Target-Triggered Catalytic Hairpin Assembly-Induced CoreâSatellite

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Target-Triggered Catalytic Hairpin Assembly-Induced Core-Satellite Nanostructures for High-Sensitive “Offto-On” SERS Detection of Intracellular MicroRNA Conghui Liu, Chao Chen, Shuzhou Li, HaiFeng Dong, Wenhao Dai, Tailin Xu, Yang Liu, Fan Yang, and Xueji Zhang Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.8b02819 • Publication Date (Web): 30 Jul 2018 Downloaded from http://pubs.acs.org on July 31, 2018

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Analytical Chemistry

Target-Triggered Catalytic Hairpin Assembly-Induced Core-Satellite Nanostructures for High-Sensitive “Off-to-On” SERS Detection of Intracellular MicroRNA Conghui Liu,

†

Chao Chen,

‡

Shuzhou Li, ‡ Haifeng Dong,

†,*

Wenhao Dai,

†

Tailin

Xu, † Yang Liu, † Fan Yang, † Xueji Zhang†,* †

Research Center for Bioengineering & Sensing Technology, School of Chemistry &

Biological Engineering, University of Science & Technology Beijing, Beijing 100083, P. R. China. ‡

School of Materials Science and Engineering, Nanyang Technological University,

Singapore 639798, Singapore

KEYWORDS: surface-enhanced Raman scattering, core-satellite, microRNA detection, catalytic hairpin assembly reactions, cell imaging

ABSTRACT: Surface-enhanced Raman scattering (SERS) technology is emerging as a powerful molecules detection method with distinct advantages of high stability, good specificity and low background signal compared with current prevailing fluorescence technique. However, the relative low sensitivity of SERS limits its wide applications. Engineered metallic nanoparticle aggregates with strong electromagnetic 1 ACS Paragon Plus Environment

Analytical Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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hot spots are urgently needed for low abundant molecules SERS detection. Herein, a microRNA

(miRNA)-triggered

catalytic

hairpin

assembly

(CHA)-induced

core-satellite (CS) nanostructure with multiple hot spots and strong electromagnetic field in nanogaps is designed. The unique plasmonic CS nanostructure is constructed by plasmonic Au nanodumbbels (Au NDs) as core and Au nanoparticles (Au NPs) as satellites, and it possesses enhanced electromagnetic field compared to that of Au NPs-Au nanorods (Au NRs) CS and Au NPs only. The “off-to-on” SERS strategy leads to a wide linear miRNA detection range from 10-19 to 10-9 M with a limit of detection (LOD) down to 0.85 aM in vitro. Intracellular accurate and sensitive miRNAs SERS imaging detection in different cell lines with distinct different miRNA expression levels are also achieved. The proposed SERS platform contributes to engineering metallic nanoparticle aggregates with strong electromagnetic intensity and has potential application in quantitative and precise detection significant intracellular molecules.

As a class of endogenously expressed, highly conserved and non-protein-coding small regulatory RNAs (19−23 nucleotides), microRNAs (miRNAs) are closely associated with almost all of biological processes, including early development, cell proliferation, differentiation, apoptosis and death.1,

2

Especially, miRNAs are

considered as emerging biomarkers for early diagnosis and prognosis of human cancers.3-5 Thus, developing miRNA detection and intracellular noninvasive monitoring strategies with high sensitivity, specificity and stability is particularly important. 2 ACS Paragon Plus Environment

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Surface-enhanced Raman scattering (SERS), a phenomenon that the Raman signals of analytes are amplified by several orders of magnitude when the analytes adsorb on the plasmonic metal (for example, copper, silver and gold) surface or locate in the nanogap of plasmonic metal nanostructures6,

7

due to the strong electromagnetic

coupling generated nearby the metal nanoparticles.6, 8 Compared to current prevailing fluorescence technology, the SERS exhibits many advantages like narrow peak width, unique fingerprint information, low photodegradation or photobleaching, label-free detection and little interference from water.7,

9-11

Nevertheless, relatively low

sensitivity compared to fluorescence method and extremely rare hot spots in substrates12 limit its further practical applications. Various controlled metallic nanoparticle aggregates with strong coupling, including dimers 16-23

12-15

or multimers11,

have been explored to create gaps or junctions (i.e., hot spots) for enhanced

sensitivity of SERS signals.7, 8

Core–satellite (CS) nanostructures are typical strongly coupled plasmonic assemblies, which can generate intense SERS signal change once the nanostructure change. Gandra et al. constructed shape-controlled CS nanostructures SERS probes of gold nanoparticles (Au NPs) containing in-built electromagnetic hot-spots through simple molecular cross-linkers.24 Xu’s group established a series of CS nanostructures through DNA hybridization with Au nanorods (Au NRs) as core and Au,16, 25 Ag,17 Ag2S26 or upconversion NPs11, 27, 28 as satellites for molecule detection and imaging.29 However, almost all of them are “turn off” CS nanosensors, and the SERS signals of these CS sensors significantly decreased with increasing concentration of targets. 3 ACS Paragon Plus Environment


