Ratiometric fluorescence sensor based on carbon dots as internal reference signal and T7 exonuclease-assisted signal amplification strategy for microRNA-21 detection

Journal Pre-proof Ratiometric fluorescence sensor based on carbon dots as internal reference signal and T7 exonuclease-assisted signal amplification strategy for microRNA-21 detection Zhenzhen Wang, Zhiqiang Xue, Xiaoli Hao, Chenfang Miao, Jianzhong Zhang, Yanjie Zheng, Zongfu Zheng, Xinhua Lin, Shaohuang Weng PII:

S0003-2670(19)31538-7

DOI:

https://doi.org/10.1016/j.aca.2019.12.068

Reference:

ACA 237351

To appear in:

Analytica Chimica Acta

Received Date: 16 September 2019 Revised Date:

19 December 2019

Accepted Date: 25 December 2019

Please cite this article as: Z. Wang, Z. Xue, X. Hao, C. Miao, J. Zhang, Y. Zheng, Z. Zheng, X. Lin, S. Weng, Ratiometric fluorescence sensor based on carbon dots as internal reference signal and T7 exonuclease-assisted signal amplification strategy for microRNA-21 detection, Analytica Chimica Acta, https://doi.org/10.1016/j.aca.2019.12.068. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier B.V.

Graphical Abstract

Using carbon dots (CDs) as internal reference and FAM-labeled ssDNA as the signal source, a turn-on ratiometric fluorescence bioassay based on the T7 exonuclease-mediated cyclic enzymatic amplification method was developed for the evaluation of the level of miRNA-21 in real blood samples.

Ratiometric fluorescence sensor based on carbon dots as internal reference signal and T7 exonuclease-assisted signal amplification strategy for microRNA-21 detection Zhenzhen Wang1, Zhiqiang Xue4, Xiaoli Hao1, Chenfang Miao1, Jianzhong Zhang3,*, Yanjie Zheng1, Zongfu Zheng2,*, Xinhua Lin1, Shaohuang Weng1,* 1. Department of Pharmaceutical Analysis, School of Pharmacy, Higher Educational Key Laboratory for Nano Biomedical Technology of Fujian Province, Fujian Medical University, Fuzhou 350122, China. 2. The 900th Hospital of PLA, Fuzhou 350002, P. R. China. 3. Department of Gastric Surgery, Fujian Medical University Union Hospital, Fuzhou 350001, China 4. Department of Orthopedic Surgery, Fujian Medical University Union Hospital, Fuzhou, 350001, China Correspondence: [email protected] (J.Z. Zhang), [email protected](Z.F. Zheng) or [email protected] (S.H. Weng)

1

Abstract The expression level of miRNA-21 is closely related to the occurrence and development of cancer, especially in gastrointestinal cancer. Monitoring miRNA-21 has clinical application in the diagnosis and evaluation of gastrointestinal cancer. A turn-on ratiometric fluorescence bioassay based on the T7 exonuclease-mediated cyclic enzymatic amplification method was developed for miRNA-21 determination by using carbon dots (CDs) and FAM-labeled ssDNA as the signal source. CDs demonstrated the triple functions of built-in internal fluorescence, probe carrier, and quencher in this strategy. In the absence of miRNA-21, FAM-labeled ssDNA would be adsorbed and quenched by CDs. The addition of miRNA-21 induced cycle hydrolysis from the 5' end by the T7 exonuclease and then released the short-cleaved FAM-labeled oligonucleotides. Then, the increased FAM signal (FFAM) and the stable CD signal (FCDs) would be tested through a ratiometric routine for the quantification of miRNA-21. The FFAM/FCDs value showed a good linear relationship with the concentration of miRNA-21 in the range of 0.05–10 nM, and the detection limit for miRNA-21 was 1 pM with excellent selectivity and reproducibility. Furthermore, this sensor successfully evaluated the expression level of miRNA-21 in clinical blood samples from healthy individuals and gastrointestinal cancer patients, and the results were highly consistent with those of qRT-PCR, suggesting the great clinical application value in the diagnosis of cancer associated with miRNA-21 expression levels. Keywords: Ratiometric fluorescence sensor; T7 exonuclease-assisted signal amplification strategy; miRNA-21; carbon dots; gastrointestinal cancer

