Copper- and Cobalt-Codoped CeO2 Nanospheres with Abundant

Jan 17, 2019 - Oxide materials with redox properties have aroused growing interest in many applications. Introducing dopants into crystal lattices pro...
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Copper- and Cobalt-Codoped CeO2 Nanospheres with Abundant Oxygen Vacancies as Highly Efficient Electrocatalysts for Dual-Mode Electrochemical Sensing of MicroRNA Shuyan Xue, Qingqing Li, Lei Wang, Wenbin You, Jie Zhang, and Renchao Che* Laboratory of Advanced Materials, Department of Materials Science and Collaborative Innovation Center of Chemistry for Energy Materials (iChem), Fudan University, Shanghai 200438, PR China

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S Supporting Information *

ABSTRACT: Oxide materials with redox properties have aroused growing interest in many applications. Introducing dopants into crystal lattices provides an effective way to optimize the catalytic activities of the oxides as well as their redox properties. Herein, CeO2 nanospheres codoped with Cu and Co (CuCo−CeO2 NSs) were first synthesized and exploited as efficient electrocatalysts for dual-mode electrochemical sensing of microRNA (miRNA). With the doping of Cu and Co into the CeO2 lattice, large amounts of extra oxygen vacancies were generated, remarkably enhancing the redox and electrocatalytic properties of the CeO2 material. The abundant oxygen vacancies of the CuCo−CeO2 NSs were further identified by X-ray photoelectron spectroscopy (XPS), H2 temperature-programmed reduction (H2-TPR), and electron-energy-loss spectroscopy (EELS). Moreover, Mg2+induced DNAzyme-assisted target recycling was introduced for ultrasensitive determination. The dual-mode sensing with generality was conducted as follows: First, the CuCo−CeO2 NSs acted as a direct redox mediator to generate a differentialpulse-voltammetry (DPV) signal, which was then greatly amplified by the efficient electrocatalysis of CuCo−CeO2 NSs toward H2O2 decomposition. Second, under the electrocatalysis of CuCo−CeO2 NSs, 3,3-diaminobenzidine (DAB) was oxidized to form nonconductive insoluble precipitates (IPs), leading to great amplification of the electrochemical-impedimetricspectroscopy (EIS) signal. The dual-mode electrochemical sensor showed a wide linear range (0.1 fM to 10 nM) with a low detection limit (33 aM), paving a new way for constructing ultrasensitive electrochemical sensors.

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vacancies,9,10 which can be adjusted by doping. Ke and coworkers noted that CeO2 nanowires doped with lanthanides show rather high catalytic activities for CO oxidation compared with those of the undoped samples.11 Zhang’s group proposed a homogeneous solid solution of Mn0.5Ce0.5O2 with high catalytic activity for the selective oxidation of hydrocarbons.12 In these cases, the introduction of dopants into the CeO2 matrix greatly facilitates the migration of lattice oxygen, which further increases the concentration of oxygen vacancies and thus the catalytic activity of the CeO2 materials.13 Meanwhile, local distortions and lattice contractions or expansions also lead to the formation of crystal defects, which helps to form the oxygen vacancies.14 Furthermore, the formation energy of oxygen vacancies can be reduced through the incorporation of dopants, resulting in the generation of extra oxygen vacancies.12 Thus, introducing foreign cations into CeO2 matrix holds great potential for improving the sensitivity of electrochemical sensors.

he acute determination of MicroRNAs (miRNAs) is of great importance in clinical diagnosis owing to the vital roles of miRNAs in heart diseases, neurological diseases, and a broad range of cancers.1 Recently, electrochemical sensors have demonstrated potential in miRNA detection owing to their relative high sensitivity, specificity, and low cost.2 To improve their sensitivity and stability, nanomaterials have been widely used for constructing electrochemical sensors on the basis of their large surface-to-volume ratios, good biocompatibility, and superior catalytic properties.3,4 Although relatively low detection limits have been achieved, more significant improvement of sensitivity are still urgently needed for developing ultrasensitive electrochemical sensors. Metal oxide nanomaterials with remarkable redox properties have received considerable interest in diverse fields, including electrochemical sensing,5 oxygen-reduction reactions (ORR),6 and CO oxidation, 7 because of their stability, good biocompatibility, and catalytic activity. Among them, ceria (CeO2) with mixed valences of Ce3+ and Ce4+ has received particular attention because of its earth abundance, low cost, and superior redox properties.8 The electrocatalytic activity of CeO2 mainly depends on the concentration of oxygen © XXXX American Chemical Society

