Near-Infrared Ag2S Quantum Dots-Based DNA Logic Gate Platform for

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Near-Infrared Ag2S Quantum Dots-Based DNA Logic Gate Platform for miRNA Diagnostics Peng Miao, Yuguo Tang, Bidou Wang, and Fanyu Meng Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.6b01044 • Publication Date (Web): 01 Jul 2016 Downloaded from http://pubs.acs.org on July 5, 2016

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Analytical Chemistry

Near-Infrared Ag2S Quantum Dots-Based DNA Logic Gate Platform for miRNA Diagnostics Peng Miao,*,†,‡ Yuguo Tang,*,†,‡ Bidou Wang,† and Fanyu Meng†

† Suzhou Institute of Biomedical Engineering and Technology, Chinese Academy of

Sciences, Suzhou 215163, P. R. China ‡ University of Chinese Academy of Sciences, Beijing 100049, P. R. China

*

Corresponding authors. Tel.: +86-512-69588279.

E-mail addresses: [email protected] (P. Miao) and [email protected] (Y. G. Tang) 1 ACS Paragon Plus Environment


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Abstract Dysregulation of miRNA expression is correlated with the development and progression of many diseases. These miRNAs are regarded as promising biomarkers. However, it is challenging to measure these low abundant molecules without employing time-consuming radioactive labelling or complex amplification strategies. Here, we present a DNA logic gate platform for miRNA diagnostics with fluorescence outputs from near-infrared (NIR) Ag2S quantum dots (QDs). Carefully designed toehold exchange-mediated strand displacements with different miRNA inputs occur on a solid-state interface, which control QDs release from solid-state interface to solution, responding to multiplex information of initial miRNAs. Excellent fluorescence emission properties of NIR Ag2S QDs certify the great prospect for amplification-free and sensitive miRNA assay. We demonstrate the potential of this platform by achieving femtomolar level miRNA analysis and the versatility of a series of logic circuits computation.

Keywords: DNA logic gate; miRNA; nanostructures; near-infrared fluorescence; quantum dots.

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INTRODUCTION miRNAs are an abundant class of noncoding small RNAs which consist of 18 to 25 nucleotides.1 These molecules are critical cogs in various biological processes, which play an important role in gene regulation.2 They can be assembled into an RNA induced silencing complex (RISC), suppressing target mRNA expression and causing reporter gene silencing.3 The discovery of stable miRNAs in peripheral blood have made these short-sequence nucleic acids as promising diagnostic biomarkers for several diseases.4 For instance, miRNA profiles may reflect the developmental lineage and differentiation state of tumors.5 Co-delivery of miRNA and drugs in a polymeric hybrid micelle demonstrates great potential in cancer therapy.6 These findings emphasize the need to make miRNA measurement a routine part of medical diagnostics.7-9 Unfortunately, the intrinsic characteristics of miRNA like small size, high sequence homology among family members and extremely low abundance in testing samples always raise great challenge to analytical methods.10 Common methods for the quantitative detection of miRNAs mainly rely on northern blotting, qRT-PCR

and

microarray-based

assays,

which

involve

wash-intensive

or

reagent-expensive processes.11-13 Currently, to meet the urgent demands for miRNA expression analysis, a variety of alternative approaches with pretty good performances have been developed,14-18 such as novel scanometric miRNA array profiling,19 electrochemical methods,20-22 fluorescent biosensors,23-25 nanopore sensors,26-27 droplet digital PCR protocols,28 surface plasmon resonance or fluorescence imaging measurements.29-31 Although these methods provide high sensitivity and multiplex 3 ACS Paragon Plus Environment


