FÃ¶rster Resonance Energy Transfer Based Soft Nanoballs for Specific

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Förster Resonance Energy Transfer Based Soft Nanoballs for Specific and Amplified Detection of MicroRNAs Yunying Cheng, Yi Fen Xie, Chun Mei Li, Yuan Fang Li, and Cheng Zhi Huang Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.9b01281 • Publication Date (Web): 03 Jul 2019 Downloaded from pubs.acs.org on July 26, 2019

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Analytical Chemistry

Förster Resonance Energy Transfer Based Soft Nanoballs for Specific and Amplified Detection of MicroRNAs Yun Ying Cheng†, Yi Fen Xie‡, Chun Mei Li ‡, *, Yuan Fang Li†, Cheng Zhi Huang†, ‡, * † Key

Laboratory of Luminescence and Real-Time Analytical Chemistry (Southwest University), Ministry of Education, College of Chemistry and Chemical Engineering, Southwest University, Chongqing 400715, P. R. China. ‡ College of Pharmaceutical Sciences, Southwest University, Chongqing 400715, P. R. China. *E-mail:

[email protected]; E-mail: [email protected]; Fax: +86 2368367257; Tel: +86 23 68254659

ABSTRACT: Förster resonance energy transfer (FRET) by using fluorescent carbon dots (CDs) as energy donors shows potential for biosensing and bioimaging. However, it remains underused and underestimated for CDs as a building block for FRET owing to the low efficiency and complex operation originating from the surface modification of CDs. To overcome these limitations, herein we develop a novel FRET soft nanoball (fretSNB) in which thousands of green CDs and black hole quencher 2 (BHQ-2) dyes are loaded, and FRET occurs from CDs to BHQ-2 dyes with the consequence of effective fluorescence quenching. These fretSNBs can be ruptured in the presence of phospholipase A2 (PLA2) released in a process of duplex-specific nuclease (DSN)-assisted target recycling amplification (TRA), making the fluorescence of CDs recovered. Thus, a dual amplification strategy is successfully developed for amplified detection of micro-ribonucleic acids (miRNAs) in the concentration range 0.025–10 nM with a limit of detection (3σ) reaching 16.5 pM which is about 515 times lower than without fretSNBs. In addition, the developed strategy exhibits high selectivity for discrimination of single nucleotide difference and capability to detect miRNAs extracted from cells, suggesting excellent potential in biomedical analysis and clinical diagnosis.

F

örster resonance energy transfer (FRET) is a powerful technique in biomedical analysis for simpleness, rapidness, high sensitivity and specificity.1-3 A series of FRET systems have been reported in terms the energy donors and acceptors, including fluorescent proteins,4,5 organic dyes,6,7 quantum dots,8,9 and carbon dots (CDs)10,11. As a rising star family in nanomaterials, CDs can be distinguished owing to their superior properties, such as non-blinking fluorescence emission, water solubility, photostability, and biosafety.12 Even though, CDs still remain underused and underappreciated building blocks for FRET owing to the limitations on their surface modification. Presently, almost of the existing FRET system based on CDs for biosensing generally involve non-covalent and covalent surface modification of CDs. For non-covalent modifications, the methods mainly including electrostatic bonding,13 π-π stacking,14 hydrogen bonding10 and coordination,15 are simple in operation but limited by poor selectivity. As for the covalent modifications, they are not only restricted to specific functional groups (such as amino and carboxyl groups) and increase the operation steps,16-21 but also exhibit low modification efficiency and possible change of the fluorescence properties of CDs, 22,23 which greatly confines the practical application of CDs. So, a best way for the utilization of CDs as a building block is to develop strategies of free from surface modification. Liposomes have been widely utilized to encapsulate numerous chemicals24-26 and nanocomponents27,28 based on their unique properties,29-32 which offer an opportunity to overcome the limitations of CDs on the following basis: 1) liposomes can synchronously entrap donors and acceptors within a single interior bringing them together in close proximity, and enhancing FRET efficiency from donors to acceptors; 2) liposomes can be employed for sensitivity improvement as a signal amplification strategy. Thus,

liposomes can be utilized to form FRET-based soft nanoballs (fretSNBs) by encapsulating thousands of CDs and black hole quencher 2 (BHQ-2) dyes into a single liposome interior, allowing the use of CDs as a building block without surface modification. To confirm the feasibility of the proposed strategy, micro-ribonucleic acids (miRNAs) were chosen as a proof-of-concept target. MiRNA are short single-stranded regulatory RNAs with a length of 18−25 nucleotides, which have become the subject of intensive investigation due to their critical roles in the regulation of a vast range of physiologic and pathologic processes.33 Dysregulated expression of miRNA is typically related to the development of various diseases in humans, such as cancer.34,35 Therefore, the expression profiles of miRNAs have been widely used as a class of accurate prognostic biomarkers, predictors of patient response to therapies and tools for investigating their function.36-39 Although several standard technological methods, such as Northern blotting,40 quantitative real time polymerase chain reaction (qPCR),41 microarray 42 hybridization and next-generation sequencing43, have been developed to investigate the expression profiles of miRNAs. In addition to the complex operation and requirement of specialized expertise, these techniques also display poor specificity. Nanomaterials-based FRET, however, brings new opportunities for developing next-generation miRNA detection. Among them, CDs have shown great potentials in the FRET-based miRNA detection20,44-48. In principle, these CD-based methods for miRNA detection are based on signal enhancement which is usually achieved through the assembly of capture DNAs on the surface of a single CD, thus the selectivity, which principally depends on the efficiency of specific hybridization and modification, is limited. Duplex-specific nuclease (DSN), a highly stable endonuclease, specifically hydrolyzes DNA in DNA/RNA

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heteroduplexes, releasing intact RNA and perfectly recognizing one-nucleotide mismatched duplexes.49 As a result, DSN-assisted target recycling amplification (TRA) was developed with simplicity, high selectivity and sensitivity. Inspired by the benefits referred above, we herein report a dual-amplification strategy combined with fretSNBs and DSN-assisted TRA for specific and amplified detection of miRNAs.

