Low Background Cascade Signal Amplification Electrochemical

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Low Background Cascade Signal Amplification Electrochemical Sensing Platform for Tumor-Related mRNA Quantification by Target Activated Hybridization Chain Reaction and Electroactive Cargo Release Hong Cheng, Jinquan Liu, Wenjie Ma, Shuangdi Duan, Jin Huang, Xiaoxiao He, and Kemin Wang Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.8b02470 • Publication Date (Web): 28 Sep 2018 Downloaded from http://pubs.acs.org on September 28, 2018

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Analytical Chemistry

Low Background Cascade Signal Amplification Electrochemical Sensing Platform for Tumor-Related mRNA Quantification by Target Activated Hybridization Chain Reaction and Electroactive Cargo Release Hong Cheng, Jinquan Liu, Wenjie Ma, Shuangdi Duan, Jin Huang, Xiaoxiao He∗, Kemin Wang∗ State Key Laboratory of Chemo/Biosensing and Chemometrics, College of Biology, College of Chemistry and Chemical Engineering, Hunan University, Key Laboratory for Bio-Nanotechnology and Molecule Engineering of Hunan Province, Changsha 410082, China ABSTRACT: Herein, a low background cascade signal amplification electrochemical sensing platform has been proposed for the ultrasensitive detection of messenger RNA (mRNA) by coupling the target activated hybridization chain reaction and electroactive cargo release from mesoporous silica nanocontainers (MSNs). In this sensing platform, the 5’-phosphate terminated DNA (5’-PO4 cDNA) complement to target mRNA is hybridized with the trigger DNA and anchor DNA on the surface of MSNs, aiming at forming a double-stranded DNA gate molecules and sealing the methylene blue (MB) in the inner pores of MSNs. In the presence of target mRNA, the 5’-PO4 cDNA is displaced from the MSNs and competitively hybridizes with mRNA, which led to the liberation of the trigger DNA and the opening of the MSNs pore. The liberated trigger DNA can be then immobilized onto the electrode surface through hybridization with the capture DNA, triggering HCR on the electrode surface. In the same time, the MB released from the MSNs will selectively intercalate into the HCR long dsDNA polymers, giving rise to significant electrochemical response. In addition, due to the λ-exonuclease (λ-Exo) cleavage reaction assisted target recycling, more amounts of trigger DNA will be liberated and trigger HCR, as well as numerous MB are uncapped and intercalates into the HCR products. As proof of concept, thymidine kinase 1 (TK1) mRNA was used as a model target. Featured with amplification efficiency, label-free capability and low background signal, the strategy could quantitatively detect TK1 mRNA down to 2.0 aM with a linear calibration range from 0.1 fM to 1 pM. We have also demonstrated the practical application of our proposed sensing platform for detecting TK1 mRNA in real samples, opening up new avenues for highly sensitive quantification of biomarkers in bioanalysis and clinical diagnosis.

Messenger RNA (mRNA), a large family of RNA molecules, is produced from deoxyribonucleic acid (DNA) in a biological process called transcription.1-4 It conveys genetic information from DNA to protein through specifying the amino acid sequence of the protein products of gene expression in the ribosome. The expression level of mRNA can provide valuable information for biological study, medical diagnosis, disease treatment, and drug discovery.5 For example, there are increasing reports to indicate that some specific mRNA expression level is correlated with tumor burden, malignant progression and metastasis.6 In this case, effectively quantifying the mRNAs expression level, especially mRNA with low expression levels, holds great promise for biology study, as well as for disease diagnosis and treatment. Up to now, the most commonly methods used to measure mRNA levels following its extraction from cell lysate are as follows: northern blotting,7 ribonuclease protection analysis,8 reverse transcription polymerase chain reaction (RT-PCR)9 and quantitative

polymerase chain reaction (qPCR).10 These methods are effective but they are often difficult in a clinical setting by the drawback of cumbersome, time-consuming, and labor-intensive. Northern blotting is able to detect small changes in gene expression, while it has a low sensitivity, which requires micrograms of total RNA.11 By compared with northern blotting, the detection sensitivity of ribonuclease protection assay is higher than that of northern blotting because the loss of sample and probe will be reduced, while radiolabeled probes are usually required. The nucleic acid amplification method of RTPCR or qPCR can theoretically amplify a single mRNA target molecule millions of times and thus is very powerful for quantification of mRNA with low expression levels. However, the RT-PCR or qPCR is complex and the introduced contaminating nucleic acid can be also amplified during the high sensitive test, thereby resulting in false-positive results.12, 13 Some of the recent advances in the field of biosensing methods for nucleic acid analysis open up new

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opportunities for mRNA quantification, including atomic force microscopy,14 surface plasmon resonance,15 surface enhanced Raman spectroscopy,16 colorimetry,17 18 19 fluorescence, electrochemistry, etc. Among them, the electrochemical nucleic acid biosensing techniques have received particular attention owing to its intrinsic advantages of low-cost manufacture, high sensitivity, simple instrumentation, ease of miniaturization, and fast response. However, among those many reported electrochemical nucleic acid biosensing techniques, only a few attempts have been used for tumor-related mRNA expressing levels quantification. One of the pivotal issues needs to be addressed when using electrochemical nucleic acid biosensing techniques for the quantification of mRNA with low expression levels is the improvement of electrochemical detection sensitivity. Until now, many efforts have been devoted to the design of high sensitive electrochemical nucleic acid biosensing techniques by use of target amplification, probe amplification or signal amplification.20 Target amplification allows replication or recycling of the target nucleic acid fragment to achieve target concentration high enough to be detected using conventional electrochemical. By compared with classic target amplification of polymerase chain reaction (PCR),9 the recently emerged isothermal target amplification methods such as strand displacement amplification (SDA),21 catalyzed hairpin assembly (CHA),22 loopmediated isothermal amplification (LAMP), 23 and nucleic acid tool enzyme-assisted target recycling (EATR), 24 are simple and do not require an expensive thermal cycler. For probe amplification strategy, the probe sequence is replicated while the amount of the nucleic acid fragment remains the same. A typical example of probe amplification method is the rolling circle amplification (RCA).25 Apart from target amplification and probe amplification, hybridization chain reaction (HCR),26 branched DNA (bDNA) technology,27 nanomaterials28 labels and other chemical approaches have been used to amplify the electrochemical signal triggered by the presence of a target nucleic acid.20 Although the three kinds of amplification technique have its own unique, the single amplification technique often may not meet the need for detection of target nucleic acid in a very small amount. Thus, the cascade amplification technique by combining two or three of them can induce higher order amplification in the electrochemical nucleic acid biosensing. For example, Yang et al. designed an ultrasensitive and specific electrochemiluminescence (ECL) biosensor for the p53 DNA sequence detection, which was based on cascade signal amplification of nicking endonuclease assisted target recycling and hyperbranched rolling circle amplification (hRCA). Using this cascade amplification strategy, it could reach a low detection limit of 20 aM.29 Qiu et al. achieved the ultrasensitive detection of miRNA 141 with the detection limit of 11 aM through cascade amplification of duplex-

