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Discriminating a Single Nucleotide Difference for Enhanced miRNA Detection Using Tunable Graphene and Oligonucleotide Nanodevices Neil M. Robertson,† Mustafa Salih Hizir,† Mustafa Balcioglu,† Rui Wang,†,‡ Mustafa Selman Yavuz,§ Hasan Yumak,∥ Birol Ozturk,⊥ Jia Sheng,†,‡ and Mehmet V. Yigit*,†,‡ †

Department of Chemistry and ‡The RNA Institute, University at Albany, State University of New York, 1400 Washington Avenue, Albany, New York 12222, United States § Department of Metallurgy and Materials Engineering, Advanced Technology Research and Application Center, Selcuk University, Konya, Turkey ∥ Department of Science, BMCC, City University of New York, 199 Chambers Street, New York, New York 10007, United States ⊥ Department of Physics and Engineering Physics, Morgan State University, 1700 E. Cold Spring Lane, Baltimore, Maryland 21251, United States S Supporting Information *

ABSTRACT: In this study we have reported our efforts to address some of the challenges in the detection of miRNAs using water-soluble graphene oxide and DNA nanoassemblies. Purposefully inserting mismatches at specific positions in our DNA (probe) strands shows increasing specificity against our target miRNA, miR-10b, over miR-10a which varies by only a single nucleotide. This increased specificity came at a loss of signal intensity within the system, but we demonstrated that this could be addressed with the use of DNase I, an endonuclease capable of cleaving the DNA strands of the RNA/ DNA heteroduplex and recycling the RNA target to hybridize to another probe strand. As we previously demonstrated, this enzymatic signal also comes with an inherent activity of the enzyme on the surface-adsorbed probe strands. To remove this activity of DNase I and the steady nonspecific increase in the fluorescence signal without compromising the recovered signal, we attached a thermoresponsive PEGMA polymer (poly(ethylene glycol) methyl ether methacrylate) to nGO. This smart polymer is able to shield the probes adsorbed on the nGO surface from the DNase I activity and is capable of tuning the detection capacity of the nGO nanoassembly with a thermoswitch at 39 °C. By utilizing probes with multiple mismatches, DNase I cleavage of the DNA probe strands, and the attachment of PEGMA polymers to graphene oxide to block undesired DNase I activity, we were able to detect miR-10b from liquid biopsy mimics and breast cancer cell lines. Overall we have reported our efforts to improve the specificity, increase the sensitivity, and eliminate the undesired enzymatic activity of DNase I on surface-adsorbed probes for miR10b detection using water-soluble graphene nanodevices. Even though we have demonstrated only the discrimination of miR-10b from miR-10a, our approach can be extended to other short RNA molecules which differ by a single nucleotide.

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INTRODUCTION

there are several challenges to overcome in order to assess miRNAs for diagnosis and therapy, particularly with respect to discriminating miRNAs with high degrees of similarities.13 For instance, miR-10a and miR-10b, which are in the same miRNA family, differ from each other by only a single nucleotide. Therefore, rapid and cost-efficient identification of miR-10b in solid or liquid breast tumor biopsies and discriminating it from miR-10a could be critical for diagnosis. Nanomaterials and their covalent or noncovalent assemblies with theranostic oligonucleotides have the potential to fulfill such a necessity due to

miRNAs are small noncoding RNA molecules which regulate gene expression at the posttranscriptional level.1 Certain miRNAs are drastically overexpressed in the tumor tissues and liquid biopsies (blood, urine, saliva, etc.) of cancer patients. These oncogene miRNAs, referred to as oncomiRs, are assessed as signatures of the presence and status of the disease.2−8 miR10b is one of these oncomiRs overexpressed in metastatic breast cancer, and the invasiveness of the tumor cells is highly correlated with its expression.9 Regulating the expression of miR-10b using synthetic antisense oligonucleotides controls the metastatic behavior of the disease, which makes miR-10b not only a potential diagnostic marker but also a promising therapeutic target for metastatic breast cancer.10−12 However, © 2015 American Chemical Society

Received: June 2, 2015 Revised: August 6, 2015 Published: August 25, 2015 9943

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Scheme 1. Schematic Illustration of Amplified miRNA Detection and Enzymatic Activities of DNase I before and after the Attachment of PEGMAa

a

Step 1 illustrates the hybridization-induced miRNA detection. In step 2, arrow A illustrates the activity of DNase I against the DNA:miRNA heteroduplex, and arrow B illustrates the enzyme activity on the surface-adsorbed DNA probes. Step 3 illustrates the inhibition of the enzyme activity on the surface-adsorbed DNA probes after PEGMA attachment.