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Under some circumstances, positive SERS signals (turned on) may be preferred to negative

SERS

signal

(turned

off)

upon

target

binding,

7

and

the

“one-target-one-triggered signal” model cannot satisfy the low-abundant miRNA detection (about 0.01% of the mass in total RNA sample2). Engineered plasmonic metal

CS

aggregates

with

strong

electromagnetic

field

combined

with

signal-amplified strategies are urgently needed.

Herein, we designed a new assembly strategy of rational CS nanostructures constructed by plasmonic Au nanodumbbels (Au NDs) as core and Au NPs as satellites using target miRNA-triggered recycling amplification for sensitive miRNA SERS detection and intracellular miRNA imaging. The finite difference time domain (FDTD) simulations confirmed the significantly enhanced electromagnetic intensity of the Au NPs-Au NDs CS than that of Au NPs-Au NRs CS and Au NPs only. As hybridization catalysts, the target miRNA induced the assembly of the proposed CS nanostructures where the newly generated hot spots provided intensely enhanced SERS signals and led to a highly sensitive “off-to-on” miRNAs SERS detection. Intracellular accurate and sensitive miRNAs SERS imaging detection in different cell lines were also demonstrated.

EXPERIMENTAL SECTION

Materials: All of the chemical reagents were used without further purification. Hydrogen tetrachloroaurate (III) trihydrate (HAuCl4) (99%), tris (carboxyethyl) phosphinehydrochloride

(TCEP),

and

3-(4,

5-dimethyl-2-thiazolyl)-2, 4

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5-diphenyl-2-H-

tetrazoliumbromide

(MTT),

Tris(hydroxymethyl)methyl

aminomethane (Tris) were purchased from Sigma-Aldrich (China). Sodium citrate, hydrochloric acid (HCl), sodium borohydride (NaBH4), silver nitrate (AgNO3), hydroxylamine (NH2OH), Cetyltrimethylammonium bromide (CTAB, 99%) and L-ascorbic acid were obtained from Sinopharm Chemical Reagent Co., Ltd (Beijing, China). PBS (pH 7.4, 10 mM), fetal bovine serum (FBS), Dulbecco’s modified Eagle’s medium (DMEM), Opti-MEM, trypsin-EDTA and penicillin-streptomycin were

purchased

from

Gibco

Life

Technologies

(AG,

Switzerland).

4%

paraformaldehyde fix solution was obtained from Beyotime. All water used in this study were ultrapure water (≥18 MΩ, Milli-Q, Millipore, Billerica, MA). The oligonucleotides were synthesized by Sangon Biological Engineering Technology & Co., Ltd (Shanghai, P. R. China) and purified using high-performance liquid chromatography. The sequences of oligonucleotides used in this study were as follows:

5PSA20 DNA:

5’-A*A*A*A*A*AAAAAAAAAAAAAAA-3’ (the * means PS modification)

Hairpin 1 (H1):

5’-CCT GCT CCA AAA ATC CAT TGT GGT GTA AAT GGA TTT TT-3’

5’-SH (CH2)6 CCT GCT CCA AAA ATC CAT TGT GGT GTA AAT GGA TTT TT -Rox-3’ 5 ACS Paragon Plus Environment


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Hairpin 2 (H2):

5’-CCA TTT ACA CCA CAA TGG ATT TTT GTG GTG TA-3’

5’-SH (CH2)6 CCA TTT ACA CCA CAA TGG ATT TTT GTG GTG TA-3’

All the RNA sequences were purchased from Shanghai Gene Pharma Co., Ltd. (Shanghai, PRC), purified using high-performance liquid chromatography. The sequence was listed as follows:

Target mir-1246:

5’-AAU GGA UUU UUG GAG CAG G-3’

1-bp mismatch mir-1246:

5’-AAU GGA UUU UUG GAG UAG G-3’

2-bp mismatch mir-1246:

5’-AAU AGA UUU UUG GAG UAG G-3’

mir-1246 mimics (duplex):

5’-AAU GGA UUU UUG GAG CAG G-3’

3’-UUUUA CCU AAA AAC CUC GU-5’

mir-1246 inhibitor:

5’-CCU GCU CCA AAA AUC CAU U-3’ 6 ACS Paragon Plus Environment

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Instruments: The morphologies of Au NPs, Au NRs, Au NDs and core-satellite structure were examined with transmission electron microscopy (TEM) (HT7700, Hitachi, Japan) at an acceleration voltage of 200 kV. The UV-visible (UV-vis) absorption was acquired with a UV-1800 spectrophotometer (Shimadzu, Japan). A Veriti 96-Well Thermal Cycler-PCR machine (Applied Biosystems, USA) was used for temperature control involved in the hybridization reaction. Raman spectra were recorded on an InVia-Reflex Raman microscope (Renishaw) system equipped with a 633 nm diode laser through a 50×, 0.75 NA objective (NPLAN EPI; Leica). Raman imaging were obtained by Laser scanning Raman microscope (Raman-11, Nanophoton Corporation). Preparation of 15 nm gold Nanoparticles (Au NPs)12：10 mL of sodium citrate (1%) was added to 100 mL of HAuCl4 solution (1 mM) at boiling point to obtain the Au nanoparticles (~15 nm). The concentration of Au NPs was estimated about 4.5 nM.

Preparation of gold nanorods (Au NRs): Au NRs were prepared according to previous reports

30-32

. Firstly, a mixture with HAuCl4 (0.25 mM) and CTAB (0.1 M)

solution was prepared. Then, freshly prepared NaBH4 solution (600 uL, 10mM) at 0 ℃ was added to the HAuCl4/CTAB solution (10 mL) stirred vigorously for 10 min to achieve gold seed solution. The growth solution was prepared by adding hydrochloric acid (0.35 mL, 1 M) and ascorbic acid (0.55 mL, 0.1 M) to a mixture solution of CTAB (95 mL, 0.1 M), AgNO3 (1 mL, 10 mM) and HAuCl4 (5 mL, 10 mM) with 7 ACS Paragon Plus Environment


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gentle stirring. Finally, 0.12 mL of gold seed solution was added to the growth solution. After standing overnight undisturbedly for 14-16 h, the colored Au NRs solution was purified by centrifugation to remove excess CTAB (twice at 14 000 rpm, 3 min each), and then were redispersed in CTAB (100 mL, 5mM) solution.

Preparation of gold nanodumbbells (Au NDs): Au NDs were prepared according to previous reports

31

. Briefly, 900 uL of gold nanorods were incubated with five

phosphorothioate (PS)-modified DNA (5PSA20, 29.75 µL, 100µM) for 1 hour. Then 2.5 µL of 400 mM NH2OH solution (pH was adjusted to 5.0 by HCl) was added and mixed. Finally, 10 µL of 1% HAuCl4 was added into the former mixture and vortexed. After standing for 1.5 hours at room temperature, the resulting Au NDs solution was washed by water and redispersed in 1 mL ultrapure water. Preparation of Au NPs-H1 probes16: 500 µL of Au NPs were centrifuged at 13 000 rpm for 10 min and redispersed in 1 mL ultrapure water contained 0.01% sodium dodecylsulfate (SDS). The thiol- and Rox-functionalized H1 (12 µL, 100 µM) was activated by 12 µL of 0.1 mM TCEP (1:100 molar ratio) for 1 hour at room temperature in order to cleave the disulfide bond, incubated with 100 µL of treated Au NPs solution at room temperature for 20 min, and aged for another 12 h with 0.7 M NaCl at room temperature. Finally, the mixed solution was washed by centrifugation at 13000 rpm for 10 min with ultrapure water for three times to remove the excess of H1 and stored in 100µL of 25 mM Tris-HCl (pH=7.4) buffer at 4 oC.

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Preparation of Au NDs-H2

16, 33

: 1 mL of Au NRs were centrifuged at 7500 rpm

for 15 min and redispersed in 100 µL 25 mM Tris-HCl (pH=7.4) buffer in order to remove the excess ascorbic acid, AgNO3, and small spherical particles. Then, thiolatemodified H2 (40 µL, 100 µM, activated by TCEP) was added and reacted at room temperature for 12 h with gentle shaking. Finally, the mixed solution was washed by centrifugation at 6000 rpm for 10 min with ultrapure water for three times to remove the excess of H2 and stored in 100 µL of 25 mM Tris-HCl (pH=7.4) buffer at 4 oC. Au NRs-H2 probes were prepared in the same steps. Preparation of core-satellite assemblies11, 16, 17: 5 µL of Au NPs-H1 probes were mixed with 1 µL of Au NDs-H2 probes and 6 µL of 25 mM Tris-HCl (pH=7.4) buffer containing various concentrations of mir-1246 with gentle shaking for several minutes. After incubated for 12 h with gentle shaking at room temperature, the mixture was washed by centrifugation at 4500 rpm for 10 min with ultrapure water to remove the excess of Au NPs-H1 nanoprobes and stored in 12 µL of 25 mM Tris-HCl (pH=7.4) buffer at 4 oC. The resulting mixture of catalytic hairpin assembly (CHA) reactions was named Au NPs-Au NDs core-satellite assemblies (Au NPs-Au NDs CS). Au NPs-Au NRs core-satellite assemblies by CHA (Au NPs-Au NRs CS) were prepared in the same steps by mixing Au NPs-H1 probes, Au NRs-H2 probes and mir-1246. After hybridization procedure, the Raman spectra of the CS assemblies were collected by a confocal Raman spectrometer under excitation at 633 nm. Five measurements were obtained from different positions of each sample and the mean values were used in further analysis and calculation. Each experimental group were 9 ACS Paragon Plus Environment


