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Letter Cite This: Nano Lett. 2018, 18, 5116−5123

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Photoactivated Specific mRNA Detection in Single Living Cells by Coupling “Signal-on” Fluorescence and “Signal-off” Electrochemical Signals Fujian Huang,† Meihua Lin,† Ruilin Duan,† Xiaoding Lou,† Fan Xia,*,†,‡ and Itamar Willner*,§

Nano Lett. 2018.18:5116-5123. Downloaded from pubs.acs.org by UNIV OF SOUTH DAKOTA on 08/26/18. For personal use only.

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Engineering Research Center of Nano-Geomaterials of Ministry of Education, Faculty of Materials Science and Chemistry, China University of Geosciences, Wuhan 430074, China ‡ Hubei Key Laboratory of Bioinorganic Chemistry & Materia Medica, School of Chemistry and Chemical Engineering, Huazhong University of Science and Technology, Wuhan 430074, China § Institute of Chemistry, Center for Nanoscience and Nanotechnology, The Hebrew University of Jerusalem, Jerusalem 91904, Israel S Supporting Information *

ABSTRACT: The spatiotemporal detection of a target mRNA in a single living cell is a major challenge in nanoscience and nanomedicine. We introduce a versatile method to detect mRNA at a single living cell level that uses photocleavable hairpin probes as functional units for the optical (fluorescent) and electrochemical (voltammetric) detection of MnSOD mRNA in single MCF-7 cancer cells. The fluorescent probe is composed of an ortho-nitrophenylphosphate ester functionalized hairpin that includes the FAM fluorophore in a caged configuration quenched by Dabcyl. The fluorescent probe is further modified with the AS1411 aptamer to facilitate the targeting and internalization of the probe into the MCF-7 cells. Under UV irradiation, the hairpin is cleaved, leading to the intracellular mRNA toehold-stimulated displacement of the FAM-functionalized strand resulting in a switched-on fluorescence signal upon the detection of the mRNA in a single cell. In addition, a nanoelectrode functionalized with a methylene blue (MB) redox-active photocleavable hairpin is inserted into the cytoplasm of a single MCF-7 cell. Photocleavage of the hairpin leads to the mRNA-mediated toehold displacement of the redox-active strand associated with the probe, leading to the depletion of the voltammetric response of the probe. The parallel optical and electrochemical detection of the mRNA at a single cell level is demonstrated. KEYWORDS: Sensor, photoprotective group, photocleavable, modified electrode, caged hairpin, light-induced activate the mRNA sensing events at a desired time by “ondemand” auxiliary triggers. This calls for the development of probes that upon internalization are quiescent but can be activated by an external stimulus in a manner that enables ondemand sensing and detection of mRNA in living cells at a desired time point. Such mRNA sensing platforms at the single-cell level would allow the monitoring of mRNA levels at dictated time-intervals of the cell life-cycle. Several studies demonstrated the imaging of mRNA at single-cell levels using fluorescence in situ hybridization (FISH) probes and flow cytometry22,23 or mRNA-induced rolling circle amplification.24 Nonetheless, these methods are time-consuming, prohibit temporal detection of mRNA in the cells, and error-prone due to a single readout signal for the analysis. To achieve this goal, light25−27 and magnetic field28 provide useful physical stimuli due to their clean characteristics. Light has been successfully used to activate caged molecular beacons

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ver the past decades, the imaging and detection of target mRNA in living cells have gained substantial interest having important implications in biology, biomedicine, diagnosis, drug discovery and cancer treatment.1−3 In particular, single-cell analysis allows better resolution of specific cellular components and their time-dependent changes within the cell cycle, thus providing important information on progressive diseases.4−13 The expression level and subcellular distribution of mRNA is precisely controlled with high spatiotemporal resolution. To study the expression level of mRNA at both high spatial and temporal resolution would prove valuable in elucidating the activity of genes. Accordingly, developing a method for on-demand mRNA detection at defined time points at the single-cell level would be desirable in the field of mRNA analysis. To date, a variety of strategies have been developed for specific mRNA detection and imaging in living cells, including DNA cascade reactions,14,15 aptamerbased systems,16 nanoparticle-based approaches15,17−19 and the application of DNA molecular beacons.20,21 However, the probes used in these methods interact directly with the target mRNA inside living cells, making it impossible to selectively © 2018 American Chemical Society