their distinct physical properties at the bionano interfaces.14−25 Graphene oxide and fluorescently labeled antisense DNA nanoassemblies, which are inexpensive, easy to assemble, and simple to operate, have gained significant attention in the past decade.26−29 These water-soluble diagnostic nanodevices have the potential to detect miRNAs specifically from complex biological matrices; therefore, they are potential candidates for point-of-care testing for personalized medicine.30−34 Graphene is a one-atom-thick two-dimensional carbon material with a honeycomb structure.35−37 Water-soluble nanoscale graphene oxide (nGO) is easy to prepare by the ultrasonication of multilayered graphene oxide. nGO has been successfully utilized for several environmental, biological, and biomedical applications including the detection of cellular and circulating RNA molecules in biological systems.26,28,29,38−50 nGO has two central properties which have been remarkable for the detection of sequence-specific oligonucleotides using fluorescence spectroscopy.51 First, single stranded (ss) short oligonucleotides (probes) can be noncovalently adsorbed on the graphene surface; however, double-stranded ones cannot.52,53 In the presence of complementary target oligonucleotides, the probes hybridize to target strands and are released from the surface.54−56 Second, the fluorescence of the probe strands labeled with a molecular fluorescent dye can be quenched almost completely by the graphene oxide surface but is recovered with target molecules through hybridization.51,54 These two properties have been very instrumental in detecting miRNAs from complex biological matrices.30,31,33 However, there are still several challenges remaining to be addressed in order for nGO to be used for miRNA detection in a diagnostic setting, which include issues with selectivity, specificity, and background signals.27,30−32 Here we will summarize our efforts to address these challenges in a systematic manner. In a typical ss oligonucleotide detection scenario, fully complementary and fluorophore-labeled antisense DNA molecules (probes) are assembled with nGO; however, oligonucleotides with single or double mismatches also generate a relatively high fluorescence recovery due to imperfect duplex formation.32,57,58 This is a critical challenge to overcome for identifying a specific miRNA from a biological matrix. We and others have hypothesized that by purposefully inserting the mismatches in the probe strands, the specificity

against the target oligonucleotide over other strands with high degrees of sequence similarity can be increased.57,59,60 Here we have tested the specificity of miR-10b over miR-10a by the systematic insertion of mismatches in the fully complementary probe. However, since imperfect hybridization decreases the observed fluorescence, the sensitivity is diminished and has to be improved both to recover the sacrificed signal and to meet the sensitivity requirement for potential diagnostic use. In a standard hybridization-based approach, the observed fluorescence signal is solely due to hybridization between the probe strand and the miRNA molecule (Scheme 1, step 1).30,31 However, spectrofluorometers are often not sensitive enough to detect fluorescence readings generated by low copies of miRNAs. Multiple approaches have been used to improve the sensitivity of graphene nanoassemblies, including hybridization chain reactions and both enzyme-free and enzyme amplification methods.34,61−65 Among many studies, DNA-specific endonucleases have the potential to increase the limit of detection drastically (Scheme 1, step 2).63,65,66 We have used DNase I to amplify the fluorescence signal (Scheme 1, step 3A) and have reported our efforts to reduce the background fluorescence increase by blocking DNase I access to the surface (Scheme 1, step 3B). This inhibition was achieved by incorporating antifouling PEGMA (poly(ethylene glycol) methyl ether methacrylate) polymers on the nGO edges (nGO-PEGMA).31 Here we have combined multiple approaches to address three different challengesspecificity, sensitivity, and an increase in background fluorescencein miRNA detection using a graphene nanodevice. We have detected miR-10b using an nGO-PEGMA and DNase I combination in three different matrices and have discriminated it from miR-10a. First, isolated miR-10b was detected in a buffered solution. Later, exogenously and endogenously expressed miR-10b was detected in an RNA pool isolated from liquid biopsy mimics and metastatic breast cancer cells, respectively. Our results demonstrate that graphene oxide possesses remarkable potential in miRNA detection and can be used for engineering rapid, inexpensive, easy-to-operate, and point-of-care nanodevices for cancer screening. 9944

