Single-Molecule Analysis of MicroRNA and Logic Operations Using a

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Single-molecule analysis of microRNA and logic operations using a smart plasmonic nanobiosensor Ying Zhang, Zhenhua Shuai, Hao Zhou, Zhimin Luo, Bing Liu, Yinan Zhang, Lei Zhang, Shufen Chen, Jie Chao, Lixing Weng, Quli Fan, Chunhai Fan, Wei Huang, and Lianhui Wang J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.7b12772 • Publication Date (Web): 05 Mar 2018 Downloaded from http://pubs.acs.org on March 5, 2018

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Journal of the American Chemical Society

Single-molecule analysis of microRNA and logic operations using a smart plasmonic nanobiosensor Ying Zhang, Zhenhua Shuai, Hao Zhou, Zhimin Luo, Bing Liu, Yinan Zhang, Lei Zhang,*, Shufen Chen,† Jie Chao,† Lixing Weng,† Quli Fan,† Chunhai Fan,†,‡ Wei Huang,†,⊥ and Lianhui Wang*,† †

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Key Laboratory for Organic Electronics and Information Displays (KLOEID) & Jiangsu Key Laboratory for Biosensor, Institute of Advanced Materials (IAM), Jiangsu National Synergistic Innovation Center for Advanced Materials (SICAM), College of Electronic and optical Engineering & College of Microelectronic, Nanjing University of Posts and Telecommunications (NUPT), 9 Wenyuan Road, Nanjing 210023, China ‡ Division of Physical Biology & Bioimaging Center, Shanghai Synchrotron Radiation Facility, CAS Key Laboratory of Interfacial Physics and Technology, Shanghai Institute of Applied Physics, Chinese Academy of Sciences, Shanghai 201800, China ⊥ Key Laboratory of Flexible Electronics (KLOFE) & Institute of Advanced Materials (IAM), Jiangsu National Synergistic Innovation Center for Advanced Materials (SICAM), Nanjing Tech University (NanjingTech), 30 South Puzhu Road, Nanjing 211816, China KEYWORDS: localized surface plasmon resonance, tetrahedron-structured DNA, single-molecule level, logic operations, bio-memory ABSTRACT: Analysis of biomolecules at the single-molecule level is a great challenge in molecular diagnostics, gene profiling, and environmental monitoring. In this work, we design a smart plasmonic nanobiosensor based on individual Au@Ag core-shell nanocube (Au@Ag NC) modified with tetrahedron-structured DNA (tsDNA) for detecting microRNA 21 (miR-21) at the single-molecule level. An average localized surface plasmon resonance (LSPR) scattering spectral wavelength shift of approximately 0.4 nm can be obtained for a single miR-21 hybridization event on the nanobiosensor. In addition, the sensing mechanism of the individual Au@Ag NC is further verified by the three-dimensional finitedifference time-domain (3D-FDTD) simulations. Notably, this system not only allows the real-time detection of miR-21 with an aM level sensitivity over a large dynamic range from 1 aM to 1 nM, but also enables DNA-based logic operations as well as bio-memory by exploiting miR-21, KpnI, and StuI-responsive assays. Our study opens a unique method for singlemolecule detection of biomolecules and thus holds great promise in a variety of biological and biomedical applications.

particle can provide even more detailed information. Therefore, we sought to explore its novel plasmonic applications based on measuring biomolecular interactions near the metal nanoparticle surface by the singlenanoparticle level. Bioanalysis at the single-molecule level holds great promise for studying molecular interactions, kinetics and conformational changes in various biomolecular systems.26-35 However, most available methods often require fluorescent labeling, which is prone to photobleaching or blinking, low signal-to-noise ratio and unsuitable for direct analysis of subtle structures.36-39 Due to the advantages of their light sensitivity and facile modification, the individual nanoparticle-based plasmonic nanoprobes provide a unique opportunity to single-molecule analysis.