EXPERIMENTAL SECTION Materials and Reagents. Ethanediamine, p-benzoquinone, 1,2-diacyl-sn-glycero-3-phosphocholine (PC) and β-cholestanol were purchased from Aladdin Reagent Co., Ltd (Shanghai, China). 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methox y (polyethylene glycol)-2000] (DSPE-PEG2000) was obtained from Ponsure Biotechnology Co., Ltd. (Shanghai, China). HPLC-purified DNA oligonucleotides and miRNAs were commercially synthesized by Sangon Biological Engineering Technology and Services Co., Ltd. (Shanghai, China), and the sequences were listed in Table S1. Streptavidin-coated magnetic beads (MBs) of 1 μm diameter and phospholipase A2 from honey bee venom were obtained from Sigma Aldrich. BHQ-2 amines were purchased from Biosearch Technologies (Novato, CA, USA). Duplex-specific nuclease (DSN) was obtained from Evrogen Joint Stock Company (Russia). One Step PrimeScript® miRNA cDNA synthesis kit and SYBR® Premix Ex TaqTM II kit were purchased from Takara Biotechnology Co. Ltd. (Dalian, China). All other reagents were of analytical grade. Preparation of CDs. The CDs were prepared through a self-exothermic reaction, as previously reported50 with slight modifications. Briefly, 50 mg p-benzoquinone and 200 μL ethanediamine were codissolved in 5 mL ultrapure water at ambient temperature and reacted for 16 h, from which a deep brown-colored solution was obtained. The non-luminous components were precipitated using methyl alcohol, and the supernatant was speared out for further purification on a silica gel column. Finally, the water soluble and photostable CDs solution was retrieved by rotary evaporation. Preparation of FRET soft nanoballs. Three lipids, PC, cholestenol and DSPE-PEG 2000 with a molar ratio of 20 : 10 : 2 were codissolved in 2 mL chloroform and mixed well at 40°C in a flask. The chloroform was evaporated using a rotary evaporator under vacuum for 30 min, with all traces eliminated by continuing evaporation under vacuum overnight. The dried lipid film was then hydrated in 2 mL 4-(2-hydroxyethyl)-1-piperazine-ethanesulphonic acid (HEPES) buffer containing 0.5 mg/mL CDs and 0.6 mg/mL BHQ-2 dye, which were mixed for 30 min in a shaker at 37°C. In order to obtain a homogeneous suspension of liposomes of uniform size, the hydrated solution was extruded 15 times through a polycarbonate membrane with the diameter pores of 200 nm. Untrapped CDs and BHQ-2 were removed by dialysis (Mwco of the membrane: 300 kDa) for 24 h at 4°C. Ultimately, fretSNBs obtained using this method were stored at 4°C. The FRET efficiency (E) between CDs and BHQ-2 in the fretSNBs was estimated according to the equation (1).51

  E  1   da   d 

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in the presence and absence of BHQ-2, respectively. Encapsulation efficiency (EE) of CDs and BHQ-2 in the fretSNBs. First, the absorbance of CDs and BHQ-2 with different concentrations at 408 and 556 nm, respectively, were measured, and then the corresponding standard curves of absorbance were plotted using Origin 8.6 software (Figure S2). The concentration of CDs and BHQ-2 in fretSNBs was determined after complete rupture of fretSNBs by measuring absorbance at 408 and 556 nm, respectively, and fitting to the corresponding standard curve. Finally, the EE was calculated according to the equation (2): c1

EE = c0 × 100 %

(2)

Where c1 is the concentrations of encapsulated CDs or BHQ-2 in the fretSNBs, and c0 represents the concentrations of CDs or BHQ-2 in the initial solution before the formation of fretSNBs. Preparation of MBs–DNA-PLA2 Conjugate. DNA-PLA2 conjugate was prepared by the maleimide-thiol reaction using hetero-bifunctional linker sulfosuccinimidyl 4-(N-maleimidomethyl) cyclohexane-1-carboxylate (sulfo-SMCC) according to the reported methods.52 Briefly, 30 μL of thiol-DNA (1 mM), 2 μL of 1 M sodium phosphate buffer (pH 5.5), and 2 μL TCEP (30 mM) were mixed and incubated at room temperature for 1 hour. Then the mixture was purified using tubular ultrafiltration membranes (10K) prior to washing with 0.1 M NaCl and 0.1 M sodium phosphate buffer PBS (Buffer A, pH 7.3) by 8 times. Concurrently, 1 mg sulfo-SMCC was added to 200 μL PLA2 (5 mg/mL) in Buffer A, followed by vortexing for 5 minutes and then shaking for 1 hour at room temperature. The mixture was purified using tubular ultrafiltration membranes (10K) and then washed with Buffer A for 8 times. The purified sulfo-SMCC-activated PLA2 was then incubated with the purified thiol-DNA at room temperature for 48 hours. The solution was purified using tubular ultrafiltration membranes (30K), and washed with Buffer A for 8 times in order to remove unreacted thiol-DNA and PLA2. Finally, 200 μL of DNA-PLA2 conjugates were incubated with 1 mL of streptavidin-coated magnetic beads (1 mg/mL) at room temperature for 30 min. The solution was rinsed three times with PBS (pH 7.4) to remove excess DNA-PLA2, and then resuspended in 1 mL of PBS. In this assay, the PBS was prepared with DEPC-treated water. Procedure for detection of miRNAs. Firstly, 50 μL of a reaction mixture containing MB-DNA-PLA2 probes (5 nM), 25 μL target miRNAs (of varying concentrations), 2 μL 1×DSN buffer (50 mM Tris-HCl, pH 8.0, 5 mM MgCl2, 1 mM DTT), 0.08 U DSN and 20 U RNase inhibitor were incubated at 50ºC for 2 h, and then cooled to ambient temperature. After a magnetic separation step, the supernatant was added to 150 μL fretSNBs and incubated at 37ºC for 50 min. The fluorescence intensity of the solution obtained was measured using a Hitachi Model F-2500 fluorescence spectrophotometer (Tokyo, Japan).

