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Analytica Chimica Acta 1021 (2018) 129e139

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Analytica Chimica Acta

journal homepage: www.elsevier .com/locate/aca

In situ monitoring of cytoplasmic precursor and mature microRNAusing gold nanoparticle and graphene oxide composite probes

Min Hong a, b, Hongxiao Sun a, Lidan Xu a, Qiaoli Yue a, Guodong Shen a, Meifang Li d,Bo Tang b, *, Chen-Zhong Li a, c, **

a School of Chemistry and Chemical Engineering, Liaocheng University, Liaocheng, 252059, PR Chinab College of Chemistry, Chemical Engineering and Materials Science, Collaborative Innovation Center of Functionalized Probes for Chemical Imaging inUniversities of Shandong, Key Laboratory of Molecular and Nano Probes, Ministry of Education, Shandong Normal University, Jinan, 250014, PR Chinac Nanobioengineering/Bioelectronics Laboratory, Department of Biomedical Engineering, Florida International University, Miami, 33174, USAd School of Life Science, Liaocheng University, Liaocheng, 252059, PR China

h i g h l i g h t s

* Corresponding author.** Corresponding author. School of Chemistry and C

E-mail addresses: tangb@sdnu.edu.cn (B. Tang), lic

https://doi.org/10.1016/j.aca.2018.03.0100003-2670/© 2018 Elsevier B.V. All rights reserved.

g r a p h i c a l a b s t r a c t

� Simultaneous fluorescence imagingof cytoplasmic precursor and maturemicroRNA using gold nanoparticleand graphene oxide compositeprobes.

� Reducing the interference of precur-sor miRNA to the intracellularsensing of mature miRNA.

� Comparative analysis of the relativeexpression levels of precursor andmature forms of miRNA-21 and let-7a.

� In situ monitoring the inhibition ef-fects of small-molecule inhibitors ofmiRNA-21.

a r t i c l e i n f o

Article history:Received 22 January 2018Received in revised form7 March 2018Accepted 9 March 2018Available online 19 March 2018

Keywords:Fluorescence imagingNanocomposite probePrecursor microRNAsMature microRNAs

a b s t r a c t

This study strategically fabricates a nucleic acid functionalized gold nanoparticle and graphene oxidecomposite probe (AuNP/GO probe) to achieve both the recognition and in situ monitoring of cytoplasmictarget precursor microRNAs (pre-miRNAs) and mature microRNAs (miRNAs) in living cells. The pre-miRNA-21 detection with AuNP probes has a good linear range of 0e300 nM and a limit of detection(LOD) of 4.5 nM, whereas the GO probe has a linear relationship with mature miRNA-21 from 0.1 to10 nM with a LOD of 1.74 nM. This assay was utilized to directly visualize the relative expression levels ofpre- and mature forms of miRNA-21 and let-7a. The results suggested that the expression levels ofprecursor miRNAs remain constant in cancer cells and normal cells. However, the expression levels ofmature miRNAs vary widely, demonstrating the “up-regulation” of miRNA-21 and “down-regulation” oflet-7a in cancer cells in contrast to that in normal cells. The practicality of this strategy was verified by insitu monitoring changes in cytoplasmic pre-miRNA-21 and mature miRNA-21 in response to small-molecule inhibitors of miRNA-21.

© 2018 Elsevier B.V. All rights reserved.

hemical Engineering, Liaocheng University, Liaocheng 252059, Shandong, PR China.z@fiu.edu (C.-Z. Li).

mailto:tangb@sdnu.edu.cnmailto:licz@fiu.eduhttp://crossmark.crossref.org/dialog/?doi=10.1016/j.aca.2018.03.010&domain=pdfwww.sciencedirect.com/science/journal/00032670www.elsevier.com/locate/acahttps://doi.org/10.1016/j.aca.2018.03.010https://doi.org/10.1016/j.aca.2018.03.010https://doi.org/10.1016/j.aca.2018.03.010

M. Hong et al. / Analytica Chimica Acta 1021 (2018) 129e139130

1. Introduction

The study of microRNAs (miRNAs) is critical for understandingbasic biology and for identifying diagnostic targets. MiRNAs havethree distinct forms in cells: primary miRNAs (pri-miRNAs), pre-cursor miRNAs (pre-miRNAs) and mature miRNAs. Mature miRNAsare a class of evolutionally conserved, single-stranded, short(19e23 nucleotides (nt)), endogenously expressed, non-codingRNAs that regulate a series of cellular functions by inducingtarget mRNA degradation and translational repression [1,2].

Mature miRNA molecules are initially transcribed as primarymiRNAs (pri-miRNAs) by miRNA genes [3]. Pri-miRNAs are furtherprocessed in the nucleus by the enzyme Drosha, which transformspri-miRNAs into precursor miRNAs (pre-miRNAs) [4]. A pre-miRNAis ~70 nt in length and contains a characteristic hairpin structure.Then, pre-miRNAs are transported from the nucleus to the cyto-plasm by exportin-5, and are subsequently cleaved by Dicer into~21-nt mature miRNAs [5]. Several studies have explored theregulation of miRNA biogenesis in normal tissues, tumors, and celllines [6,7]. These studies revealed that a large number of miRNAsare present at the same level as their primary and precursor formsbut are not processed to mature miRNAs, such as miRNA-21, innormal tissues or cells [8]. Once an organism is disordered, theconversion of pre-miRNAs to mature miRNAs is accelerated [9,10].In contrast, for other miRNAs, such as let-7a, expression of theirmature forms is inhibited in abnormal tissues or cells [11]. There-fore, the simultaneous detection and real-time tracking of pre-miRNAs and mature miRNAs in living cells will help to elucidat avariety of physiological changes.

