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A Ratiometric Fluorescent Bioprobe Based on Carbon Dots and Acridone Derivate for Signal Amplification Detection Exosomal microRNA Yaokun Xia, Liangliang Wang, Juan Li, Xiangqi Chen, Jianming Lan, An Yan, Yun Lei, Sheng Yang, Huang-Hao Yang, and Jing-Hua Chen Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.8b01143 • Publication Date (Web): 05 Jul 2018 Downloaded from http://pubs.acs.org on July 6, 2018

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Analytical Chemistry

A Ratiometric Fluorescent Bioprobe Based on Carbon Dots and Acridone Derivate for Signal Amplification Detection Exosomal microRNA ∥

Yaokun Xia†, Liangliang Wang†, Juan Li‡, Xiangqi Chen§, Jianming Lan†, An Yan†, Yun Lei†, Sheng Yang* , Huanghao Yang*‡, Jinghua Chen*† † Department of Pharmaceutical Analysis, The School of Pharmacy, Fujian Medical University, Fuzhou, Fujian Province 350108 (P. R. China) E-mail: [email protected] ‡ MOE Key Laboratory for Analytical Science of Food Safety and Biology, State Key Laboratory of Photocatalysis on Energy and Environment, College of Chemistry, Fuzhou University, Fuzhou, Fujian Province 350002 (P. R. China) E-mail: hhyang@fzu. edu.cn § Department of Respiratory Medicine, Fujian Medical University Union Hospital, Fuzhou, Fujian Province 350001 (P. R. China) ∥ Department of Medical Oncology, Fujian Medical University Union Hospital, Fuzhou, Fujian Province 350001 (P. R. China) [email protected]

ABSTRACT: Recently, sensitive and selective detection of exosomal microRNAs (miRNAs) have been garnered significant attention owing to it being related to a number of complex diseases, including cancer. Herein, we reported a ratiometric fluorescent bioprobe based on DNA-labeled carbon dots (DNA-CDs) and 5,7-dinitro-2-sulfo-acridone (DSA) coupling with target catalyzing signal amplification for detection of exosomal miRNA-21. There was high fluorescence resonance energy transfer (FRET) efficiency between CDs and DSA when the bioprobe was assembled. However, in presence of target, with disassembling of the fluorescent bioprobe, the fluorescence intensities of CDs and DSA were changed simultaneously. On account of ratio of dual fluorescence intensities, this ratiometric fluorescent bioprobe was able to cancel out environmental fluctuations by calculating emission intensity ratio at two different wavelengths, being robust and stable enough for detection of exosomal miRNA-21. In addition, we displayed that a single miRNA-21 can catalyze the disassembly of multiple CDs with DSA theoretically, yielding significantly fluorescence ratio change for miRNA-21 detecting. With this signal amplification strategy, the limit of detection (LOD) was as low as to 3.0 fM. Furthermore, due to introduction of lock nucleic acid (LNA) mediating strand displacement reaction, the selectivity of this strategy was improved remarkably even against single base mismatch sequence. More importantly, our strategy could monitor the dynamic change of exosomal miRNA-21, which maybe becomes a potential tool to distinguish cancer exosomes and nontumorigenic exosomes. In a short, this ratiometric fluorescence bioprobe possessed high stability, sensitivity and selectivity coupling with ease of operation and cost efficiency, leading to great potential for wide application. Keywords: Ratiometric fluorescence; Carbon dots; DSA; Exosomal miRNA-21

Introduction As nano-sized extracellular vesicles, exosomes have become research hotspots in recent years owing to their potential clinic diagnosis value.1 According to previous reports, exosomes are derived from and secreted by multiple cells, carrying abundant biomolecules (such as nucleic acids, proteins, and carbohydrates) that from their parental cells, and play an important role in mediating intercellular communications by conveying information.2-4 Currently, more and more evidences reveal that exosomal biomolecules were high disease-related.5 Therefore, detection of exosomal biomolecules may offer potential for the diagnosis of diseases. Especially, microRNAs (miRNAs), a group of small noncoding RNAs, are considered to have key roles in prediction of disease signature.6 Surprisingly, it is reported that miRNAs are also present in exosomes that can protect miRNAs from RNase degradation due to phospholipid bilayer structure.7 Importantly, there is high correlation between the level of exosomal miRNAs and disease development and tumor malignant progression.8 Thus, detection of exosomal miRNAs can support the better understanding of pathological process. For detection of exosomal miRNAs, the current commonly avenue is quantitative reverse transcription polymerase chain reaction (qRT-PCR), but this method needs specific primer pair for each reaction, which requires sophisticated pre-treatment and trained personnel.9 More importantly, PCR-based strategies have

