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In Situ Growth of Three-Dimensional Graphene Films for Signal-On Electrochemical Biosensing of Various Analytes Dongqing Kong, Sai Bi,* Zonghua Wang,* Jianfei Xia, and Feifei Zhang Shandong Sino-Japanese Center for Collaborative Research of Carbon Nanomaterials, College of Chemistry and Chemical Engineering, Collaborative Innovation Center for Marine Biomass Fiber, Materials and Textiles of Shandong Province, Laboratory of Fiber Materials and Modern Textiles, the Growing Base for State Key Laboratory, Qingdao University, Qingdao, Shandong 266071, China S Supporting Information *

ABSTRACT: In this work, an in situ growth protocol is introduced to fabricate three-dimensional graphene films (3D GFs) on gold substrates, which are successfully utilized as working electrode for electrochemical detection of nucleic acid (microRNA) and protein (lysozyme) based on a signal-on sensing mechanism. To realize the bridge between the gold substrate and graphene film, a monolayer of 4-aminothiophenol is selfassembled on the substrate, which is then served as connectors for the growth of 3D GFs on the gold substrate by the hydrothermal reduction (HR) technique. Moreover, given the excellent properties, such as enlarged surface area, strong binding strength between 3D GFs and gold substrate, and improved conductivity, the proposed 3D GF-fabricated gold substrate is readily employed to the construction of electrochemical biosensing platforms through introduction of magnetic nanoparticles (MNPs) as probe carriers. On the basis of the strand displacement reaction and specific binding between aptamer and its target, the developed biosensors achieve signal-on detection of microRNA155 (miR-155) and lysozyme (Lyz) with high sensitivity and selectivity and further successfully applied to human serum assay. Overall, the proposed strategy for in situ growth of 3D GFs provides a powerful tool for a wide range of applications, which is not limited to electrochemical biosensors and can be extended to other areas, such as electrocatalysis and electronic energy-related systems.

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tions, such as bio- or energy related devices, sensors, and catalysis.17−20 So far, a number of methods have been developed for the synthesis of 3D GR, which can be generally classified as selfassembly approaches (e.g., direct freeze-drying, hydrothermal reduction (HR), electrochemical deposition, etc.), templateassisted preparation (e.g., chemical vapor deposition (CVD)), and direct deposition (e.g., plasma-enhanced chemical vapor deposition (PECVD)).21−24 Among these methods, HR is considered as one of the most fascinating green techniques for the construction of 3D GR structures, since no extra chemical is required during self-assembly of graphene oxide (GO) sheets into 3D GRs and no contaminant is generated due to the simultaneous process of self-assembly and reduction of GO.25,26 In addition, it has been demonstrated that HR-induced 3D GR networks usually have much higher electrical conductivity and compressive strength than GO precursor.27 Therefore, the development of HR method for the fabrication of 3D GR architectures is in high demand in electrochemical applications.

raphene, a one-atom-thick and two-dimensional sheet of sp2-bonded carbon atoms that are densely packed in a honeycomb crystal lattice, has been widely explored for applications in versatile areas owing to its unique physical and chemical properties, such as excellent electronic and optical properties, extraordinary mechanical strength, large specific surface area, and low manufacture costs.1−3 In addition, it has been known that graphene or graphene oxide (GO) can noncovalently adsorb single-stranded nucleic acids through πstacking interactions between nucleotide bases and the carbon plane.4−7 Therefore, graphene-based materials have been successfully developed for biosensing and bioimaging applications.8−11 However, for practical applications, graphene sheets often suffer from agglomeration due to the van der Waals forces and π−π restacking, which could lead to the diminution of accessible surface area, the damage of continuous pathway for electron transport, and the suppression of highly mechanical strength of individual graphene flakes.12−14 Recently, threedimensional graphene (3D GR) with porous/hollow structures has attracted great attention, since it not only possesses intrinsic properties of graphene sheets but also owns improved performance for a wide range of applications.15,16 In particular, because of the highly compressive strength, excellent electrical conductivity, and extremely large surface area, 3D GR has emerged as promising candidates for electrochemical applica© XXXX American Chemical Society