Currently, common methods, such as northern analysis andquantitative reverse transcription-polymerase chain reaction (qRT-PCR), are conventionally utilized to probe the relationship betweenpre- and mature miRNA expression, and these two methods havedemonstrated a general correlation in the expression of pre- andmature miRNAs. However, northern analysis suffers from a short-coming, specifically the use of radioactive or hazardous agents.While qRT-PCR requires an miRNA amplification step before signaldetection and is susceptible to the same drawbacks as all PCR-basedassays, including the risk of carry-over contamination, the inhibi-tion of polymerase in clinical samples, and the requirement forexpensive equipment and reagents [12e14]. Additionally, therehave been reports concerning the use of molecular beacons toquantify specific pre- and mature miRNAs [15]. Moreover, alter-native PCR-free methods to exclusively assess miRNAs have beenactively developed, including signal amplification-based fluores-cence detection [16e18], nanopore sensors [19], LC-MS/MS-basedassays [20], hybridization chain reaction coupled electro-chemiluminescent biosensors [21], signal amplification-basedelectrochemical methods [22], and others. Although these analyt-ical methods are reliable, a large number of cell samples and lysis ofthe cells are required for the analysis, thus leading to the un-avoidable loss of cell sources. These problems are adequatelyresolved by in situ analyses, particularly nanotechnology-basedbiosensor analyses [23]. To date, several studies investigating thebioimaging of pri-miRNAs and mature miRNAs in vivo have beenreported [24e27].

Mature miRNAs are obtained directly via the degradation of pre-miRNAs in the cytoplasm, and mature miRNA sequences are con-tained in pre-miRNA [4,5]. Furthermore, pre-miRNA leads a certainlevel of interference of the intracellular in situ detection and im-aging of mature miRNA. Therefore, to reduce this disturbance, it isnecessary to design a method that detects two sequences simul-taneously; this detectionwill increase the reliability of intracellularin situ detection and imaging of mature microRNA and avoid false-positive results. To our knowledge, no study has addressed the

issue of visualizing pre-miRNAs. Thus, an in situ detection protocolto monitor cytoplasmic pre-miRNAs is urgently needed. In partic-ular, real-time observation of the relative levels of changing pre-miRNAs and mature miRNAs in living cells is highly desirable.

In the present study, we report a novel gold nanoparticle (AuNP)and graphene oxide (GO)-based nanocomposite probe (AuNP/GOprobe) for simultaneously detecting and fluorescently imaging pre-and mature miRNA in living cells. The gold nanoflare probe utilizedin this composite nanoprobe was inspired by Mirkin's reports [28]describing a probe designed to detect pre-miRNA by releasing andswitching on Cy5-labelled flare-DNA, which is first quenched byAuNPs through the formation of DNA duplexes with thiol-labelled(HS)-DNA. Hybridization chain reaction (HCR) is one of the mostattractive enzyme-free signal amplification methods [29,30]. Li andco-authors first introduced the GO-loaded HCR (HCR/GO) systemfor mature miRNA detection in solution [31]. Subsequently, ourgroup expanded this signal amplification strategy to encompass thesimultaneous imaging of two types of mature miRNA in the sameliving cell [25]. In addition, utilizing the same HCR/GO-based sys-tem, combined with AuNPs-based nanoflare probes, we have suc-ceeded in sensitively detecting telomerase in living cells [32].Considering the feasibility of the AuNP/GO composite probe for thein situ detection of biomolecules, in this study we also used theHCR/GO system as the signal output platform for the intracellularmature miRNA to monitor the relative distribution of pre-miRNAand mature miRNA in the cytoplasm. Previously studies have re-ported that miRNA-21 typically functions as an anti-apoptotic fac-tor in cancer cells, and its mature form is up-regulated in variouscancers, such as breast, ovarian, and lung cancer, as well as glio-blastomas [33,34]. Additionally, the let-7 family has attractedmuchinterest because its family members are aberrantly expressed inhuman cancers [35]. Among them,mature let-7a is down-regulatedin many cancer types, such as Burkitt's lymphoma, gastric cancer,lung cancer, colon cancer and others [36e39]. Therefore, to visu-alize the relative expression and “up- or down-regulation” ofdifferent types of miRNA in various cancer or normal cells, weselected miRNA-21 and let-7a precursors and mature forms as thetargets.

2. Experimental section

2.1. Preparation of the AuNP probe and its responses to pre-miRNA-21 and mature miRNA-21

AuNPs (13 nm) were prepared as previously described [40]. Avolume of 100 mL of HS-DNA (1 OD) (HS-DNA-1 for miRNA-21 andHS-DNA-2 for let-7a) was treatedwith 1.5 mL TCEP (100mM) for 1 h.The activated HS-DNA was mixed with 40 mL of Flare-DNA (50 mM)(Flare-DNA-1 for miRNA-21 and Flare-DNA-2 for let-7a), heated to90 �C and incubated for 5min, then slowly cooled to room tem-perature, and held in the dark for at least 12 h to allow completehybridization. Subsequently, the obtained hybridized system wasmixed with 2mL of AuNPs. After incubation overnight at roomtemperature, 0.1mL of PBS solution containing 2M NaCl was addedto the mixture stepwise to the mixture to stabilize the probe andallowed to stand for 24 h. To remove excess DNA, the solution waspurified by centrifugation for 30min at 13500 rpm and washedtwice with PBS buffer. The AuNP probe was finally re-suspended in1mL of PBS buffer and stored at 4 �C until further use.

