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A label-free luminescent light switching system for miRNA detection based on two color quantum dots
Journal of Photochemistry & Photobiology A: Chemistry 391 (2020) 112351
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Journal of Photochemistry & Photobiology A: Chemistry journal homepage: www.elsevier.com/locate/jphotochem
A label-free luminescent light switching system for miRNA detection based on two color quantum dots
T
Yasaman Sadat Borgheia, Morteza Hosseinia,*, Mohammad Reza Ganjalib,c a
Department of Life Science Engineering, Faculty of New Sciences & Technologies, University of Tehran, Tehran, Iran Center of Excellence in Electrochemistry, Faculty of Chemistry, University of Tehran, Tehran, Iran c Biosensor Research Center, Endocrinology & Metabolism Molecular-Cellular Sciences Institute, Tehran University of Medical Sciences, Tehran, Iran b
A R T I C LE I N FO
A B S T R A C T
Keywords: Fluorescent Quantum dots miRNA Ratiometric Cell
In this study, we report a novel switchable fluorescent nanobiosensor for detection of miRNA. The sensor consists of green-emitting and orange-emitting CdTe QDs exhibiting emission spectra at 525 and 599 nm respectively, under excitation wavelength of 360 nm. Following the addition of miRNA targets to the DNA probes and heteroduplex formation of the hybrid, Green fluorescence CdTe QDs were aggregated and quenched by the DNA/ miRNA hybrid via strong interactions at their metal centers. Then, with the addition of orange emitting QDs causes the fluorescence energy transfer to occur and was introduced as a ratiometric approach for miR-155 detection. The relative fluorescence intensity ratio was directly proportional to the concentration of miR-155 from 20 to 100 pM and the detection limit was determined at 14.0 pM. The novel nanobiosensor is “light-on”, rapid, and convenient while it does not require modification or separation procedures and can be applied for the detection of miRNAs in cell lysis with satisfactory results.
1. Introduction MicroRNAs (miRNAs) are a class of evolutionarily conserved, small non-coding RNAs (usually compose of 19–23 nucleotides) that control some biological processes usually after binding to the target genes. The mechanism of miRNA involvement in cancer has been proposed by two key forms: oncogenes (oncomiRs) and oncosuppressor genes (oncosuppressor-miRs). Numerous studies have clearly illustrated the determining role of miR-155 in cancer development either as an oncomiR (high expression induction) or as an oncosuppressor-miR (low expression induction) [1,2]. In another study it was shown that miR-155 overexpression could accelerate breast cancer growth and angiogenesis occurrence initiated from targeting of 3´UTR of VHL mRNA [3,4]. Multiple studies have emphasized on noteworthy potential application of miRNAs in diagnostics and prognostics as well as alternative drug targets. Standard RT-PCR, Northern blotting and microarray techniques are a few of the conventional methods used for the detection of miRNAs [5]. However, these techniques show some limitations and need development to meet current demands. In spite of recent developments, there are few examples capable of detecting miRNAs through efficient and low-cost detection techniques. It is well known that nanotechnology development has led to new progress in detection assays of DNA and miRNAs such as nanoparticle-based probes, isothermal
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amplification, electrochemical methods, and DNAzyme based reporters [6–8]. Recently semiconductor nanocrystal (NC) quantum dots (QDs) with excellent photophysical and electronic properties (such as broad absorption spectra, narrow and size-tunable emission spectra and a large effective Stokes shift) have attracted extensive application studies in many fields, particularly in sensing and biomedical imaging [9–12]. Surface interactions of the QDs are a key acting factor in their photoluminescence. These interactions perform an important part in radiative recombination efficiency that triggers fluorescence signal activation or quenching. In other words, these direct interactions between analyte and QDs surface can lead to changes in order to detect a target analyte in a complex mixture [13]. In a previous study, scientists described hydrogen-bonding interactions between nucleic acid and quantum dots capped with mercaptoacetic acid ligands [14]. These reports show that a strong interaction between CdTe QDs and double stranded nucleic acids was established [15]. The chalcogenide QDs can have strong interactions at their metal centers, Zn or Cd, featuring electrostatic interaction with the N or O atoms of DNA nucleobases due to the sub-nanometer clusters. The positively-charged sites of chalcogenide QDs (the metal ions) interact with available complimentary electronegative sites on the base molecules [13–19]. The “turn-off” switch detection systems is an attractive alternative
Corresponding author. E-mail address: [email protected] (M. Hosseini).
https://doi.org/10.1016/j.jphotochem.2019.112351 Received 12 July 2019; Received in revised form 24 December 2019; Accepted 28 December 2019 Available online 07 January 2020 1010-6030/ © 2019 Elsevier B.V. All rights reserved.
Journal of Photochemistry & Photobiology A: Chemistry 391 (2020) 112351
Y.S. Borghei, et al.
Scheme 1. Schematic representation of the fluorescent behavior of bare green and orange CdTe QDs before and after mixing and their spectra. green QDs (a), orange QDs (b) and green QDs + orange QDs (c). Photographs of them before and after mixing under UV light.
source of excitation and with the excitation and emission slits at 10 nm bandpass. (Buckinghamshire, UK). Transmission electron microscopy (TEM) images were obtained by EM10C, Zeiss at 80 KV and used for measurement of size and morphology of bare QDs. Also, Atomic Force Microscopy (AFM) () images were obtained by NT-MDT, Zelenograd, Russia instrument for further analysis. UV–vis spectroscopy measurements were conducted by a Specord 250 spectrophotometer (Analytik Jena, Germany).
method but it is often restricted due to the low signal/background ratio. Also nonspecific adsorption or interference from interfering agents and other quencher material led to false positive results. Therefore it was highly necessary to develop more efficient “off–on” switch systems. The intrinsic nature of QDs with strong fluorescence can provide very high signal/background ratio for “off–on” switch systems and have been effectively used in different biomedical labeling and sensing applications [10,20,21]. In this study, we reported a novel fluorescence “off–on” switch system and QD-based dual-emission ratiometric fluorescence sensor for miRNA detection that consists of water-soluble green and orangeemitting-mercaptoacetic acid-capped CdTe QDs (Scheme 1a and b). This nanosensor could differentiate the interaction of two applied QDs with ssDNA (Scheme 2a´ and b´) and interaction of same QDs with DNA/miRNA heteroduplex (Scheme 3a´´ and b´´). At the first step and in the absence of miRNA and in the presence of single strand DNA probes, there is no significant difference in Green QDs fluorescence emission (Scheme 2a´). After addition of orange QDs, orange QDs were insensitive to ssDNA and maintained constant fluorescence intensity (Scheme 2b´), thereby serving as a reference for ratiometric detection of miRNA. Also addition of dsDNA/miRNA hybrid to green QDs resulted to QDs aggregation and subsequently quenched the fluorescence of CdTe QDs, as a result switching the green fluorescence into an “off” state (Scheme 3a´´). Upon addition of orange QDs, they maintained their own fluorescence emission, with the difference that they were less distant (Scheme 3b´´). By applying this method to a solution containing total RNAs extracted from human breast cancer cells (MCF-7) and human normal cells (HEK 293), we obtained acceptable results in comparison with the gold standard method (qRT-PCR).
