Quantitative Detection of MicroRNA in One Step ... - ACS Publications

Jun 27, 2016 - Institute of Blood Transfusion, Chinese Academy of Medical Sciences ... ABSTRACT: One-step, quantitative and rapid detection of microRN...
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Quantitative Detection of MicroRNA in One Step via Next Generation Magnetic Relaxation Switch Sensing Wenjing Lu,†,∥ Yiping Chen,†,∥ Zhong Liu,‡,∥ Wenbo Tang,§ Qiang Feng,† Jiashu Sun,*,† and Xingyu Jiang*,† †

Beijing Engineering Research Center for BioNanotechnology & CAS Key Laboratory for Biological Effects of Nanomaterials and Nanosafety, CAS Center for Excellence in Nanoscience, National Center for NanoScience and Technology, Beijing 100190, China ‡ Institute of Blood Transfusion, Chinese Academy of Medical Sciences (CAMS)/Peking Union Medical College (PUMC), Beijing 100730, China § The Department of Hepatopancreaticobiliary Surgical Oncology, Chinese PLA General Hospital, Beijing 100853, China S Supporting Information *

ABSTRACT: One-step, quantitative and rapid detection of microRNA (miRNA) in tumor cells or tissues can provide critical information for clinical diagnosis and cancer treatment. In this work, we develop a magnetic relaxation switch sensing (MRS)-based miRNA sensor using magnetic microparticle (1 μm in diameter, MM1000)-DNA probemagnetic nanoparticle (30 nm in diameter, MN30) conjugates (MM1000DNA-MN30). In the presence of target miRNA, DSN enzyme selectively cleaves the DNA tether after miRNA/DNA hybridization to release MN30 and leaves the miRNA intact to lead to the declustering of more MN30 than before. In contrast to conventional MRS by measuring the change of transverse relaxation time (ΔT2) induced by the aggregation or dissociation of magnetic particles in the presence of target, we use the cleaved MN30 from conjugates as the direct readout of ΔT2, which is more sensitive and stable. This MRS-based assay allows for one-step detection of 5 fM of miR-21 in urine samples, quantification of miR-21 from 100 cancer cells, and differentiation of the expression of miR-21 in tumor and surrounding tissues. The merits of this assay, rapidity, ability for quantitation, high sensitivity, and one-step operation, ensure a promising future in diagnostic technology. KEYWORDS: miRNA, magnetic relaxation switch, magnetic nanoparticles, detection, tumor

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ting. The qRT-PCR for miRNA sensing is sometimes limited by the use of locked nucleic acid (LNA)-modified primers or the optimization of sequence-specific annealing temperatures. Microarray and Northern blotting require complicated procedures, long assay time, and expensive reagents.11−14 In recent years, alternative techniques with improved sensitivity and selectivity have also been developed, such as hairpinmediated quadratic enzymatic amplification,15 nonenzymatic hairpin DNA cascade amplification,16 bifunctional strand displacement amplification-mediated hyperbranched rolling circle amplification, 17 and hybridization chain reaction amplification combined with tetrahedral DNA probes.18 However, these strategies also involve the dedicated design of

icroRNAs (miRNA) are small, noncoding RNA molecules consisting of approximately 22 nucleotides that are significantly involved in the posttranscriptional regulation of gene expression.1−3 The aberrant expression of miRNA could harbor DNA amplifications or translocations, leading to tumor progression and metastasis. For example, miR-21 is often found overexpressed in various cancers, such as breast, liver, and pancreatic cancers, and associates with the proliferation and migration of cancer cells.4,5 The accurate and rapid detection of the expression level of miRNA in a noninvasive manner has shown great promise for early diagnosis of cancers or monitoring the cancer therapeutic efficacy.6,7 However, the intrinsic characteristics of miRNA such as short sequence, relatively low expression level, and highly susceptible to degradation, pose a challenge for miRNA sensing.8−10 Conventional strategies for detection of miRNA include quantitative reverse transcription polymerase chain reaction (qRT-PCR), oligonucleotide microarray, and Northern blot© 2016 American Chemical Society

