Multiplexed and Amplified Electronic Sensor for the Detection of

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Multiplexed and amplified electronic sensor for the detection of microRNAs from cancer cells Cuiyun Yang, Baoting Dou, Kai Shi, Yaqin Chai, Yun Xiang, and Ruo Yuan Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/ac503860d • Publication Date (Web): 05 Nov 2014 Downloaded from http://pubs.acs.org on November 12, 2014

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Analytical Chemistry

Multiplexed and amplified electronic sensor for the detection of microRNAs from cancer cells Cuiyun Yang, Baoting Dou, Kai Shi, Yaqin Chai, Yun Xiang,* Ruo Yuan Key Laboratory of Luminescent and Real-Time Analytical Chemistry, Ministry of Education, School of Chemistry and Chemical Engineering, Southwest University, Chongqing 400715, PR China E-mail: [email protected] (Y.X.). * Corresponding authors. Tel.: +86-23-68252277 (Y.X.).

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ABSTRACT The detection of microRNA expression profiles plays an important role in early diagnosis of different cancers. Based on the employment of redox labels with distinct potential positions and duplex specific nuclease (DSN)-assisted target recycling signal amplifications, we have developed a multiplexed and convenient electronic sensor for highly sensitive detection of microRNA (miRNA)-141 and miRNA-21. The sensor is constructed by self-assembly of thiol-modified, redox species-labeled hairpin probes on the gold sensing electrode. The hybridizations between the target miRNAs and the surface-immobilized probes lead to the formation of RNA/DNA duplexes, and DSN subsequently cleaves the redox-labeled hairpin probes of the RNA/DNA duplexes to recycle the target miRNAs and to generate significantly amplified current suppression at different potentials for multiplexed detection of miRNA-141 and miRNA-21 down to 4.2 fM and 3.0 fM, respectively. The sensor is also highly selective toward the target miRNAs and can be employed to monitor miRNAs from human prostate carcinoma (22Rv1) and breast cancer (MCF-7) cell lysates simultaneously. The sensor reported here thus holds great potential for the development of multiplexed, sensitive, selective and simple sensing platforms for simultaneous detection of a variety of miRNA biomarkers for clinic applications with careful selection of the labels.


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INTRODUCTION MicroRNAs (miRNAs), typically 19-25 nucleotides long, are single-stranded, endogenous and non-protein coding RNAs that play important roles as post-transcriptional regulators for gene expression in a broad range of animals, plants, and viruses.1,2 These small miRNAs are believed to be involved in several biological processes such as cell differentiation,3 proliferation,4,5 apoptosis6 and hematopoiesis3,7 by regulating gene expression via messenger RNA cleavage, translational repression/enhancement or deadenylation.8 Research evidences have shown that aberrant (up- or down-regulated) expression of miRNAs is closely related to the occurrence of various cancers.9,10 For example, miRNA-21 is found to be overexpressed in 80% of the tumor samples,11 while miRNA-143 and miRNA-145 are expressed at significantly lower levels in the cancer stages of colorectal neoplasia.12,13 Therefore, the miRNA expression profiles have been increasingly used as effective biomarkers for the diagnosis, classification, progression and treatment response of cancers.14-17 Quantitative reverse transcription PCR (qRT-PCR) and northern blotting represent the most commonly used methods for miRNA detection. Although qRT-PCR can offer high sensitivity for miRNA detection, the involvement of short primers due to the small size of miRNA eventually lowers the efficiency of PCR and extra primer extension techniques are required, which increases the complexity and cost of the assay method.18 The northern blotting approach, which involves size separation of RNA samples by electrophoresis and detection with complementary fluorescent or radioactive probes, however, is time/sample-consuming and has low sensitivity.19 These shortcomings have limited their applications in routine miRNA analysis and triggered the development of new approaches for sensitive, selective and convenient detection of miRNA. In recent years, a number of alternative optical20-22 and electrochemical23-25 miRNA sensing methods in connection to different signal amplification strategies based on nanomaterials2 or nucleases26 have been reported. The electrochemical miRNA assays have gained particular attention in detecting miRNAs due to the advantageous high sensitivity, low cost, speed, simplicity and portability of the electrochemical devices


