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Duplex DNA-functionalized graphene oxide: A versatile platform for miRNA sensing
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Duplex DNA-functionalized graphene oxide: A versatile platform for miRNA sensing Bomi Shina,b, Woo-Keun Kima, Seokjoo Yoona,*, Jieon Leea,* a System Toxicology Research Center, Predictive Toxicology Department, Korea Institute of Toxicology (KIT), 141 Gajeong-ro, Yuseong-gu, Daejeon, 34114, Republic of Korea b Department of Toxicology Evaluation, Konyang University, 158 Gwanjeodong-ro, Seo-gu, Daejeon, 35365, Republic of Korea
A R T I C LE I N FO
A B S T R A C T
Keywords: miRNA sensing Graphene oxide Covalent conjugation Locked nucleic acids Toehold-mediated strand displacement
MicroRNAs (miRNAs) are short, non-coding RNAs, which have emerged as promising next-generation biomarkers for clinical diagnostics. Here, we developed a graphene oxide (GO)-based fluorescent nano-sensor for miRNA sensing with high specificity and sensitivity. GO is a water-soluble derivative of graphene, which has emerged as one of the most popular nanomaterials in the bio-medical field over the last few decades. Common strategies in the design of GO-based sensors rely on the fluorescence quenching capability of GO and the strong adsorption of ssDNA probes on GO surface through pi-pi interactions or hydrogen bonding. Unfortunately, the interaction between DNA and GO is easily interrupted by other molecules present in biological samples, such as proteins, lipids, and nucleic acids, which results in the nonspecific desorption of probes. Therefore, we designed a fluorescence-labeled dsDNA probe, with an embedded locked nucleic acid (LNA). The probe was then attached onto GO by covalent coupling, instead of nonspecific adsorption. The probes on GO recognized the target miRNA sequence specifically and released the fluorescent strand via toehold-mediated strand displacement (TMSD), which furnished the recovery of quenched fluorescence. This platform displayed improved signal to noise ratio with detection limits at the pico-molar levels. We believe that this new GO-based sensor can be a suitable tool for miRNA-based practical diagnostics of diseases, and provides a valuable resource for basic and applied research.
1. Introduction MicroRNAs (miRNA) are non-coding RNAs and range in length from 19∼25 nucleotides [1–3]. miRNAs play critical roles in diverse biological processes such as cell proliferation [4,5], differentiation [6,7], angiogenesis [8,9], and apoptosis [10,11], by interrupting the translation of messenger RNAs. Recently, numerous studies have confirmed the close relationship between miRNAs and the occurrence of diseases and human cancer [12–15]. Among the various miRNA families, miRNA-21 (miR-21) is a representative oncogene, which is overexpressed in most tumor types such as lung [16,17], breast [18,19], stomach [20,21], prostate [22,23], colon [24,25], and pancreas [26,27], and has been therapeutically targeted for tumor suppression. Therefore, the profiling and evaluation of miRNAs as diagnostic and prognostic markers for human diseases, or as prominent targets for genetic modulation in disease states is an attractive and valuable prospect. Generally, the quantitative measurement of miRNAs is not feasible using traditional approaches such as microarray [28,29], northern blot [30,31], and real-time polymerase chain reaction technologies
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[32,33]. In addition, these conventional methods are typically cost- and time-intensive, laborious, and/or require expensive instrumentation. Furthermore, the performance of these methods does not provide useful detection results of miRNAs. Recently, numerous nanomaterial-based sensing strategies have been developed for miRNA detection and analysis to overcome the drawbacks of conventional methods and to broaden the applications of miRNAs [34–37]. In this study, we developed a versatile and practical platform for miRNA detection using a graphene oxide (GO)-DNA conjugate. GO is a chemically-oxidized derivative of graphene [38], and contains sp2/sp3 hybrid carbon structures and various oxygen functional groups on its basal planes and edges. GO can be easily dispersed in aqueous solutions, and importantly, it retains its original characteristics, such as the high thermal and electrical conductivity and hydrophobicity under these conditions [38–40]. These unique properties make GO a potentially valuable material for use in biosensors [41–44], drug delivery [45–52], and catalysis [53–55]. Particularly, GO has been extensively used for the development of fluorescent nucleic acid (NA)-sensing systems [56–60], which rely on the efficient fluorescent quenching capability
Corresponding authors. E-mail addresses: [email protected] (S. Yoon), [email protected] (J. Lee).
https://doi.org/10.1016/j.snb.2019.127471 Received 26 September 2019; Received in revised form 20 November 2019; Accepted 23 November 2019 0925-4005/ © 2019 Elsevier B.V. All rights reserved.
Please cite this article as: Bomi Shin, et al., Sensors & Actuators: B. Chemical, https://doi.org/10.1016/j.snb.2019.127471
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(MO, USA). Temperature control was performed using a thermocycler (BioRad, USA), and fluorescence was measured using a multi-mode microplate reader (Biotek, USA).
