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Gold Nanoparticles-Based DNA Logic Gate for miRNA Inputs Analysis Coupling Strand Displacement Reaction and Hybridization Chain Reaction Xiaoyi Ma, Liqian Gao, Yuguo Tang, and Peng Miao*
effect,[2] strong localized surface plasmon resonance (LSPR) absorption,[3] huge specific surface area,[4] facile synthesis and functionalization.[5] Since the color of AuNPs is highly sensitive to the size, capping molecule, morphology, medium refractive index, and the distribution state, AuNPs have attracted particular attention and been widely used in different colorimetric biosensors for biochemical analysis.[6–8] The information of molecular recognition events and concentration of targets can be easily acquired with the color changes observed by naked eyes.[9–11] The surface chemistry of AuNPs is significant to colorimetric assay. The past few years have witnessed the development of many successful strategies for the bioconjugation of small molecules (e.g., sulfhydryl amino acids), polymers (e.g., poly(ethylene glycol)), and biomacromolecules (e.g., nucleic acids and proteins) to the surface of AuNPs.[12] Among them, DNA have emerged as powerful and versatile materials for AuNPs based nanobiotechnology.[13] Bare AuNPs aggregate after treated with certain amount of salts. However, AuNPs with a high singlestranded DNA graft density remain stable in the presence of high concentrations of salts.[14] Moreover, DNA probes used in the colorimetric system can be further engineered by different techniques.[15,16] For example, strand displacement reaction and hybridization chain reaction have been utilized for structural transformations of nucleic acids, which can be used for signal conversion and amplification before the analysis of various targets.[17–19] miRNAs are small noncoding RNAs which are transcribed by RNA polymerase II as long primary transcripts (pri-miRNAs) and then processed into the nucleus by RNase III Drosha into pre-miRNAs. miRNAs act as critical gene regulatory elements, which have been regarded as potential noninvasive biomarkers for cancer diagnosis.[20,21] The first evidence showing the involvement of miRNAs in cancer originates from the study on chronic lymphocitic leukemia (CLL), in which Calin et al. found the tumor suppressors at chromosome 13q14 frequently deleted in CLL contained two miRNA genes (miR-15a and miR-16-1).[22] Recently, Mirkin
The molecular level DNA logic gates assisted by nanomaterials hold great promise for disease diagnosis applications. However, designing convenient and sensitive logic gates for molecular diagnostics still remains challenging. In this work, a DNA logic gate platform for miRNA inputs analysis based on the observation of localized surface plasmon resonance variation of gold nanoparticles (AuNPs) is fabricated. As a demonstration, two biomarkers to differentiate indolent and aggressive forms of prostate cancer, miR-200c and miR-605, are selected as examples of logical inputs. In addition, five DNA probes are designed according to strand displacement reaction and hybridization chain reaction which are required for DNA logical operation. Since target miRNA inputs are able to trigger DNA structural transformations, which are further used to precisely regulate salt-induced AuNPs aggregation, miRNA inputs information can be converted to the information of AuNPs states as outputs. This developed system performs amplified detection of low-abundant target miRNAs without the requirement of any enzymes. In addition, single base-pair mismatched miRNAs can be effectively differentiated. Highorder logic gates can also be developed with further modifications. Therefore, the DNA AND logic gate is successfully constructed with flexible operations, which has potential in biochemical study and disease diagnosis.
1. Introduction Gold nanoparticles (AuNPs) possess a number of unique chemical and physical properties, such as high extinction coefficients in the visible region,[1] fluorescence quenching
Dr. X. Ma, Prof. Y. Tang, Prof. P. Miao Suzhou Institute of Biomedical Engineering and Technology Chinese Academy of Sciences Suzhou 215163, China E-mail:
[email protected] Dr. X. Ma, Prof. P. Miao University of Science and Technology of China Hefei 230026, China Dr. L. Gao School of Pharmaceutical Sciences (Shenzhen) Sun Yat-sen University Guangzhou 510275, China The ORCID identification number(s) for the author(s) of this article can be found under https://doi.org/10.1002/ppsc.201700326.