Received: August 20, 2018 Accepted: January 17, 2019 Published: January 17, 2019 A

DOI: 10.1021/acs.analchem.8b03778 Anal. Chem. XXXX, XXX, XXX−XXX

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Scheme 1. Schematic Illustration of (A) Synthesis of the CuCo−CeO2 NSs, (B) Mg2+-Induced DNAzyme-Assisted TargetRecycling Amplification Strategy and (C) Dual-Mode Sensing Principle for miRNA-141 Determination

More interestingly, the coexistence of Ce3+ and Ce4+ endows the CeO2 with superior redox activity, making the doped CeO2 become a direct redox mediator to generate detectable electrochemical signal that is measurable by differential pulse voltammetry (DPV).15,16 In particular, using metal oxides as redox probes in electrochemical sensors has received little attention. On the other hand, in an impedimetric thrombin sensor reported by Li et al.,17 the oxidization of DAB was promoted by the biocatalysis of horseradish peroxidase (HRP) in the presence of H2O2, and the formed nonconductive insoluble precipitates (IPs) led to enhancement of the electrochemical-impedance-spectroscopy (EIS) signal. Considering the superior catalytic performance and chemical stability over natural enzymes,18 it is also attractive to extend the doped CeO2 for EIS-signal amplification. The combination of DPV and EIS methods avoids the limited signaling capacity of single-signaling strategies,19,20 which further endows the dualmode electrochemical strategy with lower detection limits and higher accuracy and reproducibility.21,22 To the best of our knowledge, a dual-mode electrochemical sensor for an miRNA assay in which doped CeO2 acts as a bifunctional electrocatalyst for signal amplification has not been reported yet. In this work, by using CeO2 nanospheres codoped with Cu and Co (CuCo−CeO2 NSs) as an efficient electrocatalyst, we first propose a dual-mode electrochemical platform for the ultrasensitive determination of miRNA-141 (Scheme 1). With the codoping of Cu and Co into the CeO2 lattice structure, large amounts of oxygen vacancies were successfully generated, which endowed the CuCo−CeO2 NSs with superior electrocatalytic activity compared with that of pure CeO2 or singledoped ones. With the introduction of Mg 2+ -induced DNAzyme-assisted target recycling for further signal amplification,23 numerous CuCo−CeO2 NSs were anchored on the

electrode surface. Finally, owing to the superior electrocatalytic activity of the CuCo−CeO2 NSs, high sensitivity and low detection limits of the electrochemical sensor were achieved by using DPV and EIS methods. We believe that this work holds great potential in developing an ultrasensitive biosensing platform for clinical diagnostics, medical research, and other related subjects.

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EXPERIMENTAL SECTION The reagents and materials, apparatus, and polyacrylamide-gel electrophoresis (PAGE) analysis are displayed in the Supporting Information. Preparation of CuCo−CeO2 Nanospheres (CuCo− CeO2 NSs). The uniform CuCo−CeO2 NSs were prepared by a one-pot solvothermal method. Briefly, 15.0 mM CeCl3· 7H2O, 0.31 mM CuCl2·2H2O, 0.31 mM CoCl2·6(H2O), and 1.0 g of PVP were dispersed in 34 mL of methanol with stirring for 30 min at room temperature. Then, 0.8 mL of formic acid was added into the above solution and stirred for another 10 min. Afterward, 2 mL of NH3·H2O (1 M) was added dropwise with stirring for another 30 min. The change of the solution from colorless to white suggested the successful formation of CuCo−CeO2. The resulting product was washed with ethanol and water and then dried under vacuum at 70 °C overnight. Finally, the product was calcined at 500 °C for 3 h with a heating rate of 1 °C/min. For the comparison experiment, CeO2, Cu−CeO2, and Co−CeO2 were synthesized using similar methods with minor modifications. Preparation of CuCo−CeO2 Conjugated with Complementary Strand (CuCo−CeO2−CS). First, 0.1 g of the as-synthesized CuCo−CeO2 was suspended in 10 mL of ethanol and stirred for 30 min. Afterward, 0.1 mL of APTES was added into the above mixture, followed by stirring and B

