An Ultrasensitive âFRET-SEFâ Probe for Sensing and Imaging

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An Ultrasensitive “FRET-SEF” Probe for Sensing and Imaging MicroRNAs in Living Cells Based on Gold Nano-Conjugates Jiadi Sun, Fuwei Pi, Jian Ji, Hongtao Lei, Zhixian Gao, Yinzhi Zhang, Jean de Dieu Habimana, Zaijun Li, and Xiulan Sun Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.7b04051 • Publication Date (Web): 08 Jan 2018 Downloaded from http://pubs.acs.org on January 8, 2018

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Analytical Chemistry

An Ultrasensitive “FRET-SEF” Probe for Sensing and Imaging MicroRNAs in Living Cells Based on Gold Nano-Conjugates Jiadi Suna#, Fuwei Pia#, Jian Jia, Hongtao Leib, Zhixian Gaoc, Yinzhi Zhanga, Jean de Dieu Habimanaa, Zaijun Lid, Xiulan Suna*

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

Guangdong Provincial Key Laboratory of Food Quality and Safety, South China Agricultural

University, Guangzhou 510642, Guangdong Province, P.R. China c

Institute of Hygienic and Environmental Medicine, Tianjin 300050, P.R. China

d

School of Chemical and Material Engineering, Jiangnan University, Wuxi 214122, P.R. China

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

ACS Paragon Plus Environment

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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

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ABSTRACT：：MicroRNAs (miRNAs), a kind of single-stranded small RNA molecules, play significant roles in the physiological and pathological processes of human beings. Currently, miRNAs had been demonstrated as important biomarkers critically related to many diseases and life nature，including several cancers and cell senescence. It is valuable to establish sensitive assays for monitoring the levels of intracellular up-regulated/down-regulated miRNA expression, which would contribute to the early prediction of the tumor risk and cardiovascular disease. Here, an oriented gold nanocross (AuNC)-decorated gold nanorod (AuNR) probe with “OFF-enhanced ON” fluorescence switching was developed based on fluorescence resonance energy transfer and surface enhanced fluorescence (FRET-SEF) principle. The nanoprobe was used to specifically detect miRNA in vitro, which gave two linear responses represented by the equation (F=1830.32logC+6349.27, R2=0.9901 and F=244.41logC+1916.10, R2=0.9984, respectively), along with a detection limit of 0.5 aM and 0.03 fM, respectively. Furthermore, our nanoprobe was used to dynamically monitor the expression of intracellular up-regulated miRNA-34a from the HepG2 and H9C2 cells stimulated by AFB1 and TGF-β1, and the experimental results showed that the newly probe not only could be used to quantitively evaluate miRNA oncogene in vitro, but also enabled tracking and imaging of miRNAs in living cells.

KEYWORDS：： AuNC-decorated AuNR nanoprobe, intracellular miRNA expression，OFFenhanced ON, FRET, SEF, living cells MicroRNAs (miRNAs), a class of short, endogenous, non-protein-coding single-stranded RNA molecules

with

length

from

18

to

24

nucleotides


(nt),

hold

the

ability

to

2

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modulate gene expression at post-transcription level in various of animals, plants and viruses.1-3 Currently, extensive investigations have identified that miRNAs play crucial roles in various biological processes, including cell proliferation, immune cells/systems development, repression, and apoptosis, as well as human tumor cell expressions.4,5 Furthermore, several miRNAs have emerged as important gene regulators being critically involved in carcinogenesis and cancer chemoprevention, which are commonly considered as biomarkers and therapeutic targets in cancer treatment.6-8 For example, several studies have identified miRNA-17∼92 and its paralog miRNA-106a-363 that are upregulated by estrogen in MCF-7 cells and functioned as negative regulators

of

the

ER

receptor

and

its

coactivator

AIB1,9

miRNA-21,

and

let-

7,10 while overexpression of miRNA-122 suppressed human hepatocellular carcinoma (HCC) growth and migration both in vitro and in vivo, and vice versa.11 Northern blotting and DNA microarray hybridization are two conventional strategies for profiling miRNA expression, while the low sensitivity and poor specificity largely limit both applications in-clinical settings.12,13 Another successful strategy for sensing miRNA is reverse transcription polymerase chain reaction (RT-PCR).14,15 Although RT-PCR technique is highly sensitive, the primers applied in most PCR protocol are similar to miRNAs in length, which means that very short primers would be needed for assay design, and shorter primers are typically not useful, as their low duplex melting temperature with the miRNA can introduce signal bias. 14,16 In addition, RT-PCR faces a risk of cross contamination arising from targetbased amplification. Actually, biosensing strategies, including Surface-enhanced Raman scattering (SERS),17-19electrochemical,20 microfluidic immunoarray

21

and fluorescent assays

were prospective methods.22-24 He et al.25 proposed a novel switchable DNA hydrogel SERS platform through release of Raman reporter toluidine blue (TB) for sensitive detection of


