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A Dual Energy Transfers-Based Fluorescent Nanoprobe for Imaging miR-21 in Non-Alcoholic Fatty Liver Cells with Low Background Shuiqin Chai, Wen Yi Lv, Jia Hui He, Chun Hong Li, Yuan Fang Li, Chun Mei Li, and Cheng Zhi Huang Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.9b00841 • Publication Date (Web): 25 Apr 2019 Downloaded from http://pubs.acs.org on April 25, 2019

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Analytical Chemistry

A Dual Energy Transfers-Based Fluorescent Nanoprobe for Imaging miR-21 in Non-Alcoholic Fatty Liver Cells with Low Background Shui Qin Chaia, Wen Yi Lva, Jia Hui Hea, Chun Hong Lia,Yuan Fang Lib, Chun Mei Lia*and Cheng Zhi Huanga,b* a Key

Laboratory of Luminescent and Real-Time Analytical Chemistry (Southwest University), Ministry of Education, College of Pharmaceutical Science, Southwest University, Chongqing 400715, China. b

Key Laboratory of Biomedical Analysis (Southwest University), Chongqing Science & Technology Commission, College of Chemistry and Chemical Engineering, Southwest University, Chongqing 400715, China.

ABSTRACT: Non-alcoholic fatty liver disease (NAFLD) can progress gradually to liver failure, early warning of which is critical for improving the cure rate of NAFLD. In situ imaging and monitoring of overexpressed miR-21 is an advanced strategy for NAFLD diagnosis. However, this strategy usually suffers from the high background imaging in living cells owing to the complexity of the biological system. To overcome this problem, herein, we have developed a one-donor-two-acceptors nanoprobe by assembling gold nanoparticles (AuNPs) coupled with BHQ2 (AuBHQ) and quantum dots (QDs) through DNA hybridization for imaging of miR-21 in living cells. The fluorescence of QDs was quenched up to 82.8% simultaneously by the AuNPs and the BHQ2 via nanometal surface energy transfer (NSET) and fluorescence resonance energy transfer (FRET), reducing the background signals for target imaging. This low background fluorescent nanoprobe was successfully applied for imaging the target miR-21 in non-alcoholic fatty liver cells by catalyzing the disassembly of QDs with the AuBHQ and the fluorescence recovery of QDs. In addition, the sensitivity of this nanoprobe has also been enhanced toward detecting miR-21 in the range of 2.0-15.0 nM with the detection limit (LOD, 3σ) of 0.22 nM, which was 13.5 times lower than that without BHQ2. The proposed approach provides a new way for early warning, treatments, and prognosis of NAFLD.

INTRODUCTION Nonalcoholic fatty liver disease (NAFLD), characterized by hepatocyte steatosis and lipid accumulation,1 is a pathological manifestation of metabolic syndrome in the liver. Simple steatosis can further progress to nonalcoholic steatohepatitis (NASH), fibrosis, irreversible cirrhosis and even hepatocellular carcinoma,2 ultimately deteriorate to liver failure. Therefore, there is an urgent need for early detection of NAFLD, which is significant for preventing disease progression and promising for improving its cure rate. At present, a variety of diagnostic methods for NAFLD, including serum biochemistry,3 ultrasonication,4 and histopathological examination of liver biopsy,5,6 have been developed. However, most of these approaches were limited by either large within-individual variations,3 operator- and equipment-dependent and low sensitivity,7 or tissue sampling and interpretation errors.8 Hence, it is still highly challenging to develop simple, accurate, noninvasive methods to promote the diagnosis of NAFLD. In recent years, series of studies have revealed that miR21, which is overexpressed in hepatocytes of patients with NAFLD, is a potential link between non-alcoholic fatty liver disease and hepatocellular carcinoma.9,10 Therefore, miR-21 not only plays an important role in the pathogenesis of NAFLD, but also acts as a precipitating factor for NAFLD potential progression to hepatocellular carcinoma. Monitoring of miR-21 level in hepatocytes becomes an advanced strategy for NAFLD diagnosis. However, conventional strategies for miRNA detection mainly focus on quantitative real-time polymerase chain reaction (qRT-PCR),11 Northern blotting12 and microarray hybridization.13 These strategies are usually suitable for

