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Feb 12, 2018 - In the presence of low abundance of miRNA target, the target triggered exponential amplification, producing a large quantity of stem-G-...
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Strand Displacement Amplification Reaction on Quantum DotEncoded Silica Bead for Visual Detection of Multiplex MicroRNAs Xiaojun Qu, Haojun Jin, Yuqian Liu, and Qingjiang Sun Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.7b05235 • Publication Date (Web): 12 Feb 2018 Downloaded from http://pubs.acs.org on February 12, 2018

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Analytical Chemistry

Strand Displacement Amplification Reaction on Quantum Dot-Encoded Silica Bead for Visual Detection of Multiplex MicroRNAs Xiaojun Qu, Haojun Jin, Yuqian Liu, and Qingjiang Sun* State Key Laboratory of Bioelectronics, School of Biological Science & Medical Engineering, Southeast University, Nanjing 210096, China ABSTRACT: The combination of microbead array, isothermal amplification and molecular signaling enables the continuous development of next-generation molecular diagnostic techniques. Herein we reported the implementation of nicking endonuclease-assisted strand displacement amplification reaction on quantum dotsencoded microbead (Qbead), and demonstrated its feasibility for multiplexed miRNA assay in real sample. The Qbead featured with well-defined core-shell superstructure with dual-colored quantum dots loaded in silica core and shell, respectively, exhibiting remarkably high optical encoding stability. Specially designed stem-loop-structured probes were immobilized onto the Qbead for specific target recognition and amplification. In the presence of low abundance of miRNA target, the target triggered exponential amplification, producing a large quantity of stem-G-quadruplexes, which could be selectively signaled by a fluorescent G-quadruplex intercalator. In one-step operation, the Qbeadbased isothermal amplification and signaling generated emissive “core-shell-satellite” superstructure, changing the Qbead emission-color. The target abundance-dependent emission-color changes of the Qbead allowed direct, visual detection of specific miRNA target. This visualization method achieved limit of detection at sub-femtomolar level with linear dynamic range of 4.5 logs, and point-mutation discrimination capability for precise miRNA analyses. The array of three encoded Qbeads could simultaneously quantify three miRNA biomarkers in ~500 human hepatoma carcinoma cells. With the advancements in easy operation, multiplexing and visualization capabilities, the isothermal amplification-on-Qbead assay could potentially enable the development of point-of-care diagnostics.

interest due to its inherent simplicity, stability and specificity, and have developed into many subtypes, such as polymerase-initiated SDA,13-17 nicking endonuclease (NEase)-assisted SDA (NASDA),18-21 circular SDA,22-24 toehold-mediated SDA,25 ligationmediated SDA,26 quadratic amplification27 and cascade amplification.28 These linear/exponential SDA reactions enable the quantification of specific miRNAs at femtomolar to attomolar levels, point-mutation discrimination capability and good performances comparable to RT-PCR in real samples. The SDA also has high compatibility, and its combination with functional particles such as magnetic particles, gold nanoparticles and graphene oxide has generated various biosensor methods. By making use of optical, electronic and magnetic properties of functional particles, colorimetric,29-31 fluorescent,32,33 electronic,34-36 and surface-enhanced Raman spectroscopy37 assays have been implemented for nucleic acid analysis with greater sensitivity. Despite the promising progress in sensitivity and specificity, the molecular probe/biosensor-based SDA methods lack large-scale multiplexing capability, and scalability. Oligonucleotide microarrays have provided highthroughput and multiplexing platforms for miRNA detection, but have limitations such as medium sensitivity, low specificity at the 5’-end miRNA and high-cost operations. In recent years, encoded microbeads based suspension arrays have emerged as alternative detection platforms to conventional microarrays for multiplexed bioassays, with the advantages of better reproducibility, greater sensitivity,

