- Email: [email protected]
miRNA [email protected] QDs and oxidized luminol as a fluorophore for miRNA detection
Journal of Luminescence 204 (2018) 16–23
Contents lists available at ScienceDirect
Journal of Luminescence journal homepage: www.elsevier.com/locate/jlumin
Ratiometric fluorescence biosensor based on DNA/miRNA duplex@CdTe QDs and oxidized luminol as a fluorophore for miRNA detection Yasaman Sadat Borghei, Morteza Hosseini
T
⁎
Department of Life Science Engineering, Faculty of New Sciences & Technologies, University of Tehran, Tehran, Iran
A R T I C LE I N FO
A B S T R A C T
Keywords: MicroRNAs Ratiometric Light-on Fluorescence Cancer
Breast cancer is the second most diagnosed cancer in women worldwide. Hence, the detection of prognostic and diagnostic biomarkers can improve the patient's quality of life during the course of illness and treatment. Most recently, microRNAs (miRNAs) have been widely studied for non-invasive prediction of prognosis markers in the diagnosis of breast cancer. Herein, we reported a ratiometric fluorescence nanobiosensor for detection of miR155. The biosensor comprises of 3-mercapropionic acid-coated cadmium telluride (CdTe) quantum dots and oxidized luminol (Lumox) exhibiting emission peaks at 550 and 440 nm, respectively, under single-wavelength excitation (350 nm). In the presence of miR-155, CdTe QDs were aggregated and their fluorescence was quenched via interacting strongly at their metal centers, Cd, with heteroduplex formed between the DNA probe and miR-155. Then, by addition of blue-emitting Lumox with their constant fluorescence emission, a ratiometric means of miR-155 detection was developed. The relative fluorescence intensity ration is directly proportional to the concentration of miR-155 between 20.0 and 100.0 pM. The detection limit is 12.0 pM. This novel assay is “light-on” and has been successfully applied for the detection of miRNA in MCF-7 and HEK 293 cell lysates as real samples.
1. Introduction MicroRNAs (miRNAs) are a large family of non-coding single stranded RNAs (usually19–23 nucleotides) that regulate gene expression at the post-transcriptional level via direct binding to target mRNAs. The roles of miRNAs in disease, particularly in cancer, have made miRNAs attractive tools for diagnosis and therapy. For instance, MiR155, located in chromosome 21q21, is encoded with B cell integration cluster (BIC) gene, which is up-regulated in different types of human cancers such as breast cancer [1,2]. The miR-155 binds directly to RhoA, a prometastatic gene, and plays a key role in breast cancer metastasis. In another research it has been shown that inflammatory stimulation of breast cancer cells increased the level of miR-155 which then they would bind to SOCS1 [3,4]. Also, it was found that there is an overexpression of miR-155 in invasive tumors but not in noninvasive cancer samples and it was shown that miR-155 is associated with the proliferation, invasion and apoptosis due to its action on caspase 3 [5]. These facts show that there miRNAs could have many potential applications in the diagnosis, prognosis and forecasts in therapeutic [6]. Therefore, accurate and rapid quantification of miRNAs is important. Common methods used for this purpose include microarray, next-generation sequencing, NanoString nCounter and quantitative reverse ⁎
transcriptase real-time (qRT-PCR); however, each of these methods has its own advantages and limitations [7]. For example, a major limitation of qRT-PCR, which is used as a gold standard method is that it will not generate quantitative results at the limits of its sensitivity [8]. Recently, the use of photoluminescent nanoparticles like semiconductor quantum dots (QDs) as fluorescent probes in biosensing (such as DNA, proteins, peptides, and drugs) and bio-imaging has been very much considered because of their advantages such as size-dependent tunable emission, photostability and broad excitation spectra, in comparison to organic dye. QDs are nanocrystals, which are comprised of a few hundreds to a few millions of atoms, and only a small number of free electrons (≤ 100) [9–12]. In this study for the first time, oxidized luminol (Lumox) was used as a blue-emitting fluorophore probe in the green-emitting 3-mercaptopropionic acid-capped CdTe QD-based dual-emission ratiometric fluorescence biosensor for miRNA detection. Initially, miR-155 aggregated and quenched the fluorescence of CdTe QDs [13–17] via strong interaction with DNA/miR-155 heteroduplex [18–24] switching the green fluorescence into an “off” state at 550 nm with a short response time. Meanwhile, Lumox molecules were insensitive to both CdTe QDs and DNA/miR-155 duplex and maintained constant fluorescence intensity at 440 nm, thereby serving as a reference for
Corresponding author. E-mail address: [email protected] (M. Hosseini).
https://doi.org/10.1016/j.jlumin.2018.07.034 Received 20 March 2018; Received in revised form 18 July 2018; Accepted 23 July 2018 Available online 24 July 2018 0022-2313/ © 2018 Elsevier B.V. All rights reserved.
Journal of Luminescence 204 (2018) 16–23
Y.S. Borghei, M. Hosseini
Scheme 1. Schematic representation of the fluorescence behavior of green CdTe QDs (a), DNA probe@CdTe QDs (b) and dsDNA/miR-155@CdTe QDs (c) in the presence of blue-emitting Lumox.
ratiometric detection of miRNA. On the other hand, in the absence of miR-155, CdTe QDs and Lumox exhibited emissions peaks at 550 nm and 440 nm, respectively (Scheme 1). This biosensor was successfully used for detection of miR-155 in human breast carcinoma cells (MCF-7) and human normal cells (HEK 293).
Inc., Bedford, MA) was used throughout the reactions. MCF-7 cells (human breast cancer cell line) and HEK 293 cells (from normal human embryonic kidney cell line) were used in this study.
2. Experimental
AFM imaging was performed on DNA probes and DNA/miR-155 heteroduplex samples deposited on freshly cleaved mica sheets. For this, 1 × 1 cm mica slides soaked in 5 mM MgCl2, and 2 min after, the surface was dried at room temperature. Then, 30 μL of samples in 1 mM MgCl2 were spotted onto mica plates and dried at room temperature. After washing the samples with deionized water and drying, AFM imaging was done on a Solver PRO AFM system (NT-MDT, Russia), in semi-contact (tapping) mode, using Si-gold-coated cantilevers (NTMDT, Zelenograd, Russia) with a resonance frequency of 375 kHz. Images were recorded in height mode, and Nova image processing software (NT-MDT, Zelenograd, Russia) software was used for data processing and particle analysis.
2.3. Preparation of samples for atomic force microscopy (AFM) imaging
2.1. Apparatus All fluorescence measurements were carried out using a Perkin Elmer LS-55 fluorescence spectrometer with a xenon lamp as source of excitation while the spectral band widths of monochromators for excitation and emission were 10 nm. (Buckinghamshire,UK). The size and morphology of bare QDs were measured by transmission electron microscopy (TEM) (Zeiss, EM10C, 80 kV, Germany) and atomic force microscopy (AFM) (NT-MDT, Zelenograd, Russia). UV–vis spectroscopy was performed by a Specord 250 spectrophotometer (Analytik Jena, Germany).
2.4. Preparation of oxidized luminol 2.2. Materials and reagents Reduced luminol was prepared by dissolving luminol (10-4 M) in to 10 ml of 0.1 M NaOH. In the next step, Blue-emitting luminol (Lumox) or aminodiphthalate is formed by adding a suitable amount of hydrogen peroxide (H2O2, 10-4 M).
