Gold Nanoflower @Graphene Quantum Dots

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Comet-like Heterodimers “Gold Nanoflower @Graphene Quantum Dots” Probe with FRET “off” to DNA Circuit Signal “on” for Sensing and Imaging MicroRNA in Vitro and in Vivo Jiadi Sun, Fangchao Cui, Ruyuan Zhang, Zhixian Gao, Jian Ji, Yijing Ren, Fuwei Pi, Yinzhi Zhang, and Xiulan Sun Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.8b02854 • Publication Date (Web): 05 Sep 2018 Downloaded from http://pubs.acs.org on September 8, 2018

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Analytical Chemistry

Comet-like

Heterodimers

“Gold

Nanoflower

@Graphene Quantum Dots” Probe with FRET “off” to DNA Circuit Signal “on” for Sensing and Imaging MicroRNA in Vitro and in Vivo Jiadi Suna, Fangchao Cuia#, Ruyuan Zhanga#, Zhixian Gaob, Jian Jia, Yijing Rena, Fuwei Pia, Yinzhi Zhanga, Xiulan Sun a*

a

State Key Laboratory of Food Science and Technology, School of Food Science and Technology,

National Engineering Research Center for Functional Food, Synergetic Innovation Center of Food Safety and Quality Control, Jiangnan University, Wuxi, Jiangsu 214122, P.R. China b

Tianjin Institute of Health and Environmental Medicine, Tianjin Key Laboratory of Risk

Assessment and Control Technology for Environment and Food Safety, Tianjin 300050, China

Corresponding author* E-mail: [email protected]; Tel:+86-510-85912330

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Analytical Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ABSTRACT: Cardiovascular diseases have become the number one cause of death worldwide recently and the risk of getting cardiovascular diseases is doubled as the age increases. MicroRNA34a (miRNA-34a) as an important potential sensor of ageing and cellular senescence could be used in early diagnostics. Herein, a new ultrasensitive platform on the basis of the fluorescence resonance energy transfer (FRET) “off” to DNA circuit signal “on” principle was established, termed comet-like heterodimers gold nanoflower (AuNF) @ graphene quantum dots (GQDs) probe. We discussed that the distance of 4 nm between AuNF and GQDs would increase fluorescence quenching efficiency, and light up sensitivity after the probe combined with a target miRNA initiating DNA circuit strategy. The target miRNA-34a can be quantified down to 0.1 fM, which is about two orders of magnitude lower than the existing sensing protocols. Furthermore, we constructed the ageing myocardial cell and animal model, and the nanoprobe presented low cytotoxicity and satisfied signal imaging in vitro and in vivo. Significantly, this platform herein is envisioned to provide a reliable guidance for early diagnosing cardiovascular diseases and proposing therapeutic protocols. KEYWORDS ： cardiovascular diseases, miRNA-34a, comet-like heterodimers AuNF@GQDs nanoprobe, DNA circuit, low cytotoxicity

Cardiovascular diseases remain serious problems that lead to increasing morbidity and mortality globally, and ageing is the main reason of it, acute myocardial infarction would happen with increasing ageing.1,2 MicroRNA (miRNA) as endogenous noncoding RNA, has been reported that some of them play vital roles in ageing and regulating cardiovascular functions.3,4 For example, miRNA-22 accelerates senescence and activates cardiac fibroblasts in the aging heart.5 MiRNA-34a has also been proposed by Boon et al, who showed that its expression was influenced by the ageing heart, silencing or deleting miRNA-34a could lower cardiomyocyte death and help to recover myocardial function.6 Therefore, recognizing ageing-related biomarker miRNA-34a at the cellular level would provide new perspectives on the investigation of cardiovascular disease mechanisms, as 2 ACS Paragon Plus Environment

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well as early diagnostics. Traditional method of visualizing intracellular gene expression is fluorescence in situ hybridization (FISH), through which single-molecule transcripts can be detected using multiple fluorescently-labeled oligonucleotide probe.7,-8 However, hybridization of only one probe to miRNA yielded inadequate fluorescence is hard to quantify low abundance intracellular miRNA.9,10 Moreover, shortening the probes significantly reduces their matching discrimination and thus restricts their use for detecting miRNA sequences.11 Toehold-mediated strand displacement (TMSD) was capable of quantifying highly homologous sequences in complex environment, which includes two main procedures, one is the hybridization process between the toehold domain and the fuel strand, and the other is the branch migration process.12 TMSD has been proven to be successful in highly selective recognizing single-base variations because of its predictable thermodynamics and kinetics mechanism and it was a perfect candidate in differentiating several base mismatch sequences.13,14 Up to now, some works have been reported that the control of DNA strand displacement using a toehold was combined with a wide variety of DNA nanotechnologies and obtained impressive results, such as DNA circuits and nanomachines.15,16 However, there is still much work needed to achieve the necessary sensitivity for optical imaging of low nucleic acid molecules concentration in living cells.17 Many strategies based on TMSD for high-sensitivity analysis of miRNA in vitro have been proposed, such as enzymatic amplification, nonetheless, enzymes may cost a lot and would not be a best choice for amplification because of the complicated biological environment.18,19 In this case, an ultrasensitive, simplified and stable imaging based on nonenzymatic DNA catalytic strategies would have practical and clinical significance for the quantitative analysis of miRNA. Graphene quantum dots (GQDs) as a novel fluorescent probe have ignited increasing research interest, owing to their chemical inertness, low cytotoxicity and excellent biocompatibility.20,21 However we found that GQDs as bio-probes was limited because of their low quantum yields (QYs),22-24 and combining with the toehold-mediated DNA circuit signal amplification would help 3 ACS Paragon Plus Environment