INTRODUCTION MicroRNAs (miRNAs) are a group of small, noncoding RNAs consisting of 18-25 nucleotides that are significantly involved in the post-transcriptional regulation of gene expression.1 Recently, accumulating evidence indicates that the aberrant expression of miRNAs is associated with cancer initiation, tumor stage, and tumor response to treatments.2 Thus, miRNAs have been considered as promising biomarker candidates for clinical diagnosis, prognosis and therapy.3 The biological significance has led to high demand in detection methods that enable efficient and accurate quantification of miRNAs. However, the unique characteristics of miRNAs make their precise analysis quite challenging. On the one hand, high sensitivity is a prerequisite since absolute abundance or expression variation of specific miRNA is extremely low.4 On the other hand, multiplexing capability is equally important since one gene could be regulated by multiplex miRNAs and vice versa.5 Reverse transcription polymerase chain reaction (RTPCR) and more rapid, cost-effective isothermal amplification reactions have continuously developed as powerful methods for sensitive bioassays.6 Among the reactions, isothermal strand displacement amplification (SDA) first devised by Walker and coworkers7 has found wide applications, due to its easy operation and high amplification efficiency. The SDA has high adaptability, and linear probe,8 three-way junction structured probe9,10 and stem-loop structured probe11,12 could be implemented for SDA reaction. In particular, the stemloop probe-based SDA reactions have gained intense 1

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faster reaction kinetics and cost-effectiveness.38 For the microbeads, optical encoding stands out to be an efficient approach owing both to their flexible encoding and convenient decoding.39 The Luminex has invented the organic dyes-encoded microbead array termed as xMAP array, which can simultaneously detect up to 100 analytes using one fluorescent tag.40 Quantum dots (QDs) are another class of representative fluorophores for wavelength and intensity encoding.41 The QDsencoded microbeads (Qbeads) hold significant advantages in terms of encoding capacity, photobleaching threshold and sensitivity over organic dyescoded ones.42,43 The Qbead array has thus been widely used for multiplexed detection of proteins and genomic DNAs.44,45 Notably, although a few of trials on miRNA profiling were reported by using the microbead arrays,46-48 their sensitivities are not sufficient for direct detection of ultralow abundance of miRNA biomarkers in real samples. Alternatively, Biscontin et al.49 reported target PCR pre-amplification followed by xMAP array method for miRNA detection, which however has a compromise regarding specificity, assay time and risk of carryover contamination. Therefore, it is still in urgent demand in advancing the microbead arrays for direct, sensitive, and multiplexed detection of miRNAs with easy operations. In this work, we report for the first time the implementation of NEase-assisted SDA reaction on Qbead (Qbead-NASDA) for direct, visual detection of multiplex miRNAs in cell lysate. Three encoded Qbeads featured with core-shell superstructure were generated and used to identify three miRNA biomarkers including miRNA-21, miRNA-221 and miRNA-16: miRNA-21 and miRNA-221 are upregulated, and miRNA-16 is downregulated in human hepatoma carcinoma cells.50,51 Step-loop-structured probes were specifically designed to have 6 functional regions: the 5’-linker for conjugation to Qbeads, the miRNA recognition sequence, the NEase recognition site, the sequence complementary to primer, the G-rich sequence, and the 3’-primer (Table S1 in the Supporting Information). In the absence of miRNA target, the probe on Qbead has adopted a step-loop structure with 3’-primer overhang, inactive to NASDA (Scheme 1). In the presence of miRNA target, the target (T) recognition triggers structural reformation of the probe: the 3’primer is annealed to form the stem with the nick at the NEase recognition site; the G-rich sequence forms intramolecular G-quadruplex (GQ). The nicked stemGQ structure becomes responsive to NASDA. Once initiated, the NASDA amplifies the miRNA target exponentially by two cycles of amplification reaction: the KF polymerase extends the 3’-primer and displaces the target in the first cycle, and the NEase recognizes the sequence-specific nicking site and produces the miRNA target analogue (T*) in the second cycle. In a series of cyclic chain reactions, the NASDA produces a large abundance of (nicked) stem-GQ structures on the Qbead. With a GQ-selective fluorescent intercalator, bis-2,2’-bipyridyl(dipyrido[3,2-a:2’,3-c]phenazineimidazolone)ruthenium (II) (Ru-DI), to signal the GQs, the Qbead-NASDA has generated an emissive “coreshell-satellite” superstructure. This superstructure,

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changing emission-color of the Qbead, can be used for miRNA quantification by common fluorescence microscopy. This newly developed visualization method can detect the miRNA targets at sub-femtomolar level with linear dynamic range of 4.5 orders of magnitude. By this method simultaneous detection of three miRNA biomarkers in cell lysate is simplified to observe distinct emission-color changes of three encoded Qbeads. Moreover, the Qbead-NASDA assay is performed in one step, featuring with low cost and easy operation.