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specific nuclease assisted target recycling and HCR.30 Meanwhile, Yi et al. combined molecular beacon mediated circular strand displacement polymerization (CSDP) and hRCA to develop a simple and ultrasensitive electrochemical strategy for specific detection of DNA target. Due to the advantages of target recycling and cascade signal amplification, it exhibited a low detection limit down to 8.9 aM.31 Nevertheless, as the signal is enhanced through the cascade amplification technique, the background signal may be also increased. Therefore, effective cascade signal amplification electrochemical nucleic acid biosensing strategies with the advantages of low background are being sought. Mesoporous silica nanoparticles (MSNs), a 3D network of honeycomb-like porous structure,32 have attracted much attention in developing drug delivery systems33 as well as nanoprobes34 due to its high surface area, nontoxic nature, large load capacity, tunable pore size, high chemical and thermal stability, and chemically modifiable surface. When used as nanoprobes, the signal molecules can be sealed in the mesopores of MSNs in a way that only a target can trigger the release of signal molecules.35, 36 In this case, the large load capacity and the targetresponsive controlled cargo release of the MSNs can bring signal amplification with minimal background signal. Herein, a low background cascade signal amplification electrochemical sensing platform for the ultrasensitive detection of target mRNA has been proposed by using target activated HCR and electroactive cargo release from DNA-gated MSNs. In this sensing platform, the DNA gate molecules contain the target recognition sequence and the HCR trigger sequence, respectively. Initially, the electrochemical redox-active indicator methylene blue (MB)37, 38 is sealed in the pores of the DNA gated-MSNs. Upon the target mRNA involved, the DNA gate is opened, resulting in the MB release from the pores. Meanwhile, the HCR trigger sequence is liberated and initiates HCR on the electrode surface. The released MB can then selectively intercalate into the HCR long dsDNA polymers 39, 40 and can be quantitatively determined by square wave voltammetry (SWV) method. Benefiting from a target activated MB release, the background signal of the sensing platform can be minimized effectively. More importantly, the target mRNA recycling could be achieved with λ-exonuclease (λ-Exo) cleavage reaction to release more MB and more HCR trigger sequence, respectively, thus amplifying the electrochemical signal. In this work, thymidine kinase 1 mRNA (TK1 mRNA) was chosen as a target to demonstrate the reliability of this sensing platform, which was associated with cell division and proposed to be a biomarker for tumor growth.18, 41 Under the optimal conditions, it can detect TK1 mRNA with a detection limit of 2.0 aM. It may provide a simple and powerful platform for ultrasensitive RNA quantification in bioanalysis and clinical diagnosis.

EXPERIMENTAL SECTION

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Materials and Reagents. Tris-(benzyltriazolylmethyl) amine (TBTA), Tris (2-carboxyethyl) phosphine (TCEP) and 6-Mercapto-1-hexanol (MCH) were obtained from Sigma-Aldrich Chem. Co. (St Louis, MO, USA). (3Chloropropyl) trimethoxysilane (Cl-TMS), (1-Hexadecyl) trimethyl ammonium bromide (CTAB), Tetraethylorthosilicate (TEOS, 98%), sodium ascorbate and Copper (I) bromide (CuBr, 99.9%) were obtained from Thermo Fisher Technology (China) Co., Ltd. (Shanghai, China). Sodium azide (NaN3, 99%) and methylene blue (MB) were obtained from Ding Guo Changsheng Biotechnology Co., Ltd. (Beijing, China). Dimethylformamide (DMF), Dimethylsulfoxide (DMSO), Butyl Alcohol (tBuOH), Tween-20, and methylbenzene were obtained from Xilong Chemical Co., Ltd. (Shantou, China). λ exonuclease (λ-Exo) was obtained from New England Biolabs (Beverly, MA, USA). Diethypyrocarbonate (DEPC) and all oligonucleotides were purchased from Sangon Biotech Shanghai Co. Ltd (Shanghai, China). All mRNA targets and DNA oligonucleotides used in this work were listed in Table S1. The complete TK1 mRNA sequences from NCBI Bank were listed in Table S2. All other reagents were of analytical grade and used without further purification. Aqueous solutions were prepared using ultrapure water (18.2 MΩ cm resistivity). The buffers used in this experiments were as follows: washing buffer (10 mM TrisHCl with addition of Tween 20, pH 7.4), DNA hybridization buffer (50 mM Tris-HCl containing 100 mM NaCl, 40 mM MgCl2, 100 mM KCl, pH 7.4), TBE buffer (8.9 mM Tris-Base, 8.9 mM Boric acid, 0.2 mM EDTA, pH 8.3) Characterization and Instrumentation. Transmission electron microscopy (TEM) images were taken using a Tecnai G2 20 STwin (FEI, Czech Republic) operated at 200 kV. X-ray diffraction (XRD) pattern was obtained on SIMENS D500 (Brugg, Switzerland). The FTIR spectra of different samples were detected by the TENSOR 27 spectrometer (Florida, USA). The determination of the surface area was obtained through Brunauer-Emmett-Teller (BET) method at NOVA3000/Quantachrome (Florida, USA). The gel electrophoresis on 15% nondenaturating polyacrylamide (PAGE) in 1x TBE buffer was used for determining the feasibility of λ-Exo cleavage and HCR, respectively. The PAGE was performed using 1x SYBR Gold as the stain at 100 V constant voltage for 2h at room temperature, finally photographed in Azure C600 Imaging Biosystems (California, USA). Electrochemical measurements were performed using a CHI660A electrochemistry workstation (ChenHua, Shanghai, China) with the Ag/AgCl(s)/KCl (sat) reference electrode from BASi (Bioanalytical Systems, Inc.) and a gold working electrode. All potentials are reported relatively to the saturated Ag/AgCl reference electrode. Square wave voltammetry (SWV) was carried out in DNA hybridization buffer with a 50 mV amplitude signal, over