INTRODUCTION Plasmonic materials have aroused much attention and been found many promising applications in chemical and biological sensing,1-21 due to their unique size, shape, composition, and the local environment-dependent optical properties. These sensing applications are based on the localized surface plasmon resonance (LSPR) of noble metal nanoparticles (e.g., gold or silver nanoparticles) by employing the shifts of the LSPR scattering spectrum peak (λmax) as the output signal. Recently, LSPR sensing for measuring molecular interactions near the metal nanoparticles surface has attracted much attention, and different strategies have been developed for immobilizing biomolecules on the surface of metal nanoparticles.22-25 Furthermore, signals obtained from a single metal nano-

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Figure 1. (a) A smart plasmonic nanobiosensor, comprised of tsDNA17-modified Au@Ag NCs immobilized on the surface of a glass surface, able to capture miR-21 with high specificity. Inset: the corresponding LSPR scattering spectra of the selected Au@Ag NC-tsDNA17 and Au@Ag NC-tsDNA17-miR-21 in (d). (b) Schematic illustration of the construction of tsDNA17. (c) Typical TEM image of Au@Ag NCs with average diameter of ~55 nm. Scale bar, 100 nm. (d) Typical dark-field microscopy (DFM) images of Au@Ag NC-tsDNA17 (d-I) and Au@Ag NC-tsDNA17-miR-21 (d-II) on glass. (e) In situ SEM images of the selected Au@Ag NCtsDNA17 (e-I) and Au@Ag NC-tsDNA17-miR-21 (e-II) in (d). Scale bars, 1 μm (Inset: 200 nm).

Among those nanoparticles, Au and Ag nanocubes are very attractive candidates for plasmonic optical applications due to their strong electromagnetic field enhancements at their sharp corners, which can enhance scattering cross sections of molecules for sensing.6,40-42 For example, 55 nm Ag nanocubes as near-field plasmonic nanoprobes show high SPR capability for ultrasensitive detection of the lung cancer-associated miRNAs.23 Moreover, Au@Ag core-shell nanocubes (Au@Ag NCs) are allowed to better tune the scattering efficiency and absorption wavelength range through Ag shell and Au core owning to the plasmonic coupling from the surfaces of Au and Ag,42 resulting in more abundant SPR modes and broader shift region from visible to near infrared.43 Here, we develop a smart plasmonic nanobiosensor by using single tetrahedronstructured DNA (tsDNA)-modified 55 nm Au@Ag NC for detecting microRNA 21 (miR-21) at the single-molecule level and also achieve DNA-based logic operations as well as biomemory (Figure 1a). Three-dimensional finite-difference timedomain (3D-FDTD) simulations are used to simulate the LSPR scattering spectra and calculate the electric field distributions for a single Au@Ag NC during the hybridization pro-

cess. The good agreement between the results of simulation and experiment indicates that the interaction between tsDNA molecules and its targets leads to the change of refractive index (RI) near the Au@Ag NC surface, which induces a color change of the Au@Ag NC in dark-field microscopy (DFM) and a scattering spectral shift.23,25 The shifts of peak position in LSPR scattering spectra can provide a quantitative detection for the targets with ultra-sensitivity. The employed oligonucleotides sequences are listed in Table S1.

RESULTS AND DISCUSSION In this study, we chose the “pyramidal” DNA tetrahedral structure as the recognition probe for miR-21 and the enzymatic substrate in our model system for four reasons: (I) The tetrahedral DNA structure, with excellent mechanical rigidity and structural stability, provides more possibilities for the controlled positioning of probes on a 3D surface as compared to the previously reported linear or stem-loop DNA structures.44-46 (II) The recognition


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Figure 2. (a) 3D model for FDTD simulation. A core AuNP with a diameter of 30 nm covered by 13 nm AgNC shell was simulated. (Layer A: tsDNA17 molecules layer with a thickness of 5 nm; Layer B: tsDNA17-miR-21 molecules layer with a thickness of 5 nm; Layer C: remaining miR-21 molecules layer with a thickness of 4 nm). (b) Corresponding LSPR scattering spectra of the selected Au@Ag NC-tsDNA17 (b-I) and Au@Ag NC-tsDNA17-miR-21 (b-II) in Figure 1d. (c) The simulated LSPR scattering spectra of the model in (a). (d) 3D-FDTD simulated results showing the electric field distributions of the Au@Ag NC-tsDNA17 (d-I) and Au@Ag NC-tsDNA17-miR-21 (d-II) of the model in (a), respectively. The incident light is circularly polarized with the wavelength ranging from 300 to 800 nm.