Received: May 17, 2018 Revised: July 11, 2018 Published: July 12, 2018 5116

DOI: 10.1021/acs.nanolett.8b02004 Nano Lett. 2018, 18, 5116−5123

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Scheme 1. Schematic Illustration of the Optical (Switched-on Fluorescence) and Electrochemical (Switched-off Voltammetric) Detection of mRNA Using Photocleavable Probes and the Photocativated Toehold-Mediated Displacement Process

for mRNA sensing in living cells.29−32 Recently, a magnetic field-assisted mRNA sensing approach was developed for remote and noninvasive mRNA-specific detection in living cells.33 Despite the recent advances in the use of light or magnetic field to detect mRNA in living cells, these methods provide output signals originating from a collective ensemble of cells. In fact, methods to detect mRNA in single cells with temporal and spatial resolution are scarce. Particularly, multiple-mode signal outputs for the detection of mRNA in single cells are unprecedented. The development of multimode detection platforms in single cells is important to prevent “false positive” or “false negative” results that are often encountered in single cell “signal-on” or “signal-off” transduction means.34 Within an effort to develop a superior method to monitor mRNA in single cells, we herein report on a photoactivated single-cell sensing platform that uses two complementary readout signals for detecting the target mRNA, namely a switched-on fluorescence signal, and a switched-off electrochemical signal. The method makes use of a previously reported photoactivated toehold-mediated reaction.35 By applying the UV-light photoactivated toehold-mediated mechanism and using an optical micro/nano fiber, we were able to target single cells and monitor the mRNA at a desired time. We demonstrate the incorporation of a quiescent DNA probe into cells and the on-demand photoactivation of the target mRNA by two output signals that include a switched-on fluorescence and switched-off electrochemical outputs. As illustrated in Scheme 1, the mRNA detecting process in single living cells depends on the photocontrollable toeholdmediated displacement reaction. To generate the fluorescent

signal, the DNA probe is composed of two parts: A carrier probe (CPF) and a signal probe (SPF). The carrier probe is modified with a fluorescent quencher (Dabcyl) at its 3′-end and conjugated to an aptamer (AS1411) single-strand tether, which can specifically interacts with the Nucleoline receptor associated with the cancer cells.36,37 The binding of the aptamer to the Nucleoline receptor units facilitates the internalization of the carrier probe into the cancer cells via ligand-induced endocytosis.38,39 The signal probe is modified with a fluorophore (FAM) at its 5′-end and it includes a photocleavable o-nitrobezylphosphate ester unit (PC-linker). It is a photoresponsive hairpin DNA with a single-strand tail (detailed sequences are shown in Table S1). The single-strand tail of CPF hybridizes with the single-strand tail of SPF, forming a CPF/SPF probe complex in which the fluorescence of FAM is quenched. This CPF/SPF probe complex is internalized into the cytoplasm, and it stays in the quiescent state until activated by UV irradiation. Subjecting the photoresponsive hairpin probe to UV light leads to the cleavage of the hairpin domain of the CPF/SPF complex resulting in the FAM-functionalized toehold single stranded tether that included the complementary sequence to bind to the mRNA. That is, in the presence of the mRNA the toehold-mediated displacement process proceeds, leading to the formation of the mRNA/FAMmodified strand and to the FAM switched-on fluorescence of the separated duplex. In addition to the fluorescent photocleavable probe for the single cell detection of mRNA an electrochemical photocleavable probe for the detection of mRNA was developed, Scheme 1. Toward this goal, a thiolated capture probe (CPE) 5117