the range from −0.6 V to +0.1 V versus Ag/AgCl reference. The oxidative peak of MB was detected by SWV at −200 mV (vs Ag/AgCl). Loading of MB in the DNA Gated-MSNs. To load MB in the DNA gated-MSNs, the MSNs were firstly synthesized according to the previous reported approach.35 Firstly, 0.25 g of surfactant CTAB was dissolved in 120 mL of ultrapure water, subsequently mixed with 875 μL of NaOH (2.0 M). Then the mixture of CTAB and sodium hydroxide was stirred for 10 min at 80 °C. 1.25 mL of TEOS was dropped and stirred for 2 h under 80 °C afterwards. The obtained white precipitates of MSNs were filtered and washed with ultrapure water and methanol sequentially, then dried at 55 °C under vacuum. The prepared MSNs were further activated according to the following steps. 0.35 g of the MSNs products were heated to reflux for at least 20 h in 60.0 mL of methylbenzene with Cl-TMS (0.70 mL) to yield the 3chloropropyl-modified MSNs material (MSNs-Cl). The products MSNs-Cl were then purified by the procedures with centrifugal subsidence, methanol and ultrapure water washing, followed vacuum drying. The MSNs-Cl powders (0.50 g) were heated and stirred for 6 h in ethyl alcohol (80 mL) including hydrochloric acid (1 mL, 37.2%) for the purpose of surfactant template (CTAB) removing. The MSNs-Cl without CTAB were obtained after the same purification procedure. Next, the MSNs-Cl (0.30 g) were added in the DMF (60 mL) solution with NaN3 (0.50 g) and stirred at 90 °C for 12 h. Afterwards, the azide functionalized nanoparticles (MSNs-N3) were successfully prepared, redispersed in Tris-HCl buffer (pH 7.4). The residual organic solvent could be washed off from MSNsN3 after stirring for 6 h, then the MSNs-N3 powers were obtained after purification and vacuum drying. At this moment, the loading of MB in the MSNs-N3 can be performed. The MSNs-N3 powers were added into 40 mg/mL of MB solution (Tris-HCl buffer, pH 7.4), and then the resulting mixture (1 mL, C[MSN] ≈ 10 mg/mL) was slightly rotated overnight in room temperature. In the meanwhile, major MB molecules were stirred into the pores of MSNs-N3. After the supernatant was removed, the obtained MSNs-N3 loading with MB (designated as MB@MSNs-N3) was washed several times using pH 7.4 Tris-HCl until a low background signal was achieved to remove any physically adsorbed MB on the surface of MSNs. After loading MB, the pore of the MSNs-N3 was capped through click chemistry. 2.0 mg of MB@MSNs-N3 particles were dispersed in Tris-HCl buffer (200 μL, pH 7.4) including anchor-DNA, 5’-PO4 cDNA, and trigger DNA (40.0 μM). In a separated centrifuge tube, 1 μL sodium ascorbate treated Copper (I) solution (100 mM in tBuOH/DMSO, 1:3) and 2 μL TBTA solution (100 mM in tBuOH/DMSO, 1:3) were vortexed and dropped into the mixture of MB@MSNs-N3 particles and DNA solution, which were gently rotated overnight. The obtained products were purified with Tris-HCl (pH 7.4) solution

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through the same procedure to yield the DNA gatedMSNs with loading of MB (MB@MSNs-DNA). Preparation of Capture DNA Probe Modified Gold Electrodes. The capture DNA probe for hybridization with the trigger DNA of HCR was modified on the gold electrodes by the reaction of facile gold-thiol bond.42 Before modification, the gold electrodes were pretreated according to the following procedure.43 The gold electrodes were first cleaned by soaking in a fresh piranha solution (H2O2: H2SO4 =1:3, v/v) for at least 15 min. After rinsing thoroughly with ultrapure water, the electrodes were polished with 1.0, 0.3 and 0.05 μm alumina slurry separately and then sonicated sequentially in ultrapure water and ethanol for 5 min, respectively. Then the electrodes were further electrochemically cleaned in 0.1 M H2SO4 with potential scanning from -0.2 V to 1.55 V until a remarkable voltammetric peak was obtained, followed by sonication and drying with nitrogen. Prior to immobilization on the gold electrode, the thiol group modified capture probe (SH-CP) must be treated with TCEP to reduce the disulfide bonds of the SH-CP for 60 min, then heated to 95 °C for 5 min. After cooling down slowly to room temperature, a droplet of 20 μL SHCP (1 μM) was cast onto the pretreated electrode and incubated overnight in a humidity chamber at room temperature. At last, the SH-CP/MCH modified electrodes were obtained after immersing in 20 μL of 1 mM freshly prepared MCH for 30 min TK1 mRNA Activated MB Release Behavior Investigation. To investigate the MB sealing effect and the target activated release, 1 mg of MB@MSNs-DNA were suspended in 1 mL of the Tris-HCl buffer (pH 7.4). Subsequently, 0.2 mL of the MB@MSNs-DNA suspension was taken out. Then, 10 μL 100 nM TK1 mRNA was added into the MB@MSNs-DNA suspension. After mixing thoroughly, aliquots of 20 μL of the mixture of MB@MSNs-DNA and target were taken out and incubated with the well prepared electrodes for different time. Then the incubated Au electrode was washed and used for SWV measurement. Procedures for TK1 mRNA Detection. 1.0 mg of MB@MSNs-DNA was suspended in 1.0 mL Tris-HCl buffer (pH 7.4). Several aliquots of 100 μL of this MB@MSNsDNA suspension were respectively taken out and 76 μL of hybridization buffer was added into each aliquot. Thereafter, the target recognition reaction and λ-Exo cleavage reaction can be performed. Briefly, 2 μL 100 nM target TK1 mRNA sample with different concentration were added into the MB@MSNs-DNA suspension. After sufficient mixing, λ-Exo (2 μL, 4 U/μL) and 20 μL 10 × λExo buffer (670 mM Glycine-KOH, 25 mM MgCl2, 500 μg/mL BSA, pH 9.4) were added in the mixture and incubated at 37 °C to perform the target recognition reaction and digestion reaction. When the recognition reaction and digestion reaction was allowed to proceed for 2 h, 20 μL of the above reaction products were taken