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miRNA-155 with DSN enzyme cleaved amplifying strategy, and this way not only avoided labeling signal molecule but also improved the sensitivity of miRNA detection due to immobilization of abundant TB. Miao et al.26 used a novel redox and catalytic “all-in-one” mechanism with an iridium(III) complexes as a catalyst for electrochemical miRNA-21 detection, which could selectively interact with G-quadruplex DNA and obtained a low detection limit of 1.6 fM. Du et al.27 described a microfluidic sample preparation multiplexer and assay procedure to improve amplification-free detection of Ebola virus RNA from blood, which could run up to 80 assays in parallel using a pneumatic multiplexing architecture, and the strategy would be an important step toward a useful POC device and assay. However, such methods require samples to be extracted from cells or in the blood, and considerable efforts need to be directed toward developing methods to in situ identify and quantify miRNAs. Recently, many efforts have been made to develop in situ RNA fluorescence analysis strategy using DNA-templated, even sensing diverse biological samples. Ma et al.28 developed a universal platform using DNA-programmed gold nanorod (AuNR) dimer upconversion nanoparticles (UCNP) core-satellite assemblies as SERS- and luminescence based probes for the simultaneous in situ quantification of miRNA and telomerase, which the limit of detection (LOD) for intracellular miRNA was determined to be 0.011 amol/ng RNA and 3.2 × 10-13 IU for intracellular telomerase. Yang et al.29 used bicolor fluorescent nanoprobe (Cy3 and Cy5) to dynamically monitor the conversion process of alcohol-induced fatty liver to steatohepatitis in vivo through simultaneous imaging of microRNA-155 and osteopontin mRNA. These methods have implied that fluorescence imaging for biomarker diagnostic provides a new way to obtain reliable information for early warning and treatment of corresponding diseases, and visualization


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in cancer cells or tissues is a trend, therefore, there is an urgent need to develop more stable optical output signal, less toxicity probe in bio-applications. Fluorescence resonance energy transfer (FRET), in which the electronic excitation energy of an energy donor chromophore is transferred to an energy acceptor in the presence of a target, possesses high prospects in miRNA detection due to its sensitivity and simplicity, which could advance many homogeneous and sensitive nucleic acid detections.30-32 He et al.33 developed a class of DNA-templated gold nanoparticle (GNP)-quantum dot (QD) assembly-based probes for catalytic imaging of cancer-related miRNA in living cells through FRET, and the novelty strategy could be used for high-sensitive imaging of low-abundance nucleic acid biomarkers that are difficult to tackle using conventional probes. Liu et al.34 investigated a novel strategy for the detection of multiplexed miRNAs miRNA-155, miRNA-182, and miRNA-197, which are significant for the early diagnosis of lung cancer, based on FRET from one nucleic acid stain TOTO-1 to three different organic dyes (Cy3, Cy3.5, and Cy5), using a single excitation wavelength with a sophisticated spectral crosstalk correction. However, the traditional FRET probes are exposed to analytes, the separation between the donor and quencher/acceptor leads to readable signals, the single fluorescent signal turn-on can only provide limited information, which is sometimes insufficient for accurate detection.35 Therefore, the two different morphology nanomaterials were used to be as FRET pairs, which would be helpful in improving detecting sensitivity and expanding sensing range. As is known to all, well-designed nanoconjugates with regular geometry shapes hold strong optical activity. Metallic nanomaterials, such as AuNRs and AuNCs, influence the quantum yield and lifetime of adjacent fluorophores in a manner dependent on their nanostructural properties36 and they exhibit transverse and longitudinal surface plasmon resonance which corresponds to


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electron oscillations perpendicular and parallel to the rod length direction, respectively, and their longitudinal surface plasmon wavelengths (LSPWs) are tunable from the visible to infrared regions.37 The fluorescence enhancement to a nanorod end-linked fluorophore takes place due to a combination of factors including the local electric field intensity contour around the nanorods, radiative decay rate modification, and the local orientations of dye molecules.38 The SEF, which occurs between the intense plasmon-induced electricfield and the excited-state of fluorophores located in close proximity to AuNRs or AuNCs, is often exploited to improve fluorescence measurements sensitivity.39 Furthermore, AuNRs or AuNCs have prolonged circulation and permeate barriers including cell membrane and have low osmolality,40 and are supposed to be easily modified by using specific biomarkers, such as peptides, aptamers, antibodies, and so on. If they are applied in fluorescent sensing, they would present low background, high efficiency, resulting in ultra-sensitivity.41,42 On the basis of the above analysis, a general “FRET-SEF” probe with “OFF-enhanced ON” fluorescence switching strategy basing on two gold nano-conjugates (termed AuNC-decorated AuNR probe) was proposed for quantitative sensing and imaging of miRNAs in living cells with ultrasensitive and specific recognition. As summarized in Scheme 1, in our strategy, conventional AuNR and AuNC was used as pre-auto fluorescence quencher and post-emission fluorescence enhancer, respectively, for avoiding false positive signal and obtaining ultrasensitive sensing of miRNAs. That is, the representing fluorophore of Cy5 was first at the “OFF” state when AuNR-ssDNA1 co-hybridized with AuNC-ssDNA2-Cy5 due to the FRET processes. In the presence of target miRNA, AuNR-ssDNA1 was detached from AuNCssDNA2-Cy5, and was complementary with miRNA, meanwhile, the Cy5 fluorophore was defined as at the “ON” state according to SEF effect, the above discussion was where the term