To confirm the presence of hybridized-DNA assembled on theAuNPs, a series of pre-miRNA-21 or mature miRNA-21 concentra-tions (0, 25 50,100,150, 200, 250, 300, 350, and 400 nM)wasmixedwith the AuNP probe (1.5 nM) in a 200-mL final reaction volume. Allreaction mixtures contained 4 U of RNase inhibitor. After incuba-tion at 37 �C for 6 h, the fluorescence intensities of the mixtures

M. Hong et al. / Analytica Chimica Acta 1021 (2018) 129e139 131

were determined.

2.2. Preparation of the HCR/GO system and its response to pre-miRNA-21 and mature miRNA-21

The HCR/GO system was prepared as previously described [32].First, the FAM-labelled hairpin DNA sequences (H1-DNA and H2-DNA for miRNA-21, H3-DNA and H4-DNA for let-7a) were sepa-rately heated at 75 �C for 10min and then cooled to room tem-perature for 1 h before use. Subsequently, GO (final concentration:25 mgmL�1) was added to the hairpin DNA (5 nM each) systems,and the final mixture were further incubated for 12 h to obtain theHCR/GO system. Then, a pre-miRNA-21 or mature miRNA-21 series(0, 5, 10, 20, 50,100,150, 200, 300, and 400 nM)wasmixedwith theHCR/GO probe in a final 200-mL reaction volume. All reactionmixtures contained 4 U of RNase inhibitor. After incubation at 37 �Cfor 6 h, the fluorescence signals were recorded on aspectrofluorometer.

2.3. Fluorescence detection of pre-miRNA-21 and mature miRNA-21with an AuNP/GO composite probe

Fluorescence detection of pre-miRNA-21 and mature miRNA-21with an AuNP/GO composite probe was performed using a HitachiF-7000 fluorospectrometer. Each reaction contained 200 mL of PBSwith the AuNP/GO composite probe (AuNPs 1.5 nM and GO25 mgmL�1) and different concentrations of pre-miRNA-21 andmature miRNA-21 (0, 50, 100, 200, 300, and 400 nM). All reactionmixtures contained 4 U of RNase inhibitor. Cy5 and FAM wereexcited at 630 and 488 nm, respectively. The assay systems wereincubated with the AuNP/GO composite probe for 6 h before thefluorescence spectra were recorded.

2.4. In situ imaging of pre-miRNA-21 and mature miRNA-21 inHepG2, HL-7702, MCF-7, HBL-100, HeLa, A549 and Caco-2 cells ordrug treated HeLa cells

In situ imaging of pre-miRNA-21 and mature miRNA-21 wasperformed with five cancer cell lines, specifically HeLa (humancervical cancer cells), A549 (human lung cancer cells), Caco-2(human colon cancer cells), HepG2 (human liver cancer cells),and MCF-7 cells (human breast cancer cells) as well as two normalhuman cell lines (HL-7702 liver cells and HBL-100 breast cells) byconfocal laser scanning microscopy (CLSM). After all cells (0.4mL,1� 106mL�1) were seeded into a 20-mm confocal dish for 24 h,AuNP probe (1.5 nM), GO probe (GO 25 mgmL�1), or AuNP/GO probe(AuNPs 1.5 nM and GO 25 mgmL�1) was added into each cell-adherent dish and incubated for 4 h at 37 �C. The cells wereexamined by CLSMwith different laser transmitters. Pre-miRNA-21was recorded as Cy5 in the red channel with excitation at 633 nmand mature miRNA-21 was recorded as FAM in the green channelwith excitation at 488 nm.

In the experiments showing changes in pre-miRNA-21 andmature miRNA-21 expression levels, two cancer cell lines (A549and HepG2) and one normal cell line (HL-7702) was treated withdifferent concentrations of (4-phenylazobenzoyl)propargylamine(PBPA, 2, 5 or 10 mgmL�1) for 40 h. Other steps were performed asdescribed above. Subsequently, the cells were examined by CLSM.

2.5. In situ imaging of pre-let-7a and mature let-7a in HepG2, HL-7702, MCF-7 and HBL-100 cells

In situ imaging of pre-let-7a and mature let-7a was performedby CLSM with the same method described above.

3. Results and discussion

3.1. Principle of the proposed method

Here, we designed an AuNP- and GO-based nanocompositeprobe to achieve simultaneous imaging of cytoplasmic pre-miRNAand mature miRNA. This proposed strategy includes two parts: andAuNP-based nanoflare probe designed for imaging pre-miRNA anda GO-based HCR/GO system designed for imaging mature miRNA;their underlying mechanisms are illustrated in Scheme 1.