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out and incubated with the prepared electrodes for 30 min in atmosphere ambient. Subsequently, 20 μL mixture solution of 1 μM H1 probe and 1 μM H2 probe were also dropped onto the gold electrode and further incubated for 160 min. Finally, the electrode was washed thoroughly with washing buffer and measured by SWV method. Cell Culture and Lysate Preparation. To test the detection capability of the developed sensing platform in real samples, HepG2 cells (Human liver hepatocellular carcinoma cell), MCF-7 cells (Human mammary epithelial cell) and L02 cells (Normal human hepatocyte cancer cell) were cultured in DMEM-H medium supplemented with 10% fetal bovine serum (FBS), 100 U/mL penicillin 100 μg/mL streptomycin in an incubator with 5% CO2 at 37 °C. When the cells were grown at the density of 1× 107 cells/well, the medium was removed and the cells were washed twice. Subsequently, the cells were lysated in 1 mL of Trizol reagent and total RNA was prepared according to the manufacturer's instructions, followed by incubation at room temperature for 5 min to ensure complete cell disruption. The obtained total RNA sample was then divided into two equal parts and stored at −80 °C for quantitative real time polymerase chain reaction (qRT-PCR, Sangon Co. Ltd, Shanghai, China) detection and electrochemical detection, respectively. Then one piece of the obtained cell lysates were diluted with 10-fold DEPC-treated water and then used for the electrochemical detection without other treatment. RESULTS AND DISCUSSION Principle of Detection. As shown in Scheme 1, the low background cascade signal amplification electrochemical sensing platform for the ultrasensitive detection of mRNA was just developed based on the target activated HCR on the electrode and electroactive cargo release from DNA gated-MSNs. In this strategy, in the absence of target mRNA, the signal molecules MB were sealed in the mesopores of MSNs nanoparticles through the capping of DNA gate molecules. Therefore, the background signal of the sensing platform can be minimized effectively. It is worth noting that the DNA gate molecules were skillfully designed to be composed of an anchor DNA immobilized on the surface of MSNs (anchor DNA), a 5’-phosphate terminated target mRNA recognition probe (5’-PO4 cDNA) and the HCR trigger sequence (trigger DNA). However, in the presence of mRNA, the 5’-PO4 cDNA would competitively hybridize to the target mRNA and be displaced from the MSNs. At the same time, the trigger DNA was also liberated and hybridized with the capture DNA (SH-CP) on the electrode surface and initiated the following HCR on the electrode surface. Because of the departure of 5’-PO4 cDNA and trigger DNA from the MSNs, the MB was released and then intercalated into the HCR long dsDNA polymers. More importantly, target mRNA could hybridize with 5’-PO4 cDNA to form a 5’PO4 duplex DNA and thus initiate the λ-Exo cleavage

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Scheme 1. Schematic Diagram for the Low background Cascade Signal Amplification Electrochemical Sensing Platform for mRNA Quantification by Target Activated HCR and Electroactive Cargo Release.

process to realize the target mRNA recycling, accompanied by opening more DNA gates and releasing more MB. As a result, a low background amplified oxidation peak current of MB could be detected by SWV at −200 mV, which was related to the quantification of the target mRNA. As proof of concept, TK1 mRNA was used for mRNA electrochemical quantification studies. Characterization of the DNA Gated-MSNs with MB Loading. To insure the design of a target-induced cargo release from the DNA gated-MSNs, the successful synthesis of MCM-41 type mesoporous structure would be crucial. The TEM characterization showed the obtained MSNs-Cl particles were spherical with a diameter around 80 nm (Figure 1A). The X-ray powder diffraction (XRD) results (Figure 1B) of the siliceous MSNs-Cl showed three well-defined peaks in the region 2θ = 2−6° that could be indexed as (100), (110), and (200) Bragg reflections, demonstrating that the MSNs-Cl had a hexagonal array porous structure. The application of the BET method displayed a specific surface of 983 m2 g-1, with a BJH average pore diameter of 2.34 nm (Figure 1C, inset). Subsequently, MSNs-Cl was then reacted with NaN3 in DMF solvent to obtain the azide-functionalized particles (MSNs-N3). The MSNs-N3 particles were then used to load MB. In this process, the MB can enter into the pores of MSNs-Cl through diffusion. After MB loading, the DNA gate molecules were attached to the MB@MSNs-N3 surface through a copper (I)-catalyzed cycloaddition approach. FTIR spectrum was used to monitor the successful conversion of MSNs surface with DNA gate molecules modification (Figure 1D). As was well known, the MSNs-Cl (a) only showed the silica framework vibrations whereas the MSNs-N3 (b) exhibited the absorption band around 2110 cm-1 (the labeled green circle in Figure 1D), which is assigned to the azide stretch. (c)

After successful grafting of DNA, this absorption band was apparent decline. There results could be considered to be a success for the DNA grafting on the MSNs surface. The density of DNA anchored on MSNs surface was calculated to be nearly 2.8 μmol g-1 SiO2 by UV-vis spectrum. According to the design, the loading and target activated releasing of MB are very important to the construction of the low background cascade signal amplification electrochemical sensing platform. Therefore, the loading of MB in the MSNs-N3 was first detected using the UV–vis spectrum. Figure S1 showed that the MB@MSNs-DNA exhibited an apparent absorption peak around 663 nm corresponding to the same peak of free MB (Figure S1). While the MSNs-DNA had no absorption peak around 663 nm. Moreover, it was obvious that the white-light image of MB@MSNs-DNA displayed characteristic blue color of MB (Figure S2). The UV–vis spectrum and white-light image results demonstrated the success of MB loaded into the MSNs. The loading amount was determined to be 8 mmol g− 1 SiO2 by UV–vis spectrum. Having confirmed the successful loading of MB, we next investigated the MB sealing effect and the target activated release by using SWV detection. As shown in Figure S3, the Au electrode treated with MB@MSNs-DNA displayed weak SWV response (curve a). It demonstrated that MB can be well sealed in the MSNs through capping of high concentration of DNA gate molecules. Notably, the SWV peak current of the Au electrode dropped with the mixture of MB@MSNs-DNA and TK1 mRNA increased obviously with extending of incubation time. The current intensity increase attributed to the TK1 mRNA induced DNA gate opening and the release of MB from MSNs (curve b). As a control, the release behavior of MB@MSNs in the absence of target mRNA was also investigated, the SWV signals also increased greatly (curve c). It was because the pores of MSNs were not sealed and the MB molecules could be released quickly. Validation of the λ-Exo Cleavage Reaction Assisted Target Recycling and HCR Amplification. The conception of the λ-Exo cleavage reaction assisted target recycling the HCR amplification were respectively verified by gel electrophoresis before conducting them on the electrochemical sensing platform. Considering the stability of mRNA during electrophoresis, the synthetic target DNA was used as target model, instead of TK1 mRNA. We first performed PAGE to investigate the λ-Exo cleavage reaction assisted target recycling. The results from PAGE (Figure 2A) revealed that the DNA duplex of synthetic target DNA and 5’-PO4 cDNA could be cleaved by λ-Exo, and free synthetic target DNA band was observed (line 4). However, the λ-Exo has no activity on the DNA gate for the five-base overhang of 5’phosphorylated end in 5’-PO4 cDNA (line 6). In the coexistence of synthetic target DNA and λ-Exo in line 7, the primary DNA gate band disappeared and obvious trigger

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Figure 1. (A) TEM image of MSNs-Cl; (B) Powder X-ray pattern of MSNs-Cl; (C) Nitrogen adsorption-desorption isotherms and pore size distributions (inset) of MSNs-Cl; (D) FTIR spectra of MSNs-Cl (a), MSNs-N3 (b), MSNs-DNA (c)

