Sandwich-type microRNA biosensor based on magnesium oxide nanoflower and graphene oxide–gold nanoparticles hybrids coupling with enzyme signal amplification

Accepted Manuscript Title: Sandwich-type microRNA biosensor based on magnesium oxide nanoflower and graphene oxide–gold nanoparticles hybrids coupling with enzyme signal amplification Author: Hong-Lei Shuai Ke-Jing Huang Wen-Jing Zhang Xiaoyu Cao Meng-Pei Jia PII: DOI: Reference:

S0925-4005(16)31963-3 http://dx.doi.org/doi:10.1016/j.snb.2016.12.001 SNB 21375

To appear in:

Sensors and Actuators B

Received date: Revised date: Accepted date:

7-9-2016 15-11-2016 1-12-2016

Please cite this article as: Hong-Lei Shuai, Ke-Jing Huang, Wen-Jing Zhang, Xiaoyu Cao, Meng-Pei Jia, Sandwich-type microRNA biosensor based on magnesium oxide nanoflower and graphene oxide–gold nanoparticles hybrids coupling with enzyme signal amplification, Sensors and Actuators B: Chemical http://dx.doi.org/10.1016/j.snb.2016.12.001 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Sandwich-type microRNA biosensor based on magnesium oxide nanoflower and graphene oxide–gold nanoparticles hybrids coupling with enzyme signal amplification

Hong-Lei Shuai a, Ke-Jing Huang a,b,*, Wen-Jing Zhang a, Xiaoyu Cao c, Meng-Pei Jia a

a

College of Chemistry and Chemical Engineering, Xinyang Normal University, Xinyang 464000,

China b

Institute for Conservation and Utilization of Agro-bioresources in Dabie Mountains, Xinyang

Normal University, Xinyang 464000, China c

School of Chemistry and Chemical Engineering, Henan University of Technology, Zhengzhou

450001, China *

Corresponding author. Tel.: +86-376-6390611

E-mail address: [email protected] (K.J. Huang)

Highlights • Facile approach was used for the synthesis of MgO nanoflowers. • MgO nanoflowers/AuNPs were used as sensing platform and GO–AuNPs hybrids as signal carriers. • Electrochemical–chemical–chemical redox cycling was used as a sensitive detection system. • A sandwich-type assay showed a low detection limit of 0.05 fM high specificity. 1

Abstract An ultrasensitive sandwich-type electrochemical biosensor for microRNA (miRNA) detection is developed based on magnesium oxide (MgO) nanoflower and graphene oxide–gold nanoparticles (GO–AuNPs) hybrids coupling with electrochemical–chemical–chemical (ECC) detection system. In this bioassay system, MgO nanoflowers and AuNPs are modified on electrode to act as sensing platform. The thiolated capture probe is then self-assembled onto AuNPs/MgO substrate via formation of Au-S bonds. Subsequently, a biotinylated DNA signal probe is conjugated to GO–AuNPs hybrids. When miRNA-21 is added, a sandwich complex is formed and a lot of signal indicators streptavidin-conjugated alkaline phosphatases (SA-ALP) are immobilized upon electrode by the specific reaction between avidin and biotin. Finally, ECC reaction is performed in the system to improve detection signal. The proposed sandwich-type assay benefits from advantages of sandwich-type structure for enhanced sensitivity and specificity, MgO nanoflowers/AuNPs as sensing platform and GO–AuNPs hybrids as signal carriers for signal amplification, and ECC as a sensitive detection system for low detection limit. This biosensor exhibits a good dynamic ranging from 0.1 to 100 fM and a low detection limit of 50 aM (S/N=3) toward target miRNA-21.

