A Facile Electrochemical Biosensor for the Detection of microRNA Based on Graphene Sheets/Polyanilin

www.ijm‐me.org International Journal of Material and Mechanical Engineering (IJMME), Volume 4, 2015 doi: 10.14355/ijmme.2015.04.004

A Facile Electrochemical Biosensor for the Detection of microRNA Based on Graphene Sheets/Polyaniline/AuNPs Xueping Chen1, Dandan Zhou2, Huawei Shen1, Wenli Feng1*, Hui Chen2* and Guoming Xie1* Department of Laboratory Medicine, Chongqing Medical University, Chongqing 400016, P R China

1

Clinical Laboratories, The First Affiliated Hospital of Chongqing Medical University, Chongqing 400016, P R China 2

*fengwlcqmu@sina.com; *guomingxie@cqmu.edu.cn; *huichen@cqmu.edu.cn

Abstract A facile, highly sensitive electrochemical biosensor was designed to determine microRNA based on graphene sheets/polyaniline/AuNPs (GS/PANI/AuNPs) nanocomposites. Firstly, GS/PANI/AuNPs was synthesized using a one‐step and green approach by polymerizing aniline with chloroaurate acid as the oxidant. Then, GS/PANI/AuNPs was re‐suspended in chitosan (CS) solution and fabricated onto the surface of glassy carbon electrode (GCE) to enhance the conductivity and immobilize hairpin capture probe (S). In the presence of target RNA, the RNA hybridized with the loop of S, followed by covalently binding of streptavidin‐alkaline phosphatase (SA‐ALP). Current signal was gained by differential pulse voltammetry (DPV) in the working buffer containing 1‐naphthyl phosphate (1‐NP). The biosensor exhibited high sensitivity toward microRNA, resulting in a wide linear range from 1.25×10 ‐12 M to 5×10 ‐8 M with a detection limit of 0.25 pM (S/N = 3).

Keywords microRNA; Electrochemical Biosensor; Graphene Sheets; Polyaniline; AuNPs; Nanocomposites

Introduction Nowadays, nanomaterials have been explored intensively for biosensing applications, owing to their unique chemical and physical properties [1, 2, 3]. Nanomaterials such as grapheme sheet (GS), polyaniling (PANI), and AuNPs are frequently used in biosensor fabrication. Graphene, a 2D sp 2‐hybridized carbon sheet with high surface area, electrical conductivity, high flexibility, and mechanical strength, has attracted extensive interests [4].

However, graphene has high tendency to agglomerate and even restack via van der Waals interactions. Various conducting polymers have been used as a matrix for graphene dispersion, which can further increase the sensitivity of biosesnors [5]. PANI, as the most studied conjugated polymer, has been extensively explored in the construction of graphene/PANI nanocomposites due to its high electrical conductivity, superior enviromental stability, and low toxicity. Gold nanoparticles (AuNPs), which possess good conductivity and biocompatibility, can form covalent bonds and combine with materials with functional groups [6]. Nanomaterials based on grapheme sheet (GS), PANI, or AuNPs have been widely investigated [7, 8]. However, the grapheme sheet/poplyaniline/ AuNPs nanocomposites (GS/PANI/AuNPs) are usually synthesized by complex process and the polymerization of PANI needs additional oxidant.

SCHEME 1. SCHEMATIC ILLUSTRATION OF THE SENSING STRATEGY FOR DETECTION OF TARGET RNA BASED ON THE ECTROCHEMICAL BIOSENSOR

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Over the last years, electrochemical biosensors have drawn considerable attentions due to its simplicity, low cost instrumentation, good portability, high sensitivity, and rapid response [9, 10]. Recently, a number of electrochemical approaches for detecting microRNA based on nanomaterials and oligonucleotide probes have been developed. In this work, we developed a facile, green, and one‐pot route to synthesize GS/PANI/AuNPs nanocomposites and fabricate an amplified electrochemical biosensor based on GS/PANI/AuNPs. Experimental Reagents and Materials Oligonucleotides were ordered from Shanghai Sangon Biotechnology Co. Ltd. (Shanghai, China). Their sequences were as follows: Capture probe: 5ʹ‐SH‐(CH2)6‐GCCTGCGTAATAATGAAAACCATGG CAGGC‐biotin ‐3ʹ.

MiRNA‐126: 5ʹ‐CAU UAU UAC UUU UGG UAC‐3ʹ.

