Peptide nucleic acid-based electrochemical biosensor for simultaneous detection of multiple microRNAs from cancer cells with catalytic hairpin assembly amplification

Journal Pre-proof Peptide Nucleic Acid-based Electrochemical Biosensor for Simultaneous Detection of Multiple microRNAs from Cancer Cells with Catalytic Hairpin Assembly Amplification Pan Fu, Shu Xing, Mengjia Xu, Yang Zhao, Chao Zhao

PII:

S0925-4005(19)31744-7

DOI:

https://doi.org/10.1016/j.snb.2019.127545

Reference:

SNB 127545

To appear in:

Sensors and Actuators: B. Chemical

Received Date:

26 August 2019

Revised Date:

2 December 2019

Accepted Date:

5 December 2019

Please cite this article as: Fu P, Xing S, Xu M, Zhao Y, Zhao C, Peptide Nucleic Acid-based Electrochemical Biosensor for Simultaneous Detection of Multiple microRNAs from Cancer Cells with Catalytic Hairpin Assembly Amplification, Sensors and Actuators: B. Chemical (2019), doi: https://doi.org/10.1016/j.snb.2019.127545

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Peptide Nucleic Acid-based Electrochemical Biosensor for Simultaneous Detection of Multiple microRNAs from Cancer Cells with Catalytic Hairpin Assembly Amplification Pan Fua,c, Shu Xinga, Mengjia Xua,c, Yang Zhao*,b, and Chao Zhao*,a

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Cixi Institute of Biomedical Engineering, Ningbo Institute of Materials Technology

and Engineering, Chinese Academy of Sciences, Ningbo 315201, P. R. China

College of Science and Technology, Ningbo University, Ningbo 315212, P. R. China

c

University of Chinese Academy of Sciences, Beijing 100049, P. R. China

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b

*

E-mail address: [email protected] (Chao Zhao)

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[email protected] (Yang Zhao)

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Corresponding author:

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Graphical abstract

Highlights

An electrochemical biosensor for simultaneous detection of miRNAs was

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developed using PNA and CHA.

This assay was highly selective for discriminating miRNAs with similar sequences.

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It has a low LOD of 2.49 fM and 11.63 fM for miRNA21 and miRNA155,

This assay can be used to detect a variety of miRNAs from human cancer cells at the same time.

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

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respectively.

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ABSTRACT Herein we demonstrated a facile electrochemical method for simultaneous detection of miRNA21 and miRNA155 using a peptide nucleic acids (PNAs)-modified gold electrode coupled with the target-catalyzed hairpin assembly (CHA) strategy. In the presence of the target miRNA, the CHA was triggered selectively between two hairpins with one ferrocene (Fc) or methylene blue (MB) labelled. The resulting redox-active group modified CHA products (Fc-CHA21 or MB-CHA155) were then specifically captured by the PNA probes (PNA21 or PNA155) attached on the surface

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of a gold electrode, which bring the Fc and MB labels into close proximity to generate apparently enhanced electrochemical signals for sensitive and simultaneous detecting

of low amount miRNA21 and miRNA155 in cancer cells. This assay was highly selective for discriminating miRNAs with similar sequences and has detection limits

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of 2.49 fM and 11.63 fM for miRNA21 and miRNA155, respectively. The feasibility of the method for sensitive determination of miRNA21 and miRNA155 from human

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cancer cells was also demonstrated. This method thus has great potential to be applied for simultaneous detecting of a variety of miRNA biomarkers for clinic applications

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due to its simple, sensitive and accurate features.

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Keywords: Peptide nucleic acid (PNA); Electrochemical biosensor; Catalytic hairpin

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assembly (CHA); Multiplexed miRNA detection

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1. Introduction MicroRNAs (miRNAs) are a class of short single-stranded endogenous non-coding RNAs [1], which are critical indicators of oncogenes or cancer suppressors for gene expression, gene therapy and tumor diagnosis or prognosis, etc [2]. Thus, monitoring expression levels of miRNAs contributes to reveal vital information about tumor-related medical diagnosis [3], Alzheimer's disease [4], drug delivery [5], and prognosis evaluation [6]. Towards this goal, a number of quantitative reverse transcription polymerase chain reaction (qRT-PCR) [7], DNA-structure probes

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such as molecular beacon [8], sandwich [9], tetrahedron [10], and three dimensional structure [11], non-DNA nanostructures with the properties of electrochemistry

[12,13], fluorescence [14,15], and surface enhanced Raman scattering (SERS) [16,17]

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are commonly used for the quantification of miRNAs. However, sophisticated

optimization, poor stability, and spontaneous autoxidation always limit their utility

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[18]. Furthermore, it is not suitable to recognize relatively low abundance miRNAs in typical cancer cells (HeLa, MCF-7, etc.) [17]. Moreover, miRNAs are also high

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sequence homology and short length, restricting the practical application of these technologies. Therefore, to address many of these limitations, it is imperative to introduce an amplification program to reduce sample usage.