Keywords: Sandwich-type biosensor; Magnesium oxide nanoflowers; Graphene oxide–gold nanoparticles hybrids; Enzyme signal amplification; MiRNA-21

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1. Introduction MiRNAs are non-coding small molecules that are frequently dysregulated expression in different kinds of human cancers and play critical functions in many physiological processes [1], such as developmental regulation, proliferation and differentiation [2,3]. Accumulative evidence has indicated that miRNAs are highly correlated to the cancer initiation, oncogenesis, and tumor response to treatments [4-6]. Thus, it is important to develop assays for miRNAs detection [7-9]. In the past decade, many methods have been developed to enhance the reliability and sensitivity of miRNA biosensor [4,7,8,10-13]. However, on account of some intrinsic properties of miRNA, such as short size, high sequence similarity, low-level concentration in samples, there are still significant challenges in the ultrasensitive and specific determination of miRNAs [14-16]. An effective approach for overcoming above issues is combining smart nanomaterials and enzyme signal amplification. Carbon materials is a kind of promising material for electrochemical sensor construction because they usually have outstanding stability, big specific surface area, superior electro-conductivity, and low cost [17,18]. As a famous representative, graphene (Gr) has an excellent conductivity and huge theoretical specific surface area. It has been widely used in energy storage, catalyst, sensor, and so on. Graphene oxide (GO) displays good water dispersibility and biocompatibility except all the properties of Gr, thus is widely used in electrochemical biosensor [19,20]. Au nanoparticles (AuNPs) have been recognized as an excellent mediator in the development of electrochemical biosensors due to their good conductivity and biocompatibility.

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Furthermore, they can easily introduce bio-elements on electrode by Au–S bond [21,22]. In this work, we prepare GO–AuNPs hybrid and use it as signal carrier. The material can improve signal molecular amount on the electrode and electrical conductivity of the biosensor. Under the influence of Gr, two-dimensional (2D) materials have widely been paid attention in various research fields, and Gr-like 2D material of elements other than carbon has especially generated vast entrust interest around the world [23]. MgO nanostructures have good physical and chemical properties and have attracted significant attention in different fields, such as water treatment, chemicals catalyzed and gas sensing [24-26]. Electrochemical–chemical–chemical (ECC) redox cycling has been reported as an effective signal-amplification system for electrochemical sensor [27]. Herein, ascorbic acid 2-phosphate (AAP), ferrocene methanol (FcM), and tris (carboxyethyl)phosphine (TCEP) are used as the enzyme substrate, redox intermediary and reductant, respectively. Ferrocene derivatives that have been used as the mediators to the enzyme reaction center are relatively stable in air. Ascorbic acid (AA) produced from AAP facilitates the regeneration of FcM from its electrochemical oxidation product, which results in an increase of FcM during the electrochemical scanning. While TCEP is used as a stabilized and suitable chemical reductant to regenerate AA from dehydroascorbic acid, thus amplifying the electrochemical signal. In this work, an ultrasensitive sandwich-type electrochemical biosensor for detecting miRNA is first developed based on MgO nanoflower and GO–AuNPs hybrids coupling with enzyme catalysis and ECC redox cycling. MgO nanoflowers

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were synthesized by a facile and low-cost precipitation-aging-calcination method and were applied as an outstanding sensing substrate. The nanoflowers had large specific surface area and could load more AuNPs, which not only allowed more biomolecules (capture probe) to be immobilized at the electrode surface but also accelerated electron transfer. GO–AuNPs hybrids acted as carrier were used to load vast signal indicators to amplify signal respond. ECC redox cycling was used to detect miRNAs for improving signal readout. Therefore, this assay effectively combined MgO nanoflowers, GO–AuNPs hybrids, ECC redox cycling system and sandwich-type strategy for highly sensitively and selectively detecting target miRNA-21.

2. Experimental 2.1. Apparatus and reagents All electrochemical experiments were carried out on an electrochemical workstation (EC550, Chenhua Instruments, China). EIS measurements were performed at open circuit potential in the frequency ranging from 100 kHz to 1 Hz. The morphologies and structures were examined using a field emission scanning electron microscope (JEOL, S-4800, Japan) and a transmission electron microscopy (JEOL, JEM-2100F, Japan). The chemical composition of the product was characterized by a X-ray diffractometer (XRD) (Rigaku D/Max-rA, Rigaku Corporation, Japan) equipped with graphite monochromatized high-intensity Cu Ka radiation (l=1.54178Å). Magnesium chloride hexahydrates (MgCl2.6H2O), bovine serum albumin (BSA),