(1)

where τda and τd represent the fluorescence lifetimes of CDs

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Scheme 1. Illustration of fretSNBs-based dual amplification strategy for miRNA detection. Cell culture and RNA extraction. The human breast cancer cell line (MDA-MB231), cervical cancer cell line (HeLa) and normal cell line (human umbilical vein endothelial cells, HUVEC) were cultured in Dulbecco’s modified eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 100 U/mL penicillin-streptomycin at 37ºC in a humidified atmosphere containing 5% CO2. The cells were harvested by trypsinization. Total RNAs from all cell lines were isolated using Trizol reagent according to the manufacturer's instructions.

RESULTS AND DISCUSSION Principle of dual-amplification strategy for miRNA detection. The strategy for detection of miRNA on the basis of DSN-assisted TRA and fretSNBs is illustrated in Scheme 1. DNA oligonucleotides, comprising the complementary sequences for the target miRNAs and spacer sequence, were modified with a thiol and biotin group at their 5’ and 3’-termini, respectively (Table S1). After conjugation of the surface amino groups on the PLA2 with linker DNA using the heterobifunctional reagent sulfo-SMCC, the DNA-PLA2 was bound to the streptavidin coated magnetic beads through a biotin–streptavidin linkage. In this way, the MB–DNA– PLA2 probe was obtained, integrating the functions of magnetic separation, target recognition and signal induction. In the presence of miRNAs, the probes form DNA/RNA heteroduplexes. DSN, which can discriminate between perfectly and imperfectly matched short duplexes (even one-nucleotide mismatch), is able to hydrolyze DNA in DNA/RNA heteroduplexes with no effect on the RNA, resulting in the release of miRNA and PLA2 from the MB-DNA-PLA2 probes. On one hand, the released miRNAs bind to other MB-DNA-PLA2 probes, catalyzing the next round of hydrolysis for many cycles, and triggering a large release of PLA2. On the other hand, hundreds of CDs and BHQ-2 dye molecules were entrapped in the interior of a single liposome, enforcing them into close proximity that is enough to quench the fluorescence of CDs through FRET and self-quenching. Ultimately, the released PLA2 ruptured the liposomes, enlarging the distance between CDs and BHQ-2 dye, preventing FRET and consequently recovering the fluorescence of CDs. Each liposome was able to entrap a large number of CDs and BHQ-2 molecules, thus, the liposomes served as a tool for the second round of amplification. As a result, this dual-amplification strategy can sensitively and selectively detect miRNAs. Preparation and characterization of fretSNBs. To test

our hypothesis, we utilized CDs as energy donors and BHQ-2 as acceptors. The size and morphology of the prepared CDs were characterized by transmission electron microscopy (TEM). The as-prepared CDs were well mono-dispersed with a mean diameter of 2.02 ± 0.47 nm (Figure S1) and lattice spacing of 0.21 nm and 0.34 nm, which is consistent with previously reported results.50 The emission spectrum of the CDs peaked at 530 nm (green), which is close to the typical peak absorption of BHQ-2 at 556 nm. Notably, the emission spectrum of the CDs overlapped well with the absorption spectrum of BHQ-2 (Figure 1a), demonstrating that these CDs and BHQ-2 are promising candidates for construction of FRET-based probes.

Figure 1. Characterization of fretSNBs. (a) Overlap between

the absorption spectrum of BHQ-2 and emission spectrum of CDs under excitation at 400 nm. (b) Hydrodynamic diameter determined by DLS. (c) SEM (left) and TEM images (right). Scale bar, 200 nm (left) and 100 nm (right). (d) UV-vis absorption spectra. (e) Fluorescence spectra. (f) Fluorescence lifetime decay curves of encapsulated CDs in absence and presence of BHQ-2 in the SNBs (λ=400 nm). The TEM images showed that the as-prepared fretSNBs


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were spherical with a mean diameter of ∼170 nm, which is further confirmed by the DLS result (Figures 1b, c). Both the TEM images and absorption spectra of fretSNBs with two characteristic peaks at 408 nm and 556 nm corresponding to CDs and BHQ-2, respectively, indicated the successful encapsulation of CDs and BHQ-2 in the fretSNBs (Figures 1c, d). The encapsulation efficiency of CDs and BHQ-2 were calculated to be approximately 36.8% and 43.6%, respectively (Figure S2). Moreover, strong fluorescence quenching and recovery of CDs before or after rupture of fretSNBs and the change in fluorescence lifetime (τ) in the presence or absence of BHQ-2 dye further confirmed the happening of FRET during the encapsulation of CDs and BHQ-2 within a single fretSNB (Figures 1e, f). The long-term stability of fretSNBs is an important factor for reproducibility of the detection assay in this system. Here, variation in the fluorescence intensity of fretSNBs was monitored to characterize their ability to retain entrapped CDs and BHQ-2 over 27 days. As shown in Figure S3, no apparent change was observed, suggesting that minimal leakage had occurred over the course of 27 days. This indicated that the fretSNBs exhibited excellent stability in appropriate storage conditions. Characterization of DNA-PLA2 conjugates. To precisely control the release of PLA2 which can hydrolyze the ester bond at the sn-2 position of dietary phospholipids53 and rupture the liposomes, DNA-PLA2 conjugates were constructed using the heterobifunctional linker sulfo-SMCC.54 As depicted in Figure 2a, the migration rate of DNA-PLA2 on the SDS-PAGE gel was clearly slower than that of free PLA2. Furthermore, the DNA-PLA2 conjugates slowly migrated with ladder-like bands ranging from ~25-100 kDa owing to the binding of one or multiple DNA strands to one PLA2, while free PLA2 or a simple mixture of DNA and free PLA2 (DNA+PLA2) exhibited only one band at ~18 kDa, corresponding to the molecular weight of PLA2. These observations indicated the successful conjugation of DNA and PLA2, which was further confirmed by UV absorption spectra (Figure 2b). The addition of DNA-PLA2 induced a change in the hydrodynamic diameter of fretSNBs and enhanced fluorescence intensity, indicating the rupture of fretSNBs (Figures 2c, d). This suggests that the DNA-PLA2 conjugate still retained partial enzyme activity, which may attribute to the lack of relevance of surface amine groups in PLA2 and enzymatic active sites.52 To verify the efficacy of the proposed method, miR-141, a candidate prostate and breast cancer biomarker,55,56 was chosen as a proof-of-concept target. The fluorescence emission spectra of the CDs were measured under different conditions. As shown in Figure S4, in the absence of miR-141 and DSN, the MB–DNA–PLA2 probes remained intact and were easily removed by magnetic separation, resulting in the weak fluorescence signals. Similarly, the fluorescence intensity of the system remained unchanged after the addition of miR-141 or DSN alone. Only in the presence of both miR-141 and DSN can cause a strong fluorescence signal. These results indicated that the proposed strategy could specifically detect miRNA.