Fetal bovine serum (FBS), Dulbecco's modified eagle medium (DMEM), and penicillin/streptomycin were purchased from Gibco (USA). Oligonucleotides were synthesized and purified by Shanghai Generay Biotech Co (Shanghai, China), and their sequences were listed in Table.S1. All oligonucleotide samples were purified by PAGE and prepared with TE buffer (1 M Tris-HCl, 0.5 M EDTA). Cd(NO3)2, tellurium powder, thioglycolic acid (TGA) and sodium borohydride (NaBH4) were purchased from Merck and cell culture lysis reagent (CCLR) was purchased from Sigma Aldrich. All other reagents were of analytical reagent grade and ultrapure water (Milli-Q plus, Millipore
2.5. Preparation of MAA capped-CdTe Quantum Dots The experimental procedure was based on previous works [11, 25, 26]. In summary, Cd solution (0.4 mmol) and thioglycolic acid (TGA) (1.4 mmol) were solvated in 80 ml distilled water with pH adjusted to 10.0 using NaOH solution. Next, sodium borohydrate (0.8 mmol) and 17
Journal of Luminescence 204 (2018) 16–23
Y.S. Borghei, M. Hosseini
Te powder were diluted in 10 ml distilled water in a flask, with vigorous stirring under argon flow. The mixture was heated to 80 °C to get a clear red NaHTe solution. Cd-TGA solution was heated at 100 °C under argon flow in a 250 ml three-neck flask. Then the freshly prepared NaHTe solution (4.0 ml) was added to the flask, and the resulting solution was refluxed at 100 °C for different times. The characterization of CdTe quantum dots was carried out through transmission electron microscopy and spectrofluorometry.
normal human embryonic kidney) were cultured in 25 cm2 tissue culture flasks (SPL, Korea) with 5 ml Dulbecco's modified Eagle's medium (Sigma, UK) containing 10% heat inactivated fetal bovine serum (Gibco), 100 U/ml penicillin (Sigma, UK). Cell lines were grown at 37 °C in a humidified atmosphere of 5% CO2 and 95% air for 5 days until the cell monolayer became confluent. Growth medium was replaced with fresh media every 2 days or as required, indicated by color change due to production of lactic acid and CO2, which leads to low pH. Upon reaching at least 80% confluency, the cells were washed with phosphate buffered saline (PBS) and trypsinized for 10 min at 37 °C with 0.05% trypsin.
2.6. Cell culture MCF-7 cells (human breast cancer cell line) and HEK 293 cells (from
Fig. 1. (A) Absorbance (B) and fluorescence spectra of Lumox and CdTe QDs. Photographs of them under Visible and UV light. (C) TEM image of CdTe QDs. 18
Journal of Luminescence 204 (2018) 16–23
Y.S. Borghei, M. Hosseini
3.3. Effect of miR-155 and heteroduplex formation on ratiometric fluorescence
2.7. Total RNA extraction Cell samples were disrupted and total RNA was extracted from MCF7 and HEK 293 cell lines using Cell Culture LysisReagent (CCLR). Approximately 2.0 × 106 cells collected by low-speed centrifugation at 1000 rpm for 10 min. Culture medium was carefully removed and the pellet was washed twice with PBS. The PBS was carefully removed and 600 μL CCLR buffer was added. Cells were gently resuspended in CCLR buffer with a vortex and incubated for 20 min. Subsequently, 0.2 ml chloroform was added and the mixture was vortexed for 20 s violently. Next, the mixture was centrifuged for 20 min at 13,000 rpm at 4 °C by refrigerated centrifugation. Same volume of isopropyl alcohol was added to the upper water which has been taken out and mixed evenly followed by overnight precipitation at − 20 °C. Afterward, centrifugation was carried out for 20 min at 13,000 rpm at 4 °C. Then the supernatant was removed and the precipitant was washed by 80% ethanol with DEPC water and centrifuged for 20 min at 13,000 rpm at 4 °C. The ethanol was volatilized (Letting the tube dry) and the purified RNA was dissolved in an appropriate volume of DEPC water.
Addition of DNA/miR-155 heteroduplex to CdTe QDs solution reduced the fluorescence intensity of CdTe–especially the emission peak at 550 nm–while that of blue-emitting Lumox remained constant at 440 nm. On the other hand in the absence of miR-155, QDs and Lumox still have fluorescence emission peak at 550 nm and 440 nm, respectively (Fig. 3). 3.4. Optimization We evaluated the effect of pH and different concentrations of CdTe QD on quenching effect of the DNA/miRNA duplex on CdTe QDs. Firstly, as shown in Fig. S1A the effect of different concentrations of CdTe QDs (10 pM, 100 pM and 10 nM) was tested by comparing the fluorescence spectra of CdTe QDs alone, DNA@CdTe QDs and DNA/ miR-155 duplex. It was observed that the highest decrease in fluorescence emission occurred at concentration 100 pM and therefore, this concentration was used at all stages of the experiment. We also investigated the effect of pH on quenching of CdTe QDs in the presence of DNA/miR-155 duplex and found that the fluorescence intensity of QDs at pH 7.5 is minimal (Fig. S1B).
2.8. Procedures for fluorescence detection of miR-155 The test started with incubating the target miR-155 and its complementary DNA probes (10 μL of the 100 pM) in 20 mM of phosphate buffer (pH 7.5) at 90 °C for 10 min. Then the obtained mixture solution was incubated at 37 °C for 1 h, in which the complementary DNA probes recognize the specific miR-155 targets in order to create the formations of DNA/miR-155 heteroduplex. Afterwards, 10 μL of green QDs (100 pM) was added to reaction solutions. These samples were applied to the fluorescence quenching phenomenon investigation. Subsequently, the Lumox (2 μL, 10-4 M) was used for analyzing dualemission of the solution system.
3.5. Ratiometric fluorescence detection of miR-155 We explored the capability of using the CdTe QDs and Lumox system for miRNA detection. Fig. 4A displays the typical quenching of QD fluorescence spectra at 550 nm by DNA/miR-155 heteroduplex at different concentrations of miR-155 from 20 pM to 100 pM (Fig. 4A). We attribute the aggregation to double stranded nucleic acid (DNA/miRNA duplex) binding to the chalcogenide QDs [18–24] and the quenching to intra-aggregate energy transfer between QDs [13–17]. The decrease fluorescence intensities are linearly proportional to the concentrations of target miR-155. To obtain signal “ON” and dual-emission sensor, lumox was added to the reaction solutions. Following the addition of blue-emitting Lumox, the fluorescence intensity remained at 440 nm and the fluorescence intensity at 550 nm was decreased by adding increasing amounts of miR-155 to the system (20–100 pM) (Fig. 4B). Because the Lumox was not sensitive to both CdTe QDs and DNA/miR-155 duplex and maintained constant fluorescence intensity at 440 nm, thereby serving as a reference for dual-emission assay for miRNA. The fluorescence intensity ratio of 550/440 nm showed a linear response to miR-155 concentration in the range of 20–100 pM. The detection limit was estimated at 12.0 pM. according to the definition of three times the deviation of the blank signal. The proposed method was compared with other methods [28–33] that use one or more fluorophore materials, and as shown in Table 1, this method has low sensitivity along with ease and cost-effectiveness.