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which can meet the requirements for point-of-care analysis. Indeed, the electronic DNA (E-DNA) sensors first reported by the Plaxco group27 have significantly advanced convenient detection of different sequence-specific nucleic acids. These E-DNA sensors are simply based on the conformational changes of the redox molecule-labeled DNA probes in the presence of the target sequences, which alters the distance between the redox molecules and the sensor surface and generates either increased or decreased current response for detection. Despite the significant simplicity of the E-DNA sensors, the application of this type of sensors for the monitoring of miRNA encounters two major challenges: highly sensitive detection of low levels of cellular miRNA (the average concentration of individual cellular miRNA is around ~1000 molecules per cell28) and multiplexed detection of miRNA expression profiles. To address these challenges, we report herein a highly sensitive and multiplexed electronic senor for simultaneous detection of miRNAs from different cancer cells by using electroactive labels with distinct voltammograms and duplex-specific nuclease (DSN)-assisted target recycling amplifications. We have previously demonstrated the application of electroactive labels with distinct voltammograms for simultaneous detection of different types of molecules in one single assay.29,30 In addition, nuclease-assisted target recycling can offer effective signal amplifications by cleaving the signal probes upon binding to the target to release and reuse the target.31 Further, we show here that the coupling of different electrochemical labels with DSN-assisted target recycling amplification enables us to simultaneously detect miRNA-21 and miRNA-141 from cancer cell extracts at low femtomolar level in a convenient format. EXPERIMENTAL SECTION Chemicals

and

Materials:

Tris

(2-carboxyethy)

phosphine

hydrochloride

(TCEP)

and

6-mercaptohexanol (MCH) were purchased from Sigma (St. Louis, MO, USA). 4-(2-hydroxyethyl) piperazine-1 ethanesulfonic acid sodium salt (HEPES) was obtained from Shanghai Sangon Biological Engineering Technology & Services Co., Ltd (Shanghai, China). Sodium perchlorate monohydrate (NaClO4·H2O) was obtained from Tianjin Guangfu Fine Chemical Research Institute (Tianjin, China). ACS Paragon Plus Environment

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DSN and 10×DSN master buffer (500 mM Tris-HCl, 50 mM MgCl2, 10 mM DTT, pH 8.0) were purchased from Axxora, LLC (Farmingdale, NY, USA). HPLC-purified, synthetic miRNAs and all other oligonucleotides were purchased from Invitrogen Biotechnology Co., Ltd (Shanghai, China). The sequences of the oligonucleotides were listed in Table 1. Other reagents were of analytical grade and used as received. Aqueous solutions were prepared using ultrapure water (specific resistance of 18 MΩ-cm). Table 1 Oligonucleotide sequences used in this work Synthetic oligonucleotide

Thiol - modified hairpin capture probe

Abbreviation SH-CP-141

SH-CP-21

Sequence 5’-SH-(CH2)6-GCAGTCTACCATCTTTACCAGACAGTG TTATAGACTGC-(CH2)7-MB-3’ 5’-SH-(CH2)6-CCGTTCTATCAACATCAGTCTGATAAG CTATAGAACGG-(CH2)7-Fc-3’

MiRNA-141

5’- UAACACUGUCUGGUAAAGAUGG-3’

MiRNA-21

5’-UAGCUUAUCAGACUGAUGUUGA-3’

Non-complementary

MiRNA-200b

5’-UAAUACUGCCUGGUAAUGAUGA-3’

RNA

Let-7d

5’-AGAGGUAGUAGGUUGCAUAGUU-3’