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Scheme 1. Illustration of proposed Qbead-NASDA assay for visual detection of multiplex miRNA biomarkers in crude cell lysate.

EXPERIMENTAL SECTION Materials and reagents. Tetraethyl orthosilicate (TEOS, 99.999%), ammonia (NH3·H2O, 28 wt%), 1-(3dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC, 99%), N-hydroxysuccinimide (NHS), Bovine serum albumin (BSA) and immunoglobulin G (IgG) were purchased from SigmaAldrich. Ethanol (AR) was purchased from J&K. (3Aminopropyl) triethoxysilane (APS, 97%), cis-Bis(2,2'bipyridine)dichlororuthenium(II) dihydrate (cisRu(bpy)2Cl2·2H2O) and ammonium hexafluorophosphate (NH4PF6, 99.5%) were received from Alfa Aesar. (3-mercaptopropyl)trimethoxysilane (MPS, 96%) was purchased from TCI. Klenow fragment (KF) polymerase and Nt.BbvCI NEase were purchased from New England Biolabs Ltd. The deoxynucleotide triphosphates (dNTPs) were obtained from Takara (Dalian, China). β-actin and proteinase K (PK) were purchased from Thermo Fisher. The miRNA targets, probes and molecular beacon were synthesized by Invitrogen (Shanghai, China), and their sequences are listed in Table S1 in the Supporting Information. The pure water (18.2 MΩ cm) was obtained from a Pall Cascade AN synthesis system. Preparation of encoded Qbeads. Oil soluble bQDs (ZnCdS@ZnS, EM: 431 nm) and water soluble MPAcapped gQDs (CdSe@ZnS, EM: 516 nm) were synthesized via greener chemistry method.52 The gQDdoped silica microspheres (gQD@SiO2) were first prepared through a modified Stöber method.53 Typically, 400 μL of gQDs were dispersed in 3.7 mL of ethanol/H2O followed by addition of 1 μL of APS with 2

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Analytical Chemistry abundance of miRNA target, 0.05 U μL−1 of KF, 0.4 U μL−1 of Nt.BbvCI, 250 µM of dNTPs and 20 µM of Ru-DI were added, and the reaction mixture was kept gentle shaking for 90 min. Fluorescence microscopy measurements. The miRNA detection was performed via epi-fluorescence microscopy with the excitation wavelength of 405 nm. For the sensitivity experiments, varying abundance of each of the three miRNA targets were subjected to Qbead-NASDA and signaling followed by fluorescent imaging. For the specificity experiments of singleplex detection, each of the three miRNA targets, and 21-mut (point-mutation sequence of miRNA-21) (50 pM for each miRNA) were incubated with Qbead 0:1 in amplification buffer, respectively, and subjected to NASDA and signaling followed by imaging. For the specificity experiments of multiplexing, 7 combinations of one, two or three of the three miRNA targets (50 pM for each target) were incubated with the array of three encoded Qbeads (the number of each Qbead was equal), respectively, and subjected to NASDA and signaling followed by imaging. Calculation of chromaticity coordinates. For emission-color analysis of Qbead, the XYZ tristimulus values and the (x,y) chromaticity coordinates were calculated with the following equations:

stirring for 12 h. Then 900 μL of NH3·H2O and 450 μL of TEOS were added, and the mixture was left to react for 2 h. Further, the Qbeads were constructed by thiolfunctionalization of gQD@SiO2, conjunction with bQDs and second silication.54 Schematically, 2.5 µL of MPS and 30 µL of NH3·H2O were added into 1 mL of gQD@SiO2 in ethanol, and the mixture was stirred for 12 h. The products were re-dispersed in 5 mL of toluene followed by addition of 200 μL of bQDs (1 mg mL-1), and the mixture were stirred for 20 min. Afterwards, the obtained gQD@SiO2@bQD particles were re-dispersed in 1 mL of ethanol, followed by adding 30 μL of NH3·H2O and 75 µL of TEOS. After stirred for 12 h, the Qbeads (gQD@SiO2@bQD@SiO2) were collected with centrifugation. For further bioconjugation, the Qbeads were subjected to successive surface modifications: first silanized with APS to introduce amine groups and coupled with succinic anhydride to obtain carboxyl groups.55 By tuning the amounts of bQDs in shells, the Qbeads with different blue-to-green intensity ratios were facilely prepared. Preparation of Qbead-probe. Prior to bioconjugation, the probes were annealed at 95 °C for 2 min followed by gradual cooling down and standing at 25 °C for 2 h to obtain the desired stem-loop structure. The bioconjugation was via the carbodiimide reaction between carboxyl group on Qbead and 5’-amino group of the probe. Typically, 1 mg of Qbeads were suspended in 1 mL of 2-morpholinoethanesulfonic acid buffer (pH 6.0), followed by addition of 10 μL of 50 mg mL-1 of EDC and NHS, respectively, with stirring for 0.5 h. Then 10 μL of probes (10 μM) was added with stirring for another 3 h. The bioconjugates were purified with washing buffer (10 mM Tris-HCl, 0.01% Tween, 200 mM NaCl, pH 7.4), and re-suspended in amplification buffer (20 mM Tris-HCl, pH 8.0, 100 mM KCl, 10 mM (NH4)2SO4, 2 mM MgSO4, 0.1% Triton X-100). Synthesis of Ru-DI. The Ru-DI was synthesized following an established procedure.56 Typically, 0.1 g of cis-Ru(bpy)2Cl2·2H2O and 0.1 g of DI were mixed and refluxed for 6 h in 20 mL of glycol-water (7:1, v/v). Upon cooling, the reaction mixture was diluted with 20 mL of water and filtered. The product was precipitated by addition of 1 g of NH4PF6, and then purified by alumina chromatography. 1H NMR [(CD3)2SO]: δ 11.67 (2H, s), 9.61 (2H, d), 8.89 (4H, dd), 8.22 (4H, m), 8.14 (2H, t), 8.01 (2H, t), 7.85 (2H, d), 7.75 (4H, d), 7.61 (2H, t), 7.39 (2H, t). Gel electrophoresis. 2% agarose gel and 0.5 × TBE buffer (4.5 mM Tris–HCl, 4.5 mM boric acid, 0.1 mM EDTA, pH 7.9) were used. GelRed was used as the fluorescent duplex intercalator. Electrophoresis was performed at a constant potential of 80 V for 45 min with loading of 5 µL of each sample into the lanes. The samples for NASDA were prepared by incubating 1 µM of probe, 50 pM of target, 0.05 U μL−1 of KF, 0.4 U μL−1 of Nt.BbvCI and 250 µM of dNTPs in 20 μL of amplification buffer at 37 °C for different time. The sample of 1 µM of probe hybridized with 50 pM of miRNA target was also prepared for comparison. NASDA and signaling on Qbeads. The NASDA and signaling were performed in 100 μL of amplification buffer at 37 °C. At optimized condition, certain

0.31 0.20   R   X  0.49  Y  = 1  0.17697 0.81240 0.01063 G    0.17697     Z   0.00 0.01 0.99   B

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where R, G and B values were directly obtained from the fluorescent images by ImageJ software. Direct and multiplexed detection. Varying number of HepG2 cells (10-105 cells) were lysed, respectively, with the lysing buffer. The cell lysates were incubated with the array of three encoded Qbeads in amplification buffer, respectively, for 90 min at 37 °C. The Qbead array was imaged followed by emissioncolor analysis. RT-PCR experiments were also performed for comparison. Spike/recovery experiments. Spike/recovery experiments were performed by spiking 0.05, 0.5 and 5 pM of three synthetic miRNA targets, respectively, into cell lysates (from 104 cells), followed by incubating with the Qbead array in amplification buffer. For each sample, the Qbead array was imaged followed by emission-color analysis. The recovery rate (%) was calculated as (measured level)/[(endogenous level)+(spiked level)] × 100%. Instruments. Transmission electron microscopy (TEM) images were taken on JEOL JEM-2100 microscope. Fluorescence images were taken by Olympus IX73 inverted fluorescence microscope. Confocal fluorescence images were acquired on Leica TCS SP8 confocal microscope. Gel electrophoresis was scanned by Imageio-Rad imaging system (Hercules, CA). RT-PCR was performed on StepOnePlusTM Real time3

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Analytical Chemistry PCR system. Fluorescence spectra were collected with F-7000 fluorescence spectrometer (Hitachi, Japan).