Received: March 19, 2016 Accepted: June 27, 2016 Published: June 27, 2016 6685

DOI: 10.1021/acsnano.6b01903 ACS Nano 2016, 10, 6685−6692

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ACS Nano primes or probes and multiple rounds of experimental optimization. Incorporation of nanomaterials for developing a new class of miRNA assays has attracted prominent interest. The combination of graphene oxide-protected DNA probes and a cyclic enzymatic amplification method allows for sensitive detection of miRNA in complex biological samples.19 Due to excellent photochemical stability and high quantum yield, quantum dotbased nanosensor combined with a two-stage exponential amplification reaction enables a high sensitive and specific assay for miRNA.20,21 Other nanomaterials, such as carbon nanotubes,22 nano metal−organic frameworks,23 gold nanoprism,24 and WS2 nanosheets,25 have also been involved for sensing miRNA with reduced assay time and improved sensitivity. Despite the promising applications of nanomaterials for miRNA sensing, most of them either involve the complex fabrication of fine structured nanomaterials or expensive instruments for signal detection. The real need for miRNA sensing, however, is to develop a rapid, one-step, sensitive, quantitative, and robust assay with simple operations and easy readout. To realize the one-step and rapid detection of miRNA, magnetic relaxation switch (MRS) based on magnetic nanoparticles may provide a promising approach.26−29 Compared with fluorescence assays or electrochemical sensing, MRS could significantly simplify the assay steps, reduce the assay time, increase the assay stability, and enable near background-free sensing. Due to these advantages, we develop a MRS sensor combined with magnetic separation for one-step, rapid, and sensitive immunodetection of pathogens and viruses.30 In addition to immunodetection, MRS has been applied for detection of short pieces of mRNA, which could hybridize with superparamagnetic nanoparticles and result in the change in magnetic relaxivity.31 MRS is also capable of sensing molecular interactions using functionalized magnetic particles.32 We should note that the change of transverse relaxation time in conventional MRS is induced by the state change of magnetic particles (aggregation or dissociation), which may not response to the target in some cases and lead to the low sensitivity. Moreover, the application of MRS for miRNA detection is still lacking. In this work, we present a MRS-based strategy for quantitative detection of miRNA using magnetic microparticle-DNA probe-magnetic nanoparticle conjugates (MM1000-DNA-MN30) and duplex-specific nuclease (DSN) enzyme in one step. In the presence of target miRNA, DNA in formed DNA-RNA heteroduplexes could be specifically cleaved by DSN, resulting in the release of MN30 and an enhancement of ΔT2. This direct assay allows for quantitative and sensitive detection of miR-21 in urine samples and tumor cells, without the conversion of miRNA to cDNA, nor the multiple assay steps.

Figure 1. Schematic of microRNA (miRNA) detection in one step using magnetic microparticle (1 μm, MM1000)-DNA probemagnetic nanoparticle (30 nm, MN30) conjugates (MM1000-DNAMN30). (a) The target miRNA first hybridizes with complementary DNA probe sandwiched between M1000 and MN30 to form DNARNA heteroduplexes. DNA in heteroduplexes could be specifically cleaved by duplex-specific nuclease (DSN), while the target miRNA and MN30 will be released into the solution. The target miRNA remains intact and involves in the next round of target-recycling amplification (TRA), leading to the release of more MN30. (b) The released MN30 remains suspended in supernatant under a magnetic field, resulting in the increased ΔT2 that correlates with the amount of target miRNA.