The basic concept underlying AuNP-based nanoflare probes wasintroduced byMirkin et al. who designed competitive hybridizationbetween a long target sequence (such as mRNA) and a short flaresequence [28]. However, to our knowledge, this probe has notpreviously been applied tomonitor different forms of miRNA insideliving cells. Here, we used an AuNP probe to specifically and directlyidentify longer pre-miRNAs. Mature miRNA-21 has been identifiedas the only microRNA to be over-expressed in solid tumors of thelung, breast, stomach, prostate, colon, brain, head, neck, oesoph-agus, and pancreas [41]. Thus, monitoring pre-miRNA-21 andinhibiting the processing of pre-miRNA-21 to mature miRNA-21 isemerging as a novel and promising cancer therapeutic pathway. Inview of these facts, pre- and mature miRNA-21 were selected asassay targets in the present study.

3.2. Verification of the proposed pre-miRNA-21 sensing probe

Here, the nanoflare probe consists of AuNPs functionalized witha dense shell of Cy5-labelled oligonucleotide duplexes via Au-Sbond formation. The oligonucleotide duplexes are composed oftwo segments: a long thiol-labelled sequence (HS-DNA) and a shortCy5-terminated reporter sequence (Flare-DNA), which is comple-mentary to the HS-DNA. Characterization of the AuNPs and AuNPprobes is provided in the supporting information (Fig. S1). Thesurface coverage of the oligonucleotide duplex on AuNP wasdetermined in mercaptoethanol competition experiments. Aftercentrifugation, the fluorescence of the supernatant was determined(Fig. S2A). According to the standard curve constructed for thehybridized-DNA system (Fig. S2B), the number of hybridized-DNAsystems on each AuNP was approximately 85, which is slightlylower than the amounts previously reported for other Au nanoflareprobes (~95). This difference may be attributable to the larger sterichindrance of the long sequence of the oligonucleotide duplexdesigned for the pre-miRNA. In the presence of the pre-miRNAtarget, Flare-DNA is released from the Au nanoflare probe via thecompetitive hybridization of pre-miRNAwith HS-DNA. Consideringthe instability of miRNA, competitive hybridization was first per-formed using perfectly matched DNA sequences (pre-miR-21-DNA)for the flare duplexes to evaluate the feasibility of the nanoflareprobe for the detection of the long pre-miRNA-21 sequence. Asshown in Fig. S3, fluorescence signals from the nanoflare probeincreased linearly with increasing pre-miR-21-DNA concentrationin the range of 0e250 nM, suggesting that hybridization of theAuNP probe and DNA target leads to fluorescence recovery, and thefluorescence intensity is associated with the concentration of theDNA target. The concentration of the AuNP probe was optimized,and the results are shown in Fig. S4. A previous study reported thatDNA/RNA duplexes have higher affinities than DNA/DNA duplexes[15]. Thus, AuNP probes that have been validated to respond to pre-miR-21-DNA via DNA-DNA hybridization should also respond topre-miRNA-21 via DNA-RNA hybridization. The response of theAuNP probe to pre-miRNA-21 showed a similar result as theresponse to pre-miR-21-DNA, and good linearity was obtainedusing pre-miRNA-21 at concentrations ranging from 0 to 300 nM(Fig. 1). The detection limit of the method was 4.5 nM for pre-

Scheme 1. Schematic illustration of the AuNP/GO probe-based cytoplasmic analysis of pre- and mature miRNA.

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miRNA-21 based on 3s per slope.

3.3. Specificity of the proposed pre-miRNA-21 sensing probe

The specificity of the AuNP probes for their complementarytarget was also investigated in solution using targets containingone or three base pair mismatches at the same concentration. Forthe AuNP probe, the fluorescence intensity produced by the fullycomplementary target was apporximately 3-fold of that producedby targets containing three mismatches and 1.7-fold of that pro-duced by targets containing one mismatch (Fig. S5). These resultssuggest that the proposed pre-miRNA-21 detection approach pro-vides good sequence specificity and is useful for distinguishingsequences with a single base mismatch in complex biosystems.

In many cases, pre-miRNAwill be further processed into maturemiRNA [42,43]. As mature miRNA has the same sequence as onlypart of the pre-miRNA sequence, it is expected that the AuNP probedesigned here would only target the miRNA precursor, and themature miRNA would have a very low response or almost no

Fig. 1. (A) Fluorescence spectra of the AuNP probe (1.5 nM) and pre-miRNA-21 at 0, 25, 50,concentrations of pre-miRNA-21 and mature miRNA-21.

response to the probe. Mature miRNA-21 was examined for itsresponse to the AuNP probe. There was negligible signal in thepresence of mature miRNA-21 in the range of 0e400 nM comparedto background fluorescence levels (Fig. 1B).

3.4. Verification of the proposed mature miRNA-21 sensing probe

Next, we used a previously reported HCR/GO probe as a signaltransducer of miRNA-21 [29]. According to the concept of HCR, onemiR-21-DNA target yields multiple signal outputs, thus achievingsignal amplification for a low concentration of miR-21-DNA targets.This idea was tconfirmed by the results of gel electrophoresis(Fig. S6). Here, GO was used not only as a quencher of the fluo-rophore group-modified HCR system but also as a carrier of the HCRsystem into cells. The concentration of GO was optimized withfluorescence spectra (Fig. S7). The miR-21-DNA-triggered fluores-cence recovery effect was examined by laser confocal fluorescenceimaging (Fig. S8). As shown in Fig. 2A, the fluorescence intensity ofthe detection system increased with increasing concentration of

100, 150, 200, 250, 300, 350, and 400 nM (a to i). (B) Plot of fluorescence intensity vs.

