Ultrasensitive Ratiometric Homogeneous Electrochemical MicroRNA

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Ultrasensitive Ratiometric Homogeneous Electrochemical MicroRNA Biosensing via Target-Triggered Ru(III) Release and Redox Recycling Panpan Gai, Chengcheng Gu, Haiyin Li, Xinzhi Sun, and Feng Li Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.7b03268 • Publication Date (Web): 16 Oct 2017 Downloaded from http://pubs.acs.org on October 16, 2017

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Analytical Chemistry

Ultrasensitive Ratiometric Homogeneous Electrochemical MicroRNA Biosensing via Target-Triggered Ru(III) Release and Redox Recycling

Panpan Gai, Chengcheng Gu, Haiyin Li, Xinzhi Sun and Feng Li*

College of Chemistry and Pharmaceutical Sciences, Qingdao Agricultural University, Qingdao 266109, P. R. China.

* Corresponding author. Tel/Fax: 86-532-86080855 E-mail: [email protected]

ACS Paragon Plus Environment


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ABSTRACT A new label-free and enzyme-free ratiometric homogeneous electrochemical microRNA biosensing platform was constructed via target-triggered Ru(III) release and redox recycling. To design the effective ratiometric dual-signal strategy, [Ru(NH3)6]3+ (Ru(III)) as one of electroactive probe was ingeniously entrapped in the pores of positively charged mesoporous silica nanoparticle (PMSN), as well as another electroactive probe [Fe(CN)6]3- (Fe(III)) was selected to facilitate Ru(III) redox reclycling due to their distinctly separated reduction potential and different redox properties. Owing to the liberation of the formed RNA-ssDNA complex from PMSN, the target miRNA triggered the Ru(III) release and was quickly electro-reduced to Ru (II), then the in-site generated Ru(II) could be chemically oxidized back to Ru(III) by Fe(III). Thus, with the release of Ru(III) and the consumption of Fe(III), a significant enhancement for the ratio of electro-reduction current [Ru(NH3)6]3+ over [Fe(CN)6]3- (IRu(III)/IFe(III)) value was observed, which was dependent on the concentration of the target miRNA. Consequently, a simple, accurate and ultrasensitive method for miRNAs assay was readily realized. Furthermore, the limit of detection (LOD) of our method was down to 33 aM (S/N=3), comparable or even superior to other approaches reported in literature. More importantly, it also exhibited excellent analytical performance in the complex biological matrix cell lysates. Therefore, this homogeneous biosensing strategy not only provides an ingenious idea for realizing simple, rapid, reliable, and ultrasensitive bioassays, but also has a great potential to be adopted as a powerful tool for the precision medicine.

KEYWORDS Ratiometric dual-signal strategy; Homogeneous electrochemical microRNA biosensing; Target-triggered Ru(III) release; Redox recycling


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INTRODUCTION MicroRNAs (miRNAs) have occupied a crucial position in the biological processes of cell development, metabolism, proliferation, differentiation, and apoptosis.1-3 And it has been proven that many diseases and genetic disorders are closely related with the aberrant expression levels of miRNAs.4-6 Consequently, it is urgently necessary to develop sensitive miRNAs detection methods in clinical diagnosis and therapy.4,7-10 Although various traditional techniques, including northern blot,11 quantitative real-time polymerase chain reaction (qRT-PCR),12 microarrays,13,14 fluorescence,15-18