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prepared in triplicate. The LOD was calculated by extrapolating the concentration from the signal of background plus 3SD of the background signal.34 Cytotoxicity assay: 104 A549 cells per well were seeded in 96-well plates and cultured

in

DMEM

culture

medium

containing

10%

FBS

and

1%

penicillin/streptomycin at 37 oC under 5% CO2 for 12 h. Then, the cells were treated with OPTI-MEM solution containing Au NPs-H1 nanoprobes or Au NDs-H2 nanoprobes at different concentrations and incubated for another 4 h. After replaced with fresh culture media, the cells were further incubated for 24 h. Finally, 10 µL MTT solution (5 mg/mL MTT in 10 mM PBS, pH 7.4) was added to each well. After incubated for 4 h,

the absorbance at 492 nm of cells were measured by microplate

reader. The control was the cells without addition of any nanoprobes. mir-1246 mimics and antisense mir-1246 transfection: The cells (105 cells per well) were seeded in 6-well plates and cultured in DMEM culture medium containing 10% FBS (without penicillin/streptomycin) at 37 oC under 5% CO2 for 12 h. The cells were then transfected by synthetic antisense mir-1246 or mir-1246 mimics. The transfection experiments were carried out using Lipofectamine 2000 Transfection Reagent (Life Technologies, Carlsbad, USA) following the standard procedures. 2 mL Opti-MEM containing Lipofectamine 2000 (10 µL) and mir-1246 mimics (2.5 pM, 25 pM) or antisense mir-1246 (2.5 pM, 25 pM) in each well were replaced with DMEM culture medium containing 10% FBS after incubation for 4 h, and then the cells were incubated for another continuous 18 h. 10 ACS Paragon Plus Environment

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Quantification of intracellular mir-1246 concentration: Firstly, A549 cells seeded in culture dishes were transfected with different amounts of mir-1246 followed the abovementioned steps. Then, 1.5 mL Opti-MEM containing Au NPs-H1 probes (125 µL) and Au NDs-H2 probes (25 µL) in each well were replaced with DMEM culture medium containing 10% FBS after incubated for 4 h. After incubated for another continuous 12 h, the Raman intensity of cells were measured. The intracellular mir-1246 concentration was determined by the Raman intensity of the probe-treated cells and the PCR calibration curves.

qRT-PCR procedure for mir-1246 analysis: Total cellular RNA were respectively extracted from A549, MCF-7, Hela and NHDF cells using miRcute miRNA isolation kit (DP501, Tiangen Biotech (Beijing) Co.,Ltd.) in accordance with the manufacturer’s procedure. qRT-PCR reactions were performed by miRcute miRNA first-strand cDNA kit (KR201) and qPCR detection kit (SYBR Green, FP401) from Tiangen Biotech (Beijing) Co.,Ltd. according to the manufacturer’s instructions. qRT-PCR analysis was conducted by Eco Real-Time PCR System (Illumina, USA).

Raman imaging of living cells: The MCF-7, HeLa and NHDF cells were seeded in confocal dishes (105 cells per well) and cultured in DMEM media containing 10% FBS and 1% penicillin/streptomycin at 37 oC under 5% CO2. After incubated for 12 h, Au NPs-H1 probes (125 µL) and Au NDs-H2 probes (25 µL) were added into 1.5 mL culture medium in each wells, and the cells were cultured for another 4 h at 37 °C. 11 ACS Paragon Plus Environment


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After that, the cell were washed with PBS (10 mM, pH=7.4) to remove the free probes and incubated for another 12 h. Before imaging, the cells were treated with 4% paraformaldehyde fix solution for 15 min and washed with PBS (10 mM, pH=7.4) three times. The spectra of the probe-treated single cells were collected on a Raman confocal microscope with a 633 nm laser (1 mW), an exposure time of 10 s, and a 100× objective lens. The Raman imaging of cells were acquired at an interval of 1 µm with an exposure time of 0.1 s (633 nm excitation, 10 mW, 100× objective lens).