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labeled M2 in 10 mM Tris-HCl buffer (pH 7.5, 2.5 mM MgCl2, 0.1 mM CaCl2) with 3 μg/mL nGO. Initial measurements were taken after 0, 2, or 10 μL of 1 U/μL stock DNase I was added to 100 μL of nGO/DNA, resulting in 0, 2, or 10 U of DNase I activity. Ten minutes after the addition of DNase I, 5 μL of a 1 μM stock (50 nM final concentration) target RNA was added to the same wells that contained DNase I and nGO/M2. A 1.5 h kinetic study was performed to monitor the observed fluorescence. Control experiments were performed by monitoring the fluorescence with 50 nM miR-10a and SCR. To monitor the effect of DNase I on the target and nGOPEGMA/DNA, an analogous approach was performed using 3 μg/mL nGO-PEGMA. To visualize the fluorescence difference between miR10b and miR-10a using nGO-PEGMA/M2, the incubation and kinetic studies were carried out as described above. After the 1.5 h kinetic studies were completed, the wells containing the DNase I and nGOPEGMA/M2 that had been mixed with 5 μL of 1 μM stock concentration (50 nM final) RNA, miR-10b and miR-10a, were imaged using a Bio-Rad ChemiDoc. Monitoring the Tunable Properties of nGO-PEGMA with Target miRNA. To monitor the effect of temperature on nGOPEGMA/M2 and nGO/M2 (control) with target RNA, both nanodevices were prepared as described above. The fluorescence measurements were performed at either 36 °C (below the LCST) or 42 °C (above the LCST) to monitor the observed fluorescence. Detection of Specific miRNA Targets from Liquid Biopsy Mimics. For the detection of RNA targets from liquid biopsies, 10 μL of 100 μM miR-10b or miR-10a was added to 200 μL of fetal bovine serum (FBS). Later, the RNA contents were extracted using the QIAzol method. Briefly, for each RNA preparation, 200 μL of FBS was treated with 800 μL of QIAzol in Eppendorf tubes and incubated for 5 min according to the supplier’s instructions. Later, 200 μL of chloroform was added, mixed, and again left to incubate for 5 min. The tubes were then centrifuged at 10 000g at 4 °C for 10 min, and afterward the supernatant was transferred to a new tube. An equal volume of isopropanol was added, and the tube was mixed and placed on ice to incubate for 10 min. The samples were then centrifuged at 10 000g for 20 min at 4 °C. At the end of centrifugation, the supernatant was discarded and the remaining pellet was washed with 1 mL of 70% molecular-grade ethanol before being centrifuged at 10 000g for 5 min at 4 °C. The supernatant was discarded, and the RNA pellet was left to air dry. Once dried, the pellet was resuspended in 20 μL of nuclease-free water and stored at −20 °C. The RNA content and purity were determined using the NanoDrop ND-1000 spectrophotometer. The total RNA content from each sample was determined to be 71.2 ng/μL of [serum RNA + miR-10b] and 262.8 ng/μL of [serum + miR-10a]. To detect the presence of the miR-10b target in the extracted total RNA, the nGO-PEGMA/M2 and DNase I (5U) mixture was prepared as described above. Ten minutes after, 100 or 250 pg/μL of total RNA was added to the reaction mixture. The fluorescence readings were collected for 1.5 h after the addition of the RNAs. Detection of Endogenous miRNA Targets in Breast Cancer Cell Lines. MCF-7 and 4T1 cell lines were cultured in DMEM (Invitrogen) supplemented with 10% fetal bovine serum. The cells were propagated in media supplemented with L-glutamine, 100 U/mL penicillin, and 100 μg/mL streptomycin (Life Tech Corp., Grand Island, NY) at 37 °C in a 5% CO2 incubator. The cells were lysed with the addition of QIAzol after being washed with ice-cold PBS twice according to the supplier’s (QIAGEN) instructions. The RNA from the cell lines (4T1 and MCF-7) were extracted similarly to the procedure in the liquid biopsy mimic studies described above. After extraction was completed, 10, 20, or 30 ng/μL of total RNA was added to the nGO-PEGMA/M2 in the presence of 5 U of DNase I. The fluorescence readings were collected for 1.5 h after the addition of RNAs. Gel Electrophoresis. A 15% native PAGE analysis of the (I) nGO/M2, (II) nGO/M2 with 100 nM miR-10b, and (III) nGO/M2 with 100 nM miR-10b and 10 U of DNase I was carried out in 1× TBE for 45 min. After running, fluorescent images of the gel were taken and analyzed using a Bio-Rad ChemiDoc (ext. 485, em. 520).