electrochemistry,19

electrochemiluminescence

(ECL),20-23

and

photoelectrochemistry (PEC),24 have been applied in miRNA detection and exhibited excellent performance, the shortcomings of complicated operation, relatively low sensitivity, and high cost still restricted their further development. To address the above issues, homogeneous and immobilization-free electrochemical bioassay has drawn considerable attention in terms of its advantages of simple instrumentation, rapidness, high sensitivity and excellent specificity, and has been widely applied in highly sensitive detection of heavy metal ions,25 ATP,26 cancer biomarkers,27 DNA methyltransferase activity,28,29 alkaline phosphatase activity,30 and telomerase activity.31 Recently, our group constructed a label-free and enzyme-free homogeneous electrochemical miRNA biosensor based on hybridization chain reaction (HCR),32 in which, the HCR signal amplification strategy played the key role in improving the sensitivity of the bioassay. Moreover, a series of other amplification strategies, such as, strand displacement amplification (SDA),23,33-36 rolling circle amplification (RCA),20,37,38 polymerase chain reaction (PCR),39,40 ligase chain reaction (LCR)10 have also been reported, however, they are mainly limited by the disadvantages of high background and possible false-positive readout. The fundamental reason is that the instrumental or environmental factors would cause uncertain fluctuations in absolute signal intensity, especially under low-concentration or complex biological systems.22,41 By contrast, the ratiometric dual-signal strategy based on the ratio of two detection signals is more reliable and effective than the above single-output-based bioassays, owing to its characteristics of significantly eliminating false-positive errors by the built-in correction mechanism, as well as the capability of enhancing detection signal by ratiometric as an original amplification approach at the relatively low concentration. Thus, the ratiometric dual-signal method has the merits of high



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accuracy, excellent sensitivity and good anti-interference ability. Herein, inspired by the aforementioned developments, we proposed a label-free and enzyme-free ratiometric homogeneous electrochemical miRNA biosensing platform based on the target-triggered Ru(III) release and redox recycling. To realize the as-proposed strategy, [Ru(NH3)6]3+ (Ru(III), one of the electroactive probes) was initially entrapped into the pores of mesoporous silica nanoparticle (MSN) and subsequently capped by single-stranded DNA (ssDNA) that are complementary to the target miRNA. Meanwhile, [Fe(CN)6]3- (Fe(III)) was selected as another electroactive probe to facilitate the Ru(III) redox reclycling based on the reaction of Ru(II)+Fe(III)→Ru(III)+Fe(II). As the detailed principle illustration shown in Scheme 1, in the absence of the target miRNA, there were abundant Fe(III) in the electrolyte solution and most Ru(III) were encapsulated in the pores of MSN. As such, the reduction current of Fe(III) was much larger than that of Ru(III), leading to a relatively low current ratio value of IRu(III)/IFe(III). Once the target miRNA was recognized and captured, the formed RNA-ssDNA complex with the rigid structure would be liberated from the MSN due to its lower adhesion to MSN than that of ssDNA,21 accompanied with the release of the entrapped Ru(III), which was quickly electro-reduced to Ru(II) on the electrode surface. Then the generated Ru(II) could be chemically oxidized back to Ru(III) by Fe(III). As a consequence, the reduced Fe(III) and released Ru(III) would result in an obvious enhancement for IRu(III)/IFe(III) value. Consequently, the ratiometric homogeneous electrochemical biosensing strategy for the detection of miRNA was achieved on the basis of the IRu(III)/IFe(III) value variation. This work combines the advantages of both homogenous electrochemical biosensing and ratiometric dual-signal strategy to realize the miRNA detection with high accuracy, excellent sensitivity as well as simple operation, and thus has a great potential to be adopted as the powerful tool for the precision medicine.


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Scheme 1. Schematic principle of the ratiometric homogeneous electrochemical miRNA biosensor.

EXPERIMENTAL SECTION Materials and Reagents. HPLC-purified miRNA and DNA oligonucleotides were purchased from Shanghai Sangon Biotechnology Co., Ltd. (Shanghai, China). Tetraethoxysilane (TEOS), cetyltrimethylammonium

bromide

(CTAB),

3-aminopropyltriethoxysilane

(APTES),

and

hexaammineruthenium (III) chloride ([Ru(NH3)6]3+, denoted as Ru(III)), were all obtained from Sigma-Aldrich (St. Louis, MO, U.S.A.). Tris(hydroxymethyl)aminomethane (Tris) was purchased from Shanghai Chemical Reagent Co. Ltd. (Shanghai, China). Prior to use, the miRNA and ssDNA were diluted with 100 mM Tris-HCl (pH 7.4) to give the stock solutions. Ultrapure water (resistivity > 18.2 MΩ cm at 25 °C) was obtained from a Milli-Q water purification system (Millipore Corp., Bedford, MA, U.S.A.). DEPC-treated ultrapure water was used in all experiments. All reagents were of analytical grade and used without further purification, unless otherwise indicated. The sequences of the oligonucleotides are listed in Table S1. Apparatus and Instrumentation. Transmission electron microscopy (TEM) images were recorded on a HT7700 microscope (Hitachi, Japan) operated at 100 kV. Field emission scanning electron microscopy (SEM) images and the corresponding energy dispersive spectroscopy (EDS) mapping images were measured by a HITACHI S4800 SEM (Hitachi, Japan). Differential pulse voltammetric (DPV) measurements were carried out on a CHI Model 660E electrochemical