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to UV light, and the fluorescence was collected after time intervals of 30 s of UV irradiation. As shown in Figure S2b, after 30 s of UV irradiation, the fluorescence intensity increased immediately and significantly. Fluorescence intensity reached a saturation after 6 times of UV irradiation (30 s each irradiation for a total 3 min of UV irradiation), demonstrating a 4-fold increase in fluorescence intensity over the initial value after 3 min of UV irradiation (Figure S2c). These results indicate efficient collection of the output fluorescence signal by the fluorescence collection system. We next investigated the performance of the photoactivated DNA probe for electrochemical signal generation and the in vitro electrochemical signal collection system. The performance of the photoactivated DNA probe for electrochemical signal generation was tested using a disk electrode. Figure S3a shows the working principle of photoactivated toeholdmediated displacement reaction on a disk electrode surface. The thiol-modified capture probe (CPE) was immobilized on the surface of the disk electrode through Au−S bond, followed by blocking the electrode surface with 6-mercapto-1-hexanol. Next, the methylene blue (MB)-modified, photocleavable signal probe SPE-MB was hybridized with the preimmobilized capture probe. The close proximity of MB to the electrode surface resulted in a high-electrochemical response. Without UV irradiation, no electrochemical signal decrease was observed in the presence of the target mRNA (Figure S3b). Upon UV irradiation, the photoresponsive probe was cleaved, resulting in the release of the tether that initiated the displacement reaction in the presence of target. This led to a significant decrease in the electrochemical signal. It should be noted that under UV irradiation, yet in the absence of the target mRNA, the strand-displacement could not be activated, and no significant decrease in the electrochemical signal could be detected, Figure S3c. These experiments imply that the electrochemical probe is very stable under the test conditions (Figure S4). Subsequently, the performance of the nanoelectrode was tested by using this electrochemical probe. The surface of the nanoelectrode tip was modified with CPE/SPEMB probe complex and blocked with 6-mercapto-1-hexanol (Figure S5a). The DNA probe was activated by UV irradiation that initiated the strand displacement reaction in the presence of target mRNA, resulting in a significant decrease of electrochemical signal (Figure S5d). Without UV irradiation (Figure S5b), or in the absence of target mRNA (Figure S5c), the displacement reaction on the tip surface was prohibited and no decrease of the electrochemical signal was observed compared with the blank sample. A prerequisite to follow the mRNA by the fluorescent probe SPF-FAM/CPF-Dabcyl and the integrated fluorescence signal detection system at the single cell level is the need to confirm the internalization of the probe into the cell. As our study will follow the MnSOD mRNA in single MCF-7 cancer cells the clear proof for the internalization of the fluorescent probe into the MCF-7 cells was essential. To reach this goal, the CPF labeled with TAMRA (CPF-TAMRA) and SPF labeled with FAM (SPF-FAM) were used to assess the internalization ability and to track the intracellular distribution of the probes. Confocal microscopy images showed that CPF-TAMRA (Figure S6a) and CPF-TAMRA/SPF-FAM complexes (Figure S6c) were, indeed, internalized into the cytoplasm using the AS1411 aptamer as targeting unit to the MCF-7 cells. It should be noted that SPF-FAM itself did not permeate the cell membrane, and no SPF-FAM signal was observed in the