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analysis, some drawbacks still need to be resolved including long processing time, complicated equipment for fluid or temperature controls, risk of sequence bias arising from amplification procedures, etc. In the last three decades, programmable and flexible DNA has been engineered to form two- and three-dimensional nanostructures according to Watson-Crick base-pairing principle for various applications.32 Logic systems applying this fascinating material as a building element can perform multiplex information processing at a molecular level, which has been received great attention and developing rapidly.33 However, DNA logic gate is rarely applied in clinical diagnostics due to the low concentration of analyte in biological samples and certain signal interferences in heterogeneous systems. Here, we have carefully designed a convenient, amplification-free DNA logic gate platform for miRNA diagnostics based on the principle of enzyme-free toehold exchange-mediated strand displacements.34 Simultaneous detection of multiplex low abundant miRNA inputs are achieved by recording fluorescence from Ag2S quantum dots (QDs) labeled on displaced DNA strands. Ag2S QDs belong to near-infrared (NIR) nanomaterials, which are promising candidates for bioimaging and biosensing.35-36 The synthesized QDs possess the properties of high biocompatibility, minimal autofluorescence, negligible tissue scattering in NIR region and bright photoluminescence with exceptionally large stokes shift, which are demonstrated to be excellent fluorescent probes for clinical diagnostics applications.37 In this study, we have developed an ingeniously designed DNA logic gate platform for miRNA diagnostics, taking advantages of the excellent 4 ACS Paragon Plus Environment

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properties of NIR Ag2S QDs.

EXPERIMENTAL SECTION Chemicals and Instruments Cysteine,

AgNO3,

Na2S·9H2O,

NaOH,

ethylenediaminetetraacetic

diethypyrocarbonate acid

1-ethyl-3-[3-dimethylaminopropyl]carbodiimide

(DEPC), (EDTA),

hydrochloride

(EDC),

N-hydroxyosuccinimide (NHS), hexaammineruthenium (III) chloride ([Ru(NH3)6]3+) were purchased from Sigma-Aldrich (USA). Fetal bovine serum was from Hangzhou Sijiqing Biological Engineering Material Co., Ltd. (Hangzhou, China). Quant One Step qRT-PCR Kit was purchased from TIANGEN Biotech Co., Ltd. (Beijing, China). All other chemicals were of analytical grade and used as received without further purification. Water used to prepare all solutions was purified by a Millipore system (18 MΩ·cm resistivity) and then treated with DEPC. Oligonucleotides were synthesized and purified by Sangon Biotech Co., Ltd. (Shanghai, China). miRNAs were from Takara Biotechnology Co., Ltd. (Dalian, China). The sequences with corresponding names for the logic gates were listed in Table S1. EYES, EAND, EOR, EAND* and EOR* were thiolated at the 5’ end. PYES, P2AND, P1OR, P2OR, P3AND*, P1OR*, P2OR* and P3OR* were aminated at the 5’ end. All DNA probes were prepared in 10 mM phosphate buffer (pH 7.4) containing 0.1 M NaCl. Fluorescence measurements were carried out by using a Hitachi F-4600 fluorescence spectrophotometer (Hitachi, Japan). Transmission electron microscopic 5 ACS Paragon Plus Environment


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(TEM) and high-resolution transmission electron microscopic (HRTEM) images were acquired from a FEI Tecnai G20 transmission electron microscopy (FEI, USA) with an accelerating voltage of 200 kV. UV-vis spectra were recorded by an Agilent Cary 300 Scan UV-vis absorption spectrophotometer (Agilent Technologies, USA). Fourier transform infrared (FTIR) spectra were acquired from an Agilent Cary 660 FTIR spectrometer (Agilent Technologies, USA). Fluorescence images were taken by an infrared thermal imager (Fluke Ti55FT, USA). ABI 7500 Real-Time PCR System (ABI Life Technologies, USA) was used for qRT-PCR experiments. Electrochemical experiments were conducted on a CHI 660D electrochemical workstation (CH instruments, China). A three electrode system was used throughout the experiments. Briefly, the reference electrode was a saturated calomel electrode (SCE), the counter electrode was a platinum wire electrode and the modified gold electrode was the working electrode. Chronocoulometry (CC) was conducted in 10 mM Tris−HCl with 50 µM [Ru(NH3)6]3+ (pH=7.4). The pulse period was 250 ms. Square wave voltammetry (SWV) was conducted in 20 mM Tris-HCl (pH 7.5). Synthesis of Ag2S QDs and Determination of Fluorescence Quantum Yield A facile synthesizing method for Ag2S QDs was described as follows. First, all deionized water used was deoxygenated by pumping nitrogen for 10 min. Second, 90 mg cysteine was dissolved in 75 mL H2O and the pH was adjusted to 7. Third, 42.5 mg AgNO3 was added to the above solution and the pH was also adjusted to 7. Afterward, Na2S solution was prepared by dissolving 50 mg Na2S·9H2O in 25 mL H2O and the solution was slowly added to the mixture of cysteine and AgNO3. The 6 ACS Paragon Plus Environment