All oligonucleotide strands were re‐suspended in 1× TE (10 mM Tris–HCl, 1.0 mM EDTA, pH 7.5) at 10 μM concentrations and stored at ‐20℃. Graphene sheets (GS) were purchased from Pioneer Nanotechnology Co. (Nanjing, China). Aniline (99%+) was purchased from Adamas Reagent Co. Ltd. (Brussels, Switzerland). Hydrogen tetrachloroaurate trihydrate (HAuCl4∙3H2O) was purchased from Aladdin (Shanghai, China). Streptavidin‐ Alkaline Phosphatase (SA‐ALP) and 1‐Naphthyl phosphate (1‐NP; 99%), 6‐Mercapto‐1‐hexanol (MCH) were purchased from Sigma‐Aldrich (St. Louis, MO, USA). All chemicals were of analytical reagent grade and used as received unless other indicated. All solutions were prepared with deionized water (Milli‐Q 18.2 MΩ, Millipore System Inc.). Tris‐HCl buffer (20 mM Tris, 0.10 M NaCl, 5.0 mM MgCl2, 0.005% Tween‐20, pH 7.40) was used as hybridization buffer. DEA buffer (0.10 M diethanol amine, 1.0 M MgCl2 and 10 mM KCl, pH 9.62) was used as electrochemical detection solution.

Instrumentation The electrochemical measurements were performed with a CHI660D electrochemical workstation (Shanghai Chenhua Instruments Co. Ltd., China) using a conventional three‐electrode system with an Ag/AgCl (1 M KCl) electrode as the reference electrode, a Pt wire as the counter electrode, and a modified glassy carbon electrode (GCE) as the working electrode. The morphologies of different modified electrodes were investigated using a scanning electron microscopy (SEM) system (FEI Nova‐400, USA). The GS/PANI/AuNPs nanocomposites were characterized via Energy dispersive X‐ray (EDX) spectra using a scanning electron microscope (VEGA3 SB, TESCAN, Czech Republic).

Synthesis of GS/PANI/AuNPs GS/PANI/AuNPs was prepared based on the previously reported methods [11, 12, 13] with slight modification.

Firstly, 140 μL aniline (99%+) and 1.429 mg GS (The mass ratio of aniline to grapheme was 100:1) were added to 20 ml of 2.0 M HCl solution with magnetic stir at room temperature for 30 mins. Then, 5 ml of 3 mM HAuCl4∙3H2O solution (The mole ratio of HAuCl4∙3H2O to aniline was 1:10) was added to the above solution and stirred for 5 hs under dark conditions until PANI was generated with a characteristic dark green color. The obtained solution was centrifuged, and the precipitate was washed with deionized water several times. Finally, the resulting solution was dried in vacuum at 60℃.

Preparation of Biosensor GCE was firstly polished to a mirror face with 0.3 and 0.5 μm alumina powders, and then washed in an ultrasonic cleaner in deionized water/aceton/deionized water for 5 mins respectively. Then, 1 mg of graphene/PANI/AuNPs was dissolved in 1 mL of 0.5% chitosan solution under ultrasonication to form a homogeneous dispersion. Next, 5 μL of the GS/PANI/AuNPs was deposited on the surface of the previously cleaned GCE and dried at room temperature. The modified electrode was labeled as GS/PANI/AuNPs/GCE. For immobilization, 10 μL of 1 μM

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thiolated S was dropped onto the GS/PANI/AuNPs/GCE surface and incubated overnight at 4℃. After washing with washing buffer, the electrode was immersed in 1 mM MCH for 1 h. After washing, the finally obtained biosensor was stored at 4℃ before future use.

Probe Hybridization and Electrochemical Measurements 10 μL of target RNA was placed on the modified electrode and incubated for 1 h at 37℃. And then 10μL of DEA buffer containing 0.5 μg mL‐1 of SA‐ALP and 10 mg mL‐1 BSA was cast onto the electrode surface, and then incubated for 30 mins at 37℃. After washing with DEA buffer containing 0.05% Tween‐20, differential pulse voltammetry (DPV) was performed in the DEA buffer containing 1.0 mg mL‐1 of 1‐NP. The parameters of DPV are as follows: modulation time, 0.05 s; interval time, 0.017 s; step potential, 5 mV; modulation amplitude, 70 mV and potential scan from ‐0.1 to 0.6 V. Results and Discussion Characterization of GS/PANI/Aunps In this work, GS/PANI/AuNPs was synthesized using AuCl4‐ as oxidant. The morphology and structure of GS and GS/PANI/AuNPs were first examined by SEM. As illustrated in Fig. 1(A), GS dispersed by the CS solution showed the irregularly crumpled structure which might increase the electrode effective surface. The SEM image was showed in Fig. 1(B). As it can be seen, GS and PANI joined with each other and formed branched structures or interconnected networkers, and AuNPs appeared uniformly dispersed in the GS and PANI matrix without any conglomeration. This was because that PANI provided a steric barrier against aggregation. What’s more, chitosan could bond these AuNPs via hydroxyl groups when the nanocomposites were re‐dispersed in chitosan, which also contributed to the solubility and stability of the composite particles [14]. The elemental composition of GS/PANI/AuNPs was then analyzed by EDX. As shown in Fig. 1(C), the nanocomposites were composed of mainly Au and large amounts of C and N, demonstrating the constituent of the nanocomposites in accordance with GS, PANI and AuNPs.

