Spiny Nanorod and Upconversion Nanoparticle Satellite Assemblies

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Spiny Nanorod and Upconversion Nanoparticle Satellite Assemblies for Ultrasensitive Detection of Messenger RNA in Living Cells Rui Gao, Changlong Hao, Liguang Xu, Hua Kuang, and Chuanlai Xu Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.8b00617 • Publication Date (Web): 26 Mar 2018 Downloaded from http://pubs.acs.org on March 26, 2018

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Analytical Chemistry

Spiny Nanorod and Upconversion Nanoparticle Satellite Assemblies for Ultrasensitive Detection of Messenger RNA in Living Cells Rui Gao1,2, Changlong Hao1,2, Liguang Xu1,2*, Chuanlai Xu1,2*, Hua Kuang1,2* 1 2

International Joint Research Laboratory for Biointerface and Biodetection, State Key Lab of Food Science and Technology, School of Food Science and Technology, Jiangnan University, Wuxi, Jiangsu, 214122, PRC

ABSTRACT: Quantitation and in situ monitoring of target messenger RNA (mRNA) in living cells remains a significant challenge for the chemical and biomedical communities. To quantitatively detect mRNA expression levels in living cells, we have developed DNA-driven gold nanorod coated platinum-upconversion nanoparticle satellite assemblies (termed Au NR@Pt-UCNP satellites) for intracellular thymidine kinase 1 (TK1) mRNA analysis. The nanostructures were capable of recognizing target mRNA in a sequence-specific manner as luminescence of UCNPs was effectively quenched by Au NR@Pt within the assemblies. Following recognition, UCNPs detached from Au NR@Pt, resulting in luminescence restoration to achieve effective in situ imaging and quantifiable detection of target mRNA. The upconversional luminescence intensity of confocal images showed a good linear relationship with intracellular TK1 mRNA ranging from 1.17 to 65.21 fmol/10 µg RNA and a limit of detection (LOD) of 0.67 fmol/10 µg RNA. We believe that our present assay can be broadly applied for detection of endogenous biomolecules at the cellular and tissue levels and restoration of tissue homeostasis in vivo.

Messenger RNA (mRNA) plays a key role in encoding the genetic information of DNA and ribosome-mediated translation into the corresponding proteins 1,2. Spatiotemporal distribution of specific mRNAs is constitute an important component of biological research3,4 owing to their significant utility in early diagnosis, drug discovery, biochemical pathways and treatment of disease5–8. There remains an urgent demand for sensitive, accurate, robust and quantitative detection methods to measure intracellular mRNA levels. Several methods have been developed for detection of RNA to date, ranging from simple to complex and multi-step procedures8–11. Owing to the inherent limitations of reverse transcriptase polymerase chain reaction (RT-PCR), such as tedious mRNA extraction and amplification processes and risk of contamination with genomic DNA, in situ and real time visualization as well as quantitation of the spatiotemporal variations in mRNA levels in living cells pose considerable difficulties. To meet the demand for effective and sensitive detection of intracellular mRNA, a number of live imaging methods, such as molecular beacons12–22, protein labeling methods23, catalytic hairpin assembly24, rolling circle amplification25,26, hybridization chain reaction27,28, fluorescence in situ hybridization29–31 and sticky-flare8 probes, have been evaluated in recent years. While the majority of these procedures focus on qualitative imaging of target mRNA in live cells, a few (for instance, sticky-flares with organic fluorescence dyes as the reporting signals8) have been successfully applied to achieve quantification of mRNA in live cells. However, these organic fluorescent dyes present poor photostability, broad absorbance and emission and potential interference with background autofluorescence, which limit the effectiveness of intracellular quantification of mRNA.32,33 To achieve accurate quantification as well as in situ and real time tracking of specific mRNA in live cells, the development of a new type of analysis platform