detecting extracted miRNA from cell lysate, but limited in application for in situ visualizing miRNA. Comparatively, fluorescence imaging provides a valuable means for sensing of miRNA in living cells with distinct signals, satisfactory temporal and spatial resolution, which has been widely used for detecting the occurrence and evolvement of disease.14-18 Nowadays, many fluorescence nanoprobes have been developed for imaging of intracellular miRNAs. Compared to organic dyes and fluorescent proteins, quantum dots (QDs) have provided improved sensitivity for bioimaging owing to their unique fluorescent properties, including strong fluorescence,19,20 long-term photostability,21 wide excitation spectrum and narrow emission spectrum.22 Given these properties, QDs are typically utilized as energy donors/acceptor of various fluorescence resonance energy transfer (FRET) probes.23-25 Thus, a variety of QDs-based FRET nanoprobes have been successfully designed and applied in sensing, imaging in living cells and drug delivery.26,27 Although the FRET strategy have obvious advantages, they still suffer from some limitations, such as high background signals due to the low efficiency of FRET between donor and acceptor,28 resulting in reduced sensitivity for target detection and imaging. In this study, we developed a dual energy transfer fluorescent nanoprobe containing a donor and two receptors for lowing background and high-sensitive imaging of miR-21 in nonalcoholic fatty liver cells. In order to construct the nanoprobe, QDs were choosen as donor of energy transfer and gold nanoparticles (AuNPs) were served as cellular carrier and acceptor of energy transfer. To improve the quenching efficiency, the surface of AuNP was decorated with a large

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Scheme 1. Illustration of the dual energy transfer fluorescent nanoprobe for imaging of miR-21 in NAFLD cells.

number of BHQ2 (black quencher) in advance. As shown in scheme 1, the nanoprobe comprised an thiol-terminated single strand DNA1 functionalized AuNP (AuNP-DNA1) and a biotin-terminated single strand DNA2-coupled QD (QDDNA2) which tethered to the AuNP surface through hybridization of DNA1 and DNA2. The fluorescence of QD was efficiently quenched both by AuNP and BHQ2 via nanometal surface energy transfer (NSET) and FRET, respectively. After the nanoprobe entered nonalcoholic fatty liver cells, miR-21 target could hybrid with the QD-DNA2, triggering the disassembly of QD with the AuNP, ultimately yielding significant fluorescence recovery signals. Compared with the current single-acceptor energy transfer nanoprobe, the combination of NSET and FRET would improve the efficiency of energy transfer, accompanied by reducing background signals and enhancing the sensitivity of the target detection. Furthermore, the proposed approach provides a powerful strategy for early warning and post-treatment evaluation of NAFLD.

Gene Co. Ltd. Sodium oleate and Methyl Palmitate was from Aladdin Reagent Co. Ltd (Shanghai, China). Apparatus. All fluorescence spectra were obtained with a Hitachi F-2500 fluorescence spectrophotometer (Tokyo, Japan). The UV-vis absorption spectra were carried out on a Hitachi U-3010 spectrophotometer (Tokyo, Japan). The highresolution transmission electron microscopy (HRTEM) images were taken on a Tecnai G2 F20 field emission transmission electron microscope (FEI, USA). Hydrodynamic sizes were measured by a ZEN3600 dynamic laser light scattering (DLS) (Malvern, English). The fluorescence lifetime was recorded on the FL-TCSPC fluorescence spectrophotometer (Horiba Jobin Yvon Inc., France). Fluorescence imaging was operated on a DSU live-cell confocal microscope (Olympus, Japan) system with laser excitations of QDs605. Preparation of Dual Energy Transfer Fluorescent Nanoprobe. First, AuNPs were pretreated by capping with BSPP in order to increase the stability in buffer solutions according to the literature procedure with some modifications.29 Briefly, 1 mL of AuNPs suspension was mixed with 1 mg BSPP in a glass bottle and stirred gently overnight at room temperature. The suspension was transferred to the 1 mL centrifuge tube for centrifuging at 13000 rpm for 15 min to collect the precipitated AuNPs. Secondly, SH-DNA1 (10 µL, 50 µM) was treated by TCEP (5 µL, 1 mM) for 1 h at room temperature to reduce the disulfide bond. The activated SHDNA1 was added to 1 mL of concentrated BSPP-protected AuNPs (10 nM) with a molar ratio of 50:1 and shaken overnight. Then, each 10 mM sodium chloride solution was gradually added over a 10-hour period till the final concentration reached to 100 mM. Finally, the solution was centrifuged at 13000 rpm for 15 min three times to remove the free SH-DNA1 in the supernatant, and the purified AuNPsDNA1 were resuspended in 1 mL 1×PBS. The quantitation of AuNPs-DNA1 was performed using UV-vis absorption.30 Subsequently, the purified AuNPs-DNA1 was mixed with different concentrations of BHQ2-NH2 and incubated at 37 °C for 1 h. After 1 hour, the free BHQ-NH2 was removed via