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Figure 2. Characterization of DNA-PLA2 conjugates. (a) Protein-staining image and (b) UV absorption spectrum of DNA, PLA2, and DNA-PLA2 conjugates. (c) DLS results and (d) fluorescence spectra of fretSNBs before and after adding DNA-PLA2. DNA+PLA2, simple mixture of DNA and PLA2; DNA-PLA2, conjugates of DNA and PLA2. The fretSNBs in the PBS solution was used as a control. Optimization of experimental conditions. To achieve the best performance for miRNA detection, a series of experimental conditions were optimized. Firstly, the effect of the mass ratio of BHQ-2 to CDs encapsulated in the fretSNBs on FRET efficiency was investigated, showing that the FRET efficiency reached at 90.6% when the concentration ratio of BHQ-2 and CDs increased to 4.8 (Figure S5). The value of (F/F0–1) increased with the ratio rising in the range of 0.3-1.2, but decreased when the ratio was greater than 1.2 (Figure S6a), which can be attributed to the low FRET efficiency at low mass ratio and ineffective fluorescence recovery of CDs at high ratio of BHQ-2 and CDs. Additionally, over a certain range, the concentrations of CDs and BHQ-2 molecules in a single liposome also can affect the FRET efficiency. As shown in the Figure S6b, the (F/F0–1) value reached its maximum value when the concentrations of BHQ-2 and CDs were 0.6 and 0.5 mg/mL, respectively. Given that the number of oligonucleotide per MB affected hybridization efficiency of DNA/RNA heteroduplexes, various concentrations of MBs were assessed (Figure S7a). At a fixed concentration of DNA-PLA2, the highest (F/F0–1) value was obtained when the concentration of MBs is 50 μg/mL. That might be attributed to insufficient PLA2 released from MBs and steric effects at low and high concentrations, respectively. A significant enhancement in (F/F0–1) was observed for increasing MB-DNA-PLA2 concentrations in the range 0.25 to 5 nM, followed by a decrease for concentrations greater than 5 nM, owing to the influence of an excess of probe (Figure S7b). As an amplifier, the quantity of DSN is also an important factor for the sensitivity and selectivity of this assay system. The (F/F0–1) value increased with concentration of DSN increasing in the range 0.02–0.08 U because of the DSN-assisted amplification (Figure S7c). To economize on enzyme consumption, 0.08 U of DSN was selected as the optimum concentration. The fluorescence signal was enhanced in the temperature range from 25°C to 50°C and then sharply decreased (Figure S7d), possibly resulting from the partial inactivation of PLA2


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Analytical Chemistry and denaturation of DNA/RNA heteroduplexes at high temperatures. In addition, the experimental results (Figures S7e and f) showed that the optimal reaction time for DSN and PLA2 were 120 min and 50 min, respectively. High sensitivity and selectivity of miR-141 detection. The sensitivity of the proposed strategy was investigated under optimum conditions. As shown in Figure 3a, a gradual increase in fluorescence intensity was observed as concentrations of miR-141 increased from 0.01 to 75 nM. A linear correlation of (F/F0–1) values was observed for miR-141 concentration over 3 orders of magnitude from 0.025 to 10 nM (Figures 3b, c). The calibration equation was (F/F0– 1) = 0.14312 + 0.09457c with a correlation coefficient R2 = 0.995, where F and F0 represents fluorescence intensity in the presence and absence of miR-141, respectively and c is the concentration of miR-141. The limit of detection (LOD) for miR-141 was 16.5 pM by evaluating the mean response of the negative control plus 3 times standard deviation (3σ, n=11). The LOD of this method for miRNA detection is comparable with or even more sensitive than that of some reported methods (Table S2).

homology, including miR-141, one-nucleotide difference (mis-1), two-nucleotide difference (mis-2), four-nucleotide difference (mis-4), and miR-21 under the same optimal conditions (Table S1). As shown in Figure 4, (F/F0–1) values in the presence of miR-141 were approximately 5.9, 22.8, 50.2 and 96.7-fold higher than that of mis-1, mis-2, mis-4 and miR-21, respectively, indicating that the selectivity of the proposed method is high enough to discriminate single-nucleotide difference. The high selectivity can be principally attributed to the immobilization stability of PLA2 on the MB-DNA-PLA2 probe, magnetic separation and the DSN-assisted step, which critically depends on a perfect match between the miRNA and probe DNA. Therefore, the specificity of the proposed two-step method is better than the previously reported one-step method.57-59

Figure 4. Specificity of miR-141 detection. Concentrations: miR-141, mis-1, mis-2, mis-4, and miR-21, 8 nM. Error bars indicate standard deviation (n=3).