2.2. Materials and reagents Dulbecco's modified eagle medium (DMEM), Fetal bovine serum (FBS) and penicillin/streptomycin were bought from Gibco (USA). The oligonucleotides were synthesized and PAGE purified by Shanghai Generay Biotech Co (Shanghai, China), and their sequences are listed in Table S1. All oligonucleotidestock solutions were prepared with TE buffer (1 M Tris-HCl, 0.5 M EDTA). Cadmium nitrate Cd (NO3)2, tellurium powder, sodium borohydride (NaBH4) and thioglycolic acid (TGA) were purchased from Merck and Cell Culture Lysis Reagent (CCLR) was purchased from Sigma Aldrich. Ultrapure water (Milli-Q plus, Millipore Inc., Bedford, MA) was used throughout the experiments. All other reagents were of analytical reagent grade. TheMCF-7 (human breast cancer) and HEK 293 (from normal human embryonic kidney) cell lines were used in this study. 2.3. Sample preparation before AFM imaging The AFM images of DNA probes and DNA/miR-155 hybrid samples was prepared after their deposition on freshly cleaved mica sheets. So, 1 × 1 cm mica slide soaked in 5 mM MgCl2for 2 min, then the surface was dried at room temperature. Following, 30 μL of sample was spotted on mica plates after addition of 1 mM MgCl2 and dried at room temperature. The slide were washed with deionized water and dried. Next, AFM imaging was done on a Solver PRO AFM system (NT-MDT, Russia), in a semi-contact (tapping) mode using Si-gold-coated cantilevers (NTMDT, Zelenograd, Russia) with a resonance frequency of 375 kHz. Nova
2. Experimental 2.1. Apparatus Fluorescence spectroscopy analysis was carried out using a Perkin Elmer LS-55 fluorescence spectrometer equipped with a xenon lamp as 2
Journal of Photochemistry & Photobiology A: Chemistry 391 (2020) 112351
Y.S. Borghei, et al.
Scheme 2. Schematic representation of the fluorescent behavior of ssDNA-QDs complex before (a´) and after (b) addition of orange QDs and their spectra. Photographs of them under UV light.
[22,23]. Initially, Cd(NO3)2 solution (0.4 mmol) and thioglycolic acid (TGA) (1.4 mmol) were solvated in 80 mL distilled water and pH was adjusted to 10.0 by gradual addition of NaOH solution. Then, sodium borohydride (0.8 mmol) and Tellurium powder were diluted in 10 ml distilled water in a flask, under argon flow and with vigorous stirring. The mixture was heated to 80 °C to change the solution color to clear
image processing software (NT-MDT, Zelenograd, Russia) was used for data processing and particle analysis. 2.4. Synthesis of MAA capped-CdTe QDs The synthesis protocol was performed based on previous literature
Scheme 3. Schematic representation of the fluorescent behavior of dsDNA/miR-155-gQDs complex before (a´´) and after (b´´) addition of orange QDs and their spectra. Photographs of them under UV light. 3
Journal of Photochemistry & Photobiology A: Chemistry 391 (2020) 112351
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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. Transmission electron microscopy and spectrofluorimetry was used for characterization of CdTe quantum dots.
and it was run for 20 min.The well stained gel was visualized by a UV trans-illuminator (312 nm). The gel lanes included ssDNA, hybrid and their QDs-interacted forms in order to compare the electrophoretic behavior of the dsDNA/miRNA-QDs complexes.
2.5. Cell culture
3.1. Characterization of MAA capped-CdTe QDs
MCF-7 (human breast cancer) and HEK 293 (from normal human embryonic kidney) cell lines were cultured in 25 cm2 tissue culture flasks (SPL, Korea) with 5 ml Dulbecco's modified Eagle's medium (Sigma, UK) which contained 10 % heat treated and inactivated fetal bovine serum (Gibco)including 100 U/ml penicillin (Sigma, UK). Cell lines were incubated at 37 °C in a humidified, concentrated CO2 (5 %) for 5 days. As cells reached approximately 90 % confluency, the media removed and substituted with fresh media every 2 days or as required which could be realized by color change due to production of lactic acid and CO2, resulting in low pH. Finally, the cells were rinsed twice with phosphate buffered saline (PBS) and trypsinized by 0.05 % trypsin at 37 °C for 10 min.
QDs are known to be nanomaterials with size-dependent properties. QDs size can be controlled during the synthesis, which makes it possible obtain a desirable absorbance and emission spectra for their further application (Fig. S1A). The photograph of the synthesized QDs solution, under UV and visible light illumination is shown in the inset in Fig. S1A. In this work for DNA interaction with QDs, QDs with desired 2 nm sized, were synthesized according to the method described elsewhere. The size and morphology of the QDs is shown in TEM images in Fig. S1B. Afterwards, their fluorescent properties were examined. The emission maximum of QDs after excitation by 360 nm light is at 525, 599 nm (Scheme 1a and b spectra).
3. Results and discussion
3.2. Atomic Force Microscopy (AFM) analysis 2.6. Total RNA extraction In order to survey the of green CdTe QDs aggregation through interaction of double stranded nucleic acid (DNA/miR-155 heteroduplex) and also determination of orange QDs trapped between them, AFM has been carried out for the samples. AFM spectroscopy was expected to confirm above mentioned interpretations by direct imaging of surface topographies. As shown in Fig. 1B, the AFM results indicate that orange CdTe QDs are insensitive to DNAprobe@QDs assembly and free orange QDs can provide a larger dimension with a mean diameter of 120 nm compared to DNAprobe@QDs (Fig. 1A) with a mean diameter of 40 nm. The addition of miR-155 target resulted to duplex formation with DNA probe and subsequently QDs aggregated and induced supermolecular structure with a mean diameter of 400 nm (Fig. 1C). But after addition of orange QDs, the orange QDs trapped between aggregated green QDs and provide a short inter QDs distance with a mean diameter of 450 nm (Fig. 1D). The obtained results were in good agreement with fluorescence emission expectation in Scheme 2 and 3.