DOI: 10.1002/ppsc.201700326
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and co-workers have identified five miRNA biomarkers to differentiate indolent and aggressive forms of prostate cancer (miR-200c, miR-605, miR-135a*, miR-433, and miR106a).[23] The identification and clinical application of multiple cancer biomarkers are a pressing medical need. In this study, we have developed AuNPs-based DNA logic gate for the analysis of different miRNA inputs. Unlike traditional electronic devices which use electric current as inputs and outputs, nucleic acids are employed as inputs and the resulted LSPR absorption variations of AuNPs are utilized as outputs. The predictability of Watson–Crick base pairing makes it easy for the implementation of DNA-based architecture system. Currently, a variety of complex DNA logic gates have been constructed,[24,25] which can be activated by different external stimuli with specific design and modulation, such as ions,[26,27] DNA,[28] RNA,[29] and protein.[30] Herein, strand displacement reaction is designed for computational analysis of miRNA inputs and hybridization chain reaction is involved for signal amplification. Basic logic gate with label-free output is successfully constructed. This developed strategy also provides additional design flexibility for potential AuNPs-based dynamic DNA devices.
2. Results and Discussion 2.1. Working Principle Scheme 1 outlines the mechanism of the AuNPs-based DNA logic gate, which relies on structural transformations of nucleic acids. Two miRNAs, miR-200c and miR-605, function as inputs of the DNA logic gate; DNA probe 1, 2, and 3 function as the recognition and logical operation platform; H1 and H2 probes coupled with AuNPs function as output device. To realize the majority logical function of AND gate, DNA probe 1, 2, and 3 with partially complementary sequences hybridize with each other. The formed DNA duplex contains a toehold, which accelerates its hybridization with miRNA input 1 and the subsequent release of DNA probe 2. The first strand displacement exposes another toehold, and miRNA input 2 initiated second strand displacement reaction occurs. The released DNA probe 3 opens the hairpin structure of H1, which further partially hybridizes with H2. Furthermore, the remained single-stranded part of H2 also contains complementary sequence of H1. Thus, a chain reaction of hybridization events between alternating H1 and H2 occur to form nicked long
Scheme 1. a) Schematic illustration of the DNA logic gate with two miRNA inputs. b) Schematic illustration of the colorimetric analysis of miRNAs based on hybridization chain reaction.
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Figure 1. TEM images of a) freshly prepared AuNPs and b) AuNPs after treated with 50 × 10−3 m Mg2+. c) UV–vis absorption spectra of AuNPs in the (i) presence and (ii) absence of 50 × 10−3 m Mg2+. Inset is the corresponding photograph. d) Gel electrophoresis image for nucleic acids samples at different stages: (i) DNA probe 1; (ii) DNA duplex (probe 1, 2, and 3); (iii) after strand displacement in the presence of miRNA input 1 and 2; (iv) mixture of H1 and H2; (v) mixture of nucleic acids samples (iii) and (iv); (vi) DNA Ladder.
double-stranded DNA. In this way, each copy of miRNA input 1 and 2 consumes a larger number of H1 and H2 fuel strands with single-stranded parts. Since double-stranded DNA is stiffer and the exposed phosphate backbone contributes to the strong repulsion between DNA and negatively charged AuNPs, the salt-induced AuNPs aggregation cannot be effectively prevented.[31] As a result, the color of AuNPs changes and LSPR absorption is recorded to represent the distribution state of AuNPs. After challenging different combinations of miRNA inputs, AND gate is successfully constructed as the basic work unit.
2.2. Characterization of AuNPs From the transmission electron microscope (TEM) image in Figure 1a, the size of as-prepared AuNPs is estimated to be 13 nm, which is also well-dispersed in water. Salt with high concentration will induce aggregation of AuNPs, which can be clearly observed in Figure 1b. The reason is that the addition of salt compresses the outer shell of the electrical double layer and lowers the ζ-potential. The decreased electrostatic repulsions among AuNPs thus lead to the aggregation.[32] We have further characterized AuNPs by UV–vis absorption spectra since LSPR of AuNPs is strongly correlated with their distribution states. As shown in Figure 1c, an absorbance peak at 520 nm is observed. After the treatment of salt, the AuNPs aggregate and the peak at 520 decreases. Meanwhile, a new peak around 700 nm arises. The ratio of the peak value
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at 700 nm to that at 520 nm (defined as R700/520) is used to reveal the distribution state of AuNPs in the following experiments.[33] In addition, freshly prepared AuNPs are wine red, which can be kept stable for at least 4 months. However, with the addition of certain amount of salt, AuNPs aggregate and the color of the colloid turns to pale purple (Inset in Figure 1c).