EXPERIMENTAL SECTION Materials. 10 nm AuNPs was purchased from BBI Solutions (UK). Tris (2-carboxyethyl) phosphine hydrochloride (TCEP) was obtained from Sangon BiotechnologyCo. Ltd. (Shanghai, China). Bis(p-sulfonatophenyl)phenylphosphine dipotassium salt (BSPP) was from Sigma-Aldrich, Inc. (Saint Louis, MO, USA). All of the oligonucleotide sequences (Table S1) and the 20×nuclease-free PBS (phosphate buffered saline, 2.74 M sodium chloride, 54 mM potassium chloride, 200 mM phosphate buffer, and 40 mM potassium phosphate) were supplied by Sangon Biotechnology Co., Ltd (Shanghai, China). Diethypyrocarbonate (DEPC) water was purchased from Dingguo Chang sheng Biotechnology Co., Ltd. (Beijing, China). Streptavidin modified quantum dots (SA-QDs) were ordered from WuHan JiaYuan Quantum Dots Co., Ltd (China). BHQ2 Amine (BHQ2-NH2) was from Biosearch Technologies, Inc. (USA). Agarose powder was purchased from Hongkong

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centrifugation at 13000 rpm for 10 min. The purified AuNPsDNA1 decorated with BHQ2 (AuBHQ-DNA1) was resuspended in 1× PBS. The water-soluble SA-QDs were dispersed in 1×PBS buffer. Subsequently, the biotin-DNA2 were added to 1 mL of the SAQDs solution (100 nM) at a molar ratio of 1:1 and the mixed solution was shaken gently at 37 °C for 1 h. Purified QDsDNA2 were resuspended in 1 mL 1×PBS after ultrafiltration to remove the free biotin-DNA2. Finally, the AuBHQ-DNA1 mixed with the equal concentration of QDs-DNA2 in the 1×PBS buffer was shaken gently at 37 °C for 1 h. The QDDNA-AuBHQ nanoassembly was obtained via centrifugation at 13000 rpm for 10 min and resuspended in 1× PBS three times for the following experiments. Cell Culture and Treatment. Human hepatocyte cell line LO2 cultured in DMEM medium supplemented with 10% fetal bovine serum and 1% antibiotics penicillin/streptomycin (100 U/mL) were incubated in a humidified atmosphere at 37 °C containing 5% CO2. The cell model of NAFLD was established according to the previous report.31 Briefly, cells were seeded in 6-well plates with a cover slide per well at a density of 105 cells/mL. When 80%~90% confluence was reached on the cover slide and cells were cultured in FBS-free medium for 24 h, then cells were treated with or without long-chain FFA (2:1 oleate/palmitate) in 2% FBS medium for another 24 h to develop a NAFLD model. Subsequently, cells were washed three times with PBS, and fixed with 4% paraformaldehyde solution for 15 min. Finally, the lipid droplet accumulation in the cells was determined by Oil Red O Staining and quantified on the basis of the integrated optical density (IOD) by the Image-Pro Plus 6.0 software. Confocal Fluorescence Imaging. LO2 cells (105 cells per well) were seeded into the 35 mm glass-bottom dishes to establish a steatosis model as the method mentioned above. After treatment by different levels of FFA for 24 h, the cells were washed with PBS three times. As control experiments, the cells were incubated with 0.2 nM QD-DNA-Au or QD-DNAAuBHQ probes in 2% FBS medium for 6 h. After removing medium and washing with PBS three times, the cells were imaged with a DSU live-cell confocal microscope (Olympus, Japan) system at laser excitations of QDs605.