without genomic DNA, nuclease and autoflorescence interference is essential. Owing to their unexpected optical features34–36, intracellular stability and ease of entry into cells37, nanoscale assemblies have attracted significant research attention for use as biosensors10,38–43 as well as drug delivery42,44–47 and cancer therapy agents48–54. In particular, rare earth upconversion nanoparticles (UCNP) present a type of anti-Stokes material with attractive optical features, such as low photodamage and high photostability. The most significant feature of UCNPs is the ability to avoid autofluorescence interference47,54–60. These structures thus present excellent potential building blocks to form assembled nanoprobes that can be applied for intracellular target mRNA detection. Here, we constructed DNA-driven core-satellite selfassembled compositions (denoted Au NR@Pt-UCNP satellites) by coating gold nanorods with a layer of thorn-like platinum and UCNPs. In the presence of target mRNAs, those can compete to recognize the DNA framework that forms the Au NR@Pt-UCNPs satellites, resulting in disruption of the satellite structure and UCNPs are dissociated from the satellites along with those luminescence signals restored. The Au NR@Pt-UCNPs satellite assemblies generated in this study are fully capable of in situ imaging and achieve accurate and highly selective detection of target mRNAs in live cells using upconversional luminescence signal responses. EXPERIMENTAL SECTION DNA-functionalized Au NR@Pt Au NR@Pt nanoparticles were re-suspended in 5 mM hexadecyltrimethylammonium bromide (CTAB). Polyethylene glycol (PEG MW=5000) was added at a molar ratio of 120:1. Au NR@Pt particles were centrifuged (7000 rpm for 10 min) after incubation at 37°C for 10 h and re-suspended in 5 mM

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CTAB. DNA1 was added to Au NR@Pt at a DNA/Au NR@Pt molar ratio of 400:1. After reaction for 12 h at 37°C, Au NR@Pt particles were centrifuged in 5 mM CTAB (7000 rpm) for use. UCNP modification with DNA UCNPs were added into Tris-HCl buffer and DNA2 added to the mixture at a DNA2/UCNP molar ratio of 40:1. After reaction for 12 h at 37°C, the solution was centrifuged at 9000 rpm with 10 K UF tube for 10 min and concentrated by TrisHCl buffer, ultrafiltration three times for use. Synthesis of satellite assemblies Au NR@Pt was functionalized with DNA1 in CTAB solution mixed with UCNPs decorated with DNA2 in Tris-HCl at a volume ratio of 1:1 and reacted with linker DNA3 (2 µL, 100 µM) at 60°C for 10 min. The reaction was transferred from 60°C to 37°C for 2 h. The mixture was cooled to room temperature and shaken overnight. TK1 mRNA detection in buffer Core-satellite assembled structures were added to six centrifuge tubes (400 µL each). Mimic TK1 mRNA (100 µM) was added to the tubes to final concentrations of 0, 5, 10, 50, 100 and 200 pM. Following incubation for 8 h at 37°C, centrifugation was performed at 7000 rpm for 10 min. The supernatant was collected for testing on a fluorescence spectrometer with 980 nm laser excitation (scanning range 500-700 nm). Optimization of incubation times To determine the optimal incubation times, MCF-7 cells were seeded into 96-well plates (200 µL cell culture solution per well with six wells for each incubation time of coresatellite) for 0, 4, 8, 16, 24 and 48 h. After washing with DPBS, cells were evaluated with the Cell Counting Kit-8 (CCK-8) for 4 h. Absorption was measured on a microplate reader at 450 nm. TK1 mRNA detection in MCF-7 and other cell lines MCF-7 cells with different expression levels of TK1 mRNA were obtained by treatment with different concentrations of βestradiol (100, 10 and 1 nM) and tamoxifen (10, 1 and 0.1 µM) for 24 h. Cells to which no regulatory agents were added served as the control group. MCF-7, primary uterine fibroblast cells (PCS-460-010) and human cervical cancer cells (HeLa) were used to examine the reliability of the developed method. MCF-7 and PCS-460-010 cells were cultured in DMEM supplemented with 10% fetal bovine serum, 50 units/mL penicillin, 50 µg/mL streptomycin at 37°C in a humidified incubator containing 5% CO2. HeLa cells were cultured in Roswell Park Memorial Institute (RPMI) 1640 supplemented with 10% fetal bovine serum, 50 units/mL penicillin and 50 µg/mL streptomycin at 37°C in a humidified incubator containing 5% CO2. Next, all three cell types were trypsinized and seeded in a small laser confocal dish at a density of 30,000 cells/dish, with two dishes per cell type. Core-satellite assemblies (5 nM) were added for 8 h at 37°C after incubation of cells in culture medium for 24 h at 37°C. Cells were washed with DPBS and subjected to confocal imaging (980 nm laser excitation). And three cell types that incubated with satellite (5 nM) for 8 h at 37°C, then, cells were treated with trypsin and collected for analysis of UCL intensity spectra (980 nm laser excitation). RESULTS AND DISCUSSION Principles of Intracellular mRNA Quantitation