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Catalytic hairpin assembly (CHA) is an enzyme-free isothermal amplification strategy that originally invented by the Pierce’s group [19], which uses cascade DNA

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amplification and DNA strand-displacement reaction to recycle target and achieve attractive detection sensitivity of biomarkers [20,21]. The enzyme-free CHA reaction

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has shown great potential for miRNAs analysis by building programmable DNA nanomachines [22,23], biosensors [24-27], DNA circuits [28,29], and chemical reaction networks [30,31]. For example, Liu et al. recently reported an efficient flowerlike nanovector based on polydopamine-modified gold nanoflower, which could transport CHA probes into cells for intracellular miRNA imaging [22]. Gu et al. developed a highly sensitive assay for miRNA based on CHA-hybridization chain reaction (HCR) amplification cascades and a silver coated gold nanorods (Au@Ag 4

NRs) etching process accompanied by surface plasmon resonance (SPR) shift [28]. Although the CHA enables the detection system to be with good turnover rates and ultrahigh sensitivity [32], it only achieved one target detection, which may cause false positive results for clinical diagnosis because of insufficient information [33]. Furthermore, these methods require expensive instruments, specialized reagents, and sophisticated readout system, which increase the cost and complexity. Convenient and economic devices with high sensitivity and specificity are still valuable for routine miRNAs analysis.

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Electrochemical biosensor is considered to be the most promising device for miRNA detection due to its advantages of high sensitivity, low cost, simplicity and easy miniaturization, which could meet the requirements for point-of-care analysis. In recent years, many electrochemical biosensors have been reported for simultaneous

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detection of multiple miRNAs by employing different signal amplification strategies,

such as enzyme amplification [34], duplex specific nuclease-assisted target recycling

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signal amplification [35], DNA tetrahedral nanostructure capture probes [36], ligase chain reaction and the electrochemical quantum dots barcodes [37], magnetic and

HCR

[38],

magnetic

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nanoprobes

microbeads

and

diblock

oligonucleotide-modified gold nanoparticles [39]. Although acceptable testing

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sensitivity and specificity were obtained for the determination of multiple miRNAs, the use of unstable enzyme, signal attenuation strategy, large detection volume, or sophisticatedly prepared nanomaterials makes them difficult to analyze clinic samples

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especially in resource-limited settings. More importantly, the use of conducting nanomaterials or negatively-charged DNA probes often resulted in non-specific

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reaction and high background noise. To address these challenges, we report herein a facile electrochemical method for

simultaneous detection of multiple miRNAs by taking advantage of peptide nucleic acid (PNA) and CHA. PNA is an analogue of DNA that consisting of repeated N-(2-aminoethyl) glycine units linked by peptide bonds [40]. In the past decades, a number of ingenious chemical modified PNA reagents have been selected and converted into chemistry and biology usage, including electrochemical sensing 5

[41,42], antisense reagent [43], polymeric carrier [44] and other biochemistry reaction [45,46]. Recently, the electrochemical PNA (E-PNA) sensors have been widely developed because (a) E-PNA binds to its complementary sequence more strictly than that of DNA or RNA, and compared with a duplex of oligonucleotides, a PNA-DNA (or PNA-RNA) duplex possesses higher thermal stability and is less tolerant to mismatched base pairs [47,48], (b) PNA can maintain the rigid structure and stretch state on the electrode, (c) PNA has an electroneutral framework, (d) PNA cannot be degraded enzymatically [49,50]. As a consequence, the E-PNA sensor has a relatively

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high signal-noise ratio compared with the E-DNA sensor for the reduced non-specific adsorption caused by electrostatic interaction, and the target DNA has less secondary structure and is more accessible to the probe molecules [51].

In this work, with the use of miRNA21 and miRNA155 as the proof-of-principle

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analytes, we develop a simple, sensitive and multiplexed electrochemical biosensor for simultaneous detection of miRNAs from different cancer cells by using different

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PNA capture probes and target-triggered CHA amplifications for the first time. MiRNA21, one of the most abundant miRNAs inside common cancer cells, is an

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anti-apoptotic and pro-survival factor that involved in many biological functions [52]. MiRNA155, as an oncogenic miRNA, is over-expressed in various solid tumors and

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upregulated in B-cell lymphomas and chronic lymphocytic leukemia [53]. Two PNA capture probes (PNA21 and PNA155) were designed to anchor on the gold electrode surface by the Au−S bond. Four hairpin probes (H1, H2, H3 and H4) with two

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electroactive group labelled (ferrocene (Fc)-H2 and methylene blue (MB)-H4) were designed for the CHA reaction. Target miRNAs were first incubated with H1, Fc-H2,