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workstation (Shanghai, China) employing a three-electrode system: an indium tin oxide (ITO) electrode, an Ag/AgCl electrode and a platinum wire as the working electrode, the reference electrode and the counter electrode, respectively. Synthesis of Amino-Functionalized MSN. MSN was prepared according to our previous work.42 First, 25.0 mg of the as-prepared MSN was suspended in 1.0 mL of ethanol by sonication. Subsequently, 0.8 mL of APTES was dropped and stirred continuously for 6 h. During this process, the amine groups were immobilized on the surface of MSN, thus resulting in the formation of positively charged amino-functionalized MSN (PMSN). Finally, the mixture were separated by centrifugation, washed at least three cycles with ethanol and ultrapure water, respectively, and dried to afford PMSN. Loading of [Ru(NH3)6]3+ into PMSN and capped by ssDNA. The as-synthesized PMSN was dispersed into 1.0 mL of [Ru(NH3)6]3+ solution to form a homogeneous suspension, followed by gently shaking at room temperature overnight to achieve the maximum loading in the pores of the PMSN scaffolding. Following that, 100 µL of 1 µM ssDNA was incubated with PMSN under gentle stirring for 4 h to form the DNA bio-gates via electrostatic interaction. To remove the unloaded [Ru(NH3)6]3+, the mixture was centrifuged (3000 rpm, 2 min) and rinsed with Tris-HCl buffer at least three times. Finally, the resulting precipitates, i.e. ssDNA-capped PMSN loaded with [Ru(NH3)6]3+ (denoted as ssDNA-capped PMSN), were collected carefully, suspended into pH 7.4 1.0 mL of Tris-HCl buffer, which was placed at 4 °C for further use. As a control, random DNA sequence (rDNA)-capped PMSN were prepared in a similar process except that rDNA was used instead of ssDNA. Homogenous Ratiometric Electrochemical Measurements. The homogenous ratiometric electrochemical measurements were performed by using the aforementioned three-electrode system, with ITO electrode as the substrate electrode, Ru(III) and Fe(III) as two electroactive probes. Initially, as the control experiment, 5 µL of [Fe(CN)6]3- was mixed with 50 µL of ssDNA-capped PMSN and diluted with pH 7.4 Tris-HCl buffer to 100 µL, and then the electrochemical signals of the above electroactive probes were recorded by DPV experiments. In contrast, 5 µL of the target miRNA-21 with different concentrations was firstly incubated with 50 µL of ssDNA capped PMSN at 37 °C for 2 h. Then 5 µL of [Fe(CN)6]3- was added to the resulting mixture and diluted with Tris-HCl buffer to 100 µL, and then the electrochemical signals were


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obtained similar to the above procedure. Cell Culture and Cell Lysis Human cervical cancer cells (HeLa cells), human breast adenocarcinoma cell line (MCF-7), and human lung carcinoma cells (A549) were seeded in DMEM (Dulbecco’s Modified Eagle Medium, Gibco, Grand Island, NY) supplemented with 10% (v/v) fetal bovine serum (FBS), 100 µg mL-1 penicillin, and 100 µg mL-1 streptomycin in 5% CO2 37°C incubator. All types of cells were collected in the exponential phase of growth and washed three times with PBS. Then the cell number was counted by hemocytometer. A 1.0 mL of 106 suspended cells was added into a 6-well plate and incubated for 12 h. Total RNAs from the aforementioned cells were extracted using TRIzol reagent referring to the manufacturer’s recommended protocol. miRNA detection by qRT-PCR. The first-strand cDNA was generated using AMV reverse transcriptase (Takara Bio, Shiga, Japan) and a stem-loop RT primer (Invitrogen, U. S. A.). Quantitative PCR was carried out with an ABI 7300 Sequence Detection System (Applied Biosystems, ABI, U. S. A.) using SYBR Green PCR Master Mix. The relative expression of miRNA was calculated using the 2-∆CT method, in which ∆CT = CTmiRNA-21-CTU6. All qRT-PCR reactions were performed in triplicate. The related DNA oligonucleotides for miRNA-21 analysis by qRT-PCR were listed in Table S2.