M. Hong et al. / Analytica Chimica Acta 1021 (2018) 129e139 133

the mature miRNA-21 from 0 to 400 nM. The limit of detection was1.74 nM, and the linear relationship was within the concentrationrange of 0.1e10 nM (Fig. S9). These results suggest that the fluo-rescence signal quenched by GO is turned-on after the addition ofmature miRNA-21, and the fluorescence signal from the triggeredHCR system was immobilized on the GO.

3.5. Specificity of the proposed mature miRNA-21 sensing probe

Because mature miRNA-21 is part of pre-miRNA-21, it isnecessary to test the response of pre-miRNA-21 to the HCR/GOsystem. As shown in Fig. 2B, the HCR system designed for maturemiRNA-21 detection also responded to pre-miRNA-21, suggestingthat the fluorescence signal for intracellular mature miRNA-21obtained with the HCR/GO system using probes as described Liand co-authors [25] was partly derived from the presence of pre-miRNA-21. Fortunately, the HCR detection system produced a lowfluorescence signal when incubated with solution containing onlypre-miRNA-21 (in the range of 0e400 nM). These data clearlydemonstrate that although the designed HCR system has a betterresponse to mature miRNA-21, the response to pre-miRNA-21should not be neglected.

3.6. Specificity of the proposed nanocomposite probe

As previously mentioned, Li and co-authors did not consider theeffects of pre-miRNA-21 on the detection of mature miRNA-21 [25].To reduce this interference, due to the use of HCR/GO probes, herewe simultaneously introduced AuNP probes, specifically designedto combine pre-miRNA-21. Studies revealed an obvious Cy5 signalincrease when the AuNP/GO composite probes were incubatedwith different concentrations of pre-miRNA-21, whereas FAM sig-nals showed a slight increase, particularly at the low concentrationrange (Fig. 3A). In the presence of two coexisting probes, pre-miRNA-21 preferentially combines with the AuNP probe insteadof the HCR/GO probe. When the AuNP/GO composite probes wereused, we primarily considered the response of HCR/GO probes tomature miRNA-21 in the presence of pre-miRNA-21. Based on theseresults, we concluded that the fluorescence intensities of Cy5 andFAM mainly corresponded to their respective targets when theAuNP/GO composite probes were incubated with the mixture ofpre-miRNA-21 and mature miRNA-21. As shown in Fig. 3B, the Cy5fluorescence signal derived from the response to pre-miRNA-21

Fig. 2. (A) Fluorescence spectra of the HCR/GO probe (25 mgmL�1) and mature miRNA-21 forvs. concentrations of the pre-miRNA-21 and mature miRNA-21.

and the FAM fluorescence signal responded to mature miRNA-21based on the comparison of their fluorescence intensities, asshown in Figs. 1B and 2B. Additionally, to exclude the false-positivesignal of the AuNP/GO composite probe designed specifically forpre- and mature miRNA-21, we tested the response to pre-let-7aand mature let-7a. As shown in Fig. 3B, there was no notablefluorescence enhancement when pre-let-7a and mature let-7awere simultaneously incubated with non-complementary probesas negative controls. The designed probes exhibited good speci-ficity for their corresponding target miRNAs, and the fluorescencesignals generated from pre-miRNA-21 and mature miRNA-21 werenot influenced by pre-let-7a and mature let-7a. This method per-mits two forms of miRNAs to be monitored simultaneously, with noobvious cross-reactivity between targets and non-complementaryprobes. In addition, the stability of the AuNP/GO composite probewas tested in a previous study [32], thus excluding fluorescenceleakage by the probes.

3.7. Imaging capability of the proposed pre-miRNA-21 and maturemiRNA-21 sensing platform

Having demonstrated the feasibility of simultaneously detectingtwo forms of miRNA in solution, we next explored the potential useof two probes to visualize pre-miRNA and mature miRNA in situ inliving cells. To our knowledge, there are two different forms ofmiRNA in the cytoplasm of living cells: long pre-miRNA with ahairpin structure and short mature miRNA, and the latter isinvolved in RNA interference to target mRNA for degradation ortranscriptional repression [44,45]. First, we uses the designed AuNPprobe to monitor the expression of pre-miRNA-21 in four differentcancer cell lines, specifically HepG2, HeLa, A549, and Caco-2, andone human normal cell lines (HL-7702). The response of the probeto its target versus time (Fig. S10), revealed that the fluorescenceintensity plateaued at apporoximately 3 h. To achieve the bestimaging effects, all samples were observed after the cells wereincubated with the probes for 4 h. As shown in Fig. 4, the redfluorescence of Cy5-labelled Flare-DNA was located in the cyto-plasm. Pri-miRNA is processed in the cell nucleus to produce pre-miRNA, which is subsequently transported to the cytoplasm [6,7].Consequently, pre-miRNA exists in both the cell nucleus andcytoplasm. However, the AuNPs used here (~13 nm) only enter thecytoplasm via endocytosis and are unable to penetrate the cellnucleus [46]. Therefore, it is only possible tomonitor pre-miRNA-21

0, 5, 10, 20, 50, 100, 150, 200, 300, and 400 nM (a to i). (B) Plot of fluorescence intensity

Fig. 3. (A) Responses of the AuNP/GO composite probes to pre-miRNA-21 at different concentrations (0, 50, 100, 200, 300, and 400 nM). (B) Fluorescence intensities produced by themixture of pre-miRNA-21 and mature miRNA-21 or the mixture of pre-let-7a and mature let-7a with the AuNP/GO composite probes specifically designed according to the pre-miRNA-21 and mature miRNA-21 sequences.