DOI: 10.1021/acs.analchem.8b03778 Anal. Chem. XXXX, XXX, XXX−XXX

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Analytical Chemistry refluxing at 70 °C for 1.5 h. The obtained amino-functionalized CuCo−CeO2 (CuCo−CeO2−NH2) was centrifugated out, washed with ethanol and water, and redispersed in 1 mL of water. Then, 100 μL of complementary strand (CS, 2.5 μM) and 100 μL of GA (1%, w/w) were injected into 1 mL of the above CuCo−CeO2−NH2 solution and stirred overnight at 4 °C; this was followed by the addition of 20 μL of BSA solution for 40 min. As a result, CS with −NH2 groups was attached on the surface of CuCo−CeO2−NH2 with GA serving as a linking agent. The product was collected by centrifugation, resuspended in 1 mL of water, and stored at 4 °C for further use. Other bioconjugates including CeO2−CS, Cu−CeO2−CS, and Co−CeO2−CS were also prepared by using similar procedures. Fabrication of the Electrochemical Sensor. The stepwise fabrication process of the electrochemical sensor is shown in Scheme 1. A bare GCE (Φ = 4 mm) was first polished with 0.3 and 0.05 μm of Al2O3 powder and sonicated with distilled water to get a mirror-like surface. Afterward, the electrode was electrodeposited in HAuCl4·4H2O (1%, w/w) solution at −0.2 V for 30 s to obtain a layer of Au nanoparticles (Au NPs). Then, 20 μL of 2.5 μM DNAzyme solution was incubated onto the sensing surface. Thereby, the DNAzyme was anchored onto the electrode through Au−N bonds after 12 h. After the incubation of 20 μL of BSA (1%, w/w) for 40 min to block the remaining active sites, 20 μL of 20 mM MgCl2 solution containing different concentrations of miRNA141 was coated onto the electrode for 8 h. Finally, the resultant electrode was incubated with 20 μL of CuCo−CeO2−CS solution for 1 h. The electrochemical sensor was produced and stored at 4 °C for further use. Furthermore, electrochemical sensors using other bioconjugates, including CeO2−CS, Cu− CeO2−CS, and Co−CeO2−CS, were also prepared using similar procedures. Electrochemical Measurements. DPV experiments were conducted in 1 mL of 0.1 M PBS (pH 7.0) with a certain concentration of H2O2 being optimized. The tested potential was from 0.5 to 0.1 V with a 0.05 s pulse width and 50 mV amplitude. CV and EIS were carried out in 1 mL of 5 mM Fe(CN)63−/4− as the redox probe. CV measurements were performed at a 100 mV/s scan rate with the potential from −0.2 to 0.6 V, and EIS signals were recorded with an excitation signal of 5 mV, a formal potential of 220 mV, and a frequency range of 101 to 105 Hz. The impedance (Z) of electron transfer in the EIS signal was denoted as its real (Zre) and imaginary (Zim) components.

ization reaction. Then, dual-mode electrochemical sensing was conducted as follows: First, the CuCo−CeO2 NSs acted as direct redox mediators and displayed an observable DPV signal, avoiding the involvement of traditional electron mediators. The detectable DPV signal was greatly amplified by the improved electrocatalysis of CuCo−CeO2 NSs toward H2O2 decomposition. Second, upon the introduction of 3,3diaminobenzidine (DAB) and H2O2, the oxidization of DAB was dramatically promoted by the electrocatalysis of CuCo− CeO2 NSs in the presence of H2O2, which resulted in substantial deposition of IPs and led to great amplification of the EIS signal. Thus, with the use of CuCo−CeO2 NSs and Mg2+-induced DNAzyme-assisted target recycling for signal amplification, both DPV and EIS signals were greatly amplified, achieving the ultrasensitive determination of miRNA-141. In order to characterize the step-by-step construction of the sensing electrode, CV and EIS measurements were conducted (Figure S1A,B). Furthermore, polyacrylamide-gel electrophoresis (PAGE) was also carried out to validate the feasibility of the Mg2+-induced DNAzyme-assisted target recycling (Figure S1C). All the results demonstrate the successful preparation as well as the feasibility of the proposed sensor. Morphological and Structural Characterization of CuCo− CeO2. The morphologies of the CuCo−CeO2 NPs were first characterized by field-emission scanning electron microscopy (FESEM). As shown in Figure 1A,B, CuCo−CeO2 NSs