(TRA), as well as the cleavage of more MN30 into solution. Due to this DSN enzyme-assisted TRA, a small amount of miRNA could result in the release of considerable amounts of MN30 from conjugates. The cleaved MN30 gives rise to a significant change of transverse relaxation time (ΔT2) of supernatant, which is measured by a 1.5 T cute nuclear magnetic resonance spectrometry. Preparation and Characterization of the MM1000-DNAMN30 Conjugates. To fabricate the MM1000-DNA-MN30 conjugates, we first modify the DNA probes (amine modified on 5′ and biotin modified on 3′) onto the surface of carboxylic cabrodyrate-modified MM1000 via 1-ethyl-3-[3(dimethylamino)propyl] carbodiimide hydrochloride (EDC) activation and reaction. The streptavidin-modified MN30 is conjugated onto MM1000/DNA probe complex by biotin− avidin interaction (Figure 2a). The prepared MM1000-DNAMN30 conjugates are characterized by transmission electron microscopy (TEM). In comparison with MM1000 of smooth surface, we observe several black dots on the surface of MM1000DNA-MN30 conjugates (Figure 2b). The black dots of around 30 nm in diameter are MN30 conjugated onto the surface of MM1000/DNA probe complex. The quantification result indicates that one MM1000 can conjugate with 303 of small MN30 after modification (Table S1). We next evaluate the separation efficiency of small MN30 from large MM1000, as we choose ΔT2 of the water molecules in the presence of MN30 as the signal readout after separation. We monitor the separation speed of MM1000 and MN30 under the same magnetic field (0.01 T) in a time period of 30 min. We find that the large MM1000 could be rapidly accumulated toward the vial wall within 3 min under 0.01 T due to its high saturation magnetization, whereas MB30 is well dispersed even

RESULTS AND DISSCUSIONS Principle of miRNA Detection by MRS. To develop a MRS-based miRNA assay, we adopt the MM1000 (1 μm in diameter, MM1000)-DNA-MN30 (30 nm in diameter, MN30) conjugates for miRNA hybridization. The formed miRNA/ DNA heteroduplexes sandwiched between MM1000 and MN30 will immediately become the substrate for duplex-specific nuclease (DSN) enzyme digestion, resulting in the release of miRNA and MN30 into solution (Figure 1). The released miRNA could bind to another MM1000-DNA-MN30 conjugate and initiate a next round of target-recycling amplification 6686

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The difference of ΔT2 between MM1000 and MN30 within 30 min also proves the different magnetic separation speeds between MM1000 and MN30 under the same magnetic field of 0.01 T. This result indicates that we can efficiently separate the released MN30 from MM1000 under 0.01 T after the cleavage of miRNA/DNA heteroduplexes by DSN enzyme. The correlation between the ΔT2 value and the concentration of MN30 is investigated, by measuring the ΔT2 of water spiked with different concentrations of MN30. A linear relationship between ΔT2 (92−1545 ms) and logarithmic concentration of MN30 from 1.35 × 107 to 1.35 × 1011 mL−1 is observed, and the linear equation is y = 388.1x − 2726.9 (x = lg c[MN30], R2 = 0.964, Figure S1). We thus adopt the ΔT2 value of supernatant containing MN30 as the signal readout, since the amount of cleaved MN30 from MM1000-DNA-MN30 conjugates is related to the concentration of miRNA during DSN enzymeassisted TRA. Although previous studies have designed MRS nanoassemblies for screening of DNA-cleaving agents, these methods still measure the T2 based on the state change between nanoassembly and dispersed nanoparticles.33 In comparison, our approach first separates the small magnetic nanoparticles from large ones and measures the ΔT2 of small magnetic nanoparticles as the signal readout, which is more sensitive and stable. Optimization of MRS-Based miRNA Assay. We further optimize the experimental conditions for miRNA detection by MRS sensing. The design feature of our miRNA assay is the use of DSN enzyme that selectively cleaves the DNA tether after miRNA/DNA hybridization and leaves the miRNA intact to lead to the declustering of more MN30. After magnetic separation, ΔT2 of declustered MN30 in supernatant is measured for quantitative determination of miRNA. To achieve a high sensitivity of detection, the temperature of reaction, the concentration of DSN enzyme, and the reaction time are optimized (Figure 4a−c). The reaction temperature will influence the hybridization efficiency between the DNA probe and target miRNA as well as the activity of DSN enzyme. The hybridization temperature higher than Tm (temperature of half dissociation of DNA/miRNA) leads to an ineffective hybridization, and a low hybridization temperature may result in the nonspecific hybridization.34 In most cases, the optimal hybridization temperature should be 5−10 °C lower than Tm (53 °C). In our experiments, after TRA enabled by DSN at different constant temperatures (40−60 °C) for 60 min, a maximum value of ΔT2 (574 ms) is obtained at 45 °C, while a higher or lower temperature yields the decreased ΔT2 due to the inefficient hybridization between DNA and miRNA or reduced activity of DSN enzyme (Figure 4a). We next evaluate the optimized concentration of DSN enzyme which is 0.5 U (Figure 4b). As to the optimization of reaction time, the value of ΔT2 increases gradually with the prolonged reaction time and reaches a plateau after 90 min reaction with the presence of 1 nM of miRNA (Figure 4c). The saturated reaction indicates that no more small magnetic nanoparticles could be cleaved off from large nanoparticles after 90 min. For later time points such as 105 and 120 min, the value of ΔT2 does not differ from that at 90 min. We therefore choose 45 °C as the temperature of reaction, 0.5 U as the concentration of DSN enzyme, and 90 min as the reaction time for the following miRNA detection. Quantitative Detection of miR-21. Quantitative detection of miR-21 spiked in buffer (Tris-HCl) under these optimized conditions (45 °C, 0.5 U DSN enzyme, and 90 min)