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broaden bioapplication of GQDs, and obtain a sensitive signal response. Gold nanoflowers (AuNFs) are multibranched nanoparticles with sharp tips, edges and good colloid stability. Their unique anisotropic morphology gives rise to stronger fluorescence resonance energy transfer (FRET) behavior,25 and AuNFs could be regarded as a theoretical explanation for the turn-on signal readout model.26,27 Here we proposed a comet-like heterodimers AuNF@GQDs probe based on FRET “off” to DNA circuit amplification signal “on” platform for specific ultrasensitive recognition of miRNA34a in vitro and in vivo (Scheme 1). GQDs as the fluorescence emission group was first in the “off” state, when multibranched AuNF-ssDNA1 as a pre-auto fluorescence quencher was co-hybridized with GQDs-ssDNA2. The distance effect on FRET between AuNF and GQDs was discussed firstly, which would help enhance the quenching efficiency and provide conditions for the subsequent sensing. Then, the presence of target miRNA-34a triggered disassembly of the heterodimers AuNF@GQDs probe via the toehold-mediated DNA strand displacement reaction, leading to fluorescence signal recovery of the GQDs. And with addition of the Fuel DNA, the reaction initiated another round of branch migration reactions with miRNA-34a catalysting repeatedly, thus the platform strategy would yield a significantly amplified and higher sensitivity signal, which was suitable for low abundance biomolecules detections. The low cytoxicity probe was also applied to imaging of TGF-β1-treated living rat myocardial (H9C2) cells so that in situ observation of miRNA-34a expression, as a proof of concept, an animal model with ageing heart of 12-month C57BL/6J mice was utilized to ascertain the feasibility of the proposed approach. EXPERIMENTAL SECTION Materials and Reagents. All purified oligonucleotides (Table S1) were from Shanghai Sangon Biological Engineering Technology & Services Co. Ltd, microRNA-34a (miRNA-34a) was from TAKARA BIOTECHNOLOGY(DALIAN) CO., LTD. TGF-β1 was bought from Sigma-Aldrich, Inc. (St. Louis, MO, USA). Rat myocardial cells (H9C2) were from the Cell Bank of Chinese Academy of Sciences (Shanghai, China) and all the regents for cell experiments were from Gibco 4 ACS Paragon Plus Environment

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Invitrogen Corporation (CA, USA), and other chemicals were all from Sigma-Aldrich (Shanghai, China). Apparatus. The Flow cytometry experiment were conducted on Becton Dickinson FACS Calibur. IVIS SpectrumCT (PerkinElmer, U.S.) was used for animal use, besides these instruments, others we used were completely same as our previously reported work.28 Synthesis of Gold Nanoflowers (AuNFs) and Red Fluorescent Graphene Quantum Dots (RF-GQDs). AuNFs were prepared using a surfactant-free biocompatible method.29 First, the gold seed solution was prepared. Sodium citrate solution (1% [w/v], 2 mL) was added rapidly into boiling HAuCl4 (0.01% [w/v], 50 mL) solution with vigorous stirring. After 15 min of boiling while keeping the solution volume stable, the solution was cooled and dialyzed against water to remove some electrolyte ions, and then stored at 4 °C.30 For gold nanoflowers synthesis, the above citratestabilized seed solution (A520: 2.81, 100 µL) was added to HAuCl4 solution (0.25 mM, 10 mL) with HCl (1 M, 10 µL) at 25 °C with moderate stirring (700 rpm). Then, AgNO3 solution (100 µL) of different concentrations (0.5-4 mM) and ascorbic acid (25-250 µL) were injected quickly into the solution, which was then stirred for 30 s as its color rapidly turned from light red to blue or greenish-black. The reaction products were isolated by centrifugation at 5000 rpm for 15 min followed by removal of the supernatant. The precipitate was resuspended in Ultra-Pure DI, filtered by a 0.22 µm nitrocellulose membrane, and then stored at 4 °C for long-term storage. The RF-GQDs was synthesized by a facile bottom-up method.31 Briefly, 2.0 g L-glutamic acid was heated to 210 oC using a heating mantle. After the solid L-glutamic acid turned into liquid, the boiling colorless liquid changed to brown in 45 s, and the GQDs was formed. Then, 10.0 mL water was added into the solution and the reaction system stirred continuously for 30 min. When the solution cooled to room temperature, the solution was centrifuged at 10000 rpm for 30 min. The obtained RF-GQDs could be stored at room temperature for in vivo imaging. Modification of AuNFs and GQDs. For AuNFs modification, according to a previously reported work,28 the S-S bond of thiolated ssDNA1 was reduced by adding TCEP to ssDNA1 stock 5 ACS Paragon Plus Environment