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H3 and MB-H4 probes, and then the resulting products containing CHA21 and CHA155 were selectively captured by PNA21 and PNA155 for signal amplification. Specificity of this sensing system was also verified by using mismatched miRNA targets. This sensor can achieve detection of multiple miRNAs simultaneously with good sensitivity and anti-interference capability, even in the cell lysates. Furthermore, this work provides a simple, universal and convenient strategy for multiplexed detection of miRNAs using CHA amplification by the E-PNA biosensor. 6

2. Experimental section 2.1. Materials All DNA oligonucleotides were synthesized by Jie Li Biology Inc. (Shanghai, China), and all miRNAs were purchased from Shanghai GenePharma Co. Ltd. (Shanghai, P.R. China). PNA oligomers were synthesized using Fmoc-solid phase peptide synthesis protocols as described previously [47,49,50]. The sequences of hairpin DNA probes, PNA probes and miRNAs were listed in Table S1 (mismatched

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bases underlined). 2.2. Target-triggered CHA amplification

The CHA reactions were prepared by mixing H1, H2 with miRNA21 or H3, H4

with miRNA155 at the molar ratio of 4:4:1 (the final concentration are 400 nM, 400

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nM and 100 nM, respectively) at RT for 2 h. The CHA products (CHA21 and CHA155) were analyzed by native 10% (w/w) polyacrylamide gel electrophoresis

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(PAGE) in 1 × TBE buffer consisting of Tris (40 mM), acetic acid (20 mM), and EDTA (1 mM) (pH 8.3) at 150 V for 40 min. After washing, the gel was stained by

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gel-red at RT for 15 min and imaged via a Tanon 2500 Gel Imaging System. 2.3. Fabrication of the E-PNA biosensor

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To prepare the PNA-modified gold electrode, the gold disk electrode with diameter 2 mm was first cleaned by immersing in a fresh piranha solution (98% H2SO4 and 30% H2O2, 3:1 V/V) for 30 min. Then, the electrode was polished with 1.0,

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0.3 and 0.05 μm alumina oxide slurries, respectively, to get a mirror-like surface. After sequentially sonicated in ultrapure water, ethyl alcohol and ultrapure water for

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at least 10 min, the electrode was electrochemically cleaned through successive potential scans between −0.33 V and 1.55 V in a fresh 0.5 M H2SO4 solution for 20 times. The cleaned gold electrode was dried with nitrogen. Before probe immobilization, the thiol-modified PNA probes (100 μL, 1 μM) was treated with TCEP (1 μL, 100 mM) at RT for 1 h to reduce the disulfide bonds. Then, 5 μL of the PNA solution (1 μM) was dropped onto the surface of the cleaned gold electrode and incubated at 37 °C for 2 h for immobilization. The resulting 7

PNA-coated electrode was rinsed with PBS buffer, followed by blocking with 2 mM MCH (dissolved in 60% ethanol) for 30 min. Then, the surface of the electrode was washed with 60% ethanol and ultrapure water, respectively, to obtain the E-PNA biosensor. 2.4. Simultaneous detection of multiple miRNAs 5 μL of CHA21 (containing different concentrations of target miRNA21) and 5 μL of CHA155 (containing different concentrations of target miRNA155) were dropped onto the prepared electrode surface and incubated at 37 °C for 2 h. The

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resulting electrode was then rinsed with PBS and used for electrochemical measurements.

Electrochemical measurements were performed on a CHI 660E workstation (CH Instruments Inc., Shanghai, China) with a conventional three-electrode system, which

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was consisted of a gold working electrode (Φ = 2.0 mm), an Ag/AgCl reference

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electrode, and a platinum wire auxiliary electrode. Square wave voltammetric (SWV) curves were acquired by scanning the potential from -0.45 V to +0.6 V with a step

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potential of 4 mV, a frequency of 25 HZ and an amplitude potential of 25 mV. Cyclic voltammetry (CV) measurements were collected between −0.2 V and +0.6 V in 5.0 mM [Fe(CN)6]3-/4-. Electrochemical impedance spectra (EIS) was performed within

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the frequency of 0.1 Hz to 100 KHz with an open circuit potential of 0.18 V and an amplitude potential of 5.0 mV in [Fe(CN)6]3-/4- standard solution. Chronocoulometry

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was conducted at a pulse period of 250 ms and stepped from -0.1 V to +0.5 V in PBS buffer containing 50 µM [Ru(NH3)6]3+ (RuHex).