sequence of miR-21 (l1) and cleavage sites for the endonucleases KpnI (l2) and StuI (l3) can be designed at three vertical edges of the tetrahedral DNA structure, respectively (Figure 1b and Table S1). (III) Meanwhile, the tsDNA can be readily assembled on the surface of a Au@Ag NC through three thiols at the base of the “pyramid” with high stability (Figure 1c and Figure S1). (IV) The density of probe molecules on a Au@Ag NC surface can be well-controlled by adjusting the size of the tsDNA. We designed three types of tsDNAs with different sizes: tsDNA7, tsDNA17, and tsDNA26, in which each edge of the tsDNA contains 7, 17, and 26 base pairs, respectively. The combinations of tsDNA strands were shown in Table S2 and the assembly of tsDNAs was verified by using polyacrylamide gel electrophoresis (PAGE). The tsDNA moved more slowly than either a single strand DNA or any other combinations lacking one or two strands, which is consistent with previous reports47 and suggests the successful assembly of the nanostructure (Figures S2, S3).

DFM images presented a color transition from green (Figure 1d-I) to orange (Figure 1d-II), indicating the hybridization between the tsDNA17 probes and miR-21. Of note, the in situ SEM images of the selected Au@Ag NC showed that the shape of the Au@Ag NC remained unchanged during the hybridization process (Figure 1e). In order to prove the role of the RI change in miRNA sensing of a single Au@Ag NC, 3D-FDTD simulations were utilized to simulate the LSPR scattering spectra and electric field distributions of the Au@Ag NC.48,49 Figure 2b, c showed the LSPR scattering spectra of the selected Au@Ag NC-tsDNA17 and Au@Ag NC-tsDNA17-miR-21 in Figure 1d and Figure 2a obtained in experiments and 3DFDTD simulations, respectively. The scattering spectra calculated from theoretical models using 3D-FDTD simulations fit those observed from experiments very well. Therefore, the redshift of λmax (∆λmax-red) can be attributed to the increase of the RI on the surface of the Au@Ag NCs when the miR-21 was hybridized with the tsDNA17 probes and replaced H2O molecules.23 As shown in Figure 2d, the electric field distributions for the single Au@Ag NC with a 5 nm tsDNA17 molecules layer (Layer A, Figure 2a-I) and the single Au@Ag NC with both a 5 nm tsDNA17-miR-21 molecules layer (Layer B) and a 4 nm miR-21 molecules layer (Layer C, Figure 2a-II) under their corresponding resonant wavelengths were calculated using 3D-FDTD simulations. These results exhibited that the Au@Ag NC has strong electric field at the four corners, suggesting that the ∆λmax-red might be caused by nucleic acids molecules attached to the surface of the Au@Ag NC. The timedependent redshift of λmax on a selected single Au@Ag NC-tsDNA17 probe is shown in Figure S5. In addition, we

As shown in Figure 1a, the single nanoparticle-based plasmonic probes were constructed through assembly of tsDNA17 on the surface of Au@Ag NCs (see Supporting Information for details). A thin film composed of tsDNA17 and H2O molecules was formed, and its RI value lies in the range between that of tsDNA17 and water (Figure 2aI). With the help of LSPR spectrometer and DFM, the hybridization process between tsDNA17 and miR-21 can be monitored in real time at the single-nanoparticle level. After 3 h treatment of the Au@Ag NC-tsDNA17 probes with 1 pM of miR-21, the λmax of the probes redshifted from 556 nm to 587 nm (Figure 1a and Figure S4), and the