Finite difference time domain (FDTD) simulation: The field enhancement of the nanostructures was calculated by MIT Electromagnetic Equation Propagation (Meep).35-37 Meep is an open-sourced simulation package that adopt FDTD to solve Maxwell equations. The simulation region was a cuboid space, the edge-length of this region was 400 nm, and the optical media inside was vacuum, with its permittivity equaled to 1.7. A plane wave source was placed inside the simulation region with size of 400 nm × 400 nm in positive z-axis. The mesh of FDTD simulation region was set to be 1.0 nm in all the three directions, and the courant factor was 0.5.

The permittivity of gold was expressed by Lorentz model as shown below, ω = 1 + ∑

(1)

Totally six Lorentzian terms were used to fit the published data in 1972 from Johnson and Christy38.


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For near field spectra, the wave length of the plane wave source was set to be 542 nm (for Au NPs-Au NDs CS), 531 nm (for Au NPs-Au NRs CS) and 523 nm (for single Au NP), respectively. The chosen wavelength equaled the LSPR peak value calculated in far field simulation for each system under study.

RESULTS AND DISCUSSION

Scheme 1A showed the fabrication route of CS nanostructures assembled by Au NPs and Au NDs. The Au NDs were synthesized from DNA-mediated growth of Au NRs.31 Compared with Au NRs, the irregular Au NDs with sharp tips presented more hot spots and enhanced electromagnetic intensity.7 Using oncogenic microRNA-1246 (mir-1246) in non-small cell lung cancer A549 cells as a model, two types of programmable oligonucleotide hairpins were designed (Figure S1A), a 5’-thiolated and 3’-Rox labeled hairpins H1 (containing a mir-1246 complete complementary sequence at 5’ terminal) and a 5’-thiolated hairpins H2 (containing a sequence complementary to H1 at 5’ terminal), and respectively anchored on the surface of Au NPs and Au NDs via Au−S bonds to form Au NPs-H1 and Au NDs-H2 nanoprobes. After being delivered to target cells (Scheme 1B), the hybridization between H1 and mir-1246 induced the exposure of the hidden toehold in the stem of H1 to recognize H2, which further displaced the target miRNAs from H1 to form stable H1-H2 duplex. The released target miRNA could induce next cycle, which finally led to the periphery of Au NDs anchored with an abundance of Au NPs to form CS nanostructures. 13 ACS Paragon Plus Environment


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During the assembly process, the SERS signal molecules in the 3’ terminal of H1 was taken close to the surface of Au NDs and located at the nanogaps of CS nanostructures, which produced a strong SERS signal for detection (Figure 1A).

Agarose gel electrophoresis analysis confirmed feasibility of the target miRNAs-mediated catalytic hairpin assembly (CHA) process (Figure S1B). Au NPs and Au NDs were synthesized followed previous protocol.12, 31 The mean diameter of Au NPs was ~15 nm (Figure S2A). The Au NDs were constructed by using phosphorothioate (PS)-modified DNA sequences (5PS-A20) to control the overgrowth of Au NRs (length 53 ± 1 nm × width 15 ± 0.8 nm, Figure S2B). The DNA 5PS-A20 has strong binding affinity to the side walls but sparsely distribute on the tips of Au NRs due to the higher surface curvature of end facets, resulting in less growth on the sides and preferentially growth at the ends of the Au NRs thus dumbbell shape formation.31 The average diameter of spherical ends of achieved Au NDs was 41 ± 4 nm, and the gap distance between the two spherical ends was 50 ± 2 nm (Figure 1B). The successfully synthesized Au NPs-H1 and Au NDs-H2 nanoprobes were demonstrated by the UV-vis spectra (Figure S3 and S4), and their assembly Au NPs-Au NDs CS were displayed by TEM (Figure 1C). The much larger hydrodynamic diameter of the Au NPs-Au NDs CS nanostructures than that of the individual components also confirmed their assembly (Figure 1D). Compared to the Au NRs, the longitudinal and transverse localized surface plasmon resonance (L-LSPR and T-LSPR) band of Au NDs were significantly red-shifted to 1005 nm and slightly red-shifted to 535 nm, respectively (Figure 1E). After forming Au 14 ACS Paragon Plus Environment

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NPs-Au NDs CS structure, the L-LSPR and T-LSPR band displayed redshift to 1042 nm and 548 nm respectively, in agreement with previous reports.16