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Figure 1. (A) TEM images of Qbead preparation: (a) gQD, (b) gQD@SiO2, (c) gQD@SiO2@bQD, and (d) gQD@SiO2@bQD@SiO2. Scale bar: 100 nm. (B) Blue-togreen emission intensity ratios and confocal fluorescence images of three encoded Qbeads. Scale bar: 0.5 μm.

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Core-shell-superstructured Qbeads for encoding miRNAs. The Qbeads were constructed by silica core doping with green-emssive QDs (gQDs) followed by surface conjugation with blue-emissive QDs (bQDs), and encapsulation with outer silica shell, each of which was investigated in detail. As shown in Figure 1A, the formed gQD@SiO2 core by Stöber method had an average diameter of ~386 nm, with a smooth surface. The subsequent surface conjugation enabled multiple bQDs be loaded onto one gQD@SiO2, forming the coresatellite structure. By second silication, smooth outer silica shell was coated to encapsulate the bQDs at the surface of gQD@SiO2 core. The formed Qbead had the average diameter increased to be ~480 nm, indicating that the outer silica shell was ~47 nm thick. To encode three miRNA targets, Qbeads with blue-to-green emission intensity ratios of 0:1, 1:1 and 2:1 were prepared, simply by tuning the amount of bQDs in the surface conjugation process. Figure 1B shows microscopic fluorescent images of the three encoded Qbeads. Under excitation at 405 nm, the three Qbeads exhibited welldefined emissive core-shell superstructure: green core and blue shell with varying brightnesses. Because of the respective loading in core and shell, possible energy transfer between dual-colored QDs were eliminated. Simultaneously, the silica core/shell shielded the doped QDs from the (bio-)environment. Therefore, the superstructured Qbeads featured with high fluorescent encoding stability. Indeed, it was found that encoding signal of the Qbead was tolerant of a wide range of pH (6-12) and reaction time, and also insensitive to various crosslinking agents for bioconjugation, enzymes and dNTPs for NASDA, and the signaling molecule (Figure S1 in the Supporting Information). As such, the encoded Qbeads were amenable to conjugate with the respective probes for recognizing specific miRNA targets. The immobilized probes per Qbead were estimated to be ~9100 strands that are sufficient for NASDA at the bead surface (Fig. S2 in the Supporting Information).

Figure 2. (A) Structure of stem-loop probe and its reformation upon target recognition. (B) Gel electrophoresis (lanes: M: 20-bp DNA ladder; 1: probe (1 μM); 2: probe (1 μM)/target (50 pM); 3-6: NASDA products of probe (1 μM)/target (50 pM) with amplification time of 30, 60, 90, 120 min, respectively). Stem-loop probe-based, target-triggered NASDA. The probe structure was essential to target recognition and NASDA. Being ~100-base long, the probe spontaneously self-assembled into multiple secondary structures (Figure S3 in the Supporting Information). To get the favorable stem-loop structure, the probes were subjected to typical thermal denaturation and annealing process. By this process, the probes had predominantly adopted the stem-loop structure (Tm=48.5℃, ΔG = -12.9 kcal mol-1) as shown in Figure 2A. This was confirmed by fluorescence resonance energy transfer based molecular beacon (MB) tecnnique. The main fragment of the probe (wothout 5’-linker and 3’-primer) were labeled with FAM and DABYCL at two ends, respectively, and labeled with Cy3 at 20-poitioned thymine base in the region of miRNA recognition sequence. As shown in Figure S4 in the Supporting Information, the pristine MBs exhibited intense Cy3 emission with weak FAM emission. At 95 °C, intense dual emissions from Cy3 and FAM were observed, indicating that all the MBs were denatured. After annealing, the Cy3 emission remained intense while the FAM emission was fully quenched by DABYCL. In comparison with the observed FAM emssion of pristine MBs, the fully quenched FAM emission indicated the reduction of unfavorable secondary structures and the predominance of favorable stem-loop structure. For the stem-loop-structured probes, the miRNA recognition sequence (red color) located at the loop region, ready to recognizing the target. The G-rich 4