Figure 3. (a) Time-dependent spectral peak redshifts for the selected Au@Ag NC-tsDNA17 probes in different concentrations of 2 3 4 5 6 7 8 9 miR-21 (1, 10, 10 , 10 , 10 , 10 , 10 , 10 , 10 and 10 aM). The peak position obtained by fitting each smoothed spectral data. (b) Time dynamics for the selected Au@Ag NC-tsDNA17 probe after addition of single miR-21 molecules. (c) Relationship of spectral peak shift in different concentrations of miR-21 corresponding to (a). The limit of detection of this sensor was analyzed to 0.1 aM when the signal to noise ratio was 3. (d) Peak shifts of three typical Au@Ag NC-tsDNA17 probes under different interference conditions: 1, complementary target (1 pM); 2, single-base mismatch miRNA (1 pM); 3, random miRNA (1 pM). The error bars represent the standard deviation in the measurements of eighty nanoparticles. The final concentration of tsDNA17 in all used samples is 1 pM.

also used tsDNA7 and tsDNA26 as the recognition probes for miR-21 under the same experimental conditions. Compared to the tsDNA17 probe, these two probes showed much less redshifts of λmax (Figure S6). This difference in ∆λmax-red can be attributed to the different densities, orientations and/or entanglements of the different recognition probes when DNA strands with different lengths were used, which was also observed previously in DNA-based programmable “soft lithography”.50

rupt spectral jumps at 113 min and 117 min with a ∆λmax-red = 0.4 nm strongly indicate the single irreversible miR-21 to tsDNA17 hybridization events. The relationship between the mean ∆λmax-red value and miR-21 concentrations is plotted in Figure 3c. A 4 nm redshift in the λmax distribution is obtained when a single Au@Ag NC-tsDNA17 probe is hybridized with 1 aM of miR-21. Significantly, the mean value of ∆λmax-red scales up linearly with the logarithm of the applied miR-21 concentration from 1 aM to 1 nM. To the best of our knowledge, such a large dynamic range has never been reported for spectrally responsive single-molecule or single-particle probes. Importantly, the biosensor based on Au@Ag NC-tsDNA17 probe exhibits a higher sensitivity for detecting miRNA than those of previously reported biosensors based on single nanoparticle probes.6,25

To investigate the sensitivity and dynamic range of our sensing system, we used a single Au@Ag NC-tsDNA17 with an initial λmax at around 556 nm as the probe for monitoring the hybridization process with different concentration of miR-21 (1 aM to 1 nM) for 3 h and recorded the spectral images in time. The peak position was obtained by fitting each smoothed spectral data (Figure 3a). The experimental results showed that the ∆λmax-red increased rapidly initially and then slowed down in all cases. A plot segment in Figure 3a (highlighted in the rectangle) is enlarged and shown in Figure 3b, and a representative time trace is shown in Figure S7, which reflects the hybridization process between tsDNA17 and 1 pM miR-21 during the interval from 110 to 120 min. These enlarged time traces reveal discrete on/off events with a spectral amplitude of ∆λmax-red = 0.2 nm, which is attributable to a consecutive attachment/detachment of miR-21 to tsDNA17.1 The ab-

The selectivity of the single Au@Ag NC-tsDNA17 probes was further studied by measuring the change in the LSPR scattering spectra. Compared to the fully complementary target (miR-21, 1 pM), much smaller increasement of the ∆λmax-red was collected for single-base mismatch miRNA (SM, 1 pM) and almost no change was obtained for random miRNA (R, 1 pM) (Figure 3d). This result suggests that this nanobiosensor holds excellent selectivity for target miRNA, and even the single nucleotide mutation in miRNA can be distinguished. In addition, this plasmonic