Received: August 10, 2016 Accepted: October 7, 2016

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BaseLine ChromTech Research Centre (Tianjin, China). Lysozyme (Lyz), thrombin, BSA, and hemin were obtained from Sangon Biotechnology Co., Ltd. (Shanghai, China). The oligonucleotides used in this work were synthesized and purified by Sangon Biotechnology Co., Ltd. (Shanghai, China), and the sequences are listed in Table S1. The oligonucleotide solutions were prepared with TE buffer (10 mM Tris-HCl, 1 mM EDTA-2Na) supplemented with 12.5 mM MgCl2 (pH 8.0). All other reagents were of analytical grade and were used without further purification. Doubly distilled and RNase-free water was used throughout the experiments. Preparation of Graphene Oxide (GO). GO was synthesized from natural graphite powder using a modified Hummers’ method as the previous work.41 Briefly, 35 mL of concentrated H2SO4 was added to the mixture of 0.6 g of graphite and 1.0 g of NaNO3 slowly under stirring and kept stirring for 30 min. Then, 3.0 g of KMnO4 was very slowly added when the temperature was between 5 and 15 °C. Afterward, the reaction system was transferred to a 35 °C water bath and reacted for another 30 min. Subsequently, 150 mL of deionized water was gradually added and the resulting solution was stirred for 15 min, while the temperature was raised to 98 °C. The reaction mixture was slowly transferred to 200 mL of 60 °C deionized water with stirring. Hydrogen peroxide was then added slowly until the solution color changed from brown to yellow. The resulting product was filtered and washed with diluted HCl (5%) to remove metal ions, followed by washing with deionized water thoroughly and centrifuging to remove the acid. Finally, the solid product of GO was obtained after freeze-drying and dispersed in water by ultrasonication. In Situ Growth of 3D GFs on Gold Substrate. Before modification, the gold substrate was treated with piranha solution. Then, 3D GFs were in situ fabricated on gold substrate according to the reported work with slight modification.14 Briefly, the freshly cleaned gold substrate was immersed into a 4-aminothiophenol (0.5 mM) solution for 2 h. Then, the self-assembly monolayer (SAM)-modified gold substrate was rinsed with ethanol and vertically placed into 2 mL of GO dispersion (2 mg/mL) containing coupling agents of EDC and NHS. After standing at room temperature for 6 h, the reaction system was transferred to a Teflon-line autoclave, followed by reaction at 160 °C for another 6 h. The resulting 3D GFs-fabricated gold substrate was rinsed with deionized water and then treated with freeze-drying. The morphologies of the as-prepared GO and 3D GFs on gold substrate were characterized by scanning electron microscopy (SEM, JSM-6700F, Japan). X-ray diffraction (XRD) was carried out on an X-ray diffractometer (DX-2700, China) using CuKα radition (λ = 1.5418 Å). Preparation of DNA Probe-Modified Magnetic Nanoparticles (MNPs). First of all, 60 μL of carboxyl-modified magnetic nanoparticles (MNPs) was thoroughly wash with 300 μL of PBS buffer (0.01 M, pH 7.4) containing NaCl (0.3 M) three times and then redispersed in 120 μL of PBS buffer for further use. A 500 μL of a mixture of EDC (0.1 M) and NHS (0.01 M) that was dissolved with imidazole-HCl (0.1 M, pH 6.8) was then added and incubated at room temperature for 30 min to activate carboxyl groups on MNPs. The activated MNPs were washed with PBS buffer twice and redispersed in 120 μL of PBS buffer. Subsequently, 300 μL of amino-modified capture probe (CP, 1 μM) was introduced into the activated MNP dispersions and reacted for 12 h at 37 °C. The resulting CP/ MNPs conjugates were magnetically washed with PBS buffer

Electrochemical sensors have emerged as promising tools for the detection of a wide variety of analytes in clinical, environmental, and food safety applications, due to their unique advantages of high sensitivity and specificity, rapid response, good stability, and low cost.28−30 In the past decade, graphene-based signal amplification strategies have been developed for the construction of electrochemical transducing platforms.31,32 Especially, the fabrication of novel electrode materials with 3D structures holds great promise in realizing high sensitivity and selectivity due to the large surface area of 3D structures and improved reaction sites to enhance the signal acquisition.33,34 Moreover, since the signaling mechanism is related to specific conformational change upon target recognition, such as the hybridization of nucleic acids35,36 and aptamer binding to analytes37 (e.g., small molecules, proteins, and even entire cells), another important consideration to improve the performance of electrochemical sensors is the choice of an effective strategy to signal the target events. Given the relationship between signal generation and target binding, “signal-off”, and “signal-on” electrochemical sensors have been reported. In a “signal-off” sensor, the presence of target decreases the signal intensity, which thus suffers from the problem of limited signal gain because only a maximum of 100% signal can be suppressed under any experimental conditions.38,39 Thus, the “signal-off” architecture is not ideal for most biosensing applications.40 Alternatively, “signal-on” sensors can readily circumvent this limitation that can provide an increased signal upon target recognition.41,42 Therefore, the design of “signal-on” electrochemical sensors for sensitive and selective detection of biomolecules is highly desirable. In this work, an in situ growth of three-dimensional graphene films (3D GFs) on gold substrates is achieved by HR reaction. A monolayer of 4-aminothiophenol is self-assembled on gold substrates to address the binding issue between the 3D GFs and substrate. Moreover, by utilizing the 3D GFs-fabricated gold substrate as working electrode and HRP-mimicking DNAzyme for signal amplification, a robust, sensitive, and specific electrochemical sensor has been built based on conformational shifts of biomolecular receptors upon DNA/RNA hybridization and aptamer-protein recognition, and π−π stacking between nucleotide bases of single-stranded DNA and carbon plane of graphene, achieving “signal-on” electrochemical detection of nucleic acid and protein. In this assay, we choose microRNA155 (miR-155) and lysozyme (Lyz) as model analytes. As one of the most important circulating microRNAs, the overexpression of miR-155 is associated the process of carcinogenesis. Thus, miR-155 can be considered as a biomarker for diagnosis, staging, progression, and prognosis of cancers.43 In addition, Lyz has physiological and pharmaceutical effects on many diseases, such as cancers and renal diseases.44