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sequence (blue color) was locked in the stem, inactive to the Ru-DI intercalating. The 3’-primer overhang and its complementary sequence (green color) were far separated from each other by the stem, inactive to the polymerase extension. The NEase recognition site (purple color) was mismatched, inactive to the nicking. When miRNA target was recognized, the probe underwent structural reformation, and formed nicked stem-GQ (ΔG = -38.1 kcal mol-1). The GQ portion facilitated the Ru-DI intercalating. The 3’-primer annealed with its complementary sequence, responsive to the polymerase extension and target displacement. Once extended, the newly synthesized GCTGAGG sequence (fully matched with the NEase recognition site) was responsive to specific nicking by the NEase. Therefore, the stem-loop probe-based NASDA could only be triggered by the target, offering high specificity for the miRNA assay. Once triggered, the stem-loop probe-based NASDA amplified miRNA target exponentially and produced a large quantity of (nicked) stem-GQs. The amplification time was optimized by using gel electrophoresis (Figure 2B). Without amplification, the recognition of low abundance of miRNA target (lane 2) did not cause the changes in band migration and brightness with respect to the probe only (lane 1). In contrast, with the amplification (the time extended from 30 min to 90 min), there are gradual lags observed in band migration, and gradual increase in band brightness (lanes 3-5), owing to the production of increased quantity of stemGQs. At 120 min, the band migration and brightness (lane 6) was observed to be nearly identical to that of 90 min (lane 5), indicating that 90 min was sufficient for the stem-loop probe-based NASDA, which was further used in the Qbead-NASDA assays.

mRNA. Accordingly, the Ru-DI could selectively signal the stem-GQ-structured product of target-triggered NASDA rather than the stem-loop-structured probe where the G-rich sequence was locked (Figure 3A). With the selective “light switch” property, the Ru-DI was used to signal target recognition and NASDA events in the Qbead-NASDA assay. As shown in Figure 3B, the Ru-DI signal increased with the extended time of Qbead-NASDA until it reached the maximum at 90 min. This result is in high coincidence with that of gel electrophoresis for the free probe-based NASDA, indicative of high amplification efficiency of the NASDA on Qbead. The time-dependent Qbead-NASDA was further confirmed by microscopic fluorescent imaging. Strikingly, emissive “core-shell-satellite” superstructures were observed: 30 min of target-triggered NASDA on Qbead (Qbead 1:1) generated a few of red “satellites” on green core-blue shell; 60 min of amplification significantly increased the number of red “satellite”; 90 min of amplification expanded the “satellites” to a red circle around the green core-blue shell (Figure 3B).

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Figure 4. (A) Fluorescent images of the Qbead-NASDA assay for miRNA-21 detection. Scale bar: 2 μm. (B) CIE chromaticity diagram showing color shifts of three encoded Qbeads with varying abundance of miRNA targets. (C) Relationship between the color-shift distance and the target abundance. The error bars represent standard deviation of three replicated duplicates.

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Visual miRNA detection by the Qbead-NASDA assay. The target abundance-dependent NASDA generated varying numbers of red “satellite” on emissive core-shell, which could continuously change the emission-color of Qbeads, was used for visual detection of miRNA targets. Figure 4A displays fluorescent images of Qbead 0:1 as a function of miRNA-21 abundance in the Qbead-NASDA assay. In the absence of the target, the Qbead was green emissive (green coredark shell). In the presence of increased abundance of miRNA-21, the Qbeads exhibited continuous emissioncolor changes from green to light green and then to

Molecular “light switch” and emissive “core-shellsatellite” superstructure. The fluorescence of Ru-DI behaved as a molecular “light switch”: it was nonfluorescent in buffer solution; upon intercalating into GQ, it emitted intense red fluorescence (Fig. S5 in the Supporting Information). Moreover, the Ru-DI “light switch” had high selectivity against nucleic acids: it was also nonfluorescent in the presence of single/double-stranded DNAs and secondary structured 5