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Figure 4. (a) Schematic representation of measurement of endonuclease activity and OR gate based on the RI decrease induced by endonuclease reactions on the surface of a single Au@Ag NC. Right: Corresponding DFM images of the selected single Au@Ag NC-tsDNA17-miR-21 nanoconjugates before (I, T3-state) and after cleavage by endonucleases KpnI (II, R2-state), StuI (III, R3-state) and KpnI@StuI (IV, R2/3-state), showing the color change from orange (I) to yellow-green (IV). (b) Relative signal output of the OR gate. Inset: Truth table for the OR gate. (c) Schematic representation of XOR gate based on the RI change which was attributed to miR-21 specific hybridization and endonuclease reactions on the surface of a single Au@Ag NC. Right: Corresponding DFM images of the selected Au@Ag NC-tsDNA17 probes before (I, T0-state) and after addition of either miR-21 (II, T3state) or endonucleases (KpnI and StuI) (III, R0-state) as well as both miR-21 and endonucleases (IV, R2/3-state), showing that the colors of Au@Ag NCs are green (I), orange (II), yellow (III), and yellow-green (IV), respectively. (d) Relative signal output of the XOR gate. Inset: Truth table for the XOR gate. Error bars indicate the standard deviation of measuments from eighty nanoparticles. The final concentration of tsDNA17 and miR-21 in all used solutions is 1 pM.

nanobiosensor is also suitable for the analysis of heterogeneous samples, as confirmed by the successful detection of miR-21 (1 pM) in 50% fetal bovine serum (FBS) and 50% human serum (HS) (Figure S8). Note that our sensor in blank serum or buffer solution showed negligible signals. Importantly, our sensor showed the similar spectral response towards 1 pM miR-21 in buffer solution, 50% FBS, and 50% HS, respectively, indicating that successful analysis could be attained in these complex matrixes. On the basis of these results, we speculate the aforementioned single tsDNA-modified Au@Ag NC-based plasmonic nanobiosensor can also be used for measuring nuclease activity. Herein, as a proof-of-concept study, the activity measurement of endonucleases KpnI and StuI was carried out (Figure 4a). PAGE analysis demonstrated the successful cleavage of tsDNA17 by using endonucleases KpnI and StuI (Figure S9). About eighty Au@Ag NCtsDNA17-miR-21 nanoconjugates were examined by using LSPR spectrometer and DFM. As expected, in the absence of the endonuclease, there was no significant change of the λmax of the single nanoconjugate. After addition of KpnI or StuI, the λmax exhibited a blueshift (∆λmax-blue-KpnI = 8 nm and ∆λmax-blue-StuI = 10 nm) and the DFM images pre-

sented the color transition from orange to light-yellow. Upon addition of two endonucleases simultaneously, the λmax of Au@Ag NC-tsDNA17-miR-21 nanoconjugates showed an obvious blueshift from 587 nm to 569 nm (∆λmax-blue-KpnI@StuI = 18 nm), and the orange spot in DFM images gradually changed to yellow-green (Figure 4a and Figure S10). These results suggest that the addition of either KpnI or StuI, or both could cleave the edge(s) of tsDNA17 (l2 or l3, or l2 & l3), and the configuration of tsDNA17-miR-21 turned from a taut (T-) state to a relaxed (R-) state that the originally bound miR-21 was detached from the tsDNA17 and replaced by H2O molecules, causing a decrease of RI on the surface of the Au@Ag NCs and thus a blueshift of λmax.23 In addition, control experiments were carried out to confirm that the blueshift of λmax was due to the change of tsDNA17-miR-21’s configuration cleaved by KpnI and StuI. Two other types of endonucleases (HindIII and SalI) were tested with the same assay protocol, but neither of them could induce the blueshift of λmax at the same concentration used for KpnI and StuI. This result proved that the plasmonic nanobiosensor exhibits excellent selectivity for endonucleases KpnI and StuI (Figure S11).



Figure 5. Four-level bio-memory based on the Au@Ag NC-tsDNA17. The ∆λmax-red (output) was defined to value 0 at the initial state (Logic 0: ∆λmax-red < 5 nm, T0-state). After the tsDNA17 hybridized with miR-21, the ∆λmax redshifted to 31 nm corresponding to the output value 3 (Logic 3: 29 nm < ∆λmax-red < 35 nm, T3-state). The addition of KpnI or StuI could damage one edge of tsDNA17 (l2 or l3), the LSPR scattering blueshifted to a stable state (Logic 2: 19 nm < ∆λmax-red < 25 nm, R2-state or R3-state) with ∆λmax-KpnI was 23 nm and ∆λmax-StuI was 21 nm. Both the edge l2 and l3 would be broken at the presence of two kinds of endonucleases at the same time, ∆λmax-KpnI@StuI would further blueshift to 13 nm corresponding to the output value 1 (Logic 1: 10 nm < ∆λmaxred< 15 nm, R2/3-state). The final concentration of tsDNA17 and miR-21 in all used solutions is 1 pM. Error bars indicate the standard deviation in the measurements of eighty nanoparticles.