Figure 2. 15% PAGE monitoring of the λ-Exo cleavage reaction assisted target recycling (A): Lane M, 20 bp DNA Marker; Lane 1, 200 nM synthetic target DNA; Lane 2, 200 nM 5’-PO4 cDNA; Lane 3, 200 nM target DNA + 200 nM 5’PO4 cDNA; Lane 4, 200 nM target DNA + 200 nM 5’-PO4 cDNA + 4 U/μL λ Exo; Lane 5, 200 nM trigger DNA + 200 nM anchor DNA + 200 nM 5’-PO4 cDNA; Lane 6, 200 nM trigger DNA + 200 nM anchor DNA + 200 nM 5’-PO4 cDNA + 4 U/μL λ Exo; Lane 7, 200 nM trigger DNA + 200 nM anchor DNA + 200 nM 5’-PO4 cDNA + 200 nM target DNA; Lane 8, 200 nM trigger DNA + 200 nM anchor DNA + 200 nM 200 nM 5’-PO4 cDNA + 200 nM target DNA + 4 U/μL λ Exo; Lane 9, 200 nM trigger DNA. The process of the trigger DNA initiated HCR (B): Lane M, 20 bp marker; Lane 1, 400 nM trigger DNA; Lane 2, 400 nM H2; Lane 3, 400 nM H2; Lane 4, the mixture of 400 nM H1 and 400 nM H2, Lane 5, 100 nM trigger DNA with the mixture of 400 nM H1 and 400 nM H2. SYBR gold was used and mixed with all the samples.

DNA band and synthetic target DNA band emerged. The results demonstrated that the synthetic target DNA could hybridize with 5’-PO4 cDNA and liberate the DNA gate. At this time, the new formed DNA duplex between target DNA and 5’-PO4 cDNA could be cleaved specifically by λExo to release target DNA again. Next, we tested the HCR amplification process between the trigger DNA and the two hairpin probes.

Denaturation (95 °C for 2 min) and annealing steps are essential to the formation of HCR products. As indicated in Figure 2B, from lane 1 to lane 5, they denote trigger DNA (400 nM), H1 Probes (400 nM), H2 Probes (400 nM), mixture of H1 Probes (400 nM) and H2 Probes (400 nM), and mixture of H1 Probes (400 nM), H2 Probes (400 nM) and trigger DNA (200 nM), respectively. Only line 5 could show many bright bands, confirming the assembling of many of H1 and H2 to form chain-like DNA polymers. Characterization of the Modified Electrode. The electrochemical impedance spectra (EIS) were utilized to monitor the sequential fabrication of the modified electrodes, including the modification on gold electrode and HCR process on its surface. [Fe(CN)6]3-/4- was used as a negatively charged redox label and the semicircle diameter of impedance corresponded with the electrontransfer resistance (Ret). As displayed in Figure 3, the bare gold electrode displayed an excruciatingly narrow semicircle (curve a) attributing to a fast electron-transfer process itself. When treated with the SH-CP, the Ret of the gold electrode was enhanced (curve b), which could be attributed to the further increased (curve c) for the electron transfer blocking of MCH. Subsequently, the SHCP/MCH gold electrode was treated with the mixture of trigger DNA, H1 Probes and H2 Probes. As expected, the Ret kept significant increasing (curve d) due to the introduction of more negative charges on the gold surface upon formation of the dsDNA polymers by the HCR on the electrode. To reveal the TK1 mRNA can liberate the trigger DNA from the DNA-gate and further trigger HCR on the electrode surface, Ret of the SH-CP/MCH gold electrode surface with different treatment was investigated. After treated with the mixture of MB@MSNs-DNA, TK1 mRNA, H1 Probes and H2 Probes, a

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significantly increased Ret was also observed (curve e). In contrast, a weak increase in Ret was displayed if the SHCP/MCH gold electrode was incubated with H1 Probes and H2 Probes or with MB@MSNs-DNA, H1 Probes and H2 Probes (curve f and g). The EIS results from curve a to g not only confirmed that the gold electrode had been modified with SH-CP and capped by the MCH, but also proved that the target mRNA sequence could liberate the trigger DNA from the MB@MSNs-DNA and then initiated HCR on the electrode surface. Feasibility of the Sensing Platform for TK1 mRNA Detection. As we demonstrated above, the conception of target activated electroactive cargo release from MB@MSNs-DNA, λ-Exo cleavage reaction assisted target recycling and HCR on electrode surface have been both verified. We then paid our attention on testing the feasibility of the sensing platform for TK1 mRNA detection by using SWV method. At first, the superior low background capability of platform was investigated by comparing the signal output of the MB@MSNs-DNA incubated SH-CP/MCH gold electrode and the equal MB incubated SH-CP/MCH gold electrode in the absence of target. As shown in Figure 4, the background signal of the MB@MSNs-DNA incubated SH-CP/MCH gold electrode was almost negligible (curve a), whereas noticeable signal was presented in the MB incubated SHCP/MCH gold electrode (curve b). Subsequently, the feasibility of TK1 mRNA detection has been further investigated. Upon addition of TK1 mRNA and λ-Exo to the MB@MSNs-DNA incubated SH-CP/MCH gold electrode, the SWV peak current increased, which was attributed to the hybridization between SH-CP and trigger DNA liberated from the MB@MSNs-DNA (curve c). At this time, the released MB intercalated into the duplex DNA of SH-CP/trigger DNA. Curve d is the signal of the SH-CP/MCH gold electrode incubated with the mixture of MB@MSNs-DNA, TK1 mRNA, H1 Probes and H2 Probes.

b a

0 -.4

Z'/ohm

Figure 3. EIS responses of the gold electrode stepwise modification process in 10 mL of Tris-HCl buffer and 5 mM 3-/4Fe(CN)6 , respectively. (a) bare gold electrode; (b) SH-CP modified gold Electrode; (c) SH-CP/MCH gold electrode; (d) SH-CP/MCH Au Electrode + trigger DNA + H1 + H2; (e) SHCP/MCH gold electrode + MB@MSNs-DNA + H1 + H2 + TK1 mRNA; (f) SH-CP/MCH gold electrode + H1 + H2; (g) SHCP/MCH gold electrode + H1 + H2 + MB@MSNs-DNA.

8 7 6 5 4 3 2 1 0

MB@MSNs MB

2

c

e S/B

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-.2 0.0 Potential/V

.2

Figure 4. The feasibility testing of the sensing platform for TK1 mRNA detection by using SWV method. (a) SHCP/MCH gold electrode + MB@MSNs-DNA + λ Exo + H1 + H2; (b) SH-CP/MCH modified gold electrode + MB + DNAgate + λ Exo + H1 + H2; (c) SH-CP/MCH gold electrode + MB@MSNs-DNA + λ Exo + TK1 mRNA; (d) SH-CP/MCH gold electrode + MB@MSNs-DNA + λ Exo + H1 + H2 + TK1 mRNA; (e) SH-CP/MCH gold electrode+ MB + DNA-gate + λ Exo + H1 + H2 + TK1 mRNA. (Inset image reflects the signal background ratio of different methods between MB@MSNsDNA controlling and MSN-free, respectively).