hinerance effect. Otherwise, when the concentration ratio was smaller than 50, the AuNPs aggregation was visible. Thus, the molar ratio of 50:1 was used to functionalize AuNPs. Next, AuNPs-DNA1 was coupled with BHQ2 via gold-ammonia bond. Finally, QDs-DNA2 were also grafted to AuBHQ-DNA1 solution via DNA1 and DNA2 hybridization. As shown in agarose gel electrophoresis (Figure 1a,b), successful functionalization of AuNPs with DNA1 resulted in the retarded mobility of AuNPs (lane 4), which was much slower than single AuNPs (lane 2) or the mixture of QDs and AuNPs (lane 3). When BHQ2 and

Figure 1. Characterization of QD-DNA-AuBHQ probes. (a,b) Agarose gel electrophoresis image of QDs (lane 1); AuNPs (lane 2); the mixture of QDs and AuNPs (lane 3); AuNPs-DNA1 (lane 4); QD-DNA-AuBHQ (lane 5) under daylight (a) and UV light (b). (c,d) TEM/HRTEM image of assembled QD-DNA-AuBHQ.

QDs were added, we observed a distinct band shift due to a larger molecular weight assembly formation of AuNPs-DNA1 with BHQ2 and QDs (lane 5). The TEM image (Figure 1c) of QD-DNA-AuBHQ probe showed that the ratio between AuBHQ and QD was about 1:1, with the average distance of about 1.35 nm as indicated by HRTEM image (Figure 1d). This close proximity may be attributed to the DNA hybridization between the donor of QD and the acceptor of AuBHQ complex. In addition, both the increased negative charge and hydrodynamic size of AuBHQ-DNA1 upon the addition of QDs-DNA2 further demonstrated the successful preparation of QD-DNA-AuBHQ (Figure S3a, b). Studies of the Dual Energy Transfer Fluorescent Nanoprobe. Traditional single energy transfer between QDs and AuNPs displayed high fluorescence background owing to the low quenching efficiency. To reduce the background, BHQ2 was used as another acceptor to further quench the fluorescence of QDs donor. It was found the fluorescence intensity was further decreased and quenching efficiency was gradually enhanced with an increase in the molar ratio of BHQ2 and AuNPs from 100:1 to 900:1 (Figure 2a,b). When the molar ratio was larger than 900:1, the aggregation was visible, which might be owing to the gradually reduced surface charge of AuBHQ

RESULTS AND DISCUSSIONS Characterization of the Dual Energy Transfer Fluorescent Nanoprobe. To construct the dual energy transfer fluorescent nanoprobes, first, 10 nm AuNPs were functionalized with thiol-terminated DNA1, as demonstrated by the red-shift of UV-vis absorption spectrum (Figure S1a) and increased hydrodynamic diameter (Figure S1b). In addition, the concentration of DNA1 loading on AuNPs was optimized before coupling BHQ2-NH2 to AuNPs (Figure S2). Different concentrations of DNA1 were used to functionalize AuNPs at a molar ratio of 50:1, 100:1, 200:1, 300:1 respectively. When adding the QDs-DNA2, the fluorescence quenching of QDs donor gradually reduced with the increasing concentration of DNA1, indicating lower energy transfer efficiency due to steric-



with the increasing concentrations of BHQ2 (Figure S4). Compared with single AuNPs, the quenching efficiency of QDs could be improved from 56.5% to 82.8% after coupling with BHQ2 (Figure 2b). What’s more, the maximum absorption of AuNPs was gradually red-shifted from 518 nm to 533 nm with the increasing concentration of BHQ2 (Figure 2c), and the coupling of BHQ2 on AuNPs promoted the spectral overlap between QDs donor photoluminescence (PL) and the AuBHQ composite acceptor

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dual energy transfer can be determined from the Equation (1) and (2). Therefore, R0 increases with greater overlap between the QDs donor emission spectra and the AuBHQ composite acceptor absorption spectra, which is benefit for improving the energy transfer efficiency. Moreover, the optimized molar ratio of BHQ2 and AuNPs was found to be 700 by comparing the fluorescence signal/background ratio (F/F0) with or without miR-21 target (Figure S5) (the inset Figure S5). Thus, 700 folds BHQ2 was used to couple on AuNPs surface for detecting and imaging miR-21. The energy transfer was verified by measuring the donor QDs lifetime, which was fitted to a biexponential decay in aqueous solution, showing a short lifetime component τ1 and a long lifetime component τ2 (Table 1).37 The fluorescence lifetime obviously shortened from 32.9 ns to 13.0 ns with the addition of BHQ2 and subsequently recovered to 31.7 ns upon the addition of miR-21 (Figure S6). In the experimental process, fluorescence lifetime of the energy donor is usually used to calculate the energy transfer efficiency in the presence and absence of the energy acceptor, which is expressed as in Equation (3):37,38