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solutions with a molar ratio of 200:1, and incubating it without sunlight for 3 h. Excessive TCEP was removed with a G25 spin column obtained from GM Healthcare and the purified thiolated ssDNA1 was completed. 1X TBE and 0.1% SDS were mixed together and shaken gently, and HCl was used to adjust the mixture pH to 3.0. The purified thiolated ssDNA1 was then added to the above solution with a molar ratio of thiolated ssDNA1 to AuNFs of more than 200:1. Incubating them for 12 h, the supernatant was discarded after centrifugation,32 and the precipitate was suspended in PBS buffer for characterization by dynamic light scattering (DLS). The hydrodynamic size of the ssDNA1-modified AuNFs increased by the end of the process, which demonstrated that ssDNA1 was indeed successfully conjugated to the surface of the AuNFs and could be used for the subsequent assembly. The purchased GQD solution (1 mg/mL) were ultrasonicated for 10 min. EDC (16 mg) and NHS (16 mg) were added to the GQD suspension and the resulting mixture stirred for 5 h at 4 oC to activate the surface carboxylic groups. Subsequently, the activated GQDs and ssDNA2 (20 µL, 100 µM) were mixed together and allowed to react for 24 h at room temperature with continuous stirring. The GQDs-ssDNA2 bioconjugates were obtained by centrifugation and washing three times. And the RF-GODs were also used the above method to modified ssDNA2 on the surface. Preparation of the Comet-Like Heterodimers AuNF@GQDs Probe. AuNFs-ssDNA1 (500 µL) were treated with MCH (100 µM) in a 1:1500 molar ratio for 1 hour at room temperature. The resulting MCH-treated AuNFs-ssDNA1 was purified via centrifugation at 10000 rpm for 10 min and recovered in PBS (1×, 500 µL). This was then mixed with Linker DNA in a 1:200 molar ratio in the presence of NaCl (50 mM) and MgCl2 (2.5 mM) solutions. The solution was heated at 75 °C for 10 min and then slowly cooled to room temperature and left for 12 hours at 37 °C. Free DNA was removed via centrifugation twice at 10000 rpm for 10 min. The obtained AuNFs were recovered in PBS (1×, 500 µL) and mixed with GQDs-ssDNA2 at a molar ratio of 1:100 in the presence of NaCl (50 mM) and MgCl2 (2.5 mM) solutions. All the ssDNAs contained modified phosphorothioate bonds. The solution was incubated at 37 °C for 12 hours and then slowly cooled to room 6 ACS Paragon Plus Environment

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temperature. The AuNF@GQDs nanoassembly was purified twice via centrifugation at 10000 rpm for 10 min and resuspended in PBS (1×, 500 µL). Catalytic Disassembly of the Comet-Like Heterodimers AuNF@GQDs Probe. Catalytic disassembly was performed by incubation of the AuNF@GQDs nanoassembly with Fuel DNA (100 nM) and different concentrations of target miRNA-34a C’ at 37 °C for 12 hours. Samples were then diluted to 500 µL with 1×PBS and centrifuged once at 10000 rpm for 10 min. The fluorescence spectra of the disassembled GQDs in the supernatant were recorded. Cytotoxicity for Cell Labeling. The effect of nanoprobe transfer on cell survival was examined by evaluating apoptosis via fluorescence-activated cell-sorting analysis, including an apoptosis detection kit (Nanjing KeyGen Biotech Co., Ltd., Shanghai, China). Briefly, H9C2 cells in 12-well plates were harvested by trypsinization at 24 hours post infection with the comet-like heterodimers AuNF@GQDs probe. After neutralization by washing with culture media containing fetal calf serum (FBS) and PBS, the cells were resuspended in annexin V-FITC binding buffer (100 µL) and stained in the dark with apoptosis marker annexin V-FITC (4 µL) and necrosis marker propidium iodide (PI, 4 µL) for 15 min at room temperature as a control. Annexin V-FITC binding buffer (400 µL) was then added, and the samples were analyzed by flow cytometry. Cell Counting Kit-8 (CCK-8) colorimetric assay was used for cellular toxicity evaluation of the nanoprobe. H9C2 cells at 1.0 × 104 cells/well were seeded in a 96-well plate overnight. The cells were treated with a serial of the probe for 12 h. After that, the cells were treated with CCK-8 at 37 °C for 3 h, and absorption was measured from a microplate reader at 450 nm. Colocalization Assay. H9C2 cells were seeded in Petri dishes at a density of 1 x 105 cells/dish and cultured for 12 h before use. The cells treated with TGF-β1 (500 ng/mL) were then incubated with the nanoprobe, after which they were washed three times with PBS to remove the extracellular heterodimers AuNF@GQDs probe. The transfected cells were then treated with DNA indicator