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“OFF-enhanced ON” came in. In addition, such probe was applied in living cell imaging, and has been confirmed that the probe was efficiently delivered into cells without the help of cationic liposomes, and the probe also existed low cytotoxicity and maintained longer viability, more than 6 h, which would contribute to in situ observation of variations of miRNAs expression in living cells. With the fluorescence signal of AuNC-decorated AuNR probe, an efficient way for the specific, sensitive and quantitative miRNAs detection in living cells was developed successfully. miRNA-21, one of the first identified and most prevalent miRNAs in human cells, has been studied in various diseases including cancers, so the potential of miRNA-21 as a cancer biomarker has been widely studied for the past few years.43-45 Furthermore, what calls for special attention is that some tumor suppressor genes also play key roles in cell growth. Such as miRNA-34a, which is a star molecule and has been found to participate in the regulation of p53 pathways as well as the tumour suppressor activity.46-48 Boon et al.49 reported that knocking on miRNA-34a contributes to reducing the death rate of myocardial cells and improve its survival rate, and miRNA-34a has been regarded as a special biomarker for estimating the ageing myocardial cells. Therefore, recognizing cancer-related miRNAs at the cellular level before cancerization occurs holds great promise for enhancing the survival rates of carcinoma patients, on the other hand, the detection of cancer-related miRNAs in tumor cells offers an appealing tool for medical research, which would help researchers investigate the relationship between the food contaminants/drugs and cancer morbidities. Based on the above discussion, miRNA-21/-34a was used as the model miRNAs in the following experiments. EXPERIMENTAL SECTION


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Materials and Reagents. Hydrogen tetrachloroaurate trihydrate (HAuCl4; 99%), 5bromosalicylic acid (>98.0%), sodium salicylate (>98.0%), sodium borohydride (NaBH4,99%), cetyltrimethylammonium bromide (CTAB), L-ascorbic acid (>99.5%) and silver nitrate (AgNO3, >99%) were purchased from Sigma-Aldrich (Shanghai, China). Thiolated PEG5000 and tris(2-carboxyethyl) phosphine hydrochloride (TCEP) were obtained from Sigma-Aldrich (Shanghai, China). AFB1 standard was obtained from South China Agricultural University, and TGF-β1 standard was obtained from Sigma-Aldrich, Inc. (St. Louis, MO, USA). G25 spin column was bought from GM Healthcare. Human hepatocellular carcinoma (HepG2) cells, BRL cells, and H9C2 cells were purchased from the Cell Bank of Chinese Academy of Sciences (Shanghai, China); Dulbecco's Modified Eagle's Medium (DMEM) and fetal bovine serum were obtained from Gibco Invitrogen Corporation (CA, USA). Ultrapure water obtained from a Millipore water purification system (18 MΩ, Milli-Q, Millipore) was used in all runs. All ssDNA sequences (Table S1) purified by high-performance liquid chromatography (HPLC) was purchased from Shanghai Sangon Biological Engineering Technology & Services Co. Ltd, microRNA-21/34a (miRNA-21/34a) and mismatched ones were bought from TAKARA BIOTECHNOLOGY(DALIAN) CO., LTD., and all suspended in TE buffer. Apparatus. UV-vis spectra was obtained on an Avaspec-2048 UV-vis spectrophotometer. Transmission electron microscopy images were obtained from a JEOL JEM-2100 (HR). The zeta potential and size distribution were measured using a Zetasizer Nano ZS system (Malvern) with 632.8 nm laser. Fluorescence spectra was obtained with a fluorescence spectrophotometer (F7000, Hitachi) and the conditions were as follows: Excitation wavelength, 633 nm; Emission wavelength, 645-700 nm. The cells were incubated in a CO2 incubator (Thermo Scientific Forma Series II Water Jacket, Thermo Fisher Scientific Inc., Rockford, IL, USA). The CCK-8 assay and