Figure 1. Schematic illustration of Au NR@Pt-UCNP satellites for TK1 mRNA Detection.

The basic principles of quantitative detection, in situ and real time tracking of intracellular mRNA with Au NR@PtUCNPs satellite assemblies are illustrated in Figure 1. Thiolated ssDNA (DNA1: TTT TTT CGC TAC AGC A) was modified onto the surface of Au NR@Pt, and another thiolated ssDNA (DNA2 with a sequence of TTT TTT GCT CAG TAC A) covalently conjugated to polyethylene glycol (PEG) modified UCNPs via a “click” reaction between the maleimide group on the particle surface and the thiol group. Target mRNA recognition sequences (DNA3) were used as a bridge to connect Au NR@Pt and UCNP to form satellite assemblies. Generation of these assembled nanostructures was accompanied by upconversional luminescence quenching. In theory, the target mRNA should competitively combine with DNA to induce disassembly of satellite superstructures, facilitating UCNP detachment from Au NR@Pt and consequent luminescence recovery of UCNPs. Intracellular specific mRNAs are quantified based on the intensity of the upconversion luminescent signal. Self-assembly and characterization of spiny nanorod and upconversion nanoparticles Firstly, gold nanorods (15 nm in diameter ×60 nm in length) with an aspect ratio of 4, denoted Au NRs) were synthesized using documented seed-mediated growth methods59,60 (Figure S1A). Subsequently, bimetallic spiny platinum (Pt) shell coated Au NRs, Au NR@Pt, were prepared via heterogeneous seed-mediated growth of Pt nanostructures over Au NR seeds by reducing chloroplatinic acid using vitamin C with the assistance of cetyltrimethylammonium bromide, silver and iodide ions52. The spiny shell of Au NR@Pt was composed of ~4-5 nm dendritic Pt nanoparticles. The thickness of the shell was calculated as 5 nm based on statistical analysis of the dimensions of Au NRs@Pt structures (Figures 2A, S1B). We employed thiolated DNA1 to modify the surface of Au NR@Pt at a molar ratio of DNA to NR@Pt of ~400. Next, we used a previously described method to synthesize UCNPs with a diameter of ~10 nm modified with PEG 5000 bearing a maleimide group at one end38,61 (Figure S1C). Thiolated DNA2 was modified onto the surface of UCNPs at a molar ratio of DNA:UCNP of ~ 40 via the “click” reaction. Using DNA3 as a bridge, Au NR@Pt and UCNPs were assembled into core/satellite-like superstructures through specific base-pair recognition of complementary DNAs.


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Analytical Chemistry be attributed to the uneven surface of spiky Au NR@Pt structures, which leads to an increase in the specific surface area52. In view of their high upconversional luminescent quenching efficiency, we selected Au NR@Pt-UCNP satellites as the probe for subsequent intracellular mRNA imaging and detection.

Figure 2. TEM images of Pt-decorated Au NR (A) and satellite assemblies built by Au NR@Pt and UCNPs (B) in PBS buffer. EDX mapping (C)and UCL spectra (D)of satellite assemblies.