3. The fluorescence response at different concentrations of miR-141. (a) The changes in the (F/F0–1) values of fretSNBs at different concentrations range from 0.01 to 75 nM. (b) Fluorescence emission spectra of fretSNBs upon addition of miR-141 (0 nM, 0.025 nM, 1 nM, 2.5 nM, 5 nM, 10 nM). (c) Linear relationship between Figure

(F/F0-1) value and the concentration of miR-141 with fretSNBs or (d) with CDs-SNBs. Error bars indicate standard deviation (n=3). λex=400 nm; λem=530 nm. In addition, the fluorescence responses at different concentrations of miR-141 without FRET, namely in the absence of BHQ-2 (CDs-SNBs), were also measured (Figure 3d). The (F/F0–1) value of this system was linearly dependent on the concentration of miR-141 in the range of 10–50 nM, with the linear regression equation: (F/F0–1) = 0.009c – 0.05276 (R2 = 0.975). The LOD was calculated to be 8.5 nM (3σ, n=11), considerably higher than that of fretSNBs. These observations indicate that fretSNBs not only are feasible for the detection of miRNAs, but also can act as an amplification carrier, which improved the detection sensitivity with approximately 10.5-fold, and reduced the LOD with approximately 515-fold. Owing to high sequence homology, a great challenge for specific miRNA detection is selective discrimination. Thus, the specificity of the proposed method was verified by discrimination of five different miRNA sequences with high

Detection of endogenous miRNAs from human cancer cells. It is well-known that miRNAs are often overexpressed in cancer cells, which has been proven valuable for early phase diagnosis.55,59 Therefore, to verify the capability of the proposed method for the detection of miRNAs in cell extracts, the expressions of miR-141 in three cell types, including human umbilical vein endothelial cells (HUVEC, normal cell line), breast cancer cells (MDA-MB-231) and cervical cancer cells (HeLa), were quantified by the proposed method and a commercial real-time quantitative PCR (qPCR) kit simultaneously (Figure 5). The results showed different expression levels in these three cell lines with the highest expression of miR-141 in MDA-MB-231 cells, and extremely low expression in HUVECs and HeLa cells. These observations are in good agreement with data reported previously.49,55 In addition, the result was consistent with that obtained by qPCR, suggesting the reliability of the proposed method for miRNA quantification. These results clearly demonstrated that the proposed method can be used for miRNA detection in complex samples.

Figure 5. Detection of miR-141 in different cell lines as measured by using our method and qPCR. Error bars indicate standard

deviation (n=3).


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CONCLUSIONS In summary, the fretSNBs were formed by the simultaneous encapsulation of CDs (energy donor) and BHQ-2 (energy acceptor) in a single liposome, which not only can provide a platform to construct CD-based FRET without surface modification of CDs, but also can act as an amplifier in biochemical analysis by carrying thousands of CDs and BHQ-2 molecules. With the combination of fretSNBs and DSN-assisted TRA, a dual amplification strategy has been developed for specific and amplified detection of miRNAs with the limit of detection reaching 16.5 pM, which is 515 fold lower than that of the method without fretSNBs. Moreover, the proposed method is selective enough to discriminate homologous sequences with single-nucleotide difference. Therefore, the proposed method has great potential in biomedical analysis and clinical diagnosis, and affords a new opportunity to extend the application of CDs in FRET systems without covalent modification. Besides the fretSNBs, other types of the liposomes-based SNBs have been developed, such as photothermal soft nanoballs for immunoassay with high sensitivity without the use of expensive instruments60, suggesting the versatility and potential of SNBs.

ASSOCIATED CONTENT Supporting Information Additional data and information as noted in the text. This material is available free of charge via the Internet at http://pubs.acs.org. Oligonucleotide sequences; HRTEM images; standard curves; stability of fretSNBs; optimization of experimental conditions (PDF)

AUTHOR INFORMATION Corresponding Author *E-mail: [email protected]； *E-mail: [email protected], Tel: (+86) 23 68254659, Fax: (+86) 23 68367257. Notes The authors declare no competing financial interest.

ACKNOWLEDGEMENTS We are grateful for the financial support from the National Natural Science Foundation of China (NSFC, Grant no. 21535006 and No. 21705131) and the Fundamental Research Funds for the Central Universities (XDJK2018C088).

REFERENCE (1) Ray, P. C.; Fan, Z.; Crouch, R. A.; Sinha, S. S.; Pramanik, A. Nanoscopic Optical Rulers Beyond the FRET Distance Limit: Fundamentals and Applications. Chem. Soc. Rev. 2014, 43, 6370-6404. (2) Hohng, S.; Lee, S.; Lee, J.; Jo, M. H. Maximizing Information Content of Single-Molecule FRET Experiments: Multi-Color FRET and FRET Combined with Force or Torque. Chem. Soc. Rev. 2014, 43, 1007-1013. (3) Chen, G.; Song, F.; Xiong, X.; Peng, X. Fluorescent Nanosensors Based on Fluorescence Resonance Energy Transfer (FRET). Ind. Eng. Chem. Res. 2013, 52, 11228-11245. (4) Haga, Y.; Ishii, K.; Hibino, K.; Sako, Y.; Ito, Y.;