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EXPERIMENTAL SECTION Materials and Reagents. Graphite powder (99.95%, 325 mesh) was provided by Qingdao Jin Rilai Graphite Co., Ltd. (Qingdao, China). 4-Aminothiophenol, 1-ethyl-(3-3′-dimethylaminopropyl) carbodiimide (EDC), and N-hydroxysuccinimide (NHS) were ordered from Aladdin Reagent Co., Ltd. (Shanghai, China). Potassium permanganate (KMnO4), sulfuric acid (H2SO4), hydrochloric acid (HCl), sodium nitrate (NaNO3) and methanol were purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). Carboxylmodified magnetic nanoparticles (MNPs) were ordered from B

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Analytical Chemistry twice and redispersed in 120 μL of PBS buffer. Afterward, 300 μL of reporter probe (RP, 1 μM) was added and reacted at room temperature for 1 h, followed by washing with PBS buffer twice. After magnetic separation, 300 μL of hemin (1 μM) was added to the resulting RP/CP/MNPs and incubated at room temperature for another 1 h to form hemin/G-quadruplex in RP. The excess hemin can be easily removed by magnetic separation, and the hemin/G-quadruplex-conjugated RP/CP/ MNP complexes were thoroughly washed with PBS buffer and redispersed in 120 μL of PBS buffer. Construction of 3D GFs Based Biosensors. First, different concentrations of miR-155 or Lyz in buffer or human serum were added to the hemin/G-quadruplexconjugated RP/CP/MNP complexes and incubated at 37 °C for 1 h. After magnetic separation, the above constructed 3D GF-modified gold substrate was dipped into the resulting supernatant and reacted at room temperature for 50 min. After rinsing with PBS buffer three times, the resulting gold substrate was employed as working electrode to perform electrochemical experiments using square wave voltammetry (SWV). Electrochemical Measurements. All electrochemical measurements were carried out on a Chenhua CHI 660C electrochemical workstation (Shanghai, China) with a threeelectrode system consisting of a 3D GFs-modified gold substrate working electrode, a Ag/AgCl (saturated KCl) reference electrode, and a Pt wire counter electrode. The cyclic voltammograms (CVs) were conducted at a scan rate of 100 mV s−1. The potential was scanned in the range from −0.4 to 0.8 V and the sample interval was 1 mV. The electrochemical impedance spectroscopy (EIS) was carried out with the frequency changed from 0.01 Hz to 100 kHz and signal amplitude of 5 mV. The chronocoulometry (CC) was conducted in 1 mM K3[Fe(CN)6] aqueous solution containing 2 M KCl with the potential ranging from 0 to 0.3 V and the step number of 1. The square wave voltammetry (SWV) measurements were performed in 10 mM Tris-HCl containing 2 mM H2O2 (pH 7.4) with a step potential of 2 mV, a frequency of 10 Hz, and an amplitude of 25 mV by scanning the potential in the range from −0.6 to 0 V versus Ag/AgCl references.

Scheme 1. Schematic Illustration of (A) the Proposed Strategy for in Situ Growth of 3D GFs on Gold Substrate and (B) the 3D GFs Based Electrochemical Biosensors for the Detection of Nucleic Acid (miR-155) and Protein (Lyz)

Meanwhile, hemin/G-quadruplex-conjugated reporter probe (RP)/capture probe (CP)/magnetic nanoparticle (MNP) complexes are constructed as Scheme 1B. The CP is first anchored on MNPs through the condensation reaction between amino group at 5′ terminus of CP and carboxyl group on MNP, which further partially hybridizes with RP that is functionalized with electroactive methylene blue (MB) and G-rich sequence at 5′ terminus. After addition of hemin, G-rich sequence is intercalated with hemin to form the hemin/Gquadruplex structure. Herein, two important tumor biomarkers, microRNA (miR-155) and lysozyme (Lyz), are selected as examples, and the CP is designed as the DNA sequence that is complementary to miR-155 or is the aptamer of Lyz. Therefore, upon the introduction of analytes (miR-155 or Lyz), the CP will specifically bind to its target based on strand displacement reaction or aptamer recognition, resulting in the disassociation of RP from MNPs into solution. Subsequently, based on the π−π interaction between oligonucleotides and graphene, the released RP can be tightly adsorbed on the porous graphene surface, which thus brings the hemin/G-quadruplex and MB into the proximity of the graphene surface. The hemin/Gquadruplex then acts as horseradish peroxidase (HRP)mimicking DNAzyme to catalyze the reduction of H2O2 with the aid of MB, thus leading to an amplified electronic signal that is proportional to the concentration of analyte. It should be noted that the employment of MNPs cannot only simplify the experimental operations but also facilitate a “signal-on” response mechanism, in which the electrochemical signal increases upon target recognition. More importantly, because of the enlarged surface area and high conductivity of the 3D GFs-modified gold substrate, the performance of electrochemical biosensor would be remarkably improved for sensitive and specific detection of target biomolecules. Characterization of the Fabricated 3D GFs on Gold Substrate. The morphologies of the precursor GO sheets and the as-fabricated 3D GFs on gold substrate are characterized by SEM (Figure 1). The flexible GO sheets exhibit twodimensional structure with wrinkles (Figure 1A). In contrast, large-scale and homogeneous 3D GFs with micrometer-sized porous framework are successfully fabricated on the gold substrates by HR technique (Figure 1B). It can be obviously seen that the pore walls are constructed of thin layers of stacked graphene sheets, which could offer highly efficient channels for electron transfer in the 3D GFs.12 The crystal structures of the products are verified by X-ray diffraction (XRD) (Figure S2).