In order to extract total RNA from MCF-7 and HEK 293 cell lines the cell samples were disrupted and total RNA was extracted using Cell Culture LysisReagent (CCLR). Initially 2.0 × 106 cells were precipitated using low-speed centrifugation at 1000 rpm for 10 min. The supernatant medium was aspirated and the pellet was washed twice with ice cold PBS. The PBS was carefully removed with a pipette and following 600 μl CCLR buffer was added. Cells were gently re-suspended in CCLR buffer with a vortex and incubated for 20 min. Subsequently, 0.2 ml chloroform was added and shaken vigorously for about 20 s and left for 5 min at room temperature. Next, the mixture was centrifuged for 20 min at 13,000 rpm at 4 °C by refrigerated centrifugation. At this point the same volume of isopropyl alcohol was added to the aqueous phase (top layer) and mixed gently overnight. Then the mixture was centrifuged at maximal speed (13,000 rpm) for 20 min at 4 °C. The isopropanol supernatant was removed and the precipitate was treated by 80 % ethanol in DEPC water and recentrifuged at 13,000 rpm in 4 °C for 20 min. The ethanol was volatilized (letting the tube to dry) and the purified RNA was dissolved in an appropriate volume of DEPC water.
3.3. Effect of DNA/miR-155 duplex on green CdTe QDs Addition of DNA/miR-155 heteroduplex to CdTe QDs solution resulted to fluorescence quenching of CdTe (emission spectrum at 525 nm). It was found that 100 pM miR-155 presented a quenching effect of ∼ 90 % (Fig. 2(A)). The observed quenching effect by DNA/miR-155 hybrid was attributed to the aggregation of chalcogenide QDs [24–28] because the quantum dots were able to strongly interact with double stranded nucleic acids, resulting in the self-quenching of QD photoluminescence emission originated from electron transfer. As shown in Fig. 2(A), the hybridization with increasing concentrations of target miR-155 could specifically drop the fluorescence intensity of the CdTe QDs. The fluorescence intensities reduction trend were linearly related to the concentrations of target miR-155.
2.7. Analytical procedure In order to determine the hybridization effects of miRNA targets on fluorescence signal, 10 μL of different concentration of miR-155 targets (from 20 to 100 pM) was added to 10 μL of the 100 pM of DNA probe in 40 μL of phosphate buffer (20 mM, pH = 6.5). For hybridization process the solution was heated up to 90 °C for 10 min and then incubated at 37 °C for 1 h. After hybridization, 10 μL of green QDs (100 pM)was added to the reaction solutions. The fluorescence quenching behavior of samples were recorded. Furthermore, 10 μL of orange CdTe QDs (100 pM) solution was used for monitoring the increasing trend of fluorescence in the solution system. Finally, the fluorescence spectra were recorded from 400 to 700 nm, and fluorescence intensities of green CdTe QDs at 525 nm and orange CdTe QDs at 599 nm were used for quantitative analysis of miRNA.
3.4. Ratiometric fluorescence detection of miR-155 Upon addition of orange CdTe QDs, the fluorescence intensity at 599 nm was increased and the fluorescence intensity at 525 nm was decreased by adding increasing amounts of miR-155 to the system (20–100 pM) (Fig. 2(B)). This was due to the strong and specific binding of green QDs to DNA/miR-155hybrid [13–19]. These changes in distance causes the fluorescence resonance energy transfer (FRET) processto occur, which leads to the gradual decrease of the fluorescence emission intensity of green QDs and the synchronous increase of orange QDs fluorescence emission [29,30]. The fluorescence intensity ratio of
2.8. Agarose gel electrophoresis analysis Agarose gel (3 % v/v, medium fragments, high melt, Chemos CZ, Czech Republic) was prepared in 1 × TAE buffer (40 mMTris, 1 mM ethylene diamine tetraacetic acid and 20 mM acetic acid). 5 μL of samples with5 % (v/v) bromophenol blue and 3 % (v/v) glycerol, were loaded to the wells. The electrophoresis power supply was set at 60 V 4
Journal of Photochemistry & Photobiology A: Chemistry 391 (2020) 112351
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Fig. 1. 3D images of CdTe QDs by Atomic Force Microscopy (AFM) scanning of mica surface prepared by the solution of (A) single strand DNA + green QDs, (B) single strand DNA + green QDs after addition of orange QDs, (C) double stranded DNA/miR-155 + green QDs, (D) double stranded DNA/miR-155 + green QDs after addition of orange QDs.
The lane c was loaded by hybridized dsDNA formed by ssDNA probe and miR-155 target and finally in the lane d there was a sample including mixture of lane c with QDs in ratio of 1:1. After software investigation of the gel image, the low brightness of the signal was observed in lane d compared to other 3 lines. Regarding to the observed results it was concluded that the DNA-QDs interactions are preventing the DNA staining and gel electrophoresis analysis could help us to distinguish between DNA probes and DNA/miRNA hybrids.
two CdTe QDs showed a linear response to miR-155 concentration in the range of 20−90 pM.The calibration curve was plotted as I525/I599 = −0.0288x + 2.8933 (x = pM [miR-155] (Fig. 2(B)). Ratiometric fluorescence detection by two color of QDs showed a linear response with a correlation coefficient of 0.969. The detection limit was estimated at14.0 pM according to the definition of three times the deviation of the blank signal. 3.5. Optimization
3.7. Selectivity To get the highest performance, some effective factors such as pH and concentration ratio of DNA to QDs were investigated and optimized. Fig. S1(A) exhibits the fluorescent emission spectra at different molar ratios of QDs to DNA (10, 1 and 0.1 M). Molar ratio of 1:1was chosen as the optimum ratio, because at this ratio the fluorescence signal of QDs was almost completely quenched after incubating with DNA/miR-155 hybrid. We also investigated the effect of pH on CdTe QDs (Fig. S2 (B)) and found that QDs had weak fluorescence under acidic pH values. To establish the optimal conditions for CdTe QDs interacted with dsDNA/ miR-155 hybrid, the pH was varied between 6.0 and 12.0 (20 mM phosphate buffer with 150 mM NaCl) and the fluorescence quenching was determined (Fig. S2(A)). Under slightly alkaline conditions (pH optimum of 7.4), PL ssDNA probe represented the maximum difference in fluorescence intensity of green CdTe QDs in the absence and presence of miR-155.