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tris (carboxyethyl)phosphine (TCEP), chloroauric acid and sodium carbonate (Na2CO3) were obtained from Shanghai Chemicals Company. Ferrocene methanol (FcM), diethylpyrocarbonate and streptavidin-modified alkaline phosphatase (SA-ALP) were obtained from Shanghai Linc-Bio Science Company. Ultrapure water (18.2 MΩ cm-1) used was processed with an ultrapure water system (Milli-Q Reference Water Purification System, Millipore, US). All MiRNAs and DNA were prepared from Shanghai Sangon Biological Engineering Technology Company. DNA sequences used were as follows: Signal probe: 5’-biotin-TCAACATCAGTCTGATAAGCTATTT-(CH2)6-SH-3’ Capture probe: 5’-TTTTTTTTTTTTCAACATCAGT-(CH2)6-SH-3’ MiRNA: 5’-UAGCUUAUCAGACUGAUGUUGA-3’ One-base mismatched: 5’-UAGCUUAUCGGACUGAUGUUGA-3’ Three-base mismatched: 5’-UUGCUUAUCGGACUGAUCUUGA-3’ Non-complementary: 5’-GUAAGGCAUCUGACCGAAGGCA-3’

2.2. Synthesis of MgO nanoflowers MgO nanoflowers were prepared as follows: 25 mL Na2CO3 solution (1 M) was added dropwise into 25 mL MgCl2·6H2O solution (1 M) at room temperature to form white precipitation. The precipitate was aged at 80 °C for 2 h in static condition and then washed with water for several times. Subsequently, the precipitate was annealed in a conventional tube furnace at 500 °C with a ramp rate of 2 °C min−1 for 5 h.

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2.3. Preparation of GO–AuNPs hybrids GO–AuNPs carrier was prepared according to a previous protocol [28]. First, 25 mL HAuCl4 solution (0.48 mM) was mixed with 1.25 mL GO suspension (1 mg mL-1) then stirred for 30 min at room temperature. After heated to 80 °C, 470 μL sodium citrate (85 mM) was added slowly in above mixture, and stirred for 60 min. Finally, the mixture was centrifuged (5 min, 8000 rpm) and washed with water. Immobilization of DNA on GO–AuNPs hybrids were obtained according to previous report [29]. Firstly, signal probe (20 μL, 100 μM) were mingled with 1 mL GO–AuNPs solution for 48 h at 4 °C. Next, the mixture was centrifuged under 80000 rpm for 600 seconds. The black solid was collected and washed with 10 mM PBS (pH= 7.0) for several times and then dispersed in 1mL 10 mM PBS (pH= 7.0).

2.4. Construction of miRNA electrochemical sensor Firstly, the GCEs were polished with alumina slurry to a mirror-like surface. Then, 8 μL MgO nanoflower (1 mg mL-1) was applied on GCE and dried in air. The modified electrode was then steeped into 0.1% HAuCl4 solution to electrodeposit AuNPs. The potential was -0.2 V and the electrodeposition time was 50 s. After that, the obtained electrode was washed with water. Subsequently, 8 μL of 1 nM thiolated capture probes were applied on electrode and incubated at 4 ºC for 12 hours. After washed with PBS, the obtained electrode was immersed in 1 mM 6-mercaptohexanol (MCH). After half an hour, 8 μL miRNA-21 (1 nM) was added on the electrode and kept at 37 ◦C for 1h. Subsequently, 8 μL DNA-linked GO–AuNPs hybrids was applied

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on the electrode, and kept at 37 ◦C for 70 min. After that, 6 μL SA-ALP (0.1 mg mL-1) was applied on the electrode and placed for 50 min. Finally, the electrode was incubated in 5 mL Tris buffer solution (10 mM, pH 8.0) containing 5 mM AAP and 1 mM MgCl2 for half an hour. After washed with water, the resulting electrode was kept at 4 ◦C before use.