The enhanced electromagnetic intensity of the CS assemblies were further experimentally and theoretically investigated. As show in Figure 2A, the Rox exhibited three characteristic Raman peaks located at 1348, 1501, and 1646 cm−1 respectively, which attributed to the stretching vibrations of ring C–C in Rox.39 The Au NDs-H2 and the mixture of H1, H2 and mir-1246 displayed hardly any SERS signals, while Au NPs-H1 and the mixture of Au NPs-H1 and Au NDs-H2 generated obvious SERS signals with similar intensity due to the electromagnetic coupling generated on the Au NPs surface. Notably, the Au NPs-Au NDs CS generated the strongest SERS intensity, as a result of the interparticle plasmonic coupled hot spots in CS nanostructures. The stability and specificity of the nanoprobes and the CS nanostructures were further investigated. As demonstrated in Figure 2B, C, 10% fetal bovine serum (FBS) and dulbecco's modified eagle medium (DMEM) had negligible effect on the Raman signal of the Au NPs-Au NDs CS, and the Raman intensity of the CS nanostructures showed little change in the wide pH range of 5~9 (Figure S5). Furthermore, the Raman intensity of the Au NPs-Au NDs CS nanostructures to 1-bp mismatched mir-1246 (0.5 pM) and 2-bp mismatched mir-1246 (0.5 pM) were similar to the control and much less than that to target mir-1246. Therefore, the probes possessed great stability and base-mismatched discrimination ability. The FDTD simulations were conducted to theoretically reveal the electromagnetic field distribution of Au NPs, Au NPs-Au NRs CS and Au NPs-Au NDs CS 15 ACS Paragon Plus Environment


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nanostructures (Figure 3A-C). It was found that the hot regions (orange and red color) of Au NPs-Au NDs CS mainly distributed in the junctions between the spherical ends of Au NDs and the Au NPs (Figure 3C), which was stronger than that of Au NPs surface (Figure 3A) and Au NPs-Au NRs CS (Figure 3B). The corresponding maximum electromagnetic field intensity (Ex) of three nanostructures was 3.73 V/m at the surface of Au NPs, 5.24 V/m and 12.46 V/m in the hot spots of Au NPs-Au NRs CS and Au NPs-Au NDs CS, respectively (Figure 3D). The significantly higher electromagnetic field intensity contributed to stronger SERS signals, theoretically confirmed the rational design of Au NPs-Au NDs CS. The strong electromagnetic intensity of Au NPs-Au NDs CS inspired us to further investigate its application in miRNA SERS detection. In the presence of target mir-1246, the Au NPs-H1 and Au NDs-H2 nanoprobes assembled into Au NPs-Au NDs CS nanostructure by CHA reaction (Figure 1A). The Raman intensity increased with the increasing mir-1246 concentration from 0.5 aM to 5 nM (Figure 4A), in consistent with the result of TEM images that the number of Au NPs in Au NPs-Au NDs CS nanostructures increased with the increasing mir-1246 concentration (Figure S6). The corresponding quasi-linear relationship between the Raman intensity at 1501 cm−1 and the logarithm of mir-1246 concentrations with an estimated limit of detection (LOD) of 0.85 aM calculated by extrapolating the concentration from the signal of background plus three folds standard deviation (SD) of the background signal (Figure 4B). As controls, the performance of Au NPs-Au NRs CS nanostructures (Figure 4C, Figure S7) was also explored, and the detectable linear 16 ACS Paragon Plus Environment

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range was 5 aM to 5 nM with a LOD of 20.1 aM (Figure 4D). These results confirmed the advanced SERS sensitivity of the proposed Au NPs-Au NDs CS nanostructures. It also exhibited superior performance than previously reported SERS miRNA biosensors with signal amplification (Table 1). The greatly enhanced sensitivity of the proposed SERS biosensors was attributed primarily to the following unique features: (1) the target miRNA-triggered CHA reaction exhibited high amplification efficiency, which induced numerous assembly between Au NPs and Au NDs and generated abundant hot spots to obtain enhanced SERS signal. (2) The irregular Au NDs with sharp tips provided more hot spots and enhanced electromagnetic intensity in the Au NPs-Au NDs CS nanostructures compared to Au NPs-Au NRs CS nanostructures as SERS substrates.7 The high sensitivity of the proposed system satisfied the low-abundant intracellular miRNA detection, which was further explored. The MTT assay was first employed to evaluate the cytotoxicity of two nanoprobes. The two nanoprobes showed almost no cytotoxicity toward living cells at the applied concentration (Figure S8). Figure 5A presented the Raman images of A549 cells treated with different nanostructures. The outline of the measured A549 cells in red color was mainly attributed to the C-H stretching vibrations of cells in the 2800-3200 cm-1 range,40 and the green color of measured mir-1246 was found to focused on the cytoplasm (Figure S9) came from the stretching vibrations of ring C–C in Rox at 1501 cm-1,39 which was consistent with the fact that mature single-stranded miRNAs mainly distributed in the cytoplasm.2 Compared to the control group without 17 ACS Paragon Plus Environment