Figure 1. (A−F) Morphological characterization of CuCo−CeO2. (A,B) SEM images, (C) cross-section TEM image, (D,E) TEM images, and (F) STEM image of CuCo−CeO2. (G−J) STEM-EDS elemental mapping of (G) Cu, (H) Co, (I) Ce, and (J) O of the single CuCo−CeO2 shown in (F).

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RESULTS AND DISCUSSION Sensing Principle of the Proposed Electrochemical Sensor. The principle of the electrochemical sensor for miRNA-141 is illustrated in Scheme 1. Mg2+-induced DNAzyme-assisted target recycling was first conducted according to the method reported by Yang’s group.23 In the presence of miRNA-141, the hairpin-locked DNAzyme on the electrode was opened through target-probe hybridization, yielding the “active” DNAzyme, which was then specifically cleaved by Mg2+ at the cleavage site. Because of the lower affinity, the two cleaved, shorter DNAzyme fragments were separated with the target. The released target can bind to an adjacent DNAzyme strand to drive another cycle of activation. After N cycles, the remaining fragment on the electrode surface can capture numerous bioconjugates of CuCo−CeO2 NSs immobilized with single-stranded DNA through a hybrid-

showed quite uniform sphere morphologies with diameters of about 2 μm and rough surfaces, which were assembled by numerous small nanorods. Cross-section transmission-electron-microscopy (TEM) images revealed that the CuCo− CeO2 NSs possessed solid inner nanostructures. TEM pictures at different magnifications (Figure 1D,E) indicated the CuCo− CeO2 NSs were uniform in size, which agrees well with the SEM and cross-section TEM results. Moreover, the scanning− transmission electron microscopy (STEM) and corresponding energy-dispersive-X-ray-spectroscopy (EDS) mapping confirmed the existence and homogeneous distributions of Cu, Co, Ce, and O in a single CuCo−CeO2 NS (Figure 1F,G). The real contents of Cu, Co, and Ce elements were C

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Figure 2. (A−D) Ce 3d XPS spectra and (E−H) O 1s XPS spectra of CeO2, Co−CeO2, Cu−CeO2, and CuCo−CeO2, respectively. (I) H2-TPR profiles of CeO2, Co−CeO2, Cu−CeO2, and CuCo−CeO2 samples. (J) EELS-spectrum-image-acquisition schematic diagram. (K) Ce M5,4 EELS spectra before (black line) and after (red line) doping with Cu and Co.

0.79, 0.89, and 0.98 for CeO2, Co−CeO2, and Cu−CeO2, respectively. The result indicates that codoping of Cu and Co atoms into the CeO2 lattice can change the chemical environment of Ce ions, thus generating more active Oa species. H2-TPR was further performed to investigate the reducibility of oxygen species of different CeO2 samples. As shown in Figure 2I, the reduction peaks can be divided into two parts, labeled zones I and II, which can be assigned to the surface and bulk reduction of CeO2, respectively.10 The peaks in zone II remain almost unchanged for all samples, indicating the reduction of CeO2 undergoes the same reduction process under high temperature.26,28 Generally, it was proposed that the oxygen vacancies generated in the CeO2 lattice can adsorb oxygen easily. Thus, many active oxygen species were formed, which can be reduced easily by H2 at low temperature.29 For peaks in area I, CuCo−CeO2 NSs possessed a lower reduction temperature and a higher H2-consumption value (0.295 mmol/ gcat) compared with those of CeO2, Cu−CeO2, and Co−CeO2 (0.247, 0.292, and 0.283 mmol/gcat, respectively), which indicates the significant increase of oxygen vacancies in the CuCo−CeO2 surface and agrees well with the results of the XPS spectra. The results from Raman, XRD, XPS, and H2-TPR indicate that the redox properties of CeO2 had significantly changed after codoping with Cu and Co. Particularly, higher Ce3+ numbers and abundant oxygen vacancies were found in CuCo−CeO2 compared with those of pure or single-doped CeO2. All these catalytically favorable properties are close related to each other. Moreover, EELS experiments were also conducted to further investigate the valences of cerium ions on the CeO2 surface. As shown in Figure 2K, the Ce M5,4 EELS spectra of CuCo−CeO2 and CeO2 show the presence of two Ce white lines, which are located at 884 and 902 eV because of the 3d5/2 → 4f7/2 (M5) and 3d3/2 → 4f5/2 (M4) electron transitions, respectively.30 The chemical shift of the Ce M5, 4