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Analytical Chemistry Figure 5. (A) Fluorescent images of the Qbead-NASDA assay by the presence of miRNA-21, 21-mut, miRNA-221, miRNA-16 and their mixture, respectively, and histogram on the color-shift distance of Qbead 0:1. Scale bar: 2 μm. (B) Fluorescent images of the Qbead arrayNASDA assay toward 7 combinations of three miRNA targets. Scale bar: 2 μm. Symbols of “+/-” from left to right: the presence/absence of miRNA-16, miRNA-221 ” and and miRNA-21, respectively. Red symbols of “○”, “△ “☐ ” highlight color changes of Qbeads 2:1, 1:1 and 0:1, respectively. (C) Histogram on the color-shift distances of three encoded Qbeads in (B)

yellow. The visual detection of miRNA-16 and miRNA221 was also facilely achieved by the Qbead-NASDA assay (Fig. S6 in the Supporting Information). In contrast, without NASDA, the emission-color changes of Qbeads were not readily detectable in the presence of low abundance of miRNA targets (Fig. S7 in the Supporting Information). These results demonstrate that NASDA-based target/signal amplification played the key role in visualization of miRNAs. To quantify the emission-color change of Qbeads, their (x,y) chromaticity coordinates were calculated by Eqns. 1-3 (experimental section), and plotted in the CIE chromaticity diagram as shown in Figure 4B. The green core (gQD), blue shell (bQD), and red “satellite” (Ru-DI) composed a RGB color triangle. With the increased abundance of miRNA targets, chromaticity coordinates of three encoded Qbeads shifted bitonically toward that of Ru-DI, respectively. For each of the Qbeads, the chromaticity coordinates composed the calibration curve that could quantify specific miRNA target. To further quantitatively determine linear dynamic range (LDR) and limit of detection (LOD), the relationships between the color-shift distance (square root of the sum of Δx square and Δy square, Δx and Δy are shift distances of chromaticity coordinate in the x axis and the y axis, respectively) and the abundance of three miRNA targets were plotted (Figure 4C). LDRs of 4.5 logs (1 fM-50 pM) were defined, and LODs of ~0.4 fM were estimated, indicative of high sensitivity of the Qbead-NASDA assay. The sensitivity was comparable to other SDA assays based on magnetic microbeads and nanoparticles (Table S2 in the Supporting Information). Distinctly, the visual detection of Qbead-NASDA assay offered a simple and direct quantification method for miRNA analysis with respect to the intensity-based assay methods. A

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Specificity of the Qbead-NASDA assay. The specificity of miRNA detection was first evaluated by challenging Qbead 0:1 (for identifying miRNA-21) with identical abundance of three miRNA targets, 21-mut (point-mutation sequence of miRNA-21), and their mixture, respectively. As shown in Figure 5A, only the presence of miRNA-21 target (miRNA-21 only or the mixture) changed emission-color of the Qbead from green to yellow. The presence of miRNA-221, miRNA-16 or even 21-mut could not cause obvious emission-color change of the Qbead. Quantitatively, the color-shift distance by the presence of miRNA-21 target was 22 times of those by miRNA-221/miRNA-16, and 7 times of that by 21-mut, demonstrating high specificity of the singleplex assay. The specificity of multiplexed detection was further evaluated by challenging an array of three encoded Qbeads with 7 combinations of three synthetic miRNA targets, where identical abundance of one, two or three of the three targets were presented, respectively. The blank amplification buffer was used as the negative control. As highlighted in Figure 5B, the presence of the miRNA target(s) caused distinct emission-color change(s) of the encoded Qbead(s): the presence of miRNA-16 changed the emission-color of Qbead 2:1 from blue to pink (circle); the presence of miRNA-221 changed the emission-color of Qbead 1:1 from cyan to white (triangle); the presence of miRNA-21 changed the emission-color of Qbead 0:1 from green to yellow (square). As shown in Figure 5C, the color-shift distance of each Qbead by the presence of the target only was identical to those by the presence of the target coexisted with one or two other miRNAs, indicative of the high specificity in multiplexing. This high specificity was primarily ascribed to stem-loop structure of the probes, which governed the specific target recognition. In addition, the NASDA exponentially amplified the RuDI signal and consequently improved the detection specificity. The anti-interference capability was also evaluated by spiking proteins/proteinase such as BSA, β-actin, PK and IgG, respectively, into the amplification buffer, for Qbead-NASDA assays. It was found that the interference from nonspecific adsorption of the protein/proteinase was negligible: with/without spiking 7 μM of the protein/proteinase, 50 pM of miRNA-221 target consistently shifted the emission-color of Qbead 1:1 from cyan to white (Figure S8 in the Supporting Information). Clearly, the good specificity and antiinterference ability enables the Qbead-NASDA assay for