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Taniguchi, N.; Suzuki, T. Visualizing Specific Protein Glycoforms by Transmembrane Fluorescence Resonance Energy Transfer. Nat. Commun. 2012, 3, 907. (5) Bindels, D. S.; Haarbosch, L.; van Weeren, L.; Postma, M.; Wiese, K. E.; Mastop, M.; Aumonier, S.; Gotthard, G.; Royant, A.; Hink, M. A.; Gadella Jr, T. W. J. Mscarlet: A Bright Monomeric Red Fluorescent Protein for Cellular Imaging. Nat. Methods 2016, 14, 53. (6) Imamura, H.; Huynh Nhat, K. P.; Togawa, H.; Saito, K.; Iino, R.; Kato-Yamada, Y.; Nagai, T.; Noji, H. Visualization of Atp Levels inside Single Living Cells with Fluorescence Resonance Energy Transfer-Based Genetically Encoded Indicators. Proc. Natl. Acad. Sci. 2009, 106, 15651. (7) Lai, J.; Shah, B. P.; Garfunkel, E.; Lee, K. B. Versatile Fluorescence Resonance Energy Transfer-Based Mesoporous Silica Nanoparticles for Real-Time Monitoring of Drug Release. ACS Nano 2013, 7, 2741-2750. (8) Hu, J.; Liu, M.h.; Zhang, C.y. Integration of Isothermal Amplification with Quantum Dot-Based Fluorescence Resonance Energy Transfer for Simultaneous Detection of Multiple MicroRNAs. Chem. Sci. 2018, 9, 4258-4267. (9) Zhen, S. J.; Zhuang, H. L.; Wang, J.; Huang, C. Z. Dual-Aptamer-Based Sensitive and Selective Detection of Prion Protein through the Fluorescence Resonance Energy Transfer between Quantum Dots and Graphene Oxide. Anal. Methods 2013, 5, 6904-6907. (10) Wei, W.; Xu, C.; Ren, J.; Xu, B.; Qu, X. Sensing Metal Ions with Ion Selectivity of a Crown Ether and Fluorescence Resonance Energy Transfer between Carbon Dots and Graphene. Chem. Commun. 2012, 48, 1284-1286. (11) Tang, J.; Kong, B.; Wu, H.; Xu, M.; Wang, Y.; Wang, Y.; Zhao, D.; Zheng, G. Carbon Nanodots Featuring Efficient FRET for Real-Time Monitoring of Drug Delivery and Two-Photon Imaging. Adv. Mater. 2013, 25, 6569-6574. (12) Sun, Y. P.; Zhou, B.; Lin, Y.; Wang, W.; Fernando, K. A. S.; Pathak, P.; Meziani, M. J.; Harruff, B. A.; Wang, X.; Wang, H.; Luo, P. G.; Yang, H.; Kose, M. E.; Chen, B.; Veca, L. M.; Xie, S. Y. Quantum-Sized Carbon Dots for Bright and Colorful Photoluminescence. J. Am. Chem. Soc. 2006, 128, 7756-7757. (13) Yang, W.; Ni, J.; Luo, F.; Weng, W.; Wei, Q.; Lin, Z.; Chen, G. Cationic Carbon Dots for Modification-Free Detection of Hyaluronidase Via an Electrostatic-Controlled Ratiometric Fluorescence Assay. Anal. Chem. 2017, 89, 8384-8390. (14) Li, R. S.; Yuan, B.; Liu, J. H.; Liu, M. L.; Gao, P. F.; Li, Y. F.; Li, M.; Huang, C. Z. Boron and Nitrogen Co-Doped Single-Layered Graphene Quantum Dots: A High-Affinity Platform for Visualizing the Dynamic Invasion of HIV DNA into Living Cells through Fluorescence Resonance Energy Transfer. J. Mater. Chem. B 2017, 5, 8719-8724. (15) Xu, M.; Gao, Z.; Zhou, Q.; Lin, Y.; Lu, M.; Tang, D. Terbium Ion-Coordinated Carbon Dots for Fluorescent Aptasensing of Adenosine 5′-Triphosphate with Unmodified Gold Nanoparticles. Biosens. Bioelectron. 2016, 86, 978-984. (16) Wang, Y.; Ma, T.; Ma, S.; Liu, Y.; Tian, Y.; Wang, R.; Jiang, Y.; Hou, D.; Wang, J. Fluorometric Determination of the Antibiotic Kanamycin by Aptamer-Induced FRET Quenching and Recovery between Mos2 Nanosheets and Carbon Dots. Microchim. Acta 2017, 184, 203-210. (17) Qian, Z. S.; Shan, X. Y.; Chai, L. J.; Ma, J. J.; Chen, J. R.; Feng, H. A Universal Fluorescence Sensing Strategy Based on Biocompatible Graphene Quantum Dots and Graphene Oxide for the Detection of DNA. Nanoscale 2014, 6, 5671-5674.


6

Page 7 of 9 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

Analytical Chemistry (18) Duan, N.; Wu, S.; Dai, S.; Miao, T.; Chen, J.; Wang, Z. Simultaneous Detection of Pathogenic Bacteria Using an Aptamer Based Biosensor and Dual Fluorescence Resonance Energy Transfer from Quantum Dots to Carbon Nanoparticles. Microchim. Acta 2015, 182, 917-923. (19) Qaddare, S. H.; Salimi, A. Amplified Fluorescent Sensing of DNA Using Luminescent Carbon Dots and AuNPs/GO as a Sensing Platform: A Novel Coupling of FRET and DNA Hybridization for Homogeneous HIV-1 Gene Detection at Femtomolar Level. Biosens. Bioelectron. 2017, 89, 773-780. (20) Zhang, H.; Wang, Y.; Zhao, D.; Zeng, D.; Xia, J.; Aldalbahi, A.; Wang, C.; San, L.; Fan, C.; Zuo, X.; Mi, X. Universal Fluorescence Biosensor Platform Based on Graphene Quantum Dots and Pyrene-Functionalized Molecular Beacons for Detection of MicroRNAs. ACS Appl. Mater. Inter. 2015, 7, 16152-16156. (21) Wu, X.; Sun, S.; Wang, Y.; Zhu, J.; Jiang, K.; Leng, Y.; Shu, Q.; Lin, H. A Fluorescent Carbon-Dots-Based Mitochondria-Targetable Nanoprobe for Peroxynitrite Sensing in Living Cells. Biosens. Bioelectron. 2017, 90, 501-507. (22) Cheng, L.; Li, Y.; Zhai, X.; Xu, B.; Cao, Z.; Liu, W. Polycation-B-Polyzwitterion Copolymer Grafted Luminescent Carbon Dots as a Multifunctional Platform for Serum-Resistant Gene Delivery and Bioimaging. ACS Appl. Mater. Inter. 2014, 6, 20487-20497. (23) Gao, M. X.; Yang, L.; Zheng, Y.; Yang, X. X.; Zou, H. Y.; Han, J.; Liu, Z. X.; Li, Y. F.; Huang, C. Z. "Click" on Alkynylated Carbon Quantum Dots: An Efficient Surface Functionalization for Specific Biosensing and Bioimaging. Chem. Eur. J. 2017, 23, 2171-2178. (24) Tian, X.; Liu, X.; Wang, A.; Lau, C.; Lu, J. Bioluminescence Imaging of Carbon Monoxide in Living Cells and Nude Mice Based on Pd0-Mediated Tsuji–Trost Reaction. Anal. Chem. 2018, 90, 5951-5958. (25) Lin, Y.; Zhou, Q.; Tang, D. Dopamine-Loaded Liposomes for in-Situ Amplified Photoelectrochemical Immunoassay of AFB1 to Enhance Photocurrent of Mn2+-Doped Zn3(OH)2V2O7 Nanobelts. Anal. Chem. 2017, 89, 11803-11810. (26) Chen, W.Y.; Chen, L.Y.; Ou, C.M.; Huang, C.C.; Wei, S.C.; Chang, H.T. Synthesis of Fluorescent Gold Nanodot– Liposome Hybrids for Detection of Phospholipase C and Its Inhibitor. Anal. Chem. 2013, 85, 8834-8840. (27) Zhou, J.; Wang, Q.; Zhang, C. Liposome–Quantum Dot Complexes Enable Multiplexed Detection of Attomolar Dnas without Target Amplification. J. Am. Chem. Soc. 2013, 135, 2056-2059. (28) Chen, L.J.; Yang, C.X.; Yan, X.P. Liposome-Coated Persistent Luminescence Nanoparticles as Luminescence Trackable Drug Carrier for Chemotherapy. Anal. Chem. 2017, 89, 6936-6939. (29) Al-Jamal, W. T.; Kostarelos, K. Liposomes: From a Clinically Established Drug Delivery System to a Nanoparticle Platform for Theranostic Nanomedicine. Acc Chem Res 2011, 44, 1094-1104. (30) Zhou, F.; Li, B. Exonuclease Iii-Assisted Target Recycling Amplification Coupled with Liposome-Assisted Amplification: One-Step and Dual-Amplification Strategy for Highly Sensitive Fluorescence Detection of DNA. Anal. Chem. 2015, 87, 7156-7162. (31) Mukthavaram, R.; Wrasidlo, W.; Hall, D.; Kesari, S.; Makale, M. Assembly and Targeting of Liposomal Nanoparticles Encapsulating Quantum Dots. Bioconjugate Chem. 2011, 22, 1638-1644. (32) Peer, D.; Karp, J. M.; Hong, S.; Farokhzad, O. C.;