2.5. Live subject statement All experiments were in accordance with the guidelines of the National Institute of Food and Drug, Xinyang, China, and approved by the institutional ethical committee (IEC) of Xinyang Normal University. The authors also state that informed consent was obtained for any experimentation with human subjects and the Henan University of Engineering is committed to the protection and safety of human subjects involved in research.

3. Results and discussion 3.1. Design principle of miRNA biosensor The sandwich-type and signal-amplification detection strategy for miRNA-21 consist of a substrate of MgO nanoflowers/AuNPs, signal carrier of GO–AuNPs hybrids, indicator of SA-ALP and ECC detection system for signal enhancement (Scheme 1). In this system, thiol modified capture probes that is partial-complementary with miRNA-21 are firstly fixed on AuNPs/MgO modified electrode. Then, MCH is applied on above electrode to block the unreacted gold

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surface. Subsequently, DNA-linked GO–AuNPs hybrids are introduced to hybridize with miRNA and a sandwich-type structure is formed. GO–AuNPs hybrids bring large amounts biotinylated-signal probes on electrode, which strands on AuNPs by immobilizing SA-ALP through the streptavidin–biotin reaction to amplify the signal. Finally, ECC redox cycling is used as detection system with FcM as redox mediator and TCEP as chemical reducing reagent. In this system, AA produced from ALP triggers the ECC redox cycling to generate a great electrochemical response. The most noteworthy is AA can regenerate at once in detection system. Thus, the proposed strategy effectively combines the advantages of MgO nanoflowers, GO–AuNPs hybrids, ECC detection system and sandwich-type structure and shows high sensitivity and good selectivity for target miRNA-21.

3.2. As-prepared materials characterization The morphologies of MgO nanoflowers and GO-AuNPs hybrids were characterized in Fig. 1. The SEM images of MgO nanoflowers are displayed in Fig. 1A and B. The figures show that MgO has a high uniformity of flower-like nanostructure and the average diameter of the flower is about 500 nm (Fig. 1A). From Fig. 1B, it is clearly observed that many ultrathin nanosheets extend to the outside flowers shaping a hierarchical structure. This hierarchical structure affords the MgO nanoflowers huge specific surface area and poriferous channel, which is useful to development of biosensor. Fig. 1C reveals that GO is 2D structure with the typical crumpled and wrinkled nanosheets. The TEM images of MgO nanoflowers are shown

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in Fig. 1D and E. It shows that the flower-like morphology is assembled by ultrathin nanosheets (Fig. 1D). A closer examination (Fig. 1E) reveals the existence of intact MgO nanoflowers consist of sheets with thickness of a few nanometers. From Fig. 1F, it exhibits AuNPs well distribute on GO with average diameter of ca 17 nm. The XRD patterns of MgO nanoflowers are shown in Fig. 1G. The diffraction peaks are sharp and indexes to MgO periclase phase (JCPDS 87-0653), where the diffraction peaks at 37.2°, 43.1°, 62.4°, 74.7° and 78.6° correspond to (111), (200), (220), (311), and (222) planes, respectively. The clear and sharp diffraction peaks indicate the good crystallinity of the material.

3.3. Electrochemical characterization Different electrodes were characterized by cyclic voltammetry (CV) (Fig. 2). As shown in Fig. 2A, the peak currents slightly increase when MgO nanoflowers applied on GCE due to their semiconductor characters. The current respond enhances greatly after further potentiostatic electrodeposition of AuNPs on MgO electrode (curve c), owing to large specific surface area of MgO nanoflowers and the conductive AuNPs. In addition, as shown in Fig. 2B, peak current decreases dramaticlly after capture probe is modified upon above electrode, accounting for the fact that capture probe impedes electron transfer (curve d). The peak current piece by piece decrease when MCH and miRNA-21 are applied on the electrode (curves e and f) because MCH and negative-charge miRNA-21 insulate the conductive support and the electron transfer become more difficult. However, the peak currents prodigiously increase after fixing