The truth is that the introduce of H1 Probes and H2 Probes led to a bigger increase of SWV response by compared with that of the SH-CP/MCH gold electrode in the presence of TK1 mRNA, MB@MSNs-DNA and λ-Exo, suggesting that the cascade signal amplification of our λExo cleavage reaction assisted target recycling and target activated HCR-based sensing platform. Without using of MSNs for loading MB (MSNs free cascade signal amplification electrochemical sensing platform), a significant SWV response increase could also observed for the SH-CP/MCH gold electrode treated with mixture of equal MB, DNA gate molecules, TK1 mRNA, H1 Probes and H2 Probes. However, we could find the signal-tobackground ratio of the MSNs free sensing platform is lower than that of MB@MSNs-DNA based cascade signal amplification electrochemical sensing platform (see inset of Figure 4). As a result, our developed sensing platform could lower background and improve signal-tobackground ratio. Optimization of Detection Conditions. To attain the best analytical performance of the asproposed sensing platform, the experimental conditions were carefully optimized, including MB loading concentration, λ-Exo amount and cleavage time as well as HCR reaction time in this work to obtain the best response. As demonstrated in the principle, the electrochemical signal was come from the MB. Therefore, the loading amount of MB in the MSNs, which depended on its concentration used for preparation of MB@MSNsDNA, was first optimized. Here, the concentration of TK1 mRNA we used was 10 nM. The effect of MB concentration on this sensing was shown in Figure S4A. With increasing concentration of MB from 10 to 50 mg

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mL−1, the SWV signal of the released MB increased and trended to plateau at 40 mg mL−1. We then chose 40 mg mL−1 of MB to prepare MB@MSNs-DNA. To amplify the electrochemical signal, one strategy of the proposed sensing platform is employing of λ-Exo cleavage reaction assisted target recycling. Therefore, the concentration of λ-Exo and the cleavage reaction time should be optimized respectively. As shown in Figure S4B, the electrochemical signal in the presence of variable amounts of λ-Exo increased and reached a plateau at 12 U. In addition, the current response increased with the increase of cleavage reaction time and got to a maximum signal at 125 min (Figure S4C). Therefore, 12 U of λ-Exo and 125 min of reaction time were used for the subsequent experiments. HCR is the other strategy used for amplifying the electrochemical signal. The HCR hybridization time is an important parameter affecting the analytical performance because the dsDNA polymers resulted from HCR would directly influenced the amount of MB intercalated. Thus the HCR hybridization time was investigated. Figure S4D shows the effect of different HCR hybridization time on the SWV signal. It suggested that the signal response increased with the extending of hybridization time and reached a stable value at 160 min. There was no enhancement happened on the electrochemical signal even much longer time. Hence, 160 min was utilized as the optimum time for HCR. Detection Performance. Under the optimal experimental conditions, the developed low background cascade signal amplification electrochemical sensing platform was employed for quantifying TK1 mRNA standards with various concentrations based on the target activated HCR and MB release from MB@MSNs-DNA. The relationship between the responses of the SWV peak current and different concentration of TK1 mRNA is illustrated in Figure 5. It was clear that the oxidation peak current of MB increased with the extending of TK1 mRNA concentration in the range of 0 to 1 pM. A linear relationship between the SWV peak current and the concentration of TK1 mRNA was obtained in the range of 0.1 fM to 1 pM with a correlation coefficient of 0.9843. The linear regression equation was Y = 8.0451 + 2.9043 Lg(x) [x=C (mRNA), 10-14 M]. The detection limit corresponding to a signal-to-noise ratio was 2.0 aM (S/N = 3), which was much lower than that of previously reported electrochemical detection methods (Table S3). Further, the selectivity of the proposed sensing platform was estimated by comparing the SWV peak current to diverse DNA sequences including TK1 mRNA, one-base, two-base, three-base, four-base even five-base mismatch DNA under the same concentration (1 pM). As seen in Figure S5, the SWV peak current response to TK1 mRNA indicated an evident increase than that of the blank solution. The SWV peak current response to four-

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Figure 5. (A) Responses of SWV peak current to the different concentration of TK1 mRNA from 0 to 1 pM. (B) The relationship of electrochemical signal and target TK1 mRNA. The range of TK1 mRNA concentration is from 0 to 1 pM. The inset is the linear relationship between SWV peak current intensity and the TK1 mRNA concentration. The illustrated error bars represent the standard error of at least three independent measurements.

base mismatch DNA and five-base mismatch DNA were approaching the blank solution. Indeed, both of the onebase mismatch DNA and two-base mismatch DNA had a higher SWV peak current response than those of fourbase mismatch DNA and five-base mismatch DNA. The results could be explained by the disadvantage of the designed DNA-gates molecules. Despite all this, the developed sensing platform possessed an acceptable selectivity. The reproducibility of this proposed sensing platform was investigated by treating with the same concentration of TK1 mRNA (1 pM) on four independent electrodes in the same conditions. The relative standard deviation (RSD) of SWV response was 6.62%, giving an acceptable reproducibility (Figure S6A). To further explore its stability, this sensing platform was stored at 4 °C when not in use. The electrochemical intensity of the sensing platform did not change too much after 15 days, which indicated an excellent stability (Figure S6B). Detection of TK1 mRNA in Cell Samples. Inspired by the high sensitivity and relatively good selectivity, we used this sensing platform to detect TK1 mRNA in cell samples, and the assay results were compared with the commercial qRT-PCR testing. Here, HepG2 cells and MCF-7 cells were selected as the positive samples as it was reported that the TK1 mRNA was overexpressed in both of them. 18 The relative expression of TK1 mRNA was calculated using the 2-∆∆Ct method, in which ∆Ct=CtTK1 mRNA -CtGAPDH. Consistent with the previous literature, a comparatively higher expression level of TK1 mRNA in HepG2 cells and MCF-7 cells than that of L02 cell lines could be observed with the present sensing platform, as shown in Figure 6. These cell samples results are in obvious agreement with that of qRT-PCR testing (see inset of Figure 6), which shows the practicality of our sensing strategy and the potential application in bioanalysis and clinical diagnosis.

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Analytical Chemistry Corresponding Author * Tel: 86-731-88821566; Fax: 86-731-88821566; E-mail: [email protected]. * Tel: 86-731-88821566; Fax: 86-731-88821566; E-mail: [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. Figure 6. TK1 mRNA detection of different cell lysates including HepG2, MCF-7, and L02 cells. The inset image shows the relative expression of TK1 mRNA through qRTPCR analysis.