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pH value was readjusted to 7. The solution was then heated to 100 °C under vigorous mechanical stirring for 4 h. After it was cooled to room temperature, the solution was placed in a bag filter for 24 h dialysis. The as-prepared Ag2S QDs could be further modified with aminated DNA probes. DNA-Ag2S QDs conjugates were achieved by interacting Ag2S QDs with carboxyl group activation solution (200 mM EDC and 50 mM NHS) for 30 min and then 5 µM DNA probes for another 1 h. Fluorescence quantum yield (QY) measurements of the as-prepared Ag2S QDs were carried out according to a previously established method.38 Quinine sulfate was used as a standard which was dispersed in 0.1 M H2SO4. QY of Ag2S QDs was calculated by comparing the integrated fluorescence peak intensities and the absorbency values with those of quinine sulfate. Six concentrations of both Ag2S QDs and quinine sulfate were prepared with their absorbance less than 0.1. The corresponding fluorescence peak intensities were recorded for the calculation. Gold Electrode Modification The substrate electrode was a disk gold electrode (2 mm in diameter), which was cleaned according to the protocol as previously reported.39 It was firstly dipped in piranha solution (98% H2SO4/30% H2O2 = 3:1) for 5 min (Caution: Piranha solution dangerously attacks organic matter!) and rinsed with double-distilled water. Then, the electrode was polished on microcloth (Buehler) with alumina suspensions (1, 0.3, 0.05 µm), each for 5 min. Residual alumina powder could be removed by sonicating in ethanol and then water for 5 min, respectively. Next, it was electrochemically cleaned with 0.5 M H2SO4 to remove any remaining impurities on the electrode 7 ACS Paragon Plus Environment


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surface. After being dried with nitrogen, the electrode was immediately incubated with thiolated DNA probes for 6 h and then 1 mM MCH for 30 min. Logic Gate Operations and NIR Fluorescence Emission Spectra Measurement For YES gate, the electrode was modified with EYES, which was then incubated with 1 µM PYES-Ag2S QDs for 30 min to achieve double-stranded DNA with a toehold region. Later, the electrode was immersed in the sample containing miRNA input at room temperature for 30 min, during which strand displacement induced PYES-Ag2S QDs release occurred.40 For two- and three-input AND gates, the electrodes were modified with EAND and EAND*, respectively. The electrodes were then incubated with DNA probe mixtures (P1AND and P2AND-Ag2S QDs / P1AND*, P2AND* and P3AND*-Ag2S QDs) for 30 min. Afterward, samples containing miRNA inputs were used to initiate successive strand displacements and QDs release (30 min). For twoand three-input OR gates, the electrodes were modified with EOR and EOR* instead. The DNA probe mixtures in the second step were also changed (P1OR-Ag2S QDs and P2OR-Ag2S QDs / P1OR*-Ag2S QDs, P2OR*-Ag2S QDs and P3OR*-Ag2S QDs). The last step of sample induced strand displacements and QDs release was the same as that of AND gates. After the logic gate operations, the electrodes were pulled out and the solutions were transferred to be measured by the fluorescence spectrophotometer. The fluorescence emission spectra were recorded from 750 to 850 nm.