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with DNA-linked GO-AuNPs hybrids (curve g). Electrochemical impedance spectroscopy (EIS) was applied to confirm construction of miRNA-21 sensor. Fig. 2C displays the feature of EIS identifying with the gradually modification processes. The bare GCE displays a largest electron transfer resistance (Ret) value (curve a). When MgO nanoflowers are assembled on the GCE surface, the Ret obviously decreases (curve b). When AuNPs are applied on MgO/GCE, Ret greatly decreases and exhibits almost a line because of good electro-conductivity of AuNPs/MgO film (curve c). As shown in Fig. 2D, Ret value obviously increases after capture probe is modified the electrode surface on account of its hindering the electron transfer (curve d). When MCH and miRNA (curves e, f ) applied on the electrode, respectively, Ret value obviously increases, which maybe due to the clogging electron transfer of MCH and negative-charge miRNA. However, Ret value obviously decreases after introduction of DNA-linked GO-AuNPs hybrids (curve g) because of their good conductivity.

In order to verify MgO film can greatly improve active area of electrode, two electrodes were evaluated by chronocoulumetry in 0.1 mM potassium ferricyanide containing 1.0 M potassium chloride. The value of effective surface area was computed using following equation:

Q  2ncFA Dt  Qdl  Qads

(1)

where A, n, F, c, Qdl and Qads (C) are the effective surface area, number of electron transferred, Faraday constant, substrate concentration, diffusion coefficient, double

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layer charge and adsorption charge, respectively. A was computed as 0.018 cm2 for GCE and 0.062 cm2 for AuNPs/MgO/GCE based on the data in Fig. 2E and Fig. 2F. The results indicated that AuNPs/MgO film can greatly improve the active site of electrode. As shown in Fig. 2G, the signal-amplification effect of MgO nanoflowers was investigated. Differential pulse voltammetry (DPV) respond enhances markedly when MgO nanoflowers are applied in biosensor development (curve b). When in presence of MgO nanoflowers, the current respond on the electrode is about 292.3% for that in the absence of MgO nanoflowers (curve a), demonstrating the MgO nanoflowers are conducive to improve current respond.

3.4. Experimental parameters optimization Some parameters were investigated in order to obtain the optimal experimental conditions for detection of miRNA-21. The influence of time for potentiostatic electrodeposition of Au nanoparticles on electrode was studied from10 to 120 s (Fig. 3A). The highest current response is obtained when the time is 40 s. Thus, 40 s was used in the further experiments. Fig. 3B shows current response decreases with increase of incubation time. It almost keeps unchange when it exceeds 60 min, confirming that the hybridization event between capture probe and miRNA accomplishes. Therefore, 60 min of incubation time was employed in further experiment. The effect of incubation time of miRNA and DNA-linked GO-AuNPs hybrids was studied (Fig. 3C). The peak current enhances promptly when incubation time

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grows from 20 to 160 min, and then it keeps a plateau after 70 min. Therefore, 70 min of incubation time was found to be optimum and was selected for further experiment. The effect of the pH of detection system was evaluated in the range of 5–11 (Fig. 3D). The highest DPV response is obtained at pH 8. So, pH 8 was employed in the subsequent experiments.

3.5. Signal-amplification effect of detection system Fig. 4 shows the DPV of proposed biosensor in different substrate solutions. In the detection system, AA is produced from AAP by the enzymatic reaction of ALP (equation 1). When the electrolyte solution only contains AAP or AAP/TCEP, there is no DPV respond. In the presence of FcM, a large peak current is observed (curves c and e). The electrode reaction is characteristic of an EC reaction mechanism, indicating that FcM mediates the electron transfer between AA and electrode. Thus, the result also demonstrates that FcM is regenerated after its electrochemical oxidation by the produced AA (equations 2 and 3). Furthermore, when TCEP is added, a bigger current peak is observed (curve f), which is attributed to the regeneration of AA from its oxidized product DAA by TCEP (equation 4) (curves d). All above indicate EC reaction in system is accelerated by chemical–chemical reaction of AA and TCEP. Therefore, EEC redox cycling reaction can greatly improve the electrochemical signal. AAP FcM

ALP

AA

(1)