the potential to generate false positive signal even due to a miniscule contamination. In view of these, a PCR-free technique for detection exosomal miRNAs is essential. At present, some groups developed PCR-free methods for exosomal miRNAs quantitation based on ratiometric electrochemistry,9 localized surface plasmon resonance (LSPR)10 and Surface-enhancement Raman scattering (SERS),11 respectively, but the expensive instrument and complex operation hampered their extensive application. Recently, fluorescent methods have been paid attentions due to their intrinsic advantages, including simple instrument, high sensitivity and capacity to high-throughput screening.12 Up to date, several attempts have been reported using fluorescent methods to detect exosomal miRNAs with various degrees of success, as evidenced by the cationic lipoplex nanoparticles containing molecular beacon assay,13 fluorescent dye-labeled molecular beacons strategies,14-15 fluorescence signal amplifiable biochip assay,16 and others.17 However, these methods employed solely responsive signal and were based on measuring the absolute change of the fluorescent intensity, which was readily perturbed by numerous experimental conditions, including thermodynamic fluctuations, nuclease degradation and dye photobleaching.18 To utilize exosomal miRNAs as diagnosis biomarker, a fluorescent system with antidisturbance should be developed because of complex biosystem. Surprisingly, on account of self-referencing capability, ratiometric fluorescent measurement is able to cancel out environmental fluctuations by calculating emission intensity ratio at two different

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wavelengths.19 Thus, it is great concern and significant challenges for constructing a ratiometric fluorescent bioprobe to make them more suitable for practical applications. Herein, we constructed a novel ratiometric fluorescent bioprobe based on CDs and acridone derivate called 5,7-dinitro-2-sulfoacridone (DSA) that was synthesized by our group.20 In this bioprobe, the DSA has good biocompability, and it also can bind with double strand DNA (dsDNA) through intercalate reaction, which is appropriate for biological analysis and simplifying the experimental procedure. The large Stokes shift of DSA is able to reduce the excitation interference. The most important was the high FRET efficiency between DSA and CDs, which provided a foundation for constructing the ratiometric fluorescent bioprobe. Besides, in order to raise sensitivity of this work, we combined target catalyzing signal amplification strategy based on strand displacement reaction with ratiometric fluorescence,21 developing a high sensitive and stable assay for direct detection of exosomal miRNA-21. This strategy can achieve multiple orders of magnitude signal amplification automatically and without enzyme assistant or labor-consuming washing step. Furthermore, the selectivity is the other important parameter for detection of miRNAs because of there being complex components in real samples. Supported by some previous studies including that from our group, the locked nucleic acid (LNA) was incorporated into a DNA strand displacement system, which was able to minimize unwanted reactions and improve the signal to noise.22-23 Thus, this ratiometric fluorescent bioprobe possessed high selectivity even against single base mismatch. As far as we know, it is the first time to utilize ratiometric fluorescent bioprobe and signal amplification strategy coupling with LNA mediating strand displacement reaction for detection of exosomal miRNA-21, which showed the possibility of developing a sensitive, selective and accurate method combining with chip for Point of Care Testing (POCT) of related disease.

Experimental Section Reagents and apparatus. DNA and RNA sequences were synthesized by TaKaRa (Dalian, China). All the above base sequences were illustrated in Table S1. The concentrations were quantified by OD260 based on their individual absorption coefficients. The salmon sperm DNA, a kind of double strand DNA (dsDNA), and proteinase K were purchased from Sigma-Aldrich. The RNAase and RNase inhibitor were purchased from TaKaRa. All cell lines were purchased from China Center for Type Culture Collection in Shanghai. o-phenylenediamine and acridone were purchased from Sigma. Fetal bovine serum (FBS), roswell park memorial institute (RPMI), dulbecco's modified eagle medium (DMEM) and penicillin-streptomycin were purchased from Hyclone. MEGMTM BulletKit was purchased from Lonza. Insulin was purchased from Novo Nordisk. TRIzol was purchased from Beyotime. All other chemicals were analytical grade and all aqueous solutions were prepared with MilliQ water (18 MΩ). The ultracentrifuge was Beckman optima XPN-100 (U.S.A.). The transmission electron microscopy (TEM) images of CDs were carried out on a JEM-2010 microscope (JEOL Ltd., Japan). The TEM images of MDA-MB-231 and MCF-7 exosomes were performed by Hitachi HT7700 (Japan). The fourier transform infrared (FT-IR) experiment was carried out on a Thermo Nicolet iS50 FT-IR spectrometer. The fluorescence spectra were performed on a Cary Eclipse fluorescence spectrophotometer (Agilent Technologies). UV-Vis absorbance spectra were recorded with a UV-2450 spectrophotometer (SHIMADZU, Japan) using a quartz cell with 1.0 cm optical pathway. qRT-PCR was performed on MxPro-MX3000P software (Agilent Technologies, U.S.A.). Preparation of DSA. According to the reference, the preparation of DSA included following procedures.20 (1) An amount of 0.6 g