[61–64] and high probe-loading capacity of GO [38,40]. Most of these technologies rely on the strong affinity of GO with ssDNA for forming the DNA/GO complex, which results in the generation of the signal in the presence of a complementary target. However, the lability of this complex and its reactions with competing biomolecules has been reported to cause nonspecific signals, which constitutes the key challenge while employing this sensing approach [57,65–71]. Therefore, the development of improved sensing approaches for analyzing real biological samples which contain various interfering proteins, nucleic acids, and lipids is highly desirable. To address these challenges, we report herein, the design and development of a new high-efficiency DNA probe, and its immobilization on GO via chemical conjugation for the development of practical applications. In our effort to prepare an efficient fluorescent probe for the GODNA conjugate sensor, we prepared a new duplex DNA molecular beacon (MB), in which, the sticky end is at the solution-facing terminus. Unlike the dye-labeled single-stranded probes which are employed in simple GO-DNA conjugates [66,70–73], our duplex probe comprised an upper strand for facilitating direct conjugation with GO, and a dyelabeled bottom strand which possessed the sequence complementary to that of the target. The design included a shorter upper strand, which allowed the exposure of the single-stranded region for enabling miRNA recognition. Importantly, this extended single-stranded region acts as a toehold, which participates in interactions with the target miRNA and thereby facilitates the interaction of the miRNA with the duplex probe, which is initiated by the toehold-mediated displacement mechanism (TMSD) [74–77], and leads to the eventual detachment of the dyetagged bottom strand/miRNA duplex from GO surface. Furthermore, the perfect separation of the dye-tagged bottom strand from GO allowed the fluorescence recovery with enhanced sensitivity. Our design also included the use of a locked nucleic acid (LNA) monomer in the toehold region for improving the sensing performance. LNAs are artificial RNA analogs, which have been reported to display high affinity and excellent specificity toward complementary targets [78–80]. Our LNA-containing MB (LMB) enabled faster miRNA detection and also furnished a lower detection limit (Scheme 1).
2.2. Fabrication of GO-LMB-21 conjugate To prepare LMB-21, the 100 μM amine-functionalized upper strand DNA (10 μL) was mixed with an equal amount of the 5(6)-carboxyfluorescein (FAM)-labelled bottom strand DNA in 1X PBS. Then, the mixture was annealed by heating to 90 °C for 5 min, followed by slow cooling to 25℃ over 1 h. For the conjugation of LMB and GO, 500 pmol of the annealed LMB was mixed with 100 μg of carboxylated GO in 1X PBS (pH 7.2, NaCl 137 mM, and KCl 2.7 mM) containing 10 mM EDC. The mixture was shaken at room temperature for 3 h, which was followed by washing with 1X PBS and 0.02 % HSD solutions. The washing procedure was repeated 5 times, and each supernatant was confirmed by measuring the fluorescence intensity at ex480/em 520 nm (Fig. S1). The resulting purified GO-LMB-21 conjugate was stored at 4 °C. 2.3. miRNA detection For the quantitative miRNA assay, 0.625 μg of GO-LMB-21 was incubated with various concentrations of miRNA in 1X PBS. After 2 h, the fluorescence was measured at ex480/em 520 nm. 3. Results and discussion Firstly, we confirmed the chemical conjugation between GO and LMB, and the miRNA-triggered TMSD reaction on GO by using polyacrylamide gel electrophoresis (PAGE) analysis. The LMB-21 probe was composed of a FAM-labeled bottom strand, which possessed a complementary sequence for miR-21 and an amine-terminated upper strand, which was 7 nt shorter than the bottom strand (Fig. 1a). In the toehold region, a single thymine LNA (shown in red and bold) was incorporated to enhance the binding affinity for the target miRNA as a representative example of LNA-assisted improvement. The GO and LMB conjugate for miR-21 (GO-LMB-21) was prepared by conjugating the amine-functionalized LMB-21 with the carboxylated GO using EDCcoupling chemistry. Upon mixing with miR-21, the GO-LMB-21 showed a single band, which was identified as the newly formed miR-21/ bottom duplex, whereas GO-LMB-21 without the target did not display any significant bands (Fig. 1b). These results indicated that the LMB-21 was chemically attached on the GO surface and that miR-21 successfully induced the TMSD reaction in GO-LMB-21. Importantly, the results shown in Fig. 1c indicate that the release of the miR-21/bottom duplex form the GO was quantitative and proportional to the concentration of miR-21.
2. Materials and methods 2.1. Materials RNA strands were purchased from Bioneer (Daejon, Korea). Aminefunctionalized DNA strands were purchased from Genotech (Daejon, Korea). LNA-embedded DNA strands were purchased from Integrated DNA Technologies (IA, USA). 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), herring sperm DNA (HSD), bovine serum albumin (BSA), and graphene oxide (GO) were purchased from Sigma-Aldrich
Scheme 1. Toehold-mediated strand displacement (TMSD) reaction-dependent strategy for miRNA detection using GO-LMB conjugate. 2
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Fig. 1. (a) Sequences of LMB-21 conjugated on GO and miR-21 (blue box: toehold region, underlined T base with red: inserted LNA monomer). (b) Gel electrophoresis was performed on an 18 % native-PAGE. Lanes 1–4 show 10 pmol each of LMB-21, miR-21/LMB21_B, and 20 pmol each of miR-21, miR-122. Lanes 6–9 show 5 μg of GO-LMB-21 incubated with 0, 2.5, 5, 10, and 20 pmol of miR-21. Lane 10 shows 5 μg of GO-LMB-21 incubated with 20 pmol of miR-122. (c) A Bar graph of relative band intensity of GO-LMB-21 with miR-21 (0−20 pmol). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article).