RESULTS AND DISCUSSION So far, there are few reports using aqueous synthesis of Ag2S QDs with high QY 8 ACS Paragon Plus Environment

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(>20%).41 We develop a facile route to synthesize biocompatible and bright Ag2S QDs (QY: 27%), which can be illustrated in Scheme 1. Detailed information is described in the experimental section. In a typical reaction, cysteine is used as the capping reagent. The abundant thiols behave intrinsic metal-chelating actions, which help the growth of QDs. Additionally, cysteine not only enhances the solubility and colloidal stability, but also provides reactive groups for further functionalization with a large spectra of molecules. Here, the aminated DNA probes are conjugated with Ag2S QDs and the nanocomposites are used as the signal output of the logic gate platform. From TEM images, the size of the as-prepared Ag2S QDs is estimated to be 2 nm and the crystal structures are observed well (Figure S1). The existence of Ag2S QDs could be further confirmed by EDX analysis (Figure 1a). The nanomaterials are also monitored by UV-vis absorption and fluorescence spectra (Figure 1b and c). An extraordinarily narrow full width at half-maximum (fwhm) of 6 nm with emission peak at 802 nm is found. Optical and bright NIR fluorescent images are also shown (Inset in Figure 1c). Further, functional groups on the surface of Ag2S QDs are concluded from FTIR spectrum (Figure 1d). Absorption peaks around 1100 cm-1, 1400 cm-1, 1600 cm-1 and 2500 cm-1 are ascribed to C-O, C-H, C=O, S-H, respectively. Peaks around 3400 are typical responses of O-H and N-H. QY of Ag2S QDs is determined to be 27% by calibrating against reference quinine sulfate in 0.1 M H2SO4 (Figure S2). The effect of pH condition on fluorescence emission of QDs is explored (Figure S3a). The results reveal that the QDs emit steadily from 4 to 12. To further confirm the good stability in biological samples, Ag2S QDs are spiked in serum and the fluorescence emission data 9 ACS Paragon Plus Environment


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are recorded during a long time period (Figure S3b). The emission intensities barely change and are comparable with Ag2S QDs in pure water. Therefore, these results highlight the utility of Ag2S QDs as good candidates of fluorescent probes for clinical diagnostics. The basic unit of the developed Ag2S QDs-based DNA logic gate platform for miRNA diagnostics is YES gate, the principle of which is shown in Scheme 2. Briefly, DNA probe (EYES) is firstly modified on a gold electrode via gold–sulfur chemistry.42 The concentration of EYES is optimized by chronocoulometry measurement (Figure 2) and 1 µM is used throughout the electrode modification process in this work. The surface density of EYES is calculated to be 2.31 pmol cm-2 according to a protocol as previously reported, which is spacious for hybridization events.43 EYES on the electrode can hybridize with PYES-Ag2S QDs, forming double-stranded DNA with a toehold region. Meanwhile, Ag2S QDs are localized onto the electrode surface. The electrode is then immersed in the sample to be tested. Since toehold region is also the recognition region that is complementary to target miRNA, entropy-driven, toehold exchange-mediated strand displacement reaction occurs, which could release PYES-Ag2S QDs back to the solution in the presence of target miRNA. The recorded NIR fluorescence emission intensity can be used to reveal the initial miRNA level. Melting temperatures of EYES/PYES and EYES/PYES-Ag2S QDs hybrids are approximately equal to each other, demonstrating Ag2S QDs cannot influence hybridization and strand displacement events. Moreover, we have performed control electrochemical experiments using miRNA with methylene blue (MB) modified at the 10 ACS Paragon Plus Environment