9(10H)-acridone was put into a three-necked flask, and was heated at 100 ℃ for 30 min under stirring after adding 5 mL H2SO4. (2) The solution was poured into ice water and solid precipitate appeared, and the resulting solid was filtered off, washed and further purified by recrystallization in absolute alcohol. (3) The solid was then oven-dried at 75 ℃ and 2-sulf-acridone crystal could be obtained. (4) Subsequently, an amount of 2-sulf-acridone of 0.6 g was mixed with 3 mL of 36 % acetic acid, 0.36 mL nitric acid and 0.8 mL glacial acetic acid at 58 ℃ for 2 h under stirring. (5) The solution was poured into ice water and solid precipitate appeared. The resulting solid was filtered off, washed with a few water and further purified by recrystallization in absolute alcohol. (6) The solid was then oven-dried at 80 ℃ and yellow crystal of could be obtained. CDs preparation and DNA modified. The preparation of CDs can be readily accomplished according to the reference.24 Briefly, heating o-phenylenediamine (o-PD) in ethanol solution at 180 ℃ for 12 h in an autoclave and then purification using a silica gel column chromatography. CDs were cross linked on aminomodified DNA1 and amino-modified DNA2 according to the reference.25 In this process, 1-Ethyl-3-(3-dimethyllaminopropyl) carbodiimide hydrochloride (EDC) and NHydroxysulfosuccinimide sodium salt (NHS) were added to 1 mL phosphate buffer solutions (PBS) buffer, and the mixture with the concentration of 0.01 M was obtained by stirring. Then 2 mg CDs were added to the above mixture under rapid stirring. Finally 100 µL DNA1 or DNA2 (100 µM) was added to the above mixture under vigorous stirring at room temperature, and the mixture solution was stirred sequentially for two hours. Excessive EDC and NHS were removed by dialysis. The obtained solution was preserved at 4 ℃ for the following reaction. Cell culture and exosomes isolation. MDA-MB-231 cells and MCF-7 cells were cultured in RPMI and DMEM, respectively (both containing 10 % FBS, 1 % penicillin and streptomycin and 0.2 U/mL insulin). Then, MDA-MB-231 cells and MCF-7 cells (1 ×108 cells) were cultured in RPMI and DMEM containing 5 % FBS (exosomes depleted) for 48 h, respectively. MCF-10A cells were cultured in minimal essential growth medium (MEGM) containing cholera toxin. Above cells were cultured in a humid environment of 5 % CO2 at 37 ℃. The cells culture supernatant was collected and centrifuged according to relevant literature.1 Briefly, the cells culture supernatant was centrifuged at 300 g for 10 min, 2000 g for 20 min and 11000 g for 45 min to remove intact cells, cells debris and protein, respectively. Subsequently, the supernatant was centrifuged at 110000 g for 70 min to acquire sediment exosomes. Finally, the sediment exosomes were resuspended in PBS and stored at -80 ℃. Nanoparticle tracking analysis. The exosomes dissolved in sterile PBS were injected into a Nanosight NTA NS300 instrument (Malvern). We then analyzed the size distribution and concentration of the exosomes. RNA isolate, reverse transcription and quantitative real-time PCR analysis of the exosomes. The exosomes were pretreated by proteinase K and RNase before analysis.26 In brief, for proteinase K treatment, collected exosomes were incubated with 5 mg/mL of proteinase K at 37 ℃ for 30 min, and followed by heat inactivation at 60 ℃ for 20 min. As for RNase treatment, the collected exosomes were treated by 2 mg/mL RNase at 37 ℃ for 30 min, and added RNase inhibitor to inhibit the activity of RNase. Subsequently, exosomal RNA was isolated by TRIzol reagent and quantified using nanodrop 2000C (Thermo Fisher Scientific, U.S.A.). In addition, according to manufacturer’s protocol, using miScript Ⅱ RT Kit (Qiagen), the total RNA was reversetranscribed to cDNA. Then, the reaction system was incubated at 37 ℃ for 60 min and 95 ℃ for 5 min. By using miScript SYBR