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3. Results and Discussion 3.1. Principle of the E-PNA biosensor The principle of the E-PNA biosensor coupled with target-triggered CHA

amplification for simultaneous detection of multiple miRNAs is shown in Scheme 1. Four hairpin probes H1, H2, H3 and H4 as the recognition and CHA amplification elements are rationally designed to remain metastable without the target miRNA initiators. Moreover, H2 and H4, served as reporting probes, are labelled with Fc and 8

MB at the 3’ end [54,55], respectively, for the generation of the electrochemical signals. In a typical sensing process, target miRNA21 or miRNA155 hybridize with the external toehold of H1 or H3, which will initiate the unfolding of H1 or H3 through a strand displacement process. Subsequently, the open-loop H1 or H3 hybridize with the external toehold of H2 or H4, resulting in the release of intact miRNA21 and miRNA155 and the generation of H1/H2 or H3/H4 duplexes. Then, the released miRNA21 and miRNA155 are recycled to hybridize with another H1 or H3 and initiate a new cycle of CHA reactions. Finally, the products of CHA21 and

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CHA155 that contain many and many H1/H2 or H3/H4 hybrids are specifically captured by the PNA capture probes PNA21 and PNA155, which are anchored on a gold electrode surface via Au-S bond. This will bring the Fc and MB labels into close proximity to generate apparently enhanced electrochemical signals, which could be

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used as the output signal for sensitive and simultaneous detecting of miRNA21 and

miRNA155. In this system, CHA enables each miRNA strand to generate multiple

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signal outputs by increasing the local concentration of the CHA products. And the PNA capture probe allows the direct capturing of the CHA products onto the surface

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of gold electrode without extra separation and purification steps. Therefore, the CHA-assisted E-PNA biosensor provides a robust, facile, and promising strategy for

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miRNA detection. 3.2. PAGE characterization

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The feasibility of the target-miRNA triggered CHA reactions was first verified by 10% native PAGE. As indicated in Fig. 1A, the bands of H1 (lane 2) and H2 (lane 3)

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can be clearly observed, while for the CHA system H1 and H2, there is no cross-reactive band appears when they are mixed together (lane 4). This is attributed to the intramolecular hybridization, which could effectively block the spontaneous hybridization between H1 and H2. However, upon incubating H1 or H2 with miRNA21, H1 is opened by hybridizing with miRNA21 and resulted in the formation of the miRNA21/H1 hybrid (lane 5), while H2 kept intact (lane 6). Furthermore, upon incubating miRNA21/H1 hybrid with H2, an apparent band with much lower mobility 9

(lane 7) is observed, indicating the self-assembled duplex of H1 and H2. All of the above results confirm that the hybridization between H1 and H2 is catalyzed by the target miRNA21. Same results were obtained for H3 and H4 system by using the target miRNA155 (Fig. 1B). To verify that the CHA strategy is also suitable for simultaneous detection of multiple miRNAs, the four hairpin probes were first mixed with each other, followed by incubation of miRNA21 with H3 and H4, and miRNA155 with H1 and H2, to exclude the possibility of cross hybridization. As shown in Fig. 1C, there is no cross

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hybridization band with lower mobility exists for these samples (lane 1 to lane 6), indicating that the two CHA reactions triggered by miRNA21 and miRNA 155 can be worked independently without disturbing each other. The apparent CHA products

band for the sample that containing both miRNA21 and miRNA155 (lane 7) further

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confirmed that the two CHA reactions can be worked efficiently and simultaneously.

Therefore, the proposed CHA-assisted strategy provides a feasible way to fabricate a

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signal amplification platform for multiplexed detection of miRNAs. To verify that the CHA products could be efficiently captured by PNA capture

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probes, the CHA21 or CHA155 were incubated with PNA21 and PNA155, respectively. Obviously, PNA21/CHA21 and PNA155/CHA155 exhibit an apparent

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band with lower mobility compared with PNA21/CHA155 and PNA155/CHA21 (Fig. 1D, lane 1-4), which indicates the high binding specificity of PNA probes to their corresponding complementary CHA products. In addition, the hybridization band is

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also clearly observed when mixing them all together (Fig. 1D, lane 5), suggesting that the hybridization between different PNA probes and their corresponding CHA

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products can be worked simultaneously. To eliminate the possibility of nonspecific binding, a random sequence PNA was also mixed with CHA21 and CHA155 as a negative control (lane 6). These phenomena confirmed our design that the E-PNA biosensor could be used for sensitive and selective detection of multiple miRNAs when combined with CHA. 3.3. Electrochemical characterization of the modified electrode 10

The stepwise fabrication process of the electrochemical biosensor was first characterized by EIS in a PBS solution (100 mM, pH 7.4) containing 5 mM [Fe(CN)6]3−/4−. As shown in Fig. 2A, the bare gold electrode with excellent conductivity shows a small semicircle in the impedance spectrum (curve a). After the attachment of the PNA probes onto the surface of gold electrode, the diameter of the semicircle in the impedance spectrum (curve b) increases greatly, demonstrating the successful immobilization of PNA probes through the Au-S bonds. The semicircle diameter increases continuously after blocking the nonspecific sites on the electrode

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by MCH (curve c). With the assembling of the CHA products on the electrode, a significant increase in the electrochemical impedance of the electrode is observed

(curve d), which is attributed to many H1/H2 and H3/H4 hybridized on the electrode

and the negatively charged DNA hinders the negatively charged [Fe(CN)6]3−/4− from

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accessing to the electrode surface due to the electrostatic repulsion as well as the

through PNA-DNA hybridization.