was immobilized on an Au-coated nanoelectrode through an Au−S linkage. The methylene blue (MB)-modified strand (SPE-MB) that includes an internal o-nitrobenzylphosphate ester group is complementary, in part, to the capture strand (CPE) associated with the nanoelectrode. The hybridization of CPE with the SPE-MB led to a photocleavable SPE-MB/CPE hairpin probe-functionalized electrode. The resulting probefunctionalized electrode was inserted into the single living cell using a three-axis micromanipulation system (see setup details in Scheme S1). Under these conditions the cell inserted electrode yields the electrochemical signal characteristic to MB. UV irradiation of the cell leads, however, to the cleavage of the o-nitrobenzyl ester photoactive group, resulting in the cleavage of the hairpin domain associated with the SPE-MB/ CPE probe and to the formation of a single-stranded tether, that includes the base-sequence complementary to the mRNA (for the detailed sequences see Table S2). The subsequent strand displacement of the fragmented SPE-MB by the mRNA removes the redox active MB unit from the electrode, resulting in the depletion of the electrical signal upon the detection of the mRNA. That is, SPF-FAM/CPF-Dabcyl fluorescent probe and the SPE-MB/CPE electrochemical probe provide switchON and switch-OFF signals for the detection of the mRNA in a single cell. As a first step to support the capability to apply the probes shown in Scheme 1 to monitor the mRNA at the single cell level it was essential to confirm the validity of the concept of photocleavable probes to stimulate the strand displacement reactions, and to confirm that the experimental setup has the capabilities to monitor the fluorescence/electrical output generated by the micro/nano optical fiber and the nanoelectrode, respectively. The fluorescence probe SPF/CPF functionalized with the FAM/Dabcyl fluorophore/quencher pair was subject to UV irradiation in the presence of mRNA, Figure S1a. The photoactivated toehold-mediated displacement reaction was evidenced by the increase of fluorescence. As shown in Figure S1b, the FAM fluorescence at 520 nm increased with increasing UV irradiation time. The inset in Figure S1b shows that the fluorescence reached a saturation intensity at 520 nm after about 3 min of UV irradiation, demonstrating that the photoactivated toehold-mediated displacement reaction occurs rapidly and efficiently. To confirm that the toehold-mediated displacement reaction was indeed controlled by UV light, fluorescence measurements of the CPF/SPF probe complex were performed under different conditions (Figure S1c). Without UV irradiation, no significant fluorescence enhancement was detected in the presence of target (curve b) compared with the blank sample (curve a). In turn, after UV irradiation for 3 min the FAM fluorescence of the sample in the presence of target increased significantly (curve d). In the case of UV irradiation in the absence of target, no significant FAM fluorescence increase was observed (curve c). These experiments demonstrate that the probe can be activated by UV irradiation to detect the presence of the target. Subsequently, we used this photoactivatable SPF-FAM/CPF probe to evaluate the performance of the fluorescence collection system. In a typical experiment, a 5 μL droplet containing photoactivatable CPF-Dabcyl/SPF-FAM probe complex and target mRNA was added to the cell culture dish surface. Then, the optical micro/nano fiber coupled with 495 nm external excitation light was inserted into the droplet at a designated position (Figure S2a). The droplet was exposed 5118

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Figure 1. Photoactivated toehold-mediated displacement reaction for mRNA detection in a single cell with “signal-on” fluorescent signal output. (a) Images showing the cell with optical micro/nano fiber attached to the plasma membrane with 365 and 495 nm light turning on. Scale bar is 10 μm. (b) Schematic illustration of photoactivated toehold-mediated displacement reaction in a single cell for mRNA detection. (c) Real-time monitoring of fluorescence changes in a single cell under different conditions.

TAMRA fluorescence signal, FAM fluorescence signal and the ratio of these two signals under different conditions are shown in Figure S8. For the CPF-Dabcyl-TAMRA/SPF-FAM probe complex, the TAMRA-labeled CPF fluorescence was used as a reporter for the internalized amount and distribution of probe complex, thus serving as an internal reference for ratiometric analysis. The AOD of TAMRA signals was identical under the different conditions, indicating that the treatment with LPS and cordycepin had little effect on the internalization of the CPF-Dabcyl-TAMRA/SPF-FAM probe complex. In contrast, the FAM signal intensities were quite different under different conditions in that the highest FAM signal intensity occurred with LPS treatment and UV light irradiation, whereas the lowest FAM signal intensity occurred without UV irradiation. The set of control and background experiments reveal that the internalization of the fluorescent probe into the MCF-7 cells occurred and that UV irradiation induced the intracellular separation of the hybrid probe and triggered the strand displacement process upon the detection of the MnSOD mRNA. In the next step, we have applied the micro/nano fiber system for the optical (fluorescence) detection of MnSOD mRNA in single MCF-7 cells. In a typical experiment, MCF-7 cells were first incubated with the CPF-Dabcyl/SPF-FAM probe complex for 2 h. Subsequently, the optical micro/nano fiber was precisely attached to the cell membrane by using the three-axis micromanipulation system. External UV light (λ = 365 nm) and probe excitation light (λ = 495 nm) were guided through the optical fiber to reach the single cell (Figure 1a). The UV light guided by the optical fiber was used to activate the probe in the single cell and the excitation light was used to activate the FAM signal (Figure 1b). FAM signals in a single cell, under different conditions, were collected and recorded using a photomultiplier tube (PMT). As shown in Figure 1c, without UV irradiation the FAM fluorescence signal in the