We further tested the selectivity of the presented sensing platform by examining the fluorescence responses of the assay while probe DNA interacted with non- complementary target DNA, miR-21, Let 7a sequences and mixture of all indicated sequences at the same concentration of 100 pM. As the results in Fig. S3 show, the fluorescence intensity of the DNA probe toward the perfectly matched target miR155 was much stronger than that of other sequences which were closed to that of the blank sample. These results confirm that the high selectivity for miRNA detection in this method. 3.8. Feasibility investigation to real total RNA samples extracted from human breast cancer cell lines Finally, in order to investigate the feasibility of the proposed method in real biological condition, we investigated the expression level of miR-155 in total RNA sample extracted from the MCF-7 as a human breast cancer cell lines and HEK 293 as a normal human embryonic kidney cell line. As shown in Fig. 4 the results indicated that MCF cell lines had higher miR-155 expression level than HEK 293 cell lines (Fig. 4), which was in good accordance with qRT-PCR (as a gold standard method) results. In qRT-PCR analysis, U6 small nuclear RNA (snRNA) was employed as the universal endogenous control and the relative expression was calculated by this equation: Fold change = 2−ΔCt.
3.6. Gel electrophoresis Gel electrophoresis was performed to study the interaction of CdTe QDs with dsDNA/miRNA. The agarose gel electrophoresis image were used to compare the impact of the binding of CdTe QDs with ssDNA (Lane b) and dsDNA/miR-155 (lane d) which has been shown in Fig. 3. As shown in Fig. 3, the ssDNA probe was loaded to lane a. Then the mixture of ssDNA probe with QDs was loaded in lane 2 with 1:1 ratio. 5
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Fig. 2. Emission spectra of dsDNA-green QDs complex formed with miR-155 target in a concentration ranging 20, 30, 40, 50, 60, 70, 80, 90 and 100 pM, before (A) and after (B) addition of orange QDs, and the logarithmic plot for fluorescence intensity versus target microRNA155 concentration (C).
method. Therefore, after addition of Orange-QDs, no fluorescence resonance energy transfer is observed between two CdTe QDs. The greenfluorescence decreases as a result of intercalation of those QDs into dsDNA/miR-155, while orange-emitting QDs are insensitive to dsDNA/miR-155 and their emission increases with the fluorescence resonance energy transfer (FRET) process, which enables the ratiometric detection of dsDNA/miR-155. This detection system offers a convenient procedure with simple design which is regarded as its advantages over other detection methods and allows for rapid dsDNA detection. Furthermore, CdTe QDs require no chemical modification after their synthesis, which makes this protocol cost-effective. The proposed CdTe ratiometric fluorescence sensor removes the background interference and decreases the variation in detection conditions with built-in calibration of red shift emission peaks and ensures more
The sequences of primers for qRT-PCR are listed in Table S1. Fold change for HEK-293 or MCF-7 = 2−[Ct(HEK−293 or MCF−7)− Ct(U6 snRNA)]
And in this proposed method, the amount of expression was calculated by this equation:
[HEK − 293 or MCF − 7] =
[probe] − [HEK − 293 or MCF − 7] [probe]
4. Conclusion Author report the development of a two-color water-soluble QD system for the detection of miR-155 in cellular extraction samples. DNA/miR-155 duplex quenches the green fluorescence of QDs, in this 6
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consistent results compared to single-emission signal sensors. In conclusion, these results demonstrate that CdTe QDs can be utilized for the rapid and specific detection of miRNA and common microRNAs present in biological samples with negligible interference in this process. Authors contributions Yasaman Sadat Borghei designed and performed the experiments and also wrote the manuscript. Morteza Hosseini as the corresponding author designed experiments, sponsored the study and made all the arrangements. He also edited the manuscript. Mohammad Reza Ganjali also edited the manuscript. All the authors reviewed the manuscript. Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Acknowledgments The authors are grateful to the Iran National Science Foundation (INSF 96010584), and the Tehran University, Iran for the financial support of this work. Fig. 3. Gel electrophoresis of (a) the ssDNA probe, (b) the ssDNA complex formed by QDs, (c) the dsDNA formed by miR-155 target, (d) the dsDNA/miR155 complex formed by QDs.
Appendix A. Supplementary data Supplementary material related to this article can be found, in the
Fig. 4. Total RNA extraction from HEK 293 (human normal cell line) and MCF-7 (breast cancer cell line) and show 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 realtime PCR (qRT-PCR) and the proposed method (B). 7
Journal of Photochemistry & Photobiology A: Chemistry 391 (2020) 112351
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online version, at https://doi.org/10.1016/j.jphotochem.2019.112351.