2

Introduction MicroRNAs (miRNAs) are a class of endogenous and small-sized noncoding RNAs (approximately 22 nucleotides) that directly bind their mRNA to inhibit the expression of target genes [1, 2]. This class contains regulating genes in disease environments, cell differentiation, and homeostasis [3, 4]. The level of miRNAs reflects the developmental lineage and differentiation status of the tumor. Therefore, miRNA detection can be applied in the diagnosis and evaluation of cancer [5-8]. For example, disorder modulation of miRNA-21 is closely related to the occurrence and development of gastrointestinal cancer [9, 10]. Therefore, developing a simple, fast, reproductive, and selective platform for detecting miRNAs is necessary to meet the practical demand for clinical biodiagnosis. Various methods, including northern blot [11, 12], quantitative real-time polymerase chain reaction (qRT-PCR) [13], gene microarrays [14, 15], lectrochemistry [16], electrochemiluminescence [17], photoelectrochemistry [18], and colorimetry [19], have been applied for the detection of miRNAs to date. However, such techniques still suffer weaknesses, such as intricate operation and high cost. By contrast, fluorescence detection [20-22] has been widely adopted in biomolecule analysis due to its advantages of simple fabrication, low cost, and rapid response [23, 24]. Furthermore, other effective techniques may be introduced to the fluorescent sensing procedure to improve the analytical performance in miRNA detection. Enzyme-free or enzymatic target cycle amplification methods, such as hybridization chain reaction [25] and cyclic enzymatic amplification method [26], will improve detection sensitivity for a specific miRNA remarkably [27,28]. Meanwhile, introducing the amplification method may increase the background signal and weaken the analytical performance in target genes. To address this potential problem, several functional nanomaterials, such as polydopamine [29], MnO2 nanosheets [30], and carbon quantum dots [31], were introduced as carriers and quenchers to reduce the background signal and improve the signal-to-noise ratio. Recently, a ratiometric fluorescent strategy based on two or more analyte-mediated 3

emission bands offers internal calibration for correcting possible interference factors from the environment and results in accurate and reproductive quantitative analysis [32, 33]. A dual-emission system is crucial for building ratiometric fluorescence sensors [34, 35]. For example, the combination of quantum dots and fluorescence dyes is an effective and comfortable routine for the construction of ratiometric biosensing platforms for miRNA detection [35]. However, the covalent link between quantum dots and assistant probes is somewhat complex [35]. Meanwhile, the focus on quantum dots with the multiple functions of carrier, quencher, and intrinsic signal is an interesting, facile, and effective attempt for gene detection through the ratiometric strategy [36]. The construction of ratiometric detecting methods for miRNA using simple operation procedures to make them practical in clinical diagnosis is still challenging but necessary [23, 37-39]. In this work, inspired by the aforementioned developments, we fabricated a ratiometric fluorescence strategy for miRNA-21 detection on the basis of carbon dots (CDs) with triple functions of stable built-in internal fluorescence, probe carrier, and quencher together with T7 exonuclease-assisted target amplification. As displayed in Fig. 1, the fluorescence signals of CDs and FAM represented the reference and indication signals, respectively. In the absence of target miRNA-21, FAM-labeled ssDNA would be quenched due to the adsorption and quenching effect of CDs [40]. Meanwhile, the selective target-induced cycle hydrolysis from the 5' end via the T7 exonuclease will occur once DNA–RNA heteroduplexes are formed with the presence of the target miRNA-21. Given the weak affinity between FAM or short-cleaved FAM-labeled oligonucleotides and CDs, miRNA-21 induced hydrolysis allows FAM to release in the solution. Thus, the introduction of CDs and the free FAM can illustrate their respective fluorescence behavior. Concurrently, a simple, sensitive, and highly reproducible ratiometric fluorescence sensor for miRNA-21 detection was established. The accurate detection of the level of miRNA-21 in the clinical samples of healthy donors and gastrointestinal cancer patients has further proven the simplicity and reproducibility of this method. 4