We examined the structural performance of Au NR@PtUCNP assembled particles via transmission electron microscopy (TEM) equipped with energy-dispersive X-ray spectroscopy (EDX), dynamic light scattering (DLS), and ultraviolet/visible (UV/Vis) and upconversional luminescence (UCL) spectroscopy. The representative TEM images acquired clearly indicate successful preparation of Au NR@Pt and UCNP core-satellite-like assemblies (Figure 2B and S2). The average number of satellite UCNPs around core Au NR@Pt was ~14 based on statistical analysis of the assemblies using TEM images (Figure S3). The element distribution of Au NRs@PtUCNP satellites was characterized with EDX mapping, which were consistent with the predicted assembled superstructures (Figure 2C). Due to the sensitivity of dynamic light scattering (DLS) in determining the state of dispersion, the data could be further applied to confirm that the actual species displayed in aqueous buffer were the same as those depicted in TEM images. The average DLS size of satellite assemblies was also significantly increased, compared to that of free Au NR@Pt (Figure S4). These findings were further confirmed with TEM, UV/Vis (Figure S5) and UCL spectra (Figures 2D, S6S8). Although luminescence was slightly quenched in the mixture of Au NR@Pt and UCNPs due to non-specific collision of nanocrystals (Brownian motion), the UCL intensity of satellites was predominantly quenched due to the small gaps among Au NR@Pt and UCNP and DNA molecules as bridges involved in the energy transfer process. To further investigate the behavior of dynamically assembled satellites, TEM, UCL and UV absorption spectroscopy were conducted to monitor the assembly process over time. With the progression of time, satellites gradually assembled from incomplete to complete structures (Figure 3A). Moreover, UCL intensity was gradually quenched, which tended to be consistent with the degree of assembly observed based on TEM images (Figure 3B). Importantly, we compared the quenching efficiency of upconversional luminescence between Au NR@Pt and bare Au NRs in similar assemblies (Figures 2D, S7). Quenching efficiency was improved to 12% using Au NR@Pt as the quencher compared with Au NRs, which may

Figure 3. TEM images (A) and UCL spectra (B) and extinction spectra (C) of dynamic assembly of satellite superstructures.

Quantitative and selective detection performance for mimic mRNAs in buffer

Figure 4. Au NR@Pt-UCNPs satellite assemblies for TK1 mRNA detection in vitro. (A) UCL spectra of probe 2 with addition of various targets (0, 5, 10, 50, 100 and 200 pM) in buffer solution (B) A plot of UCL intensity at 542 nm as a function of target concentrations. (C) Statistical analysis of the numbers of UCNPs on Au NR@Pt surface with different concentrations of target; (D) Selectivity of Au NR@Pt-UCNPs satellite assemblies in vitro.

Thymidine kinase 1 (TK1) mRNA is an important marker for assessing the cell cycle and estimating the tumor stage for



optimization of treatment. Accordingly, we selected TK1 mRNA as a model to evaluate the detection performance of the proposed strategy. To determine the ability of Au NR@PtUCNP satellite nanoprobes to sense mRNAs of interest, we first evaluated the response of UCL signals to synthetic DNA targets as a TK1 mRNA mimic in CTAB-Tris buffer solution. The UCL signals of nanoprobes were dynamically correlated to the concentrations of target mimic RNA within a 40-fold range from 5 to 200 pM. (Figure 4A) A regression equation was obtained for peak intensities at 542 nm (UCL542) to the target mimic RNA concentration (pM) is 1528.7 5674.3 lgCpM, 0.9972 where R2 is the correlation coefficient. The limit of detection was estimated by using three times the stand deviation of the blank, which was calculated down to 1.3 pM (Figure 4B). Our results suggest that the method developed has potential application in highly sensitive detection of intracellular mRNA. We further characterized the degree of UCNP desorption from Au NR@Pt via TEM in the presence of various concentrations of mimic mRNA. Similar to data obtained with UCL, the degree of disassembly gradually increased with increasing mimic TK1 mRNA concentrations (Figure S9). The average number of UCNPs surrounding Au NR@Pt was reduced from 14 to 3 with increasing mimic mRNA concentrations from 0 to 200 pM (Figure 4C).