Fig. 2. (a) Time-dependent fluorescence increase and b) Fluorescence spectra of the GO-LMB-21 with various concentrations of miR-21. c) Bar graph of fluorescence intensity enhancement (F/F0) and d) Fluorescence spectra of GO-LMB-21 with miR-21, and miRNAs of other families (miR-122, let-7a) and scrambled RNA (scRNA).
(Fig. 2a, b). A comparison with the conventional ssDNA conjugated GO sensors revealed that the detection sensitivity of GO-LMB was significantly enhanced due to the complete release of the probes from the GO surface (Fig. S2). We also tested the use of other miRNA families (miR-122, let 7a) and a scrambled RNA strand in our system to probe the sequence specificity of the sensing. The results of this study, which are shown in Fig. 2c and d, indicate that miR-21 induced a remarkable increase in the fluorescence of GO-LMB-21, whereas miRNAs of other families showed little changes in the fluorescence intensity, which was similar to that of the negative control. These results confirmed that the
To evaluate the performance of the developed sensor, we studied the increase in fluorescence of GO-LMB-21 in the presence of miR-21. A 12.5 μg/ml solution of GO-LMB-21 was prepared in 1X PBS and was mixed with a broad range of concentrations of miR-21. In the initial state, the fluorescence intensity of the FAM, labeled at the bottom strand of LMB-21, was quenched by FRET due to its close proximity to GO. Upon addition of miR-21, the newly formed FAM-labelled bottom/ miR-21 duplex was released, and the fluorescence intensity recovered gradually. Notably, the fluorescence intensity increased according to the increase in the concentration of miR-21 and stabilized after 2 h 3
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Fig. 3. Fluorescence intensity enhancement of a) GO-LMB-21 and b) GO-MB-21 with wide concentration ranges of miR-21 target from 0 to 100 nM (F: fluorescence intensity of GO-LMB probe hybridized with miR-21 target, Fc: without target). Inset: linear response at miR-21 concentrations lower than 1 nM. (b) GO-MB.
nM).). In addition, upon the separate addition of 500 pM of spiked miR21 to the system, the obtained fluorescence intensities were found to be in good agreement with the estimated values from the standard curve (Fig. S5). Finally, we conducted multiplex miRNA detection using the developed platform to demonstrate the capability of the developed sensor for accurate miRNA-based diagnostics. We chose the miRNAs, miR-21, miR-125b, and let-7a, which are the representative miRNA biomarkers for breast cancer for the study (Table S1) [81–87]. Each corresponding LMB probes were labeled with FAM, cyanine 5 (Cy5), and 6-carboxyl-Xrhodamine (ROX) which showed emission maxima at 520 (green), 665 nm (red), and 605 (orange), respectively, with excitations at 480, 619, and 564 nm, respectively. The multiplex sensor, termed GO-LMB-21/ 125b/7a, was prepared by conjugating GO with the three kinds of LMBs, which were FAM-LMB-21, Cy5-LMB-125b, and ROX-LMB-7a, followed by purification of the conjugates with HSD solution. For multiplex detection, GO-LMB-21/125b/7a was simply mixed with each target miRNA (100 nM) or various combinations of target miRNAs. Upon mixing, green, red, and orange fluorescence emission spectra and images were obtained only in the samples containing each of the corresponding miRNA targets (Fig. 4).
GO-LMB-21 recognized the target miRNA sequence specifically and produced a quantitative fluorescence signal with enhanced sensitivity in 2 h, which corresponded to the target concentration. Having evaluated the performance and selectivity of the developed sensor, we turned our attention to evaluating the limit of detection (LOD) of the GO-LMB-21 for the quantitative fluorometric detection of miRNA. Various concentrations of miR-21, ranging from 0 to 100 nM, were mixed with a 12.5 μg/ml solution of GO-LMB-21. The sensor showed a linear increase in the fluorescence between 0 and 25 nM, and the detection limit was determined to be 1.3 pM according to the equation, LOD = 3.3 (SD / S), where SD is standard deviation and S is the slope of the calibration curve (Fig. 3a). This LOD value is extremely lower than those of the existing GO-based miRNA sensors, and the sensor remarkably enables detection up to nano-molar levels. As a control, we repeated the same experiment using GO-MB-21, which does not have the LNA in the toehold region. While this sensor also exhibited a time-dependent increase of the fluorescence and demonstrated high selectivity in the presence of miR-21 (Fig. S3), the rate of increase of the fluorescence signal was much slower than that of GO-LMB-21, and the signal was not stabilized until 5 h. With varied concentrations of miR21, which ranged from 0 to 100 nM, GO-MB-21 exhibited smaller increases in fluorescence intensity and indicated a LOD of 99.3 pM (Fig. 3b). It is noteworthy that the incorporation of LNA in the toehold region overwhelmingly helps strengthen the binding affinity with the target and contributes to enhanced sensitivity and also lowers the detection limit. With the evaluation of the various parameters of operation of the developed sensor complete, we investigated the applicability of miR-21 in biological samples by mixing two kinds of GO-based sensors with BSA (Fig. S4a). In the case of the typical GO sensor which employed the physiologically-adsorbed cDNA probe, the fluorescence signal increased gradually with the increase in the concentration of BSA due to the competitive interaction of BSA and cDNA with GO. However, the