investigated by inductively coupled plasma mass spectrometry (ICP-MS) (Table S2), which showed the atomic ratio of Ce/ Cu/Co was 98:0.2:1.6. All the above observations suggest the successful preparation of the CuCo−CeO2 NSs. To confirm the successful synthesis of CeO2, Cu−CeO2, Co−CeO2, and CuCo−CeO2, SEM and EDS profiles were also recorded (Figure S2). The results indicate that the doping of metal ions does not cause obvious changes of the CeO2 morphology. Analysis of Oxygen Vacancies Using X-ray Photoelectron Spectroscopy (XPS), H2 Temperature-Programmed Reduction (H2-TPR), and Electron-Energy-Loss Spectroscopy (EELS). First, powder X-ray diffraction (XPS) and Raman spectroscopy were conducted to investigate the phase information and defect structure of the CeO2 materials (Figure S3). To analyze the oxygen vacancies of different CeO2 samples, XPS, H2-TPR, and EELS experiments were also conducted. Figure 2A−D is the Ce 3d XPS spectra of the CeO2, Co−CeO2, Cu−CeO2, and CuCo−CeO2, respectively. Each of the Ce 3d spectra was divided into 10 peaks, resulting from the pairs of spin−orbit doubles through the deconvolution method.24 Letters U and V represent the 3d3/2 and 3d5/2 spin−orbit components, respectively.25 Six peaks (U, U″, U‴, V, V″, V‴) are characteristic of Ce4+, whereas the other four peaks (U0, U′, V0, V′) are assigned to Ce3+,26 which indicates the defect structure of CeO2−x owing to the existence of oxygen vacancies. The peak-area ratio of Ce3+ and Ce4+ (Ce3+/ Ce4+ ratio) of CuCo−CeO2 showed a dramatic increase (0.28) compared with those of CeO2, Co−CeO2, and Cu−CeO2 (0.18, 0.22, and 0.24, respectively). This increase of Ce3+/Ce4+ ratio, driven by oxygen vacancies, can further lead to the superior catalytic performance of CuCo−CeO2. The O 1s XPS spectra of different CeO2 samples showed two peaks at about 529.15 and 531.74 eV, which can be assigned to surfaceadsorbed oxygen, Oa, and lattice oxygen, Ol11 (Figure 2E−H). The surface oxygen vacancies can be also calculated using the peak-area ratio of Oa/Ol,27 which is 1.1 for CuCo−CeO2 and D

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Figure 3. (A−D) DPV and (E−H) EIS responses of the proposed electrochemical sensor incubated with 1 nM miRNA-141 using different bioconjugates: (A,E) CeO2−CS, (B,F) Co−CeO2−CS, (C,G) Cu−CeO2−CS, and (D,H) CuCo−CeO2−CS. The DPV curves were recorded in 1 mL of 0.1 M PBS (pH 7.0) in the absence (curve a) and in the presence (curve b) of H2O2 (27 mM). The EIS plots were recorded in 1 mL of 5 mM Fe(CN)63−/4− before (curve a) and after (curve b) introduction of H2O2 and DAB under the optimized experimental conditions.