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outside the cell nucleus. Additionally, pre-miRNA-21 exhibited highexpression not only in cancer cells but also in normal human cells.

Previously, we reported the feasibility of the HCR/GO system forimaging mature miRNA-21 in human breast cancer MCF-7 cells[25]. Similarly, the expression levels of mature miRNA-21 in fourdifferent cancer cells (HeLa, A549, Caco-2, and HepG2) were alsomonitored outside the cell nucleus using the GO probe (Fig. S11).The results showed the strong fluorescence of mature miRNA-21 inall cancer cells tested. In addition, according to some previous re-ports, pre-miRNA is highly-expressed in most normal somatic cells,but its corresponding mature miRNA is not readily observed[47,48]. Here, normal HL-7702 human liver cells were selecte to testthe expression of mature miRNA-21. As shown in Fig. S11, afterincubation for 4 h with the GO probe, confocal images of HL-7702only demonstrated low FAM signals assigned to mature miRNA-21. Based on the previous studies, this response should becommonly generated by pre-miRNA-21 and mature miRNA-21.Therefore, it is difficult to conclude whether mature miRNA-21only exists in the cytoplasm in the context of the HCR/GO system.However, obviously mature miRNA-21 showed lower expression inHL-7702 cells, consistent with the results of a previous study [25].

Fig. 4. Confocal images of HeLa, A549, Caco-2, HepG2, and HL-7702 cells after incubation wibright-field images (bottom line). Scale bars are 50 mm.

Little is known about the regulation of miRNA biogenesis innormal or tumor cell lines. Several studies have shown that in somecases, miRNAs are present at the precursor level but are not pro-cessed into the mature miRNA [8]. Therefore, the simultaneousmonitoring of pre-miRNA and mature miRNA is important toadvance bioresearch and clinical diagnosis. To our knowledge, nostudies have addressed the simultaneous imaging of pre-miRNAand mature miRNA within the same living cell. As describedabove, the designed AuNP and GO probes are useful for the imagingof cytoplasmic pre-miRNA and mature miRNA, respectively. Next,we further explored the mixture of AuNP and GO probes (togetherforming the AuNP/GO composite probe) to simultaneously monitorcytoplasmic pre-miRNA and mature miRNA expression levels insitu. As shown in Fig. 5, both strong green and red fluorescenceindicative of pre-miRNA-21 and mature miRNA-21, respectively,were observed under confocal microscopy in HepG2 and MCF-7 cells after incubation with the composite probe for 4 h. Thisfinding suggests that pre-miRNA-21 andmaturemiRNA-21 co-existin the cytoplasm of these two cancer cells. The same experimentswere repeated in three other cancer cell lines (HeLa, A549 andCaco-2, see Fig. S12) to demonstrate the applicability of the AuNP/

th AuNP probes: fluorescence field images (top line), and overlapping fluorescence and

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GO composite probe in various cell types, and similar results wereobtained.

As shown above, that the HCR/GO system designed for maturemiRNA-21 detection also responds to pre-miRNA-21 but with lowerfluorescence intensity. We were unclear whether this responsedisturbs our ability to monitor mature miRNA-21. When normalHL-7702 human liver cells and normal HBL-100 human breast cellswere used as models to simultaneously determine the expressionof pre-miRNA-21 and mature miRNA-21 in normal cells, only aslight green fluorescence signal indicative of mature miRNA-21 wasobserved after incubation with the AuNP/GO composite probe,while the red fluorescence signal indicative of pre-miRNA-21 wasstrong (Fig. 5). We conclude that miRNA-21 is primarily present atthe precursor level in human normal cells but is not processed or isonly slightly processed to mature miRNA-21 compared with theresults obtained in cancer cells (HepG2 and MCF-7); furthermore,the expression of mature miRNA-21 is up-regulated in cancer cells.Thus, mature miRNA-21 is a biomarker for cancer detection, diag-nosis, and prognosis. Taken together, the above data demonstratethat the AuNP/GO composite probe designed here distinguishes thecancer cells from normal somatic cells based on mature miRNAlevels. Importantly, the presence of the AuNP probe greatly reducedthe disturbance due to the presence of pre-miRNA-21 during the

Fig. 5. Confocal images of HepG2, HL-7702, MCF-7 and HBL-100 cells after incubation withwith excitation at 633 nm; mature miRNA-21 was recorded using FAM in the green channellegend, the reader is referred to the Web version of this article.)

imaging of mature miRNA-21 compared to that in HCR/GO-basedimaging alone.