Experimental section Materials and Methods T7 exonuclease and 10 × NEBuffer 4 (500 mM Potassium acetate, 200 mM Tris-acetate buffer, 100 mM magnesium acetate,10 mM dithiothreitol, pH 7.9 at 25 ℃) were obtained from New England Biolabs (USA). The DNA capture probe was offered by Sangon Biotech (Shanghai) Co., Ltd. All miRNA sequences, RNase-free water and Recommbinant RNase Inhibitor were purchased from TaKaRa (Dalian, China). miRNAs were diluted in RNase-free water to suitable concentration. The DNA capture probe was dissolved with 10 mM TE buffer (pH 8.0, 10 mM Tris-HCl, 1 mM EDTA, 1 mM NaCl) to obtain the stock solution. The nucleotide sequences mentioned above were displayed in Table S1. The 10 mM Tris-HCl (pH 8.0) was used as detection solution. Total-RNA extraction kits were purchased from Beijing Zoman Biotechnology Co., Ltd (Beijing, China). The DEPC-treated water, RNase-free tips and tubes were used throughout the work to protect from RNase degradation. The fluorescence spectra and the fluorescence intensity were recorded by Cary Eclipse Fluorescence Spectrophotometer (Agilent Technologies). UV-vis absorption spectra were measured on UV2450 Spectrophotometer (Shimadzu Corporation). The purity of total-RNA extraction was evaluated by NanoDrop. Ratiometric fluorescence detection of miRNA-21 using the combination of CDs and cyclic enzymatic amplification reaction In an EP tube, 4 µL T7 exonuclease (10 U/µL), 4 µL NEBuffer 4, 1 µL 40 U/µL recombinant RNase inhibitor, 50 nM capture probe, and a specific amount of miRNA were mixed. After the above-mentioned solution diluted to a volume of 40 µL, the mixture was incubated to react at 37°C for 30 min. This system was called a miRNA reaction solution. CDs from pyrolysis synthesis were prepared following our reported work [36], and the detailed procedure was shown in supplementary materials. First, 10 µL CDs stock solution (80 µg/mL) and 150 µL 10 mM Tris-HCl were added into the miRNA reaction solution. Second, the fluorescence assay was tested after 10 min of 5

incubation at room temperature. The excitation and emission slits were 5 nm. For the synchronous fluorescence measurement, the constant wavelength difference value ∆λ (∆λ=λem − λex) was set to 30 nm. Total-RNA extraction from the whole blood samples The whole blood samples of gastrointestinal cancer patients and healthy donors were obtained from Fujian Medical University Union Hospital with the permission of the Ethics Committee of Fujian Medical University Union Hospital and the signed an informed consent. RNA containing miRNA-21 was extracted from the clinical whole blood samples using the total-RNA extraction kits. The quality of total RNA extraction was evaluated via NanoDrop. The content of miRNA-21 was detected and compared with the ratiometric method and qRT-PCR, respectively.

Results and Discussion Feasibility and mechanism of the sensor The optical and structure of CDs were first characterized. The CDs demonstrated a bright blue fluorescence emission centered around the maximum wavelength of 430 nm with the maximum exciting wavelength at 345 nm, as shown in the Fig. 2A. Moreover, CDs exhibited an excitation-independent photoluminescence (PL) behavior (Fig. 2B). The morphology and size distribution of CDs were characterized by TEM. The results indicated that CDs were spherical and uniform in size with an average diameter of 2.5 nm and a size range of 1.24–3.74 nm (Fig. 2C). A space lattice of 0.21 nm also confirmed the internal structure of CDs [41]. The molecular functional groups of CDs were investigated by FTIR, as shown in Fig. 2D. The two peaks around 2932 and 2840 cm−1 demonstrated the existence of C–H stretching vibrations. The peaks at 3424 and 3270 cm−1 were attributed to the stretching vibration of −OH and −NH2, whereas the band in 1653 cm−1 was related to C=O bending. C–N asymmetric stretching vibration (1124 cm−1) implied the doping effect of N. The presence of –NH2 and –NH– bonds would help the interaction between CDs and ssDNA sequences [40]. Considering the difference in exciting and emission wavelengths of CDs and FAM, 6

synchronous fluorescence with a set ∆λ was applied to monitor the simultaneous fluorescence of CDs and FAM. Compared with conventional fluorescence, synchronous spectra were simpler with narrower peaks and the peak position could move [42]. This type of spectrum has more characteristics than its conventional counterparts [42]. As shown in Fig. 3, synchronous spectra were obtained to compare the fluorescence emission spectra of parallel reaction systems under different conditions for confirming the feasibility of signal amplification for miRNA-21 detection. The signal had only one peak at 368 nm for CDs or at 498 nm for FAM-labeled ssDNA when the CDs and FAM-labeled ssDNA were tested separately (lines a and b). However, when the CDs and FAM-labeled ssDNA were mixed, emission of FAM-labeled ssDNA at 498 nm was quenched evidently, whereas that of CDs remained unchanged (line c). The mixture of CDs and FAM-labeled ssDNA exhibited stable fluorescence behavior with the addition of T7 exonuclease (line d), indicating the ignored fluctuation of the background response in the reaction process. Furthermore, considering the presence of proteins in the real blood sample, the fluorescence behavior of addition of BSA in mixture of CDs, FAM-labeled ssDNA and T7 exonuclease was also investigated (line e). The still constant fluorescence response was found with the addition of BSA, suggesting the strong anti-interference ability of this method to proteins. When miRNA-21 was added into the system containing FAM-labeled ssDNA and CDs, the fluorescence intensity of FAM increased because of the unabsorbed ability of CDs to the formed DNA/RNA heteroduplexes through hybridization (line f). By using T7 exonuclease, the FAM signal increased significantly with the still stable CDs. This phenomenon indicated the amplification of the sensing response (line g). The stable CDs and altered FAM signals in the different reaction systems confirmed the feasibility of detecting miRNA-21 by T7 exonuclease-mediated signal amplification and CDs used as the internal signal, carrier, and quencher through the ratiometric strategy. Optimization of the detection system Several experimental parameters were optimized to achieve the best response in this strategy for miRNA-21 detection. In this testing process, an appropriate situation for 7