led to greatly enhanced DPV signals. (iii) The CuCo−CeO2 efficiently catalyzed the oxidization of DAB in the presence of H2O2, which resulted in substantial deposition of nonconductive IPs and led to remarkable amplification of the EIS signal. Additionally, the possible mechanisms of H2O2 decomposition catalyzed by CuCo−CeO2 and the formation of IPs on the electrode interface were also investigated (Figures S5 and S6). Analytical Performance of the Electrochemical Sensor. The electrochemical signal of the proposed sensor relied on the fixation amount of CuCo−CeO 2 −CS, which was connected to the concentration of target miRNA-141. Under the optimal experimental conditions, the analytical performance of the electrochemical sensor was evaluated by measuring both DPV and EIS responses to miRNA-141 with different concentrations. As shown in Figure 4, both the peak current of

white lines and the relative-intensity ratio of these lines can be used to evaluate the redox properties of the CeO2 samples.10,31 Particularly, a higher intensity ratio of M5 and M4 peaks (IM5/ IM4) indicates a higher reducible nature.32,33 According to the calculation results, the IM5 /IM 4 ratio of CuCo−CeO 2 (0.8295) is higher than that of pure CeO2 (0.7972), which indicates more Ce3+ ions and a higher density of oxygen vacancies on the CeO2 surface from the introduction of the Cu and Co dopants. Investigation of the Electrochemical Sensor with Different Bioconjugates. In order to achieve the optimal analytical performance, different experimental conditions were optimized (Figure S4). Moreover, to confirm the high catalytic activity of CuCo−CeO2−CS, contrast experiments were conducted by comparing the electrochemical performance of CuCo−CeO2− CS to those of CeO2−CS, Co−CeO2−CS, and Cu−CeO2−CS under the optimized conditions. The sensors were first incubated with DNAzyme (2.5 μM) and a mixture of miRNA-141 (1 nM) and Mg2+, successively. Then, the modified electrodes were incubated with CeO2−CS, Cu− CeO2−CS, Co−CeO2−CS, and CuCo−CeO2−CS, respectively. As shown in Figure 3A−C, the sensors with CeO2−CS, Co−CeO2−CS, and Cu−CeO2−CS only gave 10.36, 15.27, and 20.71 μA for DPV signal changes, respectively. However, the sensor with CuCo−CeO2−CS showed a dramatic increase of 36.68 μA in the DPV peak current after the addition of 27 mM H2O2 (Figure 3D). Meanwhile, EIS enhancement was also measured by using the difference of Ret (ΔRet) before and after the formation of the nonconductive IPs. As shown in Figure 3E−H, the sensors with CeO2−CS, Co−CeO2−CS, and Cu− CeO2−CS only showed about 982, 1268, and 1422 Ω rising of ΔRet, whereas a much greater increase of 3020 Ω in ΔRet was obtained for the proposed sensor with CuCo−CeO2−CS as the signal enhancer. These results suggested that the catalytic ability of CuCo−CeO2 was more excellent than those of CeO2, Co−CeO2, and Cu−CeO2, resulting in dramatic amplification of both DPV and EIS signals. The good analytical performance of the sensor may be attributed to the following reasons: (i) The Mg2+-induced DNAzyme-assisted target recycling resulted in abundant immobilization of CuCo−CeO2−CS on the electrode surface, paving the way for signal amplification. (ii) The immobilized CuCo−CeO2−CS showed improved electrocatalytic activity toward H2O2 decomposition because of the codoping of Cu and Co into the CeO2 lattice, which accelerated the surface electrotransfer from Ce3+ to Ce4+ and

Figure 4. Calibration plots of (A) DPV-peak-current and (B) EIS ΔRet values of the electrochemical sensor using CuCo−CeO2 as signal enhancers under the optimized experimental conditions. The insets are the corresponding DPV curves and EIS plots. Other conditions are shown in Figure 5. Error bars are the SDs (n = 3).

DPV and ΔRet values of the EIS signal increased gradually with increases in the miRNA-141 concentration. The limit of detection (LOD, S/N = 3) for both the DPV and EIS methods was calculated to be 33 aM. It is noted that the DPV peak current of the sensor is proportional to the logarithm of the miRNA-141 concentration (lg c) in the range of 0.1 fM to 10 nM (Figure 4A). The corresponding linear equation is I = −5.382 lg c − 30.38 with a correlation coefficient of R = 0.9965. Similarly, the ΔRet values of EIS are linear with the lg c ranging from 0.1 fM to 10 nM with a linear equation of ΔRet = 387.8 lg c + 1751 and a correlation coefficient of R = 0.9967 (Figure 4B). This could attributed to the fact that the introduction of miRNA-141 caused DNAzyme-assisted target E