3.8. MTT

The safety of the nanoprobes is the key for intracellular detec-tion of biomarkers. Nucleic acid functionalized AuNP probes andGO probes have shown negligible cytotoxicity individually [25,28].Apart from our previous work [32], the mixture of the two probeshas been used in another report on the microRNA detection andshowed satisfactory low cytotoxicity [49]. To further evaluate thecytotoxicity of the complex nanoprobes, here we performed anMTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bro-mide] assay in different cells. The results indicated that the AuNP/GO composite probe showed low cytotoxicity in living cells after 4 h(Fig. S13) and confirmed the utility of the composite probe forintracellular marker imaging.

3.9. Colocalization of pre-miRNA-21 and mature miRNA-21 in thecytoplasm

As shown in Fig. S14, the medium colocalization of pre-miRNA-21 and mature miRNA-21 in the cytoplasm was visualized in

AuNP/GO composite probes. Pre-miRNA-21 was recorded using Cy5 in the red channelwith excitation at 488 nm. (For interpretation of the references to colour in this figure

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magnified merged images by using the AuNP/GO composite probefor A549 cells. Pearson's and Manders coefficients were 0.16 and0.97, respectively, for the whole cell.

3.10. Localization of nanocomposite probe in the cell

In situ trackings of cytoplasmic pre-miRNA-21 and maturemiRNA-21 were evaluated based on the Cy5 and FAM fluorescencerecovered from AuNP/GO composite probes under CLSM. As shownin Fig. S15, Cy5 and FAM fluorescence intensities graduallyincreased in the cytoplasm with increasing incubation time andexhibited a time-dependent profile similar to the fluorescenceresponse of AuNP/GO composite probes to their targets. Addition-ally, Z-stack analysis of HeLa cells was performed to confirm thelocations of the probes in cells, and the merged fluorescence in-tensity of Cy5 and FAM initially increased and subsequentlydecreased (Fig. S16 and S17). These features indicated that theprobes were present inside the cells rather than merely absorbedon the membrane. Importantly, these images revealed the efficientcellular uptake and sensitive response of the AuNP/GO compositeprobes, which are prerequisites for pre- and mature-miRNA

Fig. 6. Confocal images of HepG2, HL-7702, MCF-7 and HBL-100 cells after incubation withexcitation at 633 nm; mature let-7a was recorded using FAM in the green channel with excitreader is referred to the Web version of this article.)

detection.

3.11. Imaging capability of the proposed pre-let-7a and mature let-7a sensing platform

Mature let-7a is typically down-regulated in many cancer types,such as liver and breast cancers [48,50]. To further observe thisphenomenon, we used the AuNP/GO composite probes designedhere to simultaneously observe the relative expression levels ofpre-let-7a and mature let-7a by CLSM. For the convenience ofcomparison, the two cancer cell lines used above to detect pre-miRNA-21 and mature miRNA-21, HepG2 and MCF-7 were alsoselected here, and normal HL-7702 and HBL-100 cells were used asthe control group. As shown in Fig. 6, strong Cy5 signals assigned topre-let-7a were observed not only in the two cancer cell lines butalso in the two normal cells, whereas the FAM signals assigned tomature let-7a were only observed in the two normal cells. Thisfinding suggests that pre-let-7a exists in all cell types but maturelet-7a shows “down-regulation” in cancer cells, consistent withprevious reports [45,51,52]. These results indicated that the AuNP/GO probes are also be useful as a signal readout platform for pre-

AuNP/GO composite probes. Pre-let-7a was recorded using Cy5 in the red channel withation at 488 nm. (For interpretation of the references to colour in this figure legend, the

Fig. 7. Confocal images of HepG2 and HL-7702 cells treated with different concentrations of PBPA. Pre-miRNA-21 was recorded using Cy5 in the red channel with excitation at633 nm; mature miRNA-21 was recorded using FAM in the green channel with excitation at 488 nm. Scale bars are 50 mm. (For interpretation of the references to colour in this figurelegend, the reader is referred to the Web version of this article.)

M. Hong et al. / Analytica Chimica Acta 1021 (2018) 129e139 137

let-7a and mature let-7a.

3.12. RT-PCR verification of the contents of pre-miRNA and maturemiRNA in different cells

RT-PCR is the standard technique used to detect the expressionof different forms of miRNA. Therefore, we used this technique toexamine the ability of the AuNP/GO-based method to assess theexpression levels of pre-miRNA-21, mature miRNA-21, pre-let-7aand mature let-7a. Fig. S18, S20 and S22 show that the expressionlevel of two miRNA precursors were high for all assayed cells. Incontrast, RT-PCR showed differences in the expression of maturemiRNA between the five cancer cell lines and two normal cell linesshown in Fig. S19, S21 and S23, similar to the results obtained bySCLM. These results indicate that the fluorescence intensity corre-lates well with tumor-related mature miRNA expression levels inliving cells.