FIG. 1 (A) SEM IMAGE OF PREPARED GS (B) SEM IMAGE OF GS/PANI/AuNPs (C) EDX SPECTRUM OF GS/PANI/AuNPs

Electrochemical Characterization The stepwise reactions on the surface of the GCE were characterized by electrochemical impedance spectroscopy (EIS). Fig. 2A showed the nyquist plots of the EIS spectra for different modified electrodes in 10.0 mM Fe(CN)63‐/4‐ solution at 100mV/s scan rate. The semicircle diameter corresponded to the charge transfer resistance (Rct) on the electrode. Compared with bare GCE (curve a), the GS/PANI/AuNPs modiefied GCE (curve b) revealed an almost straight line, implying the Rct decreased, and due to the superior conductivity of GS/PANI/AuNPs nanocomposites. After S probes assembled, the Rct increased (curve c). This could attribute to the negative charged phosphoric acid backbones of oligonucleotide probe which was able to hinder the electron transmission.

Subsequently, the Rct increased significantly (curve d) after the modified electrode was blocked with MCH. When target RNA successfully hybridized with S, the Rct further increased (curve e) due to the formation of double strand with more negatively charged phosphoric acid backbones. Finally, the Rct increased continuously (curve f) when the SA‐ALP attached on the electrode surface, indicating the successful binding of SA‐ALP that retarded the

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electron transfer. The results of CV were in accordance with that of EIS.

FIG. 2 EIS (A) AND CV (B) FOR EACH FABRICATION STEP: (a) BARE GCE; (b) GS/PANI/AuNPs/GCE; (c) S/GS/PANI/AuNPs/GCE; (d) MCH/ S/GS/PANI/AuNPs/GCE; (e) AFTER ADDITION OF TARGET RNA; (f) AFTER ATTACHMENT OF SA‐ALP. EIS AND CV MEASUREMENTS WERE PERFORMED IN 0.5 mM Fe(CN)63−/4− CONTAINING 0.4 M KCl.

Sensitivity of the Biosensor The sensitivity of this method was evaluated by DPV measurements. Fig. 3(A) displayed the DPV curves of the sensing system upon the addition of different concentrations of target RNA (0, 1.25, 5, 10, 20, 40, 80, 160, 320, 640, and 50000 pM). The peak current increased with increasing concentration of C1. As shown in Fig. 3(B), the peak current varied linearly with the logarithm of the target RNA concentration in the range from 1.25 pM to 50 nM. The fitted regression equation was I=1.085 log c + 4.007 (I is the peak current (μA) and c is the concentration of the target (pM)) with a correlation coefficient (R2) of 0.9606. The detection limit was estimated to be 0.25 pM (S/N= 3).

FIG 3. (A) DPV RESPONSES OF (A) TARGET RNA CONCENTRATIONS OF 1 pM, 5pM, 10pM, 20pM, 40 pM, 80 pM, 160 pM, 320 pM, 640 pM, 50000 pM (FROM a to j). (B) CALIBRATION CURVE OF THE BIOSENSOR FOR TARGET RNA.

Conclusion In this work, we have synthesized GS/PANI/AuNPs nanocomposites in a green and one‐step method which is time‐efficient and environment‐friendly. Furthermore, GS/PANI/AuNPs was successfully applied to detect microRNA sensitively, demonstrating its great potential for clinical use.

ACKNOWLEDGMENT

This work was financially supported by Natural Science Research Foundation of China (No. 81070421 to Wenli Feng, No. 81171415 to Guoming Xie).

REFERENCES

[1]

Na N., Zhang S., Wang, S., Zhang X. “A Catalytic Nanomaterial‐Based Optical Chemo‐Sensor Array.” J. Am. Chem. Soc. 128 (2006): 14420–14421.