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RESULTS AND DISCUSSION Principle of In Situ Growth of 3D GFs on Gold Substrate for Electrochemical Biosensing. The principle of the proposed in situ fabrication of 3D GFs on gold substrate and its application in electrochemical detection of various biomolecules are illustrated in Scheme 1. The self-assembled monolayer (SAM) plays an important role in addressing the interfacial binding issue for in situ growth of 3D GFs on the gold substrate.45 Thus, as shown in Scheme 1A, a monolayer of 4-aminothiophenol is first covalently immobilized on gold substrate through the formation of Au−S bonds. The amino groups at another terminal of SAM that exposure at the surface of gold substrate can react with carboxyl groups of GO, which further facilitate the fabrication of 3D GFs with porous framework on gold substrate through the hydrothermal reduction (HR) technique as the partial overlapping of graphene sheets with the formation of physical cross-linking sites. It should be noted that the immobilization of 4aminothiophenol on the gold substrate would increase the transfer resistance of electron. Thus, the modification time of the gold substrate with 4-aminothiophenol was optimized as 2 h (see Figure S1). C

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Figure 1. SEM images of (A) the as-prepared GO and (B) 3D GFs fabricated on gold substrate. Scale bar: 10 μm.

Figure 2. (A) CVs and (B) EIS characterizations of (a) bare gold substrate, (b) graphene sheet-modified gold substrate, and (c) 3D GFs-modified gold substrate in 5 mM [Fe (CN) 6]3−/4− aqueous solution containing 0.1 M KCl.

Figure 3. Influences of (A) incubation time and (B) incubation temperature of miR-155 and Lyz reacted with hemin/G-quadruplex-conjugated RP/ CP/MNP complexes and (C) pH value and (D) concentration of H2O2 for SWV measurements. The concentrations of miR-155 and Lyz are 1 μM. Δi is the relative peak current that represents the peak current difference in the presence and absence of target. The error bars represent standard deviation by means of three independent measurements.

75.16 μA that is much higher than that obtained by bare gold substrate (12.14 μA) and even graphene sheet-modified gold substrate (35.37 μA). These results demonstrate the formation of 3D GFs on the gold substrate with porous structures, which can significantly enlarge the surface area and improve the electrochemical properties of the substrate. The electrochemically active surface area of GFs was estimated to be 0.604 cm2

Moreover, cyclic voltammograms (CVs) and electrochemical impedance spectra (EIS) were used to investigate the electrochemical behaviors of the fabricated 3D GFs on gold substrate (Figure 2). As shown in Figure 2A, when the GFmodified gold substrate are electrochemically cycled in a 5 mM [Fe(CN)6]3−/4− aqueous solution containing 0.1 M KCl solution, the oxidation peak currents obviously increase to D

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Figure 4. (A and C) SWV responses of the proposed electrochemical biosensors using 3D GF-modified gold substrate as working electrode after treated with various concentrations of miR-155 and Lyz, respectively. (B and D) The corresponding calibration curves for the analysis of miR-155 and Lyz, respectively. Δi is the relative peak current that represents the peak current difference in the presence and absence of target, and C is the concentration of target. The error bars represent standard deviation by means of three independent measurements.