3.13. In situ monitoring of the cytoplasmic pre-miRNA-21 andmature miRNA-21 after drug treatment

As reported above, the red and green fluorescence intensities ofthe AuNP/GO probes were dependent on the cytoplasmic contentsof pre-miRNA-21 and mature miRNA-21. Next, the AuNP/GO com-posite probes were further employed for in situ monitoring of thechanges in the cytoplasmic contents of pre-miRNA-21 and maturemiRNA-21 after drug treatment. (4-Phenylazobenzoyl)propargyl-amine (PBPA), a small-molecule inhibitor of miRNA-21 synthesizedaccording to the previous report [53] (Fig. S24 and S25), was used asa model drug to study the miRNA-21 inhibition in living cells. AfterHepG2, A549 and HL-7702 cells (0.5mL, 1� 106mL�1) were indi-vidually cultured with different concentrations of PBPA (2, 5, and10 mM) in a confocal dish for 40 h, 15 mL of the AuNP/GO compositeprobe was added to the dish and incubated for 4 h before confocal

images were obainted. Both fluorescence intensities correspondingto pre-miRNA-21 and mature miRNA-21 in the two cancer cell linesobviously decreased (Fig. 7 and S26) with increasing PBPA con-centrations. Similar observations were obtained for the normalliver cell line HL-7702, in which the expression of pre-miRNA-21was also inhibited by PBPA (Fig. 7). This result is consistent withthose of previous studies showing that PBPA targets the tran-scription of the miRNA-21 gene into pri-miRNA-21 therebydecreasing pre-miRNA-21 and mature miRNA-21 content accord-ingly [53]. RT-PCR further confirmed that the expression levels ofthe two forms of miRNA-21 decreased after PBPA treatment inHepG2, A549 and HL-7702 cells (Fig. S27-29). These findings sup-port the validity of the AuNP/GO probe-based method to assessmiRNA expression in living cells.

Additionally, the cytotoxicity of PBPA was tested to exclude theinfluence of the cell background fluorescence. Dose-responsestudies at concentrations of 0e40 mM for two cancer cell lines(HepG2 and A549, Fig. S30 and S31) using an MTT assay revealedthat the tested concentrations of PBPA used here (2, 5, and 10 mM)were far below the half-inhibitory concentration. In particular, asshown in Fig. S32, PBPA did not exhibit obvious cytotoxic effects at0e80 mM the human normal liver cell line (HL-7702). These resultsstrongly suggest that the AuNP/GO composite probes are useful forthe simultaneous in situ monitoring of the content changes of pre-miRNA-21 and mature miRNA-21 content changes in living cells.

4. Conclusions

In conclusion, we developed a method to simultaneouslyobserve the expression of pre-miRNA and mature miRNA in livingcells. In general, pre-miRNA is readily detectable both in normaland cancer cell lines and remains at relatively high levels. Incontrast, mature miRNA presents differential expression in normaland cancer cell lines; here, miRNA-21 was up-regulated and let-7a

M. Hong et al. / Analytica Chimica Acta 1021 (2018) 129e139138

was down-regulated. Our probe will be useful for assessing thedysregulation of mature miRNA and monitoring changes in thecytoplasmic content of pre-miRNA and mature miRNA in responseto inhibitors. More importantly, this method will effectively reducefalse-positive results for the detection of mature miRNA by intro-ducing the AuNP probes that specifically incorporate pre-miRNA.We anticipate that the ability to visualize pre-miRNAs andmature miRNAs in situ in living cells will accelerate the under-standing of the biological roles and localization of different forms ofmiRNA and provide a convenient tool for validating the effective-ness and target sites of novel small-molecule inhibitors of miRNA.Additionally, this work is important for intuitively elucidating therole of miRNAs in cellular processes and the associations betweenmiRNAs and cancer.

Acknowledgements

The present study was financially supported through grantsfrom the National Natural Science Foundation of China (21105042,91543206), the Science Foundation of China Postdoctor(2014M560572), and the Natural Science Foundation of ShandongProvince (ZR2015BM024). This work was also supported by the“TaieShan Scholar Research Fund of Shandong Province”.

Appendix A. Supplementary data

Supplementary data related to this article can be found athttps://doi.org/10.1016/j.aca.2018.03.010.

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In situ monitoring of cytoplasmic precursor and mature microRNA using gold nanoparticle and graphene oxide composite probes1. Introduction2. Experimental section2.1. Preparation of the AuNP probe and its responses to pre-miRNA-21 and mature miRNA-212.2. Preparation of the HCR/GO system and its response to pre-miRNA-21 and mature miRNA-212.3. Fluorescence detection of pre-miRNA-21 and mature miRNA-21 with an AuNP/GO composite probe2.4. In situ imaging of pre-miRNA-21 and mature miRNA-21 in HepG2, HL-7702, MCF-7, HBL-100, HeLa, A549 and Caco-2 cells or drug ...2.5. In situ imaging of pre-let-7a and mature let-7a in HepG2, HL-7702, MCF-7 and HBL-100 cells

3. Results and discussion3.1. Principle of the proposed method3.2. Verification of the proposed pre-miRNA-21 sensing probe3.3. Specificity of the proposed pre-miRNA-21 sensing probe3.4. Verification of the proposed mature miRNA-21 sensing probe3.5. Specificity of the proposed mature miRNA-21 sensing probe3.6. Specificity of the proposed nanocomposite probe3.7. Imaging capability of the proposed pre-miRNA-21 and mature miRNA-21 sensing platform3.8. MTT3.9. Colocalization of pre-miRNA-21 and mature miRNA-21 in the cytoplasm3.10. Localization of nanocomposite probe in the cell3.11. Imaging capability of the proposed pre-let-7a and mature let-7a sensing platform3.12. RT-PCR verification of the contents of pre-miRNA and mature miRNA in different cells3.13. In situ monitoring of the cytoplasmic pre-miRNA-21 and mature miRNA-21 after drug treatment

4. ConclusionsAcknowledgementsAppendix A. Supplementary dataReferences