2.3. Gel Electrophoresis Analysis DNA recognition and hybridization events are examined by gel electrophoresis. As summarized in Figure 1d, one band with low molecular weight (MW) is observed in lane i which represents DNA probe 1. After hybridized with DNA probe 2 and 3, MW of the formed DNA duplex increases (lane ii). Since MW of DNA duplex after strand displacement reactions initiated by miRNA input 1 and 2 is similar to that of DNA duplex (DNA probe 1, 2, and 3), the position of band in lane iii does not change significantly compared with that in lane ii. We have also characterized products of hybridization chain reaction in the absence and presence of miRNA inputs. Without the miRNA inputs-mediated strand displacement reactions, H1 and H2 are not consumed, and a band with low MW is observed in lane iv. On the other hand, with the aid of miRNA inputs, long double-stranded DNA duplex forms which is presented as a smear in lane v. This is because the MW of resulting polymers is not an exact numerical value, but lies in an approximate range.
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2.4. Preliminary Experiments Preliminary experiments are performed to determine optimal experimental conditions for the DNA logic gate operation. R700/520 is utilized to represent the distribution state of AuNPs. Its value for freshly prepared AuNPs is about 0.1. With the increase of the degree of AuNPs aggregation induced by salt, absorbance peak at 520 nm decreases and absorbance peak at 700 nm increases (Figure S1a, Supporting Information). Thereby, R700/520 value becomes higher. The relationship between R700/520 and the concentration of used Mg2+ is then studied (Figure S1b, Supporting Information). The fitting curve is a Boltzmann sigmoid and the equation is as follows y = A2 + ( A1 − A2 ) / (1 + exp ( x − x 0 ) /dx )
)
(1)
in which y is R700/520, x stands for the concentration of Mg2+ (mM) added to AuNPs, A1 = 0.1725, A2 = 1.0523, x0 = 13.0540, dx = 1.7392, R2 = 0.9980. Obviously, the slope of this fitting curve reaches a maximum value at the point of 13.054, indicating that AuNPs is most sensitive to the changes of Mg2+ concentration at this point. We have then used the concentration for the following colorimetric sensing. Next, we have performed experiments to optimize the concentration of H1 and H2 that protect salt-induced AuNPs aggregation. With the increase of concentration, R700/520 value decreases accordingly (Figure S1c, Supporting Information). Their relationship can be observed more clearly in Figure S1d in the Supporting Information. When the final concentrations of H1 and H2 are 25 × 10−9 m, R700/520 reaches a plateau. Thus, 25 × 10−9 m is chosen as the optimized value in the following experiments.
2.5. UV–vis Spectroscopy Analysis of miRNA Inputs and Logic Gate Operation To evaluate the working hypothesis of this AuNPs-based DNA logic gate. AuNPs after different treatment processes are characterized by UV–vis spectra. The curves are shown in Figure 2. AuNPs may aggregate in the presence of certain amount of salt. Thus, the absorbance value at 520 nm decreases and absorbance value at 700 nm increases compared with those of bare AuNPs. Due to the existence of single-stranded toehold in the DNA duplex formed by DNA probe 1, 2, and 3, salt-induce aggregation of AuNPs is inhibited in certain degree. If H1 and H2 are added, the two DNA probes are absorbed on the surface of AuNPs quickly via the interaction between gold and the exposed nitrogen-containing bases. With large amount of single-stranded tails of the two hairpin probes, AuNPs are kept stable even with high salt level. Thus, the absorbance values at 520 nm and 700 nm recover in a great extent. However, after further introduction of miRNA inputs, two strand displacement reactions occur in succession and the released DNA probe 3 initiates hybridization chain reaction which creates long double-stranded DNA and consumes numerous H1 and H2. As a result, the AuNPs cannot be protected effectively from salt-induced aggregation, which is reflected in the UV–vis spectrum. Although a number of DNA logic gates have been developed for biosensing purposes,[34–36] the sensitivity is always
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Figure 2. UV–vis spectra of a) bare AuNPs, b) treated with 13 × 10−3 m Mg2+, c) AuNPs with DNA probe 1, 2, and 3 (0.5 × 10−9 m) after treated with 13 × 10−3 m Mg2+, AuNPs with DNA probe 1, 2, 3 (0.5 × 10−9 m) and H1, H2 (25 × 10−9 m) in the d) absence and e) presence of miRNA input 1 and 2 (0.3 × 10−9 m), which are then treated with 13 × 10−3 m Mg2+.