MATERIALS AND METHODS

Materials and Reagents. All DNA and RNA sequences were purchased from Integrated DNA Technologies (IDT, USA) with the following sequence information: (1) FAM-labeled full complementary probe, 5′-/FAM/CACAAATTCGGTTCTACAGGGTA-3′ (2) FAM-labeled one mismatch probe (M1), 5′-/FAM/CACAAAATCGGTTCTACAGGGTA-3′ (3) FAM-labeled two mismatch probe (M2), 5′-/FAM/CACAAAATCGGTTCTACAGCGTA-3′ (4) miR-10b, 5′-UACCCUGUAGAACCGAAUUUGUG-3′ (5) miR-10a, 5′-UACCCUGUAGAUCCGAAUUUGUG-3′ (6) scrambled RNA (SCR), 5′-CAUCUUCCAGUACAGUGUUGGA-3′ DNase I was purchased from Thermo Fisher Scientific (Waltham, MA, USA) and supplied with 10× reaction buffer (RNase-free, 100 mM Tris-HCl, pH 7.5 at 25 °C, 25 mM MgCl2, 1 mM CaCl2). A carboxyl graphene oxide water dispersion was purchased from ACS Material (Medford, MA, USA) and sonicated for 12 h before use, which resulted in a very stable nanosized graphene oxide (nGO) solution. The average particle size and the zeta potential were determined to be 283.3 ± 15 nm and −23.7 ± 2.9 mV, respectively, using a Malvern Instruments Zetasizer Nano ZS. Absorbance measurements were performed using a Cary 60 UV−vis spectrophotometer (Agilent Technologies, Inc., USA). Nuclease-free water was used in the preparation of all solutions. Oligo(ethylene glycol) metilethermethacrylate (OEGMA, Mn = 500), di(ethylene glycol) methyl ether methacrylate (DEGMA), and 4,4′-azobis(4-cyanovaleric acid) (ACVA, initiator) were purchased from Sigma-Aldrich and used as received. Effect of Mismatches on miRNA Detection. The initial fluorescence measurements with target RNA molecules, miR-10b, miR-10a, or SCR, were performed using nanographene oxide (nGO) and FAM (ext: 495 nm, emm: 520 nm) labeled antisense DNA molecules as probe strands (full comp, M1, or M2). The nGO/DNA was prepared by mixing 50 nM probe DNA (full, M1 or M2) with 3 μg/mL nGO in 10 mM Tris-HCl buffer (pH 7.5, 2.5 mM MgCl2, 0.1 mM CaCl2). Briefly, we obtained 50 nM probe DNA by diluting a 1 μM stock solution in the Tris-HCl buffer. After, 1.50 μL of nGO stock solution (200 μg/mL) was added to each 100 μL of 50 nM probe DNA solution. The addition of nGO resulted in immediate fluorescence quenching, with the signal intensity decreased by ∼95%. Observed fluorescence measurements were performed immediately after sample preparation using a microplate reader. Ten minutes after the initial measurements, 2.5 or 5.0 μL of a 1 μM (25 and 50 nM final) stock solution of miR-10b, miR-10a, or SCR was added to nGO/full, nGO/M1, or nGO/M2. A 1.5 h kinetic study was performed to monitor the observed fluorescence and recovery kinetics. The observed fluorescence was obtained with an excitation of 495 nm and an emission of 520 nm. Effect of DNase I on nGO and nGO-PEGMA. To monitor the effect of DNase I alone on nGO and nGO-PEGMA, the nanodevices were prepared as described above. The preparation of PEGMA and functionalization of nGO with PEGMA was performed according to our recent report.31 A more detailed experimental procedure is in the Supporting Information. Briefly, 50 nM (stock solution 1 μM) probe DNA was mixed with 3 μg/mL of either nGO or nGO-PEGMA in 10 mM Tris-HCl buffer (pH 7.5, 2.5 mM MgCl2, 0.1 mM CaCl2) to assemble the nGO/DNA or nGO-PEGMA/DNA, respectively. To each of the 100 μL mixtures of nGO/DNA or nGO-PEGMA/DNA, 0, 2, 5, or 10 μL of a 1 U/μL DNase I stock was added 10 min after initial measurements. This resulted in 0, 2, 5, or 10 U of DNase I activity, and a 1.5 h kinetic study was performed to monitor the observed fluorescence. Effect of DNase I on miRNA Detection Using nGO and nGOPEGMA. For monitoring the effect of DNase I on the miRNA molecules and nGO/M2, the M2 probe was first adsorbed onto the nGO surface and then treated with the miRNA molecules at various concentrations. nGO/M2 was prepared by mixing 50 nM FAM9945

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Figure 1. Detection of miR-10b and miR-10a using graphene nanodevices: (a) Observed fluorescence recoveries in the presence of 50 nM miR-10b, miR-10a, and scrambled miRNA (SCR) using three different graphene nanodevices (nGO/full, nGO/M1, and nGO/M2) after 90 min of incubation. (b) Sequences of miR-10b and miR-10a, which differ by only a single nucleotide at the 12th nucleotide from the 5′ end (underlined) and FAM-labeled probe strands (full, M1, and M2). The highlighted bases (red) are mismatches introduced into the probe strands to increase the specificity. (c) Observed fluorescence kinetic curves with each miRNA using the three different nanodevices. Experiments were performed in triplicate.

base with A (T → A)7, and the third strand (M2) is mutated at the 7th and 20th bases from the 5′ end by replacing the T base with A (T → A)7 and the G base with C (G → C)20, respectively (Figure 1b). The mismatches are separated by 12 bases. Therefore, M1 and M2 have one and two mismatches when forming a heteroduplex with miR-10b, respectively. However, since miR-10a differs from miR-10b by only a single base in the middle of the sequence (12th base from the 5′ end, A → U), it has one, two, and three mismatches with the full, M1, and M2 probes, respectively. We investigated whether graphene oxide can discriminate miR-10b from miR-10a by increasing the number of mutations in the probe strands systematically. First, we assembled fluorescently silent nGO nanodevices using the full, M1, and M2 probes. The fluorescence readings were obtained with 50 and 25 nM miR10b, miR-10a, and scrambled RNA (SCR, used as a negative control strand) using nGO/full, nGO/M1, and nGO/M2 (Figure 1a and Supporting Information Figure S1). As seen in Figure 1a, miR-10b results in the largest fluorescence signal with nGO/full and the least recovery with nGO/M2. This was expected since the increasing number of mismatches at specified positions could reduce the hybridization efficiency and therefore the overall fluorescence recovery. A similar trend was observed with miR-10a; however, the overall observed fluorescence signals with all three nanodevices were less than those for miR-10b. Since the number of mismatches between the probe strands and miR-10a is higher than that of miR-10b, this result was predicted. Furthermore, we believe that because the inserted mismatches are separated by at least four bases from the position of single-nucleotide polymorphism (Figure 1b), the hybridization with miR-10a (23mer) was restricted. Later, the fluorescence recovery was obtained with SCR using all three nanodevices, and the observed final fluorescence signals were similar in all three cases. Since all three probe strands and the SCR are not complementary, hybridization and

Fluorescence Measurements. Fluorescence measurements were performed using a BioTek Synergy H1 microplate reader. To monitor the fluorescence of the FAM during the kinetic measurements through either nonstop reading or end-point reading, the samples were excited at 485 nm and the emission was collected at 520 nm. For each reading, the gain value was set to 100.

