorescence-labelled DNA. The DNA probes can be absorbed on the surface of SLMONs via van der Waals force.²⁹ Such interactions will be weakened upon the specific binding to the complementary target DNAs or RNAs, thus resulting in the release of the immobilized DNA probes. As shown in Fig. S4,† the fluorescence spectrum of dye-labelled hairpin DNA (hDNA, sequence information listed in Table S1†) in the absence of SLMONs had strong fluorescence emission owing to the presence of the fluorescein-based dye. However, in the presence of SLMONs, hDNA was efficiently adsorbed on the surface of SLMONs materials, resulting in the fluorescence quenching. Fig. S5† confirmed that the hDNA was successfully adsorbed on the SLMONs surface. Nevertheless, when hDNA was hybridized with an equal amount of its complementary DNA to form double-stranded DNA, the fluorescence of double-stranded DNA was largely retained in the presence of SLMONs. Moreover, to further reduce the fluorescence background signal and improve the sensitivity of the nanoprobes, the dye-labelled hDNA was functionalized with a fluorescence quencher to construct MB probe. SLMONs only assist in quenching the emission of the recognition MB probes to be deposited on its surface, thereby resulting in a compounded quenching effect. For our initial studies, MB probes that specifically recognize miR-21 miRNA target molecules are loaded onto the surface of SLMONs by gentle mixing for 1 h to form SLMONs/MB nanocomposites probes against miR-21. We then compare the fluorescence of free MB with that of nanoprobes immobilized with MB. Emission spectra show that the SLMONs/MB probes have a lower background signal than that of free MB probes (Fig. 1A), which is attributed to the fluorescence quenching ability of SLMONs. By adding the DNA analog of target miR-21 (DNA1), both free MB probes and SLMONs/MB probes are able to be activated, resulting in the recovery of the quenched fluorescence. As a control, the MB probes containing a non-recognition sequence were immobilized on the SLMONs to obtain SLMONs/cMB probes. This probe shows no obvious fluorescence recovery upon the addition of DNA1 (Fig. 1B).

Fig. 1 (A) Fluorescence emission spectra of MB recognition probes (5 nM) before and after immobilization onto the surface of SLMONs. (B) Fluorescence emission spectra of control MB probes (5 nM) containing a non-recognition sequence after immobilization onto the surface of SLMONs. (c) Fluorescence spectra of SLMONs/MB probes (5 nM loaded MB) in the presence of different concentrations (0–2000 pM) of DNA analog of miR-21 (DNA1). (d) Calibration curve for DNA1 detection. Inset: amplification of the low concentration range of the calibration curve. Excitation and emission wavelengths are 480 and 521 nm, respectively.

Based on the above-mentioned experiments, the SLMONs could serve as nanocarrier for loading miR-21-specific MB probes. In a typical experiment, SLMONs/MB nanoprobes (2 μg mL⁻¹) were treated with DNA1 at various concentrations for 10 min. As the concentration of DNA1 increased, the amount of formed MB/DNA1 duplex was increased so that the quenched fluorescence of MB probes was largely recovered (Fig. 1C). By correlating the fluorescence changes at 521 nm, a calibration curve was obtained for determining DNA1 concentration. As shown in Fig. 1D, a linear relationship between fluorescence and DNA1 concentration (R² = 0.995) was obtained over the range of 0–100 pM, with a detection limit of 1 pM (3σ).

To test the selectivity of the sensors, their capability of detecting base-mismatched DNA was investigated. In this experiment, various DNA samples including target DNA, single- and double-base mismatch DNA, and random DNA were used. All the DNA samples had the same strand length (22-base) and concentration (5 nM). With the increased mismatched bases, the fluorescence intensity of FAM dropped gradually (Fig. 2A). For the target DNA and single-base mismatch DNA, the fluorescence intensity was respectively increased by about 6 and 4 times as compared to the fluorescence background signal. This result indicates that such a nanoprobe can be used for detecting the DNA mismatch.

Fig. 2 (A) Specificity test of SLMONs/MB probes against target DNA1 (DNA analog of miR-21), DNA2 (1-base mismatched DNA), DNA3 (2-base mismatched DNA) and DNA4 (random DNA). (B) Stability test of the fixed MB probes (SLMONs/MB) and free MB probes after DNase 1 (1 unit) treatment. (C) Stability and GSH-responsive degradability of SLMONs-based nanoprobes in cell medium. (D) Viability of HeLa cells after being treated with SLMONs for 24 h.