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References [1] S.C. Eastlack, S.K. Alahari, MicroRNA and breast cancer: understanding pathogenesis improving management, Noncoding RNA 1 (2015) 17–43, https://doi.org/10. 3390/ncrna1010017. [2] K. Zhang, Y. Zhang, C. Liu, Y. Xiong, J. Zhang, MicroRNAs in the diagnosis and prognosis of breast cancer and their therapeutic potential, Int. J. Oncol. 45 (2014) 950–958, https://doi.org/10.3892/ijo.2014.2487. [3] Z. Chen, T. Ma, C. Huang, T. Hu, J. Li, The pivotal role of microRNA‐155 in the control of cancer, J. Cell. Phys. 229 (2014) 545–550, https://doi.org/10.1002/jcp. 24492. [4] J. Zhu, Z. Zheng, J. Wang, J. Sun, P. Wang, X. Cheng, L. Fu, L. Zhang, Z. Wang, Z. Li, Different miRNA expression profiles between human breast cancer tumors and serum, Front. Genet. 5 (2014), https://doi.org/10.3389/fgene.2014.00149. [5] H. Dong, J. Lei, L. Ding, Y. Wen, H. Ju, X. Zhang, MicroRNA: function.; detection.; and bioanalysis, Chem. Rev. 113 (2013) 6207–6233, https://doi.org/10.1021/ cr300362f. [6] S. Persano, M.L. Guevara, J. Wolfram, E. Blanco, H. Shen, M. Ferrari, P.P. Pompa, Label-free isothermal amplification assay for specific and highly sensitive colorimetric miRNA detection, ACS Omega 1 (2016) 448–455, https://doi.org/10.1021/ ac501505a. [7] T. Tian, J. Wang, X. Zhou, A review: microRNA detection methods, Org. Biomol. Chem. 13 (2015) 2226–2238, https://doi.org/10.1039/C4OB02104E. [8] E.A. Hunt, D. Broyles, T. Head, S.K. Deo, MicroRNA detection: current technology and research strategies, Annual Rev. Anal. Chem 8 (2015) 217–237, https://doi. org/10.1146/annurev-anchem-071114-040343. [9] K.F. Chou, A.M. Dennis, Förster resonance energy transfer between quantum dot donors and quantum dot acceptors, Sensors 15 (2015) 13288–13325, https://doi. org/10.3390/s150613288. [10] R. Zhang, D. Zhao, H.G. Ding, Y.X. Huang, H.Z. Zhong, H.Y. Xie, Sensitive singlecolor fluorescence “off–on” switch system for dsDNA detection based on quantum dots-ruthenium assembling dyads, Biosens. Bioelectron. 56 (2014) 51–57, https:// doi.org/10.1016/j.bios.2013.12.059. [11] Y.S. Borghei, M. Hosseini, M.R. Ganjali, Visual detection of miRNA using peroxidase-like catalytic activity of DNA-CuNCs and methylene blue as indicator, Clin. Chim. Acta 483 (2018) 119–125, https://doi.org/10.1016/j.cca.2018.04.031. [12] Y.S. Borghei, M. Hosseini, M.R. Ganjali, Detection of large deletion in human BRCA1 gene in human breast carcinoma MCF-7 cells by using DNA-Silver Nanoclusters, Methods Appl. Fluoresc. 6 (2017) 015001, , https://doi.org/10.1088/ 20506120/aa8988. [13] D. Cui, B. Pan, H. Zhang, F. Gao, R. Wu, J. Wang, R. He, T. Asahi, Self-assembly of quantum dots and carbon nanotubes for ultrasensitive DNA and antigen detection, Anal. Chem. 80 (2008) 7996–8001, https://doi.org/10.1021/ac800992m. [14] 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. Coll. Interf. Sci 359 (2011) 148–154 https://doi.org/ 10.1016/j.jcis.2011.03.058. [15] Z. Wang, H. He, W. Slough, R. Pandey, 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, https://doi.org/
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Journal of Photochemistry & Photobiology A: Chemistry journal homepage: www.elsevier.com/locate/jphotochem
A label-free luminescent light switching system for miRNA detection based on two color quantum dots
T
Yasaman Sadat Borgheia, Morteza Hosseinia,*, Mohammad Reza Ganjalib,c a
Department of Life Science Engineering, Faculty of New Sciences & Technologies, University of Tehran, Tehran, Iran Center of Excellence in Electrochemistry, Faculty of Chemistry, University of Tehran, Tehran, Iran c Biosensor Research Center, Endocrinology & Metabolism Molecular-Cellular Sciences Institute, Tehran University of Medical Sciences, Tehran, Iran b
A R T I C LE I N FO
A B S T R A C T
Keywords: Fluorescent Quantum dots miRNA Ratiometric Cell
In this study, we report a novel switchable fluorescent nanobiosensor for detection of miRNA. The sensor consists of green-emitting and orange-emitting CdTe QDs exhibiting emission spectra at 525 and 599 nm respectively, under excitation wavelength of 360 nm. Following the addition of miRNA targets to the DNA probes and heteroduplex formation of the hybrid, Green fluorescence CdTe QDs were aggregated and quenched by the DNA/ miRNA hybrid via strong interactions at their metal centers. Then, with the addition of orange emitting QDs causes the fluorescence energy transfer to occur and was introduced as a ratiometric approach for miR-155 detection. The relative fluorescence intensity ratio was directly proportional to the concentration of miR-155 from 20 to 100 pM and the detection limit was determined at 14.0 pM. The novel nanobiosensor is “light-on”, rapid, and convenient while it does not require modification or separation procedures and can be applied for the detection of miRNAs in cell lysis with satisfactory results.
1. Introduction MicroRNAs (miRNAs) are a class of evolutionarily conserved, small non-coding RNAs (usually compose of 19–23 nucleotides) that control some biological processes usually after binding to the target genes. The mechanism of miRNA involvement in cancer has been proposed by two key forms: oncogenes (oncomiRs) and oncosuppressor genes (oncosuppressor-miRs). Numerous studies have clearly illustrated the determining role of miR-155 in cancer development either as an oncomiR (high expression induction) or as an oncosuppressor-miR (low expression induction) [1,2]. In another study it was shown that miR-155 overexpression could accelerate breast cancer growth and angiogenesis occurrence initiated from targeting of 3´UTR of VHL mRNA [3,4]. Multiple studies have emphasized on noteworthy potential application of miRNAs in diagnostics and prognostics as well as alternative drug targets. Standard RT-PCR, Northern blotting and microarray techniques are a few of the conventional methods used for the detection of miRNAs [5]. However, these techniques show some limitations and need development to meet current demands. In spite of recent developments, there are few examples capable of detecting miRNAs through efficient and low-cost detection techniques. It is well known that nanotechnology development has led to new progress in detection assays of DNA and miRNAs such as nanoparticle-based probes, isothermal
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amplification, electrochemical methods, and DNAzyme based reporters [6–8]. Recently semiconductor nanocrystal (NC) quantum dots (QDs) with excellent photophysical and electronic properties (such as broad absorption spectra, narrow and size-tunable emission spectra and a large effective Stokes shift) have attracted extensive application studies in many fields, particularly in sensing and biomedical imaging [9–12]. Surface interactions of the QDs are a key acting factor in their photoluminescence. These interactions perform an important part in radiative recombination efficiency that triggers fluorescence signal activation or quenching. In other words, these direct interactions between analyte and QDs surface can lead to changes in order to detect a target analyte in a complex mixture [13]. In a previous study, scientists described hydrogen-bonding interactions between nucleic acid and quantum dots capped with mercaptoacetic acid ligands [14]. These reports show that a strong interaction between CdTe QDs and double stranded nucleic acids was established [15]. The chalcogenide QDs can have strong interactions at their metal centers, Zn or Cd, featuring electrostatic interaction with the N or O atoms of DNA nucleobases due to the sub-nanometer clusters. The positively-charged sites of chalcogenide QDs (the metal ions) interact with available complimentary electronegative sites on the base molecules [13–19]. The “turn-off” switch detection systems is an attractive alternative
Corresponding author. E-mail address: [email protected] (M. Hosseini).
https://doi.org/10.1016/j.jphotochem.2019.112351 Received 12 July 2019; Received in revised form 24 December 2019; Accepted 28 December 2019 Available online 07 January 2020 1010-6030/ © 2019 Elsevier B.V. All rights reserved.