In addition to the biosensing applications, we also used the single Au@Ag NC-tsDNA17-miR-21 nanoconjugate as the plasmonic label for logic operation. An OR gate means that the output is “1” when the addition of either one of the inputs, or both, is applied. Based on the aforementioned KpnI and StuI-responsive assay, an OR gate was created with KpnI and StuI as the inputs, and the ∆λmax-blue of the nanoconjugate as the output (Figure 4a,b). A single Au@Ag NC-tsDNA17 probe hybridized with miR-21 acted as the initial state of this OR gate, where the configuration of tsDNA17-miR-21 is in the T3-state. The addition of either KpnI or StuI was defined as the “1” state, and the “0” state corresponded to the absence of endonuclease. As described above, in the presence of either KpnI

or StuI, or both, the ∆λmax-blue is 8 nm (∆λmax-blue-KpnI), 10 nm (∆λmax-blue-StuI) or 18 nm (∆λmax-blue-KpnI@StuI), respectively. For the output, we defined a ∆λmax-blue of 3 nm as the threshold value, which is based on the ∆λmax-blue of the single Au@Ag NC-tsDNA17-miR-21 nanoconjugate. When the ∆λmax-blue was lower than the threshold value, the output was defined as “0”. Whereas the output = “1” when the ∆λmax-blue was higher than the threshold value. The addition of either KpnI or StuI, or both (input =1/0, 0/1 or 1/1), turned the configuration of tsDNA17-miR-21 from a T3state to a R-state (Figure 4a, R2-state, R3-state and R2/3state, respectively) and decreased the RI on the surface of Au@Ag NCs, leading to a ∆λmax-blue of the single nanoconjugates (output = 1). The output was “0” only when nei-


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Journal of the American Chemical Society ther input was applied. Comparing the ∆λmax-blue of KpnI with StuI, the ∆λmax of 2 nm was caused by the different distances between the two cleavage sites and the surface of Au@Ag NCs. Detection resolution of up to 0.5 nm was observed per DNA base pair, which could potentially be utilized to develop more complex logic operations. Similarly, an XOR logic gate could be created by employing miR-21 as one input, the addition of endonucleases (KpnI and StuI) as another input, and the ∆λmax-red of the Au@Ag NC-tsDNA17 probe as output (Figure 4c,d). The logic of an XOR gate means that the output is “1” only if one of the inputs, but not both, is applied. With respect to the input, we defined the presence of either miR-21 or endonucleases as “1” and their absence as “0”. The output was defined based on the ∆λmax-red of the Au@Ag NCtsDNA17 probe, with “1” for a ∆λmax-red greater than 20 nm. As shown in Figure 4c,d and Figure S12, introduction of miR-21 (1 pM, input=1/0) resulted in an obvious redshift of the λmax (31 nm, output = 1, T3-state). After addition of endonucleases (input = 0/1), the rigid structure of tsDNA17 could be completely destroyed and the top of “pyramidal” DNA tetrahedral structure collapsed to the surface of Au@Ag NCs, meaning that the RI on the surface of Au@Ag NCs increased when the DNAs took the place of H2O molecules near the surface of Au@Ag NCs, leading to a ∆λmax-red of the Au@Ag NC-tsDNA17 probe (28 nm, output = 1, R0-state). However, the addition of both miR-21 and endonucleases (input = 1/1, R2/3-state) resulted in a minimal redshift of the λmax (13 nm, output = 0). We also demonstrated that the single Au@Ag NCtsDNA17 probes exhibited a four-level optical memory characteristics. As shown in Figure 5, each memory state could be clearly distinguished mainly according to the configurational change of the tsDNA17 on the surface of Au@Ag NC with the treatment of miR-21, endonucleases KpnI or StuI, respectively. This bio-memory could keep its scattering ability stable in a month for all four states. These results suggest the potential of developing novel plasmonic read only memory (ROM) devices based on single Au@Ag NC-tsDNA17 probes. Importantly, in view of the resolution of DFM (~200 nm), the average dimensions of single Au@Ag NC-tsDNA17 probes can be designed in theory much smaller than those traditional ROMs such as DVD and Blu-ray Disc (Figure S13). The capacity of the tsDNA17 based bio-memory device is 3 times as large as Blu-ray Disc and 18 times to DVD.