Figure 2. (a) Schematic of assembly of MM1000-DNA-MN30 conjugates. (b) TEM images of MM1000 before modification (left) and MM1000-DNA-MN30 conjugates after modification (right). The small black dots (indicated by yellow arrows) on the surface of MM1000 are MN30.

after 30 min under the same magnetic field because of the low saturation magnetization (Figure 3a). We also measure the ΔT2 value of supernatant from MM1000 or MN30 solution at different time points under 0.01 T (Figure 3b). For the supernatant from large MM1000, the ΔT2 decreases dramatically from 1250 to 200 ms within 3 min as a result of precipitation of MM1000 under a magnetic field. In comparison, the ΔT2 of MN30 supernatant keeps almost constant (∼1250 ms) in a time period of 30 min.

Figure 3. (a) Photograph of the MM1000 solution and MN30 solution under a magnetic field (0.01 T) for different times. The MN30 is well dispersed after 30 min, while the MM1000 is precipitated toward the vial wall within 3 min under the same magnetic field. The concentrations of MM1000 and MN30 are both 1 mg/mL. (b) Measurement of ΔT2 of supernatant of MM1000 or MN30 solution at different time points under 0.01 T. 6687

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Figure 4. Optimization of (a) reaction temperature, (b) concentration of DSN enzyme, and (c) reaction time for MRS-based miRNA detection. The concentration of miRNA is 1 nM. (d) The quantitative detection of miR-21 in Tris-HCl buffer under the optimized parameters. Error bars indicate standard deviation of three replicates of different samples executing the entire assay.