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fluorescence intensity assays were performed using a microplate reader (Spectra MAX 340, Molecular Devices Co., Sunny vale, CA, USA). The Wide field High-Content Analysis System (Image Xpress Micro XLS, Molecular Devices) and Laser Confocal Fluorescence Microscopy (Carl Zeiss AG, Germany) were used to measure the fluorescence intensity and cell imaging. AuNC Decorated AuNR. AuNRs-ssDNA1 and AuNCs-ssDNA2 were mixed at a molar ratio of 2:1, which was modified phosphorothioate bonds and incubated for 4 h at 37 °C to allow hybridization. The NR pairs were formed in Tris buffer (1 mM Tris-HCL, 0.01% SDS, 20 mM MgCl2). The assembly was centrifuged at 8000 rpm for 20 min and washed with PBS buffer three times to remove the free non-conjugated complex. The final product, AuNC-decorated AuNR conjugates, was characterized by a fluorescence spectrophotometer. miRNA-21 Measurement with Newly Constructed Sensor. miRNA-21 in various concentrations was respectively added to the conjugates, to reach final concentrations of 0, 0.005, 0.1, 1, 10, 50 and 100 fM. All the solutions were incubated for 4 h at 37 °C to allow hybridization with AuNR-ssDNA1. Then, the assembly was centrifuged at 8000 rpm for 10 min and the supernatant each was measured with a fluorescence spectrophotometer. Finite-Difference Time-Domain Simulation. FDTD Solutions. (Lumerical, Inc.) was utilized to study both the near- and far-field electromagnetic responses of metal, by solving Maxwell’s curl equations on a discretized grid. The absorption cross section and near-field enhancement were calculated to evaluate the properties of plasmon-resonant local fields on the AuNRs. In our simulations, the individual nanorod was modeled as a cylinder with two hemispherical end caps. The AuNCs were composed of two AuNRs (length: 30 nm, diameter: 16 nm). A total field scattered field source with wavelength range from 400 to 1000 nm was


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launched into the boundary to simulate a propagating plane wave interacting with the targets. And the incident light polarization was parallel to the longitudinal direction of the single AuNRs. The targets and surrounding medium inside the boundary were divided into 0.5-nm meshes. The dielectric function of AuNCs was used from Johnson and Christ, and the AuNCs was assumed to be embedded in water with a refractive index of 1.33. Assay of AuNC-Decorated AuNR Cytotoxicity for Cell Labeling. Cell Counting Kit-8 (CCK-8) colorimetric assay was used to estimate the cytotoxicity of the nanoprobe. Briefly, HepG2 cells with a density of 1.0 × 104 cells/well were seeded in each well of 96-well plate containing 100 µL DMEM for 12 h. After rinsing with PBS, HepG2 cells were incubated with 100 µL fresh culture media containing serial concentrations of probe or different formulations containing nano materials were added to the culture medium for 10 h. As the control, the cells were incubated with 100 µL of culture medium without probe. At the end of the incubation, 10 µl CCK-8 was added to each well and the cells were incubated at 37 °C. After 3 h, the ultraviolet absorbance at a wavelength of 450 nm was measured with a microplate reader. The cell viability (%) was then determined by (Atest/Acontrol) × 100. Three replicates were done for each treated group and the percent viability was normalized to the cell viability in the absence of AuNCdecorated AuNR. Colocalization Assay. HepG2 cells were seeded into 96-well plate and incubated for 12 h at 37 °C. After incubated with probe for 10 h and washed with PBS three times, the cells were stained with Hoechst 33258 for 20 min. For colocalization assay of the nanoprobe transfected HepG2 cells, the fluorescence of Hoechst 33258 was collected from 400 to 500 nm with a 346nm laser.


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Automated Wide Field High-Content Analysis System. To fully exploit how the nanoprobe internalized into the cells, the real-time dynamic progress of the intracellular miRNA21 in the corresponding cells and monitoring measurements of cellular uptake of AuNCdecorated AuNR probe was carried out. And the scanning was not stopped until 6 h and the light irradiation was started every 30 min in situ, in other words, the scanning time interval was 30 min, and the total light irradiation time was 360 min. For Cy5 dye, excitation was at 633 nm and emission collected was at 645-700 nm. Evaluation the miRNA-21/34a Expressed in Living Cancer and Normal Cell Lines. The BRL cells and H9C2 cells were maintained under the standard culture conditions (atmosphere of 5% CO2 and 95% air at 37 °C) in DMEM medium, supplemented with 10% FBS (fetal calf serum), and 1% penicillin/streptomycin at 37 °C under a 5% CO2 atmosphere. What is more, the fluorescence imaging was obtained with Laser Confocal Fluorescence Microscopy for detecting intracellular miRNA-21/34a. To observe the expression of miRNA-34a, HepG2/H9C2 cells was treated with AFB1 (1 µg/mL, 2 µg/mL, 5 µg/mL, 10 µg/mL, 20 µg/mL, 40 µg/mL, 80 µg/mL, 100 µg/mL) or TGF-β1 (5 ng/mL, 10 ng/mL, 50 ng/mL, 100 ng/mL, 500 ng/mL, 1000 ng/mL), following the AuNC-decorated AuNR probe incubation. Fluorescence signal of Cy5 was monitored with a fluorescence microscope or High-Content Analysis System. RESULTS AND DISCUSSION “FRET-SEF”-Based miRNAs Sensing Protocols. A novel “FRET-SEF” probe basing on two gold nano-conjugates (termed AuNC-decorated AuNR probe) (Scheme 1c) with the switching “OFF-enhanced ON” principle was developed, through modulating the spacing distance between metal surface and the fluorescence dye, the conformational change of single strand DNA