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SYTO-59 (5 µM, excitation wavelength 622 nm, emission wavelength 645 nm, Molecular Probes) for 15 min to identify the nucleus. Intracellular miRNA Imaging using Confocal Fluorescence Microscopy. The treated H9C2 cells were seeded in Petri dishes at a density of 1 x 105 cells/dish and cultured for 12 h before use. The cells treated with TGF-β1 (500 ng/mL) were then incubated with the probe for 12 h, LysoTrack Green DND at the concentration of 50 nM was added and coincubated for 30 min before observation, after which the cells were washed three times with PBS to remove the extracellular AuNF@GQDs. Images of the treated cells were obtained using confocal fluorescence microscopy. To avoid unnecessary damage to the cells, the microscope shutter was opened only long enough to allow the laser to illuminate the bound cells while the fluorescent image was collected. The fluorescence of the probe and Lyso-Track Green DND was excited at 405 nm and 504 nm, respectively. Quantification of miRNA-34a in Living H9C2 Cells. H9C2 cells treated with TGF-β1 (500 ng/mL) were dynamically monitored for 36 h by Laser Confocal Fluorescence Microscopy, and the data was recorded every 6 h. For observing changes in intracellular miRNA-34a expression levels, H9C2 cells was treated with TGF-β1 (0, 5, 10, 50, 100, 500, 1000, 10000 ng/mL), following incubation with the AuNF@GQDs nanoprobe. The fluorescence of the probe was excited at 405 nm, and the signals were collected at 450-500 nm. Quantification of miRNAs using qRT-PCR. H9C2 cells were treated with TGF-β1 (0, 50, 500, 1000, 10000 ng/mL) for 24 h, and approximately 5.0 × 106 cells per sample were used for RNA isolation using the miRcute miRNA Isolation Kit (Tiangen, Beijing) according to the manufacturer’s protocol. RNA concentration was measured using onedrop K5500. Semiquantitative real-time PCR (qRT-PCR) was performed on total RNA extracts (200 ng) that had been polyadenylated and reverse transcribed into cDNA using first-strand cDNA via the miRcute Plus miRNA First-Strand cDNA Synthesis Kit (Tiangen, Beijing, China). The miRNA primers are listed in the Table S1. The miRcute Plus miRNA qPCR Detection Kit (SYBR green) (Tiangen, Beijing, 8 ACS Paragon Plus Environment

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China) was used for miRNA quantification and qRT-PCR was run on the PRISM 7900 Real-time PCR machine (ABI, America). Imaging in Vivo. The developed comet-like heterodimers AuNF@RF-GQDs probe was used for imaging of the heart in 12-month C57BL/6J mice. All procedures involving animals were fully compliant with the China Animal Use and Care Committee guidelines and were performed ethically and humanely. The heterodimers AuNF@RF-GQDs probe dispersed in PBS (100 µL) for in vivo fluorescence imaging was injected into the ventriculus sinister area and fluorescence images recorded on a IVIS SpectrumCT Small-animal Imaging System. The control group comprised 4 week old mice that underwent the same treatment as for the experimental group. In Vivo Toxicity Assessment. Blood serum was used for a biochemistry panel assay, 12-month C57BL/6J mice were randomly divided into experimental groups and control groups. The mice was injected with the materials or saline through ventriculus sinister area. Then some of them were killed 6 h, 36 h, 3 days and 7 days post injection respectively, for toxicity study, afterward the supernatant (serum) was taken for biochemistry analysis. Serologic parameters related to liver and kidney function were determined, such as albumin (ALB), the ratio of albumin and globulin (A/G), alanine transaminase (ALT), creatinine (CREA), globulin (GLOB), total protein (TP), aspartate transaminase (AST) and urea nitrogen (UREA). RESULTS AND DISCUSSION Principle of miRNA Sensing. Scheme 1 illustrates the working principle of the proposed method for high-sensitive miRNA detection with fluorescence resonance energy transfer (FRET) “off” to toehold-initiated DNA strand circuit signal “on”. Brifely, the system mainly consisted of a gold nanoflower (AuNF) and several graphene quantum dots (GQDs). The AuNF surface was surrounded by GQDs through the hybridization of two single strand DNAs (ssDNA 1 and ssDNA 2) with a DNA linker-strand (Linker DNA). GQDs were used as the donor, and their fluorescence intensity was quenched by nearby AuNF through FRET, indicating the fluorescence was in the “off” state. DNA fuel strand (Fuel DNA) was used to run the DNA circuit. Upon the addition of miRNA 9 ACS Paragon Plus Environment


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target, a set of reactions that drove the disassembly of multiple GQDs and AuNF was triggered through toehold-mediated DNA strand displacement reactions, yielding a significantly amplified fluorescence signal. Specifically, in the presence of miRNA, a sequence of this miRNA directly bound to the corresponding toehold 1 sequence of the Linker DNA, thus triggering a branch migration reaction and resulting in the displacement of the GQDs by the miRNA target. At this time, the intermediate product was generated, which was GQDs, and the fluorescence intensity was in the “on” state. As a result, the toehold 2 along the Linker DNA was exposed. This newly exposed sequence, along with Fuel DNA, was used to initiate another round of branch migration reactions, the Fuel DNA was hybridized onto the Linker DNA then, and the miRNA target was dissociated from the Linker DNA. The released miRNA target could further initiate another round of reactions. Therefore, toehold 1 and toehold 2 formed a circuit system. And in this work, unlike traditional probes, the miRNA target used as a catalyst to repeatedly unlock the DNA linkers to activate the probe, thus providing high sensitivity.