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steric hindrance effect [56,57], indicating the successful capture of the CHA products

Moreover, CV was also carried out to verify the feasibility of the CHA-assisted

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E-PNA biosensor under the same condition (Fig. 2B). The bare gold electrode shows a relatively high redox peak current (curve a), but as the electrode is modified with

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PNA probes and MCH via Au−S bond, the peak current decreases gradually (curves b and c). Finally, when the electrode is incubated with the CHA products, an apparent decreased current signal is recorded (curve d), demonstrating the high signal

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amplification efficiency of CHA for electrochemical detection of miRNA. The same results could be obtained for the electrode that is modified with PNA21 or PNA155,

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as shown in Fig. S1 and Fig. S2. 3.4. Validation of the CHA-assisted E-PNA biosensor for simultaneous detection of miRNA21 and miRNA155 To validate our CHA-assisted E-PNA biosensor, we monitored the SWV signal change by immersing the sensor electrode into solutions that containing different CHA products (Fig. 3). In the absence of target miRNA21 and miRNA155, a very low 11

SWV response is observed, suggesting that the CHA reaction doesn’t take place. When the sensor is incubated with the CHA solution containing 50 pM miRNA21, the peak current corresponding to Fc at +0.38 V increased by about 35-fold while the peak corresponding to MB at -0.25 V remains unchanged, indicating that the CHA reaction is initiated by miRNA21. Similarly, the presence of 50 pM miRNA155 results in the increase of the peak current at -0.25 V by about 20-fold and the peak at +0.38 V is not affected, suggesting the high selectivity of the E-PNA biosensor when combined with CHA, which could be employed to detect either miRNA21 or

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miRNA155. Significantly, when the sensor is incubated with the mixture of 50 pM miRNA21 and 50 pM miRNA155, current responses for the two SWV peaks increased dramatically and simultaneously, indicating that our E-PNA biosensor could be used for multiplexed detection of miRNAs when combine with CHA.

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To eliminate the possibility that the increased SWV signal was caused by the

direct hybridization of PNA21 or PNA155 with H2 and H4, control experiment was

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carried out by incubating the sensor with 400 nM H2 or H4 for different times. As shown in Fig. S3, only a small SWV peak was observed for both samples, and the

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signal intensity doesn’t change with the extension of the incubation time, indicating the hybridization between PNA probes and H2 or H4 is kinetically impeded, because

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their strand displacement reactivity is effectively blocked by intramolecular hybridization. In addition, the interaction between the PNA probes and H2 or H4 was further verified by PAGE. As displayed in Fig. S4, no hybridization band is observed

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with extended time of incubation. It is clearly shown that the PNA probes cannot open H2 or H4 by themselves, which ensures the relatively low background signal for the

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sensor.

3.5. Optimization of the experimental conditions To enhance the sensitivity of the E-PNA biosensor, two important parameters, the

concentration of PNA probes and the incubation time that affect the assay performance for the detection of the miRNAs was systematically studied by using target miRNAs at the concentration of 50 pM. Native PAGE was first used to 12

characterize the hybridization between the CHA products and PNA probes at different concentrations (ranging from 0.5 to 4 M). As shown in Fig. S5, the optimum hybridization efficiency was obtained at the PNA concentration of 1 M for both targets. To get an optimum signal responses for the CHA products, SWV measurements were also carried out by using gold electrodes that were assembled with different concentrations of PNA probes ranging from 0.5 to 2 M. Fig. S6 shows a ~5-fold enhancement in SWV signal after CHA21 hybridization as the concentration of PNA increased from 0.5 to 1 M. However, with further increase in PNA

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concentration, the SWV signal changed slightly ( 1-fold). Therefore, the PNA concentration of 1 M was used in the following experiments.