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(ssDNA) was an inducer. As was shown in Scheme 1b, firstly, the AuNR-ssDNA1 was hybridized with the AuNC-ssDNA2 to form a gold nano-conjugate. This resulted in the fluorescence quenching of Cy5, indicating fluorescence “OFF” state. Then, the resulted gold nano-conjugate was interacted with miRNAs and released the free AuNC-ssDNA2-Cy5. The process led to an increase of the fluorescence of Cy5, indicating a fluorescence “ON” state. Due to significant FRET between Cy5 and AuNR and SEF between the Cy5 and AuNC, the fluorescence quenching was much better than that caused by the hybridization of ssDNA1 with ssDNA2. The action would increase the change in the fluorescence intensity during the “OFF” and “ON”, which could improve sensitivity of optical detection of miRNAs, even in living cells (Scheme 1a and 1d).

Scheme 1 Schematic illustrations of the “OFF-enhanced ON” fluorescent switching system for the specific detection of miRNAs in intact cancer cells, (a) and steps involved: a, immobilization of the ssDNA1/ssDNA2 on the AuNR/AuNC, and b, fluorescence recovery due to release of the AuNCs-ssDNA2 from the AuNR-ssDNA1 surface upon that hybridization with the target


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miRNA, (b). TEM images of the AuNC-decorated AuNR, (c). Fluorescence images of HepG2 cells after 4 h incubation with nanoprobes, (d). UV/Vis and Emission Spectroscopy Analysis of Modified AuNRs and AuNCs. The detailed preparations and processes for thiolating ssDNA1 and ssDNA2 and functionalizing AuNRs or AuNCs were given in Experimental Section (ESI†). Corresponding sequences of ssDNA were listed in Table S1 (ESI†). Both the bare AuNRs and AuNCs exhibited longitudinal surface plasmon resonance (LSPR) absorbances at 785 nm and 643 nm, respectively. There is no signiﬁcant broadening was observed after the modification of AuNRs and AuNCs with SH-PEG. Whereas, a slight shift of the LSPR absorbance bands, i.e., 785 nm and 643 nm, to 795 nm and 646 nm for AuNRs and AuNCs, respectively, implies that the covalent attachment of the SHPEG occurred exclusively in the “end” positions of the AuNRs or AuNCs.51 Interestingly，after conjugation with thiolated ssDNA1 or ssDNA2, the AuNRs show distinct optical properties from AuNCs. That is, in the UV-Vis spectral region, the AuNRs underwent a blue shift to 755 nm (Figure 1a), which might be attributed to the thiolated ssDNA1 mainly attaching to the side of the AuNRs and resulting in aspect ratio reduction; while, probably due to the thiolated ssDNA2 conjugated to the end of AuNCs in priority, the AuNCs display a red shift to 651 nm (Figure 1b). The UV-vis spectra showed that the LSPR band of the AuNRs or AuNCs could be tuned from the visible to the near infrared region (NIR) of the optical spectrum, suggesting they make excellent candidates for fluorescent applications.52


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Wavelength (nm)

Figure 1. Characterizations of AuNRs and AuNCs before and after modification. UV-vis spectra of AuNRs, AuNRs-SH-PEG, AuNRs-ssDNA1 in PBS buffer, (a). UV-vis spectra of AuNCs; AuNCs-SH-PEG; AuNCs-ssDNA2-Cy5 in PBS buffer, (b). Spectral overlap between emission spectra of Cy5-ssDNA2 and absorption spectra of AuNR-ssDNA1, (c). Spectral overlap between emission spectra of Cy5-ssDNA2 and absorption spectra of AuNC-ssDNA2, (d). “OFF-enhanced ON” AuNC-Decorated AuNR Fluorescence Probe Design. Due to the various self-assembly features, the UV-vis spectra of AuNC-decorated AuNR nano-conjugates showed remarkable optical difference versus the molar ratio of AuNRs to AuNCs (Figure S3a). The occurrence of energy transfer requires a spectral overlap between the emission spectrum of the donor and the absorption spectrum of the acceptor, a spatial proximity of the donor and acceptor, and a nonperpendicular orientation of the transition dipole moments.53 In this paper, the FRET occurred due to spectral overlap (the emission spectrum of Cy5 was overlapped with the