covalently conjugated GO-LMB sensor showed little change in fluorescence response, which guaranteed the analytical quality with low background signal even in biological samples. Next, we carried out the LOD test with miR-21 spiked in protein-rich samples. In the presence of 100 μg/ml of BSA, GO-LMB-21 showed a quantitative fluorescence increase according to the increase in miR-21 concentration with a comparable LOD of 2.25 pM (Fig. S4b, c). This low pico-molar LOD demonstrated that GO-LMB could maintain the sensing capability even in protein-rich samples without appreciable loss of sensing performance. To confirm the accuracy of such measurements in real samples, we additionally attempted the quantification of spiked miR-21 at the subnanomolar level in a HepG2 lysate (10,000 cells/well). A standard curve was derived from a series of spiked miR-21 concentration (0–1
4. Conclusions We developed a practical and versatile sensing strategy for miRNA using an LNA-embedded duplex probe and GO. This platform relies on the distance-dependent fluorescence quenching capability of GO and the TMSD reaction at the LMB-21, which is conjugated on GO. In contrast to the typical GO-based sensors which use physically adsorbed ssDNA probes, the fluorescent probe was designed as a duplex and possessed a sticky end, and was conjugated onto GO by EDC coupling. The single-stranded part of the sticky end acts as a toehold to facilitate the target miRNA binding, which initiates the TMSD reaction and produces the new free duplex comprising the dye-tagged bottom strand and miRNA. Finally, the duplex is completely released from the GO surface, and thereby, the high recovery of the fluorescence intensity is achieved, which is proportional to the miRNA concentration. The conjugation of the toehold-bearing duplex probes onto GO greatly facilitates the prevention of the nonspecific noise signals from interactions with other biomolecules and enhances the detection sensitivity as well. Furthermore, we introduced an LNA monomer in the toehold region of the probe, and this moiety conferred stronger binding affinity for the miRNA, which resulted in the improvement of the LOD. Notably, this is the first application of LNA in a GO-based fluorescent method, which afforded enhanced sensitivity as well as low LOD. This LNA-GO conjugated platform is technically straightforward and suitable for 4
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Fig. 4. Multiple target detection of three miRNAs using GO-LMB-21/125b/7a in a single solution. (a) Fluorescence spectra of three dyelabeled GO-LMB-21/125b/7a in the presence of each miRNA of 100 nM concentration, and various combinations of miRNAs. (b) Fluorescence images of reaction mixtures for multiple detections. Column 1: Fluorescent signal of FAM-LMB-21. Column 2: Fluorescent signal of Cy5-LMB-125b. Column 3: Fluorescent signal of ROX-LMB-7a. (c) Bar graph showing relative fluorescence intensity of three dye-labeled LMBs conjugated on GO in the presence of each target with 100 nM concentration.
analyzing less pure biological samples. Moreover, this system could be broadly applied to sensing other miRNAs by simply varying the sequences within the duplex probe, and the sensitivity could be adjusted by controlling the number of LNAs. This smart GO-based sensor allows both sensitive and selective miRNA detection and is expected to be a suitable miRNA-based diagnostic tool for diseases, in addition to a valuable resource for basic and applied research. In our future work, we therefore aim to extend this platform to live-cell imaging and also to clinical diagnosis with real-world samples.
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Woo-keun Kim received his Ph.D. in Environmental Toxicology in 2012 from Korea University, Korea. He is currently a principal researcher scientist at KIT and a professor in Korea University of Science and Technology. His research area focuses on the development of new in vivo model and biosensor in toxicological aspect.
[85] A. Feliciano, J. Castellvi, A. Artero-Castro, J.A. Leal, C. Romagosa, J. HernándezLosa, V. Peg, A. Fabra, F. Vidal, H. Kondoh, miR-125b acts as a tumor suppressor in breast tumorigenesis via its novel direct targets ENPEP, CK2-α, CCNJ, and MEGF9, PLoS One 8 (2013) e76247. [86] K. Liu, C. Zhang, T. Li, Y. Ding, T. Tu, F. Zhou, W. Qi, H. Chen, X. Sun, Let-7a inhibits growth and migration of breast cancer cells by targeting HMGA1, Int. J. Oncol. 46 (2015) 2526–2534. [87] Y. Luo, X. Wang, W. Niu, H. Wang, Q. Wen, S. Fan, R. Zhao, Z. Li, W. Xiong, S. Peng, Z. Zeng, X. Li, G. Li, M. Tan, M. Zhou, Elevated microRNA-125b levels predict a worse prognosis in HER2-positive breast cancer patients, Oncol. Lett. 13 (2017) 867–874.
Seokjoo Yoon received his Ph.D. in Toxicology in 2000 from Hokkaido University, Japan. He, as principal researcher scientist, is currently a director of predictive toxicology department at KIT and a professor in Korea University of Science and Technology. His research interest is development of new drug and chemical toxicity prediction technology. Jieon Lee received her Ph. D. in Nano-Bio chemistry from Seoul National University in 2015. She is currently a research scientist at Korea institute of toxicology (KIT). Her research area focuses on the development of biosensing strategy using various nanomaterials.
Bomi Shin received her M.S. in Department of Toxicology Evaluation in 2019 from Konyang University, Korea. Her research area focuses on the development of biosensor in toxicological aspect.