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RESULTS AND DISCUSSION In this study, we have used nanosized graphene oxide to detect an exogenous and endogenous breast oncomiR, miR-10b, from liquid biopsy mimics and cell extracts. We have assembled and used water-soluble nanodevices composed of graphene oxide and fluorescently labeled DNA molecules to detect miR-10b and discriminate it from an miRNA, miR-10a, which differs only by a single nucleotide (12th base from the 5′ end). Even though nanosized graphene oxide (nGO) has been used for the detection of short single-stranded RNA or DNA molecules, there are still several challenges remaining. First, target strands with single or double mismatches generate a considerable fluorescence reading due to imperfect duplex formation which results in a lack of specificity. Second, the fluorescence signal is observed with target molecules at concentrations higher than their physiological level; therefore, the nanodevices are not sensitive enough for biomedical applications. Here we have combined multiple approaches to address these challenges by (1) inserting mutations into specific positions on the probe strands to improve specificity, (2) using a highly specific endonuclease to increase the limit of detection, and (3) attaching polymers to the nGO edges to reduce the steady nonspecific increase in background fluorescence caused by the enzymatic step (Scheme 1). First, we assembled three different graphene nanodevices using 3 μg/mL nGO and 50 nM antisense probe strands with variations in their sequences. The first strand (full) is fully complementary to miR-10b, the second strand (M1) is mutated only at the seventh base from the 5′ end by replacing the T 9946

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Figure 2. Monitoring the fluorescence recovery in the presence of miR-10b, miR-10a or SCR, and DNase I using nGO/M2: (a) Schematic representation of the interaction among DNase I, miRNA, and the graphene nanodevice along with the observed fluorescence recovery. (b) Observed fluorescence kinetics with 50 nM miR-10b, miR-10a, SCR, and a blank using only 10 U of DNase I. (c) The observed fluorescence recoveries with and without 2 and 10 U of DNase I after 90 min of incubation. (d) Observed fluorescence intensities with various concentrations of miR-10b with and without 5 U of DNase I after 90 min of incubation. (e) Fluorescent images of microtubes and gel studies demonstrate the formation of DNA:miRNA heteroduplexes and digestion by 10 U of DNase I.

thus a fluorescence increase were not expected. However, more importantly, this result shows that by using the M2 probe, with mutations inserted at (T → A)7 and (G → C)20 positions, even though it decreases the overall signal with miR-10b, the specificity is increased. As seen in Figure 1a (black arrows), miR-10a generates a signal similar to that of SCR, and both of these readings are significantly lower than the readings with miR-10b. The kinetics studies demonstrate that the fluorescence recoveries are observed over time with all three miRNAs using nGO/full, nGO/M1, and nGO/M2 and saturate almost an hour after the addition of the target strands (Figure 1c). The results of the kinetics studies suggest that nGO/M2 has significant discrimination power for miR-10b over miR-10a and is able to differentiate a single nucleotide difference in a 23mer RNA. We further looked into the effect of each probe strand (full, M1, and M2) on fluorescence readings with miR-10a, which needs to be minimized for a greater specificity toward miR-10b (Supporting Information Figure S2). As represented in the scheme, the fluorescence recoveries are observed due to the hybridization-induced desorption of probes from the nGO surface, which leads to the disappearance of fluorescence quenching (Supporting Information Figure S2a). Inserting mismatches (arrows and black circles in the scheme) can decrease the hybridization efficacy between the probe and the RNA molecule or completely remove it and therefore can bring miR-10a induced recovery close to the background signal. We have monitored the fluorescence recovery by miR-10a using nGO/full, nGO/M1, and nGO/M2 and have compared it to that of SCR. As the number of mismatches increases (one, two, and three with full, M1, and M2, respectively), the fluorescence readings approach those with SCR (Supporting Information Figure S2b,c). In the presence of nGO/M2, the readings with miR-10a decreases to that of the scrambled probe, which suggests that the hybridization between miR-10a and M2 is reduced significantly with three mismatches. This further suggests that the M2 probe strand is ideal for increasing the specificity of the graphene nanodevices for miR-10b detection