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absorption spectrum of AuNR-ssDNA1) (Figure 1(c)) between the AuNR and the Cy5, in addition, the FRET quenching was a short-range effect,54 the distance between Cy5 and AuNR surface was about 4 nm after AuNR-ssDNA1 co-hybridized with AuNC-ssDNA2-Cy5, which was defined as “OFF” state. Moreover, AuNRs had larger surface area and lower curvature, which could increase the quenching sites, leading to the short response time, improving the quenching efficiency. Based on the above discussion, the fluorescence intensity of AuNCdecorated AuNR probe was measured, and achieved minimal value at the optimal molar ratio of 2:1 (AuNRs: AuNCs) (Figure S3b). Furthermore, the LSPR of the AuNCs (651 nm) was closest to the emission band of Cy5 (649 nm), and the end-cup area of the AuNCs was an enhancing zone (Figure 1(d)), while the proximity of the waist of the AuNCs was a weak zone.55 In other words, if a fluorescent molecule was close to the end-cup, which was excited at the LSPR, the excitation efficiency and the SEF enhancement of the AuNCs was very large, which was defined as “enhanced ON” state. Specifically, in our “FRET-SEF” probe, thirty bases were chosen, fluorescence intensity for the probe was determined via fluorescence spectrophotometer and the maximum fluorescence intensity was obtained from the 30 bases of ssDNA2 with Cy5 spacing distance of about 10 nm from the AuNCs end-cup surface. As illustrated by FDTD Solutions (Figure S3d), the maximum intensity in electric field for AuNCs was obtained at the distance of about 10 nm in the hot spots, where indicated that the AuNCs had SEF capability to a certain extent and the near-field interaction of Cy5 with AuNCs could strongly enhance fluorescence intensity through either localized or propagating surface plasmon resonance. The amount of ssDNA2-Cy5 was also tested, and found that the fluorescence intensity did not change with the molar ratio of ssDNA2Cy5/AuNCs increased until the combination of ssDNA2-Cy5 and AuNCs had reached saturation,


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because there was no space for the ssDNA2-Cy5 to conjugate to the AuNCs (Figure S3c). The fluorescence intensity was compared between ssDNA2-Cy5, AuNCs-ssDNA2-Cy5 and AuNRAuNC as well. And the fluorescence intensity of AuNC-ssDNA2-Cy5 was obtained through AuNR-AuNC disassembly after the AuNR-ssDNA1 was combined with a certain concentration of the target. On the other hand, Figure 2 was used to illuminate the constructed “OFF-enhanced ON” probe was practical.


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Wavelength (nm) Figure 2 Fluorescence spectra of ssDNA2-Cy5, AuNCs-ssDNA2-Cy5 and AuNR-AuNC Stability and Cytotoxicity of AuNC-Decorated AuNR Probe. Before intracellular usage, the “FRET-SEF” nanoprobe with modified phosphorothioate bonds in the DNA skeleton displayed significant stability and, resisting enzymatic lysis (Figure 3a and Figure 3c). Such resistant stability of the probe shall prevent nonspecific signals produced from intracellular nuclease activities. Figure 3b showed that in the absence of the target, nanoconjugates exhibited a low fluorescence signal under both solutions of different ionic strength and there existed an increase in fluorescence with the addition of the target miRNA-21. The stability of fluorescence


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in nanoprobe under different conditions was tested as well, and from Figure 3d, in the case of the control nanoconjugates (AuNC-decorated AuNR with non-specific capture), little response was observed, however, when the nanoconjugates was modified with specific sequence, fluorescence intensity increased because there was the high expression of miRNA-21 in HepG2 cell lysis, and the nanoprobe could recognize it. Moreover, the nanoprobes incubated in HepG2 cells did not affect any cell viability with time went by more than 10 hours (Figure S5) and displayed negligible cytotoxicity (Figure S6). According to the above observations, it is reasonable to conclude that the “FRET-SEF” nanoprobe hold good activity and stability, which provide a solid foundation for real-time intracellular miRNAs sensing in living cells.

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Figure 3. Evaluation of the stability of AuNC-decorated AuNR Probe under different conditions. Structural stability of AuNC-decorated AuNR under different ionic strength measured by