pendence on miR-21 concentrations. Notably, a single miR-21 hybridization event could give rise to an average LSPR scattering spectral wavelength shift of approximately 0.4 nm on this nanobiosensor. The experimental results can be explained well by using 3D-FDTD simulations. More importantly, the tsDNA-microarray platform is competent for the assays in biological fluids because of its high protein-resistance ability. This suggests that our platform holds great promise for applications in biomedicine. In addition, the successful logic gate operations enabled by exploiting miR-21, KpnI, and StuI-responsive assay as the model provide the basis for smart biosensing and genetic control in vitro. Combined with all the aforementioned advantages, the smart plasmonic nanobiosensor developed in this contribution is expected to provide a general platform for the quantitative investigation of various biological problems, such as those in the study of biomolecular interactions and kinetics, and DNA computers that function both in vitro and in vivo.

ASSOCIATED CONTENT Supporting Information. Supporting methods and figures. This material is available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION Corresponding Author *[email protected] *[email protected]

Notes The authors declare no competing financial interest.

ACKNOWLEDGMENTS We thank the National Key Research and Development Program of China (2017YFA0205302), the National Natural Science Foundation of China (61571239, 21475064, 61378081, 61705113), the Program for Changjiang Scholars and Innovative Research Team in University (IRT_15R37), the University Science Research Project of Jiangsu Province (NY217007), and the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD, YX03001).

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CONCLUSION In summary, we reported a smart plasmonic nanobiosensor used for detecting miR-21 at aM concentrations and realized DNA-based logic operations as well as biomemory by combining LSPR spectroscopy and DFM color imaging. The single tsDNA-modified Au@Ag NC was used as a plasmonic probe to bind the targets and generate unique signals. The target-responsive, time-dependent spectral peak shifts for the probes are not only highly specific to miR-21 but also display a linear logarithmic de-



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TOC Analysis of biomolecules at the single-molecule level is a great challenge in molecular diagnostics, gene profiling, and environmental monitoring. In this work, we design a smart plasmonic nanobiosensor based on individual Au@Ag core-shell nanocube (Au@Ag NC) modified with tetrahedron-structured DNA (tsDNA) for detecting microRNA 21 (miR-21) at the single-molecule level. An average localized surface plasmon resonance (LSPR) scattering spectral wavelength shift of approximately 0.4 nm is obtained for a single miR-21 hybridization event on the nanobiosensor. In addition, the three-dimensional finite-difference time-domain (3D-FDTD) simulations are carried out to confirm the sensing mechanism of the individual Au@Ag NC. Notably, this system not only allows the real-time detection of miR-21 with the sensitivity of an aM level over a large dynamic range from 1 aM to 1 nM, but also enables DNA-based logic operations as well as bio-memory by exploiting miR-21, KpnI, and StuI-responsive assays. Our study opens a unique method for single-molecule detection of biomolecules and thus holds great promise in a variety of biological and medical applications. KEYWORDS: localized surface plasmon resonance, tetrahedron-structured DNA, single-molecule level, logic operations, bio-memory †

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Ying Zhang, Zhenhua Shuai, Hao Zhou, Zhimin Luo, Bing Liu, Yinan Zhang, Lei Zhang,*, Shufen Chen, Jie Chao, Lixing Weng, †

Quli Fan, Chunhai Fan,

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Wei Huang,

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and Lianhui Wang*,

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Single-molecule analysis of microRNA and logic operations using a smart plasmonic nanobiosensor


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Single-Molecule Analysis of MicroRNA and Logic Operations Using a

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