In addition, the stability of SLMONs-based probes was very important in biomedical applications. Thus, to investigate whether the DNA probes immobilized on the SLMONs surface could be protected from nucleases, the stability assay of SLMONs/MB against DNase 1 was performed. As shown in Fig. 2B, after the SLMONs/MB nanocomposites were treated with DNase 1 (1 unit) for 15 min, there were no obvious fluorescence change. In contrast, a strong fluorescence was detected if free MB probes were treated with DNase 1. These results suggest that the SLMONs effectively decrease enzymatic DNA degradation, possibly by spatial resistance. Furthermore, the stability of the nanoprobes in cell medium was also investigated by using the assembly of dye-labelled hDNA probes and SLMONs. After 2 h incubation with cell medium, a negligible change in fluorescence intensity was observed (Fig. 2C), showing a good stability of SLMONs/hDNA complex. However, fluorescence corresponding to FAM is highly intensified after treatment with cell medium containing glutathione (GSH). The fluorescence activation is direct correlation with the amounts of GSH (Fig. S6†). A maximum fluorescence intensity of FAM was obtained after a given amount of GSH (2 : 1 molar ratio of GSH to SLMONs) was added, further confirming that SLMONs carrier was decomposed into Mn²⁺ ions via thiol-mediated reduction.

Toxicity is a crucial factor to be taken into account in the design of an intracellular sensing system. The cytotoxicity of the SLMONs was evaluated by measuring cell viability, using MTT assay. As shown in Fig. 2D, there was found to be no apparent loss of cell viability for HeLa cells after 24 h of exposure to the SLMONs with various concentrations below 120 μg mL⁻¹. This result indicates that SLMONs have low cytotoxicity and good biocompatibility.

Subsequently, the specificity of the SLMONs/MB probes in living cells was investigated. In this experiment, HeLa cancer cells were incubated with recognition SLMONs/MB probes and control SLMONs/cMB probes and then imaged using confocal laser scanning microscopy (CLSM). The results showed that HeLa cells treated with recognition SLMONs/MB probes were highly red fluorescence as compared to those treated with SLMONs/cMB probes (Fig. S7†). It was reported that inhibition probe (IP) induced the down regulation of miR-21 expression.¹⁸ HeLa cells were divided into two groups: one group was treated with SLMONs/IP to decrease the miR-21 expression, and the other was untreated, which served as a control. Fig. 3 and S8† showed that the red fluorescence was lower in the SLMONs/IP treated HeLa cells relative to that in the untreated cells. These results indicated that the fluorescence intensity correlated well with the level of miR-21 expression in living cells. Thus, the SLMONs/MB nanoprobes are capable of detecting changes in gene expression levels in cancer cells. Considering that miR-21 is a cancer biomarker, furthermore, we used L02 cells as a negative control. The fluorescence images of miR-21 in L02 cells after being incubated with SLMONs/MB for 3 h were compared with that of positive HeLa cells. The result showed that almost no red fluorescence in L02 was detected. To correlate fluorescence intensity from our probe with actual amount of induced miR-21 in the HeLa cells, mean fluorescence obtained from flow cytometric analysis of the SLMONs/MB treated cells was plotted versus absolute amount of miR-21 per cell calculated from quantitative real-time PCR. The fluorescence intensity decreased as the amount of miR-21 increased (Fig. S9†). In addition, to provide further evidence for GSH-responsive degradation of SLMONs in living cells, HeLa cells were pre-treated with and without GSH scavenger. Then, 1,2-bis-(2-pyren-1-ylmethylamino-ethoxy) ethane (NPEY) was utilized as a fluorescent probe for sensing Mn²⁺ ions inside cells.³⁰ As shown in Fig. S10,† untreated HeLa cells incubated with SLMONs/MB and NPEY probes exhibited a strong blue fluorescence. However, addition of GSH scavenger resulted in a distinct decrease of fluorescence intensity.

Fig. 3 CLSM images of miR-21 in HeLa cells treated without (a) and with (b) SLMONs/IP, and L02 cells (c) by SLMONs/MB nanoprobes. Scale bar is 5 μm.

Conclusions

In summary, we have demonstrated that the intracellular miRNA sensing platform based on the combination of SLMONs and target-specific MB probes enabled sensitive and quantitative monitoring of miRNA targets in homogeneous solutions with high sequence specificity and low background signal. In addition, we further extended our strategy for the detection of miRNA expression levels inside live cells. The SLMONs/MB complex enhanced efficiently cell internalization of MB probes and, thus, gave sensitive response to target miRNA even in living cells. We showed that SLMONs might not only protect immobilized DNA probes from nucleases, but also allow degradation by a simple GSH treatment. We expect that the present live cell miRNA sensing platform will be readily applicable for various miRNA related investigations, for example, the discovery of small molecules which regulate certain miRNA expression levels through high-throughput chemical screening in living cells, the detection of miRNAs as biomarkers in diseased tissues, the miRNA-based therapy, and monitoring of dynamic changes in expression levels of specific miRNAs in stem cell differentiation.^2,21,31,32

Acknowledgements

This work was supported in part by the Construct Program of the Key Discipline in Hunan Province, the Research Foundation of Education Bureau of Hunan Province (13C1120, 15A023 and 15A025) and Hunan Provincial Natural Science Foundation (2015JJ6010).

Notes and references

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Footnote

† Electronic supplementary information (ESI) available: Characterization data of SLMONs, DNA sequence information, UV-vis absorption spectra, fluorescence emission spectra, and CLSM images. See DOI: 10.1039/c5ra21467j

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