Target

Cell culture and total RNA extraction: All appliances including the centrifuge tubes, spearheads, culture dishes, were soak in 0.01% DEPC solution overnight and dried in an oven after a sterilization process. The solutions used in all experiments were prepared using DEPC treated water. A human prostate carcinoma cell lines (22Rv1), human breast cancer cells (MCF-7) and human cervical cancer cell lines (Hela) were obtained from the cell bank of the type culture collection of the Chinese Academy of Sciences (Shanghai, China) and cultured in RPMI 1640 medium (Thermo Scientific Hyclone) supplemented with 10% fetal bovine serum, 100 U mL-1 penicillin and 100 μg mL-1 streptomycin at 37 °C in a humidified 5% CO2 incubator. Total RNA samples were extracted from each cell line by using Trizol Reagent (Invitrogen Biotechnology Co., Ltd) according to the manufacturer’s protocol. Briefly,


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the cell pellet containing ×106 cells was added to the appropriate amount of Trizol Reagent, followed by incubation at room temperature for 5 min to ensure complete cell disruption. Then, 2-propanol was used to precipitate the RNA extraction. Finally, the extracted RNA solution was diluted to 106 cells per 100 μl of DEPC treated water, then repackaged to 200 μl microcentrifuge tube with ten microliters of each tube and stored at -20 ºC for further quantification by using our proposed strategy. Preparation of the sensors: The gold disk electrode (AuE, 2 mm in diameter) was first cleaned by immersing in the piranha solution (mixture of 98 % H2SO4 and 30 % H2O2 at a volume ratio of 3:1) for at least 30 min, followed by rinsing thoroughly with ultrapure water. Then, prior to sensor fabrication, the AuE was polished with 0.3 μm and 0.05 μm alumina oxide slurries for 5 min separately, followed by sequentially ultrasonic treatment in water, ethanol and water for 5 min to remove the residual alumina powder. The electrode was then electrochemically cleaned through successive scans (between -0.3 V to 1.55 V) in a fresh H2SO4 solution (0.5 M). After being dried with nitrogen, the electrode should be immediately used for probe immobilization. Prior to probe immobilization, the thiol-modified hairpin capture probes (2 μL of 5 μM SH-CP-141 and 6 μL of 5 μM SH-CP-21) were mixed with 10 μL of 100 mM TCEP in a microcentrifuge tube and incubated for 60 min at room temperature to reduce the disulfide bonds of the SH-CP. The mixture was then diluted to a total volume of 100 μL with 1×PBS, and 7 μL of the diluted mixture was incubated with the clean AuE for for 2 h at room temperature in the dark. After washing by 1×PBS, the resulting electrode surface was blocked with 1 mM MCH solution for 2 h and rinsed with the water to obtain the sensor. Amplified and multiplexed detection of miRNA: Droplets of 7 µL mixtures containing a series of different concentrations of the target molecules (miRNA-141 and miRMA-21) and 0.05 U DSN in 1×DSN master buffer (50 mM Tris-HCl, 5 mM MgCl2, 1 mM DTT, pH 8.0) were incubated with the sensors for 2 h at 37 °C. The sensors were then rinsed with HEPES buffer (10 mM HEPES, 0.5 M NaClO4, pH 7.0) and transferred to electrochemical cells for measurements. Electrochemical


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measurements were performed on a CHI 621D workstation (CH Instruments Inc., Shanghai, China) with a conventional three-electrode system comprised of a platinum wire as the auxiliary electrode, a Ag/AgCl electrode as the reference electrode and the modified AuE as the working electrode. Square wave voltammetric (SWV) measurements were carried out in HEPES buffer with a step potential of 4 mV, a frequency of 25 HZ and an amplitude of 25 mV by scanning the potential from -0.50 V to +0.50 V. Data processing was made by using the “linear baseline correction” function of the CHI 621D software. RESULTS AND DISCUSSION