CDs was needed to quench the FAM-labeled ssDNA and reduce the background response of the detecting system. Thus, the concentration and incubation time of CDs for FAM-labeled ssDNA was optimized to investigate the quenching response, as shown in Fig. S1. A small amount (1 µg/mL) of CDs resulted in 91.2% quenching of FAM-labeled ssDNA. When the concentration of CDs increased to 4 µg/mL, the quenching degree slightly increased to 92%. The stable quenching efficiency was maintained even if the concentration of CDs increased continuously (Fig. S1A). Furthermore, 2 µg/mL CDs rapidly quenched FAM-labeled ssDNA in 1 min with a quenching efficiency of 91.5%. The time was extended to 10 min and longer, and the quenching efficiency was stable at around 92% (Fig. S1B). By considering the fluorescence intensity relation between CDs and FAM-labeled ssDNA for the ratiometric strategy, 4 µg/mL CDs and 10 min were chosen as the optimized quenching condition to achieve excellent performance in this method. The concentration of FAM-labeled ssDNA used as the capture probe was optimized. Varied signals increased with the increase in the capture probe, and the changed fluorescence signal reached the maximum value until the concentration reached 50 nM, and then the variety was kept relatively stable (Fig. S2A). Thus, 50 nM capture probe was chosen in the experiment. Reaction factors, including enzyme dosage, incubation temperature, and time, were optimized to maximize the enzyme performance. The amount of T7 exonuclease was optimized to evaluate the amplification efficiency. By changing the enzyme dosage from 25 U to 45 U, the signal-to-noise ratio gradually increased and then decreased, and the largest response was achieved at 40 U. The results suggested that the system containing 40 U T7 exonuclease could ensure a sufficient change in fluorescence intensity for high sensitivity detection (Fig. S2B). Furthermore, with the change in temperature, the fluorescence intensity fluctuated slightly at 20 °C to 40 °C, while it obtained the best response when the temperature was 37°C (Fig. S2C). Moreover, the fluorescence intensity gradually increased with the extending time and reached a plateau in 30 min (Fig. S2D). This finding indicated that the complete enzyme reaction took 30 min to complete. Thus, 37°C and 30 min were chosen as the 8

optimum reaction temperature and time. Detection performance of the ratiometric fluorescence method Synchronized fluorescence scanning was carried out under the optimal condition to investigate the different fluorescent signals with various concentrations of target miRNA-21. As shown in Fig. 4, the fluorescence intensity of FAM at 498 nm gradually recovered and increased with the increase in concentration of miRNA-21, whereas that of CDs centered at 368 nm and remained fairly stable. The FFAM/FCDs value was positively correlated with the miRNA-21 concentration (Fig. 4A), where FFAM is the fluorescence intensity of FAM and FCDs is the fluorescence intensity of CDs. As shown in Fig. 4B, the FFAM/FCDs value showed a good linear relationship with the concentration of miRNA-21 in the range of 0.05–10 nM. The linear equation was FFAM/FCDs = 0.0825C + 0.502 (R2 = 0.998). The detection limit of the developed method was 1 pM according to the actual testing result. Compared with other constructed ratiometric sensors (Table 1), this method illustrated the characteristics of low detection limit (1 pM) and wide linear range. Furthermore, this strategy demonstrated the advantage of only requiring a simple mixing operation without any modification or covalent process. Similar miRNAs might interfere with miRNAs during detection due to the sequence similarity of miRNAs. To evaluate the selectivity of this biosensor, control experiments were carried out under the same conditions using miRNA-21 and three mismatched