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Analytical Chemistry Green PCR Kit, the qRT-PCR was performed after following thermocycler protocol: (1) 95 ℃ for 15 min, (2) 94 ℃ for 15 s, (3) 55 ℃ for 30 s, (4) 70 ℃ for 30 s with 40 cycles. Fluorescent detection of miRNA-21. 10 µM DNA1-CDs, 10 µM DNA2-CDs and 10 µM DNA linker were mixed in PBS buffer containing 100 mM NaCl and 25 mM MgCl2. Then, the 10 µM DSA was added into the above solution. The excessive DSA was removed by dialysis. Finally, the ratiometric fluorescent bioprobe called as dsDNA-DSA-CDs was fabricated. The different concentrations of miRNA-21 were added into above solution containing 50 µM fuel DNA. The resulting solution was kept at 37 ℃ for 120 min. Then, the detection performance of this bioprobe was investigated by fluorescence spectra. The fluorescence measurement conditions were as follow: excitation wavelength of 256 nm, emission wavelength from 410 nm to 750 nm, excitation slit of 5.0 nm, emission slit of 10.0 nm and voltage of 650 V.

Results and discussion The principle of the ratiometric fluorescent bioprobe. Herein, for the first time, we presented an approach for a high-sensitive and selective ratiometric fluorescence detection of miRNA-21 in exosomes. Firstly, the exosomes were isolated from cell supernatant through ultracentrifugation (Scheme 1). Then, the ratiometric fluorescent bioprobe consists of two short strands (DNA1 and DNA2) modified with CDs, DNA linker (the orange base modified by LNA) as well as DSA. In the presence of all DNA sequences, they are able to hybridize, forming part of dsDNA structure. The DSA can bind with dsDNA through intercalating reaction, thus constructing a smart bioprobe called dsDNA-DSA-CDs. In this case, the fluorescence of DSA (donors) is quenched by the nearby CDs (acceptors) through FRET, while the enhanced emission of CDs can be detected. After the addition of miRNA-21 and fuel strand (F), the miRNA-21 with the complementary sequence binds to toehold 1 (8 nt, containing a LNA location to improve the affinity) and displaces DNA1 from the linker through branch migration to expose toehold 2 (5 nt); subsequently, the F binds to toehold 2 and liberates DNA2 and miRNA-21. Therefore, the released target further hybridizes with another toehold 1, which

induces the recycling of strand-displacement reaction. After numerous cycles, theoretically, single miRNA-21 molecule can catalytic all dsDNA-DSA-CDs disassembly, separating the donor and acceptor, inducing low FRET efficiency. Thus, the fluorescent emission of CDs (560 nm) decreases and that of DSA (420 nm) increases simultaneously. By recording the ratiometric value (F420/F560), miRNA-21 can be quantified accurately, specifically and sensitively. Characterization and Spectral Properties of DSA. Herein, an acridone derivate called DSA was designed and synthesized from acridone powder through substitution reaction according to previous method proposed by our group (Figure S1).20 The relevant data characterization about DSA was shown in S1.1 section (Support Information). The elemental analysis data of DSA was exhibited in Table S2. Intriguingly, the DSA possessed some advantages that acridone had not, such as the good water-solubility. The photographs of 1 µM acridone solution and 100 µM DSA solution were shown in Figure S1 (inset), which allowed DSA to become a better candidate in bioassay due to its clearer solution. In addition, the DSA also had following virtues: (1) The small molecule of DSA could be easily prepared with low-cost chemical materials and instruments, through a facile approach. (2) The favorable fluorescent performance was an important and interesting property for DSA to construct fluorescent bioprobe. Under continuous irradiation, the fluorescence intensity of DSA did not change significantly (Figure S2), suggesting its outstanding photostability. (3) As an excellent double-stranded DNA (dsDNA) indictor, DSA could occur to strong intercalating interaction with dsDNA, which has been confirmed by electrochemistry and UVvis (Figure S3 and Figure S4), and the relevant details were shown in S1.2 and S1.3 sections (Support Information). The binding ratio and the binding constant between DSA and dsDNA also were validated in S1.4 section (Figure S5). (4) In particular, the DSA also had excellent electrochemical activity and was utilized to detect miRNAs,20 which meant feasibility of constructing a dual functional sensor based on DSA in the future. Together with above advantages, DSA was expected having a fascinating prospect for bioanalysis.