Journal of Photochemistry & Photobiology A: Chemistry 391 (2020) 112351
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Scheme 1. Schematic representation of the fluorescent behavior of bare green and orange CdTe QDs before and after mixing and their spectra. green QDs (a), orange QDs (b) and green QDs + orange QDs (c). Photographs of them before and after mixing under UV light.
source of excitation and with the excitation and emission slits at 10 nm bandpass. (Buckinghamshire, UK). Transmission electron microscopy (TEM) images were obtained by EM10C, Zeiss at 80 KV and used for measurement of size and morphology of bare QDs. Also, Atomic Force Microscopy (AFM) () images were obtained by NT-MDT, Zelenograd, Russia instrument for further analysis. UV–vis spectroscopy measurements were conducted by a Specord 250 spectrophotometer (Analytik Jena, Germany).
method but it is often restricted due to the low signal/background ratio. Also nonspecific adsorption or interference from interfering agents and other quencher material led to false positive results. Therefore it was highly necessary to develop more efficient “off–on” switch systems. The intrinsic nature of QDs with strong fluorescence can provide very high signal/background ratio for “off–on” switch systems and have been effectively used in different biomedical labeling and sensing applications [10,20,21]. In this study, we reported a novel fluorescence “off–on” switch system and QD-based dual-emission ratiometric fluorescence sensor for miRNA detection that consists of water-soluble green and orangeemitting-mercaptoacetic acid-capped CdTe QDs (Scheme 1a and b). This nanosensor could differentiate the interaction of two applied QDs with ssDNA (Scheme 2a´ and b´) and interaction of same QDs with DNA/miRNA heteroduplex (Scheme 3a´´ and b´´). At the first step and in the absence of miRNA and in the presence of single strand DNA probes, there is no significant difference in Green QDs fluorescence emission (Scheme 2a´). After addition of orange QDs, orange QDs were insensitive to ssDNA and maintained constant fluorescence intensity (Scheme 2b´), thereby serving as a reference for ratiometric detection of miRNA. Also addition of dsDNA/miRNA hybrid to green QDs resulted to QDs aggregation and subsequently quenched the fluorescence of CdTe QDs, as a result switching the green fluorescence into an “off” state (Scheme 3a´´). Upon addition of orange QDs, they maintained their own fluorescence emission, with the difference that they were less distant (Scheme 3b´´). By applying this method to a solution containing total RNAs extracted from human breast cancer cells (MCF-7) and human normal cells (HEK 293), we obtained acceptable results in comparison with the gold standard method (qRT-PCR).
2.2. Materials and reagents Dulbecco's modified eagle medium (DMEM), Fetal bovine serum (FBS) and penicillin/streptomycin were bought from Gibco (USA). The oligonucleotides were synthesized and PAGE purified by Shanghai Generay Biotech Co (Shanghai, China), and their sequences are listed in Table S1. All oligonucleotidestock solutions were prepared with TE buffer (1 M Tris-HCl, 0.5 M EDTA). Cadmium nitrate Cd (NO3)2, tellurium powder, sodium borohydride (NaBH4) and thioglycolic acid (TGA) were purchased from Merck and Cell Culture Lysis Reagent (CCLR) was purchased from Sigma Aldrich. Ultrapure water (Milli-Q plus, Millipore Inc., Bedford, MA) was used throughout the experiments. All other reagents were of analytical reagent grade. TheMCF-7 (human breast cancer) and HEK 293 (from normal human embryonic kidney) cell lines were used in this study. 2.3. Sample preparation before AFM imaging The AFM images of DNA probes and DNA/miR-155 hybrid samples was prepared after their deposition on freshly cleaved mica sheets. So, 1 × 1 cm mica slide soaked in 5 mM MgCl2for 2 min, then the surface was dried at room temperature. Following, 30 μL of sample was spotted on mica plates after addition of 1 mM MgCl2 and dried at room temperature. The slide were washed with deionized water and dried. Next, AFM imaging was done on a Solver PRO AFM system (NT-MDT, Russia), in a semi-contact (tapping) mode using Si-gold-coated cantilevers (NTMDT, Zelenograd, Russia) with a resonance frequency of 375 kHz. Nova
2. Experimental 2.1. Apparatus Fluorescence spectroscopy analysis was carried out using a Perkin Elmer LS-55 fluorescence spectrometer equipped with a xenon lamp as 2
Journal of Photochemistry & Photobiology A: Chemistry 391 (2020) 112351
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Scheme 2. Schematic representation of the fluorescent behavior of ssDNA-QDs complex before (a´) and after (b) addition of orange QDs and their spectra. Photographs of them under UV light.
[22,23]. Initially, Cd(NO3)2 solution (0.4 mmol) and thioglycolic acid (TGA) (1.4 mmol) were solvated in 80 mL distilled water and pH was adjusted to 10.0 by gradual addition of NaOH solution. Then, sodium borohydride (0.8 mmol) and Tellurium powder were diluted in 10 ml distilled water in a flask, under argon flow and with vigorous stirring. The mixture was heated to 80 °C to change the solution color to clear
image processing software (NT-MDT, Zelenograd, Russia) was used for data processing and particle analysis. 2.4. Synthesis of MAA capped-CdTe QDs The synthesis protocol was performed based on previous literature
Scheme 3. Schematic representation of the fluorescent behavior of dsDNA/miR-155-gQDs complex before (a´´) and after (b´´) addition of orange QDs and their spectra. Photographs of them under UV light. 3
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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. Transmission electron microscopy and spectrofluorimetry was used for characterization of CdTe quantum dots.
and it was run for 20 min.The well stained gel was visualized by a UV trans-illuminator (312 nm). The gel lanes included ssDNA, hybrid and their QDs-interacted forms in order to compare the electrophoretic behavior of the dsDNA/miRNA-QDs complexes.
2.5. Cell culture
3.1. Characterization of MAA capped-CdTe QDs
MCF-7 (human breast cancer) and HEK 293 (from normal human embryonic kidney) cell lines were cultured in 25 cm2 tissue culture flasks (SPL, Korea) with 5 ml Dulbecco's modified Eagle's medium (Sigma, UK) which contained 10 % heat treated and inactivated fetal bovine serum (Gibco)including 100 U/ml penicillin (Sigma, UK). Cell lines were incubated at 37 °C in a humidified, concentrated CO2 (5 %) for 5 days. As cells reached approximately 90 % confluency, the media removed and substituted with fresh media every 2 days or as required which could be realized by color change due to production of lactic acid and CO2, resulting in low pH. Finally, the cells were rinsed twice with phosphate buffered saline (PBS) and trypsinized by 0.05 % trypsin at 37 °C for 10 min.