Scheme 1. Schematic illustration of multiplexed and amplified electrochemical detection of miRNA-141 and miRNA-21 by coupling different redox labels with DSN-assisted target recycling signal amplification. The working principle of our amplified and multiplexed elecronic sensor for simultaneous detection of miRNA-141 and miRNA-21 is illustrated in Scheme 1. The DSN employed to amplify the signal output in our experimental design is an enzyme that shows a strong preference for cleaving double-stranded (ds) DNA with at least 10 bp or DNA in DNA-RNA hybrid duplexes with at least 15 bp, and is practically inactive towards single-stranded (ss) DNA, RNA or dsRNA. The thiol-modified SH-CP hairpin probes labeled with either methylene blue (MB) or ferrocene (Fc) tags are self-assembled on the AuE surface through the formation of Au-S bonds. Subsequent surface blocking


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with MCH leads to the preparation of the sensor. In the absence of the target miRNAs, DSN is inactive to the self-assembled SH-CPs due to the short stems (8 bp) and the steric hindrance of the hairpin structures preventing DSN from accessing the dsDNA stems of the SH-CPs. Two significant current peaks (MB: -0.30 V and Fc: +0.36 V) can thus be expected because of the efficient electron transfer between the MB/Fc labels and the sensor surface. When the target miRNAs are incubated with the sensor with the addition of DSN, the miRNAs hybridize with and unfold the SH-CPs to form DNA/RNA duplexes, which are perfect substrates for DSN. The DSN then cleaves the MB/Fc-labeled DNA strands of the DNA/RNA duplexes and releases the target miRNAs, which can again hybridize with the un-folded SH-CPs to initiate cyclic cleavage of the MB/Fc-labeled DNA strands. Due to the target recycling amplification assisted by DSN, the presence of small amount of the miRNA targets can result in the removal of massive MB/Fc labels from the sensing surface, leading to highly suppressed current peaks at different potential positions (MB at -0.30 V and Fc at +0.36 V correspond to miRNA-141 and miRNA-21, respectively) for sensitive and multiplexed detection of miRNAs.

Figure 1. Typical SWV responses of the sensors for (A) the absence and the presence of (B) miRNA-141 (5.0 pM), (C) miRNA-21 (5.0 pM) and (D) miRNA-141 (5.0 pM) and miRNA-21 (5.0 pM) under the addition of DSN (0.07 U). The mixtures were incubated at 37 °C for 100 min. SWV measurements were performed in HEPES buffer with a step potential of 4 mV, a frequency of 25 HZ and an amplitude of 25 mV by scanning the potential from -0.50 V to +0.50 V. We first show the multiplex capability of our sensor for the detection of miRNA-141 and miRNA-21


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with the addition of DSN. As shown in Figure 1A, in the absence of the target miRNAs (DSN is unable to cleave the SH-CPs), the sensor exhibits two current peaks at different potential positions due to electrochemical oxidation of the MB/Fc labels (off note: the immobilization concentrations of the SH-CPs are optimized to give similar current responses). The incubation of the target miRNA-141 (5.0 pM) with the sensor leads to significant decrease in current response at -0.30 V while the peak corresponding to SH-CP-21 at 0.36 V remains unchanged (Figure 1B vs. A). Similarly, the presence of miRNA-21 (5.0 pM) results in the reduction of current peak at 0.36 V and the peak at -0.30 V is not affected (Figure 1C). This comparison indicates that our sensor can be employed to detect either miRNA-141 or miRNA-21. Importantly, when both miRNA-141 and miRNA-21 are incubated with the sensor, simultaneous and significant decrease in current responses for the two peaks are observed (Figure 1D), indicating the feasibility of the sensor for multiplexed detection.

Figure 2. Typical SWV responses of the sensors for the absence of the target miRNAs without (a) and with (b) the addition of DSN (0.07 U) and for the presence of miRNA-141 (5.0 pM) and miRNA-21 (5.0 pM) without (c) and with (d) the addition of DSN (0.07 U). Other conditions, as in Figure 1. DSN-assisted signal amplification to the sensor is also verified by comparing the current suppression with/without the addition of DSN for the presence of the target miRNAs (both at 5.0 pM). According to curve b and a in Figure 2, the incubation of the sensor with/without DSN in the absence of the target miRNAs has negligible effect on the signal output, which reveals that DSN is inactive to the SH-CPs. We can also see that the presence of the target miRNAs without the addition of DSN also causes obvious current decreases (curve c vs. a in Figure 2). Such decreases can be basically attributed to the