speciﬁcity of this strategy for TK1 mRNA detection was assessed by measuring the upcoversional luminescence intensity response to three types of bases mutations, such as single-base mismatched strand (Single base mutation, 1 nM), three-bases mismatched strand (Three bases mutation, 1 nM) and 10-bases mismatched strand (>3 bases mutation, 1 nM) in the same condition. The upcoversional luminescence intensity showed negligible changing even after addition of single base, three bases and >3 bases mutation at a concentration of 1 nM, while the TK1 mRNA exhibited a strong luminescence intensity at the concentration of 10 pM, which was ~7-fold higher than the intensity of the single, three and >3 bases mutation. (Figure 4D and S10) Those results clearly demonstrated that the proposed assay had excellent discrimination capability for a sequence with base-mismatch. Bovine serum albumin (BSA, 1 mM) and glutathione (GSH, 1 mM) were spiked into the sensing system. No obvious alterations in luminescent signals were evident, implying little or no interference of these proteins and peptides with the detection system. Optimal incubation times of probes and MCF-7 cells

Figure 6. Intracellular UCL spectra incubated with probe 1 (A) and probe 2 (B) for different time. (C) Statistics analysis of UCL intensity (542 nm) and confocal luminescence (CL) intensity (540±30 nm) of Figure 5. (D) Cytotoxicity evaluation by Au NR@Pt-UCNPs satellite assemblies (5 nM) incubated with MCF7 at various time.

Figure 5. Confocal images of probe 1 (A) and probe 2 (B) in MCF-7 cells with different incubated time. Probe 1: assembled with the sequences that had no ability to recognize TK1 mRNA. Probe 2: assembled with the sequences that could specifically hybridize with TK1 mRNA. Scale bar: 30 µm.

To assess the specificity of the developed nanoprobes, we compared the UCL restoration intensities between mimic TK1 mRNA (5 pM) and the common interference from other mRNA (including similar tumor-related mimic mRNAs, GalNAc-T and Cmyc, see sequences 8 and 9 in Table S1 of Supporting Information (SI), 1 nM). As shown in Figures 4D and S8, in the presence of mimic TK1 mRNA, the upconversional luminescence signal was restored whereas no obvious changes in the signal were observed in the other two common types of mRNA, indicative of excellent sequence specificity of the detection probe. And much more importantly, the mutation

After confirming probe activity in buffer, we investigated its performance in a breast cancer cell line (MCF-7). For in situ tracking and quantitative detection of target mRNA in specific living cells, several prerequisites must be fulfilled: 1) stability in culture medium, 2) no obvious cytotoxicity at the optimal dose and incubation time, 3) high performance delivery into the cytoplasm over a relatively short time without apparent formation of aggregates and 4) good temporal resolution of probes. Before evaluating the above criteria and the sensing performance of the probe in living cells, we attached thiolated polyethylene glycol (PEG) and the cell-penetrating peptide, TAT, onto the surface of phosphorothioate-DNA-driven satellite assemblies through covalent conjugation to enhance stability and delivery efficiency into the cytoplasm. The intact superstructures of satellite probes were maintained, even after incubation for 48 h, indicating that the probes were stable in culture medium (Figure S11). To determine the suitable concentrations of our newly developed satellite probes for application in live cells, MCF-7 cells were incubated with various concentrations of probe for 8 h. In view of the finding that cell viability remained unaffected at concentrations of ≤5 nM