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www.ijm‐me.org International Journal of Material and Mechanical Engineering (IJMME), Volume 4, 2015

[2]

Zhou M., Zhai Y., Dong S. “Electrochemical Sensing and Biosensing Platform Based on Chemically Reduced Graphene Oxide.” Anal. Chem. 81 (2009): 5603–5613.

[3]

Amjadi M., Pichitpajongkit A., Lee, S., Ryu S., Park I. “Highly Stretchable and Sensitive Strain Sensor Based on Silver Nanowire–Elastomer Nanocomposite.” ACS Nano 8 (2014): 5154–5163.

[4]

Saha K., Agasti S. S., Kim C., Li X., Rotello V. M., “Gold nanoparticles in chemical and biological sensing.” Chem. Rev. 112 (2012): 2739–2779.

[5]

Qiu J., Shi L., Liang R., Wang G., Xia X. “Controllable deposition of a platinum nanoparticle ensemble on a polyaniline/graphene hybrid as a novel electrode material for electrochemical sensing.” Chemistry‐a European Journal 18 (2012): 7950–7959.

[6]

Upadhyay S., Rao G. R., Sharma M. K., Bhattacharya, B. K., Rao V.K., Vijyaraghavan R. “Immobilization of acetylcholineesterase‐choline oxidase on a gold‐plainum bimetallic nanoparticles modified glassy carbon electrode for the sensitive detction of organophosphate pesticides, carbamates and nerve agents.” Biosens Bielectron 25 (2009): 832–838.

[7]

Wang L., Hua E., Liang M., Ma C., Liu Z., Sheng S., Liu M., Xie G., Feng W. “Graphene sheets, polyaniline and AuNPs based DNA sensor for electrochemical determination of BCR/ABL fusion gene with functional hairpin probe.” Biosens Bioelectron 51 (2014): 201‐207.

[8]

Ambrosi A., Chua C. K., Bonanni A., Pumera M. “Electrochemistry of graphene and related materials.” Chem Rev. 114 (2014): 7150‐7188.

[9]

Labib M., Ghobadloo S. M., Khan N., Kolpashchikov D. M., Berezovski M. V. “Four‐way junction formation promoting ultrasensitive electrochemical detection of microRNA.” Anal. Chem. 85 (2013): 9422‐9427.

[10] Hu J., Yu Y., Brooks J. C., Godwin L. A., Somasundaram S., Torabinejad F., Kim J., Shannon C., Easley C. J. “A reusable electrochemical proximity assay for highly selective, real‐time protein quantitation in biological matrices.” J. Am. Chem.

Soc., 136 (2014): 8467‐8474.

[11] Kong F., Gu S., Li W., Chen T., Xu Q., Wang W. “A paper disk equipped with graphene/polyaniline/Au nanoparticles/glucose oxidase biocomposite modified screen‐printed electrode: Toward whole blood glucose determination.” Biosens Bioelectron 56 (2014): 77‐82.

[12] Wang Y., Liu Z., Han B., Sun Z., Huang Y., Yang G. “Facile Synthesis of Polyaniline Nanofibers Using Chloroaurate Acid as the Oxidant.” Langmuir 21 (2005): 833‐836.

[13] Kinyanjui J. M., Hatchett D. W. “Chemical Synthesis of a Polyaniline/Gold Composite Using Tetrachloroaurate.” Chem.

Mater. 16 (2004): 3390‐3398.

[14] Han J., Ma J., Ma Z. “One‐step synthesis of graphene oxide‐thionine‐Au nanocomposites and its application for electrochemical immunosensing.” Biosens Bioelectron 47 (2013): 243‐247.

Xueping. Chen received her BS from Sun Yat‐Sen University in 2013. She is now a PhD candidate majoring in Clinical Laboratory Diagnostics under the supervision of Professor Weni Feng and Professor Guoming Xie at the Department of Laboratory Medicine of Chongqing Medical University. Her current research interests include nucleic acid probe design and synthesis of nanocomposites for cancer biomarker detection.

Dandan. Zhou received her BS from Weifang Medical College in 2014. She is now a PhD candidate majoring in Clinical Laboratory Diagnostics under the supervision of Professor Hui Chen at Department of Clinical Laboratories of The First Affiliated Hospital of Chongqing Medical University. Her current research interests include aptamer and synthesis of AuNPs nanocomposites for cancer biomarker detection.

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