Eletro-oxidization

FcM+ + AA

FcM+

(2)

FcM + DAA

(3) 13

DAA + TCEP

AA + TCEP = O

(4)

3.6. Analytical performance of designed biosensor Under optimum condition, miRNA-21 concentration was detected by DPV with proposed biosensor. Fig. 5 shows the DPV response of various miRNA-21concentration. The inset of Fig. 5 exhibits an excellent linear dependence on the logarithm of miRNA-21 concentration in range of 0.1-1.0×105 fM with correlation coefficient of 0.996. The calibration equation was i = -4.21 log(c) - 64.24 (where i and c are peak current and miRNA-21 concentration, respectively). The detection limit was 0.05 fM (S/N=3). Various assays for miRNA detection are compared in Table 1. Our method displays lower detection limit and wider linear range [30-35].

3.7. Selectivity, reproducibility and stability Trefulka et al. has reported a method for microRNA determination based on high specificity of Os(VI)bipy for ribose in nucleic acids [36, 37]. In this work, specificity of the proposed method was based on complementarity of RNA/DNA bases. The selectivity of miRNA sensor was studied by measuring peak currents variation of developed biosensor with different miRNA sequences. Fig. 6 shows peak current of complementary sequence is the highest. However, the peak current of non-complementary sequence and three-base mismatch sequence is negligible, indicating that non-complementary and three-based mismatch miRNAs do not hybridize with the capture probe. For single-base mismatched sequence, the peak 14

current is only 14.3% of that of complementary sequence, indicating good selectivity of proposed assay. Furthermore, five equally prepared electrodes were used to detect 3 pM miRNA-21 with a relative standard deviation (RSD) of 4.5%. In addition, the biosensor was kept under 4 ◦C for 10 days and it still remained 92% of the initial response to 3pM miRNA-21. Furthermore, the AuNPs/MgO/GCE was kept in air for 12 days and then used to fabricate the biosensor. Only 5% decrease of current response was observed. All these suggested good stability of biosensor and AuNPs/MgO film. Good reproducibility and stability on account of huge specific surface area of MgO nanoflowers coupling with forceful interaction between enzyme and GO-AuNPs hybrids.

3.8. Sample analysis For evaluating the application of miRNA assay, recovery experiment was carried out. Five healthy human serum samples were obtained from Xinyang Central Hospital and then diluted to 10 times with PBS. Different concentrations of miRNA-21 were then added in serum samples and detected with biosensor. The analytical results are listed in Table 2. The recoveries varies from 91.0 to 105.2% and RSDs are ranging from 3.7 to 5.2, suggesting that the assay is available for determining miRNA-21 in real samples.

4. Conclusions An ultrasensitive sandwich-type electrochemical biosensor for detecting miRNA-21 is developed based on using MgO nanoflowers as sensing platform and

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GO–AuNPs hybrids as signal carrier coupling with signal amplification of enzyme and EEC redox cycling detection system. This strategy has some excellent features. Firstly, MgO nanoflowers have large specific surface area which can load large amounts of capture probe for signal amplification. Secondly, GO–AuNPs hybrids as carrier can immobilize vast signal indicators to enhance signal respond. Thirdly, ECC detection system can markedly magnify detection signal. Finally, sandwich-type strategy greatly increases specificity and sensitivity of the assay. The proposed biosensor also displays good stability and reproducibility for target miRNA detection and exhibits a promising potential for detection of miRNA in clinic.

Acknowledgments This work was supported by the National Natural Science Foundation of China (U1304214, 21475115), Program for University Innovative Research Team of Henan (15IRTSTHN001), Henan Provincial Science and technology innovation team (C20150026), Nanhu Scholars Program of XYNU, University Graduate Students Research Innovation Fund of Xinyang Normal University (No. 2015KYJJ34) and Science and Technology Major Project of Henan province (141100310600).