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3’ end. EYES modified electrodes are firstly incubated with PYES and PYES-Ag2S QDs, respectively, following with the incubation of MB-miRNA. Square wave voltammograms probing the electrochemical responses of MB are then recorded. In the cases of PYES and PYES-Ag2S QDs incubation, extremely similar peak currents are observed. The results verify that efficient strand displacement reactions occur and neither Ag2S QD nor multiple DNA strands on them influence the hybridization and miRNA induced strand displacement events (Figure S4). To demonstrate the working hypothesis of Ag2S QDs-based miRNA assay, a series of concentrations of miR-20a (as an example) have been detected by the YES gate. As depicted in Figure 3a, fluorescence emission intensity increases with the increase of miR-20a. In the logarithmic scales, the intensity has a linear correlation with the concentration of miR-20a over the range from 10-16 to 10-11 M (Inset in Figure 3a). The large dynamic range with 5 orders of magnitude ensures the accurate quantification of miRNAs under various experimental conditions. The regression equation for the linear relationship is y = 0.1941 x + 3.1299 with a correlation coefficient of 0.99936, where y is the fluorescence emission peak intensity, x is logarithmic concentration of miR-20a. The limit of detection (LOD) for miR-20a in pure water is estimated to be 12.0 fM based on the 3σ method. A good reproducibility is also demonstrated with a relative standard deviation (RSD) of 3.5% for three repetitive measurement of 100 fM miR-20a. Figure 3b demonstrates that other miRNAs can hardly interfere the detection of target miRNA.



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The stability of this method is then studied. Compared with the miRNA analysis using freshly prepared PYES-Ag2S QDs and EYES modified gold electrodes, the sensing performance retains over 95% using these materials stored in 4°C for 1 month. A commercial qRT-PCR kit is used as a control method. Experimental results of the two methods are quite consistent with each other for the detection a series of standard miRNAs solutions, demonstrating the accuracy of the proposed method (Figure S5). Moreover, this method overcomes the drawbacks of qRT-PCR for miRNA analysis, including complex primer design and the inability to analyze multiple targets per single sample volume. We have then performed experiments to obtain LODs of different target miRNAs in the absence and presence of interfering miRNAs (Table S2). This YES gate is demonstrated to be versatile for the detection of other miRNAs and selective to distinguish target from interfering sequences. Serum samples are also employed for investigation. Different amount of miRNAs are firstly spiked in the samples (300 µL), which are then detected by the proposed method. With the increase of spiked miR-20a in serum samples, larger fluorescence emission intensity is observed (Figure S6). The LODs for miRNA diagnostics in serum samples are estimated to be 100 fM with excellent anti-jamming property (Table S3). Since many clinical cases indicate that single biomarker analysis may be insufficient for accurate disease diagnosis, we have then fabricated AND and OR logic gates, responding to multiplex information of initial miRNAs. It is reported that miR-20a, miR-21, miR-106a are enriched in colon adenocarcinoma.44 We thereby 12 ACS Paragon Plus Environment

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choose the three miRNAs as input examples of the developed logic gates, which are defined as T1, T2 and T3. The concentrations of the three miRNAs used in the AND gates are 10 pM. The principle of two-input AND gate is shown in Scheme 3a. EAND is modified on the electrode, followed by the hybridization with P1AND and P2AND-Ag2S QDs. T1 binds toehold region 1, displacing P1AND and exposes toehold region 2 for the subsequent T2 input. In the same way, P2AND-Ag2S QDs is substituted by T2 and released into the solution, which provides significant fluorescence emission. This event occurs only if both T1 and T2 are present. Three-input AND gate can be operated by using an extended EAND* and corresponding P1AND*, P2AND*, P3AND*-Ag2S QDs. Three entropy-driven, toehold exchange-mediated strand displacement reactions take place in the presence of T1, T2 and T3, which release QDs for fluorescence output (Scheme 3b). For two- or three-input OR gates, two or three independent toehold regions are generated by the hybridization of designed DNA sequences (EOR/P1OR-Ag2S QDs, P2OR-Ag2S QDs; EOR*/P1OR*-Ag2S QDs, P2OR*-Ag2S QDs, P3OR*-Ag2S QDs ) (Scheme 3c and d). Total miRNA concentrations used in OR gates are constant (10 pM). In such cases, each kind of miRNA input can bind corresponding toehold region and release Ag2S QDs for signal output after strand displacement. We have recorded fluorescence emission peak intensities for each logic gate with permutation of miRNA inputs. Two-input gates have four entries in truth tables, while three-input gates have eight. For input, we define “1” and “0” denoting the presence and absence of the target miRNAs. Fore output, we define “1” or “0” when the 13 ACS Paragon Plus Environment