RESULTS AND DISCUSSION Construction and Characterization of ssDNA-Capped PMSN. Small molecules could be effectively loaded in MSN due to its porous structure, which has been achieved in various controlled release systems by capping and gating MSN.43,44 In our design, PMSN was obtained via the functionalization of MSN by APTES, and then [Ru(NH3)6]3+ was encapsulated in the pores of PMSN through diffusion. Subsequently, the negatively charged ssDNA, which acted as gatekeepers, were attached onto the surface of PMSN through electrostatic interaction to form ssDNA-capped PMSN. To verify this issue, the morphology of MSN was characterized by TEM. As depicted in Figure 1A and the inset, MSN had a uniform size of 100~120 nm in diameter and possessed the well-ordered porous structures and well-defined pore sizes. In order to gain insights into the assembly of the bio-gate, the ssDNA-capped PMSN was characterized by SEM and EDS (Figure 1B), as well as TEM (Figure S1). The uniform distribution of Si, O, N and P elements



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throughout the entire materials was observed in EDS mapping images. In particular, the signal of P element associated only with DNA appeared, indicating that ssDNA has conjugated onto the PMSN through electrostatic attraction to form the bio-gate. Similar phenomenon was also observed in TEM image of ssDNA-capped PMSN (Figure S1A), which showed that ssDNA-capped PMSN also have good dispersity and their surface become rough as well as the pore features became undistinguishable due to the adsorption of ssDNA. Furthermore, the zeta potential analysis was also employed to confirm the assembly processes of ssDNA-capped PMSN. As shown in Figure 1C, the zeta-potential value of MSN was switched from -5.19 mV to +42.4 mV after the adsorption of APTES, which would facilitate the electrostatic interaction between PMSN and the negatively charged ssDNA. Subsequently, as expected, negative zeta-potential value was observed upon the adsorption of ssDNA on PMSN. The above results demonstrated the successful fabrication of the ssDNA-capped PMSN. After ssDNA-capped PMSN incubated with the target miRNA, the pore structure of PMSN reappeared (Figure S1B), indicating the formed ssDNA-miRNA complex successfully liberated from the surface of PMSN. All the aforementioned results lay solid foundation for acquiring greatly enhanced homogenous electrochemical signals generated by the released [Ru(NH3)6]3+.

Figure 1. (A) TEM image of the MSN. (B) SEM and EDS mapping images of ssDNA-capped PMSN and the corresponding distribution of Si, O, N and P elements. (C) Zeta-potentials of (a) MSN, (b) PMSN, and (c) ssDNA-capped PMSN.

Feasibility Investigation of the MiRNA Bioassay. To study the feasibility of the ratiometric


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strategy in miRNA bioassay, a set of pre-research DPV experiments of neat Ru(III), Fe(III), as well as the mixture were performed (Figure 2A). Firstly, the distinctly separated reduction potentials for pure Fe(III) (0.22 V) and Ru(III) (-0.16 V) could endow the separation of the dual signals (curve a and b), meanwhile, different redox properties of Ru(III) and Fe(III)