Margalit, R.; Langer, R. Nanocarriers as an Emerging Platform for Cancer Therapy. Nat. Nanotechnol. 2007, 2, 751. (33) Bartel, D. P. MicroRNAs: Genomics, Biogenesis, Mechanism, and Function. Cell 2004, 116, 281-297. (34) Kloosterman, W. P.; Plasterk, R. H. A. The Diverse Functions of MicroRNAs in Animal Development and Disease. Dev. Cell 2006, 11, 441-450. (35) Stefani, G.; Slack, F. J. Small Non-Coding RNAs in Animal Development. Nat. Rev. Mol. Cell. Biol. 2008, 9, 219-230. (36) Lagos-Quintana, M.; Rauhut, R.; Lendeckel, W.; Tuschl, T. Identification of Novel Genes Coding for Small Expressed RNAs. Science 2001, 294, 853-858. (37) Mitchell, P. S.; Parkin, R. K.; Kroh, E. M.; Fritz, B. R.; Wyman, S. K.; Pogosova-Agadjanyan, E. L.; Peterson, A.; Noteboom, J.; O'Briant, K. C.; Allen, A.; Lin, D. W.; Urban, N.; Drescher, C. W.; Knudsen, B. S.; Stirewalt, D. L.; Gentleman, R.; Vessella, R. L.; Nelson, P. S.; Martin, D. B.; Tewari, M. Circulating MicroRNAs as Stable Blood-Based Markers for Cancer Detection. Proc. Natl. Acad. Sci. USA 2008, 105, 10513-10518. (38) Esquela-Kerscher, A.; Slack, F. J. Oncomirs MicroRNAs with a Role in Cancer. Nat. Rev. Cancer 2006, 6, 259-269. (39) Dong, H.; Lei, J.; Ding, L.; Wen, Y.; Ju, H.; Zhang, X. MicroRNA: Function, Detection, and Bioanalysis. Chem. Rev. 2013, 113, 6207-6233. (40) Várallyay, É.; Burgyán, J.; Havelda, Z. MicroRNA Detection by Northern Blotting Using Locked Nucleic Acid Probes. Nat. Protoc. 2008, 3, 190-196. (41) Chen, C.; Ridzon, D. A.; Broomer, A. J.; Zhou, Z.; Lee, D. H.; Nguyen, J. T.; Barbisin, M.; Xu, N. L.; Mahuvakar, V. R.; Andersen, M. R.; Lao, K. Q.; Livak, K. J.; Guegler, K. J. Real-Time Quantification of MicroRNAs by Stem-Loop RT-PCR. Nucleic Acids Res 2005, 33, e179. (42) Git, A.; Dvinge, H.; Salmon-Divon, M.; Osborne, M.; Kutter, C.; Hadfield, J.; Bertone, P.; Caldas, C. Systematic Comparison of Microarray Profiling, Real-Time PCR, and Next-Generation Sequencing Technologies for Measuring Differential MicroRNA Expression. RNA (New York, N.Y.) 2010, 16, 991-1006. (43) Tao, W.; Sun, L.; Shi, H.; Cheng, Y.; Jiang, D.; Fu, B.; Conte, M. A.; Gammerdinger, W. J.; Kocher, T. D.; Wang, D. Integrated Analysis of miRNA and mRNA Expression Profiles in Tilapia Gonads at an Early Stage of Sex Differentiation. BMC Genomics 2016, 17, 328. (44) Cao, Y.; Dong, H.; Yang, Z.; Zhong, X.; Chen, Y.; Dai, W.; Zhang, X. Aptamer-Conjugated Graphene Quantum Dots/Porphyrin Derivative Theranostic Agent for Intracellular Cancer-Related MicroRNA Detection and Fluorescence-Guided Photothermal/Photodynamic Synergetic Therapy. ACS Appl. Mater. Inter. 2017, 9, 159-166. (45) Laurenti, M.; Paez-Perez, M.; Algarra, M.; Alonso-Cristobal, P.; Lopez-Cabarcos, E.; Mendez-Gonzalez, D.; Rubio-Retama, J. Enhancement of the Upconversion Emission by Visible-to-near-Infrared Fluorescent Graphene Quantum Dots for MiRNA Detection. ACS Appl. Mater. Inter. 2016, 8, 12644-12651. (46) Xia, Y.; Wang, L.; Li, J.; Chen, X.; Lan, J.; Yan, A.; Lei, Y.; Yang, S.; Yang, H.; Chen, J. A Ratiometric Fluorescent Bioprobe Based on Carbon Dots and Acridone Derivate for Signal Amplification Detection Exosomal MicroRNA. Anal. Chem. 2018, 90, 8969-8976. (47) Mahani, M.; Mousapour, Z.; Divsar, F.; Nomani, A.; Ju, H. X. A Carbon Dot and Molecular Beacon Based