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the endogenous three miRNA targets in each cell lysate sample were quantified (Figure 6C). For all samples, abundances of the endogenous three miRNA targets varied, and miRNA-21 ranked the highest. In 500 cells, the abundances were quantified to be 4.4×103 copies for miRNA-16, 4.9×103 copies for miRNA-221, and 5.5×105 copies for miRNA-21, respectively, paralleling the data by standard RT-PCR (4.7×103, 5×103 and 6×105 copies, respectively) (Figure S9 in the Supporting Information). To further evaluate detection accuracy of this multiplexed assay, spike/recovery experiments were performed by challenging the Qbead array with HepG2 cell lysates (from 104 cells) spiked with 0.05, 0.5 and 5 pM of three synthetic miRNAs, respectively. By the visual detection method, abundances of the three miRNA targets in each spiked sample were quantified. The calculated recovery rates ranged from 96.4 to 103.3% (Table S3 in the Supporting Information), indicative of high accuracy of the Qbead-NASDA assay.

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In summary, a robust, sensitive and multiplexed assay method was developed for miRNA detection by taking the advantages of Qbead, isothermal amplification and molecular signaling. The silica core-shell-based Qbead offered high optical encoding stability and biocompatibility. The stem-loop probe-based exponential amplification offered high specificity and amplification efficiency on Qbead. The molecular signaling offered simplicity and specificity. In combination, one-step operation of amplification and signaling on the Qbead generated emissive “core-shellsatellite” superstructure, which enabled visual detection of miRNA with high sensitivity and specificity. Facilely by the visual detection, a Qbead array could simultaneously quantify multiplex miRNA biomarkers in crude cancer cell lysates without the need of tedious RNA isolation. Together with low cost and easy operation, their capabilities of multiplexing and visual detection facilitate the translation of isothermal amplification-on-Qbead assay to point-of-care molecular diagnostics.

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Figure 6. (A) Fluorescent images of the Qbead-NASDA assay in crude lysates of varying number of HepG2 cells. Symbols of “○”, “△ ” and “☐ ” highlight color changes of Qbeads 2:1, 1:1 and 0:1, respectively. Scale bar: 2 μm. (B) Histogram on the color-shift distances of three encoded Qbeads in (A). (C) Quantification of three miRNA biomarkers in HepG2 cell lysates. Direct detection of multiplex miRNA biomarkers in crude cell lysate. The Qbead array-NASDA assay was used to quantify endogenous miRNA targets in HepG2 cells. The assays were performed by direct lysing of varying number of HepG2 cells followed by incubating with the Qbead array in amplification buffer, respectively (Scheme 1). As shown in Figure 6A, With the increased cell number (10-105 cells), the Qbeads exhibited distinct emission-color changes: Qbead 2:1 for identifying miRNA-16 shifted its color from blue to pink; Qbead 1:1 for identifying miRNA-221 shifted its color from cyan to white; Qbead 0:1 for identifying miRNA-21 shifted its color from green to yellow. The correlation of emission-color changes of the three encoded Qbeads with the cell number was further analyzed. As shown in Figure 6B, the LDR for Qbead 2:1 and Qbead 1:1 was 500-105 cells, while that for Qbead 0:1 was 10-105 cells. This result suggested that 500 cells were the LOD for quantitatively profiling the three miRNA biomarkers by the Qbead array-NASDA assay. Following by analysis with the calibration curves in Figure 4C, abundances of

ASSOCIATED CONTENT Supporting Information DNA/miRNA sequences, Qbead stability, structural simulation and characterization of probe, bioconjugation, selectivity of Ru-DI signal, images of Qbead assay with/without NASDA, anti-interference capability of Qbead-NASDA assay. This material is available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION Corresponding Author *Fax: (86) 25-83792349. Email: [email protected]

ACKNOWLEDGEMENT This work is financially supported by NSFC (Grants: 21775021, 21545006, 21375015) and priority discipline development program of Jiangsu province. The authors 7

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thank Dr. Q.Y. Ge for valuable discussions in the probe design and characterization.

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