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Analytical Chemistry probe (Figures S12 and S13), we selected 5 nM for subsequent experiments. Investigation of the effects of 5 nM satellite assemblies on cell viability over a longer time-period revealed cytotoxicity from 4 to 16 h (Figure 6D). To assess the delivery efficiency and reactive dynamics of the probes in living cells, two different types of DNA were used for preparation, specifically, probe 1 (DNA sequences do not react with TK1 mRNA) and probe 2 (DNA sequences specifically recognize TK1 mRNA). Incubation of probe 1 with MCF-7 cells for different time-periods led to very weak and even no luminescence signals while probe 2 presented strong luminescence intensity due to dissociation of UCNP from Au NR@Pt induced by inherent TK1 mRNA in living cells (Figure 5). Furthermore, the intensity of luminescence gradually increased from 0 to 8 h and began to decline beyond 8 h due to accumulation of the satellite assemblies in living cells. The intracellular luminescence signal detected with probe 2 (UCL probe 2) was stronger than that with probe 1 (UCL probe 1), and the value of ∆UCL (UCL probe 2- UCL probe 1) was higher at 8 h (Figure 6), which was accordance with the statistics analysis of the intensity of confocal luminescence images (Figure 5 and Figure 6C). Our results provide convincing evidence that probe 2 specially recognizes TK1 mRNA with excellent temporal resolution, but not probe 1. Bio-TEM images of Au NR@PtUCNP satellite assemblies in MCF-7 cells incubated for 8 h indicated excellent dispersion of the probes in living cells below the 8 h time-point (Figure S14). Consequently, 8 h was selected as the optimal incubation time for subsequent experiments. Quantitative detection of TK1 mRNA in live cells To detect the levels of TK1 mRNA in live cells, MCF-7 cells were treated with β-estradiol or tamoxifen for up/downregulating the TK1 mRNA content, respectively. After modulation of intracellular TK1 mRNA, quantitative RTPCR was employed for quantification using standard RNA extraction and detection protocols19. The linear range of threshold cycles as a function of target concentrations was 20 to 2×107 aM (Figure S15). The original concentration of TK1 mRNA in living MCF-7 cells was determined as 10.24 fmol/10 µg RNA. We used confocal imaging and upconversional luminescence spectroscopy for in situ quantitation of TK1 mRNA in living cells. The luminescence intensity of the confocal images illuminated at 980 nm was enhanced with increasing concentrations of β-estradiol. On the other hand, the luminescence signal gradually decreased with increasing concentrations of tamoxifen (Figure 7A). And also, bio-TEM images of satellite probes could further clarify that the number of UCNPs surrounding Au NRs@Pt was proportional with the concentration of TK1 mRNA. (Figure S16) These results were consistent with predicted data based on the design of the assembled nanostructure probes. Using RT-PCR to pre-quantify mRNA amounts in living MCF-7 cells, the intensity changes of confocal luminescence showed a good linear relationship with TK1 mRNA expression between 1.17 to 65.21 fmol/10 µg RNA with a limit of detection (LOD) 0.67 fmol /10µg RNA (Figure S17). Compared with the previous in situ RNA detection method16,38, the developed method has an excellent specificity and a much more sensitivity. The intensity changes of confocal luminescence were equivalent to luminescence intensity of confocal images showing up/down/no regulation of TKI mRNA in MCF-7 cells.

Figure 7. (A) The confocal images of MCF-7 cells that treated with beta-estradiol and tamoxifen with different amounts of TK1 mRNA and (B) corresponding UCL intensity spectra. Scale bar: 30 µm.



Meanwhile, we applied upconversional luminescence intensity to detect intracellular TK1 mRNA expression under various regulatory conditions. UCL intensity was gradually restored upon increasing the amounts of intracellular TK1 mRNA (Figure 7B). The intensity change of upconversional luminescence at 542 nm (∆UCL542) was used for quantitation of TK1 mRNA in live cells, with a detection limit of 1.12 fmol/10 µgRNA (Figure S18). Our results clearly demonstrated that the satellite assemblies with upconversional luminescence signals generated in this study are suitable for monitoring and quantitation of mRNAs expressed at low levels in live cells due to their ability to avoid background interference. Generality evaluation of intracellular probes