E  1-

 DA D

(3)

Figure 2. (a) Fluorescence intensity decreased with the increasing of BHQ2 concentration (the concentration ratio of BHQ2 and AuNPs varied from 100 to 900); (b) The quenching efficiency became increased with the increasing of BHQ/Au molar ratio. (c) The maximum absorption of AuNPs was gradually red-shifted with the increased BHQ2 concentration. (d) Normalized fluorescence spectra of QDs and absorption spectra of AuNPs and AuBHQ. Concentrations: QDs, 1.0 nM; AuNPs, 1.0 nM.

Where τDA and τD is the fluorescence lifetime of the energy donor in the presence and absence of an energy acceptor, respectively. In this way, we found the efficiency of single energy transfer between QDs and AuNPs was 20.7%, which could be greatly improved to 60.5% by using the proposed dual energy transfer system, further demonstrating the significance of one-donor-two-acceptors nanoprobe.

absorption (Figure 2d). As all we know, the spectral overlap is a key factor for improving the energy transfer efficiency. In general, the energy transfer efficiency (E) can be written as Equation (1): 32,33

Table 1. Fluorescence lifetimes obtained with biexponential fit of the fluorescence decay curves of the QDs, QD-DNA-Au, QDDNA-AuBHQ, QD-DNA-AuBHQ+miR-21, respectively.

E

R0n R  Rn

Sample

τ1/ns (%)

τ2/ns (%)

τave/ns

QDs

21.4 (49.04)

43.9 (50.96)

32.9

QD-DNA-Au

8.7 (25.24)

32.0 (74.76)

26.1

QD-DNA-AuBHQ

4.6 (38.90)

18.3 (61.10)

13.0

QD-DNA-AuBHQ 16.8 (28.57) +miR-21

37.6 (71.43)

31.7

(1)

n 0

Where R is the separation distance between the donor and acceptor pair. The exponent n is dependent on the nature of energy transfer.The FRET predicts an n = 6 distancedependent quenching efficiency,33 while NSET follows an n = 4 distance dependence.34,35 R0 is the Förster separation distance between donor and accepter at which 50% of the excited donor molecules decay by energy transfer, which can be calculated from the spectral properties of the donor and accepter. It is expressed as in Equation (2): 36 1/6

R0 = 9.78 103  2 n 4QD J ( ) 

 in Å 

Sensitivity and Selectivity of the miR-21 Detection. To investigate the feasibility of our design for miR-21 detection, the fluorescence spectra at different concentrations of miR-21 were recorded (Figure 3a). In the presence of miR-21 target, hybridization of the DNA2 with miR-21 resulted in the release of the QDs-DNA2 and recovery of the fluorescence signal, which displayed good linearity with the concentration of miR21 from 2.0 nM to 15.0 nM (Figure 3b). The linear regression equation (c, nM) was F/F0 = 0.092c + 1.015 (correlation coefficient, R2 = 0.991) with the detection limit of 0.22 nM

(2)

Where the factor κ2 describes the donor and acceptor transition dipole orientation. n is the refractive index of the medium. QD is the quantum yield of the donor in the absence of the acceptor and J(λ) is the overlap integral between the donor emission and the acceptor absorption. The efficiency of this


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(3/k). To demonstrate the advantage of dual energy transfer, the fluorescence spectra at different concentrations of miR-21 without BHQ2 were also measured (Figure 3c). The change in fluorescence intensity was linearly correlated with miR-21 concentration from 5.0 nM to 30.0 nM, and the linear regression equation was F/F0 = 0.051c + 1.034 (correlation coefficient, R2 = 0.995) with the detection limit of 2.96 nM (3/k) (Figure 3d). The above results indicated that the dual energy transfer containing NSET between QDs and AuNPs and FRET between QDs and BHQ2 indeed improved the detection sensitivity and reduced the detection limit about 13.5 times. A comparison of miR-21 detection between this work and other reported methods was displayed in Table S2.