3. Results and discussion 3.1. Characterization of Lumox and CdTe QDs Fig. 1 shows the absorbance (Fig. 1A) and fluorescence (Fig. 1B) spectra of Lumox and CdTe QDs. Lumox exhibits two principle absorption bands in 280 and 330 nm region, whereas, a single fluorescence emission peak appears at 440 nm. The formation of hydrogen bonds in water leads to a shift in the wavelength of emission to the larger wavelengths which is related to the stabilization of the charge transfer excited state of hydrogen bonds of water with Lumox. On the other hand, the emission peak of QDs locates at 550 nm with the characteristic absorption peak at 400 nm [27]. The TEM analysis of MAA-cappedCdTe QDs shows monodispersed particles with an average diameter of ca. 5 nm (Fig. 1C).
3.6. Selectivity 3.2. AFM and TEM analysis for CdTe QDs aggregation We investigated the selectivity of the described method here by investigative the signal responses of the probe DNA toward non-complementary target DNA, miR-21, Let 7a and miR-155 at the same concentration of 100 pM. As the results are shown in Fig. 5, the FL 550/ FL440 nm ratio of the DNA probe toward the perfectly matched target miR-155 was very different from that of other miRNAs which were closed to that of the blank sample. These results confirm that the high selectivity for miRNA detection in this described method.
In order to prove and validate the theory that in the presence of DNA/miRNA duplex, the QDs are aggregated, AFM images of the CdTe QDs were taken before and after adding the miR-155 target. Fig. 2A or B is the image taken from the sample of the DNA probe@QDs before adding the miR-155 and the Fig. 2C or D after adding the miR-155 and formation of DNA/miR-155 duplex. Comparing 3D images of A and C (or 2D images of B and D), it is easy to conclude that CdTe QDs have been aggregated in the Fig. 2C or D because they have larger dimensions and a supermolecular structure compared to that of Fig. 2A or B. In addition, TEM images were taken before (Fig. 2E) and after (Fig. 2F) adding the miR-155 target. As it is known, after adding miR-155 to the ssDNA probe@QDs, the CdTe QDs are aggregated.
3.7. MiR-155 detection in total RNA extracted from cells In order to test the selectivity and possibility of applying this dualmode fluorescent probe for miRNA determination in real samples, the 19
Journal of Luminescence 204 (2018) 16–23
Y.S. Borghei, M. Hosseini
Fig. 2. (A, C) 3D images and (B, D) 2D images of CdTe QDs by Atomic Force Microscopy (AFM) scanning of mica surface and (E, F) TEM analysis prepared by the solution of (A, B, E) single strand DNA@CdTe QDs and (C, D, F) double stranded DNA/miR-155@ CdTe QDs.
RNA extraction solution related to MCF-7 cells, while fluorescence emission of blue-emitting Lumox remains almost constant. Fluorescence emission of QDs has decreased slightly in the presence of HEK 293 cell lysate (Fig. 6A). In addition, these results are very reasonable in comparison to qRT-PCR (Fig. 6B). The relative intensity was calculated to the expression of U6 snRNA. The primers for miR-155 and U6 snRNA are shown in Table S1. However, the presence of other interfering substances such as mRNAs and miRNAs didn’t have any significant result in this dual-mode fluorescent assay. 4. Conclusion A water-soluble CdTe QDs–Lumox system was designed for the determination of miRNA in biological samples. In this suggested method, DNA probe/miR-155 duplex acted as a fluorescence “off” for greenemitting CdTe QDs through aggregation. Blue-emitting Lumox were insensitive to QDs and DNA/miR-155 duplex and no fluorescence resonance energy transfer occurred between Lumox and CdTe QDs, allowing ratiometric miRNA detection. Furthermore, the CdTe QDs and Lumox did not need chemical modification after their preparation, so making this method cost-effective. The CdTe-CDs ratiometric fluorescence sensing removed background signal and since calibration curve is based on two emission peaks, its results will be more acceptable than single-emission signal sensors.
Fig. 3. Emission spectra of CdTe QDs and Lumox in the solution of ssDNA probe and dsDNA/miR-155 in concentration of 100 pM and pH = 7.5.
Acknowledgements proposed method was used to identify miR-155 in total RNA extraction from MCF-7 (as a breast cancer cell) and HEK 293 (as a normal cell). The fluorescence spectra in Fig. 6A show that the fluorescence intensity of the QDs at 550 nm could be greatly decreased by the addition of total
The authors thank the Iran National Science Foundation (INSF 96010584) and the Research Council of University of Tehran (Grant 28645/01/02) for financial support of this work. 20
Journal of Luminescence 204 (2018) 16–23
Y.S. Borghei, M. Hosseini
Fig. 4. Emission spectra of dsDNA-green QDs complex formed with miR-155 target in concentrations of 20, 30, 40, 50, 60, 70, 80, 90 and 100 pM, before (A) and after (B) addition of Lumox. (C) the calibration curve for fluorescence intensity versus target microRNA155 concentration. (Photographs of them under UV light).
Table 1 Comparison between proposed method and other methods that use one or more fluorophore materials. Method used
Fluorophore
Detection limit
References
A magnetic fluorescence Photoinduced electron transfer (PET) Clever single-labeled fluorescence Fluorescence An “off-on” fluorescent switch assay Ratiometric fluorescence sensor Ratiometric fluorescence biosensor
dyes-loaded albumin nanoparticles DNA/AgNCs 2-aminopurine dye-labeled single-stranded DNA FAM labeled signal probes 2-aminopurine & thioflavin T CdTe QDs & Lumox
9 fM 0.06 nM 2.5 nM 0.18 nM 1 nM 72 pM 12.0 pM
[28] [29] [30] [31] [32] [33] In this work
21
Journal of Luminescence 204 (2018) 16–23
Y.S. Borghei, M. Hosseini
Fig. 5. Plot of FL 550/ FL 440 nm ratio obtained with different miRNA and non complementary DNA at the same concentration (100 pM, pH = 7.5).
Fig. 6. Total RNA extraction from HEK 293 (human normal cell line) and MCF-7 (breast cancer cell line) and the fluorescence signal of them after treatment with ssDNA probe and two color QDs (A). Photograph of them under UV light illumination. Relative signal of two methods for miR-155 detection: quantitative real-time PCR (qRT-PCR) and the proposed method (B).