by discriminating it from a very closely related miRNA in the same RNA family. After demonstrating the improvement of the specificity against miR-10b, we investigated the enhancement of the observed signal intensity of the nanodevice. To do so, we have incorporated the DNase I enzyme into our detection scheme to amplify the fluorescence signal (Figure 2a). DNase I cleaves the phosphodiester linkages in the DNA backbone and degrades the DNA molecules, but not the miRNAs, in the DNA:miRNA heteroduplexes. Therefore, after the hybridization of an miRNA molecule with the fluorescently labeled M2 probes, the M2 probes can be degraded by DNase I, freeing fragmented DNAs and fluorophores and allowing the freed, untouched miRNA to bind with another M2 probe (Figure 2a, arrow 1). This allows for a single miRNA molecule to desorb multiple M2 probes from the surface and thus increases the limit of detection.66 We first performed an experiment with 50 nM miR-10b using the nGO/M2 nanodevice and 10 U of DNase I and compared the results with the readings obtained with miR-10a and SCR. The observed fluorescence kinetic curves demonstrate that the fluorescence increases constantly in the presence of miR-10b at a higher degree and rate when compared to miR-10a and SCR. The fluorescence recovery in the presence of miR-10a was found to be lower than for miR-10b and comparable to that of SCR. This indicates that the M2 probe is ideal for bringing miR-10a-induced release to the level of SCR-induced release, even in the presence of DNase I. However, there is a steady increase in the background fluorescence in the presence of DNase I without any miRNA addition (Figure 2b orange line (blank) and Supporting Information Figure S3). This challenge is due to the fact that DNase I cleaves adsorbed ss M2 probes on the nGO surface and increases the background fluorescence at a constant rate as described in our recent report (Figure 2a, arrow 2).66 Later, we studied the signal amplification with and without two different amounts (2 and 10 U) of DNase I. The observed fluorescence signals are amplified with all three miRNAs as the amount of enzyme increases (Figure 2c and Supporting Information Figure S4). However, this amplification 9947

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Figure 3. Removing the increase in background fluorescence generated by DNase I using nGO-PEGMA: (a) Schematic representation of the degradation of the single-stranded probes by DNase I in the nGO/M2 and nGO-PEGMA/M2 nanodevices. (b) Observed fluorescence intensities with and without 50 nM miR-10b, miR-10a, and SCR and with 10 U of DNase I using nGO/M2. nGO-PEGMA/M2 removes the steady increase in background fluorescence caused by DNase I surface activity (red arrow). (c) Observed fluorescence intensities in the presence of the graphene nanodevices and different concentrations of DNase I using nGO/M2 and nGO-PEGMA/M2. Results indicate that the PEGMA molecules in nGOPEGMA/M2 block DNase I access to the M2 probes. Experiments were performed in triplicate.

miR-10b shows strong fluorescence, and the gel band (II) shows fluorescence of both the miRNA:DNA heteroduplex and ss DNA probe as predicted. On the other hand, in the presence of 10 U of DNase I, the fluorescent image of a microtube containing nGO/M2 with miR-10b shows very strong fluorescence; however, the gel band (III) shows weaker fluorescence of the miRNA:DNA heteroduplex and ss DNA probe. This is expected as fragmented DNAs and fluorophores, digested by DNase I, ran off the gel. This result further indicates that the amplified fluorescence signal in the presence of DNase I is due to the digestion of the probes with the two mechanisms illustrated in Figure 2a. Since DNase I improves the sensitivity (Figure 2a, arrow 1; Figure 2d) while increasing the background fluorescence due to its activity toward the surface-adsorbed probes (Figure 2a, arrow 2), we pursued the removal of this undesired enzymatic background signal increase by preventing DNase I from accessing the surface. In order to achieve that, we have attached PEGMA (poly(ethylene glycol) methyl ether methacrylate) polymers to the nGO edges to introduce the steric hindrance of DNase I (Figure 3a). Briefly, the PEGMA polymers were attached to the nGO edges and the M2 probes

is greater in the presence of miR-10b, 2-fold and 6-fold with 2 and 10 U of enzyme, respectively, using nGO/M2 when compared to miR-10a and SCR. Furthermore, the observed fluorescence with miR-10a and SCR is comparable in all cases, indicating that the nGO/M2 affinity for miR-10a is not significantly different than that of a noncomplementary RNA molecule. Similar studies were performed using nGO/full and nGO/M1; however, a lack of specificity of the nanodevices over miR-10a was observed (Supporting Information Figure S5). Later, various concentrations of miR-10b were tested with nGO/M2, and the limits of detection were determined to be 225 and 861 pM with and without 5 U of DNase I, respectively, (3σ/slope, Figure 2d). Later, we performed gel analysis to further characterize the hybridization-induced and DNase I-amplified fluorescence signals. The fluorescent image of a microtube containing nGO/M2 without any miR-10b addition shows weak fluorescence; however, the gel band (I) shows strong fluorescence for the ss DNA probe alone. This indicates that the probes are released from the nGO surface under the gel electrophoresis experimental conditions (Figure 2e). The fluorescent image of a microtube containing nGO/M2 with 9948

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Figure 4. Detection of miR-10b using nGO-PEGMA/full and nGO-PEGMA/M2: The observed fluorescence kinetic curves with miR-10b and miR10a with and without 5 U of DNase I using (a) nGO-PEGMA/full and (b) nGO-PEGMA/M2. (c) Overall fluorescence recoveries at the end of the 90 min of incubation with DNase I using nGO-PEGMA/full and nGO-PEGMA/M2. (d) Observed fluorescence intensities at the end of the 90 min of incubation with various concentrations of miRNAs using nGO-PEGMA/M2 and DNase I. Experiments were performed in triplicate. (e) Fluorescent images of 50 nM miR-10b and miR-10a in the nGO-PEGMA/M2 and 5 U of DNase I suspension after 90 min of incubation.