water, Tris-HCl, saline, urine, saliva, serum, and blood (Figure S2). Compared with the water, matrix effects of Tris-HCl, saline, and urine are negligible, given the similar T2 values in these matrices. In contrast, the matrix effects of saliva, serum, and blood are evident, as these matrices lead to a decreased T2 value than water. To determine the percent recovery, we spike a certain amount of miR-21 (10 fM, 500 fM, 1pM, 10 pM and 100 pM) into urine and measure the ΔT2 value after reaction. Based on the linear response between the quantity of miR-21 and the ΔT2 value, we can obtain percent recoveries (109.27%, 99.12%, 107.28%, 101.45% and 99.52%, in that order) of miR-21 detection in urine by MRS (Figure S3). Moreover, a good linear response for miR-21 detection (5 fM to 0.5 nM) in urine is observed, with the LOD of 4.55 fM (R2 = 0.998, Figure S4). To evaluate the specificity of miRNA assay, we detect three different kinds of miRNAs that differ from each other by only one or two nucleotides (Table S2) using MM1000-DNA-MN30 conjugates in which DNA probe is complementary to miR-21. The measurement of ΔT2 indicates that the presence of miR-21 (1 nM) in urine yields a maximum value of ΔT2, while the ΔT2 value for single-base mismatched miRNA (SmiR-21, 1 nM) or two-base mismatched miRNA (MmiR-21, 1 nM) is much lower than that for miR-21 (Figure S5). These results impressively demonstrate an excellent percent recovery and a high specificity of our one-step MRS-based assay for miRNA detection in urine. miRNA Detection by MRS in Cancer Cells and Tissues. The MRS-based assay is used for quantification of the expression level of miR-21 in four different cell lines (PC-3, A375, HepG2 and MCF-7). Total miRNA is extracted from each cell line, which is directly added into the reaction mixture and incubated at 45 °C for 90 min for miR-21 hybridization with DNA probes and DSN-enabled cleavage of MN30 from conjugates (details in Supporting Information). The quantification of miR-21 from different cells is realized by measuring the ΔT2 of supernatant containing released MN30 after reaction (Figure 5a). The MRS measurement reveals that the amount of

is performed by our MRS-based assay. The target miR-21 could hybridize to its complementary DNA probe in MM1000-DNAMN30 conjugates, followed by DSN digestion of formed DNA/ miRNA heteroduplex, leading to the separation of MN30 from conjugates under a magnetic field (0.01 T). The amount of released MN30 in supernatant is related to the concentration of target miRNA. The measurement of ΔT2 of supernatant after 90 min reaction shows a good linear response for miR-21 in the range from 5 fM to 0.5 nM (R2 = 0.995, Figure 4d) in Tris-HCl buffer. The limit of detection (LOD) is evaluated from the calibration curve at a signal equal to the average response of blank sample (no miRNA) plus 3 times the standard deviation. For miR-21 detection in buffer (Tris-HCl), the LOD is 3.36 fM based on the linearity plot of ΔT2 versus logarithmic concentration of miR-21 (ΔT2 of blank sample is 0 ms, and standard deviation is 5.2 ms). Previous target valency study shows that single-digit fM sensitivity is possible but only with high valency analytes.36 For a bivalent target like miRNA (one miRNA connects with two magnetic nanoparticles by two ends), the sensitivity of 3.36 fM is difficult to achieve by conventional MRS. In our miRNA assay, the key design elements are the magnetic separation for target concentration, enzymatic amplification by the nuclease, and the inherent sensitivity of the MRS design. These elements attribute to an improved sensitivity for miRNA detection. Compared with DSN-assisted miRNA detection using the florescence as the readout that has a linear detection range of 5−200 pM and LOD of 5 pM,35 our MRS-based miRNA detection exhibits a dramatically enhanced linear detection range and LOD. Moreover, we can obtain the quantity of target miRNA by measuring the value of ΔT2 of supernatant after one-pot reaction at 45 °C for 90 min, while the quantification of miRNA by florescence recovery/quenching requires real-time monitoring of fluorescence intensity during reaction. Matrix Effect, Percent Recovery, and Specificity of MRS-Based miRNA Assay. To investigate the matrix effects, we evaluate the baseline value of T2 in seven matrices including 6688

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Figure 5. (a) Detection of the expression level of miR-21 in different cell lines (PC-3, A375, HepG2, and MCF-7). (b) The linear response between the ΔT2 value and miR-21 extracted from 102−107 of MCF-7 cells. Error bars indicate standard deviation of three replicates of different samples executing the entire assay.