22
Journal of Luminescence 204 (2018) 16–23
Y.S. Borghei, M. Hosseini
Appendix A. Supporting information
[16] A. Polimeni, A. Patane, M. Henini, L. Eaves, P.C. Main, Temperature dependence of the optical properties of I n A s/A l y Ga 1− y As self-organized quantum dots, Phys. Rev. B 59 (1999) 5064. [17] A.J. Chiquito, Y.A. Pusep, S. Mergulhão, Y.G. Gobato, J.C. Galzerani, N. Moshegov, Effects of annealing on electrical and optical properties of a multilayer InAs/GaAs quantum dots system, Mater. Res. 7 (2004) 459–465. [18] S.K. Kailasa, K.H. Cheng, H.F. Wu, Semiconductor nanomaterials-based fluorescence spectroscopic and matrix-assisted laser desorption/ionization (MALDI) mass spectrometric approaches to proteome analysis, Materials 6 (2013) 5763–5795. [19] W.R. Algar, U.J. Krull, Characterization of the adsorption of oligonucleotides on mercaptopropionic acid-coated CdSe/ZnS quantum dots using fluorescence resonance energy transfer, J. Colloid Interface Sci. 359 (2011) 148–154. [20] Z. Wang, H. He, W. Slough, R. Pey, S.P. Karna, Nature of interaction between semiconducting nanostructures and biomolecules: chalcogenide QDs and BNNT with DNA molecules, J. Phys. Chem. C 119 (2015) 25965–25973. [21] W.R. Algar, U.J. Krull, Adsorption and hybridization of oligonucleotides on mercaptoacetic acid-capped CdSe/ZnS quantum dots and quantum dot? Oligonucleotide conjugates, Langmuir 22 (2006) 11346–11352. [22] S.S. Narayanan, S.S. Sinha, P.K. Verma, S.K. Pal, Ultrafast energy transfer from 3mercaptopropionic acid-capped CdSe/ZnS QDs to dye-labelled DNA, Chem. Phys. Lett. 463 (2008) 160–165. [23] Q. Xu, J.H. Wang, Z. Wang, Z.H. Yin, Q. Yang, Y.D. Zhao, Interaction of CdTe quantum dots with DNA, Electrochem. Commun. 10 (2008) 1337–1339. [24] S. Anandampillai, X. Zhang, P. Sharma, G.C. Lynch, M.A. Franchek, K.V. Larin, Quantum dot–DNA interaction: computational issues and preliminary insights on use of quantum dots as biosensors, Comput. Methods Appl. Mech. Eng. 197 (2008) 3378–3385. [25] M. Hosseini, M.R. Ganjali, Z. Vaezi, B. Arabsorkhi, M. Dadmehr, F. Faridbod, P. Norouzi, Selective recognition histidine and tryptophan by enhanced chemiluminescence ZnSe quantum dots, Sens. Actuators B 210 (2015) 349–354. [26] F.S. Sabet, M. Hosseini, H. Khabbaz, M. Dadmehr, M.R. Ganjali, FRET-based aptamer biosensor for selective and sensitive detection of aflatoxin B1 in peanut and rice, Food Chem. 220 (2017) 527–532. [27] P. Reiss, M. Protiere, L. Li, Core/shell semiconductor nanocrystals, Small 5 (2009) 154–168. [28] T. Wei, D. Du, Z. Wang, W. Zhang, Y. Lin, Z. Dai, Rapid and sensitive detection of microRNA via the capture of fluorescent dyes-loaded albumin nanoparticles around functionalized magnetic beads, Biosens. Bioelectron. 94 (2017) 56–62. [29] S. Lu, S. Wang, J. Zhao, J. Sun, X. Yang, Fluorescence light-up biosensor for microRNA based on the distance-dependent photoinduced electron transfer, Anal. Chem. 89 (2017) 8429–8436. [30] R. Liao, S. Li, H. Wang, C. Chen, X. Chen, C. Cai, Simultaneous detection of two hepatocellar carcinoma-related microRNAs using a clever single-labeled fluorescent probe, Anal. Chim. Acta 983 (2017) 181–188. [31] X. Fan, Y. Qi, Z. Shi, Y. Lv, Y. Guo, Molecular mechanism of helicase on graphenebased hybridization reaction platform for microRNA detection, RSC Adv. 7 (2017) 36444–36449. [32] Y. Li, Q. Pu, J. Li, L. Zhou, Y. Tao, Y. Li, W. Yu, G. Xie, An “off-on” fluorescent switch assay for microRNA using nonenzymatic ligation-rolling circle amplification, Microchim. Acta 184 (2017) 4323–4330. [33] Y. Liu, T. Shen, J. Li, H. Gong, C. Chen, X. Chen, C. Cai, Ratiometric fluorescence sensor for the microRNA determination by catalyzed hairpin assembly, ACS Sens. 2 (2017) 1430–1434.
Supplementary data associated with this article can be found in the online version at https://doi.org/10.1016/j.jlumin.2018.07.034. References [1] B. Smith, P. Agarwal, N.A. Bhowmick, MicroRNA applications for prostate, ovarian and breast cancer in the era of precision medicine, Endocr.-Relat. Cancer 24 (2017) 157–172. [2] S. Ding, Y. Xu, L. Shen, H. Huang, X. Yu, C. Lu, C. Zhong, MiR-155 promotes proliferation of human non-small cell lung cancer H460 cells via targeting TP53INP1, Int. J. Clin. Exp. Med. 10 (2017) 11953–11960. [3] S. O'Bryan, S. Dong, J.M. Mathis, S.K. Alahari, The roles of oncogenic miRNAs and their therapeutic importance in breast cancer, Eur. J. Cancer 72 (2017) (1-1). [4] A. Asiaf, S.T. Ahmad, W. Arjumand, M.A. Zargar, MicroRNAs in breast cancer: diagnostic and therapeutic potential, MicroRNA and Cancer, Humana Press, New York, NY, 2018, pp. 23–43. [5] H. Yu, W. Xu, F. Gong, B. Chi, J. Chen, L. Zhou, MicroRNA-155 regulates the proliferation, cell cycle, apoptosis and migration of colon cancer cells and targets CBL, Exp. Ther. Med. 14 (2017) 4053–4060. [6] M. Hemmatzadeh, H. Mohammadi, F. Jadidi-Niaragh, F. Asghari, M. Yousefi, The role of oncomirs in the pathogenesis and treatment of breast cancer, Biomed. Pharm. 78 (2016) 129–139. [7] R. Hamam, D. Hamam, K.A. Alsaleh, M. Kassem, W. Zaher, M. Alfayez, A. Aldahmash, N.M. Alajez, Circulating microRNAs in breast cancer: novel diagnostic and prognostic biomarkers, Cell Death Dis. 8 (2017) 3045. [8] S.A. Bustin, T. Nolan, Pitfalls of quantitative real-time reverse-transcription polymerase chain reaction, J. Biomol. Tech.: JBT 15 (2004) 155. [9] J. Chomoucka, J. Drbohlavova, M. Ryvolova, P. Sobrova, L. Janu, V. Adam, J. Hubalek, R. Kizek, Quantum Dots: Biological and Biomedical Application. Quantum Dots: Applications, Synthesis and Characterization, Nova Science Publishers, New York, 2012. [10] B.J. Kumar, H.M. Mahesh, Concentration-dependent optical properties of TGA stabilized CdTe Quantum dots synthesized via the single injection hydrothermal method in the ambient environment, Superlattices Microstruct. 104 (2017) 118–127. [11] Y.S. Borghei, M. Hosseini, M.R. Ganjali, Fluorometric determination of microRNA via FRET between silver nanoclusters and CdTe quantum dots, Microchim. Acta 1 (2017) 1–9. [12] Y.S. Borghei, M. Hosseini, M.R. Ganjali, S. Hosseinkhani, A novel BRCA1 gene deletion detection in human breast carcinoma MCF-7 cells through FRET between quantum dots and silver nanoclusters, J. Pharm. Biomed. Anal. 152 (2018) 81–88. [13] J. Liu, X. Yang, K. Wang, R. Yang, H. Ji, L. Yang, C. Wu, A switchable fluorescent quantum dot probe based on aggregation/disaggregation mechanism, Chem. Commun. 47 (2011) 935–937. [14] S.D. Quinn, S.W. Magennis, Optical detection of gadolinium (III) ions via quantum dot aggregation, RSC Adv. 7 (2017) 24730–24735. [15] M. Gilic, N. Romcevic, M. Romcevic, D. Stojanovic, R. Kostic, J. Trajic, W.D. Dobrowolski, G. Karczewski, R. Galazka, Optical properties of CdTe/ZnTe selfassembled quantum dots: Raman and photoluminescence spectroscopy, J. Alloy. Compd. 579 (2013) 330–335.