were loaded onto the nGO surface. The resulting fluorescently silent nGO-PEGMA/M2 nanodevice quenches the fluorescence of the fluorophore label of M2 by adsorbing the strands on the surface similar to nGO/M2. Studies were performed with 50 nM miR-10b, miR-10a, SCR, and no miRNA (blank) in the presence of 10U of DNase I using nGO/M2. Later, nGOPEGMA/M2 was treated alone under the same conditions in the presence of 10 U of DNase I (Figure 3b). The steady increase in the background fluorescence (orange line) in the blank sample using nGO/M2 was absent in studies using nGOPEGMA/M2 (Figure 3b, dark-blue line). The result shows that the steady increase in background fluorescence due to DNase I activity on the surface-adsorbed probes was removed after the attachment of PEGMA (red arrow). Later, nGO-PEGMA/M2 and nGO/M2 were incubated with various concentrations of DNase I, separately. The M2 probes in nGO/M2 were subject to DNase I cleavage due to the access, and therefore the activity, of the enzyme with respect to the surface-adsorbed M2 probes. Increasing the concentration of DNase I also increased this undesired background fluorescence signal almost 8-fold with 10 U of enzyme (Figure 3c). On the other hand, the surfaceadsorbed M2 probes in nGO-PEGMA/M2 were fully protected from DNase I degradation even after 2 h of incubation. The antifouling property of the PEGMA polymers inhibits DNase I from accessing the surface and cleaving the M2 probes, which was observed by steady weak fluorescence at all three different DNase I concentrations. These observed fluorescence signals were comparable to the inherent weak fluorescence of nGOPEGMA/M2 in the absence of the enzyme. The overall results demonstrate that the background signal generated by DNase I can be eliminated by the conjugation of PEGMA polymers to the nGO template. After demonstrating that we can improve the specificity by the incorporation of mismatches in the probe strands, increase the sensitivity and observed signal intensity with a highly specific endonuclease, and eliminate the undesired enzymatic

activity using PEGMA polymers, we tested nGO-PEGMA/M2 using 50 nM miR-10b and miR-10a with and without enzyme and compared the results to the readings obtained using nGOPEGMA/full. First, miR-10b and miR-10a were tested with nGO-PEGMA/full with and without 5 U of DNase I (Figure 4a). The fluorescence signals with miR-10b and miR-10a are comparable without any enzyme and are drastically amplified in the presence of DNase I. The miR-10b detection was amplified 5-fold and 4-fold with enzyme using nGO-PEGMA/full and nGO-PEGMA/M2, respectively (Figure 4a). However, miR10a acts similarly to miR-10b in hybridizing to the full probe, which makes nGO-PEGMA/full unable to discriminate miR10b from miR-10a (Figure 4a). The recovered fluorescence with miR-10b was only 1.4-fold greater than with miR-10a using nGO-PEGMA/full. On the other hand, when miR-10b and miR-10a were tested with nGO-PEGMA/M2 and DNase I, the miR-10a signal was reduced to the background while miR10b attained a significantly stronger fluorescence signal (6-fold greater than with miR-10a; Figure 4b). The overall final fluorescence recoveries after 90 min of incubation suggest that the M2 probe is remarkable for discriminating miR-10b from miR-10a, DNase I amplifies the signal, and the PEGMA polymers reduce the background signal by blocking the enzyme’s access to the nGO surface (Figure 4c). Later, we studied various concentrations of miR-10b (5, 10, 25, and 50 nM) with nGO-PEGMA/M2 and DNase I and compared the observed fluorescence signals to those of miR-10a and SCR. The miR-10a and SCR strands behave similarly, and the observed fluorescence signals are significantly lower than those of miR-10b (Figure 4d). Later, the limits of detection of nGOPEGMA/M2 were determined to be 0.56 and 1.66 nM for miR-10b with and without 5 U of DNase I, respectively. The fluorescent images of 50 nM miR-10b and miR-10a in the nanodevice and DNase I suspension suggest that the nGOPEGMA/M2 can discriminate a single nucleotide difference, which can be visualized by fluorescence imaging (Figure 4e). 9949

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Langmuir While the steric hindrance from PEGMA prevents DNase I from accessing the surface, it is important to note that it can also restrict the target miRNA from reaching the graphene surface and desorbing the probe strands or DNase I from accessing the heteroduplexes. An observed decrease in fluorescence recovery or specificity with nGO-PEGMA/M2 compared to nGO/M2 could be due to this steric hindrance effect (Figures 4b and 1c). Furthermore, since the PEGMA is a thermoresponsive polymer, the activity of nGO-PEGMA can be tuned with a thermoswitch at its lower critical solution temperature (LCST, 39 °C for this polymer) as demonstrated recently.31 The activity of nGO-PEGMA can be switched on and off by decreasing the temperature below and raising it above the LCST of the PEGMA, respectively. To demonstrate the tunable property of nGO-PEGMA/M2, which provides an additional feature for our nanodevice, we have tested 25 nM miR-10b with nGO-PEGMA/M2 at two temperatures, above and below the LCST, and compared it to nGO/M2. The detection of miR10b can be tuned with nGO-PEGMA/M2 with a thermoswitch but not with nGO/M2 (Figure 5 and Supporting Information