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Biographies Hong-Lei Shuai is a graduate student at Xinyang Normal University. His current researches include molecular electrochemistry and electrochemical materials. Ke-Jing Huang received his PhD in 2006 from Wuhan University. Presently, he is a professor at Xinyang Normal University. His research interests include electrochemical analysis, electrochemical sensors and biosensors. Wen-Jing Zhang is a graduate student at Xinyang Normal University. Her current researches include chemical and electrocatalytic materials. Xiaoyu Cao received his PhD in 2006 from Wuhan University. Presently, he is a professor at Henan University of Technology. His research interests include nanomaterials and biosensors. Meng-Pei Jia is an undergraduate student at Xinyang Normal University. Her current researches include electrochemical sensors and biosensors.

22

23

Fig. 1. SEM images of MgO (A, B) and GO (C); TEM images of MgO (D, E) and GO-AuNPs (F); XRD patterns of MgO (G).

24

25

Fig. 2. CVs (A, B) and EIS (C, D) of bare GCE (a), MgO/GCE (b), AuNPs/MgO/GCE (c), Capture probe/AuNPs/MgO/GCE (d), MCH/capture probe/AuNPs/MgO/GCE (e), miRNA/MCH/capture probe/AuNPs/MgO/GCE (f), DNA linked GO-AuNPs hybrid/miRNA/MCH/capture probe/AuNPs/MgO/GCE (g); (E) Q-t curve of GCE (a) and AuNPs/MgO/GCE (b) in electrolyte containing 0.1 mM K3[Fe(CN)6] and 1.0 M KCl; (F) Q-t1/2 curves on GCE (a) and AuNPs/MgO/GCE (b); (G) DPVs of AAP/SA-ALP/DNA linked GO-AuNPs hybrid/miRNA/MCH/capture probe/AuNPs/GCE (a) and AAP/SA-ALP/ DNA linked GO-AuNPs hybrid/miRNA/MCH/capture probe/AuNPs/MgO/GCE (b) in 10 mM Tri buffer solution (8.0) containing 5 mM TCEP and 2 mM FCM.

26

Fig. 3. Effects of deposition time of AuNPs (A), reaction time of capture probe and miRNA (B), incubation time of miRNA and DNA linked GO-AuNPs hybrid (C), the pH of detection system (D).

27

Fig. 4. DPVs curves in different solutions: AAP (a); AAP/TCEP (b); FcM (c); TCEP/FcM (d); AAP/FcM (e); AAP/TCEP/FcM (f).

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Fig. 5. DPVs of different miRNA-21 concentration (a to j): 0, 100, 10, 1.0, 5.0, 0.1, 0.5, 0.01, 0.001, 0.0001 pM. Inset: the calibration plots of DPV respond versus logarithm concentration of miRNA-21.

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Fig. 6. Effect of biosensor hybridized to different miRNA sequences: non-complementary sequence (a), three-base mismatched miRNA-21 (b), single-base mismatched miRNA-21 (c), target miRNA-21 (d).

30

Scheme 1. Schematic outline of the principle for miRNA-21 sensing.

31

Table 1 Comparison between different biosensors for miRNA. Linear range Methods

Analytical technique

LOD (fM)

References

(pM) Enzyme catalytic amplification based on Gr

Amperometry

0.001–100

0.14

30

DPV

1–10000

1000

31

DPV

0.002–8

0.6

32

DPV

0.1–500

84.3

33

Amperometry

0.008–10

4

34

DPV

0.0005-0.5

0.4

35

DPV

0.0001–100

0.05

This work

quantum dot Electrode modified with carbon nanotube Electrode modified with GO and gold nanorod Using methylene blue as redox indicator Os(bpy)2(API)Cl-activated GO modified electrode Using hybrid antibody and enzymatic amplification Sandwich-type sensor based on AuNPs/MgO nanoflowers and GO-AuNPs hybrids

32

Table 2 Detection of miRNA-21 in serum samples (n = 3). Samples

Added (fM)

Found (fM)

RSD (%)

Recovery (%)

1

10

9.1

5.2

91.0

2

100

98.6

3.8

98.6

3

1000

1054.3

2.7

100.4

4

10000

9784.2

2.8

99.9

5

100000

105248.6

3.6

105.2

33