23
Contents lists available at ScienceDirect
Journal of Luminescence journal homepage: www.elsevier.com/locate/jlumin
Ratiometric fluorescence biosensor based on DNA/miRNA duplex@CdTe QDs and oxidized luminol as a fluorophore for miRNA detection Yasaman Sadat Borghei, Morteza Hosseini
T
⁎
Department of Life Science Engineering, Faculty of New Sciences & Technologies, University of Tehran, Tehran, Iran
A R T I C LE I N FO
A B S T R A C T
Keywords: MicroRNAs Ratiometric Light-on Fluorescence Cancer
Breast cancer is the second most diagnosed cancer in women worldwide. Hence, the detection of prognostic and diagnostic biomarkers can improve the patient's quality of life during the course of illness and treatment. Most recently, microRNAs (miRNAs) have been widely studied for non-invasive prediction of prognosis markers in the diagnosis of breast cancer. Herein, we reported a ratiometric fluorescence nanobiosensor for detection of miR155. The biosensor comprises of 3-mercapropionic acid-coated cadmium telluride (CdTe) quantum dots and oxidized luminol (Lumox) exhibiting emission peaks at 550 and 440 nm, respectively, under single-wavelength excitation (350 nm). In the presence of miR-155, CdTe QDs were aggregated and their fluorescence was quenched via interacting strongly at their metal centers, Cd, with heteroduplex formed between the DNA probe and miR-155. Then, by addition of blue-emitting Lumox with their constant fluorescence emission, a ratiometric means of miR-155 detection was developed. The relative fluorescence intensity ration is directly proportional to the concentration of miR-155 between 20.0 and 100.0 pM. The detection limit is 12.0 pM. This novel assay is “light-on” and has been successfully applied for the detection of miRNA in MCF-7 and HEK 293 cell lysates as real samples.
1. Introduction MicroRNAs (miRNAs) are a large family of non-coding single stranded RNAs (usually19–23 nucleotides) that regulate gene expression at the post-transcriptional level via direct binding to target mRNAs. The roles of miRNAs in disease, particularly in cancer, have made miRNAs attractive tools for diagnosis and therapy. For instance, MiR155, located in chromosome 21q21, is encoded with B cell integration cluster (BIC) gene, which is up-regulated in different types of human cancers such as breast cancer [1,2]. The miR-155 binds directly to RhoA, a prometastatic gene, and plays a key role in breast cancer metastasis. In another research it has been shown that inflammatory stimulation of breast cancer cells increased the level of miR-155 which then they would bind to SOCS1 [3,4]. Also, it was found that there is an overexpression of miR-155 in invasive tumors but not in noninvasive cancer samples and it was shown that miR-155 is associated with the proliferation, invasion and apoptosis due to its action on caspase 3 [5]. These facts show that there miRNAs could have many potential applications in the diagnosis, prognosis and forecasts in therapeutic [6]. Therefore, accurate and rapid quantification of miRNAs is important. Common methods used for this purpose include microarray, next-generation sequencing, NanoString nCounter and quantitative reverse ⁎
transcriptase real-time (qRT-PCR); however, each of these methods has its own advantages and limitations [7]. For example, a major limitation of qRT-PCR, which is used as a gold standard method is that it will not generate quantitative results at the limits of its sensitivity [8]. Recently, the use of photoluminescent nanoparticles like semiconductor quantum dots (QDs) as fluorescent probes in biosensing (such as DNA, proteins, peptides, and drugs) and bio-imaging has been very much considered because of their advantages such as size-dependent tunable emission, photostability and broad excitation spectra, in comparison to organic dye. QDs are nanocrystals, which are comprised of a few hundreds to a few millions of atoms, and only a small number of free electrons (≤ 100) [9–12]. In this study for the first time, oxidized luminol (Lumox) was used as a blue-emitting fluorophore probe in the green-emitting 3-mercaptopropionic acid-capped CdTe QD-based dual-emission ratiometric fluorescence biosensor for miRNA detection. Initially, miR-155 aggregated and quenched the fluorescence of CdTe QDs [13–17] via strong interaction with DNA/miR-155 heteroduplex [18–24] switching the green fluorescence into an “off” state at 550 nm with a short response time. Meanwhile, Lumox molecules were insensitive to both CdTe QDs and DNA/miR-155 duplex and maintained constant fluorescence intensity at 440 nm, thereby serving as a reference for
Corresponding author. E-mail address: [email protected] (M. Hosseini).
https://doi.org/10.1016/j.jlumin.2018.07.034 Received 20 March 2018; Received in revised form 18 July 2018; Accepted 23 July 2018 Available online 24 July 2018 0022-2313/ © 2018 Elsevier B.V. All rights reserved.
Journal of Luminescence 204 (2018) 16–23
Y.S. Borghei, M. Hosseini
Scheme 1. Schematic representation of the fluorescence behavior of green CdTe QDs (a), DNA probe@CdTe QDs (b) and dsDNA/miR-155@CdTe QDs (c) in the presence of blue-emitting Lumox.
ratiometric detection of miRNA. On the other hand, in the absence of miR-155, CdTe QDs and Lumox exhibited emissions peaks at 550 nm and 440 nm, respectively (Scheme 1). This biosensor was successfully used for detection of miR-155 in human breast carcinoma cells (MCF-7) and human normal cells (HEK 293).
Inc., Bedford, MA) was used throughout the reactions. MCF-7 cells (human breast cancer cell line) and HEK 293 cells (from normal human embryonic kidney cell line) were used in this study.
2. Experimental
AFM imaging was performed on DNA probes and DNA/miR-155 heteroduplex samples deposited on freshly cleaved mica sheets. For this, 1 × 1 cm mica slides soaked in 5 mM MgCl2, and 2 min after, the surface was dried at room temperature. Then, 30 μL of samples in 1 mM MgCl2 were spotted onto mica plates and dried at room temperature. After washing the samples with deionized water and drying, AFM imaging was done on a Solver PRO AFM system (NT-MDT, Russia), in semi-contact (tapping) mode, using Si-gold-coated cantilevers (NTMDT, Zelenograd, Russia) with a resonance frequency of 375 kHz. Images were recorded in height mode, and Nova image processing software (NT-MDT, Zelenograd, Russia) software was used for data processing and particle analysis.
2.3. Preparation of samples for atomic force microscopy (AFM) imaging
2.1. Apparatus All fluorescence measurements were carried out using a Perkin Elmer LS-55 fluorescence spectrometer with a xenon lamp as source of excitation while the spectral band widths of monochromators for excitation and emission were 10 nm. (Buckinghamshire,UK). The size and morphology of bare QDs were measured by transmission electron microscopy (TEM) (Zeiss, EM10C, 80 kV, Germany) and atomic force microscopy (AFM) (NT-MDT, Zelenograd, Russia). UV–vis spectroscopy was performed by a Specord 250 spectrophotometer (Analytik Jena, Germany).
2.4. Preparation of oxidized luminol 2.2. Materials and reagents Reduced luminol was prepared by dissolving luminol (10-4 M) in to 10 ml of 0.1 M NaOH. In the next step, Blue-emitting luminol (Lumox) or aminodiphthalate is formed by adding a suitable amount of hydrogen peroxide (H2O2, 10-4 M).