Figure 4. Selectivity of miR-21 detection. Concentrations: QDs, 1.0 nM; AuNPs, 1.0 nM; BHQ2, 700.0 nM; miR-21, mis-1, mis-3, mis-5 and miR-34a, 10.0 nM. F and F0 represented the fluorescence intensity of QD-DNA-AuBHQ in the presence and absence of miR21, respectively.

50 μg/ml and the mixture was kept at 37 °C for 1 hour. Compared with the nanoprobe without DNase I, the fluorescence spectra of the nanoprobe treated with DNase I exhibited negligible change (Figure S7) both in the absence and presence miR-21.The results confirmed the nuclease stability of the dual energy transfer nanoprobe, which was attributed to the protection of AuNPs to oligonucleotides from enzymatic degradation. The cytotoxicity of the nanoprobe determined by Cell Counting Kit-8 (CCK-8) solution indicated that the nanoprobe had negligible cytotoxicity to both normal LO2 cells and steatosis model cells (Figure S8). Based on the above results of nuclease stability and low cytotoxicity, the nanoprobe was sufficient for application in intracellular imaging. Imaging of the Steatosis Cells. For in situ applications, the nanoprobe was used to visualize the miR-21 levels in steatosis cells via fluorescence imaging. The steatosis model cells were constructed using LO2 cells which were treated with FFA to induce steatosis. LO2 cells without FFA treatment were used as a control group. Before establishment of steatosis model cells, the effects of FFA on the viability of LO2 cells were investigated (Figure S9), which showed that FFA at the dosages of less than 200 µM had no significant toxicity on the viability of LO2 cells. Thus, 100 µM, 150 µM, 200 µM FFA were used to treat LO2 cell, respectively, and all of them resulted in lipid accumulation. The lipid contents in the cells were analyzed by using oil red O staining and quantified on the basis of the integrated optical density (IOD). Figure 5 showed that lipid accumulation was markedly aggravated with the increasing concentration of FFA. Next, we applied the nanoprobe for imaging of miR-21 in the steatosis cells. As well known, AuNPs possess many excellent properties, including protecting oligonucleotides from enzymatic degradation39 and the ability to enter cells without the assistant of another transfection reagent.40 First, we used 200 μM FFA treated-LO2 cells as a model to investigate the effect of incubation time on the cell imaging. As the time prolonged, more probes entered the cell and

Figure 3. Fluorescence spectra of the QD-DNA-AuBHQ (a) and QD-DNA-Au (c) in the presence of various concentrations of miR21. Linear relationship between F/F0 and the concentration of miR21 with (b) and without (d) BHQ2. Concentrations: QDs, 1.0 nM; AuNPs, 1.0 nM; BHQ2, 700.0 nM. Each measurement was triplicated (error bars indicate standard deviation). F and F0 represented the fluorescence intensity of QD-DNA-AuBHQ (b) or QD-DNA-Au (d) in the presence and absence of miR-21, respectively. Moreover, the QD-DNA-AuBHQ nanoprobe was highly selective, which was ensured by using five different oligonucleotides, including one-base-mismatched (mis-1), three-base-mismatched (mis-3), five-base-mismatched (mis-5), miR-21 and another high expression miRNA-34a sequences in the fatty liver cell. Even with the addition of the one-basemismatched RNAs, the fluorescence recovery was obviously lower than that of miR-21 (Figure 4), indicating the feasibility of the nanoprobe in complex biological conditions. Nuclease Stability and Cytotoxicity of the Nanoprobe. To apply the nanoprobe for imaging miRNAs in living cells, it is critical to investigate the stability and biocompatibility of the probe. Nuclease stability was carried out using enzyme deoxyribonuclease I (DNase I), which is a common endonuclease endonuclease capable of nonspecifically cleaving single-strand and double-strand DNA. DNase I was added to the probe solution to achieve a final concentration of