QDs are known to be nanomaterials with size-dependent properties. QDs size can be controlled during the synthesis, which makes it possible obtain a desirable absorbance and emission spectra for their further application (Fig. S1A). The photograph of the synthesized QDs solution, under UV and visible light illumination is shown in the inset in Fig. S1A. In this work for DNA interaction with QDs, QDs with desired 2 nm sized, were synthesized according to the method described elsewhere. The size and morphology of the QDs is shown in TEM images in Fig. S1B. Afterwards, their fluorescent properties were examined. The emission maximum of QDs after excitation by 360 nm light is at 525, 599 nm (Scheme 1a and b spectra).
3. Results and discussion
3.2. Atomic Force Microscopy (AFM) analysis 2.6. Total RNA extraction In order to survey the of green CdTe QDs aggregation through interaction of double stranded nucleic acid (DNA/miR-155 heteroduplex) and also determination of orange QDs trapped between them, AFM has been carried out for the samples. AFM spectroscopy was expected to confirm above mentioned interpretations by direct imaging of surface topographies. As shown in Fig. 1B, the AFM results indicate that orange CdTe QDs are insensitive to DNAprobe@QDs assembly and free orange QDs can provide a larger dimension with a mean diameter of 120 nm compared to DNAprobe@QDs (Fig. 1A) with a mean diameter of 40 nm. The addition of miR-155 target resulted to duplex formation with DNA probe and subsequently QDs aggregated and induced supermolecular structure with a mean diameter of 400 nm (Fig. 1C). But after addition of orange QDs, the orange QDs trapped between aggregated green QDs and provide a short inter QDs distance with a mean diameter of 450 nm (Fig. 1D). The obtained results were in good agreement with fluorescence emission expectation in Scheme 2 and 3.
In order to extract total RNA from MCF-7 and HEK 293 cell lines the cell samples were disrupted and total RNA was extracted using Cell Culture LysisReagent (CCLR). Initially 2.0 × 106 cells were precipitated using low-speed centrifugation at 1000 rpm for 10 min. The supernatant medium was aspirated and the pellet was washed twice with ice cold PBS. The PBS was carefully removed with a pipette and following 600 μl CCLR buffer was added. Cells were gently re-suspended in CCLR buffer with a vortex and incubated for 20 min. Subsequently, 0.2 ml chloroform was added and shaken vigorously for about 20 s and left for 5 min at room temperature. Next, the mixture was centrifuged for 20 min at 13,000 rpm at 4 °C by refrigerated centrifugation. At this point the same volume of isopropyl alcohol was added to the aqueous phase (top layer) and mixed gently overnight. Then the mixture was centrifuged at maximal speed (13,000 rpm) for 20 min at 4 °C. The isopropanol supernatant was removed and the precipitate was treated by 80 % ethanol in DEPC water and recentrifuged at 13,000 rpm in 4 °C for 20 min. The ethanol was volatilized (letting the tube to dry) and the purified RNA was dissolved in an appropriate volume of DEPC water.
3.3. Effect of DNA/miR-155 duplex on green CdTe QDs Addition of DNA/miR-155 heteroduplex to CdTe QDs solution resulted to fluorescence quenching of CdTe (emission spectrum at 525 nm). It was found that 100 pM miR-155 presented a quenching effect of ∼ 90 % (Fig. 2(A)). The observed quenching effect by DNA/miR-155 hybrid was attributed to the aggregation of chalcogenide QDs [24–28] because the quantum dots were able to strongly interact with double stranded nucleic acids, resulting in the self-quenching of QD photoluminescence emission originated from electron transfer. As shown in Fig. 2(A), the hybridization with increasing concentrations of target miR-155 could specifically drop the fluorescence intensity of the CdTe QDs. The fluorescence intensities reduction trend were linearly related to the concentrations of target miR-155.
2.7. Analytical procedure In order to determine the hybridization effects of miRNA targets on fluorescence signal, 10 μL of different concentration of miR-155 targets (from 20 to 100 pM) was added to 10 μL of the 100 pM of DNA probe in 40 μL of phosphate buffer (20 mM, pH = 6.5). For hybridization process the solution was heated up to 90 °C for 10 min and then incubated at 37 °C for 1 h. After hybridization, 10 μL of green QDs (100 pM)was added to the reaction solutions. The fluorescence quenching behavior of samples were recorded. Furthermore, 10 μL of orange CdTe QDs (100 pM) solution was used for monitoring the increasing trend of fluorescence in the solution system. Finally, the fluorescence spectra were recorded from 400 to 700 nm, and fluorescence intensities of green CdTe QDs at 525 nm and orange CdTe QDs at 599 nm were used for quantitative analysis of miRNA.
3.4. Ratiometric fluorescence detection of miR-155 Upon addition of orange CdTe QDs, the fluorescence intensity at 599 nm was increased and the fluorescence intensity at 525 nm was decreased by adding increasing amounts of miR-155 to the system (20–100 pM) (Fig. 2(B)). This was due to the strong and specific binding of green QDs to DNA/miR-155hybrid [13–19]. These changes in distance causes the fluorescence resonance energy transfer (FRET) processto occur, which leads to the gradual decrease of the fluorescence emission intensity of green QDs and the synchronous increase of orange QDs fluorescence emission [29,30]. The fluorescence intensity ratio of
2.8. Agarose gel electrophoresis analysis Agarose gel (3 % v/v, medium fragments, high melt, Chemos CZ, Czech Republic) was prepared in 1 × TAE buffer (40 mMTris, 1 mM ethylene diamine tetraacetic acid and 20 mM acetic acid). 5 μL of samples with5 % (v/v) bromophenol blue and 3 % (v/v) glycerol, were loaded to the wells. The electrophoresis power supply was set at 60 V 4
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Fig. 1. 3D images of CdTe QDs by Atomic Force Microscopy (AFM) scanning of mica surface prepared by the solution of (A) single strand DNA + green QDs, (B) single strand DNA + green QDs after addition of orange QDs, (C) double stranded DNA/miR-155 + green QDs, (D) double stranded DNA/miR-155 + green QDs after addition of orange QDs.