Figure 6. (a) Measurement of the absolute amount of miR-21 in MCF-7 cells by our MRS-based sensor and qRT-PCR. (b) Quantification of the expression of miR-21 in excised liver tumor and surrounding tissue by our MRS-based sensor and qRT-PCR. Error bars indicate standard deviation of three replicates of different samples executing the entire assay.

miR-21 in PC-3 cells (877 copies of miR-21 per 100 PC-3 cells) is significantly lower than that in MCF-7 (3.27 × 105 copies of miR-21 per 100 MCF-7 cells), HepG2 (2.57 × 105 copies of miR-21 per 100 HepG2 cells), and A375 cells (4.88 × 105 copies of miR-21 per 100 A375 cells). This difference in the expression of miR-21 in MCF-7 and PC-3 cells is also observed in previous studies using the PCR method for miRNA detection.37 This MRS-based assay exhibits a good linear response between the ΔT2 value and the amount of miR-21 extracted from different concentrations of MCF-7 cells (102− 107) (Figure 5b). Moreover, a good concordance of our method and qRT-PCR is obtained for qualification of miR-21 from different levels of MCF-7 cells (Figure 6a). Our MRSbased sensor is further adapted for detecting the expression of miR-21 in excised liver tumor and surrounding tissue (Figure S6, details in Supporting Information). The abundance of miR21 in tumor is about three times higher than that in normal tissue, agreeing well with the qRT-PCR result (Figure 6b). This result demonstrates that the quantification of miR-21 in tissues can provide critical information for clinical diagnosis of cancers.

proportional to the amount of target miRNA. The major reason why this design allows high sensitivity is the recycling of target miRNA assisted by DSN enzyme, which allows the smallest amounts of 5 fM of miR-21 to be detected. This one-pot assay could also quantify the expression level of miR-21 in 100 cancer cells and differentiate the abundance of miR-21 in tumor and surrounding tissues. The versatility of this MRS-based assay shows great promise in nucleic acid analysis and early clinical diagnosis.

METHODS Materials. Magnetic microparticles (1 μm in diameter, MM1000) with surface modification of carboxylic acid were purchased from Invitrogen Corporation (NY, USA, catalog number: 65011). Magnetic nanoparticles (30 nm in diameter, MN30) with surface modification of streptavidin were obtained from Ocean Nanotech (catalog number: SHS-30−05). Duplex specific nuclease kit (DSN, Evrogen Russia, catalog number: evrogen-EA002), including DSN enzyme and 10× DSN buffer, was purchased from Evrogen Joint Stock Company. HPLC-purified miRNAs including target miRNA (miR-21) and base mismatched miRNA (MmiR-21 and SmiR-21) were provided by Takara Biotechnology Co., Ltd. (Dalian, China). DNA oligonucleotides probe, with an amino-modified 5′ end and a modification of 3′ end by biotin, was synthesized by Sangon Biotech (Shanghai, China). Base sequences of miR-21, MmiR-21, SmiR-21, and DNA probe were listed in Table S2. Diethy pyrocarbonate (DEPC) treated water was used in the preparation of aqueous solutions. All other chemicals were all of analytical grade and used without pretreatment, unless mentioned otherwise. Assembly of MM1000-DNA-MN30 Conjugates. The coupling of oligonucleotide probe to MM1000 was carried out using a protocol provided by Invitrogen Corporation with small modifications. One mL

CONCLUSIONS In conclusion, we present a MRS-based assay for rapid, sensitive, quantitative, and one-step detection of miRNA with straightforward operations and signal readout, which could fit into the needs for miRNA detection. In the presence of target miRNA, the small MN30 could be cleaved off from MM1000DNA-MN30 conjugates after miRNA/DNA hybridization and DSN digestion. In contrast to conventional MRS that measures the change of ΔT2 from the state change of magnetic particles (aggregation or dissociation) induced by the target, we use the cleaved MN30 in supernatant for direct readout of ΔT2 which is 6689