Scheme 1. Schematic representation of the detection mechanism of the proposed method for exosomal miRNA-21.



Characterization and spectral properties of CDs. Transmission electron microscopy (TEM) was performed to examine the morphologies of the CDs. As shown in Figure 1A, all the CDs showed uniform nanoparticles with average sizes of about 8 nm. The FTIR spectra of the CDs (curve a) and o-PD (curve b) are compared in Figure 1B. As shown in Figure 1B, some new characteristic peaks emerged at about 2850-2940 cm-1, 1330-1386 cm-1, and can be attributed to aliphatic C-H, C-N= stretching vibrations. The broad bands around 3300 cm-1 indicated the vibrations of O-H and N-H. The peaks at 1651 cm-1, 1381 cm-1 and 1080 cm-1 were assigned to the C=O, C-N and C-O vibrations, respectively. The results showed that numerous nitrogen-containing and oxygencontaining groups existed on the CDs surface, which was responsible for high aqueous solubility and stability of CDs.

the increase of the excitation wavelength, with an excitation maximum at 420 nm and emission maximum at 560 nm. As the previous reported, it was speculated that both the quantum size effect and surface defects contribute to fluorescence emission.27 Under continuous irradiation, the fluorescence emission intensity of CDs did not change significantly (Figure 1E), suggesting their outstanding photostability. Another interesting phenomenon was the salt tolerance of the CDs. The fluorescence emission intensity of CDs at different ionic strengths was monitored when increasing the concentration of NaCl from 0.15 M to 2.0 M. As shown in Figure 1F, there were no changes of fluorescence intensity less than 5 % in NaCl solution with high concentration (2.0 M). All these features made CDs excellent candidates as a powerful tool in environmental applications as well as an appropriate sensor designed for biological applications. The characterization of exosomes. According to International Society for Extracellular Vesicles (ISEV) guidelines, the exosomes were characterized.28 At first, the morphologies of exosomes derived from MDA-MB-231 cells (triple negative human metastatic breast carcinoma) were examined by TEM. The harvested MDA-MB-231 exosomes were saucer-like and about mean diameter of 110 nm (Figure 2A), which was consistent with the characteristics of exosomes reported in the previously literature.9 Subsequently, we used nanoparticle tracking analysis (NTA) to quantify exosomes concentration and average diameter. As shown in Figure 2B, the concentration of the collected exosomes was 2.3 ×107 particles/mL, and the average diameter was 168 nm, which is consistent with previous report.29 Noteworthy, the size measured by NTA was significantly larger than that detected by TEM.

Figure 1. (A) The TEM of CDs. (B) The FT-IR spectra of CDs (curve a) and o-PD (curve b). (C) The UV/vis spectra of CDs (curve a) and o-PD (curve b). Inset: the photo of CDs under sunlight (a) and under 365 nm UV light (b). (D) The fluorescence emission spectra of CDs. (E) The CDs under continuous irradiation for 1 h, the fluorescence intensity not changed. (F) The fluorescence intensity of CDs at different ionic strengths. UV/vis absorption and fluorescence emission spectra of CDs were investigated. The UV/vis absorption spectrum of the CDs (curve a) and o-PD (curve b) was displayed in the Figure 1C. In comparison to o-PD, the higher- and lower-energy absorption bands of the CDs red-shift, that was, from λ=210 nm to 260 nm and λ=292 nm to 427 nm, which demonstrated that the CDs should contain smaller electronic band gaps than o-PD. The CDs solution was yellowish, transparent, and clear under sunlight (Inset a in Figure 1C), and exhibited bright yellow fluorescence excitation with a 365 nm UV light (Inset b in Figure 1C), indicating that the CDs had an excellent fluorescence property. Very interestingly, it was very stable without aggregating upon standing for more than several months. The high water-dispersibility of CDs can be partially ascribed to the highly negative surface charge (Zetaζ=-19.7 mV, Figure S6). The fluorescence emission spectra of CDs exhibited a typical excitation-independent feature as depicted in Figure 1D; the emission peak nearly did not shift with

Figure 2. (A) TEM of MDA-MB-231 exosomes. (B) Size and size distribution of the MDA-MB-231 exosomes as analyzed by NTA. WB images for (C) CD 63, (D) EpCAM, (E) AGO2 and (F) TSG 101 in MDA-MB-231 cell and MDA-MB-231 exosomes. According to the reference, exosomes may generally changed size or shrunk during the preparation process for TEM, which resulted in the small size for exosomes observed using TEM.30 Furthermore, the identity of the exosomes was validated through detection of CD 63, EpCAM, AGO2 and TSG 101.28, 31-33 From Figure