incubation time, with the increase of reaction time, more miR155 or Lyz was recognized by the CP, leading to the release of more hemin/G-quadruplex-conjugated RPs that was further adsorbed on GFs. As shown in Figure 3A, Δi (the difference of SWV detected in the presence and absence of miR-155 or Lyz) increases from 20 to 60 min and then reaches a plateau, suggesting the reaction is completely finished from 60 min. Therefore, 60 min was selected as the optimum incubation time for the following reaction. Figure 3B shows that Δi increases with increasing the incubation temperature up to 37 °C. In contrast, when the temperature is 45 and 55 °C, Δi decreases. Considering the melting temperature (Tm) of RP/CP DNA duplex (53.6 °C for miR-155 and 54.6 °C for Lyz), this behavior can be attributed to the dissociation of RP from the duplex with the temperature rising even in the absence of target, resulting in a high background signal. Therefore, 37 °C was selected as the optimum incubation temperature. Moreover, the influences of electrolyte pH and H2O2 concentration for SWV measurement were investigated by detecting 1 μM miR-155 under the optimized incubation conditions (60 min, 37 °C). From Figure 3C, it is clearly seen that Δi increases with increasing pH from 5.0 to 7.4, while decreases when the pH is further increased to 9.0. Thus, the SWV signal was dependent on pH of electrolyte and 7.4 was chosen to be the optimized pH. The effect of H 2 O 2 concentration was also investigated. From Figure 3D, the Δi intensely increases before the concentration of H2O2 increases to 1 mM and then it exhibits a slow increase from 1 mM to 2 mM and afterward a slight decline is followed. Since the maximum Δi was obtained when the concentration of H2O2 was at 2 mM, 2 mM H2O2 was selected as the optimal condition. Therefore, all the following SWV measurements were performed under the electrolyte pH of 7.4 and H2O2 concentration of 2 mM.

by chronocoulometry (CC), which is larger than that of bare gold substrate (0.237 cm2) (Figure S3). Thus, the constructed 3D GFs on gold substrate with enlarged surface area could provide more interfacial sites for molecular immobilization and reaction, which would benefit for the construction of electrochemical biosensors with improved performances. In addition, to evaluate the binding stability of 3D GFs grown on gold substrate via a SAM of 4-aminothiophenol, the fabricated 3D GFs were consecutively scanned for 50 cycles with CVs in the same rate of 100 mV s−1. As shown in Figure S4, compared with bare substrate, no obvious change of the current and reduction/oxidation potential is observed for the 3D GFmodified gold substrate, indicating the excellent binding strength stability at the interface between the gold substrate and graphene films. In addition, EIS was used to investigate the interfacial properties of gold substrates with different modifications (Figure 2B). In EIS, the differences in the electron transfer resistance (Ret) could be directly observed as the semicircle diameter of the Nyquist plots. Compared with a large Ret of the bare gold substrate (curve a) and a relatively decreased Ret of the graphene sheet-modified substrate (curve b), only a small impedance is detected from 3D GF-modified gold substrate (curve c), indicating a fast charge-transfer rate and excellent conductivity of the porous graphene films. Therefore, the 3D GF-modified gold substrate demonstrated outstanding properties of enlarged surface area, excellent binding strength, and high conductivity, which would make it a promising platform for a variety of advanced applications. Optimization of Conditions. To achieve better analytical performance, several experimental parameters, such as the incubation time and temperature of the reaction between hemin/G-quadruplex-conjugated RP/CP/MNP complexes and miR-155 or Lyz, and the electrolyte pH and H 2 O 2 concentration for SWV measurement were optimized. For the E

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Figure 5. (A) Specificity of the proposed miRNA biosensor toward (a) blank, (b) miR-let-7a, (c) one-base mismatched miR-155, (d) miR-155, and (e) a mixture of miR-155 and miR-let-7a, respectively. (B) Specificity of the proposed aptamer biosensor toward (a) blank, (b) thrombin, (c) BSA, (d) Lyz, and (e) a mixture of thrombin, BSA and Lyz, respectively. The concentration of each analyte is 1 μM. The error bars represent standard deviation by means of three independent measurements.

Signal-On Electrochemical Detection of Biomolecules. Nucleic acids and proteins are two kinds of biomarkers, which can be considered as indicators of disease stage and grade as well as useful for monitoring the responses to treatment and predicting the recurrence. In the present study, we chose two important biomolecules, microRNA-155 (miR-155) that is an important circulating miRNA43 and lysozyme (Lyz) that has many physiological and pharmaceutical effects,44 as target analytes to demonstrate the electrochemical performances of the proposed 3D GFs fabricated gold substrate in biosensing applications (Scheme 1B). Sensitivity is an important factor in quantitative analysis. Under the optimum experimental conditions, the sensitivity of the proposed electrochemical biosensors was evaluated by detecting miR-155 and Lyz with varied concentrations and using square wave voltammetry (SWV) to record the signal responses. As shown in Figure 4A,C, the SWV peak current increases with increasing the concentration of miR-155 (from 0.01 nM to 1.0 μM) and Lyz (from 1 pM to 1.0 μM), indicating a signal-on sensing mechanism. The corresponding calibration plots display good linear relationships between the relative peak currents (Δi) and the logarithm of target concentrations (logC) (Figure 4B,D). The limit of detections (LODs) are estimated to be 5.2 pM miR-155 and 0.67 pM Lyz using 3σ rule (σ is the standard deviation of the background), which are comparable to or even better than other graphene-based electrochemical biosensors (Table S2). This excellent sensitivity can be attributed to not only the amplified electronic signals generated by highly catalytic efficiency of HRP-mimicking DNAzymes but also the significantly improved electrochemical performances of 3D GF-modified gold substrate, such as the largely enhanced surface area and high electron transfer rate. It should be noted that a variety of electrochemical biosensors have been developed for bioanalytes detection based on graphene-based structures and achieved ultralow detection limits. However, most of these biosensing platforms were based on two or more materials.43,46 In addition, to further improve the detection sensitivity, various amplification strategies have been introduced into the systems, such as target recycling protocols and the employment of nanoparticles as signal probes.46 Although these methods could achieve ultrasensitive detection of biomolecules, they inevitably suffered from some limitations, such as complicated processing, high cost, and time-consuming operations. In contrast, our proposed biosensing platform is only based on graphene materials to improve the performance of the biosensors without any other materials or amplification techniques. Moreover, an in situ