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nanoparticles treatment, Au NPs-H1 probes treated-A549 cells exhibited slight Raman signal of Rox, while the cells treated with Au NPs-H1 and Au NRs-H2 nanoprobes presented stronger Raman signal of Rox associated with miRNAs induced the formation of Au NPs-Au NRs CS assembly and generated hot spots. Notably, the A549 cells incubated with Au NPs-H1 and Au NDs-H2 nanoprobes for 12 h showed strongest green signal resulted from the reorganization of target miRNA and formation of Au NPs-Au NDs CS assembly by miRNA triggered CHA reaction. The Raman intensity from cells with Au NPs-Au NDs CS formation was 14.1 times higher than that of Au NPs-H1-treated cells and 4.8 times higher of cells with Au NPs-Au NRs CS formation (Figure 5B). These results demonstrated the highly catalytic amplification ability of CHA-based Au NPs-Au NDs CS assembly strategy for intracellular low-abundant miRNA imaging. Next, the feasibility of the proposed strategy to monitor the change of intracellular relative expression level of mir-1246 in a single A549 cell transfected with antisense mir-1246 or mir-1246 mimics was explored. Compared to the control (Figure 5C column iii), the Raman signal of antisense mir-1246 transfected cells was obviously decreased (Figure 5C column i, ii), and the intensity of green Raman signal inversely decreased with the increase of antisense mir-1246 concentrations. On the contrary, the green Raman signal significantly increased in the mir-1246 mimics treated cells (Figure 5C column iv, v), and the intensity of green Raman signal increased with the increase of mir-1246 mimics concentrations. Figure 5D clearly presented the intensity change of the Raman signals at 1501 cm-1. These results suggested it was 18 ACS Paragon Plus Environment

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powerful to monitor the expression level change of intracellular low abundant oncogenic miRNA, holding great potential for cancer diagnosis and prognosis. Furthermore, the CHA-based CS assembly strategy for high-sensitive imaging low-abundance miRNAs in different living cells were also investigated, including two cancer cell lines of Hela and MCF-7 with high expression of mir-1246, and a normal human dermal fibroblasts (NHDF) with low expression of mir-1246. As expected, weak but distinguishable green Raman signals was observed in NHDF cells, while MCF-7 cells displayed a stronger green Raman signal, and the Hela cells exhibited strongest green signal (Figure 6A), which indicated the higher expression level of mir-1246 in Hela cells than that in MCF-7. The mir-1246 quantity were calculated to be 0.18, 0.82 and 0.30 pmol/106 cells/mL in NHDF, HeLa, and MCF-7 cells respectively according to qRT-PCR measurements (Figure 6B), which was consistent with the results obtained by Raman imaging in Figure 6A, indicating it is satisfied for sensitive Raman imaging of the low-abundance miRNA in living cells and potentially applied in early diagnosis of cancer with high accuracy. CONCLUSION

In conclusion, we have first developed a target miRNA-triggered Au NPs-Au NDs CS assembly strategy with CHA signal amplification, which led to highly efficient and sensitive miRNA SERS detection in vitro and Raman imaging of low-abundant miRNAs in living cells with “off-to-on” signal. Compared to that of Au NPs and Au NPs-Au NRs CS, experimentally and theoretically characterized the much stronger electromagnetic field generation during Au NPs-Au NDs CS nanostructures assembly 19 ACS Paragon Plus Environment


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in the presence of target miRNAs. The greatly high sensitivity of the proposed SERS biosensors was primarily attributed to the irregular Au NDs with sharp tips, which provided stronger electromagnetic intensity in Au NPs-Au NDs CS nanostructures than Au NRs in Au NPs-Au NRs as SERS substrates. Meanwhile, the target miRNA-triggered CHA reaction with high amplification efficiency generated abundant hot spots. The SERS platform is benifical to design metallic nanoparticle aggregates with strong electromagnetic field for quantitative and precise SERS detection of significant intracellular molecules. ASSOCIATED CONTENT

Supporting Information.

The following files are available free of charge. Agarose gel electrophoresis experiment, TEM of Au NPs, Au NRs and Au NPs-Au NDs CS, UV−vis spectra, MTT results, and Raman spectra acquired from different positions of cells (PDF)

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

Notes The authors declare no competing financial interest.

ACKNOWLEDGMENT 20 ACS Paragon Plus Environment

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We gratefully acknowledge support from National Key R&D Program of China (Grant No. 2016YFC0106602 and 2016YFC0106601); National Natural Science Foundation of China (Grant No. 21645005, 21475008); the Open Research Fund Program of Beijing Key Lab of Plant Resource Research and Development, Beijing Technology and Business University (PRRD-2016-YB2).