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after (I) addition of 27 mM H2O2. No obvious changes in the DPV current (ΔI = I0 − I) can be observed for the interfering miRNA sequences, whereas a dramatic change (ΔI) of the DPV signal was obtained when the proposed sensor was incubated with 1 nM miRNA-141 in a mixture with the interferences, even though the concentration of target miRNA141 was much lower than those of the interferences. Meanwhile, similar results were obtained under EIS measurements in 5 mM [Fe(CN)6]3−/4−. Almost negligible ΔRet values were observed for all the tested interferences, whereas a significant increase of ΔRet was obtained toward miRNA-141 in the mixture with the interferences (Figure 5B). These comparisons all demonstrate that miRNA-141 was highly specific to the CuCo−CeO2-based electrochemical sensor. The reproducibility of the proposed method was evaluated by measuring the precision of the intra- and interassays toward 1 nM miRNA-141. The intra-assay was conducted using the same-batch of electrochemical sensor for detection of 1 nM miRNA-141 in five parallel runs, whereas the interassay was carried out in the same way but by using five different batches of sensors to analyze each sample. Both DPV and EIS measurements were carried out under the optimized experimental conditions. For the DPV measurements, the peak current values (I) showed a 1.0% relative standard deviation (RSD) for intra-assay measurements and a 1.7% RSD for interassay measurements (Figure 5C). Meanwhile, for the EIS detection, the Ret of the EIS signal shows only a 1.4% RSD for intra-assay detection and a 2.0% RSD for interassay detection (Figure 5D). These results demonstrate that the fabricated electrochemical sensor exhibited good reproducibility. Moreover, the stability of the sensor was also investigated by checking the DPV and EIS response periodically. The electrochemical sensors were incubated with different concentrations of miRNA-141 (0.001, 1, and 1000 pM) and then the miRNA-141 was detected after 5, 10, 15, and 20 days under the optimized experimental conditions. As shown in Figure 5E, the DPV currents retained 99.1−88.5, 95.4−83.1, and 98.5− 87.2% of their initial current responses for 0.001, 1, and 1000 pM miRNA-141, respectively. Meanwhile, after incubation with 0.001, 1, and 1000 pM miRNA-141, the Ret values of the EIS signals retained 98.1−93.1, 96.2−85.2, and 99.5−86.4% of their original values, respectively (Figure 5F). These results indicate the long-term stability of the proposed sensor. Application. To demonstrate the applicability of the proposed sensor, DPV and EIS assays were performed in extraction solutions from different cancer cells, including 22 Rv1 (human prostate-cancer cells) and HeLa cells (human cervical-cancer cells). After cell counting, the cell samples were disposed using a commercial RNA-extraction kit. From Figure 6A, we can see that the extracts of 22 Rv1 cells depicted significant increases in the DPV signal with increases of 22 Rv1 cell numbers (from 102 to 106). However, the extracts of HeLa cells exhibited inconspicuous increases in DPV peak currents when the cell concentrations increased from 102 to 106 cells. These results suggest the overexpression of miRNA-141 in 22 Rv1 cells but not in HeLa cells, which agrees well with previous literature.34,35 Additionally, EIS measurements were also conducted (Figure 6B). Obviously, the lysate from HeLa cells showed no marked increase in EIS response, which was in good accordance with blank detection. However, an obvious increase in EIS signal was observed when the sensor was incubated with

recycling on the electrode surface, which led to abundant immobilization of CuCo−CeO2−CS bioconjugates on the electrode surface. The more miRNA-141 was incubated, the more DNAzyme was cleaved, and the more CuCo−CeO2−CS was anchored. Because of the superior electrocatalytic activity of CuCo−CeO2 with abundant oxygen vacancies, high sensitivity and low detection limits were achieved. Meanwhile, the DPV and EIS signals of the three other sensors with CeO2−CS, Co−CeO2−CS, and Cu−CeO2−CS were also investigated under the same experimental conditions (Figure S7). Obviously, the three sensors with CeO2−CS, Co−CeO2− CS, and Cu−CeO2−CS showed lower sensitivities, narrower dynamic linear ranges, and higher LODs, which further indicated the superior catalytic activity of the CuCo−CeO2. Comparisons with other works using the same and different detection methods are shown in Tables S3 and S4, respectively, which indicate the superior analytical performance of the proposed sensor compared with those of other methods. Specificity, Reproducibility, and Stability of the Electrochemical Sensor. To evaluate the specificity of the DPV and EIS assay for miRNA-141, the sensor was modified with different interfering miRNA sequences with the same concentration of 10 nM, including let-7d, miRNA-429, miRNA-21, three-base-mismatched miRNA-141 (t-miRNA141), single-base-mismatched miRNA-141 (s-miRNA-141), and a mixture of them with 1 nM miRNA-141. Figure 5A is the DPV signal obtained in PBS (0.1 M, pH 7.0) before (I0) and