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Scheme 1 Illustration of the FRET “off” to DNA circuit signal “on” with the comet-like heterodimers AuNF@GQDs probe for miRNA-34a sensing. Surface Characterization and Spectral Analysis of AuNFs and GQDs Before and After Modification. Figure 1a shows a representative transmission electron microscopy (TEM) image of GQDs. These GQDs were well-dispersed and showed a relatively narrow size distribution ranging from 2 to 5.5 nm with an average diameter of 2.5 nm, with no obvious morphological or size changes even for the GQDs modified on the AuNF (Figure 1b). Figure 1c shows a typical highresolution TEM (HR-TEM) image of an individual GQD. The distinct crystal lattice indicates the crystallinity of the GQD and the lattice parameter of 0.2 nm represents the (110) lattice fringe of graphene. In addition, as shown in Figure 1d, AuNFs with high monodispersion and plasmon tunability were successfully synthesized using a surfactant-free method (Figure S1 and Figure S2a in the ESI†). 11 ACS Paragon Plus Environment


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The localized surface plasmon resonance (LSPR) absorbances of AuNFs and GQDs themselves were at 734 nm (Figure S3c, ESI†) and 327 nm (Figure S3b, ESI†), and only a lightly shift in the LSPR absorbance bands to 748 nm (Figure S3c, ESI†) and 333 nm (Figure S3b, ESI†) for AuNFs and GQDs after modification, respectively, indicating that covalent binding of the ssDNA1 at the “end” part of the AuNFs,33 and that a π–π* transition of C=C occurred in the GQDs after surface modification with ssDNA2.34 Fourier-transform infrared (FT-IR) spectroscopy was also used to characterize GQDs conjugation (Figure S3d, ESI†), with characteristic absorption peaks at 1634 cm-1 and 1405 cm-1, indicating the presence of amide vibrations, which confirmed the successful formation of the amide bond between the carboxylated GQDs and amine-modified ssDNA via the EDC/NHS method. Figure S2b (ESI†) and Figure S2c (ESI†) show the TEM images of AuNF and GQDs before and after ssDNA modification.


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Figure 1. Characterization of GQDs/AuNFs before and after ssDNA modification. Representative TEM images of the GQDs, (a). HR-TEM images of the GQDs around comet-like heterodimers AuNF@GQDs probe, (b). HR-TEM images of the GQDs, (c). Representative TEM images of AuNF, (d) and comet-like heterodimers AuNF@GQDs probe, (e). 15 % denatured Polyacrylamide gel electrophoresis results for AuNF/GQDs before and after conjugated with ssDNA, (f). UV-vis spectra of AuNF-ssDNA1 and GQDs-ssDNA2, (g). Emission spectra of GQDs-ssDNA2 (red) and absorption spectra of AuNF-ssDNA1 (black), (h). Emission spectra for GQDs before (black) and after conjugated with ssDNA2 (red), and of comet-like AuNF@GQDs probe (blue) , (i). To further verify the successful conjugation of thiol-modified ssDNA1/amine-modified ssDNA2 with AuNFs/GQDs, polyacrylamide gel electrophoresis (PAGE) was performed (Figure 1f). Appearance of a bright-visible band corresponded to ssDNA1 (lane 3) and ssDNA2 (lane 5); because PAGE is a molecular weight-dependent method, low-molecular-weight ssDNA migrated much further compared with AuNFs-ssDNA1 (lane 2) and GQDs-ssDNA2 conjugates (lane 4). Comet-Like Heterodimers AuNF@GQDs Probe Based on FRET “off” to ToeholdMediated DNA Circuit Signal “on” Design. As shown in Figure 1e, the comet-like heterodimers AuNF@GQDs probe was sucessfully constructed based on DNA hybridization. The UV-vis spectra of AuNF-ssDNA1 and GQDs-ssDNA2 showed remarkable optical differences (Figure 1g). The small distance and the spectra overlap between emission band and the plasmon band are the key factors, which yields a fast nonradiative energy transfer from the excited dipole to the metal.35 In this study, the FRET occurred due to spectral overlap between the emission band of GQDs-ssDNA2 and the absorption band of AuNF-ssDNA1 (Figure 1h), the space between the GQDs and AuNF was approximately 4 nm after hybridization, and the fluorescence of GQDs was in “off” state (Figure 1i). As was discussed in Figure 2, the multibranched AuNFs structure had increased the quenching sites, and GQDs were almost conjugated with the angles of the AuNF around, and there existed the strongest quenching efficiency in the three angles of heterodimers AuNF@GQDs probe. Figure 2 (d)-(i) showed the calculated local field intensities distributed for bare AuNF@GQDs 13 ACS Paragon Plus Environment


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structures with 4 and 10 nm gap between AuNF and GQDs, respectively. With the integration of a gap in (a) was E-field 232181 V/m for 4 nm, and 231944 V/m for 10 nm, (b) was E-field 172245 V/m for 4 nm, and 158683 V/m for 10 nm, (c) was E-field 145862 V/m for 4 nm, and 144329 V/m for 10 nm. Therefore, we assumpted that nonradiative energy transfer predominates at short distances of 4 nm, which cut down the response time and increasing the quenching efficiency, both of which improved detection sensitivity after fluorescence recovery of GQDs. (a)

(b) 2

(c)

3 2 1 3

1

3 2 1

(d)

(e)

(g)

(h)

(f)

(i)