Moreover, to achieve optimal hybridization efficiency for miRNA detection, surface density of PNA probes was further investigated by chronocoulometry. The

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varied PNA monolayers were first prepared by treating gold electrode surface with

different concentrations of PNA probes ranging from 0.5 to 2 μM. With the aid of a

through

electrostatic

adsorption

in

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cationic redox marker RuHex, which can bind to anionic phosphate of nucleic acids a

stoichiometric

manner

[58,59],

the

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chronocoulometric detection was carried out by monitoring the redox charge of RuHex (Q). As shown in Fig. S7, the redox charge increased gradually with increase

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in the PNA concentration from 0.5 M to 2 M, and a sharp increase in signal change (ΔQ = QCHA – QPNA, the variation in the redox charge of RuHex before and after hybridization.) of 140% occurs at 1 μM probe concentration (Fig. S7C), whereas the

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increase ratios are below 20% with further increase in PNA concentration. To quantify the surface-immobilized PNA probes, the redox charge of RuHex, which can reflect

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the amounts of the gold electrode surface-immobilized nucleic acids, was measured by using a same sequence DNA (P-DNA) instead of PNA21, and the surface density of the 1 μM PNA assembly concentration is estimated to be ca. 1.2 × 1013 molecules/cm2 (Fig. S8) according to previous studies [58,59], which corresponds to a 2 nm intermolecular spacer, nearly equal to half of the PNA length (∼4.1 nm), rendering a favorable PNA orientation. At this optimal probe density, the sensor was also incubated with the CHA 13

solution for different times from 5 min to 8 h to get an optimum hybridization time. As displayed in Fig. S9, the SWV peak current for both targets were gradually increased as the incubation time increased from 5 min to 120 min, and with further increase in the hybridization time, the peak current almost kept unchanged, suggesting that 120 min is enough for the hybridization between PNA and the CHA products. Therefore, 120 min is selected as the optimal incubation time. 3.6. Determination of miRNA155 and miRNA21 by SWV respectively Under the optimum conditions, the target miRNA21 and miRNA155 were

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determined by the proposed CHA-assisted E-PNA biosensor, respectively. Various

concentrations of miRNA21 or miRNA155 were first added into their corresponding CHA system. After CHA reaction, the sensor electrode was incubated with CHA21 or

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CHA155, and then used for SWV measurement. As indicated in Fig. 4A, the peak

current of Fc at +0.38 V is sensitive to and increases as the concentration of

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miRNA21 increases. Meanwhile, the similar increasing trend at -0.25 V (MB) appears for miRNA155 (Fig. 4C). To quantitatively measure the sensitivity of the sensor, peak

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current was also recorded at each test concentration. Fig. 4B and 4D show the relationship between peak intensity at +0.38 V or –0.25 V and the concentrations of miRNA21 or miRNA155. A linear correlation exists between the peak current and the

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logarithm of target concentration over the range of 10 fM to 5 nM for miRNA21 (Fig. 4B) and 50 fM to 50 nM for miRNA155 (Fig. 4D). This sensor has a detection limit

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of 2.36 fM for miRNA21 and 10.56 fM for miRNA155 based on the equation LOD = 3 × (SD/S) with a 1% confidence level, where SD is the standard deviation of the

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response and S is the slope of the standard curve in the linear region, which is superior to the previously reported sensing platforms (Table S3). In particular, compared with the previous results of RNA detection without amplification, we found that owing to the CHA amplification technology, the sensitivity of RNA detection is improved by 3 orders of magnitude [60]. These data show high sensitivity for single-component detection of miRNA21 or miRNA155 by using the CHA-assisted E-PNA biosensor. 14

3.7. Analytical performance of the biosensor for simultaneous detection of miRNA21 and miRNA155 Based on the above results, the E-PNA biosensor was employed for the detection of miRNA21 and miRNA155 simultaneously (Fig. 5A). Significant increase in SWV peak currents at both +0.38 V and -0.25 V was observed with elevated concentration of the target miRNAs. The calibration plots show good linear relationship between the peak current and logcmiRNAs. The dynamic ranges are ranging from 10 fM to 5 nM for

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miRNA21 (Fig. 5B) and from 50 fM to 5 nM for miRNA155 (Fig. 5C). The low detection limits are estimated to be 2.49 fM and 11.63 fM for miRNA21 and

miRNA155, respectively. Compared with the other reported electrochemical sensing

systems for simultaneous detection of miRNAs (Table 1), the proposed E-PNA

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biosensor has a comparable or even an excellent sensitivity and a wider linear range, which can be attributed to the integration of CHA and the high binding efficiency of

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PNA.

3.8. Specificity and stability of the proposed biosensor

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To evaluate the specificity of the biosensor, control experiments were also carried out by incubating the sensor with 50 nM interfering sequences of mismatched

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miRNA21, mismatched miRNA155, miRNA141 and miRNAlet7, respectively. As shown in Fig. 6, the SWV peak currents only show negligible increase for these interfering miRNAs at even high concentrations compared with the blank (without

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miRNA). While the presence of the target miRNAs at low concentrations (10 pM miRNA21 and 10 pM miRNA155) leads to significant signal increases. These results

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verified the good discrimination ability and excellent specificity of the E-PNA biosensor toward the target miRNAs against other interfering miRNAs. To test the stability of the E-PNA biosensor for target detection, the SWV signal was monitored every 3 days, and no obvious change was observed after 2 weeks, indicating the good stability and prolonged lifetime of the sensor for simultaneous miRNAs detection. 3.9. Detection of miRNAs in real samples 15