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Analytical Chemistry 2C to Figure 2F, the western blotting (WB) images validated that there were abundant CD 63, EpCAM, AGO2 and TSG 101 in MDA-MB-231 exosomes, the obvious bindings validated the efficiency of the separation of exosomes. Similarly, the TEM and WB images of MCF-7 exosomes were also displayed in Figure S7 and Figure S8, respectively. The feasibility of the ratiometric fluorescent bioprobe. The amount of miRNA-21 in different exosomes is discrepant when the exosomes are at the same concentration. Especially, miRNA21 aberrant expression frequent association with malignancies.34 Therefore, miRNA-21 was acted as a favorable model to explore the feasibility, stability, sensitivity and selectivity of our method in this assay. Firstly, as shown in Figure 3A, the apparent spectra overlap between the ultraviolet absorption band of CDs (curve a) and fluorescence emission spectra of DSA (curve b), which resulted in a fluorescence quenching of DSA, yet the fluorescence intensity was enhancement of CDs when two fluorescent groups were approached based on FRET. Next, the fluorescence measurements were further performed to serve as a proof of concept to test the principle of our strategy. As shown in Figure 3B, the DSA exhibited strong fluorescence at 420 nm in solution (curve a). Meanwhile, in presence of DSA and dsDNA, the fluorescence intensity was declined to some extent (curve b), which indirectly demonstrated there was intercalation reaction between DSA and dsDNA.35 When the solution containing CDs-DNA1, CDs-DNA2, DNA linker and DSA, owing to FRET, the fluorescence of DSA was quenched significantly, and the CDs produce strong fluorescence (curve c). Surprisingly, once the target catalyzed the disassembly of this bioprobe, the fluorescence intensities of DSA and CDs were increased at 420 nm and decreased at 560 nm, respectively (curve d). Additionally, the disassembly kinetic was examined. As shown in Figure S9, F420/F560 was increased with increasing reaction time, and reached plateau after 120 min. At the same time, we observed that the fluorescence intensities of free CDs and DNA-labeled CDs were weak under the excitation of 256 nm (curve e and curve f) due to the excitation wavelength far away from the optimal excitation of CDs. Above results demonstrated that our method was feasible for detection of miRNA-21.

CDs, respectively; the C represents concentration of miRNA-21. The limit of detection (LOD) of the designed bioprobe based on 3σ method was 3.0 fM. The high sensitivity of this type of bioprobe can be attributed to the following factors: (1) We present a DNA strand displacement assisted target recycling system in which each copy of the target can be acted as a trigger to catalysis disassembly of multiple CDs with DSA, which eventually results in a tremendous increase in the fluorescent signals of DSA, and the fluorescence intensity of CDs is decreasing accordingly. (2) This strategy based on CDs and DSA is more biocompatible, more hydrophilic, and thus less prone to nonspecific adsorption. (3) The adoption of the LNA may increase the affinity between the probe and the perfectly matched target, thus increasing the hybridization efficiency at low target miRNA concentration. Both of the above advantages may contribute to the amazing application in the area of clinical miRNA detection. Compared to several previous methods used for exosomal miRNA-21 determination, as listed in Table 1, it can be deduced that the achieved LODs of our method are lower than some methods.11,36 However, some other methods possess the even better sensitivity.9,17 For example, He et al. reported that the signal amplification method based on hybridization chain reaction (HCR) improve the sensitivity.17 Our group developed a ratiometric electrochemical biosensor to detect exosomal miRNA-21 with LOD of 52 aM.9 For the sake of further enhancing the sensitivity of our ratiometric fluorescent bioprobe, we intended to modified the structure of DSA to improve its fluorescence efficiency.

Figure 4. (A) Fluorescence emission spectra of this bioprobe in the presence of various concentrations of miRNA-21 (from a to f): 0 fM, 10 fM, 50 fM, 100 fM, 200 fM, 500 fM. (B) The calibration plot of ratiometric of fluorescent signal versus miRNA-21 concentration. Table 1. Comparison of the sensitivity of currently available methods for the detection of exosomal miRNA-21.