were

favorable for the redox reaction of Ru(II)+Fe(III)→Ru(III)+Fe(II) when they coexisted in one system.45 The aforementioned characteristics ensured the recycling of Ru(III) via two sequential processes, namely the electro-reduction of Ru(III) and the chemical oxidization of Ru(II) by Fe(III), and thus, the DPV signals of Ru(III) barely changed (curve b and d in Figure 2A). By contrast, the signal of Fe(III) decreased due to its consumption during the redox process (curve a and d in Figure 2A). And it should be noted that with the increase of the Ru(III) concentration, the DPV signal of Ru(III) increased while that of Fe(III) further decreased (Figure S2). What’s more, because Ru(III) could intercalate into the double-stranded DNA (dsDNA) grooves by the electrostatic adsorption, the control experiments in the presence of the formed ssDNA-miRNA complex were also studied to evaluate the influence of the Ru(III) intercalation on the DPV signals. It can be deduced from the almost unchanged current signals in curve b and c, as well as curve d and e in Figure 2A that the introduction of ssDNA-miRNA complex wouldn’t interfere with the Ru(III) DPV signal. The above results demonstrated the feasibility for adopting the DPV responses of Ru(III) and Fe(III) as the dual signals in ratiometric strategy on the basis of the distinctly separated reduction potentials of these two electroactive probes and the different redox properties. By virtue of the above results, the electro-reduction signals of Ru(III) and Fe(III) were recorded in the presence and absence of the target miRNA-21. As shown in Figure 2B, in the absence of miRNA-21, the reduction currents of Ru(III) and Fe(III) were 9.3 µA and 23.0 µA (curve a), respectively, and the IRu(III)/IFe(III) value was 0.404. Whereas, when the target miRNA-21 existed, the reduction current of Ru(III) increased to 15.4 µA, accompanied with that of Fe(III) decreased to 19.3 µA, which enabled the ratio of IRu(III)/IFe(III) value to be a high value of 0.803 (curve b). It could be inferred that the miRNA-ssDNA complex with the rigid double helix structures formed and broke away from the surface of PMSN, which caused Ru(III) to be released from the pores into the reaction system and increased the reduction signal of Ru(III). Furthermore, the in-situ generated Ru(II) could be chemically oxidized back to Ru(III) by [Fe(CN)6]3-, and the



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consumption of Fe(III) would lead to a decrease of reduction currents of Fe(III). In addition, to demonstrate the signal indeed originating from the specific Watson-Crick interaction between bases not Ru(III) leaking, the control experiments rDNA as the bio-gate sealed the PMSN were measured. As shown in Figure S3, the DPV signals of the target miRNA-21 with different concentrations (0.5 fM, 10 fM and 100 fM) incubated with rDNA-capped PMSN were almost no changed. Therefore, the aforementioned results confirmed the feasibility of the as-proposed strategy for sensitive miRNA detection.

Figure 2. (A) DPV signals of 50 mM Fe(III) (a), 0.5 mM Ru(III) (b), coexisting 0.5 mM Ru(III) and 5 µM ssDNA-miRNA (c), coexisting 0.5 mM Ru(III) and 50 mM Fe(III) (d), coexisting 5 µM ssDNA-miRNA, 0.5 mM Ru(III) and 50 mM Fe(III) (e). (B) DPV signals of Ru(III) and Fe(III) before (a) and after (b) the addition of 7.5 fM miRNA-21. All DPV signals were measured at a scanning rate of 50 mV/s.

Optimization of Assay Conditions. We optimized various experimental conditions to achieve the best performance of the biosensing platform, including the amount of Ru(III) and ssDNA, the incubation time of PMSN with ssDNA, the hybridization reaction time between the target miRNA-21 and ssDNA-capped PMSN, as well as the Fe(III) concentration and its reaction time with the in-site generated Ru(II). First, since the amount of Ru(III) entrapped in PMSN directly affected the electrochemical signal, the influence of Ru(III) concentration on the electrochemical signal of the biosensing platform was investigated. As indicated in Figure S4A, the ratio values initially increased with the increase of Ru(III) concentration, demonstrating that more Ru(III) were loaded into the pores of PMSN and capped by ssDNA. When the Ru(III) concentration was higher than 10.0 mM, the signal leveled off. Thus, 10.0 mM was utilized as the optimal Ru(III)