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absorption spectra, (a). Fluorescence spectra of AuNC-decorated AuNR under different ionic strength and fluorescence spectra of AuNC-decorated AuNR under 300 mM NaCl after addition of target miRNA-21, inset: Fluorescence intensity value of AuNC-decorated AuNR probe under different ionic strength (0, 50, 100, 150, 200, 300 mM NaCl) (b). Stability of AuNC-decorated AuNR (AuNC-decorated AuNR treated with non-specific sequence) in PBS buffer, HepG2 cell lysis, cell culture medium, and nuclease stability of the probe treated with DNaseI measured by absorption spectra, (c). Fluorescence spectra of AuNC-decorated AuNR itself and Fluorescence spectra of AuNC-decorated AuNR (AuNC-decorated AuNR treated with non-specific sequence) in PBS buffer, HepG2 cell lysis, cell culture medium, nuclease stability of the probe treated with DNaseI and fluorescence spectra of AuNC-decorated AuNR with specific sequence treated with HepG2 cell lysis, inset: Fluorescence intensity value of AuNC-decorated AuNR probe itself and fluorescence intensity value of AuNC-decorated AuNR probe (treated with non-specific sequence) in PBS buffer, HepG2 cell lysis, cell culture medium, nuclease stability of the probe treated with DNaseI (d). “FRET-SEF” Nano-conjugate Based miRNA-21 Sensing in Vitro. miRNA-21 was chosen as the model miRNA to evaluate the performance of the proposed “FRET-SEF” nanoprobe in the following experiment. First, the target (miRNA-21) was used to verify the feasibility of the detection strategy in vitro, the fluorescence intensity gradually increased upon the addition of increasing amounts of miRNA-21 (Figure 4a). The fluorescence intensity of the AuNC-decorated AuNR probe was logarithmically dependent on miRNA concentration, ranging from 0.0006 to 0.0016 fM and 0.1 to 100 fM.


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Figure 4. “FRET-SEF” probe for miRNA-21 detection in vitro: fluorescence responses to the addition of various concentrations of miRNA-21 (0, 0.00001, 0.00008, 0.0002, 0.0003, 0.0006, 0.0008, 0.0013, 0.001, 0.0016, 0.005, 0.1, 1, 10, 50, 100, 150, 200, 400 fM), (a). The dependence of the fluorescence intensity on the logarithm of the miRNA-21 concentration, the linear range was from 0.0006 to 0.0016 fM and 0.1 fM to 100 fM. Each datum was the mean of three replicates (N=3) and the error bars represented the standard deviations of the measurements, (b). A plot of fluorescence intensity versus the logarithm of the miRNA-21 concentration gave two linear responses represented by the equation (F=1830.32logC+6349.27, R2=0.9901 and F=244.41logC+1916.10, R2=0.9984, respectively) (Figure 4b). The detection limit was 0.5 aM and 0.03 fM for miRNA-21, respectively, which was obtained from the signal-to-noise characteristics of these data (S/N = 3), and the result was comparable to other existing systems (Table S2, ESI†), demonstrating its satisfactory sensitivity. miRNA-21 Sensing with AuNC-Decorated AuNR “FRET-SEF” Nano-conjugate Probe in Living HepG2 Cells. miRNA-21 expression signatures could be correlated with biopathological and clinical features of Human Hepatocellular Carcinoma Cells (HepG2 Cells),56,57


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suggesting its essential role in living biology and disease. Moreover, more and more studies have shown that miRNA-21 expression is up-regulated in HepG2 cells and can promote cell cycle progression, reduce cell death, and favor angiogenesis and invasion.58,59 Therefore, HepG2 cells were selected for the intracellular miRNA-21 analysis. As expected, strong fluorescence signal was observed on a Laser Confocal Microscope when the probe was internalized into living HepG2 cells. Hoechst 33258 fluorescence dye was employed to label as a reference for colocalization assay. Bright red fluorescence signal from our probe indicated that the “FRET-SEF” probe could highly sense the intracellular miRNA in living cells (Figure 5a). Figure 5b showed the mainly nanoprobe distributions in HepG2 cells, which indicated that the nanoprobes mainly existed in cytoplasm for sensing miRNA-21. Such observations, are in accordance with the principle that the mature miRNA-21 primarily present in cytoplasm as well.60


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Figure 5. “FRET-SEF” probe for miRNA-21 analysis. Fluorescence images of AuNC-decorated AuNR probe transfected in HepG2 cells for colocalization assay of miRNA-21 by the Laser Confocal Fluorescence Microscopy. Upper: Bright field and red field; under: blue field and merged. Scale bar: 20 µm (red field, probe excitation: 633 nm; emission: 645 and 700 nm; blue field, Hoechst 33258, excitation: 346 nm; emission: 400 and 500 nm), (a). Zoom-in fluorescence images of nanoprobes in a single living HepG2 cell. front view；upper: top view; right: side view. Scale bar: 5 µm, (b). The feasibility of this newly nanoprobe for in situ tracking intracellular miRNA-21 was investigated by dynamically observing the fluorescence intensity change in living cells (seeing the Movie SI and Figure S7). With time went by, the red fluorescence intensity in HepG2 cells became stronger, and manifested the time-dependent on the intracellular detection process was not stopped until 4 h, because the intracellular fluorescence intensity did not change at this time and intracellular miRNA-21 detection reached saturation. The practical applicability of the strategy by measuring the differential expression of miRNA-21 in various cell lines was confirmed. BRL and H9C2 cells were used. A stronger red fluorescent signal was observed in the HepG2 cells than in the BRL and H9C2 cells (Figure 6), which meant the high miRNA-21 expression in HepG2 cells, while low expression in BRL and H9C2 cells. This could be attributable to the lower expression of miRNA-21 levels in normal cells than in the cancer cells.61 The above results consistently indicated that the “FRET-SEF” probe could be universally applied in living cell lines.