cytoplasm (Figure S6b). Subsequently, the CPF-DabcylTAMRA/SPF-FAM probe complex was used to confirm the photoactivation process in living cells. The probe complex was internalized into the cytoplasm of MCF-7 cells, the photoactivation of the probe was stimulated by UV irradiation to trigger the displacement reaction in the MCF-7 cytoplasm. Without UV irradiation, the CPF-Dabcyl-TAMRA/SPF-FAM probe complex remained intact, and no displacement reaction was detected. That is, only the TAMRA-labeled CPF signal was observed, while the FAM-labeled SPF signal was quenched by Dabcyl, and no FAM signal was observed in the MCF-7 cytoplasm (Figure S7a). In contrast, the CPF-Dabcyl-TAMRA/ SPF-FAM probe complex internalized into the cytoplasm of MCF-7 cell was activated under UV irradiation, and the toehold-mediated displacement proceeded, resulting in the release of FAM and the generation of its fluorescence signal as shown in Figure S7b. It should be noted that the internalization of the complex probe into the MCF-7 cells did not affect the morphology of the cells within a time-interval of 2 h, Figure S6 and Figure S7, implying that no changes in the cell morphology occurred within the sensing time-interval (30 min). Furthermore, cordycepin and lipopolysaccharide (LPS) were used to downregulate and upregulate the expression level of MnSOD mRNA in MCF-7 cells, respectively,40 and the performance of the probe under different expression levels of MnSOD mRNA were examined (for the experimental details for the up-regulation and down-regulation of MnSOD mRNA in the cells, see Supporting Information). We find that the LPS-treated MCF-7 cells generate high FAM fluorescence (Figure S7d) as compared to the FAM fluorescence generated by the cordycepin-treated MCF-7 cells (Figure S7c) or the untreated MCF-7 cells (Figure S7b), consistent with the LPSstimulated overexpressed MnSOD mRNA. The cell imaging data shown in Figure S7 were further processed with the NIH ImageJ software. The average optical density (AOD) of the 5119

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Figure 2. Confocal microscopy images of MCF-7 cells treated with CPF-Dabcyl-TAMRA/SPF-FAM probe complex and selectively activated by UV light. (a) TAMRA signal in all cells indicates that the probe complex had been internalized in all cells. (b) FAM signal is observed only in the photoactivated single cell. (c) Principle of photoactivated toehold-mediated displacement reaction for mRNA sensing in a single cell with “signal on” fluorescence. The single cell marked with the red circle was selectively activated by UV light.

Figure 3. Photostimulated toehold-mediated electrochemical detection of mRNA in a single cell: (a) Schematic configuration of the photocleavable probe-modified nanoelectrode in a single cell exposed to external UV irradiation that stimulates the photocleavage of the redox-labeled probe and photoactivated toehold-mediated displacement of the redox-labeled probe that leads to a switched off electrochemical signal. (b) Microscopic image displaying the modified nanoelectrode inserted in the cytoplasm of a single cell and the optical fiber that covers the cell and acts as an optical guide of UV light for the photocleavage process. (c) Voltammetric response of the single cell before UV irradiation (I) and after exposure to UV light for 30 min (II). (d) Voltammetric response of the cell without UV irradiation at time t = 0 (I) and after a time interval of 30 min (II).

single cell remained unchanged at extended time intervals. In contrast, upon UV irradiation, the DNA probe in the single cell was activated by UV light, resulting in a time-dependent increase in fluorescence signal intensity of FAM. After 30 min reaction, the fluorescence intensity of FAM signal in a single cell with UV irradiation increased by about 20% compared with the one without UV irradiation, curve (ii). The FAM fluorescence intensity in an LPS-treated single cell, followed by activation with UV light, increased by about 40%, curve (iii).The results highlight the successful application of the fluorophore-modified probe for the optical sensing of mRNA

in a single cell and even the monitoring of different levels of the mRNA in the cells. The time-dependent changes of the fluorescence intensities reached a saturation value after 30 min, implying complete reaction of the permeated probe with the cell-available mRNA. Accordingly, this time-interval was used for interacting the probe with the cellular mRNA in all subsequent sensing experiments. Confocal microscopy experiments further supported the integration of the CPF-Dabcyl/SPF-FAM probe into the MCF7 cells and the single cell activation of the probe by the localized UV cleavage of the probe, Figure 2. The MCF-7 cells 5120