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concentration. Meanwhile, the gatekeeper ssDNA prevented Ru(III) from diffusing into the system in the absence of the target. Therefore, to realize high encapsulation efficiency, the concentration of ssDNA was optimized for the construction of ssDNA-capped MSN. As shown in Figure S4B, the ratio values increased with the increasing ssDNA concentration, and tended to level off when the concentration was higher than 0.5 µM, which indicated that once ssDNA reached the electrochemical absorption saturation, more amount of ssDNA would be washed off. Hence, to lower the experimental cost, 0.5 µM of ssDNA was used for the entrapment of Ru(III). Next, the incubation time between ssDNA and PMSN was optimized. As ssDNA was attached on the surface of PMSN by electrostatic adsorption, the influence of different incubation time on the ratio value of IRu(III)/IFe(III) toward 0.5 µM ssDNA was evaluated. As shown in Figure S1C, a maximum ratio value was obtained after 4 h and it barely changed with even much longer incubation time. Hence, 4 h was chosen as the optimal incubation time between ssDNA and PMSN. By the same token, the dependence of electrochemical signal on the hybridization time between the target and ssDNA-capped PMSN was also monitored. As shown in Figure S4D, the ratio values increased with the increasing hybridization reaction time, indicating that more ssDNA detached from the surface of PMSN and more Ru(III) released, and then tended to reach equilibrium after 2.5 h. Thus, 2.5 h was utilized for the optimum hybridization reaction time. For the preparation of dual-signal ratiometric biosensing platform, Fe(III) acted as another electroactive probe, thus, the concentration of Fe(III) was explored. As shown in Figure S4E, the low Fe(III) concentration resulted in weak Fe(III) electro-reduction signal, since it was not sufficient to completely oxidize the in-situ generated Ru(III). While excessive Fe(III) would decrease the signal change scope and further lower the sensitivity. From Figure S4E, the optimal concentration of Fe(III) was determined to be 12 mM. Meanwhile, considering the redox reaction rate, the reaction time between Fe(III) and the in-site generated Ru(II) was investigated, which varied from 5 to 30 min. As seen in Figure S4F, the ratio value initially increased with the increase of incubation time, and then reached a plateau at 15 min, which suggested the redox reaction between Ru(III) and Fe(III) finished within 15 min. Thus, 15 min was preferable in the reaction system. Ratiometric Homogenous Electrochemical Biosensing of miRNA. On the basis of the above optimal conditions, the performance of the as-proposed biosensing platform was further evaluated. The DPV peak current of Ru(III) gradually increased and that of Fe(III) gradually decreased with



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the increase of miRNA-21 concentration from 0 to 1500 fM (Figure 3A). As a result, the IRu(III)/IFe(III) values showed positive correlation with the elevated miRNA-21 concentration (Figure 3B). Furthermore, a positive correlation was presented between the ratiometric value and logarithm of miRNA-21 concentration in the range from 0.1 to 1500 fM, with the linear equation of IRu(III)/IFe(III)=0.689+0.153logcmiRNA and the coefficient of determination (R2) of 0.9916. It should also be noted that the as-proposed ratiometric homogenous electrochemical method showed outstanding performance, with a low detection limit of 33 aM (S/N=3) and a linear range (0.1 to 1500 fM) comparable or even superior to those of the reported methods (Table S3).

Figure 3. (A) DPV of the proposed biosensor under different miRNA-21 concentrations (from a to i: 0, 0.1, 0.5, 2.5, 7.5, 12.5, 60, 300 fM, 1500 fM). (B) IRu(III)/IFe(III) values versus miRNA-21 concentration. Inset shows the linear relationship between IRu(III)/IFe(III) values and the logarithm of miRNA-21 concentration from 0.1 to 1500 fM.

Specificity and Stability. To evaluate the specificity of the as-proposed ratiometric homogeneous electrochemical biosensing platform, the interferences of some other miRNAs, including miRNA-141, miRNA-143, miRNA-155 and miRNA-199a, as well as the base mismatched strands, with the same concentration as miRNA-21, were studied as the negative-control. As depicted in Figure 4A, only in the presence of the target miRNA-21, the ∆[IRu(III)/IFe(III)] value (∆[IRu(III)/IFe(III)]= IRu(III)/IFe(III) - I0Ru(III) /I0Fe(III), in which I0 was the blank DPV signal in the absence of miRNAs) of the biosensing platform showed an elevated value due to the high specificity of base pairing reaction, whereas ∆[IRu(III)/IFe(III)] values in the presence of the interfering miRNAs, and even the one-base mismatched strand, were fairly small. The results demonstrated that the as-proposed biosensor possessed excellent sequence specificity to


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distinguish other miRNA family members and even the one-base mismatched strand. To further explore its stability, the biosensing platform was stored at 4 °C when not in use. During 20 days, DPV measurements of the platform were performed at intervals of four days. Figure 4B showed that the IRu(III)/IFe(III) value could retain its original signal with or without the target miRNA-21, implying that the as-proposed biosensing platform exhibited excellent stability.