CONCLUSION In conclusion, this work reports a low background cascade signal amplification electrochemical sensing platform for the ultrasensitive detection of tumor-related mRNA by coupling the target activated HCR and electroactive cargo release from MSNs. This platform uses MSNs to load the electroactive MB, and a skillfully designed DNA gate composed of an anchor DNA, a 5’phosphate terminated target mRNA recognition probe and a HCR trigger sequence is employed to seal the MSNs pores. When target is introduced, it can hybridize with 5’phosphate terminated target mRNA recognition probe and thus liberates the DNA gate to release the electroactive MB. At the same time, the HCR on the electrode surface and the λ-Exo cleavage reaction assisted target recycling can be initiated to achieve electrochemical signal amplification. Using TK1 mRNA as a model target, the presented strategy can quantitatively detect TK1 mRNA down to 2.0 aM with a linear calibration range from 0.1 fM to 1 pM. Compared to the reported RNA electrochemical sensing, superiority of the developed electrochemical sensing platform includes high sensitive, label-free and low background signal. We have also demonstrated the success of the practical application of our proposed sensing platform for detecting TK1 mRNA in cell samples.

ACKNOWLEDGMENT This work was supported partly by the Project of Natural Science Foundation of China (Grants 21675046, 21735002, 21521063 and 21874035). The key point research and invention program of Hunan province (2017DK2011).

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ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website. Additional information as noted in text: The UV-vis and white light imaging results, MSNs sealing and releasing behaviors, optimization of experimental conditions, the selectivity, reproducibility and stability of this sensing platform, oligonucleotides used in the work, TK1 mRNA complete sequence, the detection of limit comparing result. (PDF)

AUTHOR INFORMATION

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Slobodin, B.; Han, R.; Calderone, V.; Vrielink, J.; Puch, F.; Elkon, R.; Agami, R. Transcription Impacts the Efficiency of mRNA Translation via Co-transcriptional N6-adenosine Methylation. Cell. 2017, 169, 326-337. Jayagopal, A.; Halfpenny, K.; Perez, J.; Wright, D. Hairpin DNA-functionalized gold colloids for the imaging of mRNA in live cells. J. Am. Chem. Soc. 2010, 132, 9789-9796. Xie, N.; Huang, J.; Yang, X.; Y, Y.; Quan, K.; Ou, M.; Fang, H.; Wang, K. Competition-Mediated FRET-Switching DNA Tetrahedron Molecular Beacon for Intracellular Molecular Detection. ACS Sensors. 2016, 1, 1445-1452. Huang, J.; Wang, H.; Yang, X.; Quan, K.; Yang, Y.; Ying, L.; Xie, N.; Ou, M.; Wang, K. Fluorescence resonance energy transfer-based hybridization chain reaction for in situ visualization of tumor-related mRNA. Chem. Sci. 2016, 7, 3829-3835. Wu, C.; Cansiz, S.; Zhang, L.; Cui, L.; Tan, W. A Nonenzymatic Hairpin DNA Cascade Reaction Provides High Signal Gain of mRNA Imaging inside Live Cells. J. Am. Chem. Soc. 2015, 137, 4900-4903. Xie, N.; Huang, J.; Yang, X.; Yang, Y.; Quan, K.; Wang, H.; Ying, L.; Ou, M.; Wang, K. A DNA tetrahedron-based molecular beacon for tumor-related mRNA detection in living cells. Chem. Commun. 2016, 52, 2346-2352. Várallyay, E.; Burgyán, J.; Havelda, Z. MicroRNA detection by northern blotting using locked nucleic acid probes. Nature Protocols. 2008, 3, 190-196. Romero, L.; Barroso, D.; Menendez, P.; Herranz, B. Analysis of mRNA abundance and stability by ribonuclease protection assay. Methods in Molecular Biology. 2012, 809, 491-503. Francesco, M.; Paola, C.; Anna, T.; Jesper, T.; Sergio, A.; Riccardo, L. Normalization of circulating microRNA expression data obtained by quantitative real-time RT-PCR. Briefings in Bioniformatics. 2015, 15, 1-9. Alice, V.; Rita, G.; Elise, M.; Stella, K.; Charles, S.; Sammy, M.; Jimmy, H.; Cassian, M.; Maurice, R.; Simon, J.; Joanne, P.; Roy, M.; Thomas, B. Parasites & Vectors. 2016, 9, 32-38. Md, N.; Vinod, G.; Md, H.; Mostafa, K.; Md. S.; Yusuke, Y.; Nam, T.; Alfred, K.; Muhammad, J. A PCR-free electrochemical method for messenger RNA detection in cancer tissue samples. Biosens. Bioelectron. 2017, 98, 227-233.

9



(12) Anders, E.; Staffen, F.; Anders, P.; Malene, K.; Julie, S.; Hans, E.; Karen, D.; Martin, B. Unaccounted uncertainty from qPCR efficiency estimates entails uncontrolled false positive rates. BMC Bioinformatics. 2016, 17, 151-159. (13) Marco, F.; Paul, A.; Elena, C.; Clive, J. Avian metapneumovirus RT-nested-PCR: a novel false positive reducing inactivated control virus. Journal of Virological Methods. 2012, 186, 171-175. (14) Brian, J.; Clint, C.; Sergei, V.; Roger, P. Dual-frequency resonance-tracking atomic force microscopy. Nanotechnology. 2007, 18, 475501-475504. (15) Habib, A.; You, D.; Pierre, M. Surface Plasmon Resonance of Nanoparticles and Applications in Imaging. Arch. Rational Mech. Anal. 2016, 220, 109-153. (16) Anna,R.; Llaria, M.; Salvatore, C. Surface enhanced Raman spectroscopy based immunosensor for ultrasensitive and selective detection of wild type p53 and mutant p53R175H. Anal. Chim. Acta. 2018, 1029, 86-96. (17) Liu, S.; Xu, N.; Tan, C.; Fang, W.; Tan, Y.; Jiang, Y. A sensitive colorimetric aptasensor based on trivalent peroxidase-mimic DNAzyme and magnetic nanoparticles. Anal. Chim. Acta. 2018, 1018, 86-93. (18) Yang, Y.; Huang, J.; Yang, X.; Quan, K.; Wang, H.; Ying, L.; Xie, N.; Ou, M.; Wang, K. FRET Nano-flares for Intracellular mRNA Detection: Avoiding False Positive Signal and Minimizing Effect of System Fluctuations. J. Am. Chem. Soc. 2015, 137, 8340-8343. (19) Mao, X.; Liu, G.; Wang, S.; Lin, Y.; Zhang, A. Ultrasensitive electrochemical detection of mRNA using branched DNA amplifiers. Electrochemistry Communications. 2008, 10, 1847-1850. (20) Gerasimova, Y.; Kolpashchikov, D. Enzyme-assisted target recycling (EATR) for nucleic acid detection. Chem. Soc. Rev. 2014, 43, 6405-6409. (21) Walker, G.; Fraiser, M.; Schram, J.; Little, M.; Nadeau, J.; Malinowski, D. Multiplex detection of mycobacterium tuberculosis and differentiation from mycobacterium genus using strand displacement amplification. Nucleic Acids Res. 1992, 20, 1691-1696. (22) Yin, P.; Choi, H.; Calvert, C.; Pierce, N. Programming biomolecular self-assembly pathways. Nature, 2008, 451, 318-322. (23) Notomi, T.; Okayama, H.; Masubuchi, H.; Yonekawa, T.; Watanabe, K.; Amino, N.; Hase, T. Loop-mediated isothermal amplification of DNA. Nucleic Acids Res. 2000, 28, E63. (24) Liu, S.; Wang, C.; Zhang, C.; Wang, Y.; Tang, B. Label-free and ultrasensitive electrochemical detection of nucleic acids based on autocatalytic and exonuclease III-assisted target recycling strategy. Anal. Chem. 2013, 85, 2282-2288. (25) Zhao, W.; Ali, M.; Brook, M.; Li, Y. Rolling Circle Amplification: Applications in Nanotechnology and Biodetection with Functional Nucleic Acids. Angew. Chem. 2008, 47, 6330-6337. (26) Ge, Z.; Lin, M.; Wang, P.; Pei, H.; Yan, J.; Shi, J.; Huang, Q.; He, D.; Fan, C.; Zuo, X. Hybridization chain reaction amplification of microRNA detection with a tetrahedral DNA nanostructure-based electrochemical biosensor. Anal. Chem. 2014, 86, 2124-2130. (27) Collins, M.; Irvine, B.; Tyner, D.; Fine, E.; Zayati, C.; Chang, C.; Horn, T.; Ahle, D.; Detmer, J.; Shen, L.; Kolberg, J.; Bushnell, M.; Urdea, S.; Ho, D. A branched DNA signal amplification assay for quantification of nucleic acid targets