Fetal bovine serum (FBS), Dulbecco's modified eagle medium (DMEM), and penicillin/streptomycin were purchased from Gibco (USA). Oligonucleotides were synthesized and purified by Shanghai Generay Biotech Co (Shanghai, China), and their sequences were listed in Table.S1. All oligonucleotide samples were purified by PAGE and prepared with TE buffer (1 M Tris-HCl, 0.5 M EDTA). Cd(NO3)2, tellurium powder, thioglycolic acid (TGA) and sodium borohydride (NaBH4) were purchased from Merck and cell culture lysis reagent (CCLR) was purchased from Sigma Aldrich. All other reagents were of analytical reagent grade and ultrapure water (Milli-Q plus, Millipore
2.5. Preparation of MAA capped-CdTe Quantum Dots The experimental procedure was based on previous works [11, 25, 26]. In summary, Cd solution (0.4 mmol) and thioglycolic acid (TGA) (1.4 mmol) were solvated in 80 ml distilled water with pH adjusted to 10.0 using NaOH solution. Next, sodium borohydrate (0.8 mmol) and 17
Journal of Luminescence 204 (2018) 16–23
Y.S. Borghei, M. Hosseini
Te powder were diluted in 10 ml distilled water in a flask, with vigorous stirring under argon flow. The mixture was heated to 80 °C to get a clear red NaHTe solution. Cd-TGA solution was heated at 100 °C under argon flow in a 250 ml three-neck flask. Then the freshly prepared NaHTe solution (4.0 ml) was added to the flask, and the resulting solution was refluxed at 100 °C for different times. The characterization of CdTe quantum dots was carried out through transmission electron microscopy and spectrofluorometry.
normal human embryonic kidney) were cultured in 25 cm2 tissue culture flasks (SPL, Korea) with 5 ml Dulbecco's modified Eagle's medium (Sigma, UK) containing 10% heat inactivated fetal bovine serum (Gibco), 100 U/ml penicillin (Sigma, UK). Cell lines were grown at 37 °C in a humidified atmosphere of 5% CO2 and 95% air for 5 days until the cell monolayer became confluent. Growth medium was replaced with fresh media every 2 days or as required, indicated by color change due to production of lactic acid and CO2, which leads to low pH. Upon reaching at least 80% confluency, the cells were washed with phosphate buffered saline (PBS) and trypsinized for 10 min at 37 °C with 0.05% trypsin.
2.6. Cell culture MCF-7 cells (human breast cancer cell line) and HEK 293 cells (from
Fig. 1. (A) Absorbance (B) and fluorescence spectra of Lumox and CdTe QDs. Photographs of them under Visible and UV light. (C) TEM image of CdTe QDs. 18
Journal of Luminescence 204 (2018) 16–23
Y.S. Borghei, M. Hosseini
3.3. Effect of miR-155 and heteroduplex formation on ratiometric fluorescence
2.7. Total RNA extraction Cell samples were disrupted and total RNA was extracted from MCF7 and HEK 293 cell lines using Cell Culture LysisReagent (CCLR). Approximately 2.0 × 106 cells collected by low-speed centrifugation at 1000 rpm for 10 min. Culture medium was carefully removed and the pellet was washed twice with PBS. The PBS was carefully removed and 600 μL CCLR buffer was added. Cells were gently resuspended in CCLR buffer with a vortex and incubated for 20 min. Subsequently, 0.2 ml chloroform was added and the mixture was vortexed for 20 s violently. Next, the mixture was centrifuged for 20 min at 13,000 rpm at 4 °C by refrigerated centrifugation. Same volume of isopropyl alcohol was added to the upper water which has been taken out and mixed evenly followed by overnight precipitation at − 20 °C. Afterward, centrifugation was carried out for 20 min at 13,000 rpm at 4 °C. Then the supernatant was removed and the precipitant was washed by 80% ethanol with DEPC water and centrifuged for 20 min at 13,000 rpm at 4 °C. The ethanol was volatilized (Letting the tube dry) and the purified RNA was dissolved in an appropriate volume of DEPC water.
Addition of DNA/miR-155 heteroduplex to CdTe QDs solution reduced the fluorescence intensity of CdTe–especially the emission peak at 550 nm–while that of blue-emitting Lumox remained constant at 440 nm. On the other hand in the absence of miR-155, QDs and Lumox still have fluorescence emission peak at 550 nm and 440 nm, respectively (Fig. 3). 3.4. Optimization We evaluated the effect of pH and different concentrations of CdTe QD on quenching effect of the DNA/miRNA duplex on CdTe QDs. Firstly, as shown in Fig. S1A the effect of different concentrations of CdTe QDs (10 pM, 100 pM and 10 nM) was tested by comparing the fluorescence spectra of CdTe QDs alone, DNA@CdTe QDs and DNA/ miR-155 duplex. It was observed that the highest decrease in fluorescence emission occurred at concentration 100 pM and therefore, this concentration was used at all stages of the experiment. We also investigated the effect of pH on quenching of CdTe QDs in the presence of DNA/miR-155 duplex and found that the fluorescence intensity of QDs at pH 7.5 is minimal (Fig. S1B).
2.8. Procedures for fluorescence detection of miR-155 The test started with incubating the target miR-155 and its complementary DNA probes (10 μL of the 100 pM) in 20 mM of phosphate buffer (pH 7.5) at 90 °C for 10 min. Then the obtained mixture solution was incubated at 37 °C for 1 h, in which the complementary DNA probes recognize the specific miR-155 targets in order to create the formations of DNA/miR-155 heteroduplex. Afterwards, 10 μL of green QDs (100 pM) was added to reaction solutions. These samples were applied to the fluorescence quenching phenomenon investigation. Subsequently, the Lumox (2 μL, 10-4 M) was used for analyzing dualemission of the solution system.
3.5. Ratiometric fluorescence detection of miR-155 We explored the capability of using the CdTe QDs and Lumox system for miRNA detection. Fig. 4A displays the typical quenching of QD fluorescence spectra at 550 nm by DNA/miR-155 heteroduplex at different concentrations of miR-155 from 20 pM to 100 pM (Fig. 4A). We attribute the aggregation to double stranded nucleic acid (DNA/miRNA duplex) binding to the chalcogenide QDs [18–24] and the quenching to intra-aggregate energy transfer between QDs [13–17]. The decrease fluorescence intensities are linearly proportional to the concentrations of target miR-155. To obtain signal “ON” and dual-emission sensor, lumox was added to the reaction solutions. Following the addition of blue-emitting Lumox, the fluorescence intensity remained at 440 nm and the fluorescence intensity at 550 nm was decreased by adding increasing amounts of miR-155 to the system (20–100 pM) (Fig. 4B). Because the Lumox was not sensitive to both CdTe QDs and DNA/miR-155 duplex and maintained constant fluorescence intensity at 440 nm, thereby serving as a reference for dual-emission assay for miRNA. The fluorescence intensity ratio of 550/440 nm showed a linear response to miR-155 concentration in the range of 20–100 pM. The detection limit was estimated at 12.0 pM. according to the definition of three times the deviation of the blank signal. The proposed method was compared with other methods [28–33] that use one or more fluorophore materials, and as shown in Table 1, this method has low sensitivity along with ease and cost-effectiveness.