Figure 4. (A) Comparison of ∆[IRu(III)/IFe(III)] for the biosensor in the presence of miRNA-21 and four interfering miRNAs (a→g, miRNA-141; miRNA-143; miRNA-155; miRNA-199a; three-base mismatch strand; one-base mismatched strand; and miRNA-21), respectively, with the same concentration of 50 fM. (B) Stability analysis of the biosensor without (black column) and with 50 fM miRNA-21 (purple column). Standard deviation was obtained from three independent measurements.

miRNA Detection in Cell Lysates. To validate the practical application of this method for quantitative miRNA assay in complex biological matrix, we employed the cell lysates extracted from three tumor cells (Hela, A549, MCF-7) with high miRNA-21 expression level as the model matrix.46 When the levels of miRNA-21 concentrations in the cell lysates were over the upper limit of the calibration range, the cell lysates were appropriately diluted prior to assay. The concentrations of miRNA-21 in these cell samples were calculated from the calibration curves as 24.6 pM (MCF-7), 21.9 pM (A549), and 19.2 pM (HeLa) with estimated distributions of 14817, 13188, and 11558 copies per cell, respectively, which was in accordance with the results obtained by qRT-PCR (Figure 5). These results clearly suggested that the as-proposed method has great potential in practical miRNA detection with excellent reliability and accuracy.



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Figure 5. The amount of copies of miRNA-21 in cancer cell lysates of MCF-7, A549, and Hela cells determined using our method (ratiometric homogeneous electrochemical biosensor) and commercial kits (qRT-PCR).

CONCLUSIONS In summary, we developed a label-free and enzyme-free ratiometric homogenous electrochemical biosensing platform for ultrasensitive detection of miRNA-21 via target-triggered Ru(III) release and redox recycling. By taking full advantage of their distinctly separated reduction potential and different redox properties of the two electroactive probes, Ru(III) and Fe(III), the ratiometric dual-signal homogenous electrochemical strategy was elaborately designed. Ru(III) was entrapped into the pores of PMSN and capped by ssDNA, as well as Fe(III) was selected to facilitate the redox reclycling of Ru(III). Consequently, the as-proposed miRNA biosensing was achieved on the basis of the IRu(III)/IFe(III) value variation, which combines the advantages of both homogenous electrochemistry and ratiometric detection modes to afford the characterizes of simple operation, high accuracy as well as excellent sensitivity. It should be noted that this strategy not only efficiently decreased the systematic errors caused by instrumental or environmental factors, but also avoided the labeling of the oligonucleotides or the use of tool enzymes. Furthermore, the limit of detection LOD of our method was down to 33 aM (S/N=3), comparable or even superior to other approaches reported in literature. More importantly, it also exhibited excellent analytical performance in the complex biological matrix cell lysates, paving a new way for accurately monitoring overexpressed miRNA-21 in real samples. Therefore, this work provides new insights into the design of a simple biosensing platform for the sensitive, selective, and accurate detection of miRNAs, and is promising to be adopted as the excellent candidate in clinical applications.


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ASSOCIATED CONTENT Supporting Information Additional information as noted in the text. Experimental conditions optimization; Oligonucleotides Sequences; Comparison of miRNA detection performance between our and those reported methods.

AUTHOR INFORMATION Corresponding Author *E-mail: [email protected] (F. Li) Tel/Fax: (86) 532-86080855

Notes The authors declare no competing financial interest.

ACKNOWLEDGMENT We gratefully appreciate the financial support from the National Natural Science Foundation of China (21605092, 21605093 and 21575074), the Natural Science Foundation of Shandong Province, China (ZR2016BQ08), the Basic Research Program of Qingdao (17-1-1-36-jch), the Research Foundation for Distinguished Scholars of Qingdao Agricultural University (663-1117002), and the Special Foundation for Distinguished Taishan Scholar of Shandong Province (ts201511052)

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For TOC only


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Ultrasensitive Ratiometric Homogeneous Electrochemical MicroRNA

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