confocal imaging and 5.41±0.27 and 1.31 ±0.11 fmol/10 µg upconversional luminescence, respectively. The TK1 mRNA contents quantified via confocal and upconversional luminenscence in the corresponding cell lines were equivalent, consistent with reported results.62 Note that those results suggested that the developed probes have the excellent generality in various cell lines. Standard RT-PCR was additionally applied to quantify the intracellular TK1 mRNA content within the different cell lines. The results were in keeping with the luminescence signals obtained. Based on the collective findings, we suggest that the probes developed in this study are able to effectively distinguish and quantify a range of mRNA contents within different cell lines. Furthermore, the capability of the proposed assay for detecting the intracellular micro RNA-21 (miRNA-21) in HeLa cells as the other type of RNA to evaluate the generality of the nanoprobes to recognize other types of RNA. Firstly, the upconversional luminescence intensity increased with the increase miRNA-21 in the buffer to indicate the nanoprobes have the capability to detect the other types of RNA in the buffer. (Figure S19) And furthermore, as shown in Figure 8C-E, upon increasing the amount of miRNA-21 in living HeLa cells, the upconversional intensity of confocal images and spectroscopy also increased. And, the upconversional luminescence intensity at 542 nm measured by the luminescence spectroscopy displayed an excellent linear relationship with the logarithm of miRNA-21 concentration in the range from 0.043 to 41.25 fmol/10µg RNA with the detection limit 0.028 fmol/10µg RNA. Meanwhile, with the upconversional intensity from the confocal images, the limit of detection for miRNA-21 could be calculated to be 0.019 fmol/10µg RNA as 3 times the standard deviation of the mean of blank (Figure S20), which is superior to those of exponential ampliﬁcation assays11. These results indicated that this proposed method had high generality and promising application for quantitate detection of other types RNA in living cells. RNA using

CONCLUSIONS

Figure 8. Generality evaluation of the developed strategy. Confocal (A) and UCL (B) spectra of Au NR@Pt-UCNPs satellite assemblies incubated with MCF-7, HeLa, PCS-460-010 cells. Confocal (C), UCL spectra and standard calibration (D and E) for microRNA-21 detection in HeLa cells using the developed probes. Scale bar: 30 µm.

As the relative expression levels of TK1 mRNA vary in different cell lines, to determine the versatility and specificity of the developed probes, we selected human cervical cancer (HeLa) and primary uterine fibroblast (PCS-460-010) cells for evaluation. Confocal imaging data and UCL intensity spectra (Figure 8A, 8B) revealed that the TK1 mRNA levels in HeLa and MCF-7 cells are markedly higher than those in PCS-460010 cells. The average luminescence intensity of confocal images in MCF-7 cells was ~1.8-fold and 3.2-fold higher than that in HeLa and PCS-460-010 cells, while the signal from the UCL spectrum in live MCF-7 cells was about 1.7- and 3.3-fold that in HeLa and PCS-460-010 cell lines. The TK1 mRNA contents in HeLa and PCS-460-010 cells were determined as 5.12±0.35 fmol/10 µg RNA and 1.21±0.15 fmol/10 µg RNA using

In summary, Au NR@Pt-UCNP satellite assemblies were successfully developed as intracellular nanoprobes for in situ imaging and quantification of TK1 mRNA in live cells using confocal microscopy and upconversional luminescence spectroscopy. Disassembly of these nanoprobes was driven by TK1 mRNA recognition and accompanied by restoration of the luminescence signal with avoidance of background fluorescence of the cellular specimens. The proposed method presents a valuable potential tool for detection of intracellular mRNAs and should facilitate clarification of the specific functions of desired target mRNAs in a myriad of biological processes. The utility of this type of nanoprobe based on UCL restoration could be extended for analysis of other biomarkers in biological science.

ASSOCIATED CONTENT Supporting Information Materials and detailed experimental procedures, supplementary figures. This material is available free of charge via the Internet at http://pubs.acs.org.


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AUTHOR INFORMATION Corresponding Author *

E-mail: [email protected]; [email protected]; [email protected]

Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.

Notes The authors declare no competing financial interest.

ACKNOWLEDGMENT This work is financially supported by the National Natural Science Foundation of China (21631005, 21673104, 21522102, 21503095).

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Spiny Nanorod and Upconversion Nanoparticle Satellite Assemblies

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