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Contents lists available at ScienceDirect
Sensors and Actuators B: Chemical journal homepage: www.elsevier.com/locate/snb
Duplex DNA-functionalized graphene oxide: A versatile platform for miRNA sensing Bomi Shina,b, Woo-Keun Kima, Seokjoo Yoona,*, Jieon Leea,* a System Toxicology Research Center, Predictive Toxicology Department, Korea Institute of Toxicology (KIT), 141 Gajeong-ro, Yuseong-gu, Daejeon, 34114, Republic of Korea b Department of Toxicology Evaluation, Konyang University, 158 Gwanjeodong-ro, Seo-gu, Daejeon, 35365, Republic of Korea
A R T I C LE I N FO
A B S T R A C T
Keywords: miRNA sensing Graphene oxide Covalent conjugation Locked nucleic acids Toehold-mediated strand displacement
MicroRNAs (miRNAs) are short, non-coding RNAs, which have emerged as promising next-generation biomarkers for clinical diagnostics. Here, we developed a graphene oxide (GO)-based fluorescent nano-sensor for miRNA sensing with high specificity and sensitivity. GO is a water-soluble derivative of graphene, which has emerged as one of the most popular nanomaterials in the bio-medical field over the last few decades. Common strategies in the design of GO-based sensors rely on the fluorescence quenching capability of GO and the strong adsorption of ssDNA probes on GO surface through pi-pi interactions or hydrogen bonding. Unfortunately, the interaction between DNA and GO is easily interrupted by other molecules present in biological samples, such as proteins, lipids, and nucleic acids, which results in the nonspecific desorption of probes. Therefore, we designed a fluorescence-labeled dsDNA probe, with an embedded locked nucleic acid (LNA). The probe was then attached onto GO by covalent coupling, instead of nonspecific adsorption. The probes on GO recognized the target miRNA sequence specifically and released the fluorescent strand via toehold-mediated strand displacement (TMSD), which furnished the recovery of quenched fluorescence. This platform displayed improved signal to noise ratio with detection limits at the pico-molar levels. We believe that this new GO-based sensor can be a suitable tool for miRNA-based practical diagnostics of diseases, and provides a valuable resource for basic and applied research.
1. Introduction MicroRNAs (miRNA) are non-coding RNAs and range in length from 19∼25 nucleotides [1–3]. miRNAs play critical roles in diverse biological processes such as cell proliferation [4,5], differentiation [6,7], angiogenesis [8,9], and apoptosis [10,11], by interrupting the translation of messenger RNAs. Recently, numerous studies have confirmed the close relationship between miRNAs and the occurrence of diseases and human cancer [12–15]. Among the various miRNA families, miRNA-21 (miR-21) is a representative oncogene, which is overexpressed in most tumor types such as lung [16,17], breast [18,19], stomach [20,21], prostate [22,23], colon [24,25], and pancreas [26,27], and has been therapeutically targeted for tumor suppression. Therefore, the profiling and evaluation of miRNAs as diagnostic and prognostic markers for human diseases, or as prominent targets for genetic modulation in disease states is an attractive and valuable prospect. Generally, the quantitative measurement of miRNAs is not feasible using traditional approaches such as microarray [28,29], northern blot [30,31], and real-time polymerase chain reaction technologies
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[32,33]. In addition, these conventional methods are typically cost- and time-intensive, laborious, and/or require expensive instrumentation. Furthermore, the performance of these methods does not provide useful detection results of miRNAs. Recently, numerous nanomaterial-based sensing strategies have been developed for miRNA detection and analysis to overcome the drawbacks of conventional methods and to broaden the applications of miRNAs [34–37]. In this study, we developed a versatile and practical platform for miRNA detection using a graphene oxide (GO)-DNA conjugate. GO is a chemically-oxidized derivative of graphene [38], and contains sp2/sp3 hybrid carbon structures and various oxygen functional groups on its basal planes and edges. GO can be easily dispersed in aqueous solutions, and importantly, it retains its original characteristics, such as the high thermal and electrical conductivity and hydrophobicity under these conditions [38–40]. These unique properties make GO a potentially valuable material for use in biosensors [41–44], drug delivery [45–52], and catalysis [53–55]. Particularly, GO has been extensively used for the development of fluorescent nucleic acid (NA)-sensing systems [56–60], which rely on the efficient fluorescent quenching capability
Corresponding authors. E-mail addresses: [email protected] (S. Yoon), [email protected] (J. Lee).
https://doi.org/10.1016/j.snb.2019.127471 Received 26 September 2019; Received in revised form 20 November 2019; Accepted 23 November 2019 0925-4005/ © 2019 Elsevier B.V. All rights reserved.
Please cite this article as: Bomi Shin, et al., Sensors & Actuators: B. Chemical, https://doi.org/10.1016/j.snb.2019.127471
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(MO, USA). Temperature control was performed using a thermocycler (BioRad, USA), and fluorescence was measured using a multi-mode microplate reader (Biotek, USA).