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Scheme 1. Schematic representation of (A) the fabrication routes of Au NPs-Au NDs CS and (B) Au NPs-Au NDs core–satellite (CS) SERS sensors for miRNA detection and imaging in living cells.



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Figure 1. (A) Scheme of target miRNAs-triggered assembly of Au NPs-Au NDs CS. (B, C) TEM image of Au NDs and Au NPs-Au NDs CS (mir-1246: 0.5 µM), respectively. (D) DLS intensity and (E) UV−vis absorption spectra of Au NPs, Au NRs, Au NDs and Au NPs-Au NDs CS.


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Figure 2. (A) SERS spectra of H1+H2+mir-1246, Au NDs-H2 probe, Au NPs-H1 probe, mixture of Au NPs-H1 probes and Au NDs-H2 probes and Au NPs-Au NDs CS (mir-1246: 0.5 pM). (B) SERS spectra and (C) Raman intensity (1051 cm-1) discrimination of the nanoprobe to 1-bp mismatched mir-1246, 2-bp mismatched mir-1246, 10% FBS and DMEM (mir-1246: 0.5 pM).

Figure 3. Electromagnetic field distribution of (A) Au NPs, (B) Au NPs-Au NRs CS and (C) Au NPs-Au NDs CS obtained from FDTD simulations. (D) Maximum of field in the hot spots of Au NPs, Au NPs-Au NRs CS and Au NPs-Au NDs CS



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obtained from (A-C) respectively. Excitation wavelength is 633 nm, and the gap between the cores and satellites is 5 nm.

Figure 4. (A) Raman spectra of Au NPs-H1 probes (0.9 nM) and Au NDs-H2 probes (0.1 nM) responded to different concentrations of mir-1246 (0, 0.05 aM, 0.5 aM, 5 aM, 5 fM, 5 pM, 50 pM, 5 nM) in vitro and (B) linear relationship between the Raman intensity at 1501 cm-1 peak and the logarithm of mir-1246 concentration. (C) Raman spectra of Au NPs-H1 probes (0.9 nM) and Au NRs-H2 probes (0.1 nM) responded to different concentrations of mir-1246 (0, 0.05 aM, 0.5 aM, 5 aM, 5 fM, 5 pM, 50 pM, 5 nM) in vitro and (D) linear relationship between the Raman intensity at 1501 cm-1 peak and the logarithm of mir-1246 concentration.


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Figure 5. Bright field image and Raman images of cells based on the dual-color Raman imaging mode. (A) A549 cells were detected with Au NPs-H1 probes (0.18 nM) and Au NDs-H2 probes (0.021 nM), Au NPs-H1 probes (0.18 nM) and Au NRs-H2 probes (0.021 nM), or Au NPs-H1 probes (0.18 nM) only. (B) Raman intensity at 1501 cm-1 of A549 cells with different treatment in (A). (C) A549 cells were transfected with Lipofectamine 2000 mixed with different amounts of antisense mir-1246 (i: 25 pM, ii: 2.5 pM, iii: 0 pM) or mir-1246 mimics (iv: 2.5 pM, v: 25 pM) at 37 oC for 18 h, then detected with Au NPs-H1 probes (0.18 nM) and Au NDs-H2 probes (0.021 nM). (D) Raman intensity at 1501 cm-1 of A549 cells with different treatment in (C).

Figure 6. Bright field image and Raman images of

(A) Hela, NHDF and MCF-7

cells were treated with the mixture of Au NPs-H1 probes (0.18 nM) and Au NDs-H2 29 ACS Paragon Plus Environment


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probes (0.021 nM) respectively. (B) The expression level of mir-1246 in NHDF, HeLa and MCF-7 cells detected by qRT-PCR (left) and Raman intensity at 1501 cm-1 detected by CHA strategy (right). Scale bar: 8 µm.

Table 1. Comparison of the CHA-mediated CS assembly-based SERS miRNA Biosensors to other SERS miRNA Biosensors with signal amplification

Detection target

Amplification Method

LOD

Linear ranges Refs

mir-203

HCR[a]

0.15 fM

10-15~10-12 M

41

mir-141

HCR

0.17 fM

10-15~10-7 M

19

mir-203

SDA[b]

6.3 fM

10-14~10-8 M

42

mir-155

RCA[c]

70.2 aM

10-16~10-10 M

43

let-7b

SDA+DSN[d]

0.3 fM

10-15~10-9 M

44

mir-155

SDA+DSN

83 aM

10-16~10-10 M

45

mir-1246

CHA

0.85 aM

10-19~10-9 M

this work

[a] Hybridization chain reactions.

[b] Strand displacement amplification.

[c] Rolling circle amplification.

[d] Duplex-specific nuclease.


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