3. Results and discussion 3.1. Characterization of Lumox and CdTe QDs Fig. 1 shows the absorbance (Fig. 1A) and fluorescence (Fig. 1B) spectra of Lumox and CdTe QDs. Lumox exhibits two principle absorption bands in 280 and 330 nm region, whereas, a single fluorescence emission peak appears at 440 nm. The formation of hydrogen bonds in water leads to a shift in the wavelength of emission to the larger wavelengths which is related to the stabilization of the charge transfer excited state of hydrogen bonds of water with Lumox. On the other hand, the emission peak of QDs locates at 550 nm with the characteristic absorption peak at 400 nm [27]. The TEM analysis of MAA-cappedCdTe QDs shows monodispersed particles with an average diameter of ca. 5 nm (Fig. 1C).
3.6. Selectivity 3.2. AFM and TEM analysis for CdTe QDs aggregation We investigated the selectivity of the described method here by investigative the signal responses of the probe DNA toward non-complementary target DNA, miR-21, Let 7a and miR-155 at the same concentration of 100 pM. As the results are shown in Fig. 5, the FL 550/ FL440 nm ratio of the DNA probe toward the perfectly matched target miR-155 was very different from that of other miRNAs which were closed to that of the blank sample. These results confirm that the high selectivity for miRNA detection in this described method.
In order to prove and validate the theory that in the presence of DNA/miRNA duplex, the QDs are aggregated, AFM images of the CdTe QDs were taken before and after adding the miR-155 target. Fig. 2A or B is the image taken from the sample of the DNA probe@QDs before adding the miR-155 and the Fig. 2C or D after adding the miR-155 and formation of DNA/miR-155 duplex. Comparing 3D images of A and C (or 2D images of B and D), it is easy to conclude that CdTe QDs have been aggregated in the Fig. 2C or D because they have larger dimensions and a supermolecular structure compared to that of Fig. 2A or B. In addition, TEM images were taken before (Fig. 2E) and after (Fig. 2F) adding the miR-155 target. As it is known, after adding miR-155 to the ssDNA probe@QDs, the CdTe QDs are aggregated.
3.7. MiR-155 detection in total RNA extracted from cells In order to test the selectivity and possibility of applying this dualmode fluorescent probe for miRNA determination in real samples, the 19
Journal of Luminescence 204 (2018) 16–23
Y.S. Borghei, M. Hosseini
Fig. 2. (A, C) 3D images and (B, D) 2D images of CdTe QDs by Atomic Force Microscopy (AFM) scanning of mica surface and (E, F) TEM analysis prepared by the solution of (A, B, E) single strand DNA@CdTe QDs and (C, D, F) double stranded DNA/miR-155@ CdTe QDs.
RNA extraction solution related to MCF-7 cells, while fluorescence emission of blue-emitting Lumox remains almost constant. Fluorescence emission of QDs has decreased slightly in the presence of HEK 293 cell lysate (Fig. 6A). In addition, these results are very reasonable in comparison to qRT-PCR (Fig. 6B). The relative intensity was calculated to the expression of U6 snRNA. The primers for miR-155 and U6 snRNA are shown in Table S1. However, the presence of other interfering substances such as mRNAs and miRNAs didn’t have any significant result in this dual-mode fluorescent assay. 4. Conclusion A water-soluble CdTe QDs–Lumox system was designed for the determination of miRNA in biological samples. In this suggested method, DNA probe/miR-155 duplex acted as a fluorescence “off” for greenemitting CdTe QDs through aggregation. Blue-emitting Lumox were insensitive to QDs and DNA/miR-155 duplex and no fluorescence resonance energy transfer occurred between Lumox and CdTe QDs, allowing ratiometric miRNA detection. Furthermore, the CdTe QDs and Lumox did not need chemical modification after their preparation, so making this method cost-effective. The CdTe-CDs ratiometric fluorescence sensing removed background signal and since calibration curve is based on two emission peaks, its results will be more acceptable than single-emission signal sensors.
Fig. 3. Emission spectra of CdTe QDs and Lumox in the solution of ssDNA probe and dsDNA/miR-155 in concentration of 100 pM and pH = 7.5.
Acknowledgements proposed method was used to identify miR-155 in total RNA extraction from MCF-7 (as a breast cancer cell) and HEK 293 (as a normal cell). The fluorescence spectra in Fig. 6A show that the fluorescence intensity of the QDs at 550 nm could be greatly decreased by the addition of total
The authors thank the Iran National Science Foundation (INSF 96010584) and the Research Council of University of Tehran (Grant 28645/01/02) for financial support of this work. 20
Journal of Luminescence 204 (2018) 16–23
Y.S. Borghei, M. Hosseini
Fig. 4. Emission spectra of dsDNA-green QDs complex formed with miR-155 target in concentrations of 20, 30, 40, 50, 60, 70, 80, 90 and 100 pM, before (A) and after (B) addition of Lumox. (C) the calibration curve for fluorescence intensity versus target microRNA155 concentration. (Photographs of them under UV light).
Table 1 Comparison between proposed method and other methods that use one or more fluorophore materials. Method used
Fluorophore
Detection limit
References
A magnetic fluorescence Photoinduced electron transfer (PET) Clever single-labeled fluorescence Fluorescence An “off-on” fluorescent switch assay Ratiometric fluorescence sensor Ratiometric fluorescence biosensor
dyes-loaded albumin nanoparticles DNA/AgNCs 2-aminopurine dye-labeled single-stranded DNA FAM labeled signal probes 2-aminopurine & thioflavin T CdTe QDs & Lumox
9 fM 0.06 nM 2.5 nM 0.18 nM 1 nM 72 pM 12.0 pM
[28] [29] [30] [31] [32] [33] In this work
21
Journal of Luminescence 204 (2018) 16–23
Y.S. Borghei, M. Hosseini
Fig. 5. Plot of FL 550/ FL 440 nm ratio obtained with different miRNA and non complementary DNA at the same concentration (100 pM, pH = 7.5).
Fig. 6. Total RNA extraction from HEK 293 (human normal cell line) and MCF-7 (breast cancer cell line) and the fluorescence signal of them after treatment with ssDNA probe and two color QDs (A). Photograph of them under UV light illumination. Relative signal of two methods for miR-155 detection: quantitative real-time PCR (qRT-PCR) and the proposed method (B).