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Page 8 of 9

Fluorometric Sensor for the Cancer Marker MicroRNA-21. Microchim. Acta 2019, 186. (48) Li, N.; Li, R.; Li, Z.; Yang, Y.; Wang, G.; Gu, Z. Pentaethylenehexamine and Histidine-Functionalized Graphene Quantum Dots for Ultrasensitive Fluorescence Detection of MicroRNA with Target and Molecular Beacon Double Cycle Amplification Strategy. Sensor Actuat. B-Chem. 2019, 283, 666-676. (49) Yin, B.C.; Liu, Y.Q.; Ye, B.C. One-Step, Multiplexed Fluorescence Detection of MicroRNAs Based on Duplex-Specific Nuclease Signal Amplification. J. Am. Chem. Soc. 2012, 134, 5064-5067. (50) Zhang, Q. Q.; Yang, T.; Li, R. S.; Zou, H. Y.; Li, Y. F.; Guo, J.; Liu, X. D.; Huang, C. Z. A Functional Preservation Strategy for the Production of Highly Photoluminescent Emerald Carbon Dots for Lysosome Targeting and Lysosomal Ph Imaging. Nanoscale 2018, 10, 14705-14711. (51) Biju, V.; Itoh, T.; Baba, Y.; Ishikawa, M. Quenching of Photoluminescence in Conjugates of Quantum Dots and Single-Walled Carbon Nanotube. J. Phys. Chem. B 2006, 110, 26068-26074. (52) Xing, H.; Zhang, C. L.; Ruan, G.; Zhang, J.; Hwang, K.; Lu, Y. Multimodal Detection of a Small Molecule Target Using Stimuli-Responsive Liposome Triggered by Aptamer– Enzyme Conjugate. Anal. Chem. 2016, 88, 1506-1510. (53) Burke, J. E.; Dennis, E. A. Phospholipase A2 Biochemistry. Cardiovasc. Drugs Ther. 2009, 23, 49-59. (54) Indrasekara, A. S. D. S.; Paladini Bryan, J.; Naczynski Dominik, J.; Starovoytov, V.; Moghe Prabhas, V.; Fabris, L. Dimeric Gold Nanoparticle Assemblies as Tags for SERs-Based Cancer Detection. Adv. Healthc. Mater. 2013, 2, 1370-1376. (55) Madhavan, D.; Zucknick, M.; Wallwiener, M.; Cuk, K.; Modugno, C.; Scharpff, M.; Schott, S.; Heil, J.; Turchinovich, A.; Yang, R.; Benner, A.; Riethdorf, S.; Trumpp, A.; Sohn, C.; Pantel, K.; Schneeweiss, A.; Burwinkel, B. Circulating MiRNAs as Surrogate Markers for Circulating Tumor Cells and Prognostic Markers in Metastatic Breast Cancer. Clin. Cancer Res. 2012, 18, 5972. (56) Mitchell, P. S.; Parkin, R. K.; Kroh, E. M.; Fritz, B. R.; Wyman, S. K.; Pogosova-Agadjanyan, E. L.; Peterson, A.; Noteboom, J.; Briant, K. C.; Allen, A.; Lin, D. W.; Urban, N.; Drescher, C. W.; Knudsen, B. S.; Stirewalt, D. L.; Gentleman, R.; Vessella, R. L.; Nelson, P. S.; Martin, D. B.; Tewari, M. Circulating MicroRNAs as Stable Blood-Based Markers for Cancer Detection. Proc. Natl. Acad. Sci. 2008, 105, 10513-10518. (57) Lu, W.; Chen, Y.; Liu, Z.; Tang, W.; Feng, Q.; Sun, J.; Jiang, X. Quantitative Detection of MicroRNA in One Step Via Next Generation Magnetic Relaxation Switch Sensing. ACS Nano 2016, 10, 6685-6692. (58) Jin, Z.; Geißler, D.; Qiu, X.; Wegner, K. D.; Hildebrandt, N. A Rapid, Amplification-Free, and Sensitive Diagnostic Assay for Single-Step Multiplexed Fluorescence Detection of MicroRNA. Angew. Chem. Int. Edit. 2015, 54, 10024-10029. (59) Boeri, M.; Verri, C.; Conte, D.; Roz, L.; Modena, P.; Facchinetti, F.; Calabrò, E.; Croce, C. M.; Pastorino, U.; Sozzi, G. MicroRNA Signatures in Tissues and Plasma Predict Development and Prognosis of Computed Tomography Detected Lung Cancer. Proc. Natl. Acad. Sci. 2011, 108, 3713. (60) Li, X.; Yang, L.; Men, C.; Xie, Y. F.; Liu, J. J.; Zou, H. Y.; Li, Y. F.; Zhan, L.; Huang, C. Z. Photothermal Soft Nanoballs Developed by Loading Plasmonic Cu2–XSe Nanocrystals into Liposomes for Photothermal Immunoassay of Aflatoxin B1. Anal. Chem. 2019, 91, 4444-4450.


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For Table of Contents Only

A novel FRET soft nanoball (fretSNB), in which thousands of CDs and quencher dyes are loaded, have been developed to overcome the limitations on the surface modification of fluorescent carbon dots.


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FÃ¶rster Resonance Energy Transfer Based Soft Nanoballs for Specific

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