[61–64] and high probe-loading capacity of GO [38,40]. Most of these technologies rely on the strong affinity of GO with ssDNA for forming the DNA/GO complex, which results in the generation of the signal in the presence of a complementary target. However, the lability of this complex and its reactions with competing biomolecules has been reported to cause nonspecific signals, which constitutes the key challenge while employing this sensing approach [57,65–71]. Therefore, the development of improved sensing approaches for analyzing real biological samples which contain various interfering proteins, nucleic acids, and lipids is highly desirable. To address these challenges, we report herein, the design and development of a new high-efficiency DNA probe, and its immobilization on GO via chemical conjugation for the development of practical applications. In our effort to prepare an efficient fluorescent probe for the GODNA conjugate sensor, we prepared a new duplex DNA molecular beacon (MB), in which, the sticky end is at the solution-facing terminus. Unlike the dye-labeled single-stranded probes which are employed in simple GO-DNA conjugates [66,70–73], our duplex probe comprised an upper strand for facilitating direct conjugation with GO, and a dyelabeled bottom strand which possessed the sequence complementary to that of the target. The design included a shorter upper strand, which allowed the exposure of the single-stranded region for enabling miRNA recognition. Importantly, this extended single-stranded region acts as a toehold, which participates in interactions with the target miRNA and thereby facilitates the interaction of the miRNA with the duplex probe, which is initiated by the toehold-mediated displacement mechanism (TMSD) [74–77], and leads to the eventual detachment of the dyetagged bottom strand/miRNA duplex from GO surface. Furthermore, the perfect separation of the dye-tagged bottom strand from GO allowed the fluorescence recovery with enhanced sensitivity. Our design also included the use of a locked nucleic acid (LNA) monomer in the toehold region for improving the sensing performance. LNAs are artificial RNA analogs, which have been reported to display high affinity and excellent specificity toward complementary targets [78–80]. Our LNA-containing MB (LMB) enabled faster miRNA detection and also furnished a lower detection limit (Scheme 1).
2.2. Fabrication of GO-LMB-21 conjugate To prepare LMB-21, the 100 μM amine-functionalized upper strand DNA (10 μL) was mixed with an equal amount of the 5(6)-carboxyfluorescein (FAM)-labelled bottom strand DNA in 1X PBS. Then, the mixture was annealed by heating to 90 °C for 5 min, followed by slow cooling to 25℃ over 1 h. For the conjugation of LMB and GO, 500 pmol of the annealed LMB was mixed with 100 μg of carboxylated GO in 1X PBS (pH 7.2, NaCl 137 mM, and KCl 2.7 mM) containing 10 mM EDC. The mixture was shaken at room temperature for 3 h, which was followed by washing with 1X PBS and 0.02 % HSD solutions. The washing procedure was repeated 5 times, and each supernatant was confirmed by measuring the fluorescence intensity at ex480/em 520 nm (Fig. S1). The resulting purified GO-LMB-21 conjugate was stored at 4 °C. 2.3. miRNA detection For the quantitative miRNA assay, 0.625 μg of GO-LMB-21 was incubated with various concentrations of miRNA in 1X PBS. After 2 h, the fluorescence was measured at ex480/em 520 nm. 3. Results and discussion Firstly, we confirmed the chemical conjugation between GO and LMB, and the miRNA-triggered TMSD reaction on GO by using polyacrylamide gel electrophoresis (PAGE) analysis. The LMB-21 probe was composed of a FAM-labeled bottom strand, which possessed a complementary sequence for miR-21 and an amine-terminated upper strand, which was 7 nt shorter than the bottom strand (Fig. 1a). In the toehold region, a single thymine LNA (shown in red and bold) was incorporated to enhance the binding affinity for the target miRNA as a representative example of LNA-assisted improvement. The GO and LMB conjugate for miR-21 (GO-LMB-21) was prepared by conjugating the amine-functionalized LMB-21 with the carboxylated GO using EDCcoupling chemistry. Upon mixing with miR-21, the GO-LMB-21 showed a single band, which was identified as the newly formed miR-21/ bottom duplex, whereas GO-LMB-21 without the target did not display any significant bands (Fig. 1b). These results indicated that the LMB-21 was chemically attached on the GO surface and that miR-21 successfully induced the TMSD reaction in GO-LMB-21. Importantly, the results shown in Fig. 1c indicate that the release of the miR-21/bottom duplex form the GO was quantitative and proportional to the concentration of miR-21.
2. Materials and methods 2.1. Materials RNA strands were purchased from Bioneer (Daejon, Korea). Aminefunctionalized DNA strands were purchased from Genotech (Daejon, Korea). LNA-embedded DNA strands were purchased from Integrated DNA Technologies (IA, USA). 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), herring sperm DNA (HSD), bovine serum albumin (BSA), and graphene oxide (GO) were purchased from Sigma-Aldrich
Scheme 1. Toehold-mediated strand displacement (TMSD) reaction-dependent strategy for miRNA detection using GO-LMB conjugate. 2
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Fig. 1. (a) Sequences of LMB-21 conjugated on GO and miR-21 (blue box: toehold region, underlined T base with red: inserted LNA monomer). (b) Gel electrophoresis was performed on an 18 % native-PAGE. Lanes 1–4 show 10 pmol each of LMB-21, miR-21/LMB21_B, and 20 pmol each of miR-21, miR-122. Lanes 6–9 show 5 μg of GO-LMB-21 incubated with 0, 2.5, 5, 10, and 20 pmol of miR-21. Lane 10 shows 5 μg of GO-LMB-21 incubated with 20 pmol of miR-122. (c) A Bar graph of relative band intensity of GO-LMB-21 with miR-21 (0−20 pmol). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article).