insufficient for sensitive detection of low-abundant miRNA. To improve the analytical performance, we have introduced hybridization chain reaction in the system for signal amplification. Different amounts of miRNAs (miR-200c and miR-605) ranging from 0 to 0.5 × 10−9 m are added to the DNA logic gate platform and the UV–vis spectra are compared (Figure 3). With the increase of miRNA concentration, R700/520 value increases correspondingly. A linear range from 0.01 to 0.2 × 10−9 m is obtained. The regression equation is as follows: y = 0.270 + 0.558 x ( n = 3, R 2 = 0.982)
(2)
in which y is R700/520 value, x is the concentration of miRNA inputs (nm). The limit of detection (LOD) is as low as 1 × 10−12 m, which shows that it is a sensitive strategy for detection lowabundant miRNAs. Although the detection time is a bit long, after comparing with some representative miRNA assays, the analytical performances of the proposed system are excellent (Table S1). AuNPs based colorimetric DNA logic gates bring convenience for signal output. We have defined the presence of miRNA input 1 or 2 as “1” or “true” in the logic gate. Also, R700/520 value above the threshold of 0.4 is regarded as “1” or “true” for output. Figure 4 shows UV–vis spectra in different input cases and R700/520 values are calculated for analysis, respectively. Only in the presence of both miRNA input 1 and 2, DNA probe 3 could be released to trigger hybridization chain reaction, which then result in a “true” output; otherwise the outputs are “false” (inset in Figure 4).
2.6. Interference Study and Real Sample Analysis To demonstrate the high selectivity of the strategy, we have replaced target miRNA inputs by four single base-pair
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Figure 4. UV–vis absorption spectra of the AuNPs a) without miRNA input 1 and 2, b) with miRNA input 2, c) with miRNA input 1, d) with miRNA input 1 and 2. The concentrations of miRNAs are 1 × 10−9 m. Inset shows the AND logic gate with corresponding values of absorbance ratio (R700/520). Error bars represent the standard deviations of three independent measurements.
relative errors are less than 4%. Therefore, the practical value of the proposed work is demonstrated.
3. Conclusion
Figure 3. a) UV–vis absorption spectra for AuNP-based colorimetric detection of miRNA input 1 and 2. b) Plot of concentration of miRNAs versus the absorbance ratio (R700/520). Inset shows the linear range. Error bars represent the standard deviations of three independent measurements.
mismatched miRNAs as negative controls. As shown in Figure 5a, in the presence of mismatched miRNAs, the absorbance peaks at 520 nm drops slightly compared with that of blank curve. However, there are no new peaks around 700 nm. On the other hand, target miRNAs inputs trigger strand displacement reaction and hybridization chain reaction, in which case an obvious peak at 700 nm appears. Corresponding R700/520 values are calculated and summarized in Figure 5b, which clearly shows that R700/520 in the presence of target miRNAs is distinctly larger than that of blank or mismatched miRNAs. There results verify that the proposed method is suitable for sensitive detection of miRNA with high specificity. We have then challenged the colorimetric system with human serum samples, which are previously spiked with different amount of target miRNAs. The real samples are measured by the proposed method and a commercial qRT-PCR kit. Experimental results obtained by the two methods are in excellent accordance (Table S2, Supporting Information). The
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In summary, we have developed a DNA AND logic gate for miRNA inputs analysis based on the AuNPs colorimetric system. Nucleic acids used in this work are designed according to strand displacement reaction and hybridization chain reaction which are required for DNA logical operation. Since the initial recognition event of hybridization chain reaction is amplified with abundant supply of H1 and H2, the developed method is able to respond to multiple miRNA inputs with low concentrations. Therefore, the proposed method has potential applications in biochemical study and disease diagnosis. In addition, this DNA logic gate system also offers several other advantages. First, it only involves DNA hybridization and denaturation events. Enzymatic catalyzed reactions are avoided, which makes the detection quite simple and stable. Second, it has a high discrimination ability between perfectly matched targets and single base-pair mismatched miRNAs. Third, the strategy is also appropriate for high-order logic gates after certain modifications.