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Figure 4. Photoregulation of toehold-mediated displacement reaction in a single cell for mRNA sensing with both “signal on” fluorescence and “signal off” electrochemical signal outputs. (a) Image showing the CPE/SPE-MB DNA probe complex modified nanoelectrode inserted in the CPFDabcyl/SPF-FAM DNA probe complex treated single cell single cell, the activation light (365 nm) and excitation light (495 nm) guided by the optical fiber covering the single cell and the nanoelectrode tip. (b) Real-time monitoring of fluorescence changes in a single cell with and without UV irradiation. (c) Electrochemical signal change in the same single cell after UV irradiation.

presence of MnSOD mRNA in the cytoplasm of a single cell (Figure 3a). After displacement, the MB functionalized strand was separated from the surface of the nanoelectrode tip, resulting in the decrease of the electrochemical signal (Figure 3c). After 30 min reaction, the electrochemical signal decreased by about 33%. A control experiment revealed that without UV irradiation, the DNA probe on the tip surface remained intact, and no significant electrochemical signal decrease was observed in the single cell (Figure 3d). The bimodal (fluorescence and electrochemical) detection of the mRNA at a single cell level provides a means to apply simultaneously both methods to detect the mRNA. Beyond that scientific interest to design the photocleavable probes for the concurrent optical and electrochemical detection of the mRNA and the technological challenges accompanying the two methods on the same cell, the possibility to analyze the mRNA concomitantly by two independent methods is of analytical value as it enhances the reliability of the detection platform and eliminates false positive or negative results. Accordingly, as a proof-of-concept we analyzed, in parallel, the MnSOD mRNA in single MCF-7 cells by the two methods. The MCF-7 cells were incubated with CPF-Dabcyl/SPF-FAM DNA probe hybrid. Subsequently, the nanoelectrode modified with CPE/ SPE-MB DNA probe complex was inserted into the cytoplasm of one treated MCF-7 cell. UV light (365 nm) and the excitation light (495 nm) were guided by the same optical fiber to simultaneously reach the single cell and the tip electrode surface (Figure 4a). Similar to mRNA detection with single electrochemical output, here, an optical fiber with a relatively thick tip (about 5 μm) was used to ensure that the UV light totally covers the tip electrode surface. UV light activated the DNA probe in the single cell cytoplasm, and simultaneously opened the hairpin DNA immobilized on the surface of the nanoelectrode tip (see Scheme 1). The UV irradiation activated the fluorescence probe and the redox-active probe and stimulated the respective mRNA displacement reactions. The FAM fluorescence intensity in the single MCF-7 cell increased by about 22% ± 2% after 30 min of displacement reaction (Figure 4b). Concomitantly, a decrease in electrochemical signal, as shown in Figure 4c was observed. After 30 min displacement in the single cell, the electrochemical signal decreased by about 26% ± 2%, which is close to the electrochemical signal generated by only the microelectrode

were incubated with the CPF-Dabcyl/SPF-FAM probe for 2 h and the resulting cells were subjected to the localized UV irradiation. The resulting confocal fluorescence image revealed the FAM fluorescence only in the UV activated domain, while no fluorescence of the surrounding cells could be detected (Figure S9). This result is consistent with the localized UV light-induced cleavage of the probe and the mRNA-stimulated displacement of FAM-functionalized strand. In addition, treatment of the MCF-7 cells with the CPF-Dabcyl-TAMRA/ SPF-FAM probe for 2 h and subsequently subjecting the cells to the localized single-cell UV-induced cleavage, resulted in the fluorescence images shown in Figure 2. While all the cells displayed the TAMRA emission only the UV activated single cell (Figure 2b, marked with a red circle) showed the FAM emission while in all neighboring cells only the TAMRA fluorescence could be detected. These results further confirm that the FAM emission is selectively activated upon the UVcleavage of the probe that allows the mRNA induced displacement of the FAM-modified strand. As indicated earlier, microscale electrochemical experiments demonstrated the availability of an integrated electrochemical collection system for analyzing the mRNA by a nanoelectrode modified with a photocleavable redox-active probe, SPE-MB. Accordingly, the functionalized electrode was applied for the electrochemical detection of the mRNA in single MCF-7 cells as outlined in Figure 3a. The photocleavable DNA probe (SPEMB) was immobilized on the surface of the nanoelectrode tip via hybridization with the preimmobilized capture DNA (CPE). The tip diameter was about 78 nm, and a SEM image of the nanoelectrode is shown in Figure S10. Because the nanoelectrode tip is quite sharp, it could be inserted easily into the cell (see Movie S1). The modified nanoelectrode was precisely inserted into the cytoplasm of the single cell by using the three-axis micromanipulation system. UV light guided by the optical fiber was used to activate the DNA probe on the surface of the nanoelectrode tip. It was essential, however, to ensure that the entire nanoelectrode tip modified with the photocleavable redox probe will be exposed to the light-guided UV irradiation. To reach this goal, an optical micro/nanosized fiber with a relatively thick tip (about 5 μm) was used to guide the UV light (Figure 3b). This allowed the UV light activation of the DNA probe and the release of the caged toehold resulting in the toehold-mediated displacement reaction in the 5121