Fig. 2. (a) Time-dependent fluorescence increase and b) Fluorescence spectra of the GO-LMB-21 with various concentrations of miR-21. c) Bar graph of fluorescence intensity enhancement (F/F0) and d) Fluorescence spectra of GO-LMB-21 with miR-21, and miRNAs of other families (miR-122, let-7a) and scrambled RNA (scRNA).
(Fig. 2a, b). A comparison with the conventional ssDNA conjugated GO sensors revealed that the detection sensitivity of GO-LMB was significantly enhanced due to the complete release of the probes from the GO surface (Fig. S2). We also tested the use of other miRNA families (miR-122, let 7a) and a scrambled RNA strand in our system to probe the sequence specificity of the sensing. The results of this study, which are shown in Fig. 2c and d, indicate that miR-21 induced a remarkable increase in the fluorescence of GO-LMB-21, whereas miRNAs of other families showed little changes in the fluorescence intensity, which was similar to that of the negative control. These results confirmed that the
To evaluate the performance of the developed sensor, we studied the increase in fluorescence of GO-LMB-21 in the presence of miR-21. A 12.5 μg/ml solution of GO-LMB-21 was prepared in 1X PBS and was mixed with a broad range of concentrations of miR-21. In the initial state, the fluorescence intensity of the FAM, labeled at the bottom strand of LMB-21, was quenched by FRET due to its close proximity to GO. Upon addition of miR-21, the newly formed FAM-labelled bottom/ miR-21 duplex was released, and the fluorescence intensity recovered gradually. Notably, the fluorescence intensity increased according to the increase in the concentration of miR-21 and stabilized after 2 h 3
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Fig. 3. Fluorescence intensity enhancement of a) GO-LMB-21 and b) GO-MB-21 with wide concentration ranges of miR-21 target from 0 to 100 nM (F: fluorescence intensity of GO-LMB probe hybridized with miR-21 target, Fc: without target). Inset: linear response at miR-21 concentrations lower than 1 nM. (b) GO-MB.
nM).). In addition, upon the separate addition of 500 pM of spiked miR21 to the system, the obtained fluorescence intensities were found to be in good agreement with the estimated values from the standard curve (Fig. S5). Finally, we conducted multiplex miRNA detection using the developed platform to demonstrate the capability of the developed sensor for accurate miRNA-based diagnostics. We chose the miRNAs, miR-21, miR-125b, and let-7a, which are the representative miRNA biomarkers for breast cancer for the study (Table S1) [81–87]. Each corresponding LMB probes were labeled with FAM, cyanine 5 (Cy5), and 6-carboxyl-Xrhodamine (ROX) which showed emission maxima at 520 (green), 665 nm (red), and 605 (orange), respectively, with excitations at 480, 619, and 564 nm, respectively. The multiplex sensor, termed GO-LMB-21/ 125b/7a, was prepared by conjugating GO with the three kinds of LMBs, which were FAM-LMB-21, Cy5-LMB-125b, and ROX-LMB-7a, followed by purification of the conjugates with HSD solution. For multiplex detection, GO-LMB-21/125b/7a was simply mixed with each target miRNA (100 nM) or various combinations of target miRNAs. Upon mixing, green, red, and orange fluorescence emission spectra and images were obtained only in the samples containing each of the corresponding miRNA targets (Fig. 4).
GO-LMB-21 recognized the target miRNA sequence specifically and produced a quantitative fluorescence signal with enhanced sensitivity in 2 h, which corresponded to the target concentration. Having evaluated the performance and selectivity of the developed sensor, we turned our attention to evaluating the limit of detection (LOD) of the GO-LMB-21 for the quantitative fluorometric detection of miRNA. Various concentrations of miR-21, ranging from 0 to 100 nM, were mixed with a 12.5 μg/ml solution of GO-LMB-21. The sensor showed a linear increase in the fluorescence between 0 and 25 nM, and the detection limit was determined to be 1.3 pM according to the equation, LOD = 3.3 (SD / S), where SD is standard deviation and S is the slope of the calibration curve (Fig. 3a). This LOD value is extremely lower than those of the existing GO-based miRNA sensors, and the sensor remarkably enables detection up to nano-molar levels. As a control, we repeated the same experiment using GO-MB-21, which does not have the LNA in the toehold region. While this sensor also exhibited a time-dependent increase of the fluorescence and demonstrated high selectivity in the presence of miR-21 (Fig. S3), the rate of increase of the fluorescence signal was much slower than that of GO-LMB-21, and the signal was not stabilized until 5 h. With varied concentrations of miR21, which ranged from 0 to 100 nM, GO-MB-21 exhibited smaller increases in fluorescence intensity and indicated a LOD of 99.3 pM (Fig. 3b). It is noteworthy that the incorporation of LNA in the toehold region overwhelmingly helps strengthen the binding affinity with the target and contributes to enhanced sensitivity and also lowers the detection limit. With the evaluation of the various parameters of operation of the developed sensor complete, we investigated the applicability of miR-21 in biological samples by mixing two kinds of GO-based sensors with BSA (Fig. S4a). In the case of the typical GO sensor which employed the physiologically-adsorbed cDNA probe, the fluorescence signal increased gradually with the increase in the concentration of BSA due to the competitive interaction of BSA and cDNA with GO. However, the covalently conjugated