The lane c was loaded by hybridized dsDNA formed by ssDNA probe and miR-155 target and finally in the lane d there was a sample including mixture of lane c with QDs in ratio of 1:1. After software investigation of the gel image, the low brightness of the signal was observed in lane d compared to other 3 lines. Regarding to the observed results it was concluded that the DNA-QDs interactions are preventing the DNA staining and gel electrophoresis analysis could help us to distinguish between DNA probes and DNA/miRNA hybrids.
two CdTe QDs showed a linear response to miR-155 concentration in the range of 20−90 pM.The calibration curve was plotted as I525/I599 = −0.0288x + 2.8933 (x = pM [miR-155] (Fig. 2(B)). Ratiometric fluorescence detection by two color of QDs showed a linear response with a correlation coefficient of 0.969. The detection limit was estimated at14.0 pM according to the definition of three times the deviation of the blank signal. 3.5. Optimization
3.7. Selectivity To get the highest performance, some effective factors such as pH and concentration ratio of DNA to QDs were investigated and optimized. Fig. S1(A) exhibits the fluorescent emission spectra at different molar ratios of QDs to DNA (10, 1 and 0.1 M). Molar ratio of 1:1was chosen as the optimum ratio, because at this ratio the fluorescence signal of QDs was almost completely quenched after incubating with DNA/miR-155 hybrid. We also investigated the effect of pH on CdTe QDs (Fig. S2 (B)) and found that QDs had weak fluorescence under acidic pH values. To establish the optimal conditions for CdTe QDs interacted with dsDNA/ miR-155 hybrid, the pH was varied between 6.0 and 12.0 (20 mM phosphate buffer with 150 mM NaCl) and the fluorescence quenching was determined (Fig. S2(A)). Under slightly alkaline conditions (pH optimum of 7.4), PL ssDNA probe represented the maximum difference in fluorescence intensity of green CdTe QDs in the absence and presence of miR-155.
We further tested the selectivity of the presented sensing platform by examining the fluorescence responses of the assay while probe DNA interacted with non- complementary target DNA, miR-21, Let 7a sequences and mixture of all indicated sequences at the same concentration of 100 pM. As the results in Fig. S3 show, the fluorescence intensity of the DNA probe toward the perfectly matched target miR155 was much stronger than that of other sequences which were closed to that of the blank sample. These results confirm that the high selectivity for miRNA detection in this method. 3.8. Feasibility investigation to real total RNA samples extracted from human breast cancer cell lines Finally, in order to investigate the feasibility of the proposed method in real biological condition, we investigated the expression level of miR-155 in total RNA sample extracted from the MCF-7 as a human breast cancer cell lines and HEK 293 as a normal human embryonic kidney cell line. As shown in Fig. 4 the results indicated that MCF cell lines had higher miR-155 expression level than HEK 293 cell lines (Fig. 4), which was in good accordance with qRT-PCR (as a gold standard method) results. In qRT-PCR analysis, U6 small nuclear RNA (snRNA) was employed as the universal endogenous control and the relative expression was calculated by this equation: Fold change = 2−ΔCt.
3.6. Gel electrophoresis Gel electrophoresis was performed to study the interaction of CdTe QDs with dsDNA/miRNA. The agarose gel electrophoresis image were used to compare the impact of the binding of CdTe QDs with ssDNA (Lane b) and dsDNA/miR-155 (lane d) which has been shown in Fig. 3. As shown in Fig. 3, the ssDNA probe was loaded to lane a. Then the mixture of ssDNA probe with QDs was loaded in lane 2 with 1:1 ratio. 5
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Fig. 2. Emission spectra of dsDNA-green QDs complex formed with miR-155 target in a concentration ranging 20, 30, 40, 50, 60, 70, 80, 90 and 100 pM, before (A) and after (B) addition of orange QDs, and the logarithmic plot for fluorescence intensity versus target microRNA155 concentration (C).
method. Therefore, after addition of Orange-QDs, no fluorescence resonance energy transfer is observed between two CdTe QDs. The greenfluorescence decreases as a result of intercalation of those QDs into dsDNA/miR-155, while orange-emitting QDs are insensitive to dsDNA/miR-155 and their emission increases with the fluorescence resonance energy transfer (FRET) process, which enables the ratiometric detection of dsDNA/miR-155. This detection system offers a convenient procedure with simple design which is regarded as its advantages over other detection methods and allows for rapid dsDNA detection. Furthermore, CdTe QDs require no chemical modification after their synthesis, which makes this protocol cost-effective. The proposed CdTe ratiometric fluorescence sensor removes the background interference and decreases the variation in detection conditions with built-in calibration of red shift emission peaks and ensures more
The sequences of primers for qRT-PCR are listed in Table S1. Fold change for HEK-293 or MCF-7 = 2−[Ct(HEK−293 or MCF−7)− Ct(U6 snRNA)]
And in this proposed method, the amount of expression was calculated by this equation:
[HEK − 293 or MCF − 7] =
[probe] − [HEK − 293 or MCF − 7] [probe]
4. Conclusion Author report the development of a two-color water-soluble QD system for the detection of miR-155 in cellular extraction samples. DNA/miR-155 duplex quenches the green fluorescence of QDs, in this 6
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consistent results compared to single-emission signal sensors. In conclusion, these results demonstrate that CdTe QDs can be utilized for the rapid and specific detection of miRNA and common microRNAs present in biological samples with negligible interference in this process. Authors contributions Yasaman Sadat Borghei designed and performed the experiments and also wrote the manuscript. Morteza Hosseini as the corresponding author designed experiments, sponsored the study and made all the arrangements. He also edited the manuscript. Mohammad Reza Ganjali also edited the manuscript. All the authors reviewed the manuscript. Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Acknowledgments The authors are grateful to the Iran National Science Foundation (INSF 96010584), and the Tehran University, Iran for the financial support of this work. Fig. 3. Gel electrophoresis of (a) the ssDNA probe, (b) the ssDNA complex formed by QDs, (c) the dsDNA formed by miR-155 target, (d) the dsDNA/miR155 complex formed by QDs.
Appendix A. Supplementary data Supplementary material related to this article can be found, in the
Fig. 4. Total RNA extraction from HEK 293 (human normal cell line) and MCF-7 (breast cancer cell line) and show 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 realtime PCR (qRT-PCR) and the proposed method (B). 7
Journal of Photochemistry & Photobiology A: Chemistry 391 (2020) 112351
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online version, at https://doi.org/10.1016/j.jphotochem.2019.112351.
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