To demonstrate the applicability of the proposed sensor for multiplexed miRNA detection in real samples, miRNA21 and miRNA155 from different cancer cell lines, Hela (cervical cancer cells), MCF-7 (human breast cancer cells) and MDA-MB-231 (human breast cancer cells) were analyzed by this E-PNA biosensor. As shown in Fig. 7, apparent enhancement in the current responses was observed when incubating the sensor with miRNA samples that were extracted from HeLa, MCF-7 and MDA-MB-231, respectively, indicating that miRNA21 and miRNA155 are over-expressed in these cells, and the expression level of different cells are in the

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order of MDA-MB-231 > MCF-7 > HeLa, which is in good agreement with the previous reports in Table S4. To further verify the accuracy of the CHA based E-PNA sensing strategy, the gold standard qRT-PCRs were also carried out to quantify miRNA21 and miRNA155 in Hela, MCF-7 and MDA-MB-231 cells, respectively

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(Fig. S11 and Fig. S12). It is shown that the results determined by the proposed E-PNA biosensor are in good agreement with the qRT-PCR results (Table 2), which

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confirmed the reliability of our sensing strategy for miRNAs in real samples. These results show the great potential of this E-PNA biosensor for early cancer diagnosis by

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monitoring the expression level of miRNAs in different cancer cells. 4. Conclusions

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In summary, we demonstrated a PNA-based electrochemical biosensor for simultaneous detection of multiple miRNAs in cancer cells with high sensitivity and

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specificity. By taking the advantage of PNA probes and the target-triggered CHA strategy, we provides a simple, robust and stable sensor platform for target analysis

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under various conditions. PNA ensures the high specificity and the high signal-to-noise ratio of the sensor for target analysis. CHA ensures the high sensitivity of the analysis, and the enzyme-free feature of CHA also makes it possible for practical applications in clinic diagnosis of cancers. In addition, the employment of the electroactive labels with distinct voltammetric peak potentials provides the feasibility for multiplexed miRNAs analysis. More importantly, the ultrahigh sensitivity and specificity, isothermal conditions, and flexible design make this sensor 16

a promising platform for robust, simultaneous, ultrasensitive, and selective detection of miRNA expression profiles for clinical diagnosis of different cancers. Further studies on detecting other miRNAs that are associated with different cancers are working on.

Acknowledgements

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This work was supported by Ningbo Natural Science Foundation (2017C110020, 2018A610318, 2019C50039) and funds from Ningbo Institute of Materials Technology and Engineering, Chinese Academy of Sciences.

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Scheme 1. Schematic illustration of electrochemical and simultaneous detection of

miRNA21 and miRNA155 by coupling the E-PNA biosensor with target-triggered

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CHA amplifications.

Fig. 1. (A) Native PAGE analysis of miRNA21-triggered CHA reaction. The concentrations of miRNA21, H1, and H2 are 100 nM, 400 nM, and 400 nM, 24

respectively. (B) Native PAGE analysis of miRNA155-triggered CHA reaction. The concentrations of miRNA155, H3, and H4 are 100 nM, 400 nM, and 400 nM, respectively. (C) Native PAGE analysis of the CHA reactions in the presence of miRNA21 and miRNA155. (D) Native PAGE analysis of the hybridization between different PNA probes and the CHA products. The concentrations of all PNA probes

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are 1 μM.

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Fig. 2. (A) EIS and (B) CV characterization of modified electrode in 5 mM [Fe(CN)6]3−/4− electrolyte: (a) bare gold electrode, (b) SH-PNA21 + SH-PNA155

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modified gold Electrode, (c) SH-PNA21 + SH-PNA155/MCH gold electrode, and (d)

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SH-PNA21 + SH-PNA155/MCH gold electrode + CHA21 + CHA155.

Fig. 3. SWV curves for the E-PNA biosensor at: (black) without target miRNA; (red) in the presence of 50 pM miRNA21; (green) in the presence of 50 pM miRNA155; (blue) in the presence of 50 pM miRNA21 and 50 pM miRNA155. 25

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Fig. 4. (A) SWV responses of the E-PNA biosensor for the detection of miRNA21 at different concentrations: 10fM, 50 fM, 500 fM, 5 pM, 50 pM, 500 pM and 5 nM. (B)

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Calibration plot of SWV peak current vs. the logarithm of miRNA21 concentration. (C) SWV responses of the E-PNA biosensor for the detection of miRNA155 at different concentrations: 50 fM, 500 fM, 5 pM, 50 pM, 500 pM, 5 nM, 10 nM and 50

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nM. (D) Calibration plot of SWV peak current vs. the logarithm of miRNA155 concentration. The error bars are standard deviations of three repetitive

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measurements.