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Figure 6. Living-cell fluorescence analysis. HepG2 living cells in the presence of AuNCdecorated AuNR probe, (a). H9C2 living cells in the presence of AuNC-decorated AuNR probe, (b). BRL livings cells in the presence of AuNC-decorated AuNR probe. The reaction time is 240 min, (c). Application in miRNA-34a Detection with the New Type Probe. To test the practicability of the developed probe, miRNA-34a was used as the target one. We firstly used HepG2 cells expressing miRNA-34a in a AFB1-inducible manner. The HepG2 cells were first stimulated with different concentrations of AFB1 (1, 2, 5, 10, 20, 40, 80, 100 µg/mL) to induce different expression levels of miRNA-34a, treated with nanoprobe for 12 h, the results indicated that the fluorescence intensity was increased inside the cells as miRNA-34a expression increased induced by AFB1 (Figure 7), which was consistent with the reported one.62 The nanoprobe was


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also used to measure the senescence level of H9C2 cells after stimulated by TGF-β1 (Figure S8). The miRNA-34a expression increased with increasing concentration of TGF-β1 (5 ng/mL,10 ng/mL, 50 ng/mL, 100 ng/mL, 500 ng/mL, 1000 ng/mL), which further indicated that the nanoprobe could be used in the cell senescence prediction.

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Figure 7 Dose-dependent induction of miRNA-34a expression in HepG2 cells treated with AFB1 standard. Monitoring of miRNA-34a in AFB1-inducible HepG2 cell lines of which miRNA-34a expression increase upon treatment of AFB1 using High-Content Analysis, (a). Quantification of fluorescence intensity in HepG2 cells stimulated with AFB1 respectively, the concentrations of AFB1 from low to high in order are: 1, 2, 5, 10, 20, 40, 80 and 100 µg·mL-1,


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(b); Dose-dependent images of cell fluorescence intensity responses with Laser Confocal Fluorescence Microscopy, AFB1 was employed to stimulate HepG2 cells respectively, (c). CONCLUSIONS In this study, a newly oriented low-toxicity AuNC-decorated AuNR probe for sensing and imaging miRNA in living cells has been developed, which was ultrasensitive with a detection limit of 0.5 aM and 0.03 fM, respectively. We found that the probe could monitor the endogenous miRNA expression in living cells dynamically, and differentiate abnormal cells. The excellent performance of our strategy was attributed to the characteristic “OFF-enhanced ON” fluorescence switching signal with two gold nano-conjugates “one-one assembly”, which was in favour of energy disperse and electronic transfer, leading to improve the detection sensitivity. Our study has paved the way to sense not only miRNA-21/34a in living cancer cells, but also miRNA-34a expression levels in living H9C2 cells. We also envisage hope that the sensing miRNA platform here can provide a helpful reference for miRNA-related research. ASSOCIATED CONTENT Supporting Information Experimental section, characterization of AuNC-decorated AuNR, zeta potential of AuNR and AuNC before and after modification, spectral analysis of AuNC-decorated AuNR, selective performance in miRNA detection using AuNC-decorated AuNR probe, cytotoxicity evaluation of AuNC-decorated AuNR probe, dynamical fluorescence intensity change in HepG2 cells by High-Content Throughput Analysis, miRNA-34a detection in TGF-β1-inducible H9C2 cell lines. Table S1 Details of the ssDNA and miRNA sequences.


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Table S2 Summary of nanoprobe based on fluorescence method reported by previous papers. Movie S1 (AVI) AUTHOR INFORMATION Corresponding Author *Xiulan Sun (E-mail: [email protected]) Author Contributions Xiulan Sun and Jiadi Sun designed the project and contributed to the experimental design. Fuwei Pi guided the experiment and the data analysis. Jian Ji helped do several cell experiments. Zhixian Gao provided proposals on H9C2 cells the biological samples. Hongtao Lei provided the biological samples, such as AFB1 standard. Jean de Dieu Habimana participated in the synthesis of the nanomaterials. Zaijun Li took part in the data analysis. Yinzhi Zhang took part in the guidance of spectra analysis. NOTES The authors declare no competing financial interest. ACKNOWLEDGEMENTS. This work was supported by the National Natural Science Foundation of China (U13012141, No.31371768), the Graduate student scientific research innovation projects in Jiangsu Province (KYCX17_1404), the Program for New Century Excellent Talents in Jiangnan University (NCET-12-0877), Synergetic Innovation Center of Food Safety and Quality Control, and the Priority Academic Program Development of Jiangsu Higher Education Institutions.


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An Ultrasensitive âFRET-SEFâ Probe for Sensing and Imaging

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