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fact the miRNA targets hybridize with and unfold the hairpin SH-CPs, which push the MB/Fc labels away from the sensor surface, leading to decreased electron transfer efficiency to the electrode and thus suppressed current responses. In contrast, the addition of DSN (0.07 U) with the miRNA targets results in significant current suppression (curve d vs. a in Figure 2) due to DSN-assisted, cyclic removal of the MB/Fc labels from the sensor surface as discussed previously. This comparison clearly demonstrates the signal enhancement with the involvement of the DSN in the assay method.

Figure 3. Effect of (A) the amount of DSN (with 100 min incubation time) and (B) incubation time (with 0.05 U DSN) on the current response of the sensor for the presence of miRNA-141 (5.0 pM). Error bars, SD, n=3. Other conditions, as in Figure 1. Two important parameters, the amount of DSN and incubation time that affect the assay performance for the detection of the miRNAs were optimized by using the miRNA-141 target (5.0 pM). As displayed in Figure 3A, the current peak of MB corresponding to the presence of miRNA-141 decreases quickly with the increase of the amount of DSN from 0.01 U to 0.05 U and levels off thereafter. Thus, 0.05 U DSN is considered to be the optimal amount used in the following experiments. In Figure 3B, a quick decrease in the current peak with the extension of the incubation time from 0 to 120 min can be observed, and the current drop reaches a plateau at 120 min. Therefore, 120 min is selected as the optimal incubation time. After experimental optimization, the dependence of the current response upon the concentration of the target miRNAs was investigated to validate multiplexed miRNA detection. As displayed in Figure 4A, increased suppression of the current responses can be observed with elevated concentration of the


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target miRNAs. It is clearly shown that the addition of different concentrations target miRNAs to the sensing interface results in significant decrease in peak currents associated with the amounts of the redox-tags. The resulting calibration plots in Figure 4B and C exhibit linear relationships between Δi (the peak current difference between the absence and presence of the miRNAs) and logcmiRNAs, and the dynamic ranges are ranging from 5.0 fM to 50 pM for miRNA-141 and miRNA-21. The detection limits are estimated to be 4.2 fM and 3.0 fM, respectively, with 3 times the standard deviation of the blank. The detection limits of the developed method for multiplexed detection of miRNAs are comparable with or even more sensitive than some other reported methods (Table 2). Six repetitive measurements of miRNA-141 at 5.0 pM and miRNA-21 at 5.0 pM yielded the relative standard deviation of 6.5% and 5.9%, respectively, indicating good reproducibility of the sensor.

Figure 4. (A) Typical SWV responses of the proposed sensor for multiplexed detection of miRNA-141 and miRNA-21 at: (a) 0 fM and 0 fM, (b) 5.0 fM and 5.0 fM, (c) 50 fM and 50 fM, (d) 0.5 pM and 0.5 pM, (e) 5.0 pM and 5.0 pM, (f) 50 pM and 50 pM. (B) and (C) correspond to the resulting calibration plots of Δi vs. logcmiRNA-141 and logcmiRNA-21, respectively. Error bars, SD, n=3. Other conditions, as in Figure 1.


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Table 2 The comparison of different methods for miRNA detection Target