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ACS Nano of MM1000 solution (10 mg/mL) was transferred into a 1.5 mL centrifuge vial and washed three times with 1 mL of MES buffer (2(N-morpholino) ethanesulfonic acid, pH 4.8) under a magnetic field. The washed MM1000 was resuspended in 100 μL of MES buffer. To decorate the oligonucleotide probe (amine modified on 5′ and biotin modified on 3′) onto the surface of MM1000, we first solved oligonucleotide probe (50 nM) into 40 μL of N-ethyl-N′-(3(dimethylamino)propyl) carbodiimide hydrochloride (EDC), which was subsequently mixed with MM1000 solution and vigorously vortexed for 10 s. The mixed solution was incubated overnight on a roller mixer at room temperature to achieve an efficient coupling between the probe and MM1000. The solution was washed three times with 1 mL of TT buffer (TE buffer with 0.05% Tween-20) to remove the uncoupled probe, and the remaining MM1000-DNA probe conjugates were resuspended in 1 mL of TE buffer. We then added 0.1 mL of streptavidin-modified MN30 into MM1000-probe conjugates solution, followed by 2 h incubation at 37 °C for self-assembly of the MM1000DNA-MN30 conjugates. The prepared MM1000-DNA-MN30 conjugates are characterized by TEM (Tecnai G2 20 S-TWIN, FEI, USA, 200 kV). Characterization of the Number of MN30 onto the Surface of MM1000. To quantify the number of MN30 modified onto the surface of MM1000, we measured the initial concentrations of MN30 (CIMN30) and MM1000 (CMN1000) and the residual concentration of MN30 (CRMN30) after coupling with MM1000. Nitric acid was used to digest the MN30 into Fe ions. The content of Fe was determined by inductively coupled plasma-optical emission spectrometry (ICP-OES, Thermo ICAP 6300). The amount of MN30 modified onto per MM1000 is calculated using the following formula:

allowing for the target miRNA recycling assisted by DSN. After the reaction, we applied a magnetic field (0.01 T) for 3 min to separate the released MN30 from MM1000-DNA-MN30 conjugates. The transverse relaxation time (T2) of supernatant containing MN30 was measured by a 1.5T cute nuclear magnetic resonance spectrometry (NMR, Shanghai Shinning Globe Science and Education Equipment Co., Ltd., China). To perform miRNA detection in urine samples, we add 2 μL of 10 × DSN buffer and 10 μL of MM1000-DNA-MN30 conjugates into 18 μL of urine. Specificity of MRS-Based miRNA Assay. The specificity test was performed using three different kinds of miRNAs that differ from each other by only one or two nucleotides (miR-21; single-base mismatched miRNA, SmiR-21; two-base mismatched miRNA, MmiR-21). Base sequences of miR-21, SmiR-21, and MmiR-21 are listed in Table S2. The concentration of each kind of miRNA used in the mismatch experiments was 1 nM. Cell Culture and miRNA Extraction. The human melanoma cell line (A375), human prostate adenocarcinoma cell line (PC-3), human liver hepatocellular carcinoma (HepG2), and human breast cancer cell line (MCF-7) were cultured in Dulbecco’s modified Eagle’s medium (DMEM) with 10% fetal bovine serum (FBS) and 1% penicillin/ streptomycin at 37 °C, 5% CO2 inside an incubator (Thermo Scientific). Total RNA was extracted from each cell line using TRIzol agent (Invitrogen, Carlsbad, CA) according to the kit protocol. Compare the MRS with qRT-PCR. Total RNA was extracted from each cell line using miRNA extraction kit TRIzol agent (Invitrogen, Carlsbad, CA) according to the kit protocol. For miR21 detection by our MRS-based assay, the extracted miRNA (10 μL) was directly added into 20 μL of mixture containing 10 μL of MM1000DNA-MN30 conjugates, 2 μL of 10 × DSN buffer (50 mM Tris-HCl, pH 8.0, 5 mM MgCl2, and 1 mM DTT), 1 μL of 0.5 U DSN in 25 mM Tris-HCl (pH 8.0) and 50% glycerol, 7 μL of DEPC-treated water. The reaction was conducted in a thermal cycler at 45 °C for 90 min. For miR-21 detection by qRT-PCR, we first converted miRNA into cDNA at 37 °C for 120 min (including the label of the 3′ with poly(A) tail, and subsequent reverse transcription) using the reverse transcription kit (Tiangen Biotechnologies Co., Ltd.). The obtained cDNA was amplified using the SYBR Green real-time PCR detection kit (Tiangen Biotechnologies Co., Ltd.). A titration experiment with serial dilutions of cDNA converted from miRNA (known concentrations) was performed to obtain the linear regression of threshold cycle versus the logarithmic plot of miRNA concentration (Figure S7). Using this standard curve, we could calculate the concentration of miRNA (unknown concentration) after obtaining its threshold cycle by realtime PCR. miRNA Extraction and Detection from Tumor/Tissue Samples. The snap-frozen specimens of liver tumors and surrounding tissues were received from the General Hospital of People’s Liberation Army (301 hospital) after obtaining the patient-informed consent. Total RNA was isolated from these excised samples by TRIzol agent (Invitrogen, Carlsbad, CA), and the quality of the RNA was assessed with a NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies, Wilmington, DE). The extracted RNA was used directly for the MRS-based miRNA detection.