Page 10 of 11

below 100 molecules/ml. Nucleic Acids Res. 1997, 25, 29792984. (28) Goluch, E.; Stoeva, S.; Lee, J.; Shaikh, K.; Mirkin, C; Liu, C. A microfluidic detection system based upon a surface immobilized biobarcode assay. Biosens. Bioelectron. 2009, 24, 2397-2403. (29) Yang, L.; Tao, Y.; Yue, G.; Li, R.; Qiu, B.; Guo, L.; Lin, Z.; Yang, H. Highly Selective and Sensitive Electrochemiluminescence Biosensor for p53 DNA Sequence based on Nicking Endonuclease Assisted Target Recycling and Hyperbranched Rolling Circle Amplification. Anal. Chem. 2016, 88, 5097-5103. (30) Yuan, Y.; Chi, B.; Wen, S.; Liang, R.; Li, Z.; Qiu, J. Direct fluorescence detection of microRNA based on enzymatically engineered primer extension poly-thymine (EPEPT) reaction using copper nanoparticles as nano-dye. Biosens. Bioelectron. 2018, 102, 211-216. (31) Li, X.; Guo, J.; Zhai, Q.; Xia, J.; Yi, G. Ultrasensitive electrochemical biosensor for specific detection of DNA based on molecular beacon mediated circular strand displacement polymerization and hyperbranched rolling circle amplification. Anal. Chim. Acta. 2016, 934, 52-58. (32) Pan, W.; Zhang, T.; Yang, H.; Diao, W.; Li, N.; Tang, B. Multiplexed detection and imaging of intracellular mRNAs using a four-color nanoprobe. Anal. Chem. 2013, 85, 1058110588. (33) Zhang, Y.; Hou, Z.; Ge, Y.; Deng, K.; Liu, B.; Li, X.; Li, Q.; Cheng, Z.; Ma, P.; Li, C.; Jun, L. DNA-Hybrid-Gated Photothermal Mesoporous Silica Nanoparticles for NIRResponsive and Aptamer-Targeted Drug Delivery. ACS Appl. Mater. Interfaces. 2015, 7, 20696-20706. (34) Zhang, Y.; Yuan, Q.; Chen, T.; Zhang, X.; Chen, Y.; Tan, W. DNA-capped mesoporous silica nanoparticles as an ionresponsive release system to determine the presence of mercury in aqueous solutions. Anal. Chem. 2012, 84, 19561962. (35) He, D.; He, X.; Wang, K.; Cao, J.; Zhao, Y. A Photon-Fueled Gate-Like Delivery System Using i-Motif DNA Functionalized Mesoporous Silica Nanoparticles. Adv. Funct. Mater. 2012, 22, 4704-4710. (36) Gai, P.; Gu, C.; Li, H.; Sun, X.; Li, F. Ultrasensitive Ratiometric Homogeneous Electrochemical MicroRNA Biosensing via Target-Triggered Ru(III) Release and Redox Recycling. Anal. Chem. 2017, 89, 12293-12298. (37) Xuan, F.; Luo, X. T.; Hsing, I. M. Ultrasensitive SolutionPhase Electrochemical Molecular Beacon-Based DNA Detection with Signal Amplification by Exonuclease IIIAssisted Target Recycling. Anal. Chem. 2012, 84, 5216-5220. (38) Baker, B.; Lai, R.; Wood, M.; Doctor, E.; Heeger, A.; Plaxco, K. An electronic, aptamer-based small-molecule sensor for the rapid, label-free detection of cocaine in adulterated samples and biological fluids. J. Am. Chem. Soc. 2006, 128, 3138-3139. (39) Hou, T.; Li, W.; Liu, X. J.; Li, F. Label-Free and Enzyme-Free Homogeneous Electrochemical Biosensing Strategy Based on Hybridization Chain Reaction: A Facile, Sensitive, and Highly Specific MicroRNA Assay. Anal. Chem. 2015, 87, 11368-11374. (40) Chen, H.; Ju, H.; Xun, Y. Methylene Blue/Perfluorosulfonated Ionomer Modified Microcylinder Carbon Fiber Electrode and Its Application for the Determination of Hemoglobin. Anal. Chem. 1994, 66, 45384542.

10


Page 11 of 11 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


(41) Gao, R.; Hao, C.; Xu, L.; Xu, C.; Kuang, H. Spiny Nanorod and Upconversion Nanoparticle Satellite Assemblies for Ultrasensitive Detection of Messenger RNA in Living Cells. Anal. Chem. 2018, 90, 5414-5421. (42) Yang, H.; Liu, J.; Wang, Z.; Guo, L.; Keller, P.; Lin, B.; Sun, Y.; Zhang, X. Near-infrared-responsive gold nanorod/liquid crystalline elastomer composites prepared by sequential thiol-click chemistry. Chem. Commun. 2015, 51, 12126-12129. (43) Liu, Q.; Liu, J.; He, D.; Qing, T.; He, X.; Wang, K. Triplehelix molecular switch-induced hybridization chain reaction amplification for developing a universal and

sensitive electrochemical aptasensor. RSC Adv. 2016, 6, 90310-90317.

for TOC only

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Low Background Cascade Signal Amplification Electrochemical

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