RNAs,

including

single-

and

double-base

mismatches

and

noncomplementary (NC) RNAs (Fig. 4C). The concentration of the target miRNA-21 and mismatched RNA was set to 5 and 10 nM, respectively. The detecting system presented evident response fluorescence intensity to the addition of miRNA-21. By contrast, the mismatched sequences did not cause apparent signal changes but indicated that the system was highly specific for miRNA-21 analysis because only miRNA-21 could trigger the T7 exonuclease-assisted signal amplification reaction and recover the quenched fluorescence signal. In general, ratiometric sensors enhanced their reproducibility due to the application of internal reference to correct environmental factors unrelated to analysis. Hence, the 9

testing repeatability of this strategy was assessed. Thirty individual samples of miRNA-21 at a concentration of 0.01 nM were measured. Six samples were measured daily and within 5 days (Fig. 4D). The results demonstrated that the values were close to one other in the 30 tests. The standard deviation (SD) and relative SD (RSD) of the measurements were 0.029 and 4.92%, respectively. The low SD and RSD of the ratiometric fluorescence method using CDs suggested the high reproducibility and reliability of the measurement likely due to the triple functions of CDs as internal reference signal, adsorption carrier, and quencher. Real Sample Assays Various concentrations (10, 100, and 1000 pM) of miRNA-21 were added to 100-fold diluted blood plasma samples from four different healthy volunteers to evaluate their recovery for verifying the application potential of the proposed method for miRNA detection in clinical practice. As shown in Table 2, recovery fluctuated between 92.6% and 105.3% with the RSD less than 6.7%. The recovery indicated that the proposed sensing platform performed accurate miRNA-21detection in the potential application in clinical diagnosis. The developed method was further applied to test the relative expression level of miRNA-21 in clinical blood samples from gastrointestinal cancer patients and healthy donors. The expression level of whole blood miRNA-21 as the reference was concurrently quantified by qRT-PCR. As shown in Fig. 5, after the removal of the fluorescence response of the blank condition containing 4 µg/mL CDs, 50 nM FAM-labeled ssDNA and 40 U T7 exonuclease, the testing results of miRNA-21 obtained with this method were in good agreement with those via qRT-PCR. The consistent results indicated the testing accuracy of the developed ratiometric fluorescence sensor for clinical samples. In addition, the expression of miRNA-21 in the blood of patients with gastrointestinal cancer was remarkably higher than that of healthy donors after the two methods were statistically compared. This finding confirmed that miRNA-21 was up-regulated in the blood of gastrointestinal cancer patients [47]. Therefore, the developed ratiometric fluorescent method demonstrated excellent practicability in detecting miRNA-21 in clinical blood samples. 10

Conclusions We created a simple, fast ratiometric fluorescence sensor for high reproducibility and specific detection of miRNA-21 by using a combination of CDs and T7 exonuclease-mediated signal amplification. The proposed ratiometric strategy utilized the triple functions of CDs. The quantitative detection of miRNA-21 improved the reproducibility and reliability attributed to the application of the ratiometric approach of FFAM/FCDs. This sensor exhibited not only acceptable sensitivity but also distinguished

single-base mismatched

miRNAs.

Furthermore,

this

platform

successfully evaluated the expression level of miRNA-21 in clinical blood samples and obtained results consistent with those of qRT-PCR. This work suggested that the design of some special novel materials with multiple functions would help in fabricating a new type of bioassay with significantly improved analytical performance to meet clinical requirements.

Acknowledgements The authors gratefully acknowledge the financial support of the National Science Foundation of China (21405016 and 21705021), Joint Funds for the innovation of science and Technology, Fujian province (2017Y9121 and 2017Y9042), the Natural Science Foundation of Fujian Province of China (2017J01328), the Elite Cultivation Program of Health and Family Planning of Fujian Province (2017-ZQN-39, 2017-ZQN-61) and Fujian Provincial Key Laboratory of Featured Biochemical and Chemical Materials (Ningde Normal University) (FJKL_FBCM201806).

Abbreviations miRNAs: microRNAs; miRNA-21: microRNA-21; CDs: carbon dots; FAM: carboxyfluorescein; ssDNA: single-stranded DNA; qRT-PCR: quantitative real-time polymerase chain reaction; PL: photoluminescence; SD: standard deviation; RSD: relative standard deviation.

Conflicts of Interest The authors have declared that no competing interest exists. 11

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Figures and Figure captions

Fig. 1. Scheme of the ratiometric fluorescence-detecting platform for miRNA-21 based on CDs with the assistance of T7 exonuclease cycle signal amplification.