NMM1000/NMN30 = [CMM1000 × VMM1000 × C TMN30 × (rMN30)3 ] /[C IMN30 × VMN30 × (rMM1000)3 × (C IMN30 − C RMN30)] where VMM1000 and VMN30 are the volumes of the MM1000 and MN30 solution, rMN30 and rMM1000 are the radii of MN30 and MM1000, and NMM1000 and NMN30 are the number of MN30 and MM1000. Optimization of Temperature of Reaction. To achieve the best sensing performance, we studied the effect of temperature of reaction. Under the same concentration of target miRNA and DSN enzyme as well as the reaction time (1 nM of miR-21, 60 min, 1 U DSN), different temperatures (40, 42, 45, 47, 50, 52, 55, 57, 60 °C) were used to carry out the experiments (three replicate tests of different samples at each temperature). The mixture for reaction (total volume of 20 μL) consists of 10 μL of MM1000-DNA-MN30 conjugates, 2 μL of 10 × DSN buffer (50 mM Tris-HCl, pH 8.0, 5 mM MgCl2, and 1 mM DTT), 1 μL of 1 U DSN in 25 mM Tris-HCl (pH 8.0) and 50% glycerol, 7 μL of DEPC-treated water. After adding 10 μL of miR-21 (1 nM), the solution was incubated in a thermal cycler for 60 min. After the reaction, we applied a magnetic field (0.01 T) for 3 min to separate the released MN30 from MM1000-DNA-MN30 conjugates. The ΔT2 after reaction was recorded to obtain an optimized temperature for MRS-based miRNA sensor. Optimization of the Concentration of DSN Enzyme. Under the following conditions (1 nM of miR-21, 60 min reaction, and 45 °C), we optimized the concentration of DSN enzyme ranging from 0.05 to 0.7 U with three replicates of different samples for each DSN concentration. Optimization of the Reaction Time. Under the following conditions (1 nM of miR-21, 0.5 U DSN, and 45 °C), we optimized the reaction time from 0, 15, 30, 45, 60, 75, 90, 105, and 120 min, while each reaction time had three replicate tests of different samples. Detection of Target miRNA. The mixture for miRNA detection (total volume of 20 μL) consists of 10 μL of MM1000-DNA-MN30 conjugates, 2 μL of 10 × DSN buffer (50 mM Tris-HCl, pH 8.0, 5 mM MgCl2, and 1 mM DTT), 1 μL of 0.5 U DSN in 25 mM Tris-HCl (pH 8.0) and 50% glycerol, 7 μL of DEPC-treated water. After adding 10 μL of miRNA of different concentrations into 20 μL of mixture, the solution was incubated in a thermal cycler at 45 °C for 90 min,

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsnano.6b01903. Details about the properties of MM1000-DNA-MN30 conjugates (Table S1), nucleotide sequences of miRNA (Table S2), and characterizations and performance of MRS-based miRNA sensor (Figures S1−S7) (PDF)

AUTHOR INFORMATION Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. 6690

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These authors contributed equally.

Notes

The authors declare no competing financial interest.

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DOI: 10.1021/acsnano.6b01903 ACS Nano 2016, 10, 6685−6692