growth protocol is introduced to fabricate three-dimensional graphene films (3D GFs) on gold substrates, making this method simple, economical, and practical. Given the excellent properties of the proposed 3D GFs, such as enlarged surface area, strong binding strength between 3D GFs and gold substrate, and improved conductivity, it is believable that a ultrahigh sensitivity would readily achieve by combining the 3D GFs with other amplification strategies. Moreover, specificity is also of great importance to biosensor in analyzing biomolecules. To investigate the specificity, the proposed biosensor for miR-155 was treated with microRNAlet-7a (miR-let-7a), miR-155, one-base mismatched miR-155, and a mixture of miR-155 and miR-let-7a (the concentration of each analyte is 1 μM). Meanwhile, the specificity of the aptamer biosensor for Lyz was investigated through detection of thrombin, BSA, Lyz, and a mixture of them (the concentration of each analyte is 1 μM). The corresponding SWV results are shown in Figure 5. As expected, only the samples containing the target analytes (miR-155 or Lyz) exhibit significant increase in the peak currents. Otherwise, the current responses show negligible changes compared to the background current (in the absence of the target). The results clearly demonstrate the excellent specificity of the developed electrochemical biosensors for the detection of both nucleic acids and proteins, which could be attributed to the highly specific DNA/RNA hybridization and specific recognition of aptamer to its target. To investigate the stability of the fabricated biosensors, the 3D GF-modified gold substrates were stored at 4 °C for 1 day, 3 days, 5 days, 7 days, and 9 days, respectively, and then reacted with the supernatant produced by 1 μM miR-155 or 1 μM Lyz. From Figure S5, the biosensors retain ∼93.8% and ∼91.1% of their initial responses to miR-155 and Lyz, respectively, even after 9 days, indicating the high stability of the fabricated biosensors. Real Sample Assays. To further evaluate the analytical reliability and real applicability of our developed biosensors, a series of different concentrations of miR-155 were spiked into human serum (diluted 100 times) and detected by the proposed 3D GF-based biosensor using the standard addition method. From the results presented in Table 1, the recovery and relative standard deviation (RSD) are 97.8−101.1% and 3.2−4.3%, respectively, which are in the acceptable ranges for real sample assay. In addition, using the proposed aptamer biosensor, the concentrations of Lyz in healthy human serum samples were detected to be 0.11 ± 0.06 μM, which are consistent with the previous report.47 Therefore, the above F

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ACKNOWLEDGMENTS This work was supported by the National Natural Science Foundation of China (Grants 21275082, 21375056, 21475071, and 21405086), the Special Funds of Taishan Scholar Program for Young Expert of Shandong Province (Sai Bi), the Natural Science Foundation of Qingdao (Grant 15-9-1-107-jch), and the Taishan Scholar Program of Shandong Province (Grant ts201511027).

Table 1. Recovery Tests of miR-155 in Healthy Human Serum Samples by the Proposed 3D GF-Based Electrochemical Biosensorsa sample miR-155 miR-155 miR-155 miR-155

added (pM)

founded (pM)

recovery (%)

RSD (%)c

0 100 200 300

− 102.23 195.69 303.27

− 100.2 97.8 101.1

−b 3.2 3.9 4.3

b

b

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The human serum samples are diluted 100-fold before use. b“−” denotes that the concentration of analyte is too low to be detected. c The results are obtained from five independent experiments. a