17

Fig. 2. (A) UV-vis absorption and fluorescence spectrum of CDs, (B) FL emission spectra of CDs with different exciting wavelengths, (C) TEM image (HRTEM image (upper) and the size distribution histograms (down) in inset) of CDs, and (D)FTIR spectrum of the CDs.

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Fig. 3. Feasibility of CDs-based fluorescence platform for miRNA-21 detection assisted with T7 exonuclease signal amplification. (a) Synchronous fluorescence spectra of CDs, (b) FAM-labeled ssDNA, (c) mixture of FAM-labeled ssDNA and CDs, (d) addition of T7 exonuclease into the mixture of FAM-labeled ssDNA and CDs, (e) addition of BSA into the mixture of FAM-labeled ssDNA, CDs and T7 exonuclease, (f) addition of miRNA-21 into the mixture of FAM-labeled ssDNA and CDs, and (g) addition of miRNA-21 and T7 exonuclease into the mixture of FAM-labeled ssDNA and CDs. Inset is the amplification part of (c), (d) and (e) at 498 nm. (CDs: 4 µg/mL, FAM-labeled ssDNA: 50 nM, T7 exonuclease: 20 U, miRNA-21: 20 nM, BSA: 10 µg/mL).

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Fig. 4. (A) Synchronous fluorescence spectra of the system in the presence of different concentrations of miRNA-21 (0, 0.001, 0.01, 0.05, 0.1, 0.5, 1, 2.5, 5, 10, and 20 nM). (B) Relationship between FFAM/FCDs and miRNA-21concentration. (C) Normalized fluorescence intensity changes in FFAM/FCDs of the biosensor toward miRNA-21, single-base mismatches (Mis-1), double-base mismatches (Mis-2), and NC RNA. (D) Reproducibility of the 30 independent measurements of 0.01 nM miRNA-21. The black histogram represents 30 independent measurements. Red bars and red error bars represent the mean value and standard deviation of the 30 tests, respectively.

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Table 1 The performance comparison of this strategy and previously reported ratiometric detection of miRNAs Technique

Linear range

Detection limit

Detection method

Reference

Fluorescence

20-100 pM

12.0 pM

CdTe QDs/ oxidized luminol

[43]

Fluorescence

0.5-50 nM

72.0 pM

Fluorescence

0-500 fM

3.0 fM

Fluorescence

2-60 nM

0.68 nM

Fluorescence

1nM-5 µM

0.3 nM

Fluorescence

5-150 nM

0.38 nM

Fluorescence

0.05-10 nM

1 pM

catalyzed hairpin assembly Carbon Dots/ Acridone Derivate Zeolitic Imidazolate Framework-8 Light-up RNA aptamers MoS2 quantum dots/molecular beacon carbon dots/ T7 exonuclease

[34] [35] [44] [45] [46] This work

Table 2 Recovery results of this assay for miRNA-21 in spiked human plasma (n=3) Samples

Human plasma 1

Human plasma 2

Human plasma 3 Human plasma 4

Spiked (pM)

Found (pM)

RSD (%)

Recovery (%)

10

10.41

3.4

104.1

100

99.59

4.7

99.6

1000

925.94

2.9

92.6

10

9.90

5.2

99.0

100

105.26

0.6

105.3

1000

950.22

1.6

95.0

10

10.04

2.9

100.4

100

95.95

3.5

96.0

1000

1013.68

2.1

101.4

10

9.75

1.2

97.5

100

103.14

2.4

103.1

1000

997.26

6.7

99.7

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Fig. 5. Relative expression of miRNA-21 in the blood of three healthy donors and eight gastrointestinal cancer patients quantified by the (A) proposed method and (B) qRT-PCR, respectively, and (C–D) their corresponding statistical analysis. The error bars in (A) indicate the SD values of three replicates for the different samples.

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Highlights

A turn-on ratiometric fluorescence bioassay based on the T7 exonuclease and carbon dots (CDs) was developed for miRNA-21

CDs demonstrated the triple functions of built-in internal fluorescence, probe carrier, and quencher in this strategy

The bioassay showed a good linear relationship with low detection limit, excellent selectivity and reproducibility for miRNA-21

The expression level of miRNA-21 is closely related to the occurrence and development of gastrointestinal cancer

This method successfully evaluated miRNA-21 in clinical blood samples in accord with the results from qRT-PCR

1

Conflicts of Interest The authors have declared that no competing interests exist.