22
Journal of Luminescence 204 (2018) 16–23
Y.S. Borghei, M. Hosseini
Appendix A. Supporting information
[16] A. Polimeni, A. Patane, M. Henini, L. Eaves, P.C. Main, Temperature dependence of the optical properties of I n A s/A l y Ga 1− y As self-organized quantum dots, Phys. Rev. B 59 (1999) 5064. [17] A.J. Chiquito, Y.A. Pusep, S. Mergulhão, Y.G. Gobato, J.C. Galzerani, N. Moshegov, Effects of annealing on electrical and optical properties of a multilayer InAs/GaAs quantum dots system, Mater. Res. 7 (2004) 459–465. [18] S.K. Kailasa, K.H. Cheng, H.F. Wu, Semiconductor nanomaterials-based fluorescence spectroscopic and matrix-assisted laser desorption/ionization (MALDI) mass spectrometric approaches to proteome analysis, Materials 6 (2013) 5763–5795. [19] W.R. Algar, U.J. Krull, Characterization of the adsorption of oligonucleotides on mercaptopropionic acid-coated CdSe/ZnS quantum dots using fluorescence resonance energy transfer, J. Colloid Interface Sci. 359 (2011) 148–154. [20] Z. Wang, H. He, W. Slough, R. Pey, S.P. Karna, Nature of interaction between semiconducting nanostructures and biomolecules: chalcogenide QDs and BNNT with DNA molecules, J. Phys. Chem. C 119 (2015) 25965–25973. [21] W.R. Algar, U.J. Krull, Adsorption and hybridization of oligonucleotides on mercaptoacetic acid-capped CdSe/ZnS quantum dots and quantum dot? Oligonucleotide conjugates, Langmuir 22 (2006) 11346–11352. [22] S.S. Narayanan, S.S. Sinha, P.K. Verma, S.K. Pal, Ultrafast energy transfer from 3mercaptopropionic acid-capped CdSe/ZnS QDs to dye-labelled DNA, Chem. Phys. Lett. 463 (2008) 160–165. [23] Q. Xu, J.H. Wang, Z. Wang, Z.H. Yin, Q. Yang, Y.D. Zhao, Interaction of CdTe quantum dots with DNA, Electrochem. Commun. 10 (2008) 1337–1339. [24] S. Anandampillai, X. Zhang, P. Sharma, G.C. Lynch, M.A. Franchek, K.V. Larin, Quantum dot–DNA interaction: computational issues and preliminary insights on use of quantum dots as biosensors, Comput. Methods Appl. Mech. Eng. 197 (2008) 3378–3385. [25] M. Hosseini, M.R. Ganjali, Z. Vaezi, B. Arabsorkhi, M. Dadmehr, F. Faridbod, P. Norouzi, Selective recognition histidine and tryptophan by enhanced chemiluminescence ZnSe quantum dots, Sens. Actuators B 210 (2015) 349–354. [26] F.S. Sabet, M. Hosseini, H. Khabbaz, M. Dadmehr, M.R. Ganjali, FRET-based aptamer biosensor for selective and sensitive detection of aflatoxin B1 in peanut and rice, Food Chem. 220 (2017) 527–532. [27] P. Reiss, M. Protiere, L. Li, Core/shell semiconductor nanocrystals, Small 5 (2009) 154–168. [28] T. Wei, D. Du, Z. Wang, W. Zhang, Y. Lin, Z. Dai, Rapid and sensitive detection of microRNA via the capture of fluorescent dyes-loaded albumin nanoparticles around functionalized magnetic beads, Biosens. Bioelectron. 94 (2017) 56–62. [29] S. Lu, S. Wang, J. Zhao, J. Sun, X. Yang, Fluorescence light-up biosensor for microRNA based on the distance-dependent photoinduced electron transfer, Anal. Chem. 89 (2017) 8429–8436. [30] R. Liao, S. Li, H. Wang, C. Chen, X. Chen, C. Cai, Simultaneous detection of two hepatocellar carcinoma-related microRNAs using a clever single-labeled fluorescent probe, Anal. Chim. Acta 983 (2017) 181–188. [31] X. Fan, Y. Qi, Z. Shi, Y. Lv, Y. Guo, Molecular mechanism of helicase on graphenebased hybridization reaction platform for microRNA detection, RSC Adv. 7 (2017) 36444–36449. [32] Y. Li, Q. Pu, J. Li, L. Zhou, Y. Tao, Y. Li, W. Yu, G. Xie, An “off-on” fluorescent switch assay for microRNA using nonenzymatic ligation-rolling circle amplification, Microchim. Acta 184 (2017) 4323–4330. [33] Y. Liu, T. Shen, J. Li, H. Gong, C. Chen, X. Chen, C. Cai, Ratiometric fluorescence sensor for the microRNA determination by catalyzed hairpin assembly, ACS Sens. 2 (2017) 1430–1434.
Supplementary data associated with this article can be found in the online version at https://doi.org/10.1016/j.jlumin.2018.07.034. References [1] B. Smith, P. Agarwal, N.A. Bhowmick, MicroRNA applications for prostate, ovarian and breast cancer in the era of precision medicine, Endocr.-Relat. Cancer 24 (2017) 157–172. [2] S. Ding, Y. Xu, L. Shen, H. Huang, X. Yu, C. Lu, C. Zhong, MiR-155 promotes proliferation of human non-small cell lung cancer H460 cells via targeting TP53INP1, Int. J. Clin. Exp. Med. 10 (2017) 11953–11960. [3] S. O'Bryan, S. Dong, J.M. Mathis, S.K. Alahari, The roles of oncogenic miRNAs and their therapeutic importance in breast cancer, Eur. J. Cancer 72 (2017) (1-1). [4] A. Asiaf, S.T. Ahmad, W. Arjumand, M.A. Zargar, MicroRNAs in breast cancer: diagnostic and therapeutic potential, MicroRNA and Cancer, Humana Press, New York, NY, 2018, pp. 23–43. [5] H. Yu, W. Xu, F. Gong, B. Chi, J. Chen, L. Zhou, MicroRNA-155 regulates the proliferation, cell cycle, apoptosis and migration of colon cancer cells and targets CBL, Exp. Ther. Med. 14 (2017) 4053–4060. [6] M. Hemmatzadeh, H. Mohammadi, F. Jadidi-Niaragh, F. Asghari, M. Yousefi, The role of oncomirs in the pathogenesis and treatment of breast cancer, Biomed. Pharm. 78 (2016) 129–139. [7] R. Hamam, D. Hamam, K.A. Alsaleh, M. Kassem, W. Zaher, M. Alfayez, A. Aldahmash, N.M. Alajez, Circulating microRNAs in breast cancer: novel diagnostic and prognostic biomarkers, Cell Death Dis. 8 (2017) 3045. [8] S.A. Bustin, T. Nolan, Pitfalls of quantitative real-time reverse-transcription polymerase chain reaction, J. Biomol. Tech.: JBT 15 (2004) 155. [9] J. Chomoucka, J. Drbohlavova, M. Ryvolova, P. Sobrova, L. Janu, V. Adam, J. Hubalek, R. Kizek, Quantum Dots: Biological and Biomedical Application. Quantum Dots: Applications, Synthesis and Characterization, Nova Science Publishers, New York, 2012. [10] B.J. Kumar, H.M. Mahesh, Concentration-dependent optical properties of TGA stabilized CdTe Quantum dots synthesized via the single injection hydrothermal method in the ambient environment, Superlattices Microstruct. 104 (2017) 118–127. [11] Y.S. Borghei, M. Hosseini, M.R. Ganjali, Fluorometric determination of microRNA via FRET between silver nanoclusters and CdTe quantum dots, Microchim. Acta 1 (2017) 1–9. [12] Y.S. Borghei, M. Hosseini, M.R. Ganjali, S. Hosseinkhani, A novel BRCA1 gene deletion detection in human breast carcinoma MCF-7 cells through FRET between quantum dots and silver nanoclusters, J. Pharm. Biomed. Anal. 152 (2018) 81–88. [13] J. Liu, X. Yang, K. Wang, R. Yang, H. Ji, L. Yang, C. Wu, A switchable fluorescent quantum dot probe based on aggregation/disaggregation mechanism, Chem. Commun. 47 (2011) 935–937. [14] S.D. Quinn, S.W. Magennis, Optical detection of gadolinium (III) ions via quantum dot aggregation, RSC Adv. 7 (2017) 24730–24735. [15] M. Gilic, N. Romcevic, M. Romcevic, D. Stojanovic, R. Kostic, J. Trajic, W.D. Dobrowolski, G. Karczewski, R. Galazka, Optical properties of CdTe/ZnTe selfassembled quantum dots: Raman and photoluminescence spectroscopy, J. Alloy. Compd. 579 (2013) 330–335.
23