4. Experimental Section Materials and Chemicals: Chloroauric acid (HAuCl4) and trisodium citrate were obtained from Shanghai Jiushan Chemicals Co., Ltd. (Shanghai, China). Diethypyrocarbonate (DEPC) and ethylenediaminetetraacetic acid were from Sigma (USA). 50 bp Plus DNA Ladder (ZM202) was purchased from Beijing Zoman Biotechnology Co., Ltd. (Beijing, China). 4S Red Plus Nucleic Acid Stain (4S Red) was obtained from Sangon Biotech Company, Ltd. (Shanghai, China). DNA
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Figure 5. Selectivity of the colorimetric assay to distinguish mismatched miRNAs: a) UV–vis absorption spectra, b) corresponding values of absorbance ratio (R700/520). The concentrations of miRNAs are 1 × 10−9 m. Error bars represent the standard deviations of three independent measurements.
probes and RNA probes were synthesized and purified by Sangon Biotech Company, Ltd. (Shanghai, China) and Takara Biotechnology Co., Ltd. (Dalian, China), respectively. Detailed sequences were listed in Table S3 in the Supporting Information. Water used in this work was obtained from a Millipore water purification system with a specific resistance of 18 MΩ cm and then treated with DEPC. UV–vis absorption spectra were recorded by a Synergy HT multifunction microplate reader (BioTek Instruments, Inc., USA). TEM images were taken with a FEI Tecnai G20 transmission electron microscopy (FEI, USA). Photographs of AuNPs were taken with Canon IXUS220 HS digital camera. Gel was photographed under UV light by Gel Doc XR+ System (Bio-Rad, USA). Preparation of Bare AuNPs: Synthesis of bare AuNPs was carried out by a citrate reduction method.[37] Briefly, 3.5 mL of 1% (w/v) trisodium citrate was spiked into 100 mL of 0.01% (w/v) HAuCl4 under rapid stirring. The mixture was boiling for 15 min. Afterward, the heat was removed but the solution was under stirring for another 30 min. Then, the solution was cooled down to room temperature and was centrifuged at 12 000 rpm for 30 min for purification. AuNPs were ready for use after resuspended. Logic Gate Preparation and Colorimetric Assay: DNA probes and RNA probes were dissolved in hybridization buffer (10 × 10−3 m phosphate buffer with 0.2 m NaCl, pH 7.4). The probes were heated at 95 °C for 5 min and then slowly cooled down to ambient temperature by 1 °C min−1. DNA probe 1, 2, and 3 with the concentration of 10 × 10−9 m were mixed for 1 h at 25 °C. Then, miRNA input 1 and 2 with different concentrations were blended with the above mixture for another 1 h at 25 °C. Subsequently, H1 and H2 were added in the solution with the final concentration of 500 × 10−9 m. The hybridization chain reaction was carried out at 37 °C for 4 h. 180 µL of AuNPs was thoroughly mixed with 10 µL of nucleic acids probes, which was then blended with 10 µL of Mg2+ (260 × 10−3 m). The mixture was placed in the well of 96-well plate. UV–vis absorption spectrum was then measured immediately. Gel Electrophoresis: 4% agarose gel was stained with 4S Red. Afterward, nucleic acids samples with different treatments and 50 bp Plus DNA Ladder were injected into the gel, which were monitored by gel electrophoresis for 30 min (110 V). Finally, the gel was photographed for analysis.
Supporting Information Supporting Information is available from the Wiley Online Library or from the author.
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Acknowledgements X.M. and L.G. contributed equally to this work. This work was supported by the National Natural Science Foundation of China (Grant no. 81771929 and 31400847), the National Key Instrument Developing Project of China (Grant no. ZDYZ2013-1), and Postdoctoral Innovative Talents Program (Grant no. BX201600184).
Conflict of Interest The authors declare no conflict of interest.
Keywords biosensors, DNA logic gate, hybridization chain reaction, nanoparticles, strand displacement reaction Received: September 5, 2017 Revised: November 9, 2017 Published online:
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