Figure 3. (A) The absorption spectra of CDs (curve a) and fluorescence emission spectra of DSA (curve b). (B) Fluorescence spectra of DSA (curve a), DSA+dsDNA (curve b), CDsDNA1+CDs-DNA2+DNA linker+DSA (curve c), target+fuel DNA+ CDs-DNA1+CDs-DNA2+DNA linker+DSA (curve d), CDs (curve e), DNA-labeled CDs (curve f). The sensitivity of the ratiometric fluorescent bioprobe. In order to investigate the sensitivity of the bioprobe toward the detection of miRNA-21, the fluorescence spectra of dsDNA-DSA-CDs in the presence of different amounts of target were exhibited in Figure 4A. As shown in this figure, with increasing the concentration of miRNA-21, the fluorescence intensity of DSA increases gradually but the fluorescence intensity of CDs decreases correspondingly. At the same time, a dose-response curve was monitored by fluorescence intensity ratio between DSA and CDs. As shown in Figure 4B, F420/F560 increase with the concentration of miRNA-21 from 0 fM to 500 fM. The correlation equation could be expressed as F420/F560=0.01028C+0.591 (r=0.9954), where the F420 and F560 represent the fluorescence intensities of DSA and

No.

Method

LOD

Ref.

1

Ratiometric electrochemical biosensor

52 aM

9

2

Surface-enhancement Raman scattering (SERS) analysis strategy

5 fM

11

3

Fluorescence method based on HCR

1 fM

17

4

Amplification-free approach

electrochemical

1 pM

36

5

Ratiometric fluorescent bioprobe based on CDs and DSA

3 fM

This work

The selectivity of the ratiometric fluorescent bioprobe. To examine the selectivity of our proposed bioprobe, we performed a comparison study on mismatch targets and perfect complementary



target. Figure 5 shows the fluorescence intensities for the different kinds of targets (red bars), including the non-complementary target (NCT), two-base mismatch target (TMT) and single-base mismatch target (SMT), each with a concentration of 500 fM. As expected, compared with perfect complementary target, NMT only results in 0.75 % increasing of the F420/F560. Additionally, using the TMT causes 2.44 % increasing of the F420/F560, where as the SMT leads to only 4.51 % increasing. Meanwhile, we employed this ratiometric fluorescent bioprobe without LNA modifyed to do this assay, discovering the signal being improved significantly toward mismatch sequences (black bars). Consequently, the LNA endowed this strategy with high selectivity because of the weak hybridization of LNA and mis-matched sequence at the detection temperature.37 In a word, the proposed strategy shows high sequence specificity.

Figure 5. Selectivity of the proposed ratiometric fluorescent bioprobe with LNA modified (red bars) and without LNA modified (black bars) toward mismatched targets and miRNA-21 at the same concentrations (500 fM). Each data point represents the average value of three independent experiments with error bars indicated. Detection of miRNA-21 in exosomes. Considering free miRNAs or protein-associated miRNAs outside the exosomes is possible to influence the detection result, the proteinase K and RNase were utilized to remove the extra-exosomal miRNAs according to reference.26 After that, we used qRT-PCR to measure the Ct value of miRNA-21, in which U6 snRNA was acted as an internal control. As shown in Figure S10, when the exosomes were treated by proteinase K and RNase (gray bars), the Ct values of U6 and miRNA-21 were slightly improved compared with untreated sample (black bars), which demonstrated there were a portion of RNA in solution even after exosomes isolation, and it was necessary to remove the extra-exosomal RNA before detected the exosomal RNA in this assay. Afterwards, several reports suggested that the level of mature miRNA-21 in exosomes were dynamic change. Through monitoring the level of miRNA-21 maybe become a potential index to distinguish cancer exosomes and nontumorigenic exosomes.33 In order to validate this opinion, a time-course analysis of miRNA21 in MDA-MB-231 exosomes, MCF-7 exosomes and MCF-10A exosomes was performed by our method. When our ratiometric fluorescence bioprobe was reacted with exosomal RNA, it could produce a fluorescence intensity ratio that from DSA and CDs (F420/F560). According to the ratio and acquired correlation equation, the concentration of miRNA-21 was calculated. As shown in Figure 6A, we cultured above three different exosomes for 6, 12, 24, 36, 48, 72, and 96 h and observed that the miRNA-21 in cancer exosomes (MDA-MB-231 exosomes and MCF-7 exosomes, curve a and curve b) increased in quantity between 24 h and 72 h of culture, after which the level reached a plateau. However, the miRNA-21 in nontumorigenic exosomes (MCF-10A, curve c) was much lower and almost not change, which attributed to the low expression of miRNA-21 in nontumorigenic exosomes.33 The results were very agree with previous literature.33 According to relevant reports, above phenomenon was caused by miRNAinduced silencing complex (miRISC) in exosomes.38 This com-

plex contained double-stranded RNA binding protein TRBP (transactivating response RNA-binding protein) and RNase Ⅲ Dicer as well as pre-miRNAs and Argonaute 2 (AGO2), which was able to process pre-miRNA-21 into mature miRNA-21 in exosomes. So, our work also maybe provided an evidence to prove that the exosomes maybe a processor and carrier for miRNAs through monitoring level dynamic change of miRNA-21.