GO-LMB sensor showed little change in fluorescence response, which guaranteed the analytical quality with low background signal even in biological samples. Next, we carried out the LOD test with miR-21 spiked in protein-rich samples. In the presence of 100 μg/ml of BSA, GO-LMB-21 showed a quantitative fluorescence increase according to the increase in miR-21 concentration with a comparable LOD of 2.25 pM (Fig. S4b, c). This low pico-molar LOD demonstrated that GO-LMB could maintain the sensing capability even in protein-rich samples without appreciable loss of sensing performance. To confirm the accuracy of such measurements in real samples, we additionally attempted the quantification of spiked miR-21 at the subnanomolar level in a HepG2 lysate (10,000 cells/well). A standard curve was derived from a series of spiked miR-21 concentration (0–1
4. Conclusions We developed a practical and versatile sensing strategy for miRNA using an LNA-embedded duplex probe and GO. This platform relies on the distance-dependent fluorescence quenching capability of GO and the TMSD reaction at the LMB-21, which is conjugated on GO. In contrast to the typical GO-based sensors which use physically adsorbed ssDNA probes, the fluorescent probe was designed as a duplex and possessed a sticky end, and was conjugated onto GO by EDC coupling. The single-stranded part of the sticky end acts as a toehold to facilitate the target miRNA binding, which initiates the TMSD reaction and produces the new free duplex comprising the dye-tagged bottom strand and miRNA. Finally, the duplex is completely released from the GO surface, and thereby, the high recovery of the fluorescence intensity is achieved, which is proportional to the miRNA concentration. The conjugation of the toehold-bearing duplex probes onto GO greatly facilitates the prevention of the nonspecific noise signals from interactions with other biomolecules and enhances the detection sensitivity as well. Furthermore, we introduced an LNA monomer in the toehold region of the probe, and this moiety conferred stronger binding affinity for the miRNA, which resulted in the improvement of the LOD. Notably, this is the first application of LNA in a GO-based fluorescent method, which afforded enhanced sensitivity as well as low LOD. This LNA-GO conjugated platform is technically straightforward and suitable for 4
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Fig. 4. Multiple target detection of three miRNAs using GO-LMB-21/125b/7a in a single solution. (a) Fluorescence spectra of three dyelabeled GO-LMB-21/125b/7a in the presence of each miRNA of 100 nM concentration, and various combinations of miRNAs. (b) Fluorescence images of reaction mixtures for multiple detections. Column 1: Fluorescent signal of FAM-LMB-21. Column 2: Fluorescent signal of Cy5-LMB-125b. Column 3: Fluorescent signal of ROX-LMB-7a. (c) Bar graph showing relative fluorescence intensity of three dye-labeled LMBs conjugated on GO in the presence of each target with 100 nM concentration.
analyzing less pure biological samples. Moreover, this system could be broadly applied to sensing other miRNAs by simply varying the sequences within the duplex probe, and the sensitivity could be adjusted by controlling the number of LNAs. This smart GO-based sensor allows both sensitive and selective miRNA detection and is expected to be a suitable miRNA-based diagnostic tool for diseases, in addition to a valuable resource for basic and applied research. In our future work, we therefore aim to extend this platform to live-cell imaging and also to clinical diagnosis with real-world samples.
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Woo-keun Kim received his Ph.D. in Environmental Toxicology in 2012 from Korea University, Korea. He is currently a principal researcher scientist at KIT and a professor in Korea University of Science and Technology. His research area focuses on the development of new in vivo model and biosensor in toxicological aspect.
[85] A. Feliciano, J. Castellvi, A. Artero-Castro, J.A. Leal, C. Romagosa, J. HernándezLosa, V. Peg, A. Fabra, F. Vidal, H. Kondoh, miR-125b acts as a tumor suppressor in breast tumorigenesis via its novel direct targets ENPEP, CK2-α, CCNJ, and MEGF9, PLoS One 8 (2013) e76247. [86] K. Liu, C. Zhang, T. Li, Y. Ding, T. Tu, F. Zhou, W. Qi, H. Chen, X. Sun, Let-7a inhibits growth and migration of breast cancer cells by targeting HMGA1, Int. J. Oncol. 46 (2015) 2526–2534. [87] Y. Luo, X. Wang, W. Niu, H. Wang, Q. Wen, S. Fan, R. Zhao, Z. Li, W. Xiong, S. Peng, Z. Zeng, X. Li, G. Li, M. Tan, M. Zhou, Elevated microRNA-125b levels predict a worse prognosis in HER2-positive breast cancer patients, Oncol. Lett. 13 (2017) 867–874.
Seokjoo Yoon received his Ph.D. in Toxicology in 2000 from Hokkaido University, Japan. He, as principal researcher scientist, is currently a director of predictive toxicology department at KIT and a professor in Korea University of Science and Technology. His research interest is development of new drug and chemical toxicity prediction technology. Jieon Lee received her Ph. D. in Nano-Bio chemistry from Seoul National University in 2015. She is currently a research scientist at Korea institute of toxicology (KIT). Her research area focuses on the development of biosensing strategy using various nanomaterials.
Bomi Shin received her M.S. in Department of Toxicology Evaluation in 2019 from Konyang University, Korea. Her research area focuses on the development of biosensor in toxicological aspect.
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