Figure 6. Detection of exogenous and endogenous miR-10b in liquid biopsy mimics and cancer cell extracts. Observed fluorescence intensities with different amounts of total RNAs isolated from (a, b) serum which was spiked with miR-10b and miR-10a with and without 5 U of DNase I and (c) 4T1 and MCF-7 breast cancer cell lines in the presence of DNase I after 90 min of incubation. Experiments were performed in triplicate.

generated fluorescence signals which were almost at the background level. Later, the studies were performed in the presence of DNase I for increased sensitivity. As seen in Figure 6a, the signal with miR-10b is amplified 3-fold after incorporating 250 pg/μL RNA and 5 U of DNase I into the reaction mixture but is significantly lower with miR-10a. The results indicate that our nanodevice can identify miR-10b from a large RNA pool, discriminate it from miR-10a, and amplify the signal in the presence of DNase I. Finally, after demonstrating exogenous miRNA detection, we proceeded to detect endogenously expressed miR-10b from two different cancer cell lines. 4T1 is a highly metastatic breast cancer cell overexpressing miR-10b.10−12 We extracted cellular RNA from 4T1 cells to detect endogenous miR-10b in the total RNA pool from cancer cells. Nonmetastatic MCF-7 breast cancer cells, which do not overexpress miR-10b, were used as a control cancer cell line.11 Various amounts of total RNA extracted from both types of cancer cells were tested with nGOPEGMA/M2 and DNase I. The results demonstrate that the observed fluorescence signals with RNAs from metastatic 4T1 cells are significantly higher than those from MCF-7 at all three concentrations (Figure 6c). The overall results indicate that our diagnostic nGO-PEGMA/M2 nanodevice and DNase I system are able to detect endogenous miR-10b in a mixed RNA pool isolated from metastatic breast cancer cells.

Figure 5. Tunable miRNA detection using nGO-PEGMA/M2 and nGO/M2. The detection capacity of nGO-PEGMA/M2 can be controlled with a thermoswitch at the LCST (39 °C) of PEGMA due to its thermoresponsive properties (red line). Control bare nGO/M2 does not have such tunable properties (green line). Experiments were performed in triplicate.

Figure 6). This introduces an additional functionality into our engineered nanodevice, which, in the future, can enable us to control or avoid the detection at off-target sites in biological systems. After demonstrating that the nGO-PEGMA/M2 and DNase I combination can be used for the detection of isolated miR-10b, we moved forward to detect the miR-10b from biological samples to demonstrate the biomedical potential of our approach. First, the detection of exogenous miR-10b in an RNA pool was demonstrated using total RNA extracted from serum samples. Initially, the serum samples were enriched with miR-10b molecules to mimic a liquid biopsy. In order to determine the specificity of this water-soluble diagnostic nGOPEGMA/M2 toward miR-10b, serum samples enriched with equal concentrations of miR-10a were used for comparison studies. The extracted total RNA was used to determine the miR-10b content in the serum samples using nGO-PEGMA/ M2 alone. The results demonstrated that the observed fluorescence reading increases with increasing amount of total RNA extracted from serum spiked with miR-10b (Figure 6a). The detection limits were determined to be 1.6 and 5.5 pg/μL with and without 5 U of DNase I under these conditions, respectively (3σ/slope, Figure 6b). However, the total RNA extracted from serum which was enriched with miR-10a

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CONCLUSIONS Here we have studied how to address three different challengesspecificity, sensitivity, and an increase in background fluorescencefor the detection of miRNAs using water-soluble graphene oxide nanodevices. We have demonstrated that by the systematic insertion of mismatches, into specific positions of the probe sequences of the graphene oxide and oligonucleotide nanoassemblies, a greater specificity toward a target miRNA, miR-10b, and discrimination down to a single nucleotide difference can be achieved. Furthermore, incorporating a DNA-specific endonuclease, DNase I, into the nanodevice mixture improves the sensitivity while resulting in 9950

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an undesired background signal due to the activity of the enzyme on the surface-adsorbed probes. The attachment of PEGMA polymers eliminated the undesired enzymatic activity and steady increase in background fluorescence by blocking the access of DNase I to the nanodevice surface. Finally, the performance of the diagnostic nanodevice and DNase I combination was tested with exogenous and endogenous miRNAs in liquid biopsy mimics and metastatic breast cancer cells, respectively. This systematic study demonstrates that the challenges for the biomedical application of nGO can be addressed by a combination of several approaches. Though our method was used only to increase the specificity toward miR10b in this work, the same combination of approaches can be extended to detect other miRNAs.66

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.langmuir.5b02026. Additional experimental methods and results (PDF)

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AUTHOR INFORMATION

Corresponding Author

*Tel: (1) 518-442-3002. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank Prof. Prashanth Rangan and Dr. Siu Wah WongDeyrup for their assistance with RNA isolation. We also thank Prof. Zdravka Medarova for providing 4T1 and MCF-7 cell lines and Dr. Irfan Khan for his assistance with cell studies. Finally, we thank Vibhav A. Valsangkar and Amy E. Toscano for their help with gel electrophoresis.

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