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Figure 5. LO2 cells were treated with different doses of FFA (0,100, 150, 200 μM) for 24 h to establish a NAFLD cell model. Lipid accumulation was visualized using oil Red O staining (a) and then quantified according to the IOD value (b). Original magnification, ×200. Each measurement was triplicated (error bars indicate standard deviation). Statistical analysis is based on oneway ANOVA followed by a Dunnett’s test. ***P < 0.001 vs control group. hybridized with miR-21, resulting to the gradually increased fluorescence intensity. After 6 h of incubation, the intracellular fluorescence intensity reached its maximum (Figure S10), while the nanoprobe may run out at longer incubation time. Thus, the cells were directly incubated with the nanoprobe at 37 °C in 5% CO2 for 6 h. In order to demonstrate a low background image by dual energy transfer strategy, the QD-DNA-Au probe without BHQ2 was also used as a comparison to image miR-21 in the LO2 cells under the same conditions. As shown in Figure 6, comparing to the high background signals using only one acceptor of QD-DNA-Au probe (Figure 6a i), negligible fluorescence signals were observed in the normal LO2 cells

CONCLUSIONS

In summary, a low background fluorescence nanoprobe based on dual enery transfer of NEST and FRET is developed

(Figure 6a ii), which were attributed to the improved the energy transfer efficiency and quenching efficiency by using one donor and two acceptors. In addition, the fluorescence intensities in the steatosis cells were gradually increased as the severity of lipid accumulation increased (Figure 6a iii-v), indicating overexpression of miR-21 in the steatosis cells. The results demonstrated fluorescent signals of the QD-DNA-AuBHQ probe were correlated with the levels of the miR-21 in the steatosis cells. In conclusion, a dual energy transfer fluorescence nanoprobe can be used for the low background imaging of miR-21 in the steatosis cells based on the higher signal-to-background ratio. to image miR-21 in non-alcoholic fatty liver cells. Cell imaging technology avoids the use of any kind of enzyme or sophisticated equipments, making it more simple and cost-

Figure 6. Confocal fluorescence images of miR-21 (a) in normal LO2 cells (i,ii) and 100 μM (iii), 150 μM(iv), 200 μM (v) FFA treatedLO2 cells. The excitation wavelength was set at 365 nm, and fluorescence signals were collected at 605 nm. (b) The fluorescence intensity of QDs. Image-Pro Plus 6.0 (IPP) software (Media Cybernetics, USA) was employed to analyze the fluorescence images. 6 Statistical analysis is based on oneway ANOVA followed by a Dunnett’s test. ***P < 0.001 vs normal cells group incubated with QDDNA-AuBHQ probe.


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effective to understand the non-alcoholic fatty liver relative cellular events. In addition, the fluorescence nanoprobe exhibits high selectivity, nuclease resistance, good biocompatibility and cellular permeability. Importantly, high sensitivity is also achieved while background signal is greatly reduced through the combination of NEST and FRET between QDs and BHQ2coupled AuNPs. The current study presents a new strategy for monitoring nucleic acid molecules in a wide range of biological processes, which provides a potential tool for fundamental research and early clinical diagnostics, preventing disease from getting deteriorated.

ASSOCIATED CONTENT AUTHOR INFORMATION Corresponding Author *E-mail: [email protected]; [email protected], Tel: (+86) 23 68254659, Fax: (+86) 23 68367257.

Notes The authors declare no competing financial interest.

ACKNOWLEDGEMENTS This work was support by the National Natural Science Foundation of China (NSFC, No. 21535006 and No. 21705131) and the fund of Chongqing Research Program of Basic Research and Frontier Technology (No. cstc2017jcyjA1610). Supporting Information The sequence information; characterization of conjugation of DNA1 to AuNPs and QD-DNA-AuBHQ probes; optimization of the molar ratio between DNA1 and AuNPs or BHQ2 and AuNPs; FL lifetime measurements of QDs; nuclease stability of QDs-DNA-AuBHQ; viability of LO2 cells after incubated with QDs-DNA-AuBHQ probes; effect of FFA concentrations on the cell viability of LO2 cells; optimization of incubation time with QD-DNA-AuBHQ nanoprobe; Comparison of miR-21 detection between this work and other strategy; (PDF)

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for Table of Contents only：



Figure 5. LO2 cells were treated with different doses of FFA (0,100, 150, 200 μM) for 24 h to establish a NAFLD cell model. Lipid accumulation was visualized using oil Red O staining (a) and then quantified according to the IOD value (b). Original magnification, ×200. Each measurement was triplicated (error bars indicate standard deviation). Statistical analysis is based on oneway ANOVA followed by a Dunnett’s test. ***P < 0.001 vs control group.


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Dual Energy Transfer-Based Fluorescent ... - ACS Publications

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