Figure 6. (A) The concentration change of mature miRNA-21 of MDA-MB-231 exosomes (curve a), MCF-7 exosomes (curve b) and MCF-10A exosomes (curve c) after 6, 12, 24, 36, 48, 72, and 96 h of cell-free culture conditions. (B) Bars represent the concentration of miRNA-21 of MDA-MB-231 exosomes, MCF-7 exosomes and MCF-10A exosomes detected by the proposed bioprobe (red bars) and qRT-PCR (purple bars), respectively. Error bars represent standard deviations for measurements taken from at least three independent experiments. Afterwards, to investigate the accuracy of our proposed method, qRT-RCR and ratiometric fluorescence bioprobe were performed in parallel to test the level of miRNA-21 in various cells derived exosomes, including MDA-MB-231 cell, MCF-7 cell and MCF-10A cell after the exosomes being cultured for 72 h. As shown in Figure 6B, the final result of proposed method was good consistent with the findings of qRT-PCR, demonstrating that our ratiometric fluorescence bioprobe possessed high accuracy and practical value. Moreover, due to the sequences modification flexibility of our proposed bioprobe, the other targets including miRNAs and DNAs will also be detected through altering the sequence of bioprobe. As for proteins, through introducing aptamer and magnetic separation that is similar to previous work39, the exosomal proteins can be detected. Meanwhile, we also realized that limited by lacking of specific markers in exosomes,40 it is a difficulty and research focus to distinguish cancer and nontumorigenic exosomes, although various strategies had been constructed.41-43 Consequently, developing a high selective method to specific isolate exosomes, and combine with our versatile ratiometric fluorescence bioprobe may be provide a research foundation for exosoms analysis in the future.

Conclusion To conclude, a ratiometric fluorescent bioprobe based on CDs and DSA coupling with target catalysis signal amplification and LNA is constructed for exsomal miRNA-21 detection. Owing to the self-referencing capability of ratiometric fluorescence, this strategy is robust and stable enough for detection of exosomal miRNA-21 in real sample. With the introduction of target catalysis signal amplification, this strategy exhibited high sensitivity, and the LOD was as low as down to 3.0 fM. Moreover, the LNA endowed this method with high selectivity even against single base mismatch sequence. Furthermore, the change of miRNA-21 content was also monitored by our proposed method. More importantly, because of the versatility, our ratiometric fluorescence bioprobe has potential to detect multiple targets to provide a research foundation for exosoms analysis. In view of above merits, this proposed fluorescent bioprobe shown that possibility of developing affordable POCT devices combine with chip technology for early diagnosis of cancer in the future. Meanwhile, the per-


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Analytical Chemistry formance of this strategy is expected to be further refined. For example, finding and synthesizing more fluorescent materials with different color of fluorescence is possible to realize simultaneous measurement of multiple miRNAs, which might be beneficial to improve the detection accuracy and provide more precise information on prognostic and treatment for tumor. Consequently, we envision this proposed approach can be acted as a potentially tool for the early and accurate detection for cancer.

ASSOCIATED CONTENT Supporting Information DNA and RNA sequences (Table S1), elemental analysis of DSA (Table S2), the synthesis route, the fluorescence stability of DSA, the electrochemical activity of DSA, the interaction between DSA and DNA, the binding ratio and the binding constant between DSA and DNA, zeta potential of CDs, the TEM of MCF-7 exosomes, WB images, the disassembly kinetic, qRT-PCR (Figure S1-Figure S10). This material is available free of charge via the Internet at Http://pubs.acs.org.

AUTHOR INFORMATION Corresponding Author *Email: [email protected] *Email: hhyang@fzu. edu.cn *Email: [email protected]

Notes The authors declare no competing financial interest.

ACKNOWLEDGMENT The authors gratefully acknowledge the financial support of the Natural Science Foundation of Fujian Province (2017J07001, 2017J01226), the Fujian Science and Technology Innovation Joint Found Project (2016Y9050), the Medical Elite Cultivation Program of Fujian, P. R. C (2014-ZQN-ZD-26), the National Natural Science Foundation of China (21375017, 81372092, 81671930, 21475026, 21622502), the Foundation of Fujian Provincial Department of Science & Technology (2015Y0059).

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