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normalized fluorescence intensity is above or under the threshold value (0.3). Permutations of two inputs are (0, 0), (0, 1), (1, 0) and (1, 1). While permutations of three inputs are (0, 0, 0), (0, 0, 1), (0, 1, 0), (1, 0, 0), (0, 1, 1), (1, 0, 1), (1, 1, 0) and (1, 1, 1). Figure 4 shows the truth tables of YES gate, two- and three-input AND gates, two- and three-input OR gates for miRNA diagnostics, which are logically correct, demonstrating the Ag2S QDs-based DNA logic gate platform is workable for the analysis of one, two and three kinds of miRNAs. Moreover, this platform can be further extended to more-input systems with redesigning of sequences. Considering the efficiencies of DNA probe immobilization on electrode and the hybridization events, four to ten-input logic gates are assumed to be practically operated. Thus, this strategy is appropriate for high-order logic gate operations for miRNA diagnostics.

CONCLUSIONS In summary, we present a convenient, amplification-free DNA logic gate platform for miRNA diagnostics. To date, few DNA logic gates are used in clinical diagnostics due to the difficulty of responding to low concentration of analyte in heterogeneous biological samples. To solve this problem, we have applied NIR Ag2S QDs with excellent properties of autofluorescence, negligible tissue scattering in NIR region, bright photoluminescence, exceptionally large stokes shift, which are demonstrated to be excellent sensing materials for clinical applications. The detection strategy is simple

with

enzyme-free

toehold

exchange-mediated

strand

displacements.

Experimental results show that femtomolar level miRNA analysis performs well by 14 ACS Paragon Plus Environment

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detecting the fluorescence emission of NIR Ag2S QDs. Moreover, by carefully designing DNA sequences, YES gate, AND gate and OR gate with multiplex miRNA inputs are achieved, which demonstrate great potential of the developed DNA logic gate platform in clinical miRNA diagnostics.

CONFLICT OF INTEREST The authors declare no conﬂict of interest. ACKNOWLEDGEMENTS This work is supported by the National Key Instrument Developing Project of China (Grant no. ZDYZ2013-1) and the National Natural Science Foundation of China (Grant no. 31400847). Supporting Information Additional Figures and Tables as described in the essay. This material is available free of charge via the Internet at http://pubs.acs.org.