Figure 2. Representative characteristics of the comet-like heterodimers AuNF@GQDs probe. TEM images of three angles of AuNF combined with GQDs, (a) (b) (c). Surface electrical field distribution maps for AuNF@GQDs structures with different gaps: 4 nm (d) and 10 nm (g) between AuNF and GQDs in the structure of (a). 4 nm (e) and 10 nm (h) between AuNF and GQDs in the structure of (b). 4 nm (f) and 10 nm (i) between AuNF and GQDs in the structure of (c). 14 ACS Paragon Plus Environment

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Stability and Cytotoxicity of the Comet-Like Heterodimers AuNF@GQDs Probe. Prior to conducting intracellular investigations, the heterodimers AuNF@GQDs probe with modified phosphorothioate bonds in the DNA skeleton according to the previously work28 was found to display significant stability, including resistance to different ionic strengths, pH conditions, cell culture medium with 10% fetal bovine serum, enzymatic lysis, and GSH, in the case of the control nanoprobe (the probe with non-specific sequence), no change in the absorbance and fluorescence signals were found. But, the nanoprobe with specific modification showed increasing fluorescence signal in TGF-β1 treated H9C2 cell lysis (Figure S5 and Figure S6 in the ESI†). CCK-8 analysis was proposed to investigate potential adverse effects of the heterodimers AuNF@GQDs probe upon internalization by H9C2 cells for 24 h. Results showed that the cells still exhibited high viability (Figure S7a, ESI†) with increasing concentration of the probe: from 0.01 to 0.2 mg/mL, cell viability increased from 86.48 % to 105.30 %, respectively (Figure S7c, ESI†). In addition, apoptosis analysis was performed by flow cytometry for the heterodimers AuNF@GQDsinternalized cells (Figure S7b, ESI†), using apoptosis marker Annexin V-FITC (2.17 ± 1.95, 1.34 ± 1.20, 1.74 ± 1.58%) and necrosis marker PI (0.75 ± 0.68, 1.24 ± 1.11, 1.62 ± 1.45%). Together, the results suggested that the comet-like heterodimers AuNF@GQDs probe had no significant effect on cell viability. Comet-Like Heterodimers AuNF@GQDs Probe Based FRET “off” to DNA Circuit Signal “on” for Sensing miRNA-34a in Vitro. Disassembly of the comet-like heterodimers AuNF@GQDs probe resulted in fluorecence recovery when the target miRNA affected the toeholdmediated DNA displacement reaction of GQDs (Figure. S8, ESI†). GQDs fluorescence intensity was enhanced 4.29-, 12.2-, 28.5- and 42.84-fold by catalytic disassembly at miRNA-34a/Linker DNA (C’/L) molar ratios of 0.001, 0.004, 0.012, 0.03 and 0.05 respectively, revealing the high responsivity of the heterodimers AuNF@GQDs probe to low target concentrations (Figure S8a and Figure S8b). In contrast, activation of GQDs fluorescence intensity in the absence of Fuel DNA was only detected at C’/L molar ratios above 0.15 (Figure S8c and Figure S8d in the ESI†). In addition, 15 ACS Paragon Plus Environment


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the selective performance of the comet-like heterodimers AuNF@GQDs probe was evaluated, we found that the highest fluorescence signal was produced by the target miRNA-34a, compared to other mismatched sequences (Figure S9, ESI†), manifested the proposed method was specific enough to identify miRNA, even distinguishing at one nucleotide mismatch level. It was found that the increase in value of fluorescence intensity with increasing concentrations of miRNA-34a (Figure 3a), and a near-linear relationship was obtained between 0.4 - 4 fM (Figure 3b). The detection limit of this protocol was 0.1 fM, as represented by the equation: Y= (2.13×102) x + 1963, R2 = 0.9921

Figure 3. Heterodimers AuNF@GQDs probe for sensing miRNA-34a in vitro: Fluorescence spectra in response to the different concentrations of miRNA-34a (0, 0.15, 0.2, 0.4, 0.8, 1.6, 2, 4, 8 fM), (a). The fluorescence intensity was dependent on the concentration of miRNA-34a (0, 0.15, 0.2, 0.4, 0.8, 1.6, 2, 4, 8 fM), the linear range was from 0.4 to 4 fM (inset), (b). Fluorescence Characterization of the Comet-Like Heterodimers AuNF@GQDs Probe in Living Cells. TGF-β1 as one of the pleiotropic cytokines is a prevalent isoform among the TGF-β, and is upregulated early in myocardial damage.36,37 In this work, we used TGF-β1 to treat H9C2 cells, so that ageing rat myocardial cell model was sucessfully constructed, and prominent fluorescence signal was identified with a Laser Confocal Microscope when the heterodimers probe 16 ACS Paragon Plus Environment

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internalized. SYTO-59 red fluorescence dye was used as a marker for the colocalization assay. Blue fluorescence signal indicated sensitive detection of intracellular miRNA-34a in ageing myocardial cells through heterodimers AuNF@GQDs probe (Figure 4). This observation comfirmed the theory that the miRNA-34a exists in cytoplasm.38 The importance of Fuel DNA was also investigated, with results showing that in the Figure S10 (ESI†) and Figure S11 (ESI†).