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CONCLUSION In summary, a reliable and robust approach for in situ growth of 3D GFs on gold substrate with excellent properties has been demonstrated in this work. Taking advantages of enlarged surface area, strong binding strength of the 3GFs to gold substrate and high conductivity, the proposed 3D GF-modified gold substrate is readily applied to the construction of electrochemical biosensors, showing high sensitivity and selectivity toward detection of nucleic acid (miR-155) and protein (Lyz) and excellent performances in real sample assays. The detection limits of 5.2 pM miR-155 and 0.67 pM Lyz are achieved, respectively. In biosensing analysis, the employment of MNPs cannot only simplify the experimental operations but also facilitate a signal-on response mechanism. On the basis of different recognition elements, such as DNA/RNA hybridization and specific recognition of aptamer to its target, novel electrochemical biosensors, especially aptasensors, are expected to be established for the analysis of a broad range of target molecules of interest. Therefore, this 3D GF-based strategy offers a feasible and powerful tool for the development of biosensing platforms and further versatile applications, ranging from material science to electronic devices. ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.analchem.6b03112. Sequences of oligonucleotides used in this study, optimization of SAM modification time, XRD characterization, calculation of active surface area and binding stability of 3D GFs on gold substrate, comparison of the proposed 3D GF-based electrochemical biosensor with other graphene-based electrochemical biosensors for the determination of bioanalytes and stability of the proposed 3D GF-based electrochemical biosensors (PDF)

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REFERENCES

(1) Jariwala, D.; Sangwan, V. K.; Lauhon, L. J.; Marks, T. J.; Hersam, M. C. Chem. Soc. Rev. 2013, 42, 2824−2860. (2) Sun, M.; Liu, H. J.; Liu, Y.; Qu, J. H.; Li, J. H. Nanoscale 2015, 7, 1250−1269. (3) Estevez, L.; Kelarakis, A.; Gong, Q. M.; Da’as, E. H.; Giannelis, E. P. J. Am. Chem. Soc. 2011, 133, 6122−6125. (4) Tang, L. H.; Wang, Y.; Liu, Y.; Li, J. H. ACS Nano 2011, 5, 3817−3822. (5) Tang, L. H.; Wang, Y.; Li, J. H. Chem. Soc. Rev. 2015, 44, 6954− 6980. (6) Lu, C. H.; Yang, H. H.; Zhu, C. L.; Chen, X.; Chen, G. N. Angew. Chem., Int. Ed. 2009, 48, 4785−4787. (7) Wang, Y.; Li, Z. H.; Hu, D. H.; Lin, C. T.; Li, J. H.; Lin, Y. H. J. Am. Chem. Soc. 2010, 132, 9274−9276. (8) Zhang, C. L.; Xu, J. Q.; Li, Y. T.; Huang, L.; Pang, D. W.; Ning, Y.; Huang, W. H.; Zhang, Z. Y.; Zhang, G. J. Anal. Chem. 2016, 88, 4048−4054. (9) Li, G. X.; Yu, X. X.; Liu, D. Q.; Liu, X. Y.; Li, F.; Cui, H. Anal. Chem. 2015, 87, 10976−10981. (10) Yang, G. H.; Zhu, C. Z.; Du, D.; Zhu, J. J.; Lin, Y. H. Nanoscale 2015, 7, 14217−14231. (11) Zhang, P.; Zhuo, Y.; Chang, Y. Y.; Yuan, R.; Chai, Y. Q. Anal. Chem. 2015, 87, 10385−10391. (12) Hao, J. N.; Liao, Y. Q.; Zhong, Y. Y.; Shu, D.; He, C.; Guo, S. T.; Huang, Y. L.; Zhong, J.; Hu, L. L. Carbon 2015, 94, 879−887. (13) Chen, D.; Tang, L. H.; Li, J. H. Chem. Soc. Rev. 2010, 39, 3157− 3180. (14) Shi, L.; Chu, Z. Y.; Liu, Y.; Jin, W. Q.; Xu, N. P. Adv. Funct. Mater. 2014, 24, 7032−7041. (15) Cao, X. H.; Zheng, B.; Rui, X. H.; Shi, W. H.; Yan, Q. Y.; Zhang, H. Angew. Chem., Int. Ed. 2014, 53, 1404−1409. (16) Zhang, C. F.; Kuila, T.; Kim, N. H.; Lee, S. H.; Lee, J. H. Carbon 2015, 89, 328−339. (17) Li, C.; Shi, G. Q. Nanoscale 2012, 4, 5549−5563. (18) Lee, S. H.; Kim, H. W.; Hwang, J. O.; Lee, W. J.; Kwon, J.; Bielawski, C. W.; Ruoff, R. S.; Kim, S. O. Angew. Chem., Int. Ed. 2010, 49, 10084−10088. (19) Yu, M.; Ma, Y. X.; Liu, J. H.; Li, S. M. Carbon 2015, 87, 98−105. (20) Worsley, M. A.; Pauzauskie, P. J.; Olson, T. Y.; Biener, J.; Satcher, J. H., Jr.; Baumann, T. F. J. Am. Chem. Soc. 2010, 132, 14067− 14069. (21) Yin, S. Y.; Niu, Z. Q.; Chen, X. D. Small 2012, 8, 2458−2463. (22) Cao, X. H.; Yin, Z. Y.; Zhang, H. Energy Environ. Sci. 2014, 7, 1850−1865. (23) Chen, K. F.; Song, S. Y.; Liu, F.; Xue, D. F. Chem. Soc. Rev. 2015, 44, 6230−6257. (24) Zhan, H. L.; Garrett, D. J.; Apollo, N. V.; Ganesan, K.; Lau, D.; Prawer, S.; Cervenka, J. Sci. Rep. 2016, 6, 19822−19830. (25) Li, T. T.; Li, N.; Liu, J. W.; Cai, K.; Foda, M. F.; Lei, X. M.; Han, H. Y. Nanoscale 2015, 7, 659−669. (26) Xu, Y. X.; Sheng, K. X.; Li, C.; Shi, G. Q. ACS Nano 2010, 4, 4324−4330. (27) Mao, S.; Lu, G. H.; Chen, J. H. Nanoscale 2015, 7, 6924−6943. (28) Xu, J.; Jiang, B. Y.; Su, J.; Xiang, Y.; Yuan, R.; Chai, Y. Q. Chem. Commun. 2012, 48, 3309−3311. (29) Sheikhzadeh, E.; Chamsaz, M.; Turner, A. P. F.; Jager, E. W. H.; Beni, V. Biosens. Bioelectron. 2016, 80, 194−200.