Figure 5. (A,B) Specificity of the electrochemical sensor with (A) DPV and (B) EIS measurements for 1 nM miRNA-141 against interfering miRNA sequences: blank, let-7d (10 nM), miRNA-429 (10 nM), miRNA-21 (10 nM), t-miRNA-141 (10 nM), s-miRNA-141 (10 nM), and their mixture with 1 nM miRNA-141. (C,D) Reproducibility of the proposed sensor under (C) DPV and (D) EIS modes. (E,F) Stability of the proposed sensor with 1000, 1, and 0.001 pM miRNA-141 under (E) DPV and (F) EIS measurements. Error bars are SDs (n = 3). F

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The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the Ministry of Science and Technology of China (973 Project No. 2018YFA0209102), the National Natural Science Foundation of China (11727807, 51725101, 51672050, and 61790581), and the Science and Technology Commission of Shanghai Municipality (16DZ2260600). The authors also acknowledge Dr. Chengyi Xiong, Benhao Li, and Junlai Yu for discussions.

Figure 6. (A) DPV and (B) EIS detection of miRNA-141 from different numbers of HeLa (red bars) and 22 Rv1 (yellow bars) cell lysates. Other experimental conditions are shown in Figures 4 and 5. Error bars are SDs (n = 3).

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the extracts of 22 Rv1 cells. Both DPV and EIS measurements indicate the great potential of the proposed electrochemical sensor for detection of miRNA-141 in real biological samples. Additionally, to further investigate the potential application of the developed sensor in real samples, recovery experiments were also conducted, and the results are shown in Tables S5 and S6. The results indicate that our proposed method can serve as a potential platform in clinical samples.

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CONCLUSIONS To summarize, we have developed a novel dual-mode electrochemical sensor using CuCo−CeO2 NSs as efficient electrocatalysts for ultrasensitive detection of miRNA. With the codoping of Cu and Co into the CeO2 lattice structure, abundant oxygen vacancies were successfully generated, which were identified and confirmed by XPS, H2-TPR, and EELS. Compared with pure or single-doped CeO2, more significant enhancement of electrocatalytic activity was achieved in CuCo−CeO2 NSs, which resulted in dramatic improvement of the electrochemical analytical performance. Furthermore, using the CuCo−CeO2 NSs as bifunctional electrocatalysts, the dual-mode electrochemical sensor exhibited a wider linear range and lower detection limit than conventional singleapproach sensing methods. The successful establishment of the dual-mode electrochemical sensor provides a versatile strategy for monitoring other biomolecules, including metal ions, proteins, miRNAs, and small molecules, at ultratrace levels.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.analchem.8b03778. Reagents and materials; apparatus; polyacrylamide-gelelectrophoresis (PAGE) analysis; ICP-MS results; EDS, XRD, and Raman characterizations; characterization of the modified electrochemical sensor; optimal conditions; possible mechanisms of H2O2 decomposition and IP formation; DPV and EIS signals of electrochemical sensors with different bioconjugates; and comparisons with reported miRNA-detection methods (PDF)

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REFERENCES

AUTHOR INFORMATION

Corresponding Author

*Tel. and Fax: +86-21-51630213. E-mail: [email protected]. ORCID

Renchao Che: 0000-0003-4192-7278 G

DOI: 10.1021/acs.analchem.8b03778 Anal. Chem. XXXX, XXX, XXX−XXX

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DOI: 10.1021/acs.analchem.8b03778 Anal. Chem. XXXX, XXX, XXX−XXX