Figure 4. Fluorescence images of H9C2 cells with comet-like heterodimers AuNF@GQDs probe stimulated by TGF-β1 for colocalization assay (Laser Confocal Fluorescence Microscope, red field, SYTO-59 red, excitation: 622 nm; blue field, excitation: 405 nm). Previous studies say that nanoparticles are prone to localize in subcellular compartments such as the cytosol, endosomes and lysosomes, the subcellular location of comet-like heterodimers AuNF@GQDs probe is also of crucial importance for miRNA-34a detection, because the nanoprobe need to arrive at the target site in the cell interior. Herein the distribution of nanoprobe was investigated. To trace the lysosomes, Lysotracker was used, and the position of the nanoprobe and labeled lysosomes were visualized by Laser Confocal Fluorescence Microscopy. As was shown in Figure S12 (ESI†), most heterodimers AuNF@GQDs probe transfected in H9C2 cells treated by TGF-β1 in 24 h and 36 h, respectively, could escape from lysosome and release to the cytoplasm for 17 ACS Paragon Plus Environment


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miRNA-34a detection, and few overlay dots we could observe. Human hepatocellular carcinoma (HepG2) cells with low miRNA-34a expression was used as a control group. Figure S13 (ESI†) showed that the developed nanoprobe was stable and would not be digested by lysosomes, because little blue fluorescent signal could be observed and the developed probe could be exploited for efficiently measuring miRNA. Detection of miRNA-34a Expression Level using Heterodimers AuNF@GQDs in Living Cells. The time-dependant on the intracellular miRNA-34a detection was firstly investigated. The intracellular blue fluorescence signal increased, and it remained stable at 24 h, indicating that intracellular miRNA-34a detection had reached saturation (Figure S14 and Figure S15 in the ESI†). Therefore, different concentrations of TGF-β1 (0, 5, 10, 50, 100, 500, 1000, 10000 ng/mL) to induce various miRNA-34a expression levels were used in conjunction with the probe over 24 h. The intracellular fluorescence intensity increased as increasing concentrations of TGF-β1 induced greater miRNA-34a expression (Figure 5 and Figure S16 (ESI†)), which verified that the nanoprobe could predict cell senescence.


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Figure 5. Dose-dependent of miRNA-34a expression in H9C2 cells with comet-like heterodimers AuNF@GQDs probe treated with different concentrations of TGF-β1 (0, 5, 10, 50, 100, 500, 1000 and 10000 ng/mL) using Laser Scanning Confocal Microscope. Scale bar: 50 µm. In the concentration range of 5-500 ng/mL, the fluorescence intensity dependent on the logarithm of the concentration of TGF-β1, the equation was y = 1.37 x + 1.04, R2 = 0.9330. To verify the reliability of the proposed strategy for sensing miRNA-34a expression in TGF-β1 treating H9C2 cells, the miRNA-34a in cell extractions was measured. The cell extractions were processed by a commercial miRNA extraction kit after cell counting and were measured simultaneously using the probe method and also using a commercial kit based on quantitative Real Time-Polymerase Chain Reaction (qRT-PCR). As shown in Figure S17 (ESI†), intracellular miRNA-34a expression levels increased with the increasing TGF-β1 treatment, in other words, the gradual changes in intracellular fluorescence signals with increasing concentrations of TGF-β1 19 ACS Paragon Plus Environment


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treatment indeed matched well with the increasing trend of miRNA-34a expression obtained in our qRT-PCR analysis. These results indicated that the probe was able to potentially be applied for miRNA-34a detection in the ageing heart for clinical diagnosis. The applicability of the heterodimers probe in diverse cell lines was measured using human breast cancer cells (MCF-7) and human hepatocellular carcinoma (HepG2) cells. A stronger blue fluorescence was viewed in H9C2 cells compared with MCF-7 and HepG2 cells (Figure S18, ESI†), which indicated higher miRNA-34a expression in the H9C2 cells compared with MCF-7 and HepG2 cells, consistent with the higher expression of miRNA-34a in ageing myocardial cells. MiRNA-34a Expression Level Sensing with Heterodimers AuNF@RF-GQDs for Animal Use. The red fluorescent GQDs (RF-GQDs) would be a promising candidate for in vivo fluorescence imaging.39,40 In this paper, the RF-GQDs was synthesized, whose emission band was at 608 nm, and the procedure of the comet-like heterodimers AuNF@RF-GQDs probe construction was the same as the previous one (Figure S19). Figure 6a showed time-dependent in vivo optical images after the injection of the heterodimers AuNF@RF-GQDs probe into the heart of 12-month C57BL/6J mice. Because of the ageing nature of the mice hearts, the fluorescent signal could be observed, and the red fluorescent value increased gradually within 6-72 h postinjection, after that, the fluorecent signal decreased until disappeared. To evaluate the biodistribution of GQDs after use of the probe for sensing miRNA-34a expression in the mice hearts, ex vivo images of isolated organs were observed by the optical imaging system as well. The average fluorescence intensity of RF-GQDs in most of the mice organs rapidly increased within 6-72 h postinjection, reached a maximum value at 168 h (7 days) postinjection, then decreased over time (Figure 6b and Figure 6d). These results indicated that RFGQDs were distributed over the entire body through systemic circulation and concentrated in different organs up to 168 h (7 days) postinjection.41-43 However, fluorescence intensity in the heart at 168 h postinjection was lower than those organs at 72 h postinjection, which was the same as in vivo result. Especially, strong fluorescence intensity in the liver and kidney was observed, revealing 20 ACS Paragon Plus Environment