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Fig. 5. (A) SWV responses of the E-PNA biosensor for simultaneous detection of

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miRNA21 and miRNA155 at different concentrations: (a) 0 fM and 0 fM, (b) 10 fM and 50 fM, (c) 500 fM and 500 fM , (d) 5 pM and 5 pM, (e) 50 pM and 50 pM, (f)

500 pM and 500 pM, (g) 5 nM and 5 nM. (B) and (C) The resulting calibration plots

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of SWV peak current vs. logcmiRNA21 and logcmiRNA155, respectively. The error bars are

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standard deviations of three repetitive measurements.

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Fig. 6. (A) SWV curve and (B) SWV peak current responses of the E-PNA biosensor toward target miRNA21 and miRNA155 against other interfering miRNAs:

single-base mismatched miRNA21, single-base mismatched miRNA155, miRNA141

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and miRNAlet-7a. The concentrations of miRNA21 and miRNA155 are 50 pM, while

the concentrations of other miRNAs are 5 nM. Blank sample means the condition in

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the absence of any miRNA. The error bars are standard deviations of three repetitive

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measurements.

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Fig. 7. (A) SWV curve and (B) SWV peak current responses of the E-PNA biosensor for multiplexed detection of miRNA21 and miRNA155 from different cancer cells: (a) Hela (104 cells); (b) MCF-7 (104 cells); (c) MDA-MB-231 (104 cells). The error bars are standard deviations of three repetitive measurements.

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Author Biographies Pan Fu is currently a PhD student in Ningbo Institute of Materials Technology and Engineering, Chinese Academy of Sciences. Her research interest focuses on the fabrication of electrochemical biosensors for cancer diagnosis.

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Shu Xing gained his doctor’s degree from Shanghai Institute of Applied Physics, Chinese Academy of Sciences in 2016. Now she is an assistant professor in Ningbo Institute of Materials Technology and Engineering, Chinese Academy of Sciences. Her research interest focuses on the synthesis and application of multifunctional nanomaterials in cancer diagnosis and therapy. Mengjia Xu is currently a PhD student Ningbo Institute of Materials Technology and Engineering, Chinese Academy of Sciences. Her research interest is synthesis and application of functional peptide nucleic acids in diagnosis and treatment of diseases.

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Yang Zhao was graduated from Jilin University at China in 2010 and got the Ph. D. Now she worked at College of Science and Technology, Ningbo University as a Physical Chemistry Lecturer. Her research interest is the fabrication of functional nanomaterials for analytical and biomedical applications.

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Prof. Chao Zhao received his PhD from Changchun Institute of Applied Chemistry, Chinese Academy of Sciences in 2010. He then moved to the United States and worked at the School of Medicine, Yale University and the NIDDK, National Institutes of Health. In 2016, he joined Ningbo Institute of Materials Technology and Engineering, Chinese Academy of Sciences as a principal investigator. His research mainly focused on the modification and functionalization of biomolecules, peptide nucleic acids and nanomaterials for advanced medical technology.

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Table 1. The comparison of different electrochemical detection methods for miRNA analysis. Linear range

Detection limit

Ref.

miRNA141, miRNA21

DPV

1 fM to 1 nM

0.44 fM, 0.46 fM

[38]

miRNA141, miRNA21

SWV

5.0 fM to 50 pM

4.2 fM, 3.0 fM

[35]

miRNA21, miRNA155, miRNA196a, miRNA210

CV

10 fM to 10 nM

10 fM, 10 fM, 10 fM, 10 fM

[36]

miRNA155, miRNA27b

SWV

50 fM to 30 pM, 50 fM to 1050 pM,

12 fM, 31 fM

[37]

miRNA182, miRNA381

DPV

5 fM to 600 fM, 1 fM to 800 fM

miRNA141, miRNA21

DPV

0.1 fM to 10 nM

miRNA21, miRNA155

SWV

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Detection Method

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0.20 fM, 0.12 fM

10 fM to 5 nM, 50 fM to 5 nM

[39]

25.1 aM, 25.1 aM

[61]

2.49 fM, 11.63 fM

this study

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Detection miRNAs

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Table 2. Determination of miRNA21 and miRNA155 in cells by the proposed E-PNA biosensor and qRT-PCR.

miRNA155

RSDa

qRT-PCR

RSDa

(pM)

(%)

(pM)

(%)

HeLa

2.29

3.76

2.51

5.31

MCF-7

25.12

3.83

23.98

5.05

MDA-MB-231

63.09

3.73

56.23

2.60

HeLa

0.29

5.42

0.30

4.86

MCF-7

3.20

4.87

2.88

5.31

154.88

2.54

151.36

2.63

MDA-MB-231 Relative standard deviation.

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miRNA21

E-PNA Biosensor

cell line

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miRNA

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