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J.; Hammond, S. M.; Joshua-Tor, L.; Hannon, G. J., Science 2004, 305, 1437-1441. (4). Hur, K.; Toiyama, Y.; Takahashi, M.; Balaguer, F.; Nagasaka, T.; Koike, J.; Hemmi, H.; Koi, M.; Boland, C. R.; Goel, A., Gut 2013, 62, 1315-1326. (5). Lu, J.; Getz, G.; Miska, E. A.; Alvarez-Saavedra, E.; Lamb, J.; Peck, D.; Sweet-Cordero, A.; Ebet, B. L.; Mak, R. H.; Ferrando, A. A.; Downing, J. R.; Jacks, T.; Horvitz, H. R.; Golub, T. R., Nature 2005, 435, 834-838. (6). Li, H. M.; Fu, Y.; Zhang, T.; Li, Y. P.; Hong, X. Y.; Jiang, J. Y.; Gong, T.; Zhang, Z. R.; Sun, X., Adv. Funct. Mater. 2015, 25, 7457-7469. (7). Ralfkiaer, U.; Hagedorn, P. H.; Bangsgaard, N.; Lovendorf, M. B.; Ahler, C. B.; Svensson, L.; Kopp, K. L.; Vennegaard, M. T.; Lauenborg, B.; Zibert, J. R.; Krejsgaard, T.; Bonefeld, C. M.; Sokilde, R.; Gjerdrum, L. M.; Labuda, T.; Mathiesen, A. M.; Gronbaek, K.; Wasik, M. A.; Sokolowska-Wojdylo, M.; Queille-Roussel, C.; Gniadecki, R.; Ralfkiaer, E.; Geisler, C.; Litman, T.; Woetmann, A.; Glue, C.; Ropke, M. A.; Skov, L.; Odum, N., Blood 2011, 118, 5891-5900. (8). Yang, N. N.; Ekanem, N. R.; Sakyi, C. A.; Ray, S. D., Adv. Drug Deliv. Rev. 2015, 81, 62-74. (9). Liu, R.; Chen, X.; Du, Y. Q.; Yao, W. Y.; Shen, L.; Wang, C.; Hu, Z. B.; Zhuang, R.; Ning, G.; Zhang, C. N.; Yuan, Y. Z.; Li, Z. S.; Zen, K.; Ba, Y.; Zhang, C. Y., Clin. Chem. 2012, 58, 610-618. (10). Wark, A. W.; Lee, H. J.; Corn, R. M., Angew. Chem. Int. Ed. 2008, 47, 644-652. (11). Friedlander, M. R.; Chen, W.; Adamidi, C.; Maaskola, J.; Einspanier, R.; Knespel, S.; Rajewsky, N., Nat. Biotechnol. 2008, 26, 407-415. 16 ACS Paragon Plus Environment

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Scheme 1. Schematic illustration of the synthesis routine of DNA-Ag2S QDs.

Figure 1. (a) EDX and (b) UV absorbance spectra of as-prepared Ag2S QDs. (c) Fluorescence emission spectrum of Ag2S QDs. Inset are the corresponding optical (left) and fluorescent (right) images under daylight and excitation of a home-made light emitting diode (LED). (d) FTIR spectrum of Ag2S QDs.


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Scheme 2. Principle of Ag2S QDs-based NIR fluorescent detection of miRNA.



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Figure 2. (a) Chronocoulometry curves for the gold electrodes modified with DNA probes with the concentrations of 0, 0.05, 0.20, 0.50, 1, 2, 3, 4, 5 µM (from bottom to top). Inset is the chronocoulometry curves of charge versus t1/2. (b) Calibration curve of ∆charge versus the concentration of DNA probe. The error bars represent relative standard deviations for three independent measurements.


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Figure 3. (a) Fluorescence emission spectra of Ag2S QDs released by miRNA (miR-20a as an example) with the concentrations of 10-16, 10-15, 10-14, 10-13, 10-12, 10-11 M (from right to left). Inset shows the quantitative linear dynamic range of the designed method. (b) Fluorescence emission intensities of Ag2S QDs initiated by target and interfering miRNAs with two different concentrations.



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Scheme 3. (a, b) two- and three-input And logic gates, (c, d) two- and three-input OR logic

gates

for

Ag2S

QDs

release

based

on

exchange-mediated strand displacements.


entropy-driven,

toehold

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Figure 4. DNA logic gates are displayed in closed (no Ag2S QDs release) or opened (Ag2S QDs release) configurations with various inputs of miRNAs according to the specified rules of logic gates. Detected fluorescence intensities of the released Ag2S QDs of each gate are displayed to the right: (a) YES gate, (b) two-input AND gate, (c) two-input OR gate, (d) three-input AND gate, (e) three-input OR gate. Input is defined as “1” and “0” denoting the presence and absence of the three miRNAs, respectively. Output is defined as “1” or “0” when the normalized fluorescence intensity is above or under the threshold value (0.3).



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for TOC only


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Near-Infrared Ag2S Quantum Dots-Based DNA Logic Gate Platform for

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