Detection method

Detection limit

Signal amplification strategy DSN-assisted target recycling and bio-barcode

Let-7b

EIS

1.0 fM

MiRNA-21

Chronoamperometry

0.06 pM

nanoparticles and HRP catalyzed H2O2

32

MiRNA-21

DPV

5.36 fM

Isothermal amplification reaction

33

Let-7b

Chemiluminescence

10 fM

DSN-assisted target recycling amplification with DNAzymes

34

MiRNA-141

Fluorescence

100 fM

DSN-assisted target recycling amplification

35

Let-7a

Fluorescence

0.4 pM

DSN-assisted target recycling amplification

36

MiRNA-15a

ECL

21.7 fM

CdTe QDs-based ERET

37

MiRNA-141 and miRNA-21

SWV

4.2 fM and 3.0 fM

DSN-assisted target recycling

This Work

Ref 26

Figure 5. Specificity investigation of the sensor for the target miRNA-141 (5.0 pM) and miRNA-21 (5.0 pM) against other control miRNA sequences of miRNA-200b (0.1 nM) and let-7d (0.1 nM). Other conditions, as in Figure 1. In order to investigate the interference of the control miRNAs to the sensor, experiments were performed by incubating the target miRNA-141 (5.0 pM) and miRNA-21 (5.0 pM) against the control ACS Paragon Plus Environment

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miRNAs, miRNA-200b (0.1 nM) and let-7d (0.1 nM). As shown in Figure 5, despite the existence of an large excess (20-fold) of the control miRNAs, the signal suppression is negligible compared with the blank test (in the absence of the target miRNA-141 and miRNA-21), while the presence of the target miRNAs at even low concentrations leads to significant signal drops. These results demonstrate that DSN can provide excellent discrimination ability and our multiplexed electronic sensor possesses a high selectivity toward the target miRNAs against other control miRNAs.

Figure 6. Multiplexed detection of miRNAs from different cancer cell lysates: (a) blank, (b) Hela (10000 cells), (c) 22Rv1 (100 cells) and MCF-7 (100 cells), (d) 22Rv1 (1000 cells) and MCF-7 (1000 cells) and (e) 22Rv1 (10000 cells) and MCF-7 (10000 cells). To test the applicability of the developed sensor for miRNA analysis in real samples, miRNA-141 and miRNA-21 from different cancerous cell lines, 22Rv1 (human prostate carcinoma cells), MCF-7 (human breast cancer cells) and Hela (cervical cancer cells) were monitored by this multiplexed sensor. From the results in Figure 6, we can see that the current responses of the sensor incubated with the lysate from Hela cells (10000 cells) exhibit insignificant suppression compared to the blank buffer test (b vs. a in Figure 6), indicating that neither miRNA-141 nor miRNA-21 is over-expressed in Hela cells, which is in good agreement with the previous report.35 However, in the presence of the mixture of lysates from 22Rv1 cells (overexpression of miRNA-14122) and MCF-7 cells (overexpression of miRNA-2133), the current responses decrease with increasing number of the cells (c to e in Figure 6). Based on the current responses in Figure 6, the amount of miRNA-141 found in 22Rv1 cells and miRNA-21 found in MCF-7 cells are about 2500 and 5700 copies per cell, respectively, which is in a


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comparable range with other literatures.22,38,39 These evaluations suggest a great potential of the multiplexed sensor for the monitoring of miRNAs in different cancer cells for early diagnosis of cancers. CONCLUSIONS In conclusion, we have demonstrated a multiplexed and sensitive electronic sensor for simultaneous detection of different miRNA targets in one single assay. The employment of the MB/Fc labels with distinct voltammetric peak potentials, which reflect the identity of the target miRNAs, enables the multiplexed capability of the sensor. Besides, the involvement of the DSN in the assay method can lead to cyclic reuse of the target miRNAs and significantly amplified signals to achieve high sensitivity for miRNA detection. More importantly, the developed electronic sensor can be used for multiplexed detection of different miRNAs from the corresponding cancer cell lysates. Although, we only show the detection of miRNA-141 and miRNA-21 with the developed method, multiplexed detection of more types of miRNA targets can be realized by careful selection of redox-tags with distinct potential positions (e.g., thionine at -0.17 V, anthraquinone at -0.45 V), which could potentially pave the way for simultaneous detection of miRNA expression profiles for point-of-care diagnostic of different cancers. ACKNOWLEDGMENT This work is supported by National Natural Science Foundation of China (Nos. 21275004 and 21275119), the New Century Excellent Talent Program of MOE (NCET-12-0932) and Fundamental Research Funds for the Central Universities (XDJK2014A012).


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