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Nano Letters (30% ± 3%). The respective error bars were derived from N = 3 experiments for each sensing configuration. The very close fluorescence intensities and current values observed upon the sensing of the mRNA by the simultaneous optical (fluorescence) and electrochemical bimodal sensing platform and by the individual optical or electrochemical modes, suggest that the sensitivity of the analysis of the intracellular mRNA is not affected by the simultaneous parallel operation of the two sensing modes. That is, the concentration of mRNA in the cell is sufficiently high to eliminate competitive perturbed binding of the mRNA to the optical probe and the monolayer-bound electrochemical probe. In conclusion, the present study introduced a method to analyze by optical and/or electrochemical means the mRNA at a single cell level. The method is based on the application of onitrophenylphosphate ester functionalized hairpin structures that are modified with the FAM fluorophore or methylene blue (MB) redox active groups and act as optical or electrochemical labels for the detection of MnSOD mRNA in a single cancer MCF-7 cancer cell. The fluorescent hairpin was caged in supramolecular quenched structure, whereas the electrochemical hairpin probe was immobilized on a nanoelectrode tip. UV irradiation of the fluorescent and redox-active probes resulted in the cleavage of the respective hairpins, and the subsequent mRNA-stimulated displacement of the cleaved product to yield a switch-ON fluorescence signal and a switchOFF electrochemical signal. Upon the development of the single-cell mRNA detection platform several important phenomena were demonstrated: (i) The modification of fluorescent probe with the AS1411 aptamer facilitated the permeation of the probe into the MCF-7 cells. (ii) A dualmode parallel optical and electrochemical detection platform was achieved. (iii) Photocleavable optical or electrochemical probes provide versatile means for the spatiotemporal detection of mRNA or microRNA in single cells. This paves the way to detect important biomarkers, for example, cancer cell biomarkers at the single-cell level. It should be noted that the AS1411 aptamer unit associated with the complex probe is introduced to target and facilitate the permeation of the probe into cancer cells, where the nucleolin receptor is often overexpressed. The concept may be, however, extended by designing appropriate aptamers to other cell-specific biomarkers.

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Xiaoding Lou: 0000-0002-6556-2034 Fan Xia: 0000-0001-7705-4638 Itamar Willner: 0000-0001-9710-9077 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work is supported by the National Basic Research Program of China (973 Program, 2015CB932600) the National Key R&D Program of China (2017YFA0208000, 2016YFF0100800), the National Natural Science Foundation of China (21525523, 21722507, 21574048), and the Fok YingTong Education Foundation, China (151011).

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.nanolett.8b02004. The experimental details and the working principle and setup of the detection system; Figure S1−S10(PDF) Video of nanoelectrode being inserted into a cell (AVI)

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*E-mail: [email protected]. Tel: 86-027-67885201. Fax: 86027-67883720. *E-mail: [email protected]. Tel: 972-2-6585272. Fax: 9722-6527715. ORCID

Fujian Huang: 0000-0002-7777-1589 Meihua Lin: 0000-0001-7616-7358 5122

DOI: 10.1021/acs.nanolett.8b02004 Nano Lett. 2018, 18, 5116−5123

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DOI: 10.1021/acs.nanolett.8b02004 Nano Lett. 2018, 18, 5116−5123