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accumulation of RF-GQDs in these organs (Figure 6d), the fluorescence intensity disappeared at 336 h (14 days), which was likely due to excretion of RF-GQDs. In addition, the major organs from mice treated with heterodimers AuNF@RF-GQDs probe were weighed (Figure 6c). Results indicated that retention was higher in the liver than in other organs, most likely due to the size of the AuNFs,44 and investigations indicated the excellent biocompatibility of the smaller RF-GQDs enabled fast clearance from the kidneys.45 However, the current, albeit limited, data suggests that gold nanoparticles are not toxic in the long term. For instance, Hainfeld and co-workers evaluated gold nanomaterials of different sizes (4, 13 and 100 nm) for their toxicity, with no apparent adverse effect being observed over 6 months. Studies from other groups have indicated that gold nanoparticles tend to accumulate in major organs over the study time-period without being effectively excreted.46

Figure 6. In vivo imaging. Real-time in vivo fluorescence images obtained after the ventriculus sinister injection of the comet-like heterodimers AuNF@RF-GQDs probe in 12-month C57BL/6J mice at different time points, (a). Ex vivo images of mouse isolated organs (from top to bottom: heart, liver, spleen, lung, kidneys) after injection, (b). The weight index of main organs (heart, liver, spleen, lung, kidneys) collected at various time points from mice injected with the probe compared to the control group (treated with saline solution, dotted lines), (c). Average fluorescence intensities 21 ACS Paragon Plus Environment


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of mouse isolate organs (heart, liver, spleen, lung, kidneys) at various time points after ventriculus sinister injection, (N=5) (d). So, a further identification on any potential toxic effect of the comet-like heterodimers AuNF@GQDs probe on the treated mice was studied, and a serum biochemistry analysis was carried out. Blood was collected from the novel probe and saline (control) treated mice at 6 h, 36 h, 3 days and 7 days of observation. The indicators of kidney function such as creatinine (CREA) and urea nitrogen (UREA) were also normal (Figure S20, ESI†). The hepatic indicators including albumin (ALB), globulin (GLOB), the ratio of albumin and globulin (A/G), alanine transaminase (ALT), total protein (TP), aspartate transaminase (AST) fluctua ted compared with the control groups, but they still remained within the normal ranges and were similar to values of control group (treated with saline solution) after 7 days injection. The results of blood biochemistry suggested no adverse effects of the AuNF@GQDs probe and the survived mice showed no damage from the materials, which was in favor of bioapplication. CONCLUSIONS In summary, a miRNA-triggered comet-like heterodimers AuNF@GQDs probe disassembly provided a round of branch migration reactions for sensing miRNA-34a in vitro and in vivo, which was high sensitive with a detection limit of 0.1 fM. The outstanding proposal was on account of the characteristic FRET “off” to DNA circuit signal “on”, respectively, the distance effect on FRET between AuNF and GQDs was discussed with surface electrical feld distribution, and 4 nm was better. The quantitative evaluation of intracellular miRNA-34a expression upon TGF-β1 treatment demonstrated its practicability in evaluating the ageing level of myocardial cells, as verified by comparison with the qRT-PCR measurement. Additionally, the comet-like heterodimers AuNF@GQDs probe showed no apparent toxicity to mice as evidenced by biochemical analysis, and exhibited excellent applicability in in vivo imaging of miRNA-34a. Therefore, this proposed monitoring strategy is highly promising, and has the potential to provide a versatile new technology for early detection of ageing heart biomarkers at low levels of their expressions. 22 ACS Paragon Plus Environment

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ASSOCIATED CONTENT Supporting Information Additional characterization data of TEM, UV-vis spectrum, emission spectra, DLS, Laser Scanning Confocal Microscope tests are presented. Table S1 List of oligonucleotides and miRNA sequences.

AUTHOR INFORMATION Corresponding Author *Xiulan Sun (E-mail: [email protected]) Author Contributions Jiadi Sun and Xiulan Sun did the detailed research. Fangchao Cui synthesized red fluorescence GQDs, Ruyuan Zhang did some cell experiment, Zhixian Gao provided some clinical instructions about myocardial experiment on animals. Jian Ji did some data analysis, Yijing Ren did some nanoparticle synthesis, Fuwei Pi and Yinzhi Zhang gave some suggestions about data processing. Notes The authors declare no competing financial interest.

ACKNOWLEDGEMENTS This work was supported by the National Natural Science Foundation of China (No. 31772069), Postgraduate Research & Practice Innovation Program of Jiangsu Province (KYCX17_1404), Synergetic Innovation Center of Food Safety and Quality Control, and the Priority Academic Program Development of Jiangsu Higher Education Institutions. Jiadi Sun thank the financial support of China Scholarship Council fellowship (No. 201706790045), and thank Prof. Xiuping Yan, School of Food Science and Technology, Jiangnan University, for experimental design of nanoprobe in vitro and in vivo bio-imaging instruction. 23 ACS Paragon Plus Environment


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TOC


Gold Nanoflower @Graphene Quantum Dots

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