results indicate the potential of the developed electrochemical biosensing platform for biomolecular analysis in real samples.

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. Phone: 86-532-85953981. *E-mail: [email protected]. Phone: 86-532-85950873. Notes

The authors declare no competing financial interest. G

DOI: 10.1021/acs.analchem.6b03112 Anal. Chem. XXXX, XXX, XXX−XXX

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Analytical Chemistry (30) Cardoso, A. R.; Moreira, F. T. C.; Fernandes, R.; Sales, M. G. F. Biosens. Bioelectron. 2016, 80, 621−630. (31) Zhang, Y. Y.; Bai, X. Y.; Wang, X. M.; Shiu, K. K.; Zhu, Y. L.; Jiang, H. Anal. Chem. 2014, 86, 9459−9465. (32) Chen, X. J.; Wang, Y. Z.; Zhang, Y. Y.; Chen, Z. H.; Liu, Y.; Li, Z. L.; Li, J. H. Anal. Chem. 2014, 86, 4278−4286. (33) Han, L.; Yang, D. P.; Liu, A. H. Biosens. Bioelectron. 2015, 63, 145−152. (34) Yang, Y. K.; Cao, Y. Y.; Wang, X. M.; Fang, G. Z.; Wang, S. Biosens. Bioelectron. 2015, 64, 247−254. (35) Yang, Z. H.; Zhuo, Y.; Yuan, R.; Chai, Y. Q. Anal. Chem. 2016, 88, 5189−5196. (36) Wang, W. J.; Li, J. J.; Rui, K.; Gai, P. P.; Zhang, J. R.; Zhu, J. J. Anal. Chem. 2015, 87, 3019−3026. (37) Dong, Y. P.; Zhou, Y.; Wang, J.; Zhu, J. J. Anal. Chem. 2016, 88, 5469−5475. (38) Li, F. Q.; Xu, Y. M.; Yu, X.; Yu, Z. G.; Ji, H. R.; Song, Y. B.; Yan, H.; Zhang, G. L. Sens. Actuators, B 2016, 234, 648−657. (39) Wang, D.; Zheng, Y. N.; Chai, Y. Q.; Yuan, Y. L.; Yuan, R. Chem. Commun. 2015, 51, 10521−10523. (40) Xie, S. B.; Zhang, J.; Yuan, Y. L.; Chai, Y. Q.; Yuan, R. Chem. Commun. 2015, 51, 3387−3390. (41) Wang, H. Z.; Wang, Y.; Liu, S.; Yu, J. H.; Guo, Y. N.; Xu, Y.; Huang, J. D. Biosens. Bioelectron. 2016, 80, 471−476. (42) Li, F. Q.; Xu, Y. M.; Yu, X.; Yu, Z. G.; He, X. J.; Ji, H. R.; Dong, J. H.; Song, Y. B.; Yan, H.; Zhang, G. L. Biosens. Bioelectron. 2016, 82, 212−216. (43) Azimzadeh, M.; Rahaie, M.; Nasirizadeh, N.; Ashtari, K.; NaderiManesh, H. Biosens. Bioelectron. 2016, 77, 99−106. (44) Arabzadeh, A.; Salimi, A. Biosens. Bioelectron. 2015, 74, 270−276. (45) Vericat, C.; Vela, M. E.; Benitez, G.; Carro, P.; Salvarezza, R. C. Chem. Soc. Rev. 2010, 39, 1805−1834. (46) Wu, X.; Chai, Y.; Zhang, P.; Yuan, R. ACS Appl. Mater. Interfaces 2015, 7, 713−720. (47) Porstmann, B.; Jung, K.; Schmechta, H.; Evers, U.; Pergande, M.; Porstmann, T.; Kramm, H.-J.; Krause, H. Clin. Biochem. 1989, 22, 349−355.

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DOI: 10.1021/acs.analchem.